Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

PubMed Central

Walter, Jens; Hertel, Christian; Tannock, Gerald W.; Lis, Claudia M.; Munro, Karen; Hammes, Walter P.

2001-01-01

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects. PMID:11375166

Association of Stremptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status.

EPA Science Inventory

Abstract Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study, we used a cultureindependent met...

SIMILARITY OF PARTICLE-ASSOCIATED AND FREE-LIVING BACTERIAL COMMUNITIES IN NORTHERN SAN FRANCISCO BAY, CALIFORNIA

EPA Science Inventory

We used denaturing gradient gel electrophoresis (DGGE) of 16S rDNA PCR amplicons to analyze the composition of Bacteria communities in samples collected during the summer, low flow season from northern San Francisco Bay, California. There were clear compositional differences in ...

Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis

PubMed Central

Temmerman, R.; Scheirlinck, I.; Huys, G.; Swings, J.

2003-01-01

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential. PMID:12513998

In vitro study of prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis.

PubMed

Bello, F D; Walter, J; Hertel, C; Hammes, W P

2001-07-01

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.

Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

PubMed Central

Meroth, Christiane B.; Walter, Jens; Hertel, Christian; Brandt, Markus J.; Hammes, Walter P.

2003-01-01

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. PMID:12514030

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens.

PubMed

Quinn, Robert A; Stevenson, Roselynn M W

2012-05-01

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.

Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis.

PubMed

Haruta, Shin; Ueno, Shintaro; Egawa, Isao; Hashiguchi, Kazunori; Fujii, Akira; Nagano, Masanobu; Ishii, Masaharu; Igarashi, Yasuo

2006-05-25

Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method.

Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis.

PubMed

Tanaka, Yasushi; Watanabe, Jun; Mogi, Yoshinobu

2012-08-01

Soy sauce is a traditional seasoning produced through the fermentation of soybeans and wheat using microbes. In this study, the microbial communities involved in the soy sauce manufacturing process were analyzed by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The bacterial DGGE profile indicated that the bacterial microbes in the koji were Weissella cibaria (Weissella confusa, Weissella kimchii, Weissella salipiscis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus iners, or Streptococcus thermophilus), Staphylococcus gallinarum (or Staphylococcus xylosus), and Staphylococcus kloosii. In addition to these bacteria, Tetragenococcus halophilus was also detected in the mash during lactic acid fermentation. The fungal DGGE profile indicated that the fungal microbes in the koji were not only Aspergillus oryzae but also several yeasts. In the mash, Zygosaccharomyces rouxii appeared in the early fermentation stage, Candida etchellsii (or Candida nodaensis) and Candida versatilis were detected at the middle fermentation stage, and Candida etchellsii was detected at the mature fermentation stage. These results suggest that the microbial communities present during the soy sauce manufacturing process change drastically throughout its production. This is the first report to reveal the microbial communities involved in the soy sauce manufacturing process using a culture-independent method. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Microbial Diversity during Fermentation of Sweet Paste, a Chinese Traditional Seasoning, Using PCR-Denaturing Gradient Gel Electrophoresis.

PubMed

Mao, Ping; Hu, Yuanliang; Liao, Tingting; Wang, Zhaoting; Zhao, Shumiao; Liang, Yunxiang; Hu, Yongmei

2017-04-28

The aim of this study was to elucidate the changes in the microbial community and biochemical properties of a traditional sweet paste during fermentation. PCR-denaturing gradient gel electrophoresis (DGGE) analysis showed that Aspergillus oryzae was the predominant species in the koji (the fungal mixture), and the majority of the fungi isolated belonged to two Zygosaccharomyces species in the mash. The bacterial DGGE profiles revealed the presence of Bacillus subtilis during fermentation, and Lactobacillus acidipiscis, Lactobacillus pubuzihii, Lactobacillus sp., Staphylococcus kloosi, and several uncultured bacteria were also detected in the mash after 14 days of main fermentation. Additionally, during main fermentation, amino-type nitrogen and total acid increased gradually to a maximum of 6.77 ± 0.25 g/kg and 19.10 ± 0.58 g/kg (30 days) respectively, and the concentration of reducing sugar increased to 337.41 ± 3.99 g/kg (7 days). The 180-day fermented sweet paste contained 261.46 ± 19.49 g/kg reducing sugar and its pH value remained at around 4.65. This study has used the PCR-DGGE technique to demonstrate the microbial community (including bacteria and fungi) in sweet paste and provides useful information (biochemical properties) about the assessment of the quality of sweet paste throughout fermentation.

Population structure and abundance of arsenite-oxidizing bacteria along an arsenic pollution gradient in waters of the upper isle River Basin, France.

PubMed

Quéméneur, Marianne; Cébron, Aurélie; Billard, Patrick; Battaglia-Brunet, Fabienne; Garrido, Francis; Leyval, Corinne; Joulian, Catherine

2010-07-01

Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB-carrying bacterial population, composed of members of the Alpha-, Beta- and Gammaproteobacteria, depending on arsenic (As) and E(h) levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters.

[Observation of genetic diversity in dental plaque of elder people with root caries].

PubMed

Ma, Shan-fen; Liang, Jing-ping; Jiang, Yun-tao; Zhu, Cai-lian

2011-08-01

Bacterial community in dental plaque of elder people was analyzed to learn about the microhabitat composition and diversity. Dental plaque samples were collected from 25 elders. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to evaluate the microbial diversity by displaying PCR-generated 16SrDNA fragments that migrate at different distances, reflecting the different sequence of fragment. SPSS12.0 software was used to analyze the variance of genotypes between different groups of bacteria. Genotypes of bacteria in dental plaques in the root caries group was significantly more than the other two groups. Crown caries group and caries-free group had no significant difference. The genetic diversity of the dental plaque microflora in the root caries group is significantly higher than coronal caries group and caries-free group.

Molecular approaches to analysing the microbial composition of raw milk and raw milk cheese.

PubMed

Quigley, Lisa; O'Sullivan, Orla; Beresford, Tom P; Ross, R Paul; Fitzgerald, Gerald F; Cotter, Paul D

2011-11-01

The availability and application of culture-independent tools that enable a detailed investigation of the microbiota and microbial biodiversity of food systems has had a major impact on food microbiology. This review focuses on the application of DNA-based technologies, such as denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE), single stranded conformation polymorphisms (SSCP), the polymerase chain reaction (PCR) and others, to investigate the diversity, dynamics and identity of microbes in dairy products from raw milk. Here, we will highlight the benefits associated with culture-independent methods which include enhanced sensitivity, rapidity and the detection of microorganisms not previously associated with such products. Copyright © 2011 Elsevier B.V. All rights reserved.

The use of PCR-DGGE to determine bacterial fingerprints for poultry and red meat abattoir effluent.

PubMed

de Smidt, O

2016-01-01

Strict legislation and chemical composition monitoring of effluent may be useful, but the data generated do not allow for source tracking, and enforcing legislation remains problematic in the South African setting. These difficulties emphasize the necessity for effluent source traceability. Denaturing gradient gel electrophoresis (DGGE) targeting the V3 region of the 16S rRNA gene was considered as fingerprinting technique for effluent originating from abattoirs slaughtering different animal species. The influence of treatment to remove excess fat from effluent prior to molecular analyses and different PCR approaches on the detection of bacterial diversity were considered. Use of a treatment option to remove fat and a nested PCR approach resulted in up to 51% difference in inter-sample diversity similarity. A robust approach with no pre-treatment to remove PCR inhibitors, such as fat, and direct amplification from genomic DNA yielded optimal/maximal bacterial diversity fingerprints. Repeatable fingerprints were obtained for poultry abattoir effluent over a 4-month period, but profiles for the red meat abattoir varied with maximum similarity detected only 33·2%. Genetic material from faecal indicators Aeromona spp and Clostridium spp were detected. Genera unique to each effluent were present; Anoxybacillus, Patulibacter and Oleispira in poultry abattoir effluent and Porphyromonas and Peptostreptococcus in red meat abattoir effluent. This study was the first to demonstrate the application of denaturing gradient gel electrophoresis (DGGE) to construct bacterial diversity fingerprints for high-throughput abattoir effluents. Proved redundancy of fat removal as PCR inhibitor and change in diversity similarity introduced by nested PCR approach. The importance of limiting excessive handling/processing which could lead to misrepresented diversity profiles was emphasized. © 2015 The Society for Applied Microbiology.

Community Analysis and Recovery of Phenol-degrading Bacteria from Drinking Water Biofilters

PubMed Central

Gu, Qihui; Wu, Qingping; Zhang, Jumei; Guo, Weipeng; Wu, Huiqing; Sun, Ming

2016-01-01

Phenol is a ubiquitous organic contaminant in drinking water. Biodegradation plays an important role in the elimination of phenol pollution in the environment, but the information about phenol removal by drinking water biofilters is still lacking. Herein, we study an acclimated bacterial community that can degrade over 80% of 300 mg/L phenol within 3 days. PCR detection of genotypes involved in bacterial phenol degradation revealed that the degradation pathways contained the initial oxidative attack by phenol hydroxylase, and subsequent ring fission by catechol 1,2-dioxygenase. Based on the PCR denatured gradient gel electrophoresis (PCR-DGGE) profiles of bacteria from biological activated carbon (BAC), the predominant bacteria in drinking water biofilters including Delftia sp., Achromobacter sp., and Agrobacterium sp., which together comprised up to 50% of the total microorganisms. In addition, a shift in bacterial community structure was observed during phenol biodegradation. Furthermore, the most effective phenol-degrading strain DW-1 that correspond to the main band in denaturing gradient gel electrophoresis (DGGE) profile was isolated and identified as Acinetobacter sp., according to phylogenetic analyses of the 16S ribosomal ribonucleic acid (rRNA) gene sequences. The strain DW-1 also produced the most important enzyme, phenol hydroxylase, and it also exhibited a good ability to degrade phenol when immobilized on granular active carbon (GAC). This study indicates that the enrichment culture has great potential application for treatment of phenol-polluted drinking water sources, and the indigenous phenol-degrading microorganism could recover from drinking water biofilters as an efficient resource for phenol removal. Therefore, the aim of this study is to draw attention to recover native phenol-degrading bacteria from drinking water biofilters, and use these native microorganisms as phenolic water remediation in drinking water sources. PMID:27148185

Community Analysis and Recovery of Phenol-degrading Bacteria from Drinking Water Biofilters.

PubMed

Gu, Qihui; Wu, Qingping; Zhang, Jumei; Guo, Weipeng; Wu, Huiqing; Sun, Ming

2016-01-01

Phenol is a ubiquitous organic contaminant in drinking water. Biodegradation plays an important role in the elimination of phenol pollution in the environment, but the information about phenol removal by drinking water biofilters is still lacking. Herein, we study an acclimated bacterial community that can degrade over 80% of 300 mg/L phenol within 3 days. PCR detection of genotypes involved in bacterial phenol degradation revealed that the degradation pathways contained the initial oxidative attack by phenol hydroxylase, and subsequent ring fission by catechol 1,2-dioxygenase. Based on the PCR denatured gradient gel electrophoresis (PCR-DGGE) profiles of bacteria from biological activated carbon (BAC), the predominant bacteria in drinking water biofilters including Delftia sp., Achromobacter sp., and Agrobacterium sp., which together comprised up to 50% of the total microorganisms. In addition, a shift in bacterial community structure was observed during phenol biodegradation. Furthermore, the most effective phenol-degrading strain DW-1 that correspond to the main band in denaturing gradient gel electrophoresis (DGGE) profile was isolated and identified as Acinetobacter sp., according to phylogenetic analyses of the 16S ribosomal ribonucleic acid (rRNA) gene sequences. The strain DW-1 also produced the most important enzyme, phenol hydroxylase, and it also exhibited a good ability to degrade phenol when immobilized on granular active carbon (GAC). This study indicates that the enrichment culture has great potential application for treatment of phenol-polluted drinking water sources, and the indigenous phenol-degrading microorganism could recover from drinking water biofilters as an efficient resource for phenol removal. Therefore, the aim of this study is to draw attention to recover native phenol-degrading bacteria from drinking water biofilters, and use these native microorganisms as phenolic water remediation in drinking water sources.

Population Structure and Abundance of Arsenite-Oxidizing Bacteria along an Arsenic Pollution Gradient in Waters of the Upper Isle River Basin, France▿ †

PubMed Central

Quéméneur, Marianne; Cébron, Aurélie; Billard, Patrick; Battaglia-Brunet, Fabienne; Garrido, Francis; Leyval, Corinne; Joulian, Catherine

2010-01-01

Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB-carrying bacterial population, composed of members of the Alpha-, Beta- and Gammaproteobacteria, depending on arsenic (As) and Eh levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters. PMID:20453153

Bacteria community study of combined periodontal-endodontic lesions using denaturing gradient gel electrophoresis and sequencing analysis.

PubMed

Li, Hong; Guan, Rui; Sun, Jinghua; Hou, Benxiang

2014-10-01

The entire microbial population and predominant microflora of root canals (RCs) and adjacent periodontal pockets (PPs) from teeth with combined periodontal-endodontic lesions were determined and compared. Pooled RC and PP samples were collected from the molars of 20 patients diagnosed with combined periodontal-endodontic lesions. DNA was extracted for polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequence analysis. A coefficient of similarity (Cs) was used to determine the similarity of the bacterial profiles from RCs and PPs. Significantly fewer bands were produced by PCR-DGGE from RCs (5.9 ± 1.7) than from PPs (8.0 ± 1.8) (P <0.001). The average Cs of the RC and PP samples was 93.81% ± 10.26%. Overall, 60 genera/species were identified by sequencing. Of these, the predominant genera in RCs were Porphyromonas sp. (13.9%), Filifactor sp. (12.5%), and Parvimonas sp. (11.1%), similar to the genera obtained from PP samples. In total, 43 genera/species were common to the RC and PP samples. The most prevalent bacteria in both the RC and PP samples were (in descending order) Filifactor alocis, Parvimonas micra, Porphyromonas gingivalis, and Tannerella forsythia. The high similarity in the sets of organisms present in both RC and PP samples in this study suggests that the pocket could be a source of RC infection. The data also demonstrate that combined periodontal-endodontic lesions consist of a diverse and complex microbial community.

Comparison of PCR-DGGE and PCR-SSCP analysis for bacterial flora of Japanese traditional fermented fish products, aji-narezushi and iwashi-nukazuke.

PubMed

An, Choa; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi

2010-08-30

The bacterial flora of two Japanese traditional fermented fish products, aji-narezushi (salted and long-fermented horse mackerel (Trachurus japonicas) with rice) and iwashi-nukazuke (salted and long-fermented sardine (Sardinops melanostica) with rice bran), was analysed using non-culture-based polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) and culture-based PCR single-strand conformation polymorphism (SSCP) methods. Viable plate counts in aji-narezushi and iwashi-nukazuke were about 6.3-6.6 and 5.7-6.9 log colony-forming units g(-1) respectively. In the PCR-DGGE analysis, Lactobacillus acidipiscis was detected as the predominant bacterium in two of three aji-narezushi samples, while Lactobacillus versmoldensis was predominant in the third sample. By the PCR-SSCP method, Lb. acidipiscis and Lactobacillus plantarum were isolated as the predominant bacteria, while Lb. versmoldensis was not detected. The predominant bacterium in two of three iwashi-nukazuke samples was Tetragenococcus muriaticus, while Tetragenococcus halophilus was predominant in the third sample. The results suggest that the detection of some predominant lactic acid bacteria species in fermented fish by cultivation methods is difficult. Copyright (c) 2010 Society of Chemical Industry.

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE).

PubMed

Yoshikawa, Shuji; Yasokawa, Daisuke; Nagashima, Koji; Yamazaki, Koji; Kurihara, Hideyuki; Ohta, Tomoki; Kawai, Yuji

2010-06-01

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 +/- 2.5 degrees C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor. 2009 Elsevier Ltd. All rights reserved.

Phylogenetic analysis of a biofilm bacterial population in a water pipeline in the Gulf of Mexico.

PubMed

López, Miguel A; Zavala-Díaz de la Serna, F Javier; Jan-Roblero, Janet; Romero, Juan M; Hernández-Rodríguez, César

2006-10-01

The aim of this study was to assess the bacterial diversity associated with a corrosive biofilm in a steel pipeline from the Gulf of Mexico used to inject marine water into the oil reservoir. Several aerobic and heterotrophic bacteria were isolated and identified by 16S rRNA gene sequence analysis. Metagenomic DNA was also extracted to perform a denaturing gradient gel electrophoresis analysis of ribosomal genes and to construct a 16S rRNA gene metagenomic library. Denaturing gradient gel electrophoresis profiles and ribosomal libraries exhibited a limited bacterial diversity. Most of the species detected in the ribosomal library or isolated from the pipeline were assigned to Proteobacteria (Halomonas spp., Idiomarina spp., Marinobacter aquaeolei, Thalassospira sp., Silicibacter sp. and Chromohalobacter sp.) and Bacilli (Bacillus spp. and Exiguobacterium spp.). This is the first report that associates some of these bacteria with a corrosive biofilm. It is relevant that no sulfate-reducing bacteria were isolated or detected by a PCR-based method. The diversity and relative abundance of bacteria from water pipeline biofilms may contribute to an understanding of the complexity and mechanisms of metal corrosion during marine water injection in oil secondary recovery.

COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing.

PubMed

Castellanos-Rizaldos, Elena; Milbury, Coren A; Guha, Minakshi; Makrigiorgos, G Mike

2014-01-01

Detection of low-level mutations is important for cancer biomarker and therapy targets discovery, but reliable detection remains a technical challenge. The newly developed method of CO-amplification at Lower Denaturation temperature PCR (COLD-PCR) helps to circumvent this issue. This PCR-based technology preferentially enriches minor known or unknown variants present in samples with a high background of wild type DNA which often hampers the accurate identification of these minority alleles. This is a simple process that consists of lowering the temperature at the denaturation step during the PCR-cycling protocol (critical denaturation temperature, T c) and inducing DNA heteroduplexing during an intermediate step. COLD-PCR in its simplest forms does not need additional reagents or specific instrumentation and thus, can easily replace conventional PCR and at the same time improve the mutation detection sensitivity limit of downstream technologies. COLD-PCR can be applied in two basic formats: fast-COLD-PCR that can enrich T m-reducing mutations and full-COLD-PCR that can enrich all mutations, though it requires an intermediate cross-hybridization step that lengthens the thermocycling program. An improved version of full-COLD-PCR (improved and complete enrichment, ice-COLD-PCR) has also been described. Finally, most recently, we developed yet another form of COLD-PCR, temperature-tolerant-COLD-PCR, which gradually increases the denaturation temperature during the COLD-PCR reaction, enriching diverse targets using a single cycling program. This report describes practical considerations for application of fast-, full-, ice-, and temperature-tolerant-COLD-PCR for enrichment of mutations prior to downstream screening.

Lessons Learned on Bioaugmentation of DNAPL Source Zone Areas

DTIC Science & Technology

2007-10-01

but rather have stringers, ganglia or blobs that can create an “effective pool length”. As the leading edge of these discontinuous DNAPL free-phases...terminal restriction fragment length polymorphism (T-RFLP), denaturing gradient gel electrophoresis (DGGE), and fluorescent in situ hybridization ( FISH ...question of interest (e.g. PCR, FISH , DGGE); (ii) sampling location(s); (iii) an appropriate sampling procedure; and (iv) an appropriate sample handling

Association of diverse bacterial communities in human bile samples with biliary tract disorders: a survey using culture and polymerase chain reaction-denaturing gradient gel electrophoresis methods.

PubMed

Tajeddin, E; Sherafat, S J; Majidi, M R S; Alebouyeh, M; Alizadeh, A H M; Zali, M R

2016-08-01

Bacterial infection is considered a predisposing factor for disorders of the biliary tract. This study aimed to determine the diversity of bacterial communities in bile samples and their involvement in the occurrence of biliary tract diseases. A total of 102 bile samples were collected during endoscopic retrograde cholangiopancreatography (ERCP). Characterization of bacteria was done using culture and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) methods. Antimicrobial susceptibility of the isolates was determined based on the Clinical and Laboratory Standards Institute (CLSI) guidelines and identity of the nucleotide sequences of differentiated bands from the DGGE gels was determined based on GenBank data. In total, 41.2 % (42/102) of the patients showed bacterial infection in their bile samples. This infection was detected in 21 % (4/19), 45.4 % (5/11), 53.5 % (15/28), and 54.5 % (24/44) of patients with common bile duct stone, microlithiasis, malignancy, and gallbladder stone, respectively. Escherichia coli showed a significant association with gallstones. Polymicrobial infection was detected in 48 % of the patients. While results of the culture method established coexistence of biofilm-forming bacteria (Pseudomonas aeruginosa, E. coli, Klebsiella pneumoniae, Enterococcus spp., and Acinetobacter spp.) in different combinations, the presence of Capnocytophaga spp., Lactococcus spp., Bacillus spp., Staphylococcus haemolyticus, Enterobacter or Citrobacter spp., Morganella spp., Salmonella spp., and Helicobacter pylori was also characterized in these samples by the PCR-DGGE method. Multidrug resistance phenotypes (87.5 %) and resistance to third- and fourth-generation cephalosporins and quinolones were common in these strains, which could evolve through their selection by bile components. Ability for biofilm formation seems to be a need for polymicrobial infection in this organ.

Detection by denaturing gradient gel electrophoresis of ammonia-oxidizing bacteria in microcosms of crude oil-contaminated mangrove sediments.

PubMed

dos Santos, A C F; Marques, E L S; Gross, E; Souza, S S; Dias, J C T; Brendel, M; Rezende, R P

2012-01-27

Currently, the effect of crude oil on ammonia-oxidizing bacterium communities from mangrove sediments is little understood. We studied the diversity of ammonia-oxidizing bacteria in mangrove microcosm experiments using mangrove sediments contaminated with 0.1, 0.5, 1, 2, and 5% crude oil as well as non-contaminated control and landfarm soil from near an oil refinery in Camamu Bay in Bahia, Brazil. The evolution of CO(2) production in all crude oil-contaminated microcosms showed potential for mineralization. Cluster analysis of denaturing gradient gel electrophoresis-derived samples generated with primers for gene amoA, which encodes the functional enzyme ammonia monooxygenase, showed differences in the sample contaminated with 5% compared to the other samples. Principal component analysis showed divergence of the non-contaminated samples from the 5% crude oil-contaminated sediment. A Venn diagram generated from the banding pattern of PCR-denaturing gradient gel electrophoresis was used to look for operational taxonomic units (OTUs) in common. Eight OTUs were found in non-contaminated sediments and in samples contaminated with 0.5, 1, or 2% crude oil. A Jaccard similarity index of 50% was found for samples contaminated with 0.1, 0.5, 1, and 2% crude oil. This is the first study that focuses on the impact of crude oil on the ammonia-oxidizing bacterium community in mangrove sediments from Camamu Bay.

Application of denaturing high-performance liquid chromatography for monitoring sulfate-reducing bacteria in oil fields.

PubMed

Priha, Outi; Nyyssönen, Mari; Bomberg, Malin; Laitila, Arja; Simell, Jaakko; Kapanen, Anu; Juvonen, Riikka

2013-09-01

Sulfate-reducing bacteria (SRB) participate in microbially induced corrosion (MIC) of equipment and H2S-driven reservoir souring in oil field sites. Successful management of industrial processes requires methods that allow robust monitoring of microbial communities. This study investigated the applicability of denaturing high-performance liquid chromatography (DHPLC) targeting the dissimilatory sulfite reductase ß-subunit (dsrB) gene for monitoring SRB communities in oil field samples from the North Sea, the United States, and Brazil. Fifteen of the 28 screened samples gave a positive result in real-time PCR assays, containing 9 × 10(1) to 6 × 10(5) dsrB gene copies ml(-1). DHPLC and denaturing gradient gel electrophoresis (DGGE) community profiles of the PCR-positive samples shared an overall similarity; both methods revealed the same samples to have the lowest and highest diversity. The SRB communities were diverse, and different dsrB compositions were detected at different geographical locations. The identified dsrB gene sequences belonged to several phylogenetic groups, such as Desulfovibrio, Desulfococcus, Desulfomicrobium, Desulfobulbus, Desulfotignum, Desulfonatronovibrio, and Desulfonauticus. DHPLC showed an advantage over DGGE in that the community profiles were very reproducible from run to run, and the resolved gene fragments could be collected using an automated fraction collector and sequenced without a further purification step. DGGE, on the other hand, included casting of gradient gels, and several rounds of rerunning, excising, and reamplification of bands were needed for successful sequencing. In summary, DHPLC proved to be a suitable tool for routine monitoring of the diversity of SRB communities in oil field samples.

Molecular diagnostics of periodontitis.

PubMed

Korona-Głowniak, Izabela; Siwiec, Radosław; Berger, Marcin; Malm, Anna; Szymańska, Jolanta

2017-01-28

The microorganisms that form dental plaque are the main cause of periodontitis. Their identification and the understanding of the complex relationships and interactions that involve these microorganisms, environmental factors and the host's health status enable improvement in diagnostics and targeted therapy in patients with periodontitis. To this end, molecular diagnostics techniques (both techniques based on the polymerase chain reaction and those involving nucleic acid analysis via hybridization) come increasingly into use. On the basis of a literature review, the following methods are presented: polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), 16S rRNA-encoding gene sequencing, checkerboard and reverse-capture checkerboard hybridization, microarrays, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), as well as terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). The advantages and drawbacks of each method in the examination of periopathogens are indicated. The techniques listed above allow fast detection of even small quantities of pathogen present in diagnostic material and prove particularly useful to detect microorganisms that are difficult or impossible to grow in a laboratory.

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR.

PubMed

Garofalo, Cristiana; Bancalari, Elena; Milanović, Vesna; Cardinali, Federica; Osimani, Andrea; Sardaro, Maria Luisa Savo; Bottari, Benedetta; Bernini, Valentina; Aquilanti, Lucia; Clementi, Francesca; Neviani, Erasmo; Gatti, Monica

2017-02-02

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug -1 . Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacterial Population Changes in a Membrane Bioreactor for Graywater Treatment Monitored by Denaturing Gradient Gel Electrophoretic Analysis of 16S rRNA Gene Fragments

PubMed Central

Stamper, David M.; Walch, Marianne; Jacobs, Rachel N.

2003-01-01

The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD5), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time. PMID:12571004

Fate of a metal-resistant inoculum in contaminated and pristine soils assessed by denaturing gradient gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephen, J.R.; Chang, Y.J.; MacNaughton, S.J.

Cesium, cadmium, cobalt, and strontium are four contaminants frequently found in soils at biotoxic levels. Introduction of certain nongenetically modified bacteria has been frequently suggested as a method for the immobilization of heavy metal contaminants in soil, thereby reducing runoff and bioavailability. In this study, the authors have used the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to track the survival of the five bacterial species added to soil microcosms with and without the addition of a mixture of these metals. The PCR primers targeted conserved regions of the 165 rDNA molecular present in all bacteria. Themore » reaction products were shown to reflect the relative abundance of the bacteria both in mixtures of pure cultures and against a background of all the eubacterial species present in the soil following inoculation. Three of the species (Pseudomonas aeruginosa FRD-1, Shewanella putrifaciens 200, and Desulfovibrio vulgaris Hildenborough) decreased rapidly following inoculation into both soils. The proportion of Alcaligenes eutrophus CH34 remained at a constant level throughout the 8-week experiment in both soil treatments. Sphingomonas aromaticivorans B0695 showed toxic metal-dependent survival in that its relative abundance dropped rapidly in pristine soil but remained at approximately inoculation levels throughout the experiment in contaminated microcosms.« less

Bacterial population changes in a membrane bioreactor for graywater treatment monitored by denaturing gradient gel electrophoretic analysis of 16S rRNA gene fragments.

PubMed

Stamper, David M; Walch, Marianne; Jacobs, Rachel N

2003-02-01

The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD(5)), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time.

Bacterial analysis of combined periodontal-endodontic lesions by polymerase chain reaction-denaturing gradient gel electrophoresis.

PubMed

Xia, Minghui; Qi, Qingguo

2013-01-01

We used denaturing gradient gel electrophoresis (DGGE) to compare bacterial profiles in periodontium and root canals of teeth with combined periodontal-endodontic lesions. Samples of dental plaque and necrotic pulp were collected from thirteen extracted teeth with advanced periodontitis. Genomic DNA was extracted for polymerase chain reaction (PCR) analysis using universal bacterial primers. The PCR products were then loaded onto DGGE gels to obtain fractionated bands. Characteristic DGGE bands were excised and DNA was cloned and sequenced. The number of bands, which indicates the number of bacterial species, was compared between dental plaques and necrotic pulp tissues from the same tooth. Although the difference was statistically significant (P < 0.01), there was no positive correlation; similarity (Dice coefficient) was 13.1% to 62.5%. Some bacteria species were present in both the periodontal pockets and root canals of the same tooth; however, periodontal bacteria did not always invade the root canals, and some bacteria in root canals were not present in periodontal pockets of the same tooth. In some teeth, unique bacteria in root canals had not passed from periodontal pockets. A basic local alignment search tool (BLAST) sequence search in Genbank indicated that new bacteria species were present in periodontal pockets and root canals. Their characteristics must thus be further analyzed.

Efficacy of rifaximin vaginal tablets in treatment of bacterial vaginosis: a molecular characterization of the vaginal microbiota.

PubMed

Cruciani, Federica; Brigidi, Patrizia; Calanni, Fiorella; Lauro, Vittoria; Tacchi, Raffaella; Donders, Gilbert; Peters, Klaus; Guaschino, Secondo; Vitali, Beatrice

2012-08-01

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the vaginal bacterial morphotypes, associated with sexually transmitted infections and adverse pregnancy outcomes. The purpose of the present study was to evaluate the impact of different doses of rifaximin vaginal tablets (100 mg/day for 5 days, 25 mg/day for 5 days, and 100 mg/day for 2 days) on the vaginal microbiota of 102 European patients with BV enrolled in a multicenter, double-blind, randomized, placebo-controlled study. An integrated molecular approach based on quantitative PCR (qPCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to investigate the effects of vaginal tablets containing the antibiotic. An increase in members of the genus Lactobacillus and a decrease in the BV-related bacterial groups after the antibiotic treatment were demonstrated by qPCR. PCR-DGGE profiles confirmed the capability of rifaximin to modulate the composition of the vaginal microbial communities and to reduce their complexity. This molecular analysis supported the clinical observation that rifaximin at 25 mg/day for 5 days represents an effective treatment to be used in future pivotal studies for the treatment of BV.

Efficacy of Rifaximin Vaginal Tablets in Treatment of Bacterial Vaginosis: a Molecular Characterization of the Vaginal Microbiota

PubMed Central

Cruciani, Federica; Brigidi, Patrizia; Calanni, Fiorella; Lauro, Vittoria; Tacchi, Raffaella; Donders, Gilbert; Peters, Klaus; Guaschino, Secondo

2012-01-01

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the vaginal bacterial morphotypes, associated with sexually transmitted infections and adverse pregnancy outcomes. The purpose of the present study was to evaluate the impact of different doses of rifaximin vaginal tablets (100 mg/day for 5 days, 25 mg/day for 5 days, and 100 mg/day for 2 days) on the vaginal microbiota of 102 European patients with BV enrolled in a multicenter, double-blind, randomized, placebo-controlled study. An integrated molecular approach based on quantitative PCR (qPCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to investigate the effects of vaginal tablets containing the antibiotic. An increase in members of the genus Lactobacillus and a decrease in the BV-related bacterial groups after the antibiotic treatment were demonstrated by qPCR. PCR-DGGE profiles confirmed the capability of rifaximin to modulate the composition of the vaginal microbial communities and to reduce their complexity. This molecular analysis supported the clinical observation that rifaximin at 25 mg/day for 5 days represents an effective treatment to be used in future pivotal studies for the treatment of BV. PMID:22585228

Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

PubMed

Flórez, Ana Belén; Mayo, Baltasar

2015-12-02

This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 μg ml(-1)) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird-Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5-1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison among amoA Primers Suited for Quantification and Diversity Analyses of Ammonia-Oxidizing Bacteria in Soil

PubMed Central

Shimomura, Yumi; Morimoto, Sho; Hoshino, Yuko Takada; Uchida, Yoshitaka; Akiyama, Hiroko; Hayatsu, Masahito

2012-01-01

Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment. PMID:22075625

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

NASA Technical Reports Server (NTRS)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

PubMed Central

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

Evaluation of different PCR primers for denaturing gradient gel electrophoresis (DGGE) analysis of fungal community structure in traditional fermentation starters used for Hong Qu glutinous rice wine.

PubMed

Lv, Xu-Cong; Jiang, Ya-Jun; Liu, Jie; Guo, Wei-Ling; Liu, Zhi-Bin; Zhang, Wen; Rao, Ping-Fan; Ni, Li

2017-08-16

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial community structure. However, when DGGE is applied to evaluate the fungal community of traditional fermentation starters, the choice of hypervariable ribosomal RNA gene regions is still controversial. In the current study, several previously published fungal PCR primer sets were compared and evaluated using PCR-DGGE, with the purpose of screening a suitable primer set to study the fungal community of traditional fermentation starters for Hong Qu glutinous rice wine. Firstly, different primer sets were used to amplify different hypervariable regions from pure fungal cultures. Except NS1/FR1+ and ITS1fGC/ITS4, other primer sets (NL1+/LS2R, NL3A/NL4GC, FF390/FR1+, NS1/GCFung, NS3+/YM951r and ITS1fGC/ITS2r) amplified the target DNA sequences successfully. Secondly, the selected primer sets were further evaluated based on their resolution to distinguish different fungal cultures through DGGE fingerprints. Three primer sets (NL1+/LS2R, NS1/GCFung and ITS1fGC/ITS2r) were finally selected for investigating the fungal community structure of different traditional fermentation starters for Hong Qu glutinous rice wine. The internal transcribed spacer (ITS) region amplified by ITS1fGC/ITS2r, which is more hypervariable than the 18S rRNA gene and 26S rRNA gene, provides an excellent tool to separate amplification products of different fungal species. Results indicated that PCR-DGGE profile using ITS1fGC/ITS2r showed more abundant fungal species than that using NL1+/LS2R and NS1/GCFung. Therefore, ITS1fGC/ITS2r is the most suitable primer set for PCR-DGGE analysis of fungal community structure in traditional fermentation starters for Hong Qu glutinous rice wine. DGGE profiles based on ITS1fGC/ITS2r revealed the presence of twenty-four fungal species in traditional fermentation starter. A significant difference of fungal community can be observed directly from DGGE fingerprints and principal component analysis. The statistical analysis results based on the band intensities of fungal DGGE profile showed that Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Rhizopus oryzae, Monascus purpureus and Aspergillus niger were the dominant fungal species. In conclusion, the comparison of several primer sets for fungal PCR-DGGE would be useful to enrich our knowledge of the fungal community structures associated with traditional fermentation starters, which may facilitate the development of better starter cultures for manufacturing Chinese Hong Qu glutinous rice wine. Copyright © 2017 Elsevier B.V. All rights reserved.

Soil Microbial Community Structure across a Thermal Gradient following a Geothermal Heating Event

PubMed Central

Norris, Tracy B.; Wraith, Jon M.; Castenholz, Richard W.; McDermott, Timothy R.

2002-01-01

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65°C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50°C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50°C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them. PMID:12450855

Soil microbial community structure across a thermal gradient following a geothermal heating event.

PubMed

Norris, Tracy B; Wraith, Jon M; Castenholz, Richard W; McDermott, Timothy R

2002-12-01

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65 degrees C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50 degrees C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50 degrees C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.

COLD-PCR: improving the sensitivity of molecular diagnostics assays

PubMed Central

Milbury, Coren A; Li, Jin; Liu, Pingfang; Makrigiorgos, G Mike

2011-01-01

The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold. PMID:21405967

Human Papillomavirus Genotyping After Denaturation of Specimens for Hybrid Capture 2 Testing: Feasibility Study for the HPV Persistence and Progression Cohort†

PubMed Central

LaMere, Brandon J.; Kornegay, Janet; Fetterman, Barbara; Sadorra, Mark; Shieh, Jen; Castle, Philip E.

2009-01-01

Human papillomavirus (HPV) genotyping could be clinically useful, depending on the results of large, prospective studies like the HPV Persistence and Progression cohort. The cohort is based on genotyping and follow-up of Hybrid Capture-positive women at Kaiser Permanente, Northern California. HPV DNA testing by Hybrid Capture 2 requires denaturation with alkali, possibly damaging the DNA for optimal PCR-based genotyping. A feasibility study was conducted on paired aliquots of anonymized specimens from 100 women with low-grade intraepithelial lesion cytology. Test aliquots were left in denaturant for 10 or 18 hours at 4°C and then neutralized; comparison aliquots were not denatured but diluted to match the timing, temperature, concentration and salt conditions of the treated specimens. The masked aliquots were tested using a commercialized PCR-based assay that detects of 37 HPV genotypes. There was no overall effect of treatment on test positivity or number of types. HPV16 was marginally more likely to be detected in untreated versus treated aliquots (P = 0.09) but HPV45 was marginally more likely to be detected in treated than untreated aliquots (P = 0.07), suggesting that these differences represented chance (intra-test variability). It can be concluded that residual Hybrid Capture-positive specimens can be accurately genotyped by PCR after Hybrid Capture 2 processing. PMID:17673302

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ngo, K.Y.; Liu, D.; Lee, J.

During the past two years we have tested 2,300 Southeast Asians for alpha- and beta-thaleassemia mutations. We found the incidence of hemoglobin E ({beta}{sup 26}) to be 47% among Laotians and 38% among Cambodians. The incidence of beta thalassemia trait is 9% for Laotians and 6% for Cambodians. Thus, the risk for hemoglobin E/{beta}{sup 26} thalassemia, a transfusion-dependent disorder, is increased in these two population groups. Denaturing gradient gel electrophoresis (DGGE) has proven to be useful in testing for beta-thalassemia carriers and identifying new mutations in the beta globin gene. DNA was extracted from venous blood obtained from patients withmore » elevated Hgb A2 (>4%). Five DNA fragments, encompassing the beta globin gene cluster, were amplified by PCR and analyzed, along with known beta gene mutations as controls, by DGGE using different denaturing gradient concentrations. Different mutations at the same nucleotide position can be distinguished by migration pattern on the DGGE (e.g., in IVS-I-1, G{r_arrow}A and T). Compound heterozygotes for {beta}-thalassemia can be detected on the same gel (e.g., HbE/mutation codon 17). New mutations are identified by their migration pattern compared with controls and determined by subsequent sequencing. We have identified three new mutations: codon 82 CAA{r_arrow}AAA in one Cambodian patient; IVS-II-667, T{r_arrow}C and IVS-II-672, A{r_arrow}C in two Laotian patients. When the parent`s genotypes are known, prenatal diagnosis can be obtained within 24 hours. Thus, PCR/DGGE combination is a rapid and reliable diagnostic approach to clinically significant {beta}-thalassemia. The most important steps are carefully designed primers and predetermined gradient concentrations for DGGE.« less

Autoclave method for rapid preparation of bacterial PCR-template DNA.

PubMed

Simmon, Keith E; Steadman, Dewey D; Durkin, Sarah; Baldwin, Amy; Jeffrey, Wade H; Sheridan, Peter; Horton, Rene; Shields, Malcolm S

2004-02-01

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.

In vitro fermentation behaviors of fucosylated chondroitin sulfate from Pearsonothuria graeffei by human gut microflora.

PubMed

Wei, Chao-Yang; Liao, Ning-Bo; Zhang, Yu; Ye, Xing-Qian; Li, Shan; Hu, Ya-Qin; Liu, Dong-Hong; Linhardt, Robert J; Wang, Xin; Chen, Shi-Guo

2017-09-01

A fucosylated chondroitin sulfate (FCS-pg) with highly repeated structure from Pearsonothuria graeffei was subjected to a in vitro fermentation model to investigate its fermentability and effects on human gut microflora. High performance liquid chromatography (HPLC) measurement found FCS-pg can be fermented to short chain fatty acids (SCFAs) by gut microflora from partial human fecal samples. 16S rRNA gene-based polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) profiling and real-time quantitative PCR analysis showed that FCS-pg mainly increased the proportions of Clostridium cluster XI, Bacteriodes prevotella group, Bifidobacterium genus, Clostridium cluster I and Clostridium cluster XIVab, whereas the numbers of the Enterobacteriaceae and Lactobacillus decreased. These results indicated that FCS-pg was mainly fermented by Bacteroides, Bifidobacterium and Clostridium. It increased the content of probiotics bacteria in achieving health-enhancing effect, was slightly different than most sulfated polysaccharides from marine animals. The current study provides useful new information on the mechanism of absorption and functional activity on FCS-pg within the gastrointestinal tract of the human body. Copyright © 2017. Published by Elsevier B.V.

Pyrosequencing®-Based Identification of Low-Frequency Mutations Enriched Through Enhanced-ice-COLD-PCR.

PubMed

How-Kit, Alexandre; Tost, Jörg

2015-01-01

A number of molecular diagnostic assays have been developed in the last years for mutation detection. Although these methods have become increasingly sensitive, most of them are incompatible with a sequencing-based readout and require prior knowledge of the mutation present in the sample. Consequently, coamplification at low denaturation (COLD)-PCR-based methods have been developed and combine a high analytical sensitivity due to mutation enrichment in the sample with the identification of known or unknown mutations by downstream sequencing experiments. Among these methods, the recently developed Enhanced-ice-COLD-PCR appeared as the most powerful method as it outperformed the other COLD-PCR-based methods in terms of the mutation enrichment and due to the simplicity of the experimental setup of the assay. Indeed, E-ice-COLD-PCR is very versatile as it can be used on all types of PCR platforms and is applicable to different types of samples including fresh frozen, FFPE, and plasma samples. The technique relies on the incorporation of an LNA containing blocker probe in the PCR reaction followed by selective heteroduplex denaturation enabling amplification of the mutant allele while amplification of the wild-type allele is prevented. Combined with Pyrosequencing(®), which is a very quantitative high-resolution sequencing technology, E-ice-COLD-PCR can detect and identify mutations with a limit of detection down to 0.01 %.

Optimization of β-glucan synthase gene primers for molecular DNA fingerprinting in Pleurotus pulmonarious

NASA Astrophysics Data System (ADS)

Kadir, Zaiton Abdul; Daud, Fauzi; Mohamad, Azhar; Senafi, Sahidan; Jamaludin, Ferlynda Fazleen

2015-09-01

Pleurotus pulmonarius is an edible mushroom in Malaysia and commonly known as Oyster mushroom. The species are important not only for nutritional values but also for pharmaceutical importance related to bioactive compounds in polysaccharides such as β glucan. Hence, β-glucan synthase gene (BGS) pathways which are related to the production of the β-glucan might be useful as marker for molecular DNA fingerprinting in P. pulmonarius. Conserved regions of β-glucan gene were mined from public database and aligned. Consensus from the alignment was used to design the primers by using Primer 3 software. Eight primers were designed and a single primer pair (BGF3: 5' TCTTGGCGAGTTCGAAGAAT 3'; BGR3: 5' TTCCGATCTTGGTCTGGAAG 3') was optimized at Ta (annealing temperature) 57.1°C to produce PCR product ranging from 400-500 bp. Optimum components for PCR reactions were 5.0 µl of 10× PCR buffer, 1.5 µl of 25 mM MgCl2, 1 µl of 10 mM dNTP, 1 µl of β-glucan primers, 0.1 µl of 5 units/ml Taq polymerase and 2 µl DNA template. PCR program was set at 34 PCR cycles by using Bio-Rad T100 Thermal Cycler. Initial denaturation was set at 94°C for 2 min, denaturation at 94°C for 1 minute, primer annealing at 45°C to 60°C (gradient temperature) for 50 seconds, followed by elongation at 72°C for 1 minute and further extension 5 minutes for last cycle PCR prior to end the program cycle. Thus, this information revealed that the primer of β-glucan gene designed could be used as targeted markers in screening population strains of P. pulmonarius.

Abundance and Genetic Diversity of nifH Gene Sequences in Anthropogenically Affected Brazilian Mangrove Sediments

PubMed Central

Dias, Armando Cavalcante Franco; Pereira e Silva, Michele de Cassia; Cotta, Simone Raposo; Dini-Andreote, Francisco; Soares, Fábio Lino; Salles, Joana Falcão; Azevedo, João Lúcio; van Elsas, Jan Dirk

2012-01-01

Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies. PMID:22941088

Abundance and genetic diversity of nifH gene sequences in anthropogenically affected Brazilian mangrove sediments.

PubMed

Dias, Armando Cavalcante Franco; Pereira e Silva, Michele de Cassia; Cotta, Simone Raposo; Dini-Andreote, Francisco; Soares, Fábio Lino; Salles, Joana Falcão; Azevedo, João Lúcio; van Elsas, Jan Dirk; Andreote, Fernando Dini

2012-11-01

Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies.

Detection of the Dinozoans Pfiesteria piscicida and P. shumwayae: a review of detection methods and geographic distribution.

PubMed

Rublee, Parke A; Remington, David L; Schaefer, Eric F; Marshall, Michael M

2005-01-01

Molecular methods, including conventional PCR, real-time PCR, denaturing gradient gel electrophoresis, fluorescent fragment detection PCR, and fluorescent in situ hybridization, have all been developed for use in identifying and studying the distribution of the toxic dinoflagellates Pfiesteria piscicida and P. shumwayae. Application of the methods has demonstrated a worldwide distribution of both species and provided insight into their environmental tolerance range and temporal changes in distribution. Genetic variability among geographic locations generally appears low in rDNA genes, and detection of the organisms in ballast water is consistent with rapid dispersal or high gene flow among populations, but additional sequence data are needed to verify this hypothesis. The rapid development and application of these tools serves as a model for study of other microbial taxa and provides a basis for future development of tools that can simultaneously detect multiple targets.

Effect of nitrate injection on the bacterial community in a water-oil tank system analyzed by PCR-DGGE.

PubMed

Jurelevicius, Diogo; von der Weid, Irene; Korenblum, Elisa; Valoni, Erika; Penna, Mônica; Seldin, Lucy

2008-04-01

Sulfide production by sulfate-reducing bacteria (SRB) is a major concern for the petroleum industry since it is toxic and corrosive, and causes plugging due to the formation of insoluble iron sulfides (reservoir souring). In this study, PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) using two sets of primers based on the 16S rRNA gene and on the aps gene (adenosine-5-phosphosulfate reductase) was used to track changes in the total bacterial and SRB communities, respectively, present in the water-oil tank system on an offshore platform in Brazil in which nitrate treatment was applied for 2 months (15 nitrate injections). PCR-DGGE analysis of the total bacterial community showed the existence of a dominant population in the water-oil tank, and that the appearance and/or the increase of intensity of some bands in the gels were not permanently affected by the introduction of nitrate. On the other hand, the SRB community was stimulated following nitrate treatment. Moreover, sulfide production did not exceed the permissible exposure limit in the water-oil separation tank studied treated with nitrate. Therefore, controlling sulfide production by treating the produced water tank with nitrate could reduce the quantity of chemical biocides required to control microbial activities.

Molecular-based environmental risk assessment of three varieties of genetically engineered cows.

PubMed

Xu, Jianxiang; Zhao, Jie; Wang, Jianwu; Zhao, Yaofeng; Zhang, Lei; Chu, Mingxing; Li, Ning

2011-10-01

The development of animal biotechnology has led to an increase in attention to biosafety issues. Here we evaluated the impact of genetically engineered cows on the environment. The probability of horizontal gene transfer and the impact on the microbial communities in cow gut and soil were tested using three varieties of genetically engineered cows that were previously transformed with a human gene encoding lysozyme, lactoferrin, or human alpha lactalbumin. The results showed that the transgenes were not detectable by polymerase chain reaction (PCR) or quantitative real-time PCR in gut microbial DNA extracts of manure or microbial DNA extracts of topsoil. In addition, the transgenes had no impact on the microbial communities in cow gut or soil as assessed by PCR-denaturing gradient gel electrophoresis or 16S rDNA sequencing. Furthermore, phylogenetic analyses showed that the manure bacteria sampled during each of the four seasons belonged primarily to two groups, Firmicutes and Bacteroidetes, and the soil bacteria belonged to four groups, Firmicutes, Bacteroidetes, Actinobacteria, and α-proteobacteria. Other groups, such as β-proteobacteria, γ-proteobacteria, δ-proteobacteria, ε-proteobacteria, Spirochaetes, Acidobacteria, Chloroflexi, and Nitrospira, were not dominant in the manure or soil.

Co-amplification at lower denaturation temperature-PCR: methodology and applications.

PubMed

Liang, Hui; Chen, Guo-Jie; Yu, Yan; Xiong, Li-Kuan

2018-03-20

Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance mutations from mixtures of wild-type and mutation-containing sequences, enriching the mutation 10 to 100 folds. Due to the slightly altered melting temperature (Tm) of the double-stranded DNA and the formation of the mutation/wild-type heteroduplex DNA, COLD-PCR methods are sensitive, specific, accurate, cost-effective and easy to maneuver, and can enrich mutations of any type and at any position, even unknown mutations within amplicons. COLD-PCR and its improved methods are now applied in cancer, microorganisms, prenatal screening, animals and plants. They are extremely useful for early diagnosis, monitoring the prognosis of disease and the efficiency of the treatment, drug selection, prediction of prognosis, plant breeding and etc. In this review, we introduce the principles, key techniques, derived methods and applications of COLD-PCR.

PCR-denaturing gradient gel electrophoresis analysis of microbial community in soy-daddawa, a Nigerian fermented soybean (Glycine max (L.) Merr.) condiment.

PubMed

Ezeokoli, Obinna T; Gupta, Arvind K; Mienie, Charlotte; Popoola, Temitope O S; Bezuidenhout, Cornelius C

2016-03-02

Soy-daddawa, a fermented soybean (Glycine max (L.) Merr.) condiment, plays a significant role in the culinary practice of West Africa. It is essential to understand the microbial community of soy-daddawa for a successful starter culture application. This study investigated the microbial community structure of soy-daddawa samples collected from Nigerian markets, by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the V3-V5 region of the 16S rRNA gene of bacteria and internal transcribed spacer 2 (ITS2) region of fungi. Six bacterial and 16 fungal (nine yeasts and seven molds) operational taxonomic units (OTUs)/species were obtained at 97% sequence similarity. Taxonomic assignments revealed that bacterial OTUs belonged to the phyla Firmicutes and Actinobacteria, and included species from the genera Atopostipes, Bacillus, Brevibacterium and Nosocomiicoccus. Densitometric analysis of DGGE image/bands revealed that Bacillus spp. were the dominant OTU/species in terms of population numbers. Fungal OTUs belonged to the phyla Ascomycota and Zygomycota, and included species from the genera, Alternaria, Aspergillus, Candida, Cladosporium, Dokmaia, Issatchenkia, Kodamaea, Lecythophora, Phoma, Pichia, Rhizopus, Saccharomyces and Starmerella. The majority of fungal species have not been previously reported in soy-daddawa. Potential opportunistic human pathogens such as Atopostipes suicloacalis, Candida rugosa, Candida tropicalis, and Kodamaea ohmeri were detected. Variation in soy-daddawa microbial communities amongst samples and presence of potential opportunistic pathogens emphasises the need for starter culture employment and good handling practices in soy-daddawa processing. Copyright © 2016 Elsevier B.V. All rights reserved.

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

PubMed

Adewumi, Gbenga A; Oguntoyinbo, Folarin A; Keisam, Santosh; Romi, Wahengbam; Jeyaram, Kumaraswamy

2012-01-01

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

Senior Thai fecal microbiota comparison between vegetarians and non-vegetarians using PCR-DGGE and real-time PCR.

PubMed

Ruengsomwong, Supatjaree; Korenori, Yuki; Sakamoto, Naoshige; Wannissorn, Bhusita; Nakayama, Jiro; Nitisinprasert, Sunee

2014-08-01

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report.

PubMed

Galbiati, Silvia; Stenirri, Stefania; Sbaiz, Luca; Barberis, Marco; Cremonesi, Laura; Restagno, Gabriella; Ferrari, Maurizio

2014-04-01

Non-invasive prenatal diagnosis has found application in a limited number of genetic diseases due to the difficulty in detecting a few copies of fetal mutated sequences in the presence of a large excess of wild-type maternal alleles, even in the case of single-base mutations. We developed conditions for the enrichment of fetal mutated alleles in maternal plasma based on CO-amplification at lower denaturation temperature-PCR (COLD-PCR). In particular, we applied a full COLD-PCR protocol to the identification of a p.A87_G92del mutation in the TWIST1 gene causing craniosynostosis in a couple at risk for the disease. The use of the COLD-PCR protocol coupled with direct sequencing enabled correct identification of the fetal paternally inherited mutated allele, in accordance with the result obtained on DNA extracted from chorionic villi. COLD-PCR proved to be a simple and powerful tool for the identification of minority mutated alleles even in the case of a moderately large deletion (18 bp) and confirmed to be very suitable for non-invasive prenatal diagnosis of a variety of genetic diseases.

In vitro probiotic characteristics of Lactobacillus plantarum ZDY 2013 and its modulatory effect on gut microbiota of mice.

PubMed

Huang, Renhui; Tao, Xueying; Wan, Cuixiang; Li, Shengjie; Xu, Hengyi; Xu, Feng; Shah, Nagendra P; Wei, Hua

2015-09-01

Lactobacillus plantarum ZDY 2013, a novel strain isolated from Chinese traditional fermented acid beans, was systematically evaluated for its survival capacity under stress conditions (pH, bile salt, simulated gastrointestinal tract, and antibiotics), production of exopolysaccharide and antagonism against 8 pathogens. Its effect on mice gut microbiota was also investigated by quantitative PCR and PCR-denaturing gradient gel electrophoresis. The results showed that ZDY 2013 can grow at pH 3.5 and survive at pH 2.0 for 6 h and at 0.45% bile salt for 3 h. The exopolysaccharide yield was up to 204±7.68 mg/L. The survival rate of ZDY 2013 in a simulated gastrointestinal tract was as high as 65.84%. Antagonism test with a supernatant of ZDY 2013 showed maximum halo of 28 mm against Listeria monocytogenes. The inhibition order was as follows: Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei, Enterobacter sakazakii, and Staphylococcus aureus. Lactobacillus plantarum ZDY 2013 was sensitive to some antibiotics (e.g., macrolide, sulfonamides, aminoglycoside, tetracyclines and β-lactams), whereas it was resistant to glycopeptides, quinolones, and cephalosporins antibiotics. Denaturing gradient gel electrophoresis profile demonstrated that ZDY 2013 administration altered the composition of the microbiota at various intestinal loci of the mice. Moreover, the quantitative PCR test showed that the administration of ZDY 2013 enhanced the populations of Bifidobacterium and Lactobacillus in either the colon or cecum, and reduced the potential enteropathogenic bacteria (e.g., Enterococcus, Enterobacterium, and Clostridium perfringens). Lactobacillus plantarum ZDY 2013 exhibited high resistance against low pH, bile salt, and gastrointestinal fluid, and possessed antibacterial and gut microbiota modulation properties with a potential application in the development of dairy food and nutraceuticals. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Microbial community analysis in the autotrophic denitrification process using spent sulfidic caustic by denaturing gradient gel electrophoresis of PCR-amplified genes.

PubMed

Lee, J-H; Lee, S-M; Choi, G-C; Park, H-S; Kang, D-H; Park, J-J

2011-01-01

Spent sulfidic caustic (SSC) produced from petrochemical plants contains a high concentration of hydrogen sulfide and alkalinity, and some almost non-biodegradable organic compounds such as benzene, toluene, ethylbenzene and xylenes (BTEX). SSC is mainly incinerated with auxiliary fuel, leading to secondary pollution problems. The reuse of this waste is becoming increasingly important from economic and environmental viewpoints. To denitrify wastewater with low COD/N ratio, additional carbon sources are required. Thus, autotrophic denitrification has attracted increasing attention. In this study, SSC was injected as an electron donor for sulfur-based autotrophic denitrification in the modified Ludzack-Ettinger (MLE) process. The efficiencies of nitrification, COD, and total nitrogen (TN) removal were evaluated with varying SSC dosage. Adequate SSC injection exhibited stable autotrophic denitrification. No BTEX were detected in the monitored BTEX concentrations of the effluent. To analyse the microbial community of the MLE process, PCR-DGGE based on 16 S rDNA with EUB primers, TD primers and nirK gene with nirK primers was performed in order to elucidate the application of the MLE process to SSC.

Molecular investigation of bacterial communities during the manufacturing and ripening of semi-hard Iranian Liqvan cheese.

PubMed

Ramezani, M; Hosseini, S M; Ferrocino, I; Amoozegar, M A; Cocolin, L

2017-09-01

Liqvan (or Lighvan) is a traditional Iranian cheese from the East Azerbaijan province of Iran, which is made of raw ewe's milk without the addition of a starter. The grazing pastures, environmental conditions and the ancient regional production methods allocate a distinctive microbial ecology to this type of cheese, and these factors are consequently associated with the quality of the product. In this study, the microbiota of the milk, curd and cheese has been investigated using culture independent approaches. Denaturing gradient gel electrophoresis (DGGE) of the bacteria, 16S rRNA based high-throughput sequencing and enumeration of the live bacterial community by means of quantitative PCR (qPCR) have been used for this purpose. The results showed that the main bacterial population in the milk belonged to both microbial contaminants and lactic acid bacteria (LAB). However, both of these populations were totally replaced by LAB during ripening. The present survey contributes by describing the microbiota of this ancient cheese in more detail during fermentation and ripening. Copyright © 2017 Elsevier Ltd. All rights reserved.

Barcoded pyrosequencing analysis of the microbial community in a simulator of the human gastrointestinal tract showed a colon region-specific microbiota modulation for two plant-derived polysaccharide blends.

PubMed

Marzorati, Massimo; Maignien, Lois; Verhelst, An; Luta, Gabriela; Sinnott, Robert; Kerckhof, Frederiek Maarten; Boon, Nico; Van de Wiele, Tom; Possemiers, Sam

2013-02-01

The combination of a Simulator of the Human Intestinal Microbial Ecosystem with ad hoc molecular techniques (i.e. pyrosequencing, denaturing gradient gel electrophoresis and quantitative PCR) allowed an evaluation of the extent to which two plant polysaccharide supplements could modify a complex gut microbial community. The presence of Aloe vera gel powder and algae extract in product B as compared to the standard blend (product A) improved its fermentation along the entire simulated colon. The potential extended effect of product B in the simulated distal colon, as compared to product A, was confirmed by: (i) the separate clustering of the samples before and after the treatment in the phylogenetic-based dendrogram and OTU-based PCoA plot only for product B; (ii) a higher richness estimator (+33 vs. -36 % of product A); and (iii) a higher dynamic parameter (21 vs. 13 %). These data show that the combination of well designed in vitro simulators with barcoded pyrosequencing is a powerful tool for characterizing changes occurring in the gut microbiota following a treatment. However, for the quantification of low-abundance species-of interest because of their relationship to potential positive health effects (i.e. bifidobacteria or lactobacilli)-conventional molecular ecological approaches, such as PCR-DGGE and qPCR, still remain a very useful complementary tool.

Synergistic in vitro and in vivo antimicrobial effect of a mixture of ZnO nanoparticles and Lactobacillus fermentation liquor.

PubMed

Kuang, Huijuan; Yang, Lin; Shah, Nagendra P; Aguilar, Zoraida P; Wang, Lijun; Xu, Hengyi; Wei, Hua

2016-04-01

In this study, we investigated the antibacterial activity of ZnO nanoparticles (NPs) and Lactobacillus-fermentation liquor (LFL) against two pathogenic bacteria in vitro and in vivo. Bactericidal tests were performed on solid agar plates and quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE) techniques were used to examine the antibacterial activity of the mixture of ZnO NPs and LFL in vivo. The results showed that the mixture exhibited higher antibacterial activity against Salmonella typhimurium in vitro in comparison with ZnO NPs alone. The results showed that ZnO NPs and LFL significantly enhanced microbial diversity in mouse intestine which suggested a synergistic antibacterial activity against the tested pathogenic bacteria that could be used for the control of the spread and persistence of bacterial infections.

Unraveling the microbiota of teat apices of clinically healthy lactating dairy cows, with special emphasis on coagulase-negative staphylococci.

PubMed

Braem, G; De Vliegher, S; Verbist, B; Piessens, V; Van Coillie, E; De Vuyst, L; Leroy, F

2013-03-01

Swab samples (n=72) obtained from the teat apex of lactating dairy cows without visual signs of inflammation (n=18) were gathered on 2 well-managed Flemish dairy herds (herds 1 and 2) during the same month to assess the bacterial diversity of teat apices before milking. A combination of both culture-dependent [plating and (GTG)(5)-PCR fingerprinting of the colonies] and culture-independent [denaturing gradient gel electrophoresis (PCR-DGGE)] techniques indicated that the teat apices contain a wide diversity of bacterial genera. Despite a low bacterial load, 20 bacterial genera of 3 phyla (Actinobacteria, Firmicutes, and Proteobacteria) were present. The most prevalent bacteria were the coagulase-negative staphylococci (CNS), encompassing a total of 15 species, which were identified to the species level using a combination of (GTG)(5)-PCR fingerprinting, gene sequencing (16S ribosomal RNA and rpoB genes), and a novel PCR-DGGE technique based on the tuf-PCR amplicon. Overall bacterial diversity did not differ significantly between the herds or between noninfected and subclinically infected quarters in herd 1. In herd 1, borderline significant lower CNS species diversity was found on teat apices of noninfected quarters compared with subclinically infected quarters. The most prevalent CNS species were Staphylococcus haemolyticus and Staphylococcus equorum in both herds and Staphylococcus carnosus in herd 2. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Clinical signs and bacterial communities of deciduous necrotic root canals detected by PCR-DGGE analysis: research association.

PubMed

de Paula, Viviane Andrade Cancio; de Carvalho Ferreira, Dennis; Cavalcante, Fernanda Sampaio; do Carmo, Flávia Lima; Rosado, Alexandre Soares; Primo, Laura Guimarães; dos Santos, Kátia Regina Netto

2014-08-01

This study sought to investigate the possible association between clinical and radiographic data of the patients with the bacterial community profiles involved in cases of necrosis in primary root canals. Microbial community profiles for 25 samples from necrotic deciduous root canals were analyzed using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting approach. These results were related to the clinical and radiographic data of these patients. The analysis showed a large diversity of microbial communities in necrotic deciduous root canals. The statistical results pointed out that posterior and anterior teeth were associated with <20 bands and >20 bands in PCR-DGGE method, respectively. A relationship was verified between ages >4 years old and posterior teeth and, ages ≤4 years old and anterior teeth. The data showed a polymicrobial community and pointed out the association of age with necrosis in anterior and posterior teeth. Copyright © 2014 Elsevier Ltd. All rights reserved.

PCR-DGGE analysis of the microbial communities in three different Chinese "Baiyunbian" liquor fermentation starters.

PubMed

Xiong, Xiaomao; Hu, Yuanliang; Yan, Nanfeng; Huang, Yingna; Peng, Nan; Liang, Yunxiang; Zhao, Shumiao

2014-08-01

A systematic investigation was performed on the bacterial, Bacillus, fungal, and yeast communities of the three types of Daqu (mechanically prepared, manually prepared, and mixed prepared) used in Baiyunbian Company by reconditioning PCR-denaturing gradient gel electrophoresis (PCR-DGGE). The DGGE results showed that the microbes in the three types of Daqu were mainly thermotolerant and thermophilic microbes, and the most dominant bacterial species were Bacillus and Virgibacillus, followed by Lactobacillus and Trichococcus. Furthermore, the dominant fungi were found to be molds, such as Rasamsonia, Penicillium, Aspergillus, and Monascus, and the dominant yeasts were Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Pichia anomala, and Debaryomyces hansenii. In general, the three types of Daqu showed slight differences in microbial communities, and the Shannon indexes (H') of the manually prepared and mechanically prepared Daqu were similar. The results suggest that mechanically prepared Daqu can replace manually prepared Daqu in liquor production, and this research provides useful information for liquor production and process improvement.

Simultaneous construction of PCR-DGGE-based predictive models of Listeria monocytogenes and Vibrio parahaemolyticus on cooked shrimps.

PubMed

Liao, C; Peng, Z Y; Li, J B; Cui, X W; Zhang, Z H; Malakar, P K; Zhang, W J; Pan, Y J; Zhao, Y

2015-03-01

The aim of this study was to simultaneously construct PCR-DGGE-based predictive models of Listeria monocytogenes and Vibrio parahaemolyticus on cooked shrimps at 4 and 10°C. Calibration curves were established to correlate peak density of DGGE bands with microbial counts. Microbial counts derived from PCR-DGGE and plate methods were fitted by Baranyi model to obtain molecular and traditional predictive models. For L. monocytogenes, growing at 4 and 10°C, molecular predictive models were constructed. It showed good evaluations of correlation coefficients (R(2) > 0.92), bias factors (Bf ) and accuracy factors (Af ) (1.0 ≤ Bf ≤ Af ≤ 1.1). Moreover, no significant difference was found between molecular and traditional predictive models when analysed on lag phase (λ), maximum growth rate (μmax ) and growth data (P > 0.05). But for V. parahaemolyticus, inactivated at 4 and 10°C, molecular models show significant difference when compared with traditional models. Taken together, these results suggest that PCR-DGGE based on DNA can be used to construct growth models, but it is inappropriate for inactivation models yet. This is the first report of developing PCR-DGGE to simultaneously construct multiple molecular models. It has been known for a long time that microbial predictive models based on traditional plate methods are time-consuming and labour-intensive. Denaturing gradient gel electrophoresis (DGGE) has been widely used as a semiquantitative method to describe complex microbial community. In our study, we developed DGGE to quantify bacterial counts and simultaneously established two molecular predictive models to describe the growth and survival of two bacteria (Listeria monocytogenes and Vibrio parahaemolyticus) at 4 and 10°C. We demonstrated that PCR-DGGE could be used to construct growth models. This work provides a new approach to construct molecular predictive models and thereby facilitates predictive microbiology and QMRA (Quantitative Microbial Risk Assessment). © 2014 The Society for Applied Microbiology.

Analysis of the bacterial community in aged and aging pit mud of Chinese Luzhou-flavour liquor by combined PCR-DGGE and quantitative PCR assay.

PubMed

Liang, Huipeng; Li, Wenfang; Luo, Qingchun; Liu, Chaolan; Wu, Zhengyun; Zhang, Wenxue

2015-10-01

The community structure of bacteria in aged and aging pit mud, which was judged according to their sensory and physicochemical characteristics, was analysed using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time PCR (qPCR). The phyla Firmicutes, Actinobacteria, Proteobacteria, Synergistetes and Unclassified Bacteria were detected and the fermentative Firmicutes was predominant in both types of pit mud in the PCR-DGGE analysis. Among Firmicutes, Clostridiales was dominant in aged pit mud while Bacillales and Lactobacillales were dominant in aging pit mud. The diversity of bacterial communities in aged pit mud was higher than that in aging pit mud. In the qPCR analysis the abundance of Clostridium IV in aged pit mud was higher than that in aging pit mud and there were significant differences in the quantity of Clostridium IV between aged and aging pit mud of the same cellar (P < 0.05). There were some significant differences in the microbial community structure between aged and aging pit mud. The differences in the quantity of Clostridium IV might be involved in the distinction that the aged pit mud has a strong aroma while the aging pit mud does not. © 2014 Society of Chemical Industry.

Sulphur-oxidizing and sulphate-reducing communities in Brazilian mangrove sediments.

PubMed

Varon-Lopez, Maryeimy; Dias, Armando Cavalcante Franco; Fasanella, Cristiane Cipolla; Durrer, Ademir; Melo, Itamar Soares; Kuramae, Eiko Eurya; Andreote, Fernando Dini

2014-03-01

Mangrove soils are anaerobic environments rich in sulphate and organic matter. Although the sulphur cycle is one of the major actors in this ecosystem, little is known regarding the sulphur bacteria communities in mangrove soils. We investigated the abundance, composition and diversity of sulphur-oxidizing (SOB) and sulphate-reducing (SRB) bacteria in sediments from three Brazilian mangrove communities: two contaminated, one with oil (OilMgv) and one with urban waste and sludge (AntMgv), and one pristine (PrsMgv). The community structures were assessed using quantitative real-time polymerase chain reaction (qPCR), polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries, using genes for the enzymes adenosine-5'-phosphosulphate reductase (aprA) and sulphite reductase (Dsr) (dsrB). The abundance for qPCR showed the ratio dsrB/aprA to be variable among mangroves and higher according to the gradient observed for oil contamination in the OilMgv. The PCR-DGGE patterns analysed by Nonmetric Multidimensional Scaling revealed differences among the structures of the three mangrove communities. The clone libraries showed that Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria were the most abundant groups associated with sulphur cycling in mangrove sediments. We conclude that the microbial SOB and SRB communities in mangrove soils are different in each mangrove forest and that such microbial communities could possibly be used as a proxy for contamination in mangrove forests. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

Application of Coamplification at Lower Denaturation Temperature-PCR Sequencing for Early Detection of Antiviral Drug Resistance Mutations of Hepatitis B Virus

PubMed Central

Wong, Danny Ka-Ho; Tsoi, Ottilia; Huang, Fung-Yu; Seto, Wai-Kay; Fung, James; Lai, Ching-Lung

2014-01-01

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations. PMID:24951803

The assessment of the coke wastewater treatment efficacy in rotating biological contractor.

PubMed

Cema, G; Żabczyński, S; Ziembińska-Buczyńska, A

2016-01-01

Coke wastewater is known to be relatively difficult for biological treatment. Nonetheless, biofilm-based systems seem to be promising tool for such treatment. That is why a rotating biological contactor (RBC) system focused on the Anammox process was used in this study. The experiment was divided into two parts with synthetic and then real wastewater. It was proven that it is possible to treat coke wastewater with RBC but such a procedure requires a very long start-up period for the nitritation (190 days), as well as for the Anammox process, where stable nitrogen removal over 70% was achieved after 400 days of experiment. Interestingly, it was possible at a relatively low (20.2 ± 2.2 °C) temperature. The polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) based monitoring of the bacterial community showed that its biodiversity decreased when the real wastewater was treated and it was composed mainly of GC-rich genotypes, probably because of the modeling influence of this wastewater and the genotypes specialization.

Differences in Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient

PubMed Central

Feris, Kevin; Ramsey, Philip; Frazar, Chris; Moore, Johnnie N.; Gannon, James E.; Holben, William E.

2003-01-01

The hyporheic zone of a river is nonphotic, has steep chemical and redox gradients, and has a heterotrophic food web based on the consumption of organic carbon entrained from downwelling surface water or from upwelling groundwater. The microbial communities in the hyporheic zone are an important component of these heterotrophic food webs and perform essential functions in lotic ecosystems. Using a suite of methods (denaturing gradient gel electrophoresis, 16S rRNA phylogeny, phospholipid fatty acid analysis, direct microscopic enumeration, and quantitative PCR), we compared the microbial communities inhabiting the hyporheic zone of six different river sites that encompass a wide range of sediment metal loads resulting from large base-metal mining activity in the region. There was no correlation between sediment metal content and the total hyporheic microbial biomass present within each site. However, microbial community structure showed a significant linear relationship with the sediment metal loads. The abundances of four phylogenetic groups (groups I, II, III, and IV) most closely related to α-, β-, and γ-proteobacteria and the cyanobacteria, respectively, were determined. The sediment metal content gradient was positively correlated with group III abundance and negatively correlated with group II abundance. No correlation was apparent with regard to group I or IV abundance. This is the first documentation of a relationship between fluvially deposited heavy-metal contamination and hyporheic microbial community structure. The information presented here may be useful in predicting long-term effects of heavy-metal contamination in streams and provides a basis for further studies of metal effects on hyporheic microbial communities. PMID:12957946

[Effect of fluoride on gut microflora of silkworm (Bombyx mori)].

PubMed

Li, Guannan; Xia, Xuejuan; Sendegeya, Parfait; Zhao, Huanhuan; Long, Yaohang; Zhu, Yong

2015-07-04

We examined the effect of fluoride on gut microflora of silkworm. After DNA extraction and PCR amplification, clone libraries of 16S rRNA gene fragment were constructed. Amplified ribosomal DNA restriction analysis (ARDRA) was performed by digestion of the 16S rRNA gene, and each unique restriction fragment polymorphism pattern was designated as an operational taxonomic unit (OTU). A total of 14 OTUs were identified from intestinal samples of both T6 and 734. Phylogenetic trees of bacterial 16S rRNA nucleotide sequences were constructed and analyzed. Furthermore, the dominant bacteria were studied by the nested polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DDGE) technology. After fluorosis, the flora of Enterococcus and Bacillus reduced. However, the flora of Staphylococcus increased. Fluoride can destroy the balance of microflora in the gut of silkworm by changing the bacteria diversity and proportion, which has bigger effect to 734 than T6.

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

PubMed Central

Rölleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

1996-01-01

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings. PMID:8787403

Petroleum contamination and bioaugmentation in bacterial rhizosphere communities from Avicennia schaueriana.

PubMed

Dealtry, Simone; Ghizelini, Angela Michelato; Mendonça-Hagler, Leda C S; Chaloub, Ricardo Moreira; Reinert, Fernanda; Campos, Tácio M P de; Gomes, Newton C M; Smalla, Kornelia

2018-06-01

Anthropogenic activity, such as accidental oil spills, are typical sources of urban mangrove pollution that may affect mangrove bacterial communities as well as their mobile genetic elements. To evaluate remediation strategies, we followed over the time the effects of a petroleum hydrocarbon degrading consortium inoculated on mangrove tree Avicennia schaueriana against artificial petroleum contamination in a phytoremediation greenhouse experiment. Interestingly, despite plant protection due to the inoculation, denaturing gradient gel electrophoresis of the bacterial 16S rRNA gene fragments amplified from the total community DNA indicated that the different treatments did not significantly affect the bacterial community composition. However, while the bacterial community was rather stable, pronounced shifts were observed in the abundance of bacteria carrying plasmids. A PCR-Southern blot hybridization analysis indicated an increase in the abundance of IncP-9 catabolic plasmids. Denaturing gradient gel electrophoresis of naphthalene dioxygenase (ndo) genes amplified from cDNA (RNA) indicated the dominance of a specific ndo gene in the inoculated petroleum amendment treatment. The petroleum hydrocarbon degrading consortium characterization indicated the prevalence of bacteria assigned to Pseudomonas spp., Comamonas spp. and Ochrobactrum spp. IncP-9 plasmids were detected for the first time in Comamonas sp. and Ochrobactrum spp., which is a novelty of this study. Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Bacterial community structure and activity of sulfate reducing bacteria in a membrane aerated biofilm analyzed by microsensor and molecular techniques.

PubMed

Liu, Hong; Tan, Shuying; Sheng, Zhiya; Liu, Yang; Yu, Tong

2014-11-01

The activities and vertical spatial distribution of sulfate reducing bacteria (SRB) in an oxygen (O2 )-based membrane aerated biofilm (MAB) were investigated using microsensor (O2 and H2 S) measurements and molecular techniques (polymerase chain reaction-denaturing gradient gel electrophoresis [PCR-DGGE] and fluorescence in situ hybridization [FISH]). The O2 concentration profile revealed that O2 penetrated from the bottom (substratum) of the gas permeable membrane, and was gradually consumed within the biofilm until it was completely depleted near the biofilm/bulk liquid interface, indicating oxic and anoxic zone in the MAB. The H2 S concentration profile showed that H2 S production was found in the upper 285 µm of the biofilm, indicating a high activity of SRB in this region. The results from DGGE of the PCR-amplified dissimilatory sulfite reductase subunit B (dsrB) gene and FISH showed an uneven spatial distribution of SRB. The maximum SRB biomass was located in the upper biofilm. The information from the molecular analysis can be supplemented with that from microsensor measurements to better understand the microbial community and activity of SRB in the MAB. © 2014 Wiley Periodicals, Inc.

The effect of heavy metal contamination on the bacterial community structure at Jiaozhou Bay, China.

PubMed

Yao, Xie-Feng; Zhang, Jiu-Ming; Tian, Li; Guo, Jian-Hua

In this study, determination of heavy metal parameters and microbiological characterization of marine sediments obtained from two heavily polluted sites and one low-grade contaminated reference station at Jiaozhou Bay in China were carried out. The microbial communities found in the sampled marine sediments were studied using PCR-DGGE (denaturing gradient gel electrophoresis) fingerprinting profiles in combination with multivariate analysis. Clustering analysis of DGGE and matrix of heavy metals displayed similar occurrence patterns. On this basis, 17 samples were classified into two clusters depending on the presence or absence of the high level contamination. Moreover, the cluster of highly contaminated samples was further classified into two sub-groups based on the stations of their origin. These results showed that the composition of the bacterial community is strongly influenced by heavy metal variables present in the sediments found in the Jiaozhou Bay. This study also suggested that metagenomic techniques such as PCR-DGGE fingerprinting in combination with multivariate analysis is an efficient method to examine the effect of metal contamination on the bacterial community structure. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with atmore » least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.« less

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples.

PubMed

Mahmood, Shahid; Freitag, Thomas E; Prosser, James I

2006-06-01

PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.

PubMed

Leite, A M O; Mayo, B; Rachid, C T C C; Peixoto, R S; Silva, J T; Paschoalin, V M F; Delgado, S

2012-09-01

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem. Copyright © 2012 Elsevier Ltd. All rights reserved.

Functional Stability of a Mixed Microbial Consortium Producing PHA From Waste Carbon Sources

DOE Office of Scientific and Technical Information (OSTI.GOV)

David N. Thompson; Erik R. Coats; William A. Smith

2006-04-01

Polyhydroxyalkanoates (PHAs) represent an environmentally-effective alternative to synthetic thermoplastics; however, current production practices are not sustainable. In this study, PHA production was accomplished in sequencing batch bioreactors utilizing real wastewaters and mixed microbial consortia from municipal activated sludge as inoculum. Polymer production reached 85%, 53%, and 10% of the cell dry weight from methanol-enriched pulp-and-paper mill foul condensate, fermented municipal primary solids, and biodiesel wastewater, respectively. Employing denaturing gradient gel electrophoresis of 16S-rDNA from PCR-amplified DNA extracts, distinctly different communities were observed between and within wastewaters following enrichment. Most importantly, functional stability was maintained despite differing and contrasting microbial populations.

Airborne myxomycete spores: detection using molecular techniques

NASA Astrophysics Data System (ADS)

Kamono, Akiko; Kojima, Hisaya; Matsumoto, Jun; Kawamura, Kimitaka; Fukui, Manabu

2009-01-01

Myxomycetes are organisms characterized by a life cycle that includes a fruiting body stage. Myxomycete fruiting bodies contain spores, and wind dispersal of the spores is considered important for this organism to colonize new areas. In this study, the presence of airborne myxomycetes and the temporal changes in the myxomycete composition of atmospheric particles (aerosols) were investigated with a polymerase chain reaction (PCR)-based method for Didymiaceae and Physaraceae. Twenty-one aerosol samples were collected on the roof of a three-story building located in Sapporo, Hokkaido Island, northern Japan. PCR analysis of DNA extracts from the aerosol samples indicated the presence of airborne myxomycetes in all the samples, except for the one collected during the snowfall season. Denaturing gradient gel electrophoresis (DGGE) analysis of the PCR products showed seasonally varying banding patterns. The detected DGGE bands were subjected to sequence analyses, and four out of nine obtained sequences were identical to those of fruiting body samples collected in Hokkaido Island. It appears that the difference in the fruiting period of each species was correlated with the seasonal changes in the myxomycete composition of the aerosols. Molecular evidence shows that newly formed spores are released and dispersed in the air, suggesting that wind-driven dispersal of spores is an important process in the life history of myxomycetes. This study is the first to detect airborne myxomycetes with the use of molecular ecological analyses and to characterize their seasonal distribution.

[Effects and Biological Response on Bioremediation of Petroleum Contaminated Soil].

PubMed

Yang, Qian; Wu, Man-li; Nie, Mai-qian; Wang, Ting-ting; Zhang, Ming-hui

2015-05-01

Bioaugmentation and biostimulation were used to remediate petroleum-contaminated soil which were collected from Zichang city in North of Shaanxi. The optimal bioremediation method was obtained by determining the total petroleum hydrocarbon(TPH) using the infrared spectroscopy. During the bioremediation, number of degrading strains, TPH catabolic genes, and soil microbial community diversity were determined by Most Probable Number (MPN), polymerase chain reaction (PCR) combined agarose electrophoresis, and PCR-denaturing gradient electrophoresis (DGGE). The results in different treatments showed different biodegradation effects towards total petroleum hydrocarbon (TPH). Biostimulation by adding N and P to soils achieved the best degradation effects towards TPH, and the bioaugmentation was achieved by inoculating strain SZ-1 to soils. Further analysis indicated the positive correlation between catabolic genes and TPH removal efficiency. During the bioremediation, the number of TPH and alkanes degrading strains was higher than the number of aromatic degrading strains. The results of PCR-DGGE showed microbial inoculums could enhance microbial community functional diversity. These results contribute to understand the ecologically microbial effects during the bioremediation of petroleum-polluted soil.

Bulk soil and rhizosphere bacterial community PCR-DGGE profiles and beta-galactosidase activity as indicators of biological quality in soils contaminated by heavy metals and cultivated with Silene vulgaris (Moench) Garcke.

PubMed

Martínez-Iñigo, M J; Pérez-Sanz, A; Ortiz, I; Alonso, J; Alarcón, R; García, P; Lobo, M C

2009-06-01

The biological quality of two heavy metal contaminated soils (soil C: Typic Calcixerept, pH 8.3 and soil H: Typic Haploxeraf, pH 7.3) was investigated after growing the metal-tolerant plant Silene vulgaris (Moench) Garcke for two vegetative periods. The activity of the enzyme beta-galactosidase, which is sensitive to the presence of contaminants in soil, and the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of 16S rRNA gene fragments of culturable bacteria from bulk soil and rhizosphere were determined. The microbial enzymatic activity was higher in planted soils than in bare soils at the contamination level of 600 mg of total heavy metals kg(-1) soil. After growing S. vulgaris, beta-galactosidase activity was almost recovered in the calcareous soil. In this soil new bands appeared in the PCR-DGGE profiles of the rhizosphere bacterial community as a response to the exposure to heavy metals.

Thermal analysis of the vortex tube based thermocycler for fast DNA amplification: Experimental and two-dimensional numerical results

NASA Astrophysics Data System (ADS)

Raghavan, V.; Whitney, Scott E.; Ebmeier, Ryan J.; Padhye, Nisha V.; Nelson, Michael; Viljoen, Hendrik J.; Gogos, George

2006-09-01

In this article, experimental and numerical analyses to investigate the thermal control of an innovative vortex tube based polymerase chain reaction (VT-PCR) thermocycler are described. VT-PCR is capable of rapid DNA amplification and real-time optical detection. The device rapidly cycles six 20μl 96bp λ-DNA samples between the PCR stages (denaturation, annealing, and elongation) for 30cycles in approximately 6min. Two-dimensional numerical simulations have been carried out using computational fluid dynamics (CFD) software FLUENT v.6.2.16. Experiments and CFD simulations have been carried out to measure/predict the temperature variation between the samples and within each sample. Heat transfer rate (primarily dictated by the temperature differences between the samples and the external air heating or cooling them) governs the temperature distribution between and within the samples. Temperature variation between and within the samples during the denaturation stage has been quite uniform (maximum variation around ±0.5 and 1.6°C, respectively). During cooling, by adjusting the cold release valves in the VT-PCR during some stage of cooling, the heat transfer rate has been controlled. Improved thermal control, which increases the efficiency of the PCR process, has been obtained both experimentally and numerically by slightly decreasing the rate of cooling. Thus, almost uniform temperature distribution between and within the samples (within 1°C) has been attained for the annealing stage as well. It is shown that the VT-PCR is a fully functional PCR machine capable of amplifying specific DNA target sequences in less time than conventional PCR devices.

Comparison of benthic bacterial community composition in nine streams

Treesearch

Xueqing Gao; Ola A. Olapade; Laura G. Leff

2005-01-01

In this study, the abundance of major bacterial taxa (based on fluorescent in situ hybridization, FISH) and the structure of the bacterial community (based on denaturing gradient gel electrophoresis, DGGE) were determined in the benthos of 9 streams in the southeastern and midwestern United States and related to differences in environmental...

Camparison of benthic bacterial community composition in nine streams

Treesearch

Xuqing Gao; Ola A. Olapade; Laura G. Leff

2005-01-01

In this study, the abundance of major bacterial taxa (based on fluorescent in situ hybridization, FISH) and the structure of the bacterial community (based on denaturing gradient gel electrophoresis, DGGE) were determined in the benthos of 9 streams in the southeastern and midwestern United States and related to differences in environmental conditions. Taxa examined...

Flow-through PCR on a 3D qiandu-shaped polydimethylsiloxane (PDMS) microdevice employing a single heater: toward microscale multiplex PCR.

PubMed

Wu, Wenming; Loan, Kieu The Loan; Lee, Nae Yoon

2012-05-07

Consistent temperature control in an on-chip flow-through polymerase chain reaction (PCR) employing two or more heaters is one of the main obstacles for device miniaturization and integration when realizing micro total analysis systems (μTAS), and also leads to operational complexity. In this study, we propose a qiandu (right triangular prism)-shaped polydimethylsiloxane (PDMS) microdevice with serpentine microchannels fabricated on its slanted plane, and apply the device for an on-chip flow-through PCR employing a single heater. The inclined nature of the qiandu-shaped microdevice enables the formation of a surface temperature gradient along the slanted plane of the microdevice in a height-dependent manner by the use of a single heater, and enables liquid to traverse over wide ranges of temperatures, including the three temperature zones--denaturation, annealing, and extension temperatures--required in a typical PCR. The feasibility of the qiandu-shaped PDMS microdevice as a versatile platform for performing a flow-through PCR was examined by employing multiple templates and varying the inclination angle of the device. In addition, the potential of performing a multiplex PCR using a single qiandu-shaped PDMS microdevice was explored. A 409 bp long gene fragment effective as a marker for diagnosing lung cancer and a 230 bp long gene fragment from a plasmid vector were simultaneously amplified in less than 25 min on a single microdevice, paving the way for a microscale, multiplex PCR on a single device employing a single heater.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

PubMed

Mauger, Florence; Kernaleguen, Magali; Lallemand, Céline; Kristensen, Vessela N; Deleuze, Jean-François; Tost, Jörg

2018-05-01

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance.

PubMed

Wang, Yong-Feng; Zhang, Fang-Qiu; Gu, Ji-Dong

2014-06-01

Denaturing gradient gel electrophoresis (DGGE) is a powerful technique to reveal the community structures and composition of microorganisms in complex natural environments and samples. However, positive and reproducible polymerase chain reaction (PCR) products, which are difficult to acquire for some specific samples due to low abundance of the target microorganisms, significantly impair the effective applications of DGGE. Thus, nested PCR is often introduced to generate positive PCR products from the complex samples, but one problem is also introduced: The total number of thermocycling in nested PCR is usually unacceptably high, which results in skewed community structures by generation of random or mismatched PCR products on the DGGE gel, and this was demonstrated in this study. Furthermore, nested PCR could not resolve the uneven representative issue with PCR products of complex samples with unequal richness of microbial population. In order to solve the two problems in nested PCR, the general protocol was modified and improved in this study. Firstly, a general PCR procedure was used to amplify the target genes with the PCR primers without any guanine cytosine (GC) clamp, and then, the resultant PCR products were purified and diluted to 0.01 μg ml(-1). Subsequently, the diluted PCR products were utilized as templates to amplify again with the same PCR primers with the GC clamp for 17 cycles, and the products were finally subjected to DGGE analysis. We demonstrated that this is a much more reliable approach to obtain a high quality DGGE profile with high reproducibility. Thus, we recommend the adoption of this improved protocol in analyzing microorganisms of low abundance in complex samples when applying the DGGE fingerprinting technique to avoid biased results.

Actively Growing Bacteria in the Inland Sea of Japan, Identified by Combined Bromodeoxyuridine Immunocapture and Denaturing Gradient Gel Electrophoresis▿ †

PubMed Central

Hamasaki, Koji; Taniguchi, Akito; Tada, Yuya; Long, Richard A.; Azam, Farooq

2007-01-01

A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order to test the validity of the method for application to diverse marine assemblages. We found that 27 of 29 isolates belonging to different taxa could incorporate BrdU. BUMP-DGGE analysis revealed phylogenetic affiliations of DNA-synthesizing, presumably actively growing bacteria across a eutrophic to mesotrophic transect in the Inland Sea of Japan. We found that the BrdU-incorporating (growing) communities were substantially different from the total communities. The majority (34/56) of phylotypes incorporated BrdU and were presumably growing, and these phylotypes comprised 10 alphaproteobacteria, 1 betaproteobacterium, 11 gammaproteobacteria, 11 Cytophaga-Flavobacterium-Bacteroides group bacteria, and 1 unclassified bacterium. All BrdU-responsive alphaproteobacteria were members of the Rhodobacterales, suggesting that such bacteria were dominant in the growing alphaproteobacterial populations in our samples. The BrdU-responsive gammaproteobacteria belonged to the Oceanospirillales, the SAR86 cluster, the Pseudomonadales, the Alteromonadales, and the Vibrionales. Thus, contemporaneous cooccurrence of diverse actively growing bacterial taxa was a consistent pattern in our biogeochemically varied study area. PMID:17337555

Spatial and temporal changes in Actinobacterial dominance in experimental artificial groundwater recharge.

PubMed

Kolehmainen, Reija E; Tiirola, Marja; Puhakka, Jaakko A

2008-11-01

Artificial groundwater recharge (AGR) is used in the drinking water industry to supplement groundwater resources and to minimise the use of chemicals in water treatment. This study analysed the spatial and temporal changes of microbial communities in AGR using two test systems: a nutrient-amended fluidized-bed reactor (FBR) and a sand column. Structural changes in the feed lake water (Lake Roine), FBR, and sand column bacterial communities were determined by denaturing gradient gel electrophoresis (DGGE) and the length heterogeneity analysis of amplified 16S rRNA genes (LH-PCR). Two clone libraries were created to link the LH-PCR results to the dominant bacterial groups. The lake water bacterial community was relatively stable, with three bands dominating in all LH-PCR products. The most dominant fragment accounted for up to 72% and was derived from Actinobacteria. Based on the clone libraries and LH-PCR data, Actinobacteria also dominated in the unattached bacterial community of the FBR, whereas several Proteobacterial groups were more abundant on the FBR carrier particles. In the stabilised AGR system a major change in the community structure of the lake water bacteria took place during passage within the first 0.6m in the sand column as the community composition shifted from Actinobacteria-dominated populations to a diverse, mainly Proteobacterial communities. Concurrently, most of the dissolved organic carbon (DOC) was removed at this stage. In summary, the study showed that the make-up of microbial communities in experimental AGR systems responded to changes in their environment. LH-PCR showed potential as a method to determine microbial community dynamics in long-term studies at real-scale AGR sites. This is the first step to provide data on microbial community dynamics in AGR for drinking water production.

Occurrence and Phylogenetic Diversity of Sphingomonas Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons

PubMed Central

Leys, Natalie M. E. J.; Ryngaert, Annemie; Bastiaens, Leen; Verstraete, Willy; Top, Eva M.; Springael, Dirk

2004-01-01

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae. Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 104 CFU g of soil−1. The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques. PMID:15066784

A survey of microbial community diversity in marine sediments impacted by petroleum hydrocarbons from the Gulf of Mexico and Atlantic shorelines, Texas to Florida

USGS Publications Warehouse

Lisle, John T.; Stellick, Sarah H.

2011-01-01

Microbial community genomic DNA was extracted from sediment samples collected along the Gulf of Mexico and Atlantic coasts from Texas to Florida. Sample sites were identified as being ecologically sensitive and (or) as having high potential of being impacted by Macondo-1 (M-1) well oil from the Deepwater Horizon blowout. The diversity within the microbial communities associated with the collected sediments provides a baseline dataset to which microbial community-diversity data from impacted sites could be compared. To determine the microbial community diversity in the samples, genetic fingerprints were generated and compared. Specific sequences within the community genomic DNA were first amplified using the polymerase chain reaction (PCR) with a primer set that provides possible resolution to the species level. A second nested PCR was performed on the primary PCR products using a primer set on which a GC-clamp was attached to one of the primers. The nested PCR products were separated using denaturing-gradient gel electrophoresis (DGGE) that resolves the nested PCR products based on sequence dissimilarities (or similarities), forming a genomic fingerprint of the microbial diversity within the respective samples. Samples with similar fingerprints were grouped and compared to oil-fingerprint data from the same sites (Rosenbauer and others, 2011). The microbial community fingerprints were generally grouped into sites that had been shown to contain background concentrations of non-Deepwater Horizon oil. However, these groupings also included sites where no oil signature was detected. This report represents some of the first information on naturally occurring microbial communities in sediment from shorelines along the Gulf of Mexico and Atlantic coasts from Texas to Florida.

Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients

PubMed Central

Rabilloud, Thierry; Adessi, C.; Giraudel, A.; Lunardi, J.

2007-01-01

Summary We have carried out the separation of sparingly-soluble (membrane and nuclear) proteins by high resolution two-dimensional electrophoresis. IEF with immobilized pH gradients leads to severe quantitative losses of proteins in the resulting 2-D map, although the resolution is usually kept high. We therefore tried to improve the solubility of proteins in this technique, by using denaturing cocktails containing various detergents and chaotropes. Best results were obtained by using a denaturing solution containing urea, thiourea, and detergents (both nonionic and zwitterionic). The usefulness of thiourea-containing denaturing mixtures are shown in this article on several models including microsomal and nuclear proteins and on tubulin, a protein highly prone to aggregation. PMID:9150907

Sequence analysis of MHC class I α2 from sockeye salmon (Oncorhynchus nerka).

PubMed

McClelland, Erin K; Ming, Tobi J; Tabata, Amy; Miller, Kristina M

2011-09-01

Most studies assessing adaptive MHC diversity in salmon populations have focused on the classical class II DAB or DAA loci, as these have been most amenable to single PCR amplifications due to their relatively low level of sequence divergence. Herein, we report the characterization of the classical class I UBA α2 locus based on collections taken throughout the species range of sockeye salmon (Oncorhynchus nerka). Through use of multiple lineage-specific primer sets, denaturing gradient gel electrophoresis and sequencing, we identified thirty-four alleles from three highly divergent lineages. Sequence identity between lineages ranged from 30.0% to 56.8% but was relatively high within lineages. Allelic identity within the antigen recognition site (ARS) was greater than for the longer sequence. Global positive selection on UBA was seen at the sequence level (dN:dS = 1.012) with four codons under positive selection and 12 codons under negative selection. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Improved composting of poultry feces via supplementation with ammonia oxidizing archaea.

PubMed

Xie, Kaizhi; Jia, Xiaoshan; Xu, Peizhi; Huang, Xu; Gu, Wenjie; Zhang, Fabao; Yang, Shaohai; Tang, Shuanhu

2012-09-01

Ammonia-oxidizing archaea (AOA) play an important role in the oxidation of ammonia. However, the participation of AOA in the composting process has not been established. The addition of AOA to a compost mix was able to speed up both the onset of the hyperthermic phase and the composting time. The composition of the microflora and the relative abundance were determined by using denaturing gradient gel electrophoresis and quantitative real-time PCR, based on the presence of the archaeal amoA genes. The amplicon profiles allowed some of the major AOA species present in the final compost to be identified, and their relative abundance to be estimated from their amplification intensity. The lower pH during the lower temperature phase of compost served to enhance the nitrogen content of the final compost. The addition of AOA resulted in the expanding diversity of microflora species than that of the natural colonization. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Rumen microbial diversity in Svalbard reindeer, with particular emphasis on methanogenic archaea.

PubMed

Sundset, Monica A; Edwards, Joan E; Cheng, Yan Fen; Senosiain, Roberto S; Fraile, Maria N; Northwood, Korinne S; Praesteng, Kirsti E; Glad, Trine; Mathiesen, Svein D; Wright, André-Denis G

2009-12-01

Ruminal methanogens, bacteria and ciliate protozoa of Svalbard reindeer grazing natural pastures in October (late fall) and April (late winter) were investigated using molecular-based approaches. The appetite of the Svalbard reindeer peaks in August (summer) and is at its lowest in March (winter). Microbial numbers, quantified by real-time PCR, did not change significantly between October and April, when food intakes are at similar levels, although the numbers of methanogens tended to be higher in October (P=0.074), and ciliate numbers tended to be higher in April (P=0.055). Similarly, no change was detected in the bacterial and protozoal population composition by rRNA gene-based denaturing gradient gel electrophoresis analysis. Dominant methanogens were identified using a 16S rRNA gene library (97 clones) prepared from pooled PCR products from reindeer on October pasture (n=5). Eleven of the 22 distinct operational taxonomic units (OTUs) generated exhibited a high degree of sequence similarity to methanogens affiliated with Methanobacteriales (eight OTUs), Methanomicrobiales (one OTU) and Methanosarcinales (two OTUs). The remaining 11 OTUs (53% of the clones) were associated with a cluster of uncultivated ruminal archaea. This study has provided important insights into the rumen microbiome of a high-arctic herbivorous animal living under harsh nutritional conditions, and evidence suggesting that host type affects the population size of ruminal methanogens.

Characterization of incubation experiments and development of an enrichment culture capable of ammonium oxidation under iron-reducing conditions

NASA Astrophysics Data System (ADS)

Huang, S.; Jaffé, P. R.

2015-02-01

Incubation experiments were conducted using soil samples from a forested riparian wetland where we have previously observed anaerobic ammonium oxidation coupled to iron reduction. Production of both nitrite and ferrous iron was measured repeatedly during incubations when the soil slurry was supplied with either ferrihydrite or goethite and ammonium chloride. Significant changes in the microbial community were observed after 180 days of incubation as well as in a continuous flow membrane reactor, using 16S rRNA gene PCR-denaturing gradient gel electrophoresis, 454 pyrosequencing, and real-time quantitative PCR analysis. We be Acidimicrobiaceae bacterium A6), belonging to the Acidimicrobiaceae family, whose closest cultivated relative is Ferrimicrobium acidiphilum (with 92% identity) and Acidimicrobium ferrooxidans (with 90% identity), might play a key role in this anaerobic biological process that uses ferric iron as an electron acceptor while oxidizing ammonium to nitrite. After ammonium was oxidized to nitrite, nitrogen loss proceeded via denitrification and/or anammox.

Effects of organic loading rates on reactor performance and microbial community changes during thermophilic aerobic digestion process of high-strength food wastewater.

PubMed

Jang, Hyun Min; Lee, Jae Won; Ha, Jeong Hyub; Park, Jong Moon

2013-11-01

To evaluate the applicability of single-stage thermophilic aerobic digestion (TAD) process treating high-strength food wastewater (FWW), TAD process was operated at four organic loading rates (OLRs) from 9.2 to 37.2 kg COD/m(3)d. The effects of OLRs on microbial community changes were also examined. The highest volumetric removal rate (13.3 kg COD/m(3)d) and the highest thermo-stable protease activity (0.95 unit/mL) were detected at OLR=18.6 kg COD/m(3)d. Denaturing gradient gel electrophoresis (DGGE) profiles and quantitative PCR (qPCR) results showed significant microbial community shifts in response to changes in OLR. In particular, DGGE and phylogenetic analysis demonstrate that the presence of Bacillus sp. (phylum of Firmicutes) was strongly correlated with efficient removal of organic particulates from high-strength food wastewater. Copyright © 2013 Elsevier Ltd. All rights reserved.

Application of rDNA-PCR amplification and DGGE fingerprinting for detection of microbial diversity in a Malaysian crude oil.

PubMed

Liew, Pauline Woanying; Jong, Bor Chyan

2008-05-01

Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.

The synergy of permeable pavements and geothermal heat pumps for stormwater treatment and reuse.

PubMed

Tota-Maharaj, K; Scholz, M; Ahmed, T; French, C; Pagaling, E

2010-12-14

The use of permeable pavement systems with integrated geothermal heat pumps for the treatment and recycling of urban runoff is novel and timely. This study assesses the efficiency of the combined technology for controlled indoor and uncontrolled outdoor experimental rigs. Water quality parameters such as biochemical oxygen demand, nutrients, total viable heterotrophic bacteria and total coliforms were tested before and after treatment in both rigs. The water borne bacterial community genomic deoxyribonucleic acid (DNA) was analyzed by polymerase chain reaction (PCR) amplification followed by denaturing gradient gel electrophoresis (DGGE) and was further confirmed by DNA sequencing techniques. Despite the relatively high temperatures in the indirectly heated sub-base of the pavement, potentially pathogenic organisms such as Salmonella spp., Escherichia coli, faecal Streptococci and Legionella were not detected. Moreover, mean removal rates of 99% for biochemical oxygen demand, 97% for ammonia-nitrogen and 95% for orthophosphate-phosphates were recorded. This research also supports decision-makers in assessing public health risks based on qualitative molecular microbiological data associated with the recycling of treated urban runoff.

Early Results and Spaceflight Implications of the SWAB Flight Experiment

NASA Technical Reports Server (NTRS)

Ott, C. Mark; Pierson, Duane L.

2007-01-01

Microbial monitoring of spacecraft environments provides key information in the assessment of infectious disease risk to the crew. Monitoring aboard the Mir space station and International Space Station (ISS) has provided a tremendous informational baseline to aid in determining the types and concentrations of microorganisms during a mission. Still, current microbial monitoring hardware utilizes culture-based methodology which may not detect many medically significant organisms, such as Legionella pneumophila. We hypothesize that evaluation of the ISS environment using non-culture-based technologies would reveal microorganisms not previously reported in spacecraft, allowing for a more complete health assessment. To achieve this goal, a spaceflight experiment, operationally designated as SWAB, was designed to evaluate the DNA from environmental samples collected from ISS and vehicles destined for ISS. Results from initial samples indicate that the sample collection and return procedures were successful. Analysis of these samples using denaturing gradient gel electrophoresis and targeted PCR primers for fungal contaminants is underway. The current results of SWAB and their implication for in-flight molecular analysis of environmental samples will be discussed.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei.

PubMed

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

2016-07-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes) or TINA-DNA (Twisted Intercalating Nucleic Acids). Gene targets can be specifically labelled with at least about 20 probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3d-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. Copyright © 2016. Published by Elsevier Inc.

Distinctive archaebacterial species associated with anaerobic rumen protozoan Entodinium caudatum.

PubMed

Tóthová, T; Piknová, M; Kisidayová, S; Javorský, P; Pristas, P

2008-01-01

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.

Biofilm comprising phototrophic, diazotrophic, and hydrocarbon-utilizing bacteria: a promising consortium in the bioremediation of aquatic hydrocarbon pollutants.

PubMed

Al-Bader, Dhia; Kansour, Mayada K; Rayan, Rehab; Radwan, Samir S

2013-05-01

Biofilms harboring simultaneously anoxygenic and oxygenic phototrophic bacteria, diazotrophic bacteria, and hydrocarbon-utilizing bacteria were established on glass slides suspended in pristine and oily seawater. Via denaturing gradient gel electrophoresis analysis on PCR-amplified rRNA gene sequence fragments from the extracted DNA from biofilms, followed by band amplification, biofilm composition was determined. The biofilms contained anoxygenic phototrophs belonging to alphaproteobacteria; pico- and filamentous cyanobacteria (oxygenic phototrophs); two species of the diazotroph Azospirillum; and two hydrocarbon-utilizing gammaproteobacterial genera, Cycloclasticus and Oleibacter. The coexistence of all these microbial taxa with different physiologies in the biofilm makes the whole community nutritionally self-sufficient and adequately aerated, a condition quite suitable for the microbial biodegradation of aquatic pollutant hydrocarbons.

Microbial community dynamics in anaerobic bioreactors and algal tanks treating piggery wastewater.

PubMed

Patil, Sayali S; Kumar, Martin S; Ball, Andrew S

2010-06-01

Integrated biosystem is becoming a major aspect of wastewater management practice. Microbial communities in piggery wastewater sampled from anaerobic (thermophilic and mesophilic) and aerobic digesters (algal tanks) during waste remediation were analyzed by culture-independent techniques based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The use of Muyzer's 314F-GC, 518R bacterial primers, and archaeal A934F, 1309R primers followed by partial 16s rDNA sequence analysis of the main bands from DGGE revealed the presence of unknown and as yet uncultured microorganisms but also showed functional and ecologically significant denitrifying, acetogenic bacteria along with autotrophic, hydrogenotrophic, and acetoclastic methanogen archaea. Thermophilic digesters were dominated by gamma-Proteobacteria, Methanothermobacter sp., while mesophilic digesters showed dominance by Firmicutes, uncultured bacteria, Methanosarcina, and Methanoculleus genera. Under aerobic conditions within algal tanks, pH rose from 7.17 to 9.32, with a significant decrease in total ammonia nitrogen, chemical oxygen demand, and soluble phosphorus levels. PCR-DGGE proved a useful tool for investigating the dynamics of microbial community in the bio-processing of piggery wastewater. Knowledge of the microbial communities involved in digestion of piggery wastewater will allow optimization of integrated biosystem by removing the main pollutants like inorganic ammonium-nitrogen, phosphorus, and pathogens from intensive farming system.

Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods.

PubMed

Chen, Hsi-Chia; Wang, Sheng-Yao; Chen, Ming-Ju

2008-05-01

Lactic acid bacteria (LAB) in different original kefir grains were first assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) by a culture-dependent way, and were further confirmed by DNA sequencing techniques. Results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). Lactobacillus kefiri accounted, in the three kefir grains, for at least half of the isolated colonies while Lb. kefiranofaciens was the second most frequently isolated species. Leuconostoc mesenteroides was less frequently found but still in the three kefir grains conversely to Lactococcus lactis which based on culture-dependent isolation was only found in two of the kefir grains. It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method was also applied to detect the LAB strains. Results indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains.

Occurrence of fungi in combs of fungus-growing termites (Isoptera: Termitidae, Macrotermitinae).

PubMed

Guedegbe, Herbert J; Miambi, Edouard; Pando, Anne; Roman, Jocelyne; Houngnandan, Pascal; Rouland-Lefevre, Corinne

2009-10-01

Fungus-growing termites cultivate their mutualistic basidiomycete Termitomyces species on a substrate called a fungal comb. Here, the Suicide Polymerase Endonuclease Restriction (SuPER) method was adapted for the first time to a fungal study to determine the entire fungal community of fungal combs and to test whether fungi other than the symbiotic cultivar interact with termite hosts. Our molecular analyses show that although active combs are dominated by Termitomyces fungi isolated with direct Polymerase Endonuclease Restriction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE), they can also harbor some filamentous fungi and yeasts only revealed by SuPER PCR-DGGE. This is the first molecular evidence of the presence of non-Termitomyces species in active combs. However, because there is no evidence for a species-specific relationship between these fungi and termites, they are mere transient guests with no specialization in the symbiosis. It is however surprising to notice that termite-associated Xylaria strains were not isolated from active combs even though they are frequently retrieved when nests are abandoned by termites. This finding highlights the implication of fungus-growing termites in the regulation of fungi occurring within the combs and also suggests that they might not have any particular evolutionary-based association with Xylaria species.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented quinoa sourdoughs.

PubMed

Ruiz Rodríguez, L; Vera Pingitore, E; Rollan, G; Cocconcelli, P S; Fontana, C; Saavedra, L; Vignolo, G; Hebert, E M

2016-05-01

To analyse lactic acid bacteria (LAB) diversity and technological-functional and safety properties of strains present during spontaneous fermented quinoa sourdoughs. Fermentation was performed by daily backslopping at 30°C for 10 days. Autochthonous LAB microbiota was monitored by a biphasic approach combining random amplified polymorphic DNA (RAPD)-PCR and rRNA gene sequencing with PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Identification and intraspecies differentiation allowed to group isolates within nine LAB species belonging to four genera. A succession of LAB species occurred during 10-days backslopping; Lactobacillus plantarum and Lactobacillus brevis were detected as dominant species in the consortium. The characterization of 15 representative LAB strains was performed based on the acidifying capacity, starch and protein hydrolysis, γ-aminobutyric acid and exopolysaccharides production, antimicrobial activity and antibiotic resistance. Strains characterization led to the selection of Lact. plantarum CRL1905 and Leuconostoc mesenteroides CRL1907 as candidates to be assayed as functional starter culture for the gluten-free (GF) quinoa fermented products. Results on native LAB microbiota present during quinoa sourdough fermentation will allow the selection of strains with appropriate technological properties to be used as a novel functional starter culture for GF-fermented products. © 2016 The Society for Applied Microbiology.

Microbial diversity in a submarine carbonate edifice from the serpentinizing hydrothermal system of the Prony Bay (New Caledonia) over a 6-year period.

PubMed

Postec, Anne; Quéméneur, Marianne; Bes, Méline; Mei, Nan; Benaïssa, Fatma; Payri, Claude; Pelletier, Bernard; Monnin, Christophe; Guentas-Dombrowsky, Linda; Ollivier, Bernard; Gérard, Emmanuelle; Pisapia, Céline; Gérard, Martine; Ménez, Bénédicte; Erauso, Gaël

2015-01-01

Active carbonate chimneys from the shallow marine serpentinizing Prony Hydrothermal Field were sampled 3 times over a 6 years period at site ST09. Archaeal and bacterial communities composition was investigated using PCR-based methods (clone libraries, Denaturating Gel Gradient Electrophoresis, quantitative PCR) targeting 16S rRNA genes, methyl coenzyme M reductase A and dissimilatory sulfite reductase subunit B genes. Methanosarcinales (Euryarchaeota) and Thaumarchaea were the main archaeal members. The Methanosarcinales, also observed by epifluorescent microscopy and FISH, consisted of two phylotypes that were previously solely detected in two other serpentinitzing ecosystems (The Cedars and Lost City Hydrothermal Field). Surprisingly, members of the hyperthermophilic order Thermococcales were also found which may indicate the presence of a hot subsurface biosphere. The bacterial community mainly consisted of Firmicutes, Chloroflexi, Alpha-, Gamma-, Beta-, and Delta-proteobacteria and of the candidate division NPL-UPA2. Members of these taxa were consistently found each year and may therefore represent a stable core of the indigenous bacterial community of the PHF chimneys. Firmicutes isolates representing new bacterial taxa were obtained by cultivation under anaerobic conditions. Our study revealed diverse microbial communities in PHF ST09 related to methane and sulfur compounds that share common populations with other terrestrial or submarine serpentinizing ecosystems.

Estimation of contamination sources of human enteroviruses in a wastewater treatment and reclamation system by PCR-DGGE.

PubMed

Ji, Zheng; Wang, Xiaochang C; Xu, Limei; Zhang, Chongmiao; Funamizu, Naoyuki; Okabe, Satoshi; Sano, Daisuke

2014-06-01

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method was employed to estimate the contamination sources of human enteroviruses and understand how their dominant strains vary in a wastewater treatment and reclamation system consisting of sewage collection, wastewater treatment with membrane bioreactor and open lakes for reclaimed water storage and reuse. After PCR-DGGE using a selected primer set targeting enteroviruses, phylogenetic analysis of acquired enterovirus gene sequences was performed. Enteroviruses identified from the septic tank were much more diverse than those from grey water and kitchen wastewater. Several unique types of enterovirus different from those in wastewater samples were dominant in a biological wastewater treatment unit. Membrane filtration followed by chlorination was proved effective for physically eliminating enteroviruses; however, secondary contamination likely occurred as the reclaimed water was stored in artificial lakes. Enterovirus 71 (EV71), a hand-foot-and-mouth disease (HFMD) viral pathogen, was detected mainly from the artificial lakes, implying that wastewater effluent was not the contamination source of EV71 and that there were unidentified non-point sources of the contamination with the HFMD viral pathogen in the reclaimed water stored in the artificial lakes. The PCR-DGGE targeting enteroviruses provided robust evidence about viral contamination sources in the wastewater treatment and reclamation system.

[Effects of different catch modes on soil enzyme activities and bacterial community in the rhizosphere of cucumber].

PubMed

Li, Min; Wu, Feng-zhi

2014-12-01

Effects of different catch modes on soil enzyme activities and bacterial community in the rhizosphere of cucumber (Cucumis sativus) were analyzed by conventional chemical method, PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR methods. Pot experiment was carried out in the greenhouse for three consecutive years with cucumber as the main crop, and scallion (Allium fistulosum), wheat (Triticum aestivum) and oilseed rape (Brassica campestri) as catch crops. Results showed that, with the increase of crop planting times, soil urease, neutral phosphatase and invertase activities in the wheat treatment were significantly) higher than in the scallion and oilseed rape treatments, and these enzyme activities in the oilseed rape treatment were significantly higher than in the scallion treatment. PCR-DGGR analysis showed that cucumber rhizosphere bacterial community structures were different among treatments. Scallion and wheat treatments maintained relatively higher diversity indices of bacterial community structure. qPCR results showed that the abundance of soil bacterial community in the wheat treatment was significantly higher than in the scallion and oilseed rape treatments. In conclusion, different catch treatments affected soil enzyme activities and bacteria community and changed the soil environment. Wheat used as summer catch crop could maintain relatively higher soil enzyme activities, bacterial community diversity and abundance.

PCR-DGGE analysis of bacterial community dynamics in kava beverages during refrigeration.

PubMed

Dong, J; Kandukuru, P; Huang, A S; Li, Y

2011-07-01

Kava beverages are highly perishable even under refrigerated conditions. This study aimed to investigate the bacterial community dynamics in kava beverages during refrigeration.  Four freshly made kava beverages were obtained from kava bars and stored at 4°C. On days 0, 3 and 6, the aerobic plate count (APC), lactic acid bacteria (LAB) count and yeast and mould count (YMC) of the samples were determined. Meanwhile, bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Moreover, species-specific PCR assays were employed to identify predominant Pseudomonas spp. involved in kava spoilage. Over the storage period, the APC, LAB count and YMC of the four kava beverages all increased, whereas their pH values decreased. The DGGE profile revealed diverse bacterial populations in the samples. LAB, such as Weissella soli, Lactobacillus spp. and Lactococcus lactis, were found in the kava beverages. Species-specific PCR assays detected Pseudomonas putida and Pseudomonas fluorescens in the samples; Ps. fluorescens became dominant during refrigeration. LAB and Pseudomonas may play a significant role in the spoilage of kava beverages. This study provides important information that may be used to extend the shelf life of kava beverages. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

Microbiota of an Italian Grana-Like Cheese during Manufacture and Ripening, Unraveled by 16S rRNA-Based Approaches.

PubMed

Alessandria, Valentina; Ferrocino, Ilario; De Filippis, Francesca; Fontana, Mauro; Rantsiou, Kalliopi; Ercolini, Danilo; Cocolin, Luca

2016-07-01

The microbial ecology of cheese involves a rich and complex interaction between starter lactic acid bacteria and nonstarter lactic acid bacteria (NSLAB), mainly originating from raw milk and/or from the environment, that can contribute to the final characteristics of cheese. The aim of the present research was the exploration of the active microbiota by RNA-based approaches during the manufacturing and ripening of a Grana-like cheese. Reverse transcriptase PCR (RT-PCR)-denaturing gradient gel electrophoresis (DGGE) and RNA-based high-throughput sequencing were applied to profile microbial populations, while the enumeration of active bacteria was carried out by using quantitative PCR (qPCR). Three different cheese productions (named D, E, and F) collected in the same month from the same dairy plant were analyzed. The application of the qPCR protocol revealed the presence of 7 log CFU/ml of bacterial load in raw milk, while, during ripening, active bacterial populations ranged from <4 to 8 log CFU/ml. The natural whey starters used in the three productions showed the same microbiota composition, characterized by the presence of Lactobacillus helveticus and Lactobacillus delbrueckii Nevertheless, beta-diversity analysis of the 16S rRNA sequencing data and RT-PCR-DGGE showed a clear clustering of the samples according to the three productions, probably driven by the different milks used. Milk samples were found to be characterized by the presence of several contaminants, such as Propionibacterium acnes, Acidovorax, Acinetobacter, Pseudomonas, and NSLAB. The core genera of the starter tended to limit the development of the spoilage bacteria only in two of the three batches. This study underlines the influence of different factors that can affect the final microbiota composition of the artisanal cheese. This study highlights the importance of the quality of the raw milk in the production of a hard cheese. Independent from the use of a starter culture, raw milk with low microbiological quality can negatively affect the populations of lactic acid bacteria and, as a consequence, impact the quality of the final product due to metabolic processes associated with spoilage bacteria. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A survey of alterations in microbial community diversity in marine sediments in response to oil from the Deepwater Horizon spill: Northern Gulf of Mexico shoreline, Texas to Florida

USGS Publications Warehouse

Lisle, John T.

2011-01-01

Microbial community genomic DNA was extracted from sediment samples collected from the northern Gulf of Mexico (NGOM) coast. These samples had a high probability of being impacted by Macondo-1 (M-1) well oil from the Deepwater Horizon (DWH) drilling site. The hypothesis for this project was that presence of M-1 oil in coastal sediments would significantly alter the diversity within the microbial communities associated with the impacted sediments. To determine if community-level changes did or did not occur following exposure to M-1 oil, microbial community-diversity fingerprints were generated and compared. Specific sequences within the community's genomic DNA were first amplified using the polymerase chain reaction (PCR) using a primer set that provides possible resolution to the species level. A second nested PCR that was performed on the primary PCR products using a primer set on which a GC-clamp was attached to one of the primers. These nested PCR products were separated using denaturing-gradient gel electrophoresis (DGGE) that resolves the nested PCR products based on sequence dissimilarities (or similarities), forming a genomic fingerprint of the microbial diversity within the respective samples. Sediment samples with similar fingerprints were grouped and compared to oil-fingerprint data from Rosenbauer and others (2010). The microbial community fingerprints grouped closely when identifying those sites that had been impacted by M-1 oil (N=12) and/or some mixture of M-1 and other oil (N=4), based upon the oil fingerprints. This report represents some of the first information on naturally occurring microbial communities in sediment from shorelines along the NGOM coast. These communities contain microbes capable of degrading oil and related hydrocarbons, making this information relevant to response and recovery of the NGOM from the DWH incident.

Microbial fouling community analysis of the cooling water system of a nuclear test reactor with emphasis on sulphate reducing bacteria.

PubMed

Balamurugan, P; Joshi, M Hiren; Rao, T S

2011-10-01

Culture and molecular-based techniques were used to characterize bacterial diversity in the cooling water system of a fast breeder test reactor (FBTR). Techniques were selected for special emphasis on sulphate-reducing bacteria (SRB). Water samples from different locations of the FBTR cooling water system, in addition to biofilm scrapings from carbon steel coupons and a control SRB sample were characterized. Whole genome extraction of the water samples and SRB diversity by group specific primers were analysed using nested PCR and denaturing gradient gel electrophoresis (DGGE). The results of the bacterial assay in the cooling water showed that the total culturable bacteria (TCB) ranged from 10(3) to 10(5) cfu ml(-1); iron-reducing bacteria, 10(3) to 10(5) cfu ml(-1); iron oxidizing bacteria, 10(2) to 10(3) cfu ml(-1) and SRB, 2-29 cfu ml(-1). However, the counts of the various bacterial types in the biofilm sample were 2-3 orders of magnitude higher. SRB diversity by the nested PCR-DGGE approach showed the presence of groups 1, 5 and 6 in the FBTR cooling water system; however, groups 2, 3 and 4 were not detected. The study demonstrated that the PCR protocol influenced the results of the diversity analysis. The paper further discusses the microbiota of the cooling water system and its relevance in biofouling.

Behavior of variable V3 region from 16S rDNA of lactic acid bacteria in denaturing gradient gel electrophoresis.

PubMed

Ercolini, D; Moschetti, G; Blaiotta, G; Coppola, S

2001-03-01

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects.

Salecan Enhances the Activities of β-1,3-Glucanase and Decreases the Biomass of Soil-Borne Fungi

PubMed Central

Chen, Yunmei; Xu, Haiyang; Zhou, Mengyi; Wang, Yang; Wang, Shiming; Zhang, Jianfa

2015-01-01

Salecan, a linear extracellular polysaccharide consisting of β-1,3-D-glucan, has potential applications in the food, pharmaceutical and cosmetic industries. The objective of this study was to evaluate the effects of salecan on soil microbial communities in a vegetable patch. Compositional shifts in the genetic structure of indigenous soil bacterial and fungal communities were monitored using culture-dependent dilution plating, culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR. After 60 days, soil microorganism counts showed no significant variation in bacterial density and a marked decrease in the numbers of fungi. The DGGE profiles revealed that salecan changed the composition of the microbial community in soil by increasing the amount of Bacillus strains and decreasing the amount of Fusarium strains. Quantitative PCR confirmed that the populations of the soil-borne fungi Fusarium oxysporum and Trichoderma spp. were decreased approximately 6- and 2-fold, respectively, in soil containing salecan. This decrease in the amount of fungi can be explained by salecan inducing an increase in the activities of β-1,3-glucanase in the soil. These results suggest the promising application of salecan for biological control of pathogens of soil-borne fungi. PMID:26247592

Microbiological and fermentative properties of baker's yeast starter used in breadmaking.

PubMed

Reale, A; Di Renzo, T; Succi, M; Tremonte, P; Coppola, R; Sorrentino, E

2013-08-01

This study assessed the levels of microbial contaminants in liquid, compressed and dry commercial baker's yeasts used as starters in breadmaking. Eumycetes, Enterobacteriaceae, total and fecal coliforms, Bacillus spp., and lactic acid bacteria (LAB), in particular enterococci, were quantified. Results obtained in this study highlighted that baker's yeast could represent a potential vehicle of spoilage and undesirable microorganisms into the baking environment, even if these do not influence the leavening activity in the dough, as ascertained by rheofermentometer analysis. Different microbial groups, such as spore-forming bacteria and moulds, were found in baker's yeast starters. Moreover, different species of LAB, which are considered the main contaminants in large-scale yeast fermentations, were isolated and identified by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rDNA sequencing. The most recurrent species were Lactobacillus plantarum, Enterococcus faecalis, and Enterococcus durans, isolated from both compressed and dry starters, whereas strains belonging to Leuconostoc and Pediococcus genera were found only in dry ones. Nested-Polymerase Chain Reaction (Nested-PCR) and Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR) were also used to highlight the biodiversity of the different commercial yeast strains, and to ascertain the culture purity. © 2013 Institute of Food Technologists®

Decreased microbial diversity and Lactobacillus group in the intestine of geriatric giant pandas (Ailuropoda melanoleuca).

PubMed

Peng, Zhirong; Zeng, Dong; Wang, Qiang; Niu, Lili; Ni, Xueqin; Zou, Fuqin; Yang, Mingyue; Sun, Hao; Zhou, Yi; Liu, Qian; Yin, Zhongqiong; Pan, Kangcheng; Jing, Bo

2016-05-01

It has been established beyond doubt that giant panda genome lacks lignin-degrading related enzyme, gastrointestinal microbes may play a vital role in digestion of highly fibrous bamboo diet. However, there is not much information available about the intestinal bacteria composition in captive giant pandas with different ages. In this study, we compared the intestinal bacterial community of 12 captive giant pandas from three different age groups (subadults, adults, and geriatrics) through PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR analysis. Results indicated that microbial diversity in the intestine of adults was significantly higher than that of the geriatrics (p < 0.05), but not significant compared to the subadults (p > 0.05). The predominant bands in DGGE patterns shared by the twelve pandas were related to Firmicutes and Proteobacteria. Additionally, in comparison to healthy individuals, antibiotic-treated animals showed partial microbial dysbiosis. Real-time PCR analyses confirmed a significantly higher abundance of the Lactobacillus in the fecal microbiota of adults (p < 0.05), while other bacterial groups and species detected did not significantly differ among the three age groups (p > 0.05). This study revealed that captive giant pandas with different ages showed different intestinal bacteria composition.

Characterisation of microbial communities in Chinese liquor fermentation starters Daqu using nested PCR-DGGE.

PubMed

Zhang, Liqiang; Wu, Chongde; Ding, Xiaofei; Zheng, Jia; Zhou, Rongqing

2014-12-01

In this study, characterises of the microbial community structures of three typical Chinese liquor Daqu, as well as different kinds of light flavour Daqu were investigated using nested PCR-denaturing gradient gel electrophoresis (DGGE). The results showed that microbial diversity was considerably different, and the microfloral compositions were highly variable among various Daqu. Lactic acid bacteria, which accounted for 30.95 % of all identified bacteria, were dominant in all Daqu samples, whereas Bacillus species were also predominant in the Luzhou (14.8 %) and Langjiu Daqu (18.2 %). Citrobacter and Burkholderia were first identified in light flavour Daqu. Aspergillus was the dominant moulds, and the non-Saccharomyces yeast species, Saccharomycopsis fibuligera, Wallemia sebi, Wallemia muriae, and Pichia subpelliculosa, were the dominant yeasts. Rasamsonia, Galactomyces, Geotrichum and Wallemia were first identified using nested PCR-DGGE. Cluster analysis indicated that the microbial community structures of different Daqu samples exhibited some differences. These may be ascribed to the different peak production temperatures, raw material constituents and microhabitats around the liquor enterprises. The current study provides insights into the microbial community structures of three typical Daqu samples, and may facilitate the development of starter cultures for manufacturing Chinese liquor.

Competing factors of compost concentration and proximity to root affect the distribution of streptomycetes.

PubMed

Inbar, Ehud; Green, Stefan J; Hadar, Yitzhak; Minz, Dror

2005-07-01

Streptomycetes are important members of soil microbial communities and are particularly active in the degradation of recalcitrant macromolecules and have been implicated in biological control of plant disease. Using a streptomycetes-specific polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (PCR-DGGE) methodology coupled with band excision and sequence analysis, we examined the effect of grape marc compost amendment to soil on cucumber plant-associated streptomycetes community composition. We observed that both compost amendment and proximity to the root surface influenced the streptomycetes community composition. A strong root selection for a soil-derived Streptomycete, most closely related to Streptomyces thermotolerans, S. iakyrus, and S. thermocarboxydus, was independent of compost amendment rate. However, while the impact of compost amendment was mitigated with increasing proximity to the root, high levels of compost amendment resulted in the detection of compost-derived species on the root surface. Conversely, in rhizosphere and non-rhizosphere soils, the community composition of streptomycetes was affected strongly even by modest compost amendment. The application of a streptomycetes-specific PCR primer set combined with DGGE analysis provided a rapid means of examining the distribution and ecology of streptomycetes in soils and plant-associated environments.

[Application of rapid PCR to authenticate medicinal snakes].

PubMed

Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-Qi; Li, Man

2014-10-01

To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

Evaluation of soil bioremediation techniques in an aged diesel spill at the Antarctic Peninsula.

PubMed

de Jesus, Hugo E; Peixoto, Raquel S; Cury, Juliano C; van Elsas, Jan D; Rosado, Alexandre S

2015-12-01

Many areas on the Antarctic continent already suffer from the direct and indirect influences of human activities. The main cause of contamination is petroleum hydrocarbons because this compound is used as a source of energy at the many research stations around the continent. Thus, the current study aims to evaluate treatments for bioremediation (biostimulation, bioaugmentation, and bioaugmentation + biostimulation) using soils from around the Brazilian Antarctic Station "Comandante Ferraz" (EACF), King George Island, Antarctic Peninsula. The experiment lasted for 45 days, and at the end of this period, chemical and molecular analyses were performed. Those analyses included the quantification of carbon and nitrogen, denaturing gradient gel electrophoresis (DGGE) analysis (with gradient denaturation), real-time PCR, and quantification of total hydrocarbons and polyaromatics. Molecular tests evaluated changes in the profile and quantity of the rrs genes of archaea and bacteria and also the alkB gene. The influence of the treatments tested was directly related to the type of soil used. The work confirmed that despite the extreme conditions found in Antarctic soils, the bacterial strains degraded hydrocarbons and bioremediation treatments directly influenced the microbial communities present in these soils even in short periods. Although the majority of the previous studies demonstrate that the addition of fertilizer seems to be most effective at promoting bioremediation, our results show that for some conditions, autochthonous bioaugmentation (ABA) treatment is indicated. This work highlights the importance of understanding the processes of recovery of contaminated environments in polar regions because time is crucial to the soil recovery and to choosing the appropriate treatment.

Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed

PubMed Central

Smalla, K.; Wieland, G.; Buchner, A.; Zock, A.; Parzy, J.; Kaiser, S.; Roskot, N.; Heuer, H.; Berg, G.

2001-01-01

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands. PMID:11571180

Case Study of the Distribution of Mucosa-Associated Bifidobacterium Species, Lactobacillus Species, and Other Lactic Acid Bacteria in the Human Colon

PubMed Central

Nielsen, D. S.; Møller, P. L.; Rosenfeldt, V.; Pærregaard, A.; Michaelsen, K. F.; Jakobsen, M.

2003-01-01

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site. PMID:14660412

Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

PubMed

Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

2016-09-29

The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

PubMed Central

Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

2016-01-01

The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi. PMID:27600711

Effect of Elevated Salt Concentrations on the Aerobic Granular Sludge Process: Linking Microbial Activity with Microbial Community Structure▿

PubMed Central

Bassin, J. P.; Pronk, M.; Muyzer, G.; Kleerebezem, R.; Dezotti, M.; van Loosdrecht, M. C. M.

2011-01-01

The long- and short-term effects of salt on biological nitrogen and phosphorus removal processes were studied in an aerobic granular sludge reactor. The microbial community structure was investigated by PCR-denaturing gradient gel electrophoresis (DGGE) on 16S rRNA and amoA genes. PCR products obtained from genomic DNA and from rRNA after reverse transcription were compared to determine the presence of bacteria as well as the metabolically active fraction of bacteria. Fluorescence in situ hybridization (FISH) was used to validate the PCR-based results and to quantify the dominant bacterial populations. The results demonstrated that ammonium removal efficiency was not affected by salt concentrations up to 33 g/liter NaCl. Conversely, a high accumulation of nitrite was observed above 22 g/liter NaCl, which coincided with the disappearance of Nitrospira sp. Phosphorus removal was severely affected by gradual salt increase. No P release or uptake was observed at steady-state operation at 33 g/liter NaCl, exactly when the polyphosphate-accumulating organisms (PAOs), “Candidatus Accumulibacter phosphatis” bacteria, were no longer detected by PCR-DGGE or FISH. Batch experiments confirmed that P removal still could occur at 30 g/liter NaCl, but the long exposure of the biomass to this salinity level was detrimental for PAOs, which were outcompeted by glycogen-accumulating organisms (GAOs) in the bioreactor. GAOs became the dominant microorganisms at increasing salt concentrations, especially at 33 g/liter NaCl. In the comparative analysis of the diversity (DNA-derived pattern) and the activity (cDNA-derived pattern) of the microbial population, the highly metabolically active microorganisms were observed to be those related to ammonia (Nitrosomonas sp.) and phosphate removal (“Candidatus Accumulibacter”). PMID:21926194

Nanoscale superstructures assembled by polymerase chain reaction (PCR): programmable construction, structural diversity, and emerging applications.

PubMed

Kuang, Hua; Ma, Wei; Xu, Liguang; Wang, Libing; Xu, Chuanlai

2013-11-19

Polymerase chain reaction (PCR) is an essential tool in biotechnology laboratories and is becoming increasingly important in other areas of research. Extensive data obtained over the last 12 years has shown that the combination of PCR with nanoscale dispersions can resolve issues in the preparation DNA-based materials that include both inorganic and organic nanoscale components. Unlike conventional DNA hybridization and antibody-antigen complexes, PCR provides a new, effective assembly platform that both increases the yield of DNA-based nanomaterials and allows researchers to program and control assembly with predesigned parameters including those assisted and automated by computers. As a result, this method allows researchers to optimize to the combinatorial selection of the DNA strands for their nanoparticle conjugates. We have developed a PCR approach for producing various nanoscale assemblies including organic motifs such as small molecules, macromolecules, and inorganic building blocks, such as nanorods (NRs), metal, semiconductor, and magnetic nanoparticles (NPs). We start with a nanoscale primer and then modify that building block using the automated steps of PCR-based assembly including initialization, denaturation, annealing, extension, final elongation, and final hold. The intermediate steps of denaturation, annealing, and extension are cyclic, and we use computer control so that the assembled superstructures reach their predetermined complexity. The structures assembled using a small number of PCR cycles show a lower polydispersity than similar discrete structures obtained by direct hybridization between the nanoscale building blocks. Using different building blocks, we assembled the following structural motifs by PCR: (1) discrete nanostructures (NP dimers, NP multimers including trimers, pyramids, tetramers or hexamers, etc.), (2) branched NP superstructures and heterochains, (3) NP satellite-like superstructures, (4) Y-shaped nanostructures and DNA networks, (5) protein-DNA co-assembly structures, and (6) DNA block copolymers including trimers and pentamers. These results affirm that this method can produce a variety of chemical structures and in yields that are tunable. Using PCR-based preparation of DNA-bridged nanostructures, we can program the assembly of the nanoscale blocks through the adjustment of the primer intensity on the assembled units, the number of PCR cycles, or both. The resulting structures are highly complex and diverse and have interesting dynamics and collective properties. Potential applications of these materials include chirooptical materials, probe fabrication, and environmental and biomedical sensors.

Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

PubMed Central

Kowalchuk, George A.; Naoumenko, Zinaida S.; Derikx, Piet J. L.; Felske, Andreas; Stephen, John R.; Arkhipchenko, Irina A.

1999-01-01

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms. PMID:9925559

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana▿

PubMed Central

Camu, Nicholas; De Winter, Tom; Verbrugghe, Kristof; Cleenwerck, Ilse; Vandamme, Peter; Takrama, Jemmy S.; Vancanneyt, Marc; De Vuyst, Luc

2007-01-01

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as “Weissella ghanaensis,” was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named “Acetobacter senegalensis” (A. tropicalis-like) and “Acetobacter ghanaensis” (A. syzygii-like). PMID:17277227

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patrick, N.; Miyakawa, F.; Hunt, J.A.

The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5more » of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].« less

Diversity of Bacterial Communities in Container Habitats of Mosquitoes

PubMed Central

Ponnusamy, Loganathan; Xu, Ning; Stav, Gil; Wesson, Dawn M.; Schal, Coby

2010-01-01

We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n=12), cemetery urns (n=23), and miscellaneous containers that included two tree holes (n=19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species. PMID:18373113

Effect of bioaugmentation and biostimulation on sulfate-reducing column startup captured by functional gene profiling.

PubMed

Pereyra, Luciana P; Hiibel, Sage R; Perrault, Elizabeth M; Reardon, Kenneth F; Pruden, Amy

2012-10-01

Sulfate-reducing permeable reactive zones (SR-PRZs) depend upon a complex microbial community to utilize a lignocellulosic substrate and produce sulfides, which remediate mine drainage by binding heavy metals. To gain insight into the impact of the microbial community composition on the startup time and pseudo-steady-state performance, functional genes corresponding to cellulose-degrading (CD), fermentative, sulfate-reducing, and methanogenic microorganisms were characterized in columns simulating SR-PRZs using quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). Duplicate columns were bioaugmented with sulfate-reducing or CD bacteria or biostimulated with ethanol or carboxymethyl cellulose and compared with baseline dairy manure inoculum and uninoculated controls. Sulfate removal began after ~ 15 days for all columns and pseudo-steady state was achieved by Day 30. Despite similar performance, DGGE profiles of 16S rRNA gene and functional genes at pseudo-steady state were distinct among the column treatments, suggesting the potential to control ultimate microbial community composition via bioaugmentation and biostimulation. qPCR revealed enrichment of functional genes in all columns between the initial and pseudo-steady-state time points. This is the first functional gene-based study of CD, fermentative and sulfate-reducing bacteria and methanogenic archaea in a lignocellulose-based environment and provides new qualitative and quantitative insight into startup of a complex microbial system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments

PubMed Central

Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

2006-01-01

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained. PMID:16391020

Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene.

PubMed

Dobrowolski, Steven F; McKinney, Jason T; Amat di San Filippo, Cristina; Giak Sim, Keow; Wilcken, Bridget; Longo, Nicola

2005-03-01

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate dye-binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA binding dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate dye-binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration. (c) 2005 Wiley-Liss, Inc.

Variation of bacterial communities and expression of Toll-like receptor genes in the rumen of steers differing in susceptibility to subacute ruminal acidosis.

PubMed

Chen, Yanhong; Oba, Masahito; Guan, Le Luo

2012-10-12

In order to determine differences in the ruminal bacterial community and host Toll-like receptor (TLR) gene expression of beef cattle with different susceptibility to acidosis, rumen papillae and content were collected from acidosis-susceptible (AS, n=3) and acidosis-resistant (AR, n=3) steers. The ruminal bacterial community was characterized using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time PCR (qRT-PCR) analysis. Global R analysis of bacterial profile similarity revealed that bacterial diversity was significantly different between AR and AS groups for both rumen content (P=0.001) and epithelial (P=0.002) communities. The copy number of total bacterial 16S rRNA genes in content of AS steers was 10-fold higher than that of AR steers, and the copy number of total 16S rRNA genes of epimural bacteria in AR steers was positively correlated with ruminal pH (r=0.59, P=0.04), and negatively correlated with total VFA concentration (r=-0.59, P=0.05). The expressions of host TLR2 and 4 genes were significantly higher in AR steers compared to those in AS steers. These findings enhance our understanding about the ruminal microbial ecology and host gene expression changes that may be useful in the prevention of ruminal acidosis. Copyright © 2012 Elsevier B.V. All rights reserved.

Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches

PubMed Central

Alegría, Ángel; Szczesny, Pawel; Mayo, Baltasar; Bardowski, Jacek

2012-01-01

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures. PMID:22247135

Molecular characterization of skin microbiota between cancer cachexia patients and healthy volunteers.

PubMed

Li, Wei; Han, Lei; Yu, Pengbo; Ma, Chaofeng; Wu, Xiaokang; Moore, John E; Xu, Jiru

2014-04-01

Systemic inflammation contributes to both the development of cancer and of cachexia. The microenvironment of bacterial habitats might be changed during the progression of cancer cachexia. The aim of this study was to quantitatively and qualitatively compare the composition of the skin microbiota between cancer cachexia patients and healthy volunteers. Cutaneous bacteria were swabbed at the axillary fossa of 70 cancer cachexia patients and 34 healthy individuals from China. Nested-PCR-denaturing gradient gel electrophoresis (PCR-DGGE) with primers specifically targeting V3 region and quantitative PCR (qPCR) for total bacteria, Corynebacterium spp., Staphylococcus spp., and Staphylococcus epidermidis were performed on all samples. Barcoded 454 pyrosequencing of the V3-V4 regions was performed on 30 randomly selected samples. By comparing diversity and richness indices, we found that the skin microbiome of cachectic cancer patients is less diverse than that of healthy participants, though these differences were not significant. The main microbes that reside on human skin were divided into four phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. Staphylococcus spp. and Corynebacterium spp. were the dominant bacteria at the genus level. Significantly fewer Corynebacterium spp. had been observed in cachexia patients compared to healthy subjects. These results suggest that the presence of cancer and cachexia alters human skin bacterial communities. Understanding the changes in microbiota during cancer cachexia may lead to new insights into the syndrome.

Identification of yeasts and evaluation of their distribution in Taiwanese Kefir and Viili starters.

PubMed

Wang, S Y; Chen, H C; Liu, J R; Lin, Y C; Chen, M J

2008-10-01

The objective of the present study was to investigate yeast communities in kefir grains and viili starters in Taiwan through conventional microbiological cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The DNA sequencing was used as a validity technique to ensure that all isolates within each group belonged to just one species, and to confirm the identified results of PCR-DGGE. Results indicated that a combination of conventional microbiological cultivation with PCR-DGGE and sequencing could successfully identify 4 yeast species from both types of cultures in Taiwan. Kluyveromyces marxianus, Saccharomyces turicensis, and Pichia fermentans were found in Taiwanese kefir grains with a distribution of 76, 22, and 2%, respectively, whereas Klu. marxianus, Saccharomyces unisporus and P. fermentans were identified in viili starters corresponding to 58, 11, and 31% of the total cell counts, respectively. Furthermore, the culture-independent method was applied to identify the yeast species using DGGE. Only 2 yeast species, Klu. marxianus and S. turicensis, were found in kefir grains and 2, Klu. marxianus and P. fermentans, in viili starters. These results suggest that in samples containing multiple species, PCR-DGGE may fail to detect some species. Sequences of yeast isolates reported in this study have been deposited in the GenBank database under accession nos. DQ139802, AF398485, DQ377652, and AY007920.

Molecular identification of Mango, Mangifera indica L.var. totupura

PubMed Central

Jagarlamudi, Sankar; G, Rosaiah; Kurapati, Ravi Kumar; Pinnamaneni, Rajasekhar

2011-01-01

Mango (>Mangifera indica) belonging to Anacardiaceae family is a fruit that grows in tropical regions. It is considered as the King of fruits. The present work was taken up to identify a tool in identifying the mango species at the molecular level. The chloroplast trnL-F region was amplified from extracted total genomic DNA using the polymerase chain reaction (PCR) and sequenced. Sequence of the dominant DGGE band revealed that Mangifera indica in tested leaves was Mangifera indica (100% similarity to the ITS sequences of Mangifera indica). This sequence was deposited in NCBI with the accession no. GQ927757. Abbreviations AFLP - Amplified fragment length polymorphism , cpDNA - Chloroplast DNA, DDGE - Denaturing gradient gel electrophoresis, DNA - Deoxyribo nucleic acid, EDTA - Ethylenediamine tetraacetic acid, HCl - Hydrochloric acid, ISSR - Inter simple sequence repeats, ITS - Internal transcribed spacer, MATAB - Methyl Ammonium Bromide, Na2SO3 - Sodium sulphite, NaCl - Sodium chloride, NCBI - National Centre for Biotechnology Information, PCR - Polymerase chain reaction, PEG - Polyethylene glycol, RAPD - Randomly amplified polymorphic DNA, trnL-F - Transfer RNA genes start codon- termination codon. PMID:21423885

Characterization of incubation experiments and development of an enrichment culture capable of ammonium oxidation under iron reducing conditions

NASA Astrophysics Data System (ADS)

Huang, S.; Jaffé, P. R.

2014-08-01

Incubation experiments were conducted using soil samples from a forested riparian wetland where we have previously observed anaerobic ammonium oxidation coupled to iron reduction. Production of both nitrite and ferrous iron were measured repeatedly during incubations when the soil slurry was supplied with either ferrihydrite or goethite and ammonium chloride. Significant changes in the microbial community were observed after 180 days of incubation as well as in a continuous flow membrane reactor, using 16S rRNA gene PCR-denaturing gradient gel electrophoresis, 454-pyrosequencing, and real-time quantitative PCR analysis. We believe that one of the dominant microbial species in our system (an uncultured Acidimicrobiaceae bacterium A6), belonging to the Acidimicrobiaceae family, whose closest cultivated relative is Ferrimicrobium acidiphilum (with 92% identity) and Acidimicrobium ferrooxidans (with 90% identity), might play a key role in this anaerobic biological process that uses ferric iron as an electron acceptor while oxidizing ammonium to nitrite. After ammonium was oxidized to nitrite, nitrogen loss proceeded via denitrification and/or anammox.

Enhanced biodegradation of antibiotic combinations via the sequential treatment of the sludge resulting from pharmaceutical wastewater treatment using white-rot fungi Trametes versicolor and Bjerkandera adusta.

PubMed

Aydin, Sevcan

2016-07-01

While anaerobic treatment is capable of treating pharmaceutical wastewater and removing antibiotics in liquid phases, solid phases may still contain significant amounts of antibiotics following this treatment. The main goal of this study was to evaluate the use of white-rot fungi to remove erythromycin, sulfamethoxazole, and tetracycline combinations from biosolids. The degradation potential of Trametes versicolor and Bjerkandera adusta was evaluated via the sequential treatment of anaerobic sludge. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses were used to identify competition between the autochthonous microbial communities and white-rot fungi. Solid-phase treatment using white-rot fungi substantially reduced antibiotic concentrations and toxicity in sludge. According to PCR-DGGE results, there is an association between species of fungus and antibiotic type as a result of the different transformation pathways of fungal strains. Fungal post-treatment of sludge represents a promising method of removing antibiotic combinations, therefore holding a significant promise as an environmentally friendly means of degrading the antibiotics present in sludge.

Dynamics of oral microbial community profiling during severe early childhood caries development monitored by PCR-DGGE.

PubMed

Tao, Ye; Zhou, Yan; Ouyang, Yong; Lin, HuanCai

2013-09-01

To monitor the longitudinal changes in oral microbial diversity of children with severe early childhood caries (S-ECC) compared to caries free (CF) controls. Dental plaque samples of 12 children in each group at 8, 14, 20, 26 and 32 months of age were analysed. Total microbial genomic DNA was isolated from each sample, and PCR-denaturing gradient gel electrophoresis (DGGE) analyses were carried out. The number of bands was significantly higher in the CF group (18.17±4.91 bands) than in the S-ECC group (14.54±5.56 bands) at 32 months of age (P<0.05). A total of 21 genera were identified in all subjects, and there were no significant differences between the two groups at genus level. DGGE profiles showed that most of the clusters were constructed from one individual over time in the both groups. The onset of S-ECC is accompanied by a decrease in microbial diversity. The overall composition of the microbiota is highly similar within an individual over time. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.

PubMed

Neria-González, Isabel; Wang, En Tao; Ramírez, Florina; Romero, Juan M; Hernández-Rodríguez, César

2006-06-01

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.

Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR.

PubMed

Osimani, Andrea; Milanović, Vesna; Garofalo, Cristiana; Cardinali, Federica; Roncolini, Andrea; Sabbatini, Riccardo; De Filippis, Francesca; Ercolini, Danilo; Gabucci, Claudia; Petruzzelli, Annalisa; Tonucci, Franco; Clementi, Francesca; Aquilanti, Lucia

2018-07-02

The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of C/N ratio on nitrous oxide production from nitrification in a laboratory-scale biological aerated filter reactor.

PubMed

He, Qiang; Zhu, Yinying; Fan, Leilei; Ai, Hainan; Huangfu, Xiaoliu; Chen, Mei

2017-03-01

Emission of nitrous oxide (N 2 O) during biological wastewater treatment is of growing concern. This paper reports findings of the effects of carbon/nitrogen (C/N) ratio on N 2 O production rates in a laboratory-scale biological aerated filter (BAF) reactor, focusing on the biofilm during nitrification. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and microelectrode technology were utilized to evaluate the mechanisms associated with N 2 O production during wastewater treatment using BAF. Results indicated that the ability of N 2 O emission in biofilm at C/N ratio of 2 was much stronger than at C/N ratios of 5 and 8. PCR-DGGE analysis showed that the microbial community structures differed completely after the acclimatization at tested C/N ratios (i.e., 2, 5, and 8). Measurements of critical parameters including dissolved oxygen, oxidation reduction potential, NH 4 + -N, NO 3 - -N, and NO 2 - -N also demonstrated that the internal micro-environment of the biofilm benefit N 2 O production. DNA analysis showed that Proteobacteria comprised the majority of the bacteria, which might mainly result in N 2 O emission. Based on these results, C/N ratio is one of the parameters that play an important role in the N 2 O emission from the BAF reactors during nitrification.

Characterisation of the microbiota of rice sourdoughs and description of Lactobacillus spicheri sp. nov.

PubMed

Meroth, Christiane B; Hammes, Walter P; Hertel, Christian

2004-03-01

The microbiota of two industrially processed rice sourdoughs was characterised by bacteriological culture in combination with PCR-denaturing gradient gel electrophoresis (DGGE) and 16S/28S rDNA sequence analysis. Rice sourdough I was continuously propagated for several years by back-slopping every week, whereas sourdough II was processed by using a commercial starter culture and back-slopping daily for three days. In rice sourdough II Candida krusei and Saccharomyces cerevisiae as well as Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus kimchii, Lactobacillus plantarum, and Lactobacillus pontis dominated at the first day of fermentation. RAPD analysis of lactobacilli revealed identical profiles for each of the species except for L. fermentum and L. pontis indicating the presence of different strains. Fluctuations within the LAB community during fermentation were monitored by PCR-DGGE. L. pontis decreased in numbers over time and L. curvatus became dominant after 3 days of fermentation. Rice sourdough I contained S. cerevisiae, Lactobacillus paracasei (present with three different RAPD types), Lactobacillus paralimentarius, and a Lactobacillus strain which could not be allotted to any valid species. Phylogenetic analysis based on 16S rDNA sequences revealed Lactobacillus brevis as the closest relative (97.3% sequence similarity). Differences in some phenotypic characteristics and DNA-DNA relatedness indicated that the strain represents a new Lactobacillus species, for which the name Lactobacillus spicheri is proposed.

Effects of bioaugmentation in para-nitrophenol-contaminated soil on the abundance and community structure of ammonia-oxidizing bacteria and archaea.

PubMed

Chi, Xiang-Qun; Liu, Kun; Zhou, Ning-Yi

2015-07-01

Pseudomonas sp. strain WBC-3 mineralizes the priority pollutant para-nitrophenol (PNP) and releases nitrite (NO2 (-)), which is probably involved in the nitrification. In this study, the rate of PNP removal in soil bioaugmented with strain WBC-3 was more accelerated with more NO2 (-) accumulation than in uninoculated soils. Strain WBC-3 survived well and remained stable throughout the entire period. Real-time polymerase chain reaction (real-time PCR) indicated a higher abundance of ammonia-oxidizing bacteria (AOB) than ammonia-oxidizing archaea (AOA), suggesting that AOB played a greater role in nitrification in the original sampled soil. Real-time PCR and multivariate analysis based on the denaturing gradient gel electrophoresis showed that PNP contamination did not significantly alter the abundance and community structure of ammonia oxidizers except for inhibiting the AOB abundance. Bioaugmentation of PNP-contaminated soil showed a significant effect on AOB populations and community structure as well as AOA populations. In addition, ammonium (NH4 (+)) variation was found to be the primary factor affecting the AOB community structure, as determined by the correlation between the community structures of ammonia oxidizers and environmental factors. It is here proposed that the balance between archaeal and bacterial ammonia oxidation could be influenced significantly by the variation in NH4 (+) levels as caused by bioaugmentation of contaminated soil by a pollutant containing nitrogen.

Microbial diversity in a submarine carbonate edifice from the serpentinizing hydrothermal system of the Prony Bay (New Caledonia) over a 6-year period

PubMed Central

Postec, Anne; Quéméneur, Marianne; Bes, Méline; Mei, Nan; Benaïssa, Fatma; Payri, Claude; Pelletier, Bernard; Monnin, Christophe; Guentas-Dombrowsky, Linda; Ollivier, Bernard; Gérard, Emmanuelle; Pisapia, Céline; Gérard, Martine; Ménez, Bénédicte; Erauso, Gaël

2015-01-01

Active carbonate chimneys from the shallow marine serpentinizing Prony Hydrothermal Field were sampled 3 times over a 6 years period at site ST09. Archaeal and bacterial communities composition was investigated using PCR-based methods (clone libraries, Denaturating Gel Gradient Electrophoresis, quantitative PCR) targeting 16S rRNA genes, methyl coenzyme M reductase A and dissimilatory sulfite reductase subunit B genes. Methanosarcinales (Euryarchaeota) and Thaumarchaea were the main archaeal members. The Methanosarcinales, also observed by epifluorescent microscopy and FISH, consisted of two phylotypes that were previously solely detected in two other serpentinitzing ecosystems (The Cedars and Lost City Hydrothermal Field). Surprisingly, members of the hyperthermophilic order Thermococcales were also found which may indicate the presence of a hot subsurface biosphere. The bacterial community mainly consisted of Firmicutes, Chloroflexi, Alpha-, Gamma-, Beta-, and Delta-proteobacteria and of the candidate division NPL-UPA2. Members of these taxa were consistently found each year and may therefore represent a stable core of the indigenous bacterial community of the PHF chimneys. Firmicutes isolates representing new bacterial taxa were obtained by cultivation under anaerobic conditions. Our study revealed diverse microbial communities in PHF ST09 related to methane and sulfur compounds that share common populations with other terrestrial or submarine serpentinizing ecosystems. PMID:26379636

Development of a molecular approach to describe the composition of Trichoderma communities.

PubMed

Meincke, Remo; Weinert, Nicole; Radl, Viviane; Schloter, Michael; Smalla, Kornelia; Berg, Gabriele

2010-01-01

Trichoderma and its teleomorphic stage Hypocrea play a key role for ecosystem functioning in terrestrial habitats. However, little is known about the ecology of the fungus. In this study we developed a novel Trichoderma-specific primer pair for diversity analysis. Based on a broad range master alignment, specific Trichoderma primers (ITSTrF/ITSTrR) were designed that comprise an approximate 650bp fragment of the internal transcribed spacer region from all taxonomic clades of the genus Trichoderma. This amplicon is suitable for identification with TrichoKey and TrichoBLAST. Moreover, this primer system was successfully applied to study the Trichoderma communities in the rhizosphere of different potato genotypes grown at two field sites in Germany. Cloning and sequencing confirmed the specificity of the primer and revealed a site-dependent Trichoderma composition. Based on the new primer system a semi-nested approach was used to generate amplicons suitable for denaturing gradient gel electrophoresis (DGGE) analysis and applied to analyse Trichoderma communities in the rhizosphere of potatoes. High field heterogeneity of Trichoderma communities was revealed by both DGGE. Furthermore, qPCR showed significantly different Trichoderma copy numbers between the sites. Copyright 2009 Elsevier B.V. All rights reserved.

Estimating biodiversity of fungi in activated sludge communities using culture-independent methods.

PubMed

Evans, Tegan N; Seviour, Robert J

2012-05-01

Fungal diversity of communities in several activated sludge plants treating different influent wastes was determined by comparative sequence analyses of their 18S rRNA genes. Methods for DNA extraction and choice of primers for PCR amplification were both optimised using denaturing gradient gel electrophoresis profile patterns. Phylogenetic analysis revealed that the levels of fungal biodiversity in some communities, like those treating paper pulp wastes, were low, and most of the fungi detected in all communities examined were novel uncultured representatives of the major fungal subdivisions, in particular, the newly described clade Cryptomycota. The fungal populations in activated sludge revealed by these culture-independent methods were markedly different to those based on culture-dependent data. Members of the genera Penicillium, Cladosporium, Aspergillus and Mucor, which have been commonly identified in mixed liquor, were not identified in any of these plant communities. Non-fungal eukaryotic 18S rRNA genes were also amplified with the primer sets used. This is the first report where culture-independent methods have been applied to flocculated activated sludge biomass samples to estimate fungal community composition and, as expected, the data obtained gave a markedly different view of their population biodiversity compared to that based on culture-dependent methods.

Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

PubMed Central

Huys, Geert; Vanhoutte, Tom; Vandamme, Peter

2008-01-01

Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated. PMID:19277102

Seasonal variation of plankton communities influenced by environmental factors in an artificial lake

NASA Astrophysics Data System (ADS)

Li, Xuemei; Yu, Yuhe; Zhang, Tanglin; Feng, Weisong; Ao, Hongyi; Yan, Qingyun

2012-05-01

We evaluated the seasonal variation in plankton community composition in an artificial lake. We conducted microscopic analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA and 18S rRNA genes to characterize the plankton community. The clustering of unweighted pair group method with arithmetic mean (UPGMA) was then used to investigate the similarity of these plankton communities. DGGE fingerprinting revealed that samples collected at the different sites within a season shared high similarity and were generally grouped together. In contrast, we did not observe any seasonal variation based on microscopic analysis. Redundancy analysis (RDA) of the plankton operational taxonomic units (OTUs) in relation to environmental factors revealed that transparency was negatively correlated with the first axis ( R=-0.931), and temperature and total phosphorus (TP) were positively correlated with the first axis ( R=0.736 and R=0.660, respectively). In conclusion, plankton communities in the artificial lake exhibited significant seasonal variation. Transparency, phosphorus and temperature appear to be the major factors driving the differences in plankton composition.

The principle and application of new PCR Technologies

NASA Astrophysics Data System (ADS)

Yu, Miao; Cao, Yue; Ji, Yubin

2017-12-01

Polymerase chain reaction (PCR) is essentially a selective DNA amplification technique commonlyapplied for genetic testing and molecular diagnosis because of its high specificity and sensitivity.PCR technologies as the key of molecular biology, has realized that the qualitative detection of absolute quantitative has been changed. It has produced a variety of new PCR technologies, such as extreme PCR, photonic PCR, o-amplification at lower denaturation temperature PCR, nanoparticle PCR and so on. In this paper, the principle and application of PCR technologies are reviewed, and its development is prospected too.

Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells

PubMed Central

Lim, Eelin L.; Tomita, Aoy V.; Thilly, William G.; Polz, Martin F.

2001-01-01

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 104 cells ml−1. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml−1 by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples. PMID:11525983

Comparison of vaginal microbial community structure in healthy and endometritis dairy cows by PCR-DGGE and real-time PCR.

PubMed

Wang, Jun; Sun, Chengtao; Liu, Chang; Yang, Yujiang; Lu, Wenfa

2016-04-01

The normal vaginal microflora provides protection against infections of the reproductive tract. Previous studies have focused on the isolation and screening of probiotic strains from the vagina of cows; however, the vaginal microflora of postpartum cows is poorly characterized. The present study was conducted to evaluate and characterize the vaginal microflora of healthy postpartum cows in relation to postpartum cows with endometritis by using PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and Real-time PCR. The study population comprised 5 healthy cows and 5 cows with endometritis. The results indicated that the vaginal bacterial microflora of healthy postpartum cows was dominated by Lactobacillus sakei subsp. and Weissella koreensis, while there were no dominant bacterial species in the vaginal microflora of postpartum cows with endometritis. Common microorganisms such as Bacteroides spp., Fusobacterium spp., Enterococcus spp., Prevotella spp., Clostridium perfringens strains, and Escherichia coli were detected in both groups of cows by Real-time PCR. The bacterial diversity in the vagina of cows with endometritis was significantly higher than that in healthy cows. The results indicated that the vaginal microflora of cows with endometritis was more diverse and lacked dominant bacterial species as compared to that of the healthy cows, suggesting that disruption of the normal vaginal microflora may contribute to the onset of endometritis. This microbial community analysis provided information that might be used to develop probiotics to treat endometritis in cows; however, further investigation is needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of artifacts suggests DGGE should not be used for quantitative diversity analysis.

PubMed

Neilson, Julia W; Jordan, Fiona L; Maier, Raina M

2013-03-01

PCR-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used in microbial ecology for the analysis of comparative community structure. However, artifacts generated during PCR-DGGE of mixed template communities impede the application of this technique to quantitative analysis of community diversity. The objective of the current study was to employ an artificial bacterial community to document and analyze artifacts associated with multiband signatures and preferential template amplification and to highlight their impacts on the use of this technique for quantitative diversity analysis. Six bacterial species (three Betaproteobacteria, two Alphaproteobacteria, and one Firmicutes) were amplified individually and in combinations with primers targeting the V7/V8 region of the 16S rRNA gene. Two of the six isolates produced multiband profiles demonstrating that band number does not correlate directly with α-diversity. Analysis of the multiple bands from one of these isolates confirmed that both bands had identical sequences which lead to the hypothesis that the multiband pattern resulted from two distinct structural conformations of the same amplicon. In addition, consistent preferential amplification was demonstrated following pairwise amplifications of the six isolates. DGGE and real time PCR analysis identified primer mismatch and PCR inhibition due to 16S rDNA secondary structure as the most probable causes of preferential amplification patterns. Reproducible DGGE community profiles generated in this study confirm that PCR-DGGE provides an excellent high-throughput tool for comparative community structure analysis, but that method-specific artifacts preclude its use for accurate comparative diversity analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Monitoring the bacterial community dynamics in a petroleum refinery wastewater membrane bioreactor fed with a high phenolic load.

PubMed

Silva, Cynthia C; Viero, Aline F; Dias, Ana Carolina F; Andreote, Fernando D; Jesus, Ederson C; De Paula, Sergio O; Torres, Ana Paula R; Santiago, Vania M J; Oliveira, Valeria M

2010-01-01

The phenolic compounds are a major contaminant class often found in industrial wastewaters and the biological treatment is an alternative tool commonly employed for their removal. In this sense, monitoring microbial community dynamics is crucial for a successful wastewater treatment. This work aimed to monitor the structure and activity of the bacterial community during the operation of a laboratory-scale continuous submerged membrane bioreactor (SMBR), using PCR and RT-PCR followed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA libraries. Multivariate analyses carried out using DGGE profiles showed significant changes in the total and metabolically active dominant community members during the 4-week treatment period, explained mainly by phenol and ammonium input. Gene libraries were assembled using 16S rDNA and 16S rRNA PCR products from the fourth week of treatment. Sequencing and phylogenetic analyses of clones from 16S rDNA library revealed a high diversity of taxa for the total bacterial community, with predominance of Thauera genus (ca. 50%). On the other hand, a lower diversity was found for metabolically active bacteria, which were mostly represented by members of Betaproteobacteria (Thauera and Comamonas), suggesting that these groups have a relevant role in the phenol degradation during the final phase of the SMBR operation.

Molecular assays reveal the presence and diversity of genes encoding pea footrot pathogenicity determinants in Nectria haematococca and in agricultural soils.

PubMed

Etebu, E; Osborn, A M

2009-05-01

The aim of this study was to develop molecular assays for investigating the presence and diversity of pathogenicity genes from the pea footrot pathogen Nectria haematococca (anamorph Fusarium solani f.sp. pisi) in soils. Polymerase chain reaction (PCR) assays were developed to amplify four N. haematococca pathogenicity genes (PDA, PEP1, PEP3 and PEP5) from isolates and soil-DNA from five agricultural fields with a prior footrot history. A collection of 15 fungi isolated on medium selective for Fusarium spp. exhibited variation in their virulence to peas as assessed via a disease index (DI: 0-5; no virulence to the highest virulence). PCR analyses showed that three isolates in which all four pathogenicity genes were detected resulted in the highest DI (>3.88). All four pathogenicity genes were detected in soil-DNA obtained from all five fields with a footrot disease history, but were not amplified from soils, which had no footrot history. Denaturing gradient gel electrophoresis and/or sequence analysis revealed diversity amongst the pathogenicity genes. The PCR assays developed herein enable the specific detection of pathogenic N. haematococca in soils without recourse to culture. Molecular assays that specifically target pathogenicity genes have the capacity to assess the presence of the footrot-causing pathogen in agricultural soils.

Molecular microbiological characterization of preterm neonates at risk of bronchopulmonary dysplasia.

PubMed

Payne, Matthew S; Goss, Kevin C W; Connett, Gary J; Kollamparambil, Tanoj; Legg, Julian P; Thwaites, Richard; Ashton, Mark; Puddy, Victoria; Peacock, Janet L; Bruce, Kenneth D

2010-04-01

The role of infection in bronchopulmonary dysplasia (BPD) is unknown. We present an observational study of 55 premature infants born weighing less than 1.3 kg within two level III neonatal intensive care units. Endotracheal aspirates (ETA) and nasogastric aspirates (NGA) were studied with denaturing gradient gel electrophoresis (DGGE) profiling to elucidate the total bacterial community, and species-specific PCR was used to detect the presence of Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum. DGGE identified bacterial species in 59% of NGA and ETA samples combined. A diverse range of species were identified including several implicated in preterm labor. Species-specific PCR identified M. hominis in 25% of NGA and 11% of ETA samples. Among the 48 infants surviving up to 36 wk-postconceptual age, ordinal logistic regression showed the odds ratio for BPD or death where Ureaplasma was present/absent as 4.80 (95% CI 1.15-20.13). After adjusting for number of days ventilated, this was reduced to 2.04 (0.41-10.25). These data demonstrate how the combined use of DGGE and species-specific PCR identifies a high exposure in utero and around the time of birth to bacteria that might be causally related to preterm delivery and subsequent lung injury.

Rice root-associated bacteria – insights in community structures across ten cultivars

PubMed Central

Hardoim, Pablo Rodrigo; Andreote, Fernando Dini; Reinhold-Hurek, Barbara; Sessitsch, Angela; van Overbeek, Leonard Simon; van Elsas, Jan Dirk

2015-01-01

In this study, the effect of plant genotype, soil type and nutrient use efficiency on the composition of different bacterial communities associated with rice roots were investigated. Thus, total bacteria, Alpha- and Beta-proteobacteria, Pseudomonas and Actinobacteria were studied using PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE). Rice genotype determined to a large extent the composition of the different bacterial communities across cultivars. Several cultivars belonging to Oryza sativa subspecies indica tended to select similar bacterial communities, whereas those belonging to subspecies japonica and aromatica selected ones with divergent community structures. An effect of soil type was pronounced for the Actinobacteria communities, while a small effect of ‘improved’ and ‘traditional’ plants was noted for all communities analysed. A few dominant bands in PCR-DGGE, affiliated with Rhizobium radiobacter, Dickeya zeae, Mycobacterium bolletii and with members of the Rhizobiales, Rhodospirillaceae and Paenibacillaceae were spread across cultivars. In contrast, a majority of bands (e.g. affiliated with Enterobacter cloacae or Burkholderia kururiensis) was only present in particular cultivars or was erratically distributed amongst rice replicates. The data suggested that both bacterial adaptation and plant genotype contribute to the shaping of the dynamic bacterial communities associated with roots of rice plants. PMID:21426364

Assessing the impact of fungicide enostroburin application on bacterial community in wheat phyllosphere.

PubMed

Gu, Likun; Bai, Zhihui; Jin, Bo; Hu, Qing; Wang, Huili; Zhuang, Guoqiang; Zhang, Hongxun

2010-01-01

Fungicides have been used extensively for controlling fungal pathogens of plants. However, little is known regarding the effects that fungicides upon the indigenous bacterial communities within the plant phyllosphere. The aims of this study were to assess the impact of fungicide enostroburin upon bacterial communities in wheat phyllosphere. Culture-independent methodologies of 16S rDNA clone library and 16S rDNA directed polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE) were used for monitoring the change of bacterial community. The 16S rDNA clone library and PCR-DGGE analysis both confirmed the microbial community of wheat plant phyllosphere were predominantly of the gamma-Proteobacteria phyla. Results from PCR-DGGE analysis indicated a significant change in bacterial community structure within the phyllosphere following fungicide enostroburin application. Bands sequenced within control cultures were predominantly of Pseudomonas genus, but those bands sequenced in the treated samples were predominantly strains of Pantoea genus and Pseudomonas genus. Of interest was the appearance of two DGGE bands following fungicide treatment, one of which had sequence similarities (98%) to Pantoea sp. which might be a competitor of plant pathogens. This study revealed the wheat phyllosphere bacterial community composition and a shift in the bacterial community following fungicide enostroburin application.

Effects of oxytetracycline on archaeal community, and tetracycline resistance genes in anaerobic co-digestion of pig manure and wheat straw.

PubMed

Wang, Xiaojuan; Pan, Hongjia; Gu, Jie; Qian, Xun; Gao, Hua; Qin, Qingjun

2016-12-01

In this study, the effects of different concentrations of oxytetracycline (OTC) on biogas production, archaeal community structure, and the levels of tetracycline resistance genes (TRGs) were investigated in the anaerobic co-digestion products of pig manure and wheat straw. PCR denaturing gradient gel electrophoresis analysis and real-time quantitative polymerase chain reaction (RT-qPCR) (PCR) were used to detect the archaeal community structure and the levels of four TRGs: tet(M), tet(Q), tet(W), and tet(C). The results showed that anaerobic co-digestion with OTC at concentrations of 60, 100, and 140 mg/kg (dry weight of pig manure) reduced the cumulative biogas production levels by 9.9%, 10.4%, and 14.1%, respectively, compared with that produced by the control, which lacked the antibiotic. The addition of OTC substantially modified the structure of the archaeal community. Two orders were identified by phylogenetic analysis, that is, Pseudomonadales and Methanomicrobiales, and the methanogen present during anaerobic co-digestion with OTC may have been resistant to OTC. The abundances of tet(Q) and tet(W) genes increased as the OTC concentration increased, whereas the abundances of tet(M) and tet(C) genes decreased as the OTC concentration increased.

Occurrence and diversity of naphthalene dioxygenase genes in soil microbial communities from the Maritime Antarctic.

PubMed

Flocco, Cecilia G; Gomes, Newton C M; Mac Cormack, Walter; Smalla, Kornelia

2009-03-01

The diversity of naphthalene dioxygenase genes (ndo) in soil environments from the Maritime Antarctic was assessed, dissecting as well the influence of the two vascular plants that grow in the Antarctic: Deschampsia antarctica and Colobanthus quitensis. Total community DNA was extracted from bulk and rhizosphere soil samples from Jubany station and Potter Peninsula, South Shetland Islands. ndo genes were amplified by a nested PCR and analysed by denaturant gradient gel electrophoresis approach (PCR-DGGE) and cloning and sequencing. The ndo-DGGE fingerprints of oil-contaminated soil samples showed even and reproducible patterns, composed of four dominant bands. The presence of vascular plants did not change the relative abundance of ndo genotypes compared with bulk soil. For non-contaminated sites, amplicons were not obtained for all replicates and the variability among the fingerprints was comparatively higher, likely reflecting a lower abundance of ndo genes. The phylogenetic analyses showed that all sequences were affiliated to the nahAc genes closely related to those described for Pseudomonas species and related mobile genetic elements. This study revealed that a microdiversity of nahAc-like genes exists in microbial communities of Antarctic soils and quantitative PCR indicated that their relative abundance was increased in response to anthropogenic sources of pollution.

Insights into biodegradation through depth-resolved microbial community functional and structural profiling of a crude-oil contaminant plume

USGS Publications Warehouse

Fahrenfeld, Nicole; Cozzarelli, Isabelle M.; Bailey, Zach; Pruden, Amy

2014-01-01

Small-scale geochemical gradients are a key feature of aquifer contaminant plumes, highlighting the need for functional and structural profiling of corresponding microbial communities on a similar scale. The purpose of this study was to characterize the microbial functional and structural diversity with depth across representative redox zones of a hydrocarbon plume and an adjacent wetland, at the Bemidji Oil Spill site. A combination of quantitative PCR, denaturing gradient gel electrophoresis, and pyrosequencing were applied to vertically sampled sediment cores. Levels of the methanogenic marker gene, methyl coenzyme-M reductase A (mcrA), increased with depth near the oil body center, but were variable with depth further downgradient. Benzoate degradation N (bzdN) hydrocarbon-degradation gene, common to facultatively anaerobic Azoarcus spp., was found at all locations, but was highest near the oil body center. Microbial community structural differences were observed across sediment cores, and bacterial classes containing known hydrocarbon degraders were found to be low in relative abundance. Depth-resolved functional and structural profiling revealed the strongest gradients in the iron-reducing zone, displaying the greatest variability with depth. This study provides important insight into biogeochemical characteristics in different regions of contaminant plumes, which will aid in improving models of contaminant fate and natural attenuation rates.

Molecular characterization of microbial population dynamics during sildenafil citrate degradation.

PubMed

De Felice, Bruna; Argenziano, Carolina; Guida, Marco; Trifuoggi, Marco; Russo, Francesca; Condorelli, Valerio; Inglese, Mafalda

2009-02-01

Little is known about pharmaceutical and personal care products pollutants (PPCPs), but there is a growing interest in how they might impact the environment and microbial communities. The widespread use of Viagra (sildenafil citrate) has attracted great attention because of the high usage rate, the unpredictable disposal and the unknown potential effects on wildlife and the environment. Until now information regarding the impact of Viagra on microbial community in water environment has not been reported. In this research, for the first time, the genetic profile of the microbial community, developing in a Viagra polluted water environment, was evaluated by means of the 16S and 18S rRNA genes, for bacteria and fungi, respectively, amplified by polymerase chain reaction (PCR) and separated using the denaturing gradient gel electrophoresis (DGGE) technique. The DGGE results revealed a complex microbial community structure with most of the population persisting throughout the experimental period. DNA sequences from bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria and fungi found previously mainly in polluted environmental and treating bioreactors. Biotransformation ability of sildenafil citrate by the microbial pool was studied and the capability of these microorganisms to detoxify a polluted water ecosystem was assessed. The bacterial and fungal population was able to degrade sildenafil citrate entirely. Additionally, assays conducted on Daphnia magna, algal growth inhibition assay and cell viability determination on HepG2 human cells showed that biotransformation products obtained from the bacterial growth was not toxic. The higher removal efficiency for sildenafil citrate and the lack of toxicity by the biotransformation products obtained showed that the microbial community identified here represented a composite population that might have biotechnological relevance to retrieve sildenafil citrate contaminated sites.

A fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp.

PubMed

Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

2011-06-01

The present study aimed to establish a fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Based on the sequences of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA, we designed a set of genus-specific primers for the amplification of Fasciola ITS-2, with an estimated size of 140 bp. These primers were labelled by fluorescence dyes, and the PCR products were analyzed by capillary electrophoresis under non-denaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments displayed three different peak profiles that allowed the accurate identification of Fasciola species: one single peak specific for either Fasciola hepatica or Fasciola gigantica and a doublet peak corresponding to the "intermediate" Fasciola. Validation of our novel method was performed using Fasciola specimens from different host animals from China, Spain, Nigeria, and Egypt. This F-PCR-SSCP assay provides a rapid, simple, and robust tool for the identification and differentiation between Fasciola spp.

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples.

PubMed

Zuo, Zhuang; Chen, Su S; Chandra, Pranil K; Galbincea, John M; Soape, Matthew; Doan, Steven; Barkoh, Bedia A; Koeppen, Hartmut; Medeiros, L Jeffrey; Luthra, Rajyalakshmi

2009-08-01

KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80 degrees C (vs 94 degrees C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.

Bacterial Population in Intestines of the Black Tiger Shrimp (Penaeus monodon) under Different Growth Stages

PubMed Central

Rungrassamee, Wanilada; Klanchui, Amornpan; Chaiyapechara, Sage; Maibunkaew, Sawarot; Tangphatsornruang, Sithichoke; Jiravanichpaisal, Pikul; Karoonuthaisiri, Nitsara

2013-01-01

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities. PMID:23577162

Bacterial population in intestines of the black tiger shrimp (Penaeus monodon) under different growth stages.

PubMed

Rungrassamee, Wanilada; Klanchui, Amornpan; Chaiyapechara, Sage; Maibunkaew, Sawarot; Tangphatsornruang, Sithichoke; Jiravanichpaisal, Pikul; Karoonuthaisiri, Nitsara

2013-01-01

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities.

Structural shifts of fecal microbial communities in rats with acute rejection after liver transplantation.

PubMed

Xie, Yirui; Luo, Zhuanbo; Li, Zhengfeng; Deng, Min; Liu, Hao; Zhu, Biao; Ruan, Bing; Li, Lanjuan

2012-08-01

Bacterial translocation and the development of sepsis after orthotopic liver transplantation (OLT) may be promoted by immunological damage to the intestinal mucosa or by quantitative and qualitative changes in intestinal microbiota. This study monitored structural shifts of gut microbiota in rats with OLT using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (RT-qPCR). RT-qPCR targets six major microorganisms (Domain Bacteria, Bacteroides, Bifidobacteria, Enterobacteriaceae, Lactobacillus and Clostridium leptum subgroup). Isograft, Allograft and Sham model were studied. Bacterial translocation to host organs and plasma endotoxin were determined. Alteration in gut microbiota was associated with the elevation of plasma endotoxin and a higher rate of bacterial translocation (BT) to liver in rats with acute rejection. Dynamic analysis of DGGE fingerprints showed that the gut microbiota structure of animals in the three groups was similar before the operation. But significant alterations in the composition of fecal microbiota in Allograft group were observed at 1 and 2 weeks after the OLT. The acute rejection was accompanied by the shifts of gut microbiota towards members of Bacteroides and Ruminococcus. Results from RT-qPCR indicated that Bacteroides significantly increased at 2 weeks after the OLT, whereas numbers of Bifidobacterium spp. decreased at 1 week and recovered at 2 weeks after the OLT. In summary, our data showed that rats with acute rejection after OLT exhibited significant structure shifts in the gut microbiota which dominant by overgrowth of Bacteroides and Ruminococcus, and these were associated with elevation of plasma endotoxin and higher rate of BT.

Effect of Phenotypic Residual Feed Intake and Dietary Forage Content on the Rumen Microbial Community of Beef Cattle

PubMed Central

Carberry, Ciara A.; Kenny, David A.; Han, Sukkyan; McCabe, Matthew S.

2012-01-01

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered. PMID:22562991

Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle.

PubMed

Carberry, Ciara A; Kenny, David A; Han, Sukkyan; McCabe, Matthew S; Waters, Sinead M

2012-07-01

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered.

[Analysis of the oral microbiota in twin children].

PubMed

Du, Qin; Wang, Yan; Xu, Xin; Li, Yuqing; Li, Mingyun; Zou, Jing; Zhou, Xuedong

2014-04-01

To analyze the differences between the oral microbiota of monozygotic and dizygotic twins by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). A total of 20 pairs of twin children were included in this study, in which 10 pairs were monozygotic (MZ) twins, and 10 pairs were dizygotic (DZ) twins. Of the 20 pairs, 10 pairs of twins had primary dentition, and 10 pairs had mixed dentition; 17 children had caries, and 23 children had no caries. Genomic DNA was extracted from saliva samples. The 16s rRNA was amplified and analyzed by PCR-DGGE. The PCR-DGGE band number and Shannon index were calculated. Cluster analysis showed high similarity in the oral bacterial community seen in co-twins. However, no significant difference was seen between MZ and DZ twins. In the primary dentition, the PCR-DGGE band number and Shannon index of children with caries (11.00 +/- 1.56, 1.05 +/- 0.36) were lower than those of children without caries (14.00 +/- 2.74, 1.44 +/- 0.37) (P < 0.05). In mixed dentition, the PCR-DGGE band number and Shannon index of children with caries (11.88 +/- 4.05, 1.18 +/- 0.36) were lower than those of children without caries (14.31 +/- 5.71, 1.28 +/- 0.47), but the differences were not statistically significant (P > 0.05). Environmental factors may have a stronger effect on the constitution of oral microbiota in children compared with genetic factors. Children without caries may have a richer microbial diversity compared with children with caries.

Variability of bacterial biofilms of the "tina" wood vats used in the ragusano cheese-making process.

PubMed

Licitra, G; Ogier, J C; Parayre, S; Pediliggieri, C; Carnemolla, T M; Falentin, H; Madec, M N; Carpino, S; Lortal, S

2007-11-01

Ragusano cheese is a "protected denomination of origin" cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called "tinas" were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.

Mutation-based detection and monitoring of cell-free tumor DNA in peripheral blood of cancer patients.

PubMed

Benesova, L; Belsanova, B; Suchanek, S; Kopeckova, M; Minarikova, P; Lipska, L; Levy, M; Visokai, V; Zavoral, M; Minarik, M

2013-02-15

Prognosis of solid cancers is generally more favorable if the disease is treated early and efficiently. A key to long cancer survival is in radical surgical therapy directed at the primary tumor followed by early detection of possible progression, with swift application of subsequent therapeutic intervention reducing the risk of disease generalization. The conventional follow-up care is based on regular observation of tumor markers in combination with computed tomography/endoscopic ultrasound/magnetic resonance/positron emission tomography imaging to monitor potential tumor progression. A recent development in methodologies allowing screening for a presence of cell-free DNA (cfDNA) brings a new viable tool in early detection and management of major cancers. It is believed that cfDNA is released from tumors primarily due to necrotization, whereas the origin of nontumorous cfDNA is mostly apoptotic. The process of cfDNA detection starts with proper collection and treatment of blood and isolation and storage of blood plasma. The next important steps include cfDNA extraction from plasma and its detection and/or quantification. To distinguish tumor cfDNA from nontumorous cfDNA, specific somatic DNA mutations, previously localized in the primary tumor tissue, are identified in the extracted cfDNA. Apart from conventional mutation detection approaches, several dedicated techniques have been presented to detect low levels of cfDNA in an excess of nontumorous (nonmutated) DNA, including real-time polymerase chain reaction (PCR), "BEAMing" (beads, emulsion, amplification, and magnetics), and denaturing capillary electrophoresis. Techniques to facilitate the mutant detection, such as mutant-enriched PCR and COLD-PCR (coamplification at lower denaturation temperature PCR), are also applicable. Finally, a number of newly developed miniaturized approaches, such as single-molecule sequencing, are promising for the future. Copyright © 2012 Elsevier Inc. All rights reserved.

Changes in the rumen epimural bacterial diversity of beef cattle as affected by diet and induced ruminal acidosis.

PubMed

Petri, R M; Schwaiger, T; Penner, G B; Beauchemin, K A; Forster, R J; McKinnon, J J; McAllister, T A

2013-06-01

Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering (P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing (P ≤ 0.05) among diets. During acidosis, levels of Atopobium, Desulfocurvus, Fervidicola, Lactobacillus, and Olsenella increased, while during the recovery, Desulfocurvus, Lactobacillus, and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed (P ≤ 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium, cc142, Lactobacillus, Olsenella, RC39, Sharpea, Solobacterium, Succiniclasticum, and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis.

Changes in the Rumen Epimural Bacterial Diversity of Beef Cattle as Affected by Diet and Induced Ruminal Acidosis

PubMed Central

Petri, R. M.; Schwaiger, T.; Penner, G. B.; Beauchemin, K. A.; Forster, R. J.; McKinnon, J. J.

2013-01-01

Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering (P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing (P ≤ 0.05) among diets. During acidosis, levels of Atopobium, Desulfocurvus, Fervidicola, Lactobacillus, and Olsenella increased, while during the recovery, Desulfocurvus, Lactobacillus, and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed (P ≤ 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium, cc142, Lactobacillus, Olsenella, RC39, Sharpea, Solobacterium, Succiniclasticum, and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis. PMID:23584771

Physicochemical and microbiological characterization of chicha, a rice-based fermented beverage produced by Umutina Brazilian Amerindians.

PubMed

Puerari, Cláudia; Magalhães-Guedes, Karina Teixeira; Schwan, Rosane Freitas

2015-04-01

Chicha is a traditional, fermented rice beverage produced by the indigenous Umutina people in Brazil. Culture-dependent and independent approaches were used to investigate the microbial community dynamic. The bacterial population ranged from 0.1 to 6.83 log mL(-1). Lactic acid bacteria (LAB) and Bacillus dominated throughout the fermentation process. Representative colonies were grouped by Repetitive Extragenic Palindromic and Polymerase Chain Reaction (Rep-PCR) and by biochemical features. Genera of Lactobacillus, Bacillus, Leuconostoc, Enterococcus, Streptomyces, Enterobacter, Acinetobacter, Escherichia, Cronobacter, and Klebsiella were identified by partial 16S rRNA gene sequence. As shown by Polimerase and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analysis, uncultivable Bifidobacterium and Propioniobacterium were found throughout fermentation. Uncultured fungi composed the fungal PCR-DGGE profile. The pH values decreased from 5.2 (time 0) to 3.9 at 36 h of fermentation. Ethanol was not found. The lactic acid concentration increased rapidly throughout fermentation until it reached a high final value (1.4 g L(-1)) and the average glycerol content in the beverage was 0.425 g L(-1). Chicha fermentation might be described by the following phenomena: (i) increasing bacterial population, with lactic acid bacteria (LAB) as the largest group detected; (ii) increasing concentrations of lactic and citric acids; and (iii) the final product is characterized by a high content of acids and the absence of ethanol, therefore characterizing rice chicha an acidic and nonalcoholic beverage. First, this study characterizes the microbial population involved in the nonalcoholic fermentation of chicha, which is produced from rice by Amerindians in Brazil. This study is important for promoting the appreciation of and safeguarding this Brazilian indigenous beverage as an immaterial cultural heritage. Copyright © 2014 Elsevier Ltd. All rights reserved.

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis.

PubMed

Papalexandratou, Zoi; De Vuyst, Luc

2011-11-01

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The Primary Results of Analyses on The Archaeal and Bacterial Diversity of Active Cave Environments Settled in Limestones at Southern Turkey

NASA Astrophysics Data System (ADS)

Tok, Ezgi; Kurt, Halil; Tunga Akarsubasi, A.

2016-04-01

The microbial diversity of cave sediments which are obtained from three different caves named Insuyu, Balatini and Altınbeşik located at Southern Turkey has been investigated using molecular methods for biomineralization . The total number of 22 samples were taken in duplicates from the critical zones of the caves at where the water activity is observed all year round. Microbial communities were monitored by 16S rRNA gene based PCR-DGGE (Polymerase Chain Reaction - Denaturating Gradient Gel Electrophoresis) methodology. DNA were extracted from the samples by The PowerSoil® DNA Isolation Kit (MO BIO Laboratories inc., CA) with the modifications on the producer's protocol. The synthetic DNA molecule poly-dIdC was used to increase the yield of PCR amplification via blocking the reaction between CaCO3 and DNA molecules. Thereafter samples were amplified by using both Archaeal and Bacterial universal primers (ref). Subsequently, archaeal and bacterial diversities in cave sediments, were investigated to be able to compare with respect to their similarities by using DGGE. DGGE patterns were analysed with BioNumerics software 5.1. Similarity matrix and dendograms of the DGGE profiles were generated based on the Dice correlation coefficient (band-based) and unweighted pair-group method with arithmetic mean (UPGMA). The structural diversity of the microbial community was examined by the Shannon index of general diversity (H). Similtaneously, geochemical analyses of the sediment samples were performed within the scope of this study. Total organic carbon (TOC), x-ray diffraction spectroscopy (XRD) and x-ray fluorescence spectroscopy (XRF) analysis of sediments were also implemented. The extensive results will be obtained at the next stages of the study currently carried on.

Exploring Microbial Iron Oxidation in Wetland Soils

NASA Astrophysics Data System (ADS)

Wang, J.; Muyzer, G.; Bodelier, P. L. E.; den Oudsten, F.; Laanbroek, H. J.

2009-04-01

Iron is one of the most abundant elements on earth and is essential for life. Because of its importance, iron cycling and its interaction with other chemical and microbial processes has been the focus of many studies. Iron-oxidizing bacteria (FeOB) have been detected in a wide variety of environments. Among those is the rhizosphere of wetland plants roots which release oxygen into the soil creating suboxic conditions required by these organisms. It has been reported that in these rhizosphere microbial iron oxidation proceeds up to four orders of magnitude faster than strictly abiotic oxidation. On the roots of these wetland plants iron plaques are formed by microbial iron oxidation which are involved in the sequestering of heavy metals as well organic pollutants, which of great environmental significance.Despite their important role being catalysts of iron-cycling in wetland environments, little is known about the diversity and distribution of iron-oxidizing bacteria in various environments. This study aimed at developing a PCR-DGGE assay enabling the detection of iron oxidizers in wetland habitats. Gradient tubes were used to enrich iron-oxidizing bacteria. From these enrichments, a clone library was established based on the almost complete 16s rRNA gene using the universal bacterial primers 27f and 1492r. This clone library consisted of mainly α- and β-Proteobacteria, among which two major clusters were closely related to Gallionella spp. Specific probes and primers were developed on the basis of this 16S rRNA gene clone library. The newly designed Gallionella-specific 16S rRNA gene primer set 122f/998r was applied to community DNA obtained from three contrasting wetland environments, and the PCR products were used in denaturing gradient gel electrophoresis (DGGE) analysis. A second 16S rRNA gene clone library was constructed using the PCR products from one of our sampling sites amplified with the newly developed primer set 122f/998r. The cloned 16S rRNA gene sequences all represented novel culturable iron oxidizers most closely related to Gallionella spp. Based on their nucleotide sequences four groups could be identified, which were comparable to the DGGE banding pattern obtained before with the gradient tubes enrichments. The above mentioned nested PCR-DGGE method was used to study the distribution and community composition of Gallionella-like iron-oxidizing bacteria under the influence of plants species, soil depth, as well as season. Soil samples from Appels, Belgium, an intertidal, freshwater marsh known to hold intensive iron cycling, were taken from 5 different vegetation types in April, July and October 2007. Soil cores were sliced at 1-cm intervals and subjected to chemical and molecular analyses. The DGGE patterns showed that the community of iron-oxidizing bacteria differed with vegetation type, and sediment depth. Samples taken in autumn held lower diversity in Gallionella-related iron oxidizers than those sampled in spring and summer.

Analysis of microbial diversity in Shenqu with different fermentation times by PCR-DGGE.

PubMed

Liu, Tengfei; Jia, Tianzhu; Chen, Jiangning; Liu, Xiaoyu; Zhao, Minjie; Liu, Pengpeng

Shenqu is a fermented product that is widely used in traditional Chinese medicine (TCM) to treat indigestion; however, the microbial strains in the fermentation process are still unknown. The aim of this study was to investigate microbial diversity in Shenqu using different fermentation time periods. DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) profiles indicated that a strain of Pediococcus acidilactici (band 9) is the predominant bacteria during fermentation and that the predominant fungi were uncultured Rhizopus, Aspergillus oryzae, and Rhizopus oryzae. In addition, pathogenic bacteria, such as Enterobacter cloacae, Klebsiella oxytoca, Erwinia billingiae, and Pantoea vagan were detected in Shenqu. DGGE analysis showed that bacterial and fungal diversity declined over the course of fermentation. This determination of the predominant bacterial and fungal strains responsible for fermentation may contribute to further Shenqu research, such as optimization of the fermentation process. Copyright © 2017. Published by Elsevier Editora Ltda.

Molecular and Microscopical Investigation of the Microflora Inhabiting a Deteriorated Italian Manuscript Dated from the Thirteenth Century

PubMed Central

Michaelsen, Astrid; Piñar, Guadalupe

2010-01-01

This case study shows the application of nontraditional diagnostic methods to investigate the microbial consortia inhabiting an ancient manuscript. The manuscript was suspected to be biologically deteriorated and SEM observations showed the presence of fungal spores attached to fibers, but classic culturing methods did not succeed in isolating microbial contaminants. Therefore, molecular methods, including PCR, denaturing gradient gel electrophoresis (DGGE), and clone libraries, were used as a sensitive alternative to conventional cultivation techniques. DGGE fingerprints revealed a high biodiversity of both bacteria and fungi inhabiting the manuscript. DNA sequence analysis confirmed the existence of fungi and bacteria in manuscript samples. A number of fungal clones identified on the manuscript showed similarity to fungal species inhabiting dry or saline environments, suggesting that the manuscript environment selects for osmophilic or xerophilic fungal species. Most of the bacterial sequences retrieved from the manuscript belong to phylotypes with cellulolytic activities. PMID:20449583

The Effects of GH Transgenic Goats on the Microflora of the Intestine, Feces and Surrounding Soil.

PubMed

Bao, Zekun; Gao, Xue; Zhang, Qiang; Lin, Jian; Hu, Weiwei; Yu, Huiqing; Chen, Jianquan; Yang, Qian; Yu, Qinghua

2015-01-01

The development of genetically engineered animals has brought with it increasing concerns about biosafety issues. We therefore evaluated the risks of growth hormone from transgenic goats, including the probability of horizontal gene transfer and the impact on the microbial community of the goats' gastrointestinal tracts, feces and the surrounding soil. The results showed that neither the GH nor the neoR gene could be detected in the samples. Moreover, there was no significant change in the microbial community of the gastrointestinal tracts, feces and soil, as tested with PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing. Finally, phylogenetic analysis showed that the intestinal content, feces and soil samples all contained the same dominant group of bacteria. These results demonstrated that expression of goat growth hormone in the mammary of GH transgenic goat does not influence the microflora of the intestine, feces and surrounding soil.

The effectiveness of the biodegradation of raw and processed polystyrene by mealworms

NASA Astrophysics Data System (ADS)

Leluk, Karol; Hanus-Lorenz, Beata; Rybak, Justyna; Bożek, Magdalena

2017-11-01

In our studies biodegradation of four variants of polystyrene was performed. We tested: raw material (PS), processed polystyrene (PSr), building insulation material (EPS) and food packaging boxes (PSp). Materials were characterized by means melt flow ratio (MFR), shore hardness and gloss. The biochemical assessment of macromolecules (proteins, lipids and sugars) in the mealworms organisms fed with tested forms of polystyrene allowed us to set how efficient and beneficial the biodegradation of types of polystyrene is. We also evaluated the variability of bacterial community in larval guts by the use of denaturing gradient gel electrophoresis (DGGE) on the bacterial DNA of 16S rRNA genes amplified in polymerase chain reaction (PCR). The results suggest that EPS and PSp polystyrene are the most digestible for T. molitor larvae. The metabolic degradation of polystyrene is probably strictly connected with the changes in biodiversity of gut bacteria.

A microsampling method for genotyping coral symbionts

NASA Astrophysics Data System (ADS)

Kemp, D. W.; Fitt, W. K.; Schmidt, G. W.

2008-06-01

Genotypic characterization of Symbiodinium symbionts in hard corals has routinely involved coring, or the removal of branches or a piece of the coral colony. These methods can potentially underestimate the complexity of the Symbiodinium community structure and may produce lesions. This study demonstrates that microscale sampling of individual coral polyps provided sufficient DNA for identifying zooxanthellae clades by RFLP analyses, and subclades through the use of PCR amplification of the ITS-2 region of rDNA and denaturing-gradient gel electrophoresis. Using this technique it was possible to detect distinct ITS-2 types of Symbiodinium from two or three adjacent coral polyps. These methods can be used to intensely sample coral-symbiont population/communities while causing minimal damage. The effectiveness and fine scale capabilities of these methods were demonstrated by sampling and identifying phylotypes of Symbiodinium clades A, B, and C that co-reside within a single Montastraea faveolata colony.

Evaluation of the specificity and effectiveness of selected oral hygiene actives in salivary biofilm microcosms.

PubMed

Ledder, Ruth G; Sreenivasan, Prem K; DeVizio, William; McBain, Andrew J

2010-12-01

The microbiological effects of biocidal products used for the enhancement of oral hygiene relate to the active compound(s) as well as other formulation components. Here, we test the specificities of selected actives in the absence of multiple excipients. Salivary ecosystems were maintained in tissue culture plate-based hydroxyapatite disc models (HDMs) and modified drip-flow biofilm reactors (MDFRs). Test compounds stannous fluoride (SF), SDS, triclosan (TCS), zinc lactate (ZL) and ZL with SF in combination (ZLSF) were delivered to the HDMs once and four times daily for 6 days to MDFRs. Plaques were characterized by differential viable counting and PCR-denaturing gradient gel electrophoresis (DGGE). TCS and SDS were the most effective compounds against HDM plaques, significantly reducing total viable counts (P<0.05), whilst SF, ZL and ZLSF were comparatively ineffective. TCS exhibited specificity for streptococci (P<0.01) and Gram-negative anaerobes (P<0.01) following a single dosing and also on repeated dosing in MDFRs. In contrast to single exposures, multiple dosing with ZLSF also significantly reduced all bacterial groups, whilst SF and ZL caused significant but transient reductions. According to PCR-DGGE analyses, significant (P<0.05) reductions in eubacterial diversity occurred following 6 day dosing with both TCS and ZLSF. Concordance of MDFR eubacterial profiles with salivary inocula ranged between 58 and 97%. TCS and ZL(SF) exhibited similar specificities to those reported for formulations. TCS was the most potent antibacterial, after single and multiple dosage regimens.

Methods for MHC genotyping in non-model vertebrates.

PubMed

Babik, W

2010-03-01

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems. © 2009 Blackwell Publishing Ltd.

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

PubMed

Giannattasio, S; Bobba, A; Jurgelevicius, V; Vacca, R A; Lattanzio, P; Merafina, R S; Utkus, A; Kucinskas, V; Marra, E

2006-01-01

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.

Different effects of transgenic maize and nontransgenic maize on nitrogen-transforming archaea and bacteria in tropical soils.

PubMed

Cotta, Simone Raposo; Dias, Armando Cavalcante Franco; Marriel, Ivanildo Evódio; Andreote, Fernando Dini; Seldin, Lucy; van Elsas, Jan Dirk

2014-10-01

The composition of the rhizosphere microbiome is a result of interactions between plant roots, soil, and environmental conditions. The impact of genetic variation in plant species on the composition of the root-associated microbiota remains poorly understood. This study assessed the abundances and structures of nitrogen-transforming (ammonia-oxidizing) archaea and bacteria as well as nitrogen-fixing bacteria driven by genetic modification of their maize host plants. The data show that significant changes in the abundances (revealed by quantitative PCR) of ammonia-oxidizing bacterial and archaeal communities occurred as a result of the maize host being genetically modified. In contrast, the structures of the total communities (determined by PCR-denaturing gradient gel electrophoresis) were mainly driven by factors such as soil type and season and not by plant genotype. Thus, the abundances of ammonia-oxidizing bacterial and archaeal communities but not structures of those communities were revealed to be responsive to changes in maize genotype, allowing the suggestion that community abundances should be explored as candidate bioindicators for monitoring the possible impacts of cultivation of genetically modified plants. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Symbiodinium diversity in the soft coral Heteroxenia sp. and its nudibranch predator Phyllodesmium lizardensis

NASA Astrophysics Data System (ADS)

FitzPatrick, S. K.; Liberatore, K. L.; Garcia, J. R.; Burghardt, I.; Colman, D. R.; Moquin, S. A.; Takacs-Vesbach, C. D.; Shepherd, U. L.

2012-09-01

We examined the diversity of the photosynthetic dinoflagellate, Symbiodinium, over a 2-year period in two invertebrates from Australia's Northern Great Barrier Reef: the nudibranch Phyllodesmium lizardensis and an octocoral of the genus Heteroxenia. In years one and two, we used denaturing gradient gel electrophoresis with internal transcribed spacer 2 (ITS2) region amplicons and identified two nearly identical genotypes of clade C (C64 and a variant) in all samples of each species. We examined the secondary structure of both sequences and found that each had predicted ∆G values within the range of stable free energy values for Symbiodinium ITS2 sequences. In year two, we also used real-time quantitative polymerase chain reaction assays (qPCR) with clade-specific internal transcribed spacer 1 primers to determine whether there were cryptic clades (A, B, and/or D) associated with either host in addition to clade C. qPCR revealed that clades B, C, and D were present in all animals of both species and that all but two nudibranch samples also harbored clade A. These findings suggest that there may be more flexibility in this host/symbiont interaction than has previously been assumed.

Effects of lead and zinc mining contamination on bacterial community diversity and enzyme activities of vicinal cropland.

PubMed

Qu, Juanjuan; Ren, Guangming; Chen, Bao; Fan, Jinghua; E, Yong

2011-11-01

In the process of mining activity, many kinds of heavy metals enter into soils with dust, causing serious contamination to the environment. In this study, six soils were sampled from cropland at different distances from a lead/zinc mine in Heilongjiang Province, China. The total contents of lead and zinc in the vicinal cropland exceeded the third level of environmental quality standard for soil in China, which indicated that soils in this area were moderately contaminated. Bacterial community diversity and population were greatly decreased when the concentrations of lead and zinc were beyond 1,500 and 995 mg kg(-1), respectively, as analyzed by plate counting and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The bands of DGGE patterns varied with the degree of contamination. The activities of soil urease, phosphatase, and dehydrogenase were negatively correlated with the concentrations of lead and zinc. The highest inhibitory effect of heavy metals on soil enzyme activities was observed in urease. It was noted that PCR-DGGE patterns combined with soil enzyme activity analysis can be indices for the soil quality assessment by heavy metal contamination.

Evaluation of bacterial flora during the ripening of Kedong sufu, a typical Chinese traditional bacteria-fermented soybean product.

PubMed

Feng, Zhen; Gao, Wei; Ren, Dan; Chen, Xi; Li, Juan-juan

2013-04-01

Kedong sufu is a typical bacteria-fermented sufu in China. Isolation and identification of the autochthonous bacteria involved would allow the design of specific starters for this speciality. The purpose of the present study was to evaluate the bacterial flora during the ripening of Kedong sufu using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and culturing. In terms of bacterial diversity, 22 strains were isolated and identified and 27 strains were detected by DGGE. Regarding bacterial dynamics, the results of culturing and PCR-DGGE exhibited a similar trend towards dominant strains. Throughout the fermentation of sufu, Enterococcus avium, Enterococcus faecalis and Staphylococcus carnosus were the dominant microflora, while the secondary microflora comprised Leuconostoc mesenteroides, Staphylococcus saprophyticus, Streptococcus lutetiensis, Kocuria rosea, Kocuria kristinae, Bacillus pumilus, Bacillus cereus and Bacillus subtilis. This study is the first to reveal the bacterial flora during the ripening of Kedong sufu using both culture-dependent and culture-independent methods. This information will help in the design of autochthonous starter cultures for the production of Kedong sufu with desirable characteristic sensory profiles and shorter ripening times. © 2012 Society of Chemical Industry.

Response of rhizosphere microbial community structure and diversity to heavy metal co-pollution in arable soil.

PubMed

Deng, Linjing; Zeng, Guangming; Fan, Changzheng; Lu, Lunhui; Chen, Xunfeng; Chen, Ming; Wu, Haipeng; He, Xiaoxiao; He, Yan

2015-10-01

Due to the emerging environmental issues related to heavy metals, concern about the soil quality of farming lands near manufacturing district is increasing. Investigating the function of soil microorganisms exposed to long-term heavy metal contamination is meaningful and important for agricultural soil utilization. This article studied the potential influence of several heavy metals on microbial biomass, activity, abundance, and community composition in arable soil near industrial estate in Zhuzhou, Hunan province, China. The results showed that soil organic contents (SOC) were significantly positive correlated with heavy metals, whereas dehydrogenase activity (DHA) was greatly depressed by the heavy metal stress. Negative correlation was found between heavy metals and basal soil respiration (BSR), and no correlation was found between heavy metals and microbial biomass content (MBC). The quantitative PCR (QPCR) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis could suggest that heavy metal pollution has significantly decreased abundance of bacteria and fungi and also changed their community structure. The results could contribute to evaluate heavy metal pollution level in soil. By combining different environmental parameters, it would promote the better understanding of heavy metal effect on the size, structure, and activity of microbial community in arable soil.

Comparing the effects of three pre-treatment disintegration techniques on aerobic sludge digestion: biodegradability enhancement and microbial community monitoring by PCR-DGGE.

PubMed

Jaziri, Kais; Casellas, Magali; Dagot, Christophe

2012-06-01

The objectives of this work were to compare and investigate the effect of three activated sludge disintegration processes before aerobic sludge digestion on 1) aerobic biodegradability enhancement and 2) microbial community evolution using the polymerase chain reaction-denaturant gel gradient electrophoresis (PCR-DGGE) technique. The comparison of three disintegration processes: thermal treatment (95 degrees C, 2h), sonication (100,000 kJ/kgTS) and ozonation (0.108 g O3/gTS) showed that the disintegration processes acted differently according to the composition of the soluble phase and to the DNA damage. Thermal treatment led to significant protein solubilization and to DNA modification. Sonication and ozonation resulted in similar soluble phase compositions and did not lead to any DNA modifications. During activated sludge aerobic digestion, intrinsic biodegradability enhancement was observed for thermal and ozone activated sludge pre-treatments. The analysis of the DGGE patterns at the end of aerobic digestion showed that population diversity was affected by both the aerobic digestion and the pre-treatment. The dissimilarity percentages measured at the end of aerobic digestion in the control sample and in the treated sludge were equal to 22, 25 and 20% for thermal treatment, sonication and ozonation respectively. This study indicated that PCR-DGGE could be a useful tool for the comparison of disintegration processes before and after aerobic digestion.

Characterisation of the spoilage bacterial microbiota in oyster gills during storage at different temperatures.

PubMed

Chen, Huibin; Liu, Zhiyu; Wang, Meiying; Chen, Shaojun; Chen, Tuanwei

2013-12-01

The spoilage bacterial community in oyster gill was investigated during storage at 4, 10 and 20 °C. Aerobic plate counts and pH values were determined. Total bacterial DNA was extracted from oyster gill and bulk cells of plate count media. The major bacterial species during fresh or different temperatures storage were determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The initial aerobic plate count in oyster gill reached 6.70 log CFU g(-1). PCR-DGGE fingerprinting analysis of the 16S rRNA gene V3 region revealed that most of the strains in fresh oyster gill belonged to the genera Lactococcus and Enterobacter. The major spoilage bacteria at a storage temperature of 20 °C were Leuconostoc pseudomesenteroides, an uncultured bacterium, Cytophaga fermentans, Lactococcus lactis, Pseudoalteromonas sp., Enterococcus mundtii, Clostridium difficile and an uncultured Fusobacteria; those at 10 °C were Lactococcus spp., Lactobacillus curvatus, Weissella confusa and C. difficile; those at 4 °C were Lactococcus, Weissella, Enterobacter and Aeromonas. The other minor species were L. curvatus, Pseudomonas sp. and E. mundtii. Lactococcus spp. was the most common main spoilage bacteria in oyster gill during chilled storage. PCR-DGGE revealed the complexity of the bacterial microbiota and the major bacteria species in oyster gill for fresh and storage. © 2013 Society of Chemical Industry.

Comparative analysis of fecal microbiota and intestinal microbial metabolic activity in captive polar bears.

PubMed

Schwab, Clarissa; Gänzle, Michael

2011-03-01

The composition of the intestinal microbiota depends on gut physiology and diet. Ursidae possess a simple gastrointestinal system composed of a stomach, small intestine, and indistinct hindgut. This study determined the composition and stability of fecal microbiota of 3 captive polar bears by group-specific quantitative PCR and PCR-DGGE (denaturing gradient gel electrophoresis) using the 16S rRNA gene as target. Intestinal metabolic activity was determined by analysis of short-chain fatty acids in feces. For comparison, other Carnivora and mammals were included in this study. Total bacterial abundance was approximately log 8.5 DNA gene copies·(g feces)-1 in all 3 polar bears. Fecal polar bear microbiota was dominated by the facultative anaerobes Enterobacteriaceae and enterococci, and the Clostridium cluster I. The detection of the Clostridium perfringens α-toxin gene verified the presence of C. perfringens. Composition of the fecal bacterial population was stable on a genus level; according to results obtained by PCR-DGGE, dominant bacterial species fluctuated. The total short-chain fatty acid content of Carnivora and other mammals analysed was comparable; lactate was detected in feces of all carnivora but present only in trace amounts in other mammals. In comparison, the fecal microbiota and metabolic activity of captive polar bears mostly resembled the closely related grizzly and black bears.

Influence of pasteurization, brining conditions and production environment on the microbiota of artisan Gouda-type cheeses.

PubMed

Van Hoorde, Koenraad; Heyndrickx, Marc; Vandamme, Peter; Huys, Geert

2010-05-01

To monitor the effect of the indigenous milk microbiota and of technological and environmental parameters on the microbiota established in ripened cheese, the diversity and dynamics of the predominant microbial communities in artisan Gouda-type cheeses produced under different conditions was studied. A total of 22 cheese types differing in milk source, milk treatment, production environment and brining conditions were analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) using total DNA extracts as well as DNA extracted from culturable fractions. Through band position analysis and band sequencing, the majority of DGGE bands could be attributed to lactic acid bacteria (LAB), although a few bands also belonged to staphylococci and gamma-Proteobacteria. Aided by principal component analysis (PCA) and multivariate analysis of variance (MANOVA), cheeses produced at different locations could clearly be differentiated. The same approach also allowed to distinguish raw and pasteurized milk cheeses, the former showing a more diverse microbiota in terms of a higher species richness and number of DGGE bands. No substantial differences were found between cheeses brined at two different locations. In conclusion, the combined PCR-DGGE approach relying on both total DNA extracts and culturable fractions proved its value for analyzing the effect of technological and environmental parameters on the diversity and dynamics of the microbiota in Gouda-type cheeses. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene.

PubMed

Offre, Pierre; Prosser, James I; Nicol, Graeme W

2009-10-01

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 degrees C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.

Centrifugal Microfluidic System for Nucleic Acid Amplification and Detection.

PubMed

Miao, Baogang; Peng, Niancai; Li, Lei; Li, Zheng; Hu, Fei; Zhang, Zengming; Wang, Chaohui

2015-11-04

We report here the development of a rapid PCR microfluidic system comprising a double-shaft turntable and centrifugal-based disc that rapidly drives the PCR mixture between chambers set at different temperatures, and the bidirectional flow improved the space utilization of the disc. Three heating resistors and thermistors maintained uniform, specific temperatures for the denaturation, annealing, and extension steps of the PCR. Infrared imaging showed that there was little thermal interference between reaction chambers; the system enabled the cycle number and reaction time of each step to be independently adjusted. To validate the function and efficiency of the centrifugal microfluidic system, a 350-base pair target gene from the hepatitis B virus was amplified and quantitated by fluorescence detection. By optimizing the cycling parameters, the reaction time was reduced to 32 min as compared to 120 min for a commercial PCR machine. DNA samples with concentrations ranging from 10 to 10⁶ copies/mL could be quantitatively analyzed using this system. This centrifugal-based microfluidic platform is a useful system and possesses industrialization potential that can be used for portable diagnostics.

Centrifugal Microfluidic System for Nucleic Acid Amplification and Detection

PubMed Central

Miao, Baogang; Peng, Niancai; Li, Lei; Li, Zheng; Hu, Fei; Zhang, Zengming; Wang, Chaohui

2015-01-01

We report here the development of a rapid PCR microfluidic system comprising a double-shaft turntable and centrifugal-based disc that rapidly drives the PCR mixture between chambers set at different temperatures, and the bidirectional flow improved the space utilization of the disc. Three heating resistors and thermistors maintained uniform, specific temperatures for the denaturation, annealing, and extension steps of the PCR. Infrared imaging showed that there was little thermal interference between reaction chambers; the system enabled the cycle number and reaction time of each step to be independently adjusted. To validate the function and efficiency of the centrifugal microfluidic system, a 350-base pair target gene from the hepatitis B virus was amplified and quantitated by fluorescence detection. By optimizing the cycling parameters, the reaction time was reduced to 32 min as compared to 120 min for a commercial PCR machine. DNA samples with concentrations ranging from 10 to 106 copies/mL could be quantitatively analyzed using this system. This centrifugal-based microfluidic platform is a useful system and possesses industrialization potential that can be used for portable diagnostics. PMID:26556354

Analysis of β-Subgroup Proteobacterial Ammonia Oxidizer Populations in Soil by Denaturing Gradient Gel Electrophoresis Analysis and Hierarchical Phylogenetic Probing

PubMed Central

Stephen, John R.; Kowalchuk, George A.; Bruns, Mary-Ann V.; McCaig, Allison E.; Phillips, Carol J.; Embley, T. Martin; Prosser, James I.

1998-01-01

A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic β-subgroup proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DNA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE. Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers. A sequence specific to all β-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147–4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters. DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results were ambiguous because of overlapping banding patterns. Unambiguous band identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes. The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol 62:4147–4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor. The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of β-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils. PMID:9687457

Dramatic Increase in the Signal and Sensitivity of Detection via Self-Assembly of Branched DNA

PubMed Central

Kim, Kyung-Tae; Chae, Chi-Bom

2011-01-01

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA. PMID:21870112

Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA

PubMed Central

Heilig, Hans G.H.J.; Zoetendal, Erwin G.; Vaughan, Elaine E.; Marteau, Philippe; Akkermans, Antoon D.L.; de Vos, Willem M.

2002-01-01

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and Weissella. Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant’s life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract. PMID:11772617

Bacterial chitinolytic communities respond to chitin and pH alteration in soil.

PubMed

Kielak, Anna M; Cretoiu, Mariana Silvia; Semenov, Alexander V; Sørensen, Søren J; van Elsas, Jan Dirk

2013-01-01

Chitin amendment is a promising soil management strategy that may enhance the suppressiveness of soil toward plant pathogens. However, we understand very little of the effects of added chitin, including the putative successions that take place in the degradative process. We performed an experiment in moderately acid soil in which the level of chitin, next to the pH, was altered. Examination of chitinase activities revealed fast responses to the added crude chitin, with peaks of enzymatic activity occurring on day 7. PCR-denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA and chiA genes showed structural changes of the phylogenetically and functionally based bacterial communities following chitin addition and pH alteration. Pyrosequencing analysis indicated (i) that the diversity of chiA gene types in soil is enormous and (i) that different chiA gene types are selected by the addition of chitin at different prevailing soil pH values. Interestingly, a major role of Gram-negative bacteria versus a minor one of Actinobacteria in the immediate response to the added chitin (based on 16S rRNA gene abundance and chiA gene types) was indicated. The results of this study enhance our understanding of the response of the soil bacterial communities to chitin and are of use for both the understanding of soil suppressiveness and the possible mining of soil for novel enzymes.

A novel COLD-PCR/FMCA assay enhances the detection of low-abundance IDH1 mutations in gliomas.

PubMed

Pang, Brendan; Durso, Mary B; Hamilton, Ronald L; Nikiforova, Marina N

2013-03-01

Point mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in many gliomas. The detection of IDH1 mutations becomes challenging on suboptimal glioma biopsies when a limited number of tumor cells is available for analysis. Coamplification at lower denaturing-polymerase chain reaction (COLD-PCR) is a PCR technique that deliberately lowers the denaturing cycle temperature to selectively favor amplification of mutant alleles, allowing for the sensitive detection of low-abundance mutations. We developed a novel COLD-PCR assay on the LightCycler platform (Roche, Applied Science, Indianapolis, IN), using post-PCR fluorescent melting curve analysis (FMCA) for the detection of mutant IDH1 with a detection limit of 1%. Thirty-five WHO grade I to IV gliomas and 9 non-neoplastic brain and spinal cord biopsies were analyzed with this technique and the results were compared with the conventional real-time PCR and the Sanger sequencing analysis. COLD-PCR/FMCA was able to detect the most common IDH1 R132H mutation and rare mutation types including R132H, R132C, R132L, R132S, and R132G mutations. Twenty-five glioma cases were positive for IDH1 mutations by COLD-PCR/FMCA, and 23 gliomas were positive by the conventional real-time PCR and Sanger sequencing. A pilocytic astrocytoma (PA I) and a glioblastoma multiforme (GBM IV) showed low-abundance IDH1 mutations detected by COLD-PCR/FMCA. The remaining 10 glioma and 9 non-neoplastic samples were negative by all the 3 methods. In summary, we report a novel COLD-PCR/FMCA method that provides rapid and sensitive detection of IDH1 mutations in formalin-fixed paraffin-embedded tissue and can be used in the clinical setting to assess the small brain biopsies.

Solving traveling salesman problems with DNA molecules encoding numerical values.

PubMed

Lee, Ji Youn; Shin, Soo-Yong; Park, Tai Hyun; Zhang, Byoung-Tak

2004-12-01

We introduce a DNA encoding method to represent numerical values and a biased molecular algorithm based on the thermodynamic properties of DNA. DNA strands are designed to encode real values by variation of their melting temperatures. The thermodynamic properties of DNA are used for effective local search of optimal solutions using biochemical techniques, such as denaturation temperature gradient polymerase chain reaction and temperature gradient gel electrophoresis. The proposed method was successfully applied to the traveling salesman problem, an instance of optimization problems on weighted graphs. This work extends the capability of DNA computing to solving numerical optimization problems, which is contrasted with other DNA computing methods focusing on logical problem solving.

Beneficial Effects of Rhodotorula sp. C11 on Growth and Disease Resistance of Juvenile Japanese Spiky Sea Cucumber Apostichopus japonicus.

PubMed

Yang, ZhiPing; Sun, JianMing; Xu, Zhe

2015-06-01

The purpose of this study was to evaluate the effects of dietary administration of the live yeast, Rhodotorula sp. C11, on growth and disease resistance against Vibrio splendidus infection in juvenile Japanese spiky sea cucumber Apostichopus japonicus. Sea cucumbers were fed diets containing Rhodotorula sp. C11 at 0 (control), 10⁴, 10⁵, and 10⁶ CFU/g of feed for 45 d. There were three replicate tanks per dietary treatment. The specific growth rates were higher in all sea cucumbers treated with Rhodotorula sp. C11 than in the controls. Following a challenge with V. splendidus NB13, the cumulative prevalence and mortality of sea cucumbers fed diets supplemented with Rhodotorula sp. C11 were lower than in animals fed the basal diet. In sea cucumbers fed diets supplemented with Rhodotorula sp. C11 for 42 d, the only viable yeast found in the intestine was Rhodotorula sp. C11, which had counts of 1.58-1.98 × 10⁴CFU/g. No yeast was isolated from the intestine of animals fed the basal diet. For the colonization study, 20 sea cucumbers from each dietary treatment were removed to separate tanks and fed the control diet from day 16 to day 46. The viable yeast (Rhodotorula sp. C11) counts in the intestine decreased to 60-80 CFU/g by day 37. Moreover, as demonstrated by denaturing gradient gel electrophoresis, Rhodotorula sp. C11 colonization of the intestine could be detected until day 46. The differences in culture and PCR-denaturing gradient gel electrophoresis may be due to differences in the sensitivity of both methods. The present result showed that Rhodotorula sp. C11 was able to successfully colonize the intestine of juvenile Japanese spiky sea cucumbers by dietary supplementation, which improved its growth and disease resistance.

The effect of a multispecies probiotic on the composition of the faecal microbiota and bowel habits in chronic obstructive pulmonary disease patients treated with antibiotics.

PubMed

Koning, Catherina J M; Jonkers, Daisy; Smidt, Hauke; Rombouts, Frans; Pennings, Herman-Jan; Wouters, Emiel; Stobberingh, Ellen; Stockbrügger, Reinhold

2010-05-01

Short-term antibiotic treatment profoundly affects the intestinal microbiota, which may lead to sustained changes in microbiota composition. Probiotics may restore such a disturbance. The objective of the present study was to investigate the effect of a multispecies probiotic on the faecal microbiota during and after antibiotic intake in patients with a history of frequent antibiotic use. In this randomised, placebo-controlled, double-blind study, thirty chronic obstructive pulmonary disease (COPD) patients treated with antibiotics for a respiratory tract infection received 5 g of a multispecies probiotic or placebo twice daily for 2 weeks. Faecal samples were collected at 0, 7, 14 and 63 d. Changes in the composition of the dominant faecal microbiota were determined by PCR-denaturing gradient gel electrophoresis (DGGE). Changes in bacterial subgroups were determined by quantitative PCR and culture. Bowel movements were scored daily according to the Bristol stool form scale. During and after antibiotic treatment, DGGE-based similarity indices (SI) were high ( >/= 84 %) and band richness was relatively low, both remaining stable over time. No difference in SI was observed between patients with and without diarrhoea-like bowel movements. The multispecies probiotic had a modest effect on the bacterial subgroups. Nevertheless, it affected neither the composition of the dominant faecal microbiota nor the occurrence of diarrhoea-like bowel movements. The dominant faecal microbiota was not affected by antibiotics in this COPD population, suggesting an existing imbalance of the microbiota, which may also have contributed to the lack of effect by probiotic intake.

Molecular biodiversity of arbuscular mycorrhizal fungi in trace metal-polluted soils.

PubMed

Hassan, Saad El Din; Boon, Eva; St-Arnaud, Marc; Hijri, Mohamed

2011-08-01

We assessed the indigenous arbuscular mycorrhizal fungi (AMF) community structure from the roots and associated soil of Plantago major (plantain) plants growing on sites polluted with trace metals (TM) and on unpolluted sites. Uncontaminated and TM-contaminated sites containing As, Cd, Cu, Pb, Sn and Zn were selected based on a survey of the TM concentration in soils of community gardens in the City of Montréal. Total genomic DNA was extracted directly from these samples. PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE), augmented by cloning and sequencing, as well as direct sequencing techniques, was all used to investigate AMF community structure. We found a decreased diversity of native AMF (assessed by the number of AMF ribotypes) in soils and plant roots harvested from TM-polluted soils compared with unpolluted soils. We also found that community structure was modified by TM contamination. Various species of Glomus, Scutellospora aurigloba and S. calospora were the most abundant ribotypes detected in unpolluted soil; ribotypes of G. etunicatum, G. irregulare/G. intraradices and G. viscosum were found in both polluted and unpolluted soils, while ribotypes of G. mosseae and Glomus spp. (B9 and B13) were dominant in TM-polluted soils. The predominance of G. mosseae in metal-polluted sites suggests the tolerance of this species to TM stress, as well as its potential use for phytoremediation. These data are relevant for our understanding of how AMF microbial communities respond to natural environments that contain a broad variety of toxic inorganic compounds and will substantially expand our knowledge of AMF ecology and biodiversity. © 2011 Blackwell Publishing Ltd.

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

PubMed Central

Busti, Elena; Bordoni, Roberta; Castiglioni, Bianca; Monciardini, Paolo; Sosio, Margherita; Donadio, Stefano; Consolandi, Clarissa; Rossi Bernardi, Luigi; Battaglia, Cristina; De Bellis, Gianluca

2002-01-01

Background PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. Results Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. Conclusions The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria. PMID:12243651

The biogeography of fungal communities in wetland sediments along the Changjiang River and other sites in China

PubMed Central

Wu, Bing; Tian, Jianqing; Bai, Chunming; Xiang, Meichun; Sun, Jingzu; Liu, Xingzhong

2013-01-01

Whether fungal community structure depends more on historical factors or on contemporary factors is controversial. This study used culture-dependent and -independent (polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)) methods to assess the influence of historical and contemporary factors on the distributions of fungi in the wetland sediments at 10 locations along the Changjiang River and at 10 other locations in China. The culture-dependent approach detected greater species diversity (177 operational taxonomic units (OTUs)) than PCR-DGGE analysis (145 OTUs), and the species in the genera of Penicillium (relative frequency=16.8%), Fusarium (15.4%), Aspergillus (7.6%), Trichoderma (5.8%) and Talaromyces (4.2%) were dominant. On the basis of DGGE data, fungal diversity along the Changjiang River increased from upstream to downstream; altitude explained 44.8% of this variation in diversity. And based on the data from all 20 locations, the fungal communities were geographically clustered into three groups: Southern China, Northern China and the Qinghai-Tibetan Plateau. Multivariate regression tree analysis for data from the 20 locations indicated that the fungal community was influenced primarily by location (which explained 61.8% of the variation at a large scale), followed by total potassium (9.4%) and total nitrogen (3.5%) at a local scale. These results are consistent with the concept that geographic distance is the dominant factor driving variation in fungal diversity at a regional scale (1000–4000 km), whereas environmental factors (total potassium and total nitrogen) explain variation in fungal diversity at a local scale (<1000 km). PMID:23446835

Effects of Lactobacillus salivarius Ren on cancer prevention and intestinal microbiota in 1, 2-dimethylhydrazine-induced rat model.

PubMed

Zhang, Ming; Fan, Xing; Fang, Bing; Zhu, Chengzhen; Zhu, Jun; Ren, Fazheng

2015-06-01

Probiotics have been suggested as a prophylactic measure in colon cancer. The aim of this study was to investigate the impact of Lactobacillus salivarius Ren (Ren) in modulating colonic microbiota structure and colon cancer incidence in a rat model after injection with 1,2-dimethyl hydrazine (DMH). The results indicated that oral administration of Ren could effectively suppress DMH-induced colonic carcinogenesis. A significant decrease in cancer incidence (87.5% to 25%) was detected in rats fed with a dose of 5 × 10(10) CFU/kg bodyweight per day. Using denaturing gradient gel electrophoresis and Real-time PCR combined with multivariate statistical methods, we demonstrated that injection with DMH significantly altered the rat gut microbiota, while Ren counteracted these DMH-induced adverse effects and promoted reversion of the gut microbiota close to the healthy state. Tvalue biplots followed by band sequencing identified 21 bacterial strains as critical variables affected by DMH and Ren. Injection of DMH significantly increased the amount of Ruminococcus species (sp.) and Clostridiales bacteria, as well as decreasing the Prevotella sp. Administration of Ren reduced the amount of Ruminococcus sp., Clostridiales bacteria, and Bacteroides dorei, and increased the amount of Prevotella. Real-time PCR results were consistent with the results derived by t-value biplots. These findings suggested that Ren is a potential agent for colon cancer prevention. In conclusion, the results in the present study suggest a potential therapeutic approach based on the modulation of intestinal microflora by probiotics may be beneficial in the prevention of colorectal carcinogenesis.

Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil.

PubMed

Jousset, Alexandre; Lara, Enrique; Nikolausz, Marcell; Harms, Hauke; Chatzinotas, Antonis

2010-02-01

Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats. Copyright 2009 Elsevier B.V. All rights reserved.

Oral administration of kefiran exerts a bifidogenic effect on BALB/c mice intestinal microbiota.

PubMed

Hamet, M F; Medrano, M; Pérez, P F; Abraham, A G

2016-01-01

The activity of kefiran, the exopolysaccharide present in kefir grains, was evaluated on intestinal bacterial populations in BALB/c mice. Animals were orally administered with kefiran and Eubacteria, lactobacilli and bifidobacteria populations were monitored in faeces of mice at days 0, 2, 7, 14 and 21. Profiles obtained by Denaturing Gradient Gel Electrophoresis (DGGE) with primers for Eubacteria were compared by principal component analysis and clearly defined clusters, correlating with the time of kefiran consumption, were obtained. Furthermore, profile analysis of PCR products amplified with specific oligonucleotides for bifidobacteria showed an increment in the number of DGGE bands in the groups administered with kefiran. Fluorescent In Situ Hybridisation (FISH) with specific probes for bifidobacteria showed an increment of this population in faeces, in accordance to DGGE results. The bifidobacteria population was also studied on distal colon content after 0, 2 and 7 days of kefiran administration. Analysis of PCR products by DGGE with Eubacteria primers showed an increment in the number and intensity of bands with high GC content of mice administered with kefiran. Sequencing of DGGE bands confirmed that bifidobacteria were one of the bacterial populations modified by kefiran administration. DGGE profiles of PCR amplicons obtained by using Bifidobacterium or Lactobacillus specific primers confirmed that kefiran administration enhances bifidobacteria, however no changes were observed in Lactobacillus populations. The results of the analysis of bifidobacteria populations assessed on different sampling sites in a murine model support the use of this exopolysaccharide as a bifidogenic functional ingredient.

Impact of Feed Efficiency and Diet on Adaptive Variations in the Bacterial Community in the Rumen Fluid of Cattle

PubMed Central

Hernandez-Sanabria, Emma; Goonewardene, Laksiri A.; Wang, Zhiquan; Durunna, Obioha N.; Moore, Stephen S.

2012-01-01

Limited knowledge of the structure and activities of the ruminal bacterial community prevents the understanding of the effect of population dynamics on functional bacterial groups and on host productivity. This study aimed to identify particular bacteria associated with host feed efficiency in steers with differing diets and residual feed intake (RFI) using culture-independent methods: PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis. PCR-DGGE profiles were generated from the ruminal fluid of 55 steers fed a low-energy-density diet and then switched to a high-energy-density diet. Bacterial profile comparisons by multivariate statistical analysis showed a trend only for RFI-related clusters on the high-energy diet. When steers (n = 19) belonging to the same RFI group under both diets were used to identify specific bacterial phylotypes related to feed efficiency traits, correlations were detected between dry matter intake, average daily gain, and copy numbers of the 16S rRNA gene of Succinivibrio sp. in low-RFI (efficient) steers, whereas correlations between Robinsoniella sp. and RFI (P < 0.05) were observed for high-RFI (inefficient) animals. Eubacterium sp. differed significantly (P < 0.05) between RFI groups that were only on the high-energy diet. Our work provides a comprehensive framework to understand how particular bacterial phylotypes contribute to differences in feed efficiency and ultimately influence host productivity, which may either depend on or be independent from diet factors. PMID:22156428

Analysis of a microbial community oxidizing inorganic sulfide and mercaptans.

PubMed

Duncan, K E; Sublette, K L; Rider, P A; Stepp, A; Beitle, R R; Conner, J A; Kolhatkar, R

2001-01-01

Successful treatment of refinery spent-sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio-Sep). The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources. The starting cultures for the enrichment consisted of several different Thiobacilli spp. (T. thioparus, T. denitrificans, T. thiooxidans, and T. neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester. Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms. Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR-amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis). Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands), Gram-positive cocci (Micrococcineae), alpha proteobacteria (3 bands), and an unidentified beta proteobacterium. Culturable microbes included several genera of fungi as well as various Gram-positive and Gram-negative heterotrophic bacteria not seen in techniques using direct DNA extraction.

[Bacterial diversity in the oral cavity of adolescents with different caries susceptibilities].

PubMed

Yangyang, Zhang; Jinzhi, He; Xin, Xu; Xuedong, Zhou

2015-12-01

To analyze the differences between the bacterial diversities in the saliva of caries-free and caries-susceptible adolescents through polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Twenty adolescent subjects aged 12-18 years were recruited and subdivided into two groups: caries-free adolescents (n = 10) and caries-susceptible adolescents (n = 10). Saliva samples were collected. Total DNA was isolated directly from each sample. A portion of the 16S rRNA gene locus was PCR-amplified by using universal primers. Microbial diversity was analyzed through PCR-DGGE. Analyzing the DGGE profile, we found that the composition of the saliva microbiome exhibited great intra-individual differences; the average band numbers of the caries-free adolescent group and the caries-susceptible adolescent group were 32.5 ± 3.7 and 27.3 ± 3.4, respectively. The differences between the groups were statistically significant (P = 0.008). Shannon-Wiener's indexes of the caries-susceptible adolescent group and the caries-free adolescent group were 2.5 ± 0.2 and 2.6 ± 0.2, respectively, but the differences between the groups were not significant (P = 0.405). Clustering analysis results suggested that most of the samples in the same group clustered together; this observation showed a high community structure similarity. The microbial diversity and complexity of bacteria in saliva are significantly higher in caries-free adolescents than in caries-susceptible adolescents. During caries development, bacterial diversity in the saliva likely decreases.

First report of Sneathia sanguinegens together with Mycoplasma hominis in postpartum prosthetic valve infective endocarditis: a case report.

PubMed

Kotaskova, Iva; Nemec, Petr; Vanerkova, Martina; Malisova, Barbora; Tejkalova, Renata; Orban, Marek; Zampachova, Vita; Freiberger, Tomas

2017-08-14

The presence of more than one bacterial agent is relatively rare in infective endocarditis, although more common in prosthetic cases. Molecular diagnosis from a removed heart tissue is considered a quick and effective way to diagnose fastidious or intracellular agents. Here we describe the case of postpartum polymicrobial prosthetic valve endocarditis in a young woman. Sneathia sanguinegens and Mycoplasma hominis were simultaneously detected from the heart valve sample using broad range 16S rRNA polymerase chain reaction (PCR) followed by sequencing while culture remained negative. Results were confirmed by independent PCR combined with denaturing gradient gel electrophoresis. Before the final agent identification, the highly non-compliant patient left from the hospital against medical advice on empirical intravenous treatment with aminopenicillins, clavulanate and gentamicin switched to oral amoxycillin and clavulanate. Four months after surgery, no signs of inflammation were present despite new regurgitation and valve leaflet flail was detected. However, after another 5 months the patient died from sepsis and recurrent infective endocarditis of unclarified etiology. Mycoplasma hominis is a rare causative agent of infective endocarditis. To the best of our knowledge, presented case is the first report of Sneathia sanguinegens detected in this condition. Molecular techniques were shown to be useful even in polymicrobial infective endocarditis samples.

Biodiversity of Bacterial Ecosystems in Traditional Egyptian Domiati Cheese▿

PubMed Central

El-Baradei, Gaber; Delacroix-Buchet, Agnès; Ogier, Jean-Claude

2007-01-01

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type. PMID:17189434

Biodiversity of bacterial ecosystems in traditional Egyptian Domiati cheese.

PubMed

El-Baradei, Gaber; Delacroix-Buchet, Agnès; Ogier, Jean-Claude

2007-02-01

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type.

Effects of poly-β-hydroxybutyrate (PHB) on Siberian sturgeon (Acipenser baerii) fingerlings performance and its gastrointestinal tract microbial community.

PubMed

Najdegerami, Ebrahim H; Tran, Tiet Ngoc; Defoirdt, Tom; Marzorati, Massimo; Sorgeloos, Patrick; Boon, Nico; Bossier, Peter

2012-01-01

Poly-β-hydroxybutyrate (PHB) is a natural polymer that can be depolymerized into water-soluble short-chain fatty acid monomers. These monomers can act as microbial control agents. In this study, the effects of partially replacing the diet of Siberian sturgeon fingerlings with 2% and 5% PHB were investigated. Replacing 2% of the diet with PHB improved weight gain, specific growth rate (SGR) and survival in the sturgeon fingerlings during the 10-week experimental period. Community-level physiological profiling and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) were used to analyze the microbial community diversity and community organization in the sturgeon gastrointestinal tract. DGGE analysis revealed that PHB affected the intestinal microbial species richness and diversity. The highest species richness was observed with 2% PHB. DNA sequencing of the dominant bands in 2% and 5% PHB treatments revealed that PHB stimulated bacteria belonging to the genera Bacillus and Ruminococcaceae. Principal component analysis, Lorenz curves and the Shannon index of Biolog Ecoplate data revealed that aerobic metabolic potential of the bacterial community was different in the PHB-treated fishes as compared with the control situation. Overall, our results indicate that PHB act as microbial control agents and replacement of 2% of Siberian sturgeon fingerling diet with PHB has beneficial effects.

Incidence of Staphylococcus aureus and analysis of associated bacterial communities on food industry surfaces.

PubMed

Gutiérrez, Diana; Delgado, Susana; Vázquez-Sánchez, Daniel; Martínez, Beatriz; Cabo, Marta López; Rodríguez, Ana; Herrera, Juan J; García, Pilar

2012-12-01

Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. A study designed to determine the presence of S. aureus on food contact surfaces in dairy, meat, and seafood environments and to identify coexisting microbiota has therefore been carried out. A total of 442 samples were collected, and the presence of S. aureus was confirmed in 6.1% of samples. Sixty-three S. aureus isolates were recovered and typed by random amplification of polymorphic DNA (RAPD). Profiles were clustered into four groups which were related to specific food environments. All isolates harbored some potential virulence factors such as enterotoxin production genes, biofilm formation-associated genes, antibiotic resistance, or lysogeny. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of bacterial communities coexisting with S. aureus revealed the presence of bacteria either involved in food spoilage or of concern for food safety in all food environments. Food industry surfaces could thus be a reservoir for S. aureus forming complex communities with undesirable bacteria in multispecies biofilms. Uneven microbiological conditions were found in each food sector, which indicates the need to improve hygienic conditions in food processing facilities, particularly the removal of bacterial biofilms, to enhance the safety of food products.

PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone.

PubMed

Garofalo, C; Silvestri, G; Aquilanti, L; Clementi, F

2008-07-01

To study lactic acid bacteria (LAB) and yeast dynamics during the production processes of sweet-leavened goods manufactured with type I sourdoughs. Fourteen sourdough and dough samples were taken from a baking company in central Italy during the production lines of three varieties of Panettone. The samples underwent pH measurements and plating analysis on three solid media. The microbial DNA was extracted from both the (sour)doughs and the viable LAB and yeast cells collected in bulk, and subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The molecular fingerprinting of the cultivable plus noncultivable microbial populations provide evidence of the dominance of Lactobacillus sanfranciscensis, Lactobacillus brevis and Candida humilis in the three fermentation processes. The DGGE profiles of the cultivable communities reveal a bacterial shift in the final stages of two of the production processes, suggesting an effect of technological parameters on the selection of the dough microflora. Our findings confirm the importance of using a combined analytical approach to explore microbial communities that develop during the leavening process of sweet-leavened goods. In-depth studies of sourdough biodiversity and population dynamics occurring during sourdough fermentation are fundamental for the control of the leavening process and the manufacture of standardized, high-quality products.

Addition of allochthonous fungi to a historically contaminated soil affects both remediation efficiency and bacterial diversity.

PubMed

Federici, Ermanno; Leonardi, Vanessa; Giubilei, Maria A; Quaratino, Daniele; Spaccapelo, Roberta; D'Annibale, Alessandro; Petruccioli, Maurizio

2007-11-01

Botryosphaeria rhodina DABAC P82 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow and to degrade aromatic hydrocarbons in an aged contaminated soil. To evaluate the impact of indigenous microflora on the overall process, incubations were performed on both fumigated and nonfumigated soils. Fungal colonization by B. rhodina was unexpectedly lower in the fumigated than in the nonfumigated soil while the growth of P. pulmonarius showed an opposite response. Degradation performances and detoxification by both fungi in the nonfumigated soil were markedly higher than those observed in the fumigated one. Heterotrophic bacterial counts in nonfumigated soil augmented with either B. rhodina or P. pulmonarius were significantly higher than those of the corresponding incubation control (6.7 +/- 0.3 x 10(8) and 8.35 +/- 0.6 x 10(8), respectively, vs 9.2 +/- 0.3 x 10(7)). Bacterial communities of both incubation controls and fungal-augmented soil were compared by numerical analysis of denaturing gradient gel electrophoresis profiles of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Besides increasing overall diversity, fungal augmentation led to considerable qualitative differences with respect to the pristine soil.

Long-term impact of farm management and crops on soil microorganisms assessed by combined DGGE and PLFA analyses

PubMed Central

Stagnari, Fabio; Perpetuini, Giorgia; Tofalo, Rosanna; Campanelli, Gabriele; Leteo, Fabrizio; Della Vella, Umberto; Schirone, Maria; Suzzi, Giovanna; Pisante, Michele

2014-01-01

In the present study, long-term organic and conventional managements were compared at the experimental field of Monsampolo del Tronto (Marche region, Italy) with the aim of investigating soil chemical fertility and microbial community structure. A polyphasic approach, combining soil fertility indicators with microbiological analyses (plate counts, PCR-denaturing gradient gel electrophoresis [DGGE] and phospholipid fatty acid analysis [PLFA]) was applied. Organic matter, N as well as some important macro and micronutrients (K, P, Mg, Mn, Cu, and Zn) for crop growth, were more available under organic management. Bacterial counts were higher in organic management. A significant influence of management system and management x crop interaction was observed for total mesophilic bacteria, nitrogen fixing bacteria and actinobacteria. Interestingly, cultivable fungi were not detected in all analyzed samples. PLFA biomass was higher in the organic and Gram positive bacteria dominated the microbial community in both systems. Even if fungal biomass was higher in organic management, fungal PCR-DGGE fingerprinting revealed that the two systems were very similar in terms of fungal species suggesting that 10 years were not enough to establish a new dynamic equilibrium among ecosystem components. A better knowledge of soil biota and in particular of fungal community structure will be useful for the development of sustainable management strategies. PMID:25540640

Occurrence and Diversity of Tetracycline Resistance Genes in Lagoons and Groundwater Underlying Two Swine Production Facilities

USGS Publications Warehouse

Chee-Sanford, J. C.; Aminov, R.I.; Krapac, I.J.; Garrigues-Jeanjean, N.; Mackie, R.I.

2001-01-01

In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.

Bacterial dynamics and metabolite changes in solid-state acetic acid fermentation of Shanxi aged vinegar.

PubMed

Li, Sha; Li, Pan; Liu, Xiong; Luo, Lixin; Lin, Weifeng

2016-05-01

Solid-state acetic acid fermentation (AAF), a natural or semi-controlled fermentation process driven by reproducible microbial communities, is an important technique to produce traditional Chinese cereal vinegars. Highly complex microbial communities and metabolites are involved in traditional Chinese solid-state AAF, but the association between microbiota and metabolites during this process are still poorly understood. In this study, we performed amplicon 16S rRNA gene sequencing on the Illumina MiSeq platform, PCR-denaturing gradient gel electrophoresis, and metabolite analysis to trace the bacterial dynamics and metabolite changes under AAF process. A succession of bacterial assemblages was observed during the AAF process. Lactobacillales dominated all the stages. However, Acetobacter species in Rhodospirillales were considerably accelerated during AAF until the end of fermentation. Quantitative PCR results indicated that the biomass of total bacteria showed a "system microbe self-domestication" process in the first 3 days, and then peaked at the seventh day before gradually decreasing until the end of AAF. Moreover, a total of 88 metabolites, including 8 organic acids, 16 free amino acids, and 66 aroma compounds were detected during AAF. Principal component analysis and cluster analyses revealed the high correlation between the dynamics of bacterial community and metabolites.

Conversion from long-term cultivated wheat field to Jerusalem artichoke plantation changed soil fungal communities

NASA Astrophysics Data System (ADS)

Zhou, Xingang; Zhang, Jianhui; Gao, Danmei; Gao, Huan; Guo, Meiyu; Li, Li; Zhao, Mengliang; Wu, Fengzhi

2017-01-01

Understanding soil microbial communities in agroecosystems has the potential to contribute to the improvement of agricultural productivity and sustainability. Effects of conversion from long-term wheat plantation to Jerusalem artichoke (JA) plantation on soil fungal communities were determined by amplicon sequencing of total fungal ITS regions. Quantitative PCR and PCR-denaturing gradient gel electrophoresis were also used to analyze total fungal and Trichoderma spp. ITS regions and Fusarium spp. Ef1α genes. Results showed that soil organic carbon was higher in the first cropping of JA and Olsen P was lower in the third cropping of JA. Plantation conversion changed soil total fungal and Fusarium but not Trichoderma spp. community structures and compositions. The third cropping of JA had the lowest total fungal community diversity and Fusarium spp. community abundance, but had the highest total fungal and Trichoderma spp. community abundances. The relative abundances of potential fungal pathogens of wheat were higher in the wheat field. Fungal taxa with plant growth promoting, plant pathogen or insect antagonistic potentials were enriched in the first and second cropping of JA. Overall, short-term conversion from wheat to JA plantation changed soil fungal communities, which is related to changes in soil organic carbon and Olsen P contents.

Potential nitrification and denitrification and the corresponding composition of the bacterial communities in a compact constructed wetland treating landfill leachates.

PubMed

Sundberg, C; Tonderski, K; Lindgren, P E

2007-01-01

Constructed wetlands can be used to decrease the high ammonium concentrations in landfill leachates. We investigated nitrification/denitrification activity and the corresponding bacterial communities in landfill leachate that was treated in a compact constructed wetland, Tveta Recycling Facility, Sweden. Samples were collected at three depths in a filter bed and the sediment from a connected open pond in July, September and November 2004. Potential ammonia oxidation was measured by short-term incubation method and potential denitrification by the acetylene inhibition technique. The ammonia-oxidising and the denitrifying bacterial communities were investigated using group-specific PCR primers targeting 16S rRNA genes and the functional gene nosZ, respectively. PCR products were analysed by denaturing gradient gel electrophoresis and nucleotide sequencing. The same degree of nitrification activity was observed in the pond sediment and at all levels in the filter bed, whereas the denitrification activity decreased with filter bed depth. Denitrification rates were higher in the open pond, even though the denitrifying bacterial community was more diverse in the filter bed. The ammonia-oxidising community was also more varied in the filter bed. In the filter bed and the open pond, there was no obvious relationship between the nitrification/denitrification activities and the composition of the corresponding bacterial communities.

Conversion from long-term cultivated wheat field to Jerusalem artichoke plantation changed soil fungal communities

PubMed Central

Zhou, Xingang; Zhang, Jianhui; Gao, Danmei; Gao, Huan; Guo, Meiyu; Li, Li; Zhao, Mengliang; Wu, Fengzhi

2017-01-01

Understanding soil microbial communities in agroecosystems has the potential to contribute to the improvement of agricultural productivity and sustainability. Effects of conversion from long-term wheat plantation to Jerusalem artichoke (JA) plantation on soil fungal communities were determined by amplicon sequencing of total fungal ITS regions. Quantitative PCR and PCR-denaturing gradient gel electrophoresis were also used to analyze total fungal and Trichoderma spp. ITS regions and Fusarium spp. Ef1α genes. Results showed that soil organic carbon was higher in the first cropping of JA and Olsen P was lower in the third cropping of JA. Plantation conversion changed soil total fungal and Fusarium but not Trichoderma spp. community structures and compositions. The third cropping of JA had the lowest total fungal community diversity and Fusarium spp. community abundance, but had the highest total fungal and Trichoderma spp. community abundances. The relative abundances of potential fungal pathogens of wheat were higher in the wheat field. Fungal taxa with plant growth promoting, plant pathogen or insect antagonistic potentials were enriched in the first and second cropping of JA. Overall, short-term conversion from wheat to JA plantation changed soil fungal communities, which is related to changes in soil organic carbon and Olsen P contents. PMID:28134269

The Meselson-Stahl Experiment

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2012-01-01

Terms to be familiar with before you start to solve the test: DNA replication, nitrogen isotopes, density labeling, cesium chloride density gradient centrifugation, ultraviolet absorption, DNA denaturation, circular and linear DNA, superspiralization, superhelical DNA, and template.

Highly diverse community structure in a remote central Tibetan geothermal spring does not display monotonic variation to thermal stress.

PubMed

Yim, Lau Chui; Hongmei, Jing; Aitchison, Jonathan C; Pointing, Stephen B

2006-07-01

We report an assessment of whole-community diversity for an extremely isolated geothermal location with considerable phylogenetic and phylogeographic novelty. We further demonstrate, using multiple statistical analyses of sequence data, that the response of community diversity is not monotonic to thermal stress along a gradient of 52-83 degrees C. A combination of domain- and division-specific PCR was used to obtain a broad spectrum of community phylotypes, which were resolved by denaturing gradient gel electrophoresis. Among 58 sequences obtained from microbial mats and streamers, some 95% suggest novel archaeal and bacterial diversity at the species level or higher. Moreover, new phylogeographic and thermally defined lineages among the Cyanobacteria, Chloroflexi, Eubacterium and Thermus are identified. Shannon-Wiener diversity estimates suggest that mats at 63 degrees C supported highest diversity, but when alternate models were applied [Average Taxonomic Distinctness (AvTD) and Variation in Taxonomic Distinctness (VarTD)] that also take into account the phylogenetic relationships between phylotypes, it is evident that greatest taxonomic diversity (AvTD) occurred in streamers at 65-70 degrees C, whereas greatest phylogenetic distance between taxa (VarTD) occurred in streamers of 83 degrees C. All models demonstrated that diversity is not related to thermal stress in a linear fashion.

The expression of miR-125b regulates angiogenesis during the recovery of heat-denatured HUVECs.

PubMed

Zhou, Situo; Zhang, Pihong; Liang, Pengfei; Huang, Xiaoyuan

2015-06-01

In previous studies we found that miR-125b was down-regulated in denatured dermis of deep partial thickness burn patients. Moreover, miR-125b inhibited tumor-angiogenesis associated with the decrease of ERBB2 and VEGF expression in ovarian cancer cells and breast cancer cells, etc. In this study, we investigated the expression patterns and roles of miR-125b during the recovery of denatured dermis and heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burns in Sprague-Dawley rats and the heat-denatured cells (52°C, 35 s) were used for analysis. Western blot analysis and real-time PCR were applied to evaluate the expression of miR-125b and ERBB2 and VEGF. The ability of angiogenesis in heat-denatured HUVECs was analyzed by scratch wound healing and tube formation assay after pri-miR-125b or anti-miR-125b transfection. miR-125b expression was time-dependent during the recovery of heat-denatured dermis and HUVECs. Moreover, miR-125b regulated ERBB2 mRNA and Protein Expression and regulated angiogenesis association with regulating the expression of VEGF in heat-denatured HUVECs. Taken together our results show that the expression of miR-125b is time-dependent and miR-125b plays a regulatory role of angiogenesis during wound healing after burns. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Biological treatment of steroidal drug industrial effluent and electricity generation in the microbial fuel cells.

PubMed

Liu, Ru; Gao, Chongyang; Zhao, Yang-Guo; Wang, Aijie; Lu, Shanshan; Wang, Min; Maqbool, Farhana; Huang, Qing

2012-11-01

The single chamber microbial fuel cells (MFCs) were used to treat steroidal drug production wastewater (SPW) and generate electricity simultaneously. The results indicated that the maximum COD removal efficiency reached 82%, total nitrogen and sulfate removal rate approached 62.47% and 26.46%, respectively. The maximum power density and the Coulombic efficiency reached to 22.3Wm(-3) and 30%, respectively. The scanning electron microscope showed that the dominant microbial populations were remarkably different in morphology on the surface of SPW and acetate-fed anodes. PCR-denaturing gradient gel electrophoresis profiles revealed that the microbial community structure fed with different concentrations of SPW presented a gradual succession and unique bacterial sequences were detected on the SPW and acetate-fed anodes. This research demonstrates that MFCs fed with SPW achieved a high efficiency of power density and simultaneous nutrient removal, and the dominant microorganisms on the anode were related to the types and the concentrations of substrates. Copyright © 2012 Elsevier Ltd. All rights reserved.

Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps.

PubMed

Vigneron, Adrien; L'Haridon, Stéphane; Godfroy, Anne; Roussel, Erwan G; Cragg, Barry A; Parkes, R John; Toffin, Laurent

2015-05-15

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Removal of bisphenol A (BPA) in a nitrifying system with immobilized biomass.

PubMed

Zielińska, Magdalena; Cydzik-Kwiatkowska, Agnieszka; Bernat, Katarzyna; Bułkowska, Katarzyna; Wojnowska-Baryła, Irena

2014-11-01

The potential for bisphenol A (BPA) removal by mixed consortia of immobilized microorganisms with high nitrification activity was investigated with BPA concentrations in the influent from 2.5 to 10.0 mg/L. The presence of BPA limited ammonium oxidation; nitrification efficiency decreased from 91.2±1.3% in the control series to 47.4±9.4% when BPA concentration in wastewater was the highest. The efficiency of BPA removal rose from 87.1±5.5% to 92.9±2.9% with increased BPA concentration in the influent. Measurement of oxygen uptake rates by biomass exposed to BPA showed that BPA was mainly removed by heterotrophic bacteria. A strong negative correlation between the BPA removal efficiency and nitrification efficiency indicated the limited contribution of ammonia-oxidizing bacteria (AOB) to BPA biodegradation. Exposure of biomass to BPA changed the quantity and diversity of AOB in the biomass as shown by real-time PCR and denaturing gradient gel electrophoresis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phylogenetic characterization of a corrosive consortium isolated from a sour gas pipeline.

PubMed

Jan-Roblero, J; Romero, J M; Amaya, M; Le Borgne, S

2004-06-01

Biocorrosion is a common problem in oil and gas industry facilities. Characterization of the microbial populations responsible for biocorrosion and the interactions between different microorganisms with metallic surfaces is required in order to implement efficient monitoring and control strategies. Denaturing gradient gel electrophoresis (DGGE) analysis was used to separate PCR products and sequence analysis revealed the bacterial composition of a consortium obtained from a sour gas pipeline in the Gulf of Mexico. Only one species of sulfate-reducing bacteria (SRB) was detected in this consortium. The rest of the population consisted of enteric bacteria with different characteristics and metabolic capabilities potentially related to biocorrosion. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. The low abundance of the detected SRB was evidenced by environmental scanning electron microscopy (ESEM). In addition, the localized corrosion of pipeline steel in the presence of the consortium was clearly observed by ESEM after removing the adhered bacteria.

Biotechnological potential of microbial consortia and future perspectives.

PubMed

Bhatia, Shashi Kant; Bhatia, Ravi Kant; Choi, Yong-Keun; Kan, Eunsung; Kim, Yun-Gon; Yang, Yung-Hun

2018-05-15

Design of a microbial consortium is a newly emerging field that enables researchers to extend the frontiers of biotechnology from a pure culture to mixed cultures. A microbial consortium enables microbes to use a broad range of carbon sources. It provides microbes with robustness in response to environmental stress factors. Microbes in a consortium can perform complex functions that are impossible for a single organism. With advancement of technology, it is now possible to understand microbial interaction mechanism and construct consortia. Microbial consortia can be classified in terms of their construction, modes of interaction, and functions. Here we discuss different trends in the study of microbial functions and interactions, including single-cell genomics (SCG), microfluidics, fluorescent imaging, and membrane separation. Community profile studies using polymerase chain-reaction denaturing gradient gel electrophoresis (PCR-DGGE), amplified ribosomal DNA restriction analysis (ARDRA), and terminal restriction fragment-length polymorphism (T-RFLP) are also reviewed. We also provide a few examples of their possible applications in areas of biopolymers, bioenergy, biochemicals, and bioremediation.

Microbial community structure in a thermophilic aerobic digester used as a sludge pretreatment process for the mesophilic anaerobic digestion and the enhancement of methane production.

PubMed

Jang, Hyun Min; Park, Sang Kyu; Ha, Jeong Hyub; Park, Jong Moon

2013-10-01

An effective two-stage sewage sludge digestion process, consisting of thermophilic aerobic digestion (TAD) followed by mesophilic anaerobic digestion (MAD), was developed for efficient sludge reduction and methane production. Using TAD as a biological pretreatment, the total volatile suspended solid reduction (VSSR) and methane production rate (MPR) in the MAD reactor were significantly improved. According to denaturing gradient gel electrophoresis (DGGE) analysis, the results indicated that the dominant bacteria species such as Ureibacillus thermophiles and Bacterium thermus in TAD were major routes for enhancing soluble organic matter. TAD pretreatment using a relatively short SRT of 1 day showed highly increased soluble organic products and positively affected an increment of bacteria populations which performed interrelated microbial metabolisms with methanogenic species in the MAD; consequently, a quantitative real-time PCR indicated greatly increased Methanosarcinales (acetate-utilizing methanogens) in the MAD, resulting in enhanced methane production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Chemical and microbial changes during autothermal thermophilic aerobic digestion (ATAD) of sewage sludge.

PubMed

Liu, Shugen; Song, Fanyong; Zhu, Nanwen; Yuan, Haiping; Cheng, Jiehong

2010-12-01

Autothermal thermophilic aerobic digestion (ATAD) is a promising process for sewage sludge stabilization. Batch experiments were conducted on sewage sludge collected from a municipal wastewater treatment plant in Shanghai, China, to evaluate the effectiveness of the ATAD system by determining changes in volatile suspended solids (VSSs) and to study its microbial diversity by denaturing gradient gel electrophoresis of 16S rRNA gene sequences amplified by PCR. The digestion system achieved rapid degradation of the organic substrate at 55 degrees C. The VSS was removed by up to 45.3% and 50.4% at 216 h and 264 h, respectively, while NH(4)(+)-N, chemical oxidation demand and total organic carbon of supernatant as well as total nitrogen did not exhibit obvious declines after 168 h. The microbial diversity changed during the thermophilic process as thermophiles belonging to the Hydrogenophilaceae, Thermotogaceae, Clostridiaceae and the genus Ureibacillus replaced less temperature-tolerant microorganisms such as Sphingobacteriaceae and the genus Trichococcus. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Successive changes in community structure of an ethylbenzene-degrading sulfate-reducing consortium.

PubMed

Nakagawa, Tatsunori; Sato, Shinya; Yamamoto, Yoko; Fukui, Manabu

2002-06-01

The microbial community structure and successive changes in a mesophilic ethylbenzene-degrading sulfate-reducing consortium were for the first time clarified by the denaturing gradient gel electrophoresis (DGGE) analysis of the PCR amplified 16S rRNA gene fragments. At least ten bands on the DGGE gel were detected in the stationary phase. Phylogenetic analysis of the DGGE bands revealed that the consortium consisted of different eubacterial phyla including the delta subgroup of Proteobacteria, the order Sphingobacteriales, the order Spirochaetales, and the unknown bacterium. The most abundant band C was closely related to strain mXyS1, an m-xylene-degrading sulfate-reducing bacterium (SRB), and occurred as a sole band on DGGE gels in the logarithmic growth phase that 40% ethylbenzene was consumed accompanied by sulfide production. During further prolonged incubation, the dominancy of band C did not change. These results suggest that SRB corresponds to the most abundant band C and contributes mainly to the degradation of ethylbenzene coupled with sulfate reduction.

Effects of oxytetracycline on the abundance and community structure of nitrogen-fixing bacteria during cattle manure composting.

PubMed

Sun, Jiajun; Qian, Xun; Gu, Jie; Wang, Xiaojuan; Gao, Hua

2016-09-01

The effects of oxytetracycline (OTC) on nitrogen-fixing bacterial communities were investigated during cattle manure composting. The abundance and community structure of nitrogen-fixing bacteria were determined by qPCR and denaturing gradient gel electrophoresis (DGGE), respectively. The matrix was spiked with OTC at four levels: no OTC, 10mg/kg dry weight (DW) OTC (L), 60mg/kg DW OTC (M), and 200mg/kg DW OTC (H). The high temperature period of composting was shorter with M and H, and the decline in temperature during the cooling stage was accelerated by OTC. OTC had a concentration-dependent inhibitory effect on the nitrogenase activity during early composting, and the nifH gene abundance declined significantly during the later composting stage. The DGGE profile and statistical analysis showed that OTC changed the nitrogen-fixing bacterial community succession and reduced the community richness and dominance. The nitrogen-fixing bacterial community structure was affected greatly by the high level of OTC. Copyright © 2016. Published by Elsevier Ltd.

Characterization of nitrifying microbial community in a submerged membrane bioreactor at short solids retention times.

PubMed

Duan, Liang; Song, Yonghui; Xia, Siqing; Hermanowicz, Slawomir W

2013-12-01

This study investigated the nitrifying bacterial community in membrane bioreactor (MBR) at short solids retention times (SRTs) of 3, 5 and 10 days. The denaturing gradient gel electrophoresis results showed that different types of ammonia-oxidizing bacteria (AOB) can survive at different operating conditions. The diversity of AOB increased as the SRT increased. The real-time PCR results showed that the amoA gene concentrations were similar when MBRs were stabilized, and it can be a good indicator of stabilized nitrification. The results of clone library indicated that Nitrosomonas was the dominant group of AOB in three reactors. The microarray results showed that Nitrospira was the dominant group of nitrite-oxidizing bacteria (NOB) in the system. All groups of AOB and NOB except Nitrosolobus and Nitrococcus were found in MBR, indicated that the nitrifying bacterial community structure was more complicated. The combination of some molecular tools can provide more information of microbial communities. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms.

PubMed

Verhamme, Daniel T; Prosser, James I; Nicol, Graeme W

2011-06-01

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH(4)(+)-N per gram of soil) and high (200 μg NH(4)(+)-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil.

Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms

PubMed Central

Verhamme, Daniel T; Prosser, James I; Nicol, Graeme W

2011-01-01

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH4+-N per gram of soil) and high (200 μg NH4+-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil. PMID:21228892

The impact of shrimp farming effluent on bacterial communities in mangrove waters, Ceará, Brazil.

PubMed

Sousa, O V; Macrae, A; Menezes, F G R; Gomes, N C M; Vieira, R H S F; Mendonça-Hagler, L C S

2006-12-01

The effects of shrimp farm effluents on bacterial communities in mangroves have been infrequently reported. Classic and molecular biology methods were used to survey bacterial communities from four mangroves systems. Water temperature, salinity, pH, total heterotrophic bacteria and maximum probable numbers of Vibrio spp. were investigated. Genetic profiles of bacterial communities were also characterized by polymerase chain reaction (PCR) amplification of eubacterial and Vibrio 16S rDNA using denaturing gradient gel electrophoresis (DGGE). Highest heterotrophic counts were registered in the mangrove not directly polluted by shrimp farming. The Enterobacteriaceae and Chryseomonas luteola dominated the heterotrophic isolates. Vibrio spp. pathogenic to humans and shrimps were identified. Eubacterial genetic profiles suggest a shared community structure independent of mangrove system. Vibrio genetic profiles were mangrove specific. Neither microbial counts nor genetic profiling revealed a significant decrease in species richness associated with shrimp farm effluent. The complex nature of mangrove ecosystems and their microbial communities is discussed.

Cyanobacterial composition of microbial mats from an Australian thermal spring: a polyphasic evaluation.

PubMed

McGregor, Glenn B; Rasmussen, J Paul

2008-01-01

Cyanobacterial composition of microbial mats from an alkaline thermal spring issuing at 43-71 degrees C from tropical north-eastern Australia are described using a polyphasic approach. Eight genera and 10 species from three cyanobacterial orders were identified based on morphological characters. These represented taxa previously known as thermophilic from other continents. Ultrastructural analysis of the tower mats revealed two filamentous morphotypes contributed the majority of the biomass. Both types had ultrastructural characteristics of the family Pseudanabaenaceae. DNA extracts were made from sections of the tentaculiform towers and the microbial community analysed by 16S cyanobacteria-specific PCR and denaturing-gradient gel electrophoresis. Five significant bands were identified and sequenced. Two bands clustered closely with Oscillatoria amphigranulata isolated from New Zealand hot springs; one unique phylotype had only moderate similarity to a range of Leptolyngbya species; and one phylotype was closely related to a number of Geitlerinema species. Generally the approaches yielded complementary information, however the results suggest that species designation based on morphological and ultrastructural criteria alone often fails to recognize their true phylogenetic position. Conversely some molecular techniques may fail to detect rare taxa suggesting that the widest possible suite of techniques be applied when conducting analyses of cyanobacterial diversity of natural populations. This is the first polyphasic evaluation of thermophilic cyanobacterial communities from the Australian continent.

Microorganisms with a Taste for Vanilla: Microbial Ecology of Traditional Indonesian Vanilla Curing

PubMed Central

Röling, Wilfred F. M.; Kerler, Josef; Braster, Martin; Apriyantono, Anton; Stam, Hein; van Verseveld, Henk W.

2001-01-01

The microbial ecology of traditional postharvesting processing of vanilla beans (curing) was examined using a polyphasic approach consisting of conventional cultivation, substrate utilization-based and molecular identification of isolates, and cultivation-independent community profiling by 16S ribosomal DNA based PCR-denaturing gradient gel electrophoresis. At two different locations, a batch of curing beans was monitored. In both batches a major shift in microbial communities occurred after short-term scalding of the beans in hot water. Fungi and yeast disappeared, although regrowth of fungi occurred in one batch during a period in which process conditions were temporarily not optimal. Conventional plating showed that microbial communities consisting of thermophilic and thermotolerant bacilli (mainly closely related to Bacillus subtilis, B. licheniformis,, and B. smithii) developed under the high temperatures (up to 65°C) that were maintained for over a week after scalding. Only small changes in the communities of culturable bacteria occurred after this period. Molecular analysis revealed that a proportion of the microbial communities could not be cultured on conventional agar medium, especially during the high-temperature period. Large differences between both batches were observed in the numbers of microorganisms, in species composition, and in the enzymatic abilities of isolated bacteria. These large differences indicate that the effects of microbial activities on the development of vanilla flavor could be different for each batch of cured vanilla beans. PMID:11319073

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

PubMed Central

Hjelmsø, Mathis Hjort; Hansen, Lars Hestbjerg; Bælum, Jacob; Feld, Louise; Holben, William E.

2014-01-01

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition. PMID:24610853

Temperature effect on nitrogen removal performance and bacterial community in culture of marine anammox bacteria derived from sea-based waste disposal site.

PubMed

Kawagoshi, Yasunori; Fujisaki, Koichiro; Tomoshige, Yuki; Yamashiro, Kento; Wei, Qiaoyan; Qiao, Yanwei

2012-04-01

Anaerobic ammonium oxidation (anammox) bacteria have been detected in variety of marine environment in recent years, however, there have been only a few studies on their characteristics in the culture. The aim of this study is to reveal the effect of temperature on nitrogen removal ability and bacterial community in a culture of marine anammox bacteria (MAAOB). The MAAOB were cultured from the sediment of a sea-based waste disposal site at the North Port of Osaka Bay in Japan. The maximum nitrogen removal rate (NRR) was observed at 25°C in the MAAOB culture, and it decreased both at below 20°C and over 33°C. The activation energy of the MAAOB culture was calculated to be 54.6 kJ mol(-1) in the 5°C to 30°C range. No significant change in bacterial community according with temperature (5-37°C) was confirmed in the results of polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Meanwhile, a number of bacteria related to the oxidation-reduction reaction of sulfur were confirmed and it is speculated that they involved in the activity of MAAOB and nitrogen removal ability in the culture. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Prenatal diagnosis of cystic fibrosis: 10-years experience.

PubMed

Hadj Fredj, S; Ouali, F; Siala, H; Bibi, A; Othmani, R; Dakhlaoui, B; Zouari, F; Messaoud, T

2015-06-01

We present in this study our 10years experience in prenatal diagnosis of cystic fibrosis performed in the Tunisian population. Based on family history, 40 Tunisian couples were selected for prenatal diagnosis. Fetal DNA was isolated from amniotic fluid collected by transabdominal amniocentesis or from chronic villi by transcervical chorionic villus sampling. The genetic analysis for cystic fibrosis mutations was performed by denaturant gradient gel electrophoresis and denaturing high-pressure liquid phase chromatography. We performed microsatellites analysis by capillary electrophoresis in order to verify the absence of maternal cell contamination. Thirteen fetuses were affected, 21 were heterozygous carriers and 15 were healthy with two normal alleles of CFTR gene. Ten couples opted for therapeutic abortion. The microsatellites genotyping showed the absence of contamination of the fetal DNA by maternal DNA in 93.75%. Our diagnostic strategy provides rapid and reliable prenatal diagnosis at risk families of cystic fibrosis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.

PubMed

Gill, Pooria; Forouzandeh, Mehdi; Rahbarizadeh, Fatemeh; Ramezani, Reihaneh; Rasaee, Mohammad Javad

2006-01-01

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.

Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

PubMed

Randazzo, Cinzia L; Torriani, Sandra; Akkermans, Antoon D L; de Vos, Willem M; Vaughan, Elaine E

2002-04-01

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

Nitrogen Cycling and Community Structure of Proteobacterial β-Subgroup Ammonia-Oxidizing Bacteria within Polluted Marine Fish Farm Sediments

PubMed Central

McCaig, Allison E.; Phillips, Carol J.; Stephen, John R.; Kowalchuk, George A.; Harvey, S. Martyn; Herbert, Rodney A.; Embley, T. Martin; Prosser, James I.

1999-01-01

A multidisciplinary approach was used to study the effects of pollution from a marine fish farm on nitrification rates and on the community structure of ammonia-oxidizing bacteria in the underlying sediment. Organic content, ammonium concentrations, nitrification rates, and ammonia oxidizer most-probable-number counts were determined in samples of sediment collected from beneath a fish cage and on a transect at 20 and 40 m from the cage. The data suggest that nitrogen cycling was significantly disrupted directly beneath the fish cage, with inhibition of nitrification and denitrification. Although visual examination indicated some slight changes in sediment appearance at 20 m, all other measurements were similar to those obtained at 40 m, where the sediment was considered pristine. The community structures of proteobacterial β-subgroup ammonia-oxidizing bacteria at the sampling sites were compared by PCR amplification of 16S ribosomal DNA (rDNA), using primers which target this group. PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) and with oligonucleotide hybridization probes specific for different ammonia oxidizers. A DGGE doublet observed in PCR products from the highly polluted fish cage sediment sample was present at a lower intensity in the 20-m sample but was absent from the pristine 40-m sample station. Band migration, hybridization, and sequencing demonstrated that the doublet corresponded to a marine Nitrosomonas group which was originally observed in 16S rDNA clone libraries prepared from the same sediment samples but with different PCR primers. Our data suggest that this novel Nitrosomonas subgroup was selected for within polluted fish farm sediments and that the relative abundance of this group was influenced by the extent of pollution. PMID:9872782

Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan.

PubMed

Patrone, Vania; Molinari, Paola; Morelli, Lorenzo

2016-11-21

Probiotics are actively used for treatment of diarrhoea, respiratory infections, and prevention of infectious gastrointestinal diseases. The efficacy of probiotics is due to strain-specific features and the number of viable cells; however, several reports of deviations from the label in the actual content of strains in probiotic products are a matter of concern. Most of the available data on quality focuses on probiotic products containing lactobacilli and/or bifidobacteria, while very few data are available on spore-forming probiotics. The present study evaluates the label claims for spore count and species identification in five commercial probiotic products marketed in India and Pakistan that claim to contain Bacillus clausii: Tufpro, Ecogro, Enterogermina, Entromax, and Ospor. Bacterial enumeration from three batches was done by microbiological plating methods by two independent operators. Species identification was done using PCR amplification and sequence analysis of the 16S rRNA gene, and determination of the total amount of species present in the products was done using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by DNA sequencing of the excised bands. Plate count methods demonstrated poor correlations between quantitative label indications and bacteria recovered from plates for Tufpro, Ecogro, and Ospor. The 16S rRNA analysis performed on bacteria isolated from plate counts showed that only Enterogermina and Ospor contained homogenous B. clausii. PCR-DGGE analysis revealed that only Enterogermina had a homogenous B. clausii population while other products had mixed bacterial populations. In conclusion, the current analysis clearly demonstrates that of the five analysed commercial probiotics, only Enterogermina followed the label claims. Copyright © 2016 Elsevier B.V. All rights reserved.

Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR*

PubMed Central

Chen, Lie-zhong; Li, Yan-li; Yu, Yun-long

2014-01-01

Chlorpyrifos is a widely used insecticide in recent years, and it will produce adverse effects on soil when applied on crops or mixed with soil. In this study, nested polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) were combined to explore the bacterial and fungal community successions in soil treated with 5 and 20 mg/kg of chlorpyrifos. Furthermore, isolates capable of efficiently decomposing chlorpyrifos were molecular-typed using enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). Under the experimental conditions, degradation of chlorpyrifos in soil was interpreted with the first-order kinetics, and the half-lives of chlorpyrifos at 5 and 20 mg/kg doses were calculated to be 8.25 and 8.29 d, respectively. DGGE fingerprint and principal component analysis (PCA) indicated that the composition of the fungal community was obviously changed with the chlorpyrifos treatment, and that samples of chlorpyrifos treatment were significantly separated from those of the control from the beginning to the end. While for the bacterial community, chlorpyrifos-treated soil samples were apparently different in the first 30 d and recovered to a similar level of the control up until 60 d, and the distance in the PCA between the chlorpyrifos-treated samples and the control was getting shorter through time and was finally clustered into one group. Together, our results demonstrated that the application of chlorpyrifos could affect the fungal community structure in a quick and lasting way, while only affecting the bacterial community in a temporary way. Finally, nine typical ERIC types of chlorpyrifos-degrading isolates were screened. PMID:24711353

A Multiplex PCR for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in Clinical Specimens

DTIC Science & Technology

2005-01-24

detection of S . pneumoniae from throat swab or sputum samples may indicate colonization rather than illness, as it is often found in nonsterile sites...pertussis were considered in this paper. 1.2. Mycoplasma pneumoniae M. pneumoniae may be second only to S . pneumoniae as a causative agent of CAP, with...performed using an iCycler Thermal Cycler (Bio-Rad). Denaturation was performed for 15 min at 95°C followed by 35 cycles of denaturation at 94°C for 30 s

The study of marine corrosion of copper alloys in chlorinated condenser cooling circuits: the role of microbiological components.

PubMed

Carvalho, Maria L; Doma, Jemimah; Sztyler, Magdalena; Beech, Iwona; Cristiani, Pierangela

2014-06-01

The present paper reports the on-line monitoring of corrosion behavior of the CuNi 70:30 and Al brass alloys exposed to seawater and complementary offline microbiological analyses. An electrochemical equipment with sensors specifically set for industrial application and suitable to estimate the corrosion (by linear polarization resistance technique), the biofilm growth (by the BIOX electrochemical probe), the chlorination treatment and other physical-chemical parameters of the water has been used for the on-line monitoring. In order to identify and better characterize the bacteria community present on copper alloys, tube samples were collected after a long period (1year) and short period (2days) of exposition to treated natural seawater (TNSW) and natural seawater (NSW). From the collected samples, molecular techniques such as DNA extraction, polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and identification by sequencing were performed to better characterize and identify the microbial biodiversity present in the samples. The monitoring data confirmed the significant role played by biofouling deposition against the passivity of these Cu alloys in seawater and the positive influence of antifouling treatments based on low level dosages. Molecular analysis indicated biodiversity with the presence of Marinobacter, Alteromonas and Pseudomonas species. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of the microbial community in industrial rye sourdough upon continuous back-slopping propagation revealed Lactobacillus helveticus as the dominant species.

PubMed

Viiard, E; Mihhalevski, A; Rühka, T; Paalme, T; Sarand, I

2013-02-01

To assess the structure and stability of a dominant lactic acid bacteria (LAB) population during the propagation of rye sourdough in an industrial semi-fluid production over a period of 7 months. The sourdough was started from a 6-year-old freeze-dried sourdough originating from the same bakery. A unique microbial consortium consisting mainly of bacteria belonging to species Lactobacillus helveticus, Lactobacillus panis and Lactobacillus pontis was identified based on culture-dependent (Rep-PCR) and culture-independent [denaturing gradient gel electrophoresis (DGGE)] methods. Three of the isolated Lact. helveticus strains showed remarkable adaptation to the sourdough conditions. They differed from the type strain by the ability to ferment compounds specific to plant material, like salicin, cellobiose and sucrose, but did not ferment lactose. We showed remarkable stability of a LAB consortium in rye sourdough started from lyophilized sourdough and propagated in a large bakery for 7 months. Lactobacillus helveticus was detected as the dominant species in the consortium and was shown to be metabolically adapted to the sourdough environment. The use of an established and adapted microbial consortium as a starter is a good alternative to commercial starter strains. © 2012 The Society for Applied Microbiology.

Chemosynthetic bacteria found in bivalve species from mud volcanoes of the Gulf of Cadiz.

PubMed

Rodrigues, Clara F; Webster, Gordon; Cunha, Marina R; Duperron, Sébastien; Weightman, Andrew J

2010-09-01

As in other cold seeps, the dominant bivalves in mud volcanoes (MV) from the Gulf of Cadiz are macrofauna belonging to the families Solemyidae (Acharax sp., Petrasma sp.), Lucinidae (Lucinoma sp.), Thyasiridae (Thyasira vulcolutre) and Mytilidae (Bathymodiolus mauritanicus). The delta(13)C values measured in solemyid, lucinid and thyasirid specimens support the hypothesis of thiotrophic nutrition, whereas isotopic signatures of B. mauritanicus suggest methanotrophic nutrition. The indication by stable isotope analysis that chemosynthetic bacteria make a substantial contribution to the nutrition of the bivalves led us to investigate their associated bacteria and their phylogenetic relationships based on comparative 16S rRNA gene sequence analysis. PCR-denaturing gradient gel electrophoresis analysis and cloning of bacterial 16S rRNA-encoding genes confirmed the presence of sulfide-oxidizing symbionts within gill tissues of many of the studied specimens. Phylogenetic analysis of bacterial 16S rRNA gene sequences demonstrated that most bacteria were related to known sulfide-oxidizing endosymbionts found in other deep-sea chemosynthetic environments, with the co-occurrence of methane-oxidizing symbionts in Bathymodiolus specimens. This study confirms the presence of several chemosynthetic bivalves in the Gulf of Cadiz and further highlights the importance of sulfide- and methane-oxidizing symbionts in the trophic ecology of macrobenthic communities in MV.

Investigating Freshwater Periphyton Community Response to Uranium with Phospholipid Fatty Acid and Denaturing Gradient Gel Electrophoresis Analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Small, Jack A.; Bunn, Amoret L.; McKinstry, Craig A.

2008-04-01

Periphyton communities can be used as monitors of ecosystem health and as indicators of contamination in lotic systems. Measures of biomass, community structure and genetic diversity were used to investigate impacts of uranium exposure on periphyton. Laboratory exposures of periphyton in river water amended with uranium were performed for 5 days, followed by 2 days of uranium depuration in unamended river water. Productivity as measured by biomass was not affected by concentrations up to 100 µg L-1 uranium. Phospholipid fatty acid (PLFA) profiles and denaturing gradient gel electrophoresis (DGGE) banding patterns found no changes in community or genetic structure relatedmore » to uranium exposure. We suggest that the periphyton community as a whole is not impacted by exposures of uranium up to a dose of 100 µg L-1. These findings have significance for the assessment and prediction of uranium impacts on aquatic ecosystems.« less

Herpesvirus in the oral cavity of children with leukaemia and its impact on the oral bacterial community profile.

PubMed

Bezerra, Tacíria M; Ferreira, Dennis C; Carmo, Flávia L; Pinheiro, Raquel; Leite, Deborah C A; Cavalcante, Fernanda S; Belinho, Raquel A; Peixoto, Raquel S; Rosado, Alexandre S; dos Santos, Kátia R N; Castro, Gloria F B A

2015-03-01

This cross-sectional study investigated the association between eight herpesviruses and the bacterial community profiles from the oral cavity of children with and without leukaemia. Sixty participants (aged 3-13), divided into the leukaemia group (LG) and healthy group (HG), were evaluated. Collection of medical data, intraoral examination and collection of clinical specimens were carried out. Single PCR and nested-PCR techniques were used to identify the viral types; denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were used to evaluate the profile and abundance of bacterial communities. All the children with leukaemia were positive for at least one type of herpesvirus, compared with healthy participants (33.3%; p<0.000). Human cytomegalovirus (HCMV; 46.7%), human herpesvirus-7 (HHV-7; 20%) and HHV-8 (77.3%) were in higher prevalence in the LG (p ≤ 0.01). Children with leukaemia had positive associations with the presence of HCMV, HHV-7 and HHV-8 in the oral cavity when under chemotherapy (p<0.05). There was a qualitative (means of DGGE bands) and quantitative (means of 16S rRNA gene abundance) difference in relation to the bacterial community between the two groups (p<0.05). Based on the results, the prevalence of herpesviruses and the qualitative bacterial profiles was higher in children with leukaemia and HCMV, HHV-7 and HHV-8 were related to the use of chemotherapy. Moreover, HHV-6 was correlated with an increased bacterial community profile in patients with leukaemia (p<0.05). More attention should be paid to the oral health of these individuals, mainly those under chemotherapy, in order to prevent infections by opportunistic pathogens. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Determination of lactic microflora of kefir grains and kefir beverage by using culture-dependent and culture-independent methods.

PubMed

Kesmen, Zülal; Kacmaz, Nazife

2011-01-01

In this study, we investigated the bacterial compositions of kefir grains and kefir beverages collected from different regions of Turkey by using culture-independent and culture-dependent methods. In the culture-independent detection, 10 different species of bacteria were detected in total by using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the 16S rRNA gene V3 region. Among these species, Lactobacillus kefiranofaciens was the most dominant one in the kefir grains, while Lactococcus lactis was found to be significantly prevalent in the kefir beverages. In the culture-dependent detection, the primary differentiation and grouping of the isolates from kefir beverages and kefir grains were performed using repetitive sequence-based PCR (rep-PCR) fingerprinting, and the results were validated by 16S rDNA full-length sequencing. According to the results of culture-dependent methods, the most frequently isolated species were L. lactis, Leuconostoc mesenteroides, and Lactobacillus kefiri, respectively. Only 3 species, which are L. lactis, Lactobacillus acidophilus, and Streptococcus thermophilus, were detected with both culture-dependent and culture-independent methods. This study showed that the combination of both methods is necessary for a detailed and reliable investigation of microbial communities in kefir grains and kefir beverages. Due to their artisan- and region-dependent microflora, kefir products can be a source of interesting lactic acid bacteria, either new taxa or strains with specific functional properties, which might be used for the development of new starter cultures and innovative food products. Therefore, an increasing demand exists for new strains that show desirable effects on the product characteristics Artisan dairy products are a candidate source of such microorganisms. For this reason, in this study, the bacterial compositions of kefir grains and kefir beverages obtained from different regions of Turkey were studied using culture-dependent and culture-independent molecular methods. © 2011 Institute of Food Technologists®

The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs.

PubMed

Liang, Pengfei; Jiang, Bimei; Lv, Chunliu; Huang, Xu; Sun, Li; Zhang, Pihong; Huang, Xiaoyuan

2013-10-01

The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs). Deep partial thickness burn model in Sprague-Dawley rats and the heat denatured cell model (52°C, 35s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin. Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs. The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF. We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis. Copyright © 2013 Elsevier B.V. All rights reserved.

A simple device using magnetic transportation for droplet-based PCR.

PubMed

Ohashi, Tetsuo; Kuyama, Hiroki; Hanafusa, Nobuhiro; Togawa, Yoshiyuki

2007-10-01

The Polymerase chain reaction (PCR) was successfully and rapidly performed in a simple reaction device devoid of channels, pumps, valves, or other control elements used in conventional lab-on-a-chip technology. The basic concept of this device is the transportation of aqueous droplets containing hydrophilic magnetic beads in a flat-bottomed, tray-type reactor filled with silicone oil. The whole droplets sink to the bottom of the reactor because their specific gravity is greater than that of the silicone oil used here. The droplets follow the movement of a magnet located underneath the reactor. The notable advantage of the droplet-based PCR is the ability to switch rapidly the proposed reaction temperature by moving the droplets to the required temperature zones in the temperature gradient. The droplet-based reciprocative thermal cycling was performed by moving the droplets composed of PCR reaction mixture to the designated temperature zones on a linear temperature gradient from 50 degrees C to 94 degrees C generated on the flat bottom plate of the tray reactor. Using human-derived DNA containing the mitochondria genes as the amplification targets, the droplet-based PCR with magnetic reciprocative thermal cycling successfully provided the five PCR products ranging from 126 to 1,219 bp in 11 min with 30 cycles. More remarkably, the human genomic gene amplification targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was accomplished rapidly in 3.6 min with 40 cycles.

GeoSymbio: a hybrid, cloud-based web application of global geospatial bioinformatics and ecoinformatics for Symbiodinium-host symbioses.

PubMed

Franklin, Erik C; Stat, Michael; Pochon, Xavier; Putnam, Hollie M; Gates, Ruth D

2012-03-01

The genus Symbiodinium encompasses a group of unicellular, photosynthetic dinoflagellates that are found free living or in hospite with a wide range of marine invertebrate hosts including scleractinian corals. We present GeoSymbio, a hybrid web application that provides an online, easy to use and freely accessible interface for users to discover, explore and utilize global geospatial bioinformatic and ecoinformatic data on Symbiodinium-host symbioses. The novelty of this application lies in the combination of a variety of query and visualization tools, including dynamic searchable maps, data tables with filter and grouping functions, and interactive charts that summarize the data. Importantly, this application is hosted remotely or 'in the cloud' using Google Apps, and therefore does not require any specialty GIS, web programming or data programming expertise from the user. The current version of the application utilizes Symbiodinium data based on the ITS2 genetic marker from PCR-based techniques, including denaturing gradient gel electrophoresis, sequencing and cloning of specimens collected during 1982-2010. All data elements of the application are also downloadable as spatial files, tables and nucleic acid sequence files in common formats for desktop analysis. The application provides a unique tool set to facilitate research on the basic biology of Symbiodinium and expedite new insights into their ecology, biogeography and evolution in the face of a changing global climate. GeoSymbio can be accessed at https://sites.google.com/site/geosymbio/. © 2011 Blackwell Publishing Ltd.

Bioremediation of oil-based drill cuttings by a halophilic consortium isolated from oil-contaminated saline soil.

PubMed

Rezaei Somee, Maryam; Shavandi, Mahmoud; Dastgheib, Seyed Mohammad Mehdi; Amoozegar, Mohammad Ali

2018-05-01

Oil-based drill cuttings are hazardous wastes containing complex hydrocarbons, heavy metals, and brine. Their remediation is a crucial step before release to the environment. In this work, we enriched a halophilic consortium, from oil-polluted saline soil, which is capable of degrading diesel as the main pollutant of oil-based drill cuttings. The degradation ability of the consortium was evaluated in microcosms using two different diluting agents (fine sand and biologically active soil). During the bioremediation process, the bacterial community dynamics of the microcosms was surveyed using PCR amplification of a fragment of 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE). The diesel degradation rates were monitored by total petroleum hydrocarbon (TPH) measurement and the total count of heterotrophic and diesel-degrading bacteria. After 3 months, the microcosm containing fine sand and drill cuttings with the ratio of 1:1 (initial TPH of 36,000 mg/kg) showed the highest TPH removal (40%) and its dominant bacterial isolates belonged to the genera Dietzia, Arthrobacter , and Halomonas . DGGE results also confirmed the role of these genera in drill cuttings remediation. DGGE analysis of the bacterial diversity showed that Propionibacterium, Salinimicrobium, Marinobacter , and Dietzia are dominant in active soil microcosm; whereas Bacillus, Salinibacillus , and Marinobacter are abundant in sand microcosm. Our results suggest that the bioaugmentation strategy would be more successful if the diluting agent does not contain a complex microbial community.

Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase.

PubMed

King, J A; Ball, J K

1993-09-01

An extremely sensitive and convenient microtiter plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphate conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75 microgram.

Molecular Monitoring of the Fecal Microbiota of Healthy Human Subjects during Administration of Lactulose and Saccharomyces boulardii

PubMed Central

Vanhoutte, Tom; De Preter, Vicky; De Brandt, Evie; Verbeke, Kristin; Swings, Jean; Huys, Geert

2006-01-01

Diet is a major factor in maintaining a healthy human gastrointestinal tract, and this has triggered the development of functional foods containing a probiotic and/or prebiotic component intended to improve the host's health via modulation of the intestinal microbiota. In this study, a long-term placebo-controlled crossover feeding study in which each subject received several treatments was performed to monitor the effect of a prebiotic substrate (i.e., lactulose), a probiotic organism (i.e., Saccharomyces boulardii), and their synbiotic combination on the fecal microbiota of three groups of 10 healthy human subjects differing in prebiotic dose and/or intake of placebo versus synbiotic. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was used to detect possible changes in the overall bacterial composition using the universal V3 primer and to detect possible changes at the subpopulation level using group-specific primers targeting the Bacteroides fragilis subgroup, the genus Bifidobacterium, the Clostridium lituseburense group (cluster XI), and the Clostridium coccoides-Eubacterium rectale group (cluster XIVa). Although these populations remained fairly stable based on DGGE profiling, one pronounced change was observed in the universal fingerprint profiles after lactulose ingestion. Band position analysis and band sequencing revealed that a band appearing or intensifying following lactulose administration could be assigned to the species Bifidobacterium adolescentis. Subsequent analysis with real-time PCR (RT-PCR) indicated a statistically significant increase (P < 0.05) in total bifidobacteria in one of the three subject groups after lactulose administration, whereas a similar but nonsignificant trend was observed in the other two groups. Combined RT-PCR results from two subject groups indicated a borderline significant increase (P = 0.074) of B. adolescentis following lactulose intake. The probiotic yeast S. boulardii did not display any detectable universal changes in the DGGE profiles, nor did it influence the bifidobacterial levels. This study highlighted the capacity of an integrated approach consisting of DGGE analysis and RT-PCR to monitor and quantify pronounced changes in the fecal microbiota of healthy subjects upon functional food administration. PMID:16957220

COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications.

PubMed

Mauger, Florence; How-Kit, Alexandre; Tost, Jörg

2017-06-01

Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are therefore difficult to detect. Many assays for mutation analysis in cancer-related genes (hotspots) have been developed to improve diagnosis, prognosis, prediction of drug resistance, and monitoring of the response to treatment. Two major approaches have been developed: mutation-specific amplification methods and methods that enrich and detect mutations without prior knowledge on the exact location and identity of the mutation. CO-amplification at Lower Denaturation temperature Polymerase Chain Reaction (COLD-PCR) methods such as full-, fast-, ice- (improved and complete enrichment), enhanced-ice, and temperature-tolerant COLD-PCR make use of a critical temperature in the polymerase chain reaction to selectively denature wild-type-mutant heteroduplexes, allowing the enrichment of rare mutations. Mutations can subsequently be identified using a variety of laboratory technologies such as high-resolution melting, digital polymerase chain reaction, pyrosequencing, Sanger sequencing, or next-generation sequencing. COLD-PCR methods are sensitive, specific, and accurate if appropriately optimized and have a short time to results. A large variety of clinical samples (tumor DNA, circulating cell-free DNA, circulating cell-free fetal DNA, and circulating tumor cells) have been studied using COLD-PCR in many different applications including the detection of genetic changes in cancer and infectious diseases, non-invasive prenatal diagnosis, detection of microorganisms, or DNA methylation analysis. In this review, we describe in detail the different COLD-PCR approaches, highlighting their specificities, advantages, and inconveniences and demonstrating their use in different fields of biological and biomedical research.

Interrelations between the microbiotas in the litter and in the intestines of commercial broiler chickens.

PubMed

Cressman, Michael D; Yu, Zhongtang; Nelson, Michael C; Moeller, Steven J; Lilburn, Michael S; Zerby, Henry N

2010-10-01

The intestinal microbiota of broiler chickens and the microbiota in the litter have been well studied, but the interactions between these two microbiotas remain to be determined. Therefore, we examined their reciprocal effects by analyzing the intestinal microbiotas of broilers reared on fresh pine shavings versus reused litter, as well as the litter microbiota over a 6-week cycle. Composite ileal mucosal and cecal luminal samples from birds (n = 10) reared with both litter conditions (fresh versus reused) were collected at 7, 14, 21, and 42 days of age. Litter samples were also collected at days 7, 14, 21, and 42. The microbiotas were profiled and compared within sample types based on litter condition using PCR and denaturing gradient gel electrophoresis (PCR-DGGE). The microbiotas were further analyzed using 16S rRNA gene clone libraries constructed from microbiota DNA extracted from both chick intestinal and litter samples collected at day 7. Results showed significant reciprocal effects between the microbiotas present in the litter and those in the intestines of broilers. Fresh litter had more environmental bacteria, while reused litter contained more bacteria of intestinal origin. Lactobacillus spp. dominated the ileal mucosal microbiota of fresh-litter chicks, while a group of bacteria yet to be classified within Clostridiales dominated in the ileal mucosal microbiota in the reused-litter chicks. The Litter condition (fresh versus reused) seemed to have a more profound impact on the ileal microbiota than on the cecal microbiota. The data suggest that the influence of fresh litter on ileal microbiota decreased as broilers grew, compared with temporal changes observed under reused-litter rearing conditions.

Microbial Ecology Dynamics during Rye and Wheat Sourdough Preparation

PubMed Central

Ercolini, Danilo; Pontonio, Erica; De Filippis, Francesca; Minervini, Fabio; La Storia, Antonietta; Gobbetti, Marco

2013-01-01

The bacterial ecology during rye and wheat sourdough preparation was described by 16S rRNA gene pyrosequencing. Viable plate counts of presumptive lactic acid bacteria, the ratio between lactic acid bacteria and yeasts, the rate of acidification, a permutation analysis based on biochemical and microbial features, the number of operational taxonomic units (OTUs), and diversity indices all together demonstrated the maturity of the sourdoughs during 5 to 7 days of propagation. Flours were mainly contaminated by metabolically active genera (Acinetobacter, Pantoea, Pseudomonas, Comamonas, Enterobacter, Erwinia, and Sphingomonas) belonging to the phylum Proteobacteria or Bacteroidetes (genus Chryseobacterium). Their relative abundances varied with the flour. Soon after 1 day of propagation, this population was almost completely inhibited except for the Enterobacteriaceae. Although members of the phylum Firmicutes were present at very low or intermediate relative abundances in the flours, they became dominant soon after 1 day of propagation. Lactic acid bacteria were almost exclusively representative of the Firmicutes by this time. Weissella spp. were already dominant in rye flour and stably persisted, though they were later flanked by the Lactobacillus sakei group. There was a succession of species during 10 days of propagation of wheat sourdoughs. The fluctuation between dominating and subdominating populations of L. sakei group, Leuconostoc spp., Weissella spp., and Lactococcus lactis was demonstrated. Other subdominant species such as Lactobacillus plantarum were detectable throughout propagation. As shown by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, Saccharomyces cerevisiae dominated throughout the sourdough propagation. Notwithstanding variations due to environmental and technology determinants, the results of this study represent a clear example of how the microbial ecology evolves during sourdough preparation. PMID:24096427

[Effects of continuous application of bio-organic fertilizer on banana production and cultural microflora of bulk soil in orchard with serious disease incidence].

PubMed

Zhong, Shu-tang; Shen, Zong-zhuan; Sun, Yi-fei; Lyu, Na-na; Ruan, Yun-ze; Li, Rong; Shen, Qi-rong

2015-02-01

A field experiment was conducted for two years to investigate the effects of different fertilization applications on the suppression of banana fusarium wilt disease, crop yield, fruit quality and culturable microflora in a banana orchard which has been monocultured with banana for 12 years and suffered serious banana fusarium wilt disease. The fertilizers included chemical fertilizer (CF), cow manure compost (CM), pig manure compost (PM) and bio-organic fertilizer (BIO). The banana soil microflora was invested using plate-counting method and culture-dependent polymerase chain reaction denaturing gradient gel electrophoresis method (CD PCR-DGGE). Results showed that, compared with the other treatments, 2-year consecutive application of BIO significantly reduced the banana fusarium wilt disease incidence, and improved the banana mass per tree, crop yield, total soluble sugar content and the ratio of total soluble sugar to titratable acidity of fruits (sugar/acid ratio). Moreover, the analysis of culturable microflora showed that BIO application significantly increased the soil microbial biomass, soil culturable bacteria, bacillus and actinomycetes, and the ratio of bacteria to fungi (B/F) , while decreased the Fusarium oxysporum. Based on the CD PCR-DGGE results, the BIO application significantly altered the soil culturable bacterial structure and showed highest richness and diversity after 2 years of BIO application. The phylogenetic analysis of the selected bands showed that BIO application enriched the soil with the species of Paenibacillus sp., Burkholderia sp., uncultured Verrucomicrobia sp. and Bacillus aryabhattai, and depressed the species of Ralstonia sp., Chryseobacterium gleum, Fluviicola taffensis, Enterobacter sp. and Bacillus megaterium. These results confirmed that the continuous application of BIO effectively controlled the fusarium wilt disease, improved the crop yield and fruit quality, and modulated the soil culturable microflora under field condition.

Anaerobic Oxidation of Methane at a Marine Methane Seep in a Forearc Sediment Basin off Sumatra, Indian Ocean

PubMed Central

Siegert, Michael; Krüger, Martin; Teichert, Barbara; Wiedicke, Michael; Schippers, Axel

2011-01-01

A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by 13C-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO2 was confirmed in a 13C-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 × 109 cells cm−3 sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition–fluorescence in situ hybridization (CARD–FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD–FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea, but only small numbers of Eukarya. The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (δ-Proteobacteria, candidate division OP9, and Anaerolineaceae) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae, which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible. PMID:22207865

Analysis of Inter-Individual Bacterial Variation in Gut of Cicada Meimuna mongolica (Hemiptera: Cicadidae)

PubMed Central

Zhou, Wenting; Nan, Xiaoning; Zheng, Zhou; Wei, Cong; He, Hong

2015-01-01

Intestinal bacterial community plays a crucial role in the nutrition, development, survival, and reproduction of insects. When compared with other insects with piercing-sucking mouthparts, the habitats of cicada nymphs and adults are totally different. However, little is known about the differences in the gut bacterial communities in the nymphs and adults within any cicada species. The diversity of bacteria in the gut of nymphs and adults of both genders of Meimuna mongolica (Distant) was studied using the denaturing gradient gel electrophoresis (DGGE) method. Few inter-individual variations among gut microbiota were observed, suggesting that M. mongolica typically harbors a limited and consistent suite of bacterial species. Bacteria in the genera Pseudomonas and Enterobacter were the predominant components of the gut microflora of M. mongolica at all life stages. Bacteria of Pantoea, Streptococcus, and Uruburuella were also widespread in the cicada samples but at relatively lower concentrations. The relative stability and similarity of the PCR-DGGE patterns indicate that all individuals of this cicada species harbor a characteristic bacterial community which is independent from developmental stages and genders. Related endosymbionts that could be harbored in bacteromes of cicadas were not detected in any gut samples, which could be related to the cicada species and the distribution of these endosymbionts in the cicada cavity, or due to some of the possible limitations of PCR-DGGE community profiling. It is worthwhile to further address if related cicada endosymbiont clades distribute in the alimentary canals and other internal organs through diagnostic PCR using group-specific primer sets. PMID:26411784

[Comparison between transgenic insect-resistant cotton expressing Cry1Ac protein and its parental variety in rhizospheric fungal diversity].

PubMed

Pan, Jian-Gang; Jiao, Hai-Hua; Bai, Zhi-Hui; Qi, Hong-Yan; Ma, An-Zhou; Zhuang, Guo-qiang; Zhang, Hong-xun

2014-11-01

The dynamics of rhizospheric fungal diversity and biomass at different sampling stages associated with two transgenic insectresistant cottons expressing Cry1Ac protein and their control varieties were studied under greenhouse conditions, followed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time polymerase chain reaction (Q-PCR), in order to evaluate the ecological security of planting transgenic cotton expressing Cry1Ac protein. The results indicated that the fungal superior bands in rhizosphere of transgenic Bt cotton were similar with that of control cotton at four sampling stages, the more obvious difference in the blurred bands among transgenic Bt cotton, JM20 and SHIYUAN321 was detected. The rhizospheric fungal biomass of transgenic Bt cotton SGK321 was significantly lower than that of its parental control cotton at seedling stage, while the slight decrease in fungal biomass of transgenic Bt cotton XP188 was detected at boll forming stage, the ill-defined decrease, even growing tendency in two transgenic Bt cottons was detected at other stages. However, the difference of rhizospheric fungal community compositions and biomass was not only existed between transgenic cotton and its control, but also between SHIYUAN321 and JM20, and the same phenomenon was also detected between transgenic Bt cotton SGK321 and XP188. Hence, Bt protein is not the only incentive resulting in the difference in fungal community composition and diversity, the decrease in biomass between transgenic cotton and untransgenic cotton, different cotton varieties has an effect on them.

Fish-mediated changes in bacterioplankton community composition: an in situ mesocosm experiment

NASA Astrophysics Data System (ADS)

Luo, Congqiang; Yi, Chunlong; Ni, Leyi; Guo, Longgen

2017-06-01

We characterized variations in bacterioplankton community composition (BCC) in mesocosms subject to three different treatments. Two groups contained fish (group one: Cyprinus carpio; group two: Hypophthalmichthys molitrix); and group three, the untreated mesocosm, was the control. Samples were taken seven times over a 49-day period, and BCC was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR). Results revealed that introduction of C. carpio and H. molitrix had a remarkable impact on the composition of bacterioplankton communities, and the BCC was significantly different between each treatment. Sequencing of DGGE bands revealed that the bacterioplankton community in the different treatment groups was consistent at a taxonomic level, but differed in its abundance. H. molitrix promoted the richness of Alphaproteobacteria and Actinobacteria, while more bands affiliated to Cyanobacteria were detected inC. carpio mesocosms. The redundancy analysis (RDA) result demonstrated that the BCC was closely related to the bottom-up (total phosphorus, chlorophyll a, phytoplankton biomass) and top-down forces (biomass of copepods and cladocera) in C. carpio and control mesocosms, respectively. We found no evidence for top-down regulation of BCC by zooplankton in H. molitrix mesocosms, while grazing by protozoa (heterotrophic nanoflagellates, ciliates) became the major way to regulate BCC. Total bacterioplankton abundances were significantly higher in C. carpio mesocosms because of high nutrient concentration and suspended solids. Our study provided insights into the relationship between fish and bacterioplankton at species level, leading to a deep understanding of the function of the microbial loop and the aquatic ecosystem.

Aerobic biological treatment of low-strength synthetic wastewater in membrane-coupled bioreactors: the structure and function of bacterial enrichment cultures as the net growth rate approaches zero.

PubMed

Chen, Ruoyu; LaPara, Timothy M

2006-01-01

The goal of the current research was to determine if the stringent nutrient limitation imposed by membrane-coupled bioreactors (MBRs) could be used to force mixed bacterial communities to exhibit a zero net growth rate over an extended time period. Mechanistically, this zero net growth rate could be achieved when the amount of energy available for growth is balanced by the maintenance requirements of the bacterial community. Bench-scale MBRs were fed synthetic feed medium containing gelatin as the major organic substrate. Biomass concentrations initially increased rapidly, but subsequently declined until an asymptote was reached. Leucine aminopeptidase activities concomitantly increased by at least 10-fold, suggesting that bacterial catabolic activity remained high even while growth rates became negligible. In contrast, alpha-glucosidase and heptanoate esterase activities decreased, indicating that the bacterial community specifically adapted to the carbon source in the feed medium. Bacterial community analysis by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments (PCR-DGGE) suggested that the bacterial community structure completely changed from the beginning to the end of each MBR. Excision and nucleotide sequence analysis of prominent PCR-DGGE bands suggested that many of the dominant populations were similar to novel bacterial strains that were previously uncultivated or recently cultivated during studies specifically targeting these novel populations. This research demonstrates that MBRs have substantial practical applications for biological wastewater treatment; in addition, MBRs are a useful tool to study the ecology of slow-growing bacteria.

Anaerobic Oxidation of Methane at a Marine Methane Seep in a Forearc Sediment Basin off Sumatra, Indian Ocean.

PubMed

Siegert, Michael; Krüger, Martin; Teichert, Barbara; Wiedicke, Michael; Schippers, Axel

2011-01-01

A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by (13)C-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO(2) was confirmed in a (13)C-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 × 10(9) cells cm(-3) sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD-FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea, but only small numbers of Eukarya. The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (δ-Proteobacteria, candidate division OP9, and Anaerolineaceae) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae, which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible.

Fish-mediated changes in bacterioplankton community composition: an in situ mesocosm experiment

NASA Astrophysics Data System (ADS)

Luo, Congqiang; Yi, Chunlong; Ni, Leyi; Guo, Longgen

2018-03-01

We characterized variations in bacterioplankton community composition (BCC) in mesocosms subject to three different treatments. Two groups contained fish (group one: Cyprinus carpio; group two: Hypophthalmichthys molitrix); and group three, the untreated mesocosm, was the control. Samples were taken seven times over a 49-d period, and BCC was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR). Results revealed that introduction of C. carpio and H. molitrix had a remarkable impact on the composition of bacterioplankton communities, and the BCC was significantly different between each treatment. Sequencing of DGGE bands revealed that the bacterioplankton community in the different treatment groups was consistent at a taxonomic level, but differed in its abundance. H. molitrix promoted the richness of Alphaproteobacteria and Actinobacteria, while more bands affiliated to Cyanobacteria were detected in C. carpio mesocosms. The redundancy analysis (RDA) result demonstrated that the BCC was closely related to the bottom-up (total phosphorus, chlorophyll a, phytoplankton biomass) and top-down forces (biomass of copepods and cladocera) in C. carpio and control mesocosms, respectively. We found no evidence for top-down regulation of BCC by zooplankton in H. molitrix mesocosms, while grazing by protozoa (heterotrophic nanoflagellates, ciliates) became the major way to regulate BCC. Total bacterioplankton abundances were significantly higher in C. carpio mesocosms because of high nutrient concentration and suspended solids. Our study provided insights into the relationship between fish and bacterioplankton at species level, leading to a deep understanding of the function of the microbial loop and the aquatic ecosystem.

Autecology of an arsenite chemolithotroph: sulfide constraints on function and distribution in a geothermal spring.

PubMed

D'Imperio, Seth; Lehr, Corinne R; Breary, Michele; McDermott, Timothy R

2007-11-01

Previous studies in an acid-sulfate-chloride spring in Yellowstone National Park found that microbial arsenite [As(III)] oxidation is absent in regions of the spring outflow channel where H(2)S exceeds approximately 5 microM and served as a backdrop for continued efforts in the present study. Ex situ assays with microbial mat samples demonstrated immediate As(III) oxidation activity when H(2)S was absent or at low concentrations, suggesting the presence of As(III) oxidase enzymes that could be reactivated if H(2)S is removed. Cultivation experiments initiated with mat samples taken from along the H(2)S gradient in the outflow channel resulted in the isolation of an As(III)-oxidizing chemolithotroph from the low-H(2)S region of the gradient. The isolate was phylogenetically related to Acidicaldus and was characterized in vitro for spring-relevant properties, which were then compared to its distribution pattern in the spring as determined by denaturing gradient gel electrophoresis and quantitative PCR. While neither temperature nor oxygen requirements appeared to be related to the occurrence of this organism within the outflow channel, H(2)S concentration appeared to be an important constraint. This was verified by in vitro pure-culture modeling and kinetic experiments, which suggested that H(2)S inhibition of As(III) oxidation is uncompetitive in nature. In summary, the studies reported herein illustrate that H(2)S is a potent inhibitor of As(III) oxidation and will influence the niche opportunities and population distribution of As(III) chemolithotrophs.

Autecology of an Arsenite Chemolithotroph: Sulfide Constraints on Function and Distribution in a Geothermal Spring▿

PubMed Central

D'Imperio, Seth; Lehr, Corinne R.; Breary, Michele; McDermott, Timothy R.

2007-01-01

Previous studies in an acid-sulfate-chloride spring in Yellowstone National Park found that microbial arsenite [As(III)] oxidation is absent in regions of the spring outflow channel where H2S exceeds ∼5 μM and served as a backdrop for continued efforts in the present study. Ex situ assays with microbial mat samples demonstrated immediate As(III) oxidation activity when H2S was absent or at low concentrations, suggesting the presence of As(III) oxidase enzymes that could be reactivated if H2S is removed. Cultivation experiments initiated with mat samples taken from along the H2S gradient in the outflow channel resulted in the isolation of an As(III)-oxidizing chemolithotroph from the low-H2S region of the gradient. The isolate was phylogenetically related to Acidicaldus and was characterized in vitro for spring-relevant properties, which were then compared to its distribution pattern in the spring as determined by denaturing gradient gel electrophoresis and quantitative PCR. While neither temperature nor oxygen requirements appeared to be related to the occurrence of this organism within the outflow channel, H2S concentration appeared to be an important constraint. This was verified by in vitro pure-culture modeling and kinetic experiments, which suggested that H2S inhibition of As(III) oxidation is uncompetitive in nature. In summary, the studies reported herein illustrate that H2S is a potent inhibitor of As(III) oxidation and will influence the niche opportunities and population distribution of As(III) chemolithotrophs. PMID:17827309

Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening.

PubMed

Lovell, Charles R; Decker, Peter V; Bagwell, Christopher E; Thompson, Shelly; Matsui, George Y

2008-05-01

Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.

Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo.

PubMed

Limpiyakorn, Tawan; Shinohara, Yuko; Kurisu, Futoshi; Yagi, Osami

2005-10-01

We investigated ammonia-oxidizing bacteria in activated sludge collected from 12 sewage treatment systems, whose ammonia removal and treatment processes differed, during three different seasons. We used real-time PCR quantification to reveal total bacterial numbers and total ammonia oxidizer numbers, and used specific PCR followed by denaturing gel gradient electrophoresis, cloning, and sequencing of 16S rRNA genes to analyze ammonia-oxidizing bacterial communities. Total bacterial numbers and total ammonia oxidizer numbers were in the range of 1.6 x 10(12) - 2.4 x 10(13) and 1.0 x 10(9) - 9.2 x 10(10)cellsl(-1), respectively. Seasonal variation was observed in the total ammonia oxidizer numbers, but not in the ammonia-oxidizing bacterial communities. Members of the Nitrosomonas oligotropha cluster were found in all samples, and most sequences within this cluster grouped within two of the four sequence types identified. Members of the clusters of Nitrosomonas europaea-Nitrosococcus mobilis, Nitrosomonas cryotolerans, and unknown Nitrosomonas, occurred solely in one anaerobic/anoxic/aerobic (A2O) system. Members of the Nitrosomonas communis cluster occurred almost exclusively in association with A2O and anaerobic/aerobic systems. Solid residence time mainly influenced the total numbers of ammonia-oxidizing bacteria, whereas dissolved oxygen concentration primarily affected the ammonia-oxidizing activity per ammonia oxidizer cell.

[Distribution and Diversity of Ammonium-oxidizing Archaea and Ammonium-oxidizing Bacteria in Surface Sediments of Oujiang River].

PubMed

Li, Hu; Huang, Fu-yi; Su, Jian-qiang; Hong, You-wei; Yu, Shen

2015-12-01

Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) play important roles in the biogeochemical nitrogen cycle. Rivers are important ecosystems containing a large number of functional microbes in nitrogen cycle. In this study, denaturing gradient gel electrophoresis (DGGE ) and real-time quantitative PCR (qPCR) technology were used to analyze the distribution and diversity of AOA and AOB in sediments from Oujiang. The results showed that the AOA community structure was similar among various sites, while the AOB community structure was significantly different, in which all detected AOB sequences were classified into Nitrosospira and Nitrosomonas, and 90% affiliated to Nitrosospira. The community composition of AOA was influenced by NH₄⁺ and TS, in addition, the AOB composition was affected by NH₄⁺, EC, pH, NO₃⁻, TC and TN. Total sulfur (TS) and electrical conductivity (EC) were the major factors influencing the diversity of AOA and AOB, respectively. AOA abundance was significantly higher than that of AOB. EC, NH₄⁺-N and NO₃⁻-N were the main environmental factors affecting the abundance of AOA and AOB. This study indicated that the community composition and diversity of AOA and AOB were significantly influenced by environmental factors, and AOA might be dominant drivers in the ammonia oxidation process in Oujiang surface sediment.

Bacteria of the Candidate Phylum TM7 are Prevalent in Acidophilic Nitrifying Sequencing-Batch Reactors

PubMed Central

Hanada, Akiko; Kurogi, Takashi; Giang, Nguyen Minh; Yamada, Takeshi; Kamimoto, Yuki; Kiso, Yoshiaki; Hiraishi, Akira

2014-01-01

Laboratory-scale acidophilic nitrifying sequencing-batch reactors (ANSBRs) were constructed by seeding with sewage-activated sludge and cultivating with ammonium-containing acidic mineral medium (pH 4.0) with or without a trace amount of yeast extract. In every batch cycle, the pH varied between 2.7 and 4.0, and ammonium was completely converted to nitrate. Attempts to detect nitrifying functional genes in the fully acclimated ANSBRs by PCR with previously designed primers mostly gave negative results. 16S rRNA gene-targeted PCR and a subsequent denaturating gradient gel electrophoresis analysis revealed that a marked change occurred in the bacterial community during the overall period of operation, in which members of the candidate phylum TM7 and the class Gammaproteobacteria became predominant at the fully acclimated stage. This result was fully supported by a 16S rRNA gene clone library analysis, as the major phylogenetic groups of clones detected (>5% of the total) were TM7 (33%), Gammaproteobacteria (37%), Actinobacteria (10%), and Alphaproteobacteria (8%). Fluorescence in situ hybridization with specific probes also demonstrated the prevalence of TM7 bacteria and Gammaproteobacteria. These results suggest that previously unknown nitrifying microorganisms may play a major role in ANSBRs; however, the ecophysiological significance of the TM7 bacteria predominating in this process remains unclear. PMID:25241805

Comparison of bacterial community structures of terrestrial cyanobacterium Nostoc flagelliforme in three different regions of China using PCR-DGGE analysis.

PubMed

Han, Pei-pei; Shen, Shi-gang; Jia, Shi-ru; Wang, Hui-yan; Zhong, Cheng; Tan, Zhi-lei; Lv, He-xin

2015-07-01

Filamentous Nostoc flagelliforme form colloidal complex, with beaded cells interacting with other bacteria embedded in the complex multilayer sheath. However, the species of bacteria in the sheath and the interaction between N. flagelliforme and associated bacteria remain unclear. In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of N. flagelliforme from three regions of China. DGGE patterns showed variations in all samples, exhibiting 25 discrete bands with various intensities. The diversity index analysis of bands profiles suggested the high similarity of bacterial communities to each other but also the dependence of microbial composition on each location. Phylogenetic affiliation indicated that the majority of the sequences obtained were affiliated with Actinobacteria, Cyanobacteria, Proteobacteria, Acidobacteria, Bacteroidetes, of which Cyanobacteria was dominant, followed the Proteobacteria. Members of the genus Nostoc were the most abundant in all samples. Rhizobiales and Actinobacteria were identified, whereas, Craurococcus, Caulobacter, Pseudomonas, Terriglobus and Mucilaginibacter were also identified at low levels. Through comparing the bacterial composition of N. flagelliforme from different regions, it was revealed that N. flagelliforme could facilitate the growth of other microorganisms including both autotrophic bacteria and heterotrophic ones and positively contributed to their harsh ecosystems. The results indicated N. flagelliforme played an important role in diversifying the microbial community composition and had potential application in soil desertification.

Plant-fed versus chemicals-fed rhizobacteria of Lucerne: Plant-only teabags culture media not only increase culturability of rhizobacteria but also recover a previously uncultured Lysobacter sp., Novosphingobium sp. and Pedobacter sp.

PubMed

Hegazi, Nabil A; Sarhan, Mohamed S; Fayez, Mohamed; Patz, Sascha; Murphy, Brian R; Ruppel, Silke

2017-01-01

In an effort to axenically culture the previously uncultivable populations of the rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags culture media to mimic the nutritional matrix available in the rhizosphere. Here, we show that culture media prepared from Lucerne powder teabags substantially increased the cultivability of Lucerne rhizobacteria compared with a standard nutrient agar, where we found that the cultivable populations significantly increased by up to 60% of the total bacterial numbers as estimated by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of 16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct composition and separation of bacterial populations recovered on the plant-only teabags culture media than those developed on a standard nutrient agar. Further, the new plant medium gave preference to the micro-symbiont Sinorhizobium meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The present study may encourage other researchers to consider moving from the well-established standard culture media to the challenging new plant-only culture media. Such a move may reveal previously hidden members of rhizobacteria, and help to further explore their potential environmental impacts.

PCR-DGGE Analysis on Microbial Community Structure of Rural Household Biogas Digesters in Qinghai Plateau.

PubMed

Han, Rui; Yuan, Yongze; Cao, Qianwen; Li, Quanhui; Chen, Laisheng; Zhu, Derui; Liu, Deli

2018-05-01

To investigate contribution of environmental factor(s) to microbial community structure(s) involved in rural household biogas fermentation at Qinghai Plateau, we collected slurry samples from 15 digesters, with low-temperature working conditions (11.1-15.7 °C) and evenly distributed at three counties (Datong, Huangyuan, and Ledu) with cold plateau climate, to perform polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and further sequencing. The bacterial communities in the total 15 digesters were classified into 38 genera with Mangroviflexus (12.1%) as the first dominant, and the archaeal communities into ten genera with Methanogenium (38.5%) as the most dominant. For each county, the digesters with higher biogas production, designated as HP digesters, exclusively had 1.6-3.1 °C higher fermentation temperature and the unique bacterial structure composition related, i.e., unclassified Clostridiales for all the HP digesters and unclassified Marinilabiliaceae and Proteiniclasticum for Ledu HP digesters. Regarding archaeal structure composition, Methanogenium exhibited significantly higher abundances at all the HP digesters and Thermogymnomonas was the unique species only identified at Ledu HP digesters with higher-temperature conditions. Redundancy analysis also confirmed the most important contribution of temperature to the microbial community structures investigated. This report emphasized the correlation between temperature and specific microbial community structure(s) that would benefit biogas production of rural household digesters at Qinghai Plateau.

Diversity of ndo Genes in Mangrove Sediments Exposed to Different Sources of Polycyclic Aromatic Hydrocarbon Pollution▿

PubMed Central

Gomes, Newton C. Marcial; Borges, Ludmila R.; Paranhos, Rodolfo; Pinto, Fernando N.; Krögerrecklenfort, Ellen; Mendonça-Hagler, Leda C. S.; Smalla, Kornelia

2007-01-01

Polycyclic aromatic hydrocarbon (PAH) pollutants originating from oil spills and wood and fuel combustion are pollutants which are among the major threats to mangrove ecosystems. In this study, the composition and relative abundance in the sediment bacterial communities of naphthalene dioxygenase (ndo) genes which are important for bacterial adaptation to environmental PAH contamination were investigated. Three urban mangrove sites which had characteristic compositions and levels of PAH compounds in the sediments were selected. The diversity and relative abundance of ndo genes in total community DNA were assessed by a newly developed ndo denaturing gradient gel electrophoresis (DGGE) approach and by PCR amplification with primers targeting ndo genes with subsequent Southern blot hybridization analyses. Bacterial populations inhabiting sediments of urban mangroves under the impact of different sources of PAH contamination harbor distinct ndo genotypes. Sequencing of cloned ndo amplicons comigrating with dominant DGGE bands revealed new ndo genotypes. PCR-Southern blot analysis and ndo DGGE showed that the frequently studied nah and phn genotypes were not detected as dominant ndo types in the mangrove sediments. However, ndo genotypes related to nagAc-like genes were detected, but only in oil-contaminated mangrove sediments. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the abundance and diversity of ndo genes in sediments of urban mangroves. PMID:17905873

Diversity and dynamics of the Vibrio community in well water used for drinking in Guinea-Bissau (West Africa).

PubMed

Machado, A; Bordalo, A A

2014-09-01

Bacteria of the genus Vibrio are ubiquitous in aquatic environments and can be found either in culturable or in a viable but nonculturable (VBNC) state. The genus comprises many pathogenic species accountable for water and food-borne diseases that prove to be fatal, especially in developing countries, as in Guinea-Bissau (West Africa), where cholera is endemic. In order to ascertain the abundance and structure of Vibrio spp. community in well waters that serve as the sole source of water for the population, quantitative polymerase chain reaction (qPCR), PCR-denaturant gradient gel electrophoresis (DGGE), and cloning approaches were used. Results suggest that Vibrio spp. were present throughout the year in acidic, freshwater wells with a seasonal community composition shift. Vibrio spp. abundance was in accordance with the abundance found in coastal environments. Sequences closely related to pathogenic Vibrio species were retrieved from well water revealing exposure of the population to such pathogens. pH, ammonium, and turbidity, regulated by the rain pattern, seem to be the variables that contributed mostly to the shaping and selection of the Vibrio spp. community. These results reinforce the evidence for water monitoring with culture-independent methods and the clear need to create/recover water infrastructures and a proper water resources management in West African countries with similar environmental conditions.

Incidence of Staphylococcus aureus and Analysis of Associated Bacterial Communities on Food Industry Surfaces

PubMed Central

Gutiérrez, Diana; Delgado, Susana; Vázquez-Sánchez, Daniel; Martínez, Beatriz; Cabo, Marta López; Rodríguez, Ana; Herrera, Juan J.

2012-01-01

Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. A study designed to determine the presence of S. aureus on food contact surfaces in dairy, meat, and seafood environments and to identify coexisting microbiota has therefore been carried out. A total of 442 samples were collected, and the presence of S. aureus was confirmed in 6.1% of samples. Sixty-three S. aureus isolates were recovered and typed by random amplification of polymorphic DNA (RAPD). Profiles were clustered into four groups which were related to specific food environments. All isolates harbored some potential virulence factors such as enterotoxin production genes, biofilm formation-associated genes, antibiotic resistance, or lysogeny. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of bacterial communities coexisting with S. aureus revealed the presence of bacteria either involved in food spoilage or of concern for food safety in all food environments. Food industry surfaces could thus be a reservoir for S. aureus forming complex communities with undesirable bacteria in multispecies biofilms. Uneven microbiological conditions were found in each food sector, which indicates the need to improve hygienic conditions in food processing facilities, particularly the removal of bacterial biofilms, to enhance the safety of food products. PMID:23023749

Biodiversity of thermophilic prokaryotes with hydrolytic activities in hot springs of Uzon Caldera, Kamchatka (Russia).

PubMed

Kublanov, Ilya V; Perevalova, Anna A; Slobodkina, Galina B; Lebedinsky, Aleksander V; Bidzhieva, Salima K; Kolganova, Tatyana V; Kaliberda, Elena N; Rumsh, Lev D; Haertlé, Thomas; Bonch-Osmolovskaya, Elizaveta A

2009-01-01

Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87 degrees C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or alpha- or beta-keratin) or carbohydrate (cellulose, carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several enrichment cultures growing in situ on different polymeric substrates were obtained. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments obtained after PCR with Bacteria-specific primers showed that the bacterial communities developing on carbohydrates included the genera Caldicellulosiruptor and Dictyoglomus and that those developing on proteins contained members of the Thermotogales order. DGGE analysis performed after PCR with Archaea- and Crenarchaeota-specific primers showed that archaea related to uncultured environmental clones, particularly those of the Crenarchaeota phylum, were present in both carbohydrate- and protein-degrading communities. Five isolates obtained from in situ enrichments or corresponding natural samples of water and sediments represented the bacterial genera Dictyoglomus and Caldanaerobacter as well as new archaea of the Crenarchaeota phylum. Thus, in situ enrichment and consequent isolation showed the diversity of thermophilic prokaryotes competing for biopolymers in microbial communities of terrestrial hot springs.

Droplet-based micro oscillating-flow PCR chip

NASA Astrophysics Data System (ADS)

Wang, Wei; Li, Zhi-Xin; Luo, Rong; Lü, Shu-Hai; Xu, Ai-Dong; Yang, Yong-Jun

2005-08-01

Polymerase chain reactions (PCR), thermally activated chemical reactions which are widely used for nucleic acid amplification, have recently received much attention in microelectromechanical systems and micro total analysis systems because a wide variety of DNA/RNA molecules can be enriched by PCR for further analyses. In the present work, a droplet-based micro oscillating-flow PCR chip was designed and fabricated by the silicon microfabrication technique. Three different temperature zones, which were stable at denaturation, extension and annealing temperatures and isolated from each other by a thin-wall linkage, were integrated with a single, simple and straight microchannel to form the chip's basic functional structure. The PCR mixture was injected into the chip as a single droplet and flowed through the three temperature zones in the main microchannel in an oscillating manner to achieve the temperature maintenance and transitions. The chip's thermal performance was theoretically analyzed and numerically simulated. The results indicated that the time needed for the temperature of the droplet to change to the target value is less than 1 s, and the root mean square error of temperature is less than 0.2 °C. A droplet of 1 µl PCR mixture with standard HPV (Human Papilloma Virus)-DNA sample inside was amplified by the present chip and the results were analyzed by slab gel electrophoresis with separation of DNA markers in parallel. The electrophoresis results demonstrated that the micro oscillating-flow PCR chip successfully amplified the HPV-DNA, with a processing time of about 15 min which is significantly reduced compared to that for the conventional PCR instrument.

Intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding.

PubMed

Hutchinson, Matthew H; Chase, Howard A

2006-01-01

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

Selection of Sphingomonadaceae at the base of Laccaria proxima and Russula exalbicans fruiting bodies.

PubMed

Boersma, F G Hidde; Warmink, Jan A; Andreote, Fernando A; van Elsas, Jan Dirk

2009-04-01

The dense hyphal network directly underneath the fruiting bodies of ectomycorrhizal fungi might exert strong influences on the bacterial community of soil. Such fruiting bodies might serve as hot spots for bacterial activity, for instance by providing nutrients and colonization sites in soil. Here, we assessed the putative selection of specific members of the Sphingomonadaceae family at the bases of the fruiting bodies of the ectomycorrhizal fungi Laccaria proxima and Russula exalbicans in comparison to the adjacent bulk soil. To do so, we used a previously designed Sphingomonadaceae-specific PCR-denaturing gradient gel electrophoresis (DGGE) system and complemented this with analyses of sequences from a Sphingomonadaceae-specific clone library. The analyses showed clear selective effects of the fruiting bodies of both fungi on the Sphingomonadaceae community structures. The effect was especially prevalent with R. exalbicans. Strikingly, similar fungi sampled approximately 100 m apart showed similar DGGE patterns, while corresponding bulk soil-derived patterns differed from each other. However, the mycospheres of L. proxima and R. exalbicans still revealed divergent community structures, indicating that different fungi select for different members of the Sphingomonadaceae family. Excision of specific bands from the DGGE patterns, as well as analyses of the clone libraries generated from both habitats, revealed fruiting body-specific Sphingomonadaceae types. It further showed that major groups from the mycospheres of R. exalbicans and L. proxima did not cluster with known bacteria from the database, indicating new groups within the family of Sphingomonadaceae present in these environments.

Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning.

PubMed

Kraková, Lucia; Šoltys, Katarína; Budiš, Jaroslav; Grivalský, Tomáš; Ďuriš, František; Pangallo, Domenico; Szemes, Tomáš

2016-09-01

Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.

Significant impact of amount of PCR input templates on various PCR-based DNA methylation analysis and countermeasure.

PubMed

Liu, Zhaojun; Zhou, Jing; Gu, Liankun; Deng, Dajun

2016-08-30

Methylation changes of CpG islands can be determined using PCR-based assays. However, the exact impact of the amount of input templates (TAIT) on DNA methylation analysis has not been previously recognized. Using COL2A1 gene as an input reference, TAIT difference between human tissues with methylation-positive and -negative detection was calculated for two representative genes GFRA1 and P16. Results revealed that TAIT in GFRA1 methylation-positive frozen samples (n = 332) was significantly higher than the methylation-negative ones (n = 44) (P < 0.001). Similar difference was found in P16 methylation analysis. The TAIT-related effect was also observed in methylation-specific PCR (MSP) and denatured high performance liquid chromatography (DHPLC) analysis. Further study showed that the minimum TAIT for a successful MethyLight PCR reaction should be ≥ 9.4 ng (CtCOL2A1 ≤ 29.3), when the cutoff value of the methylated-GFRA1 proportion for methylation-positive detection was set at 1.6%. After TAIT of the methylation non-informative frozen samples (n = 94; CtCOL2A1 > 29.3) was increased above the minimum TAIT, the methylation-positive rate increased from 72.3% to 95.7% for GFRA1 and 26.6% to 54.3% for P16, respectively (Ps < 0.001). Similar results were observed in the FFPE samples. In conclusion, TAIT critically affects results of various PCR-based DNA methylation analyses. Characterization of the minimum TAIT for target CpG islands is essential to avoid false-negative results.

Analysis of DGGE profiles to explore the relationship between prokaryotic community composition and biogeochemical processes in deep subseafloor sediments from the Peru Margin.

PubMed

Fry, John C; Webster, Gordon; Cragg, Barry A; Weightman, Andrew J; Parkes, R John

2006-10-01

The aim of this work was to relate depth profiles of prokaryotic community composition with geochemical processes in the deep subseafloor biosphere at two shallow-water sites on the Peru Margin in the Pacific Ocean (ODP Leg 201, sites 1228 and 1229). Principal component analysis of denaturing gradient gel electrophoresis banding patterns of deep-sediment Bacteria, Archaea, Euryarchaeota and the novel candidate division JS1, followed by multiple regression, showed strong relationships with prokaryotic activity and geochemistry (R(2)=55-100%). Further correlation analysis, at one site, between the principal components from the community composition profiles for Bacteria and 12 other variables quantitatively confirmed their relationship with activity and geochemistry, which had previously only been implied. Comparison with previously published cell counts enumerated by fluorescent in situ hybridization with rRNA-targeted probes confirmed that these denaturing gradient gel electrophoresis profiles described an active prokaryotic community.

Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis

PubMed Central

Ferris, Michael J; Masztal, Alicia; Aldridge, Kenneth E; Fortenberry, J Dennis; Fidel, Paul L; Martin, David H

2004-01-01

Background Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a change in vaginal flora away from predominantly Lactobacillus species. The cause of BV is unknown, but the condition has been implicated in diverse medical outcomes. The bacterium Atopobium vaginae has been recognized only recently. It is not readily identified by commercial diagnostic kits. Its clinical significance is unknown but it has recently been isolated from a tuboovarian abcess. Methods Nucleotide sequencing of PCR amplified 16S rRNA gene segments, that were separated into bands within lanes on polyacrylamide gels by denaturing gradient gel electrophoresis (DGGE), was used to examine bacterial vaginal flora in 46 patients clinically described as having normal (Lactobacillus spp. predominant; Nugent score ≤ 3) and abnormal flora (Nugent score ≥ 4). These women ranged in age from 14 to 48 and 82% were African American. Results The DGGE banding patterns of normal and BV-positive patients were recognizably distinct. Those of normal patients contained 1 to 4 bands that were focused in the centre region of the gel lane, while those of BV positive patients contained bands that were not all focused in the center region of the gel lane. More detailed analysis of patterns revealed that bands identified as Atopobium vaginae were present in a majority (12/22) of BV positive patients, while corresponding bands were rare (2/24) in normal patients. (P < 0.001) Two A. vaginae isolates were cultivated from two patients whose DGGE analyses indicated the presence of this organism. Two A. vaginae 16S rRNA gene sequences were identified among the clinical isolates. The same two sequences were obtained from DGGE bands of the corresponding vaginal flora. The sequences differed by one nucleotide over the short (~300 bp) segment used for DGGE analysis and migrated to slightly different points in denaturing gradient gels. Both isolates were strict anaerobes and highly metronidazole resistant. Conclusion The results suggest that A. vaginae may be an important component of the complex bacterial ecology that constitutes abnormal vaginal flora. This organism could play a role in treatment failure if further studies confirm it is consistently metronidozole resistant. PMID:15018635

Structure of nitrogen-converting communities induced by hydraulic retention time and COD/N ratio in constantly aerated granular sludge reactors treating digester supernatant.

PubMed

Cydzik-Kwiatkowska, Agnieszka; Rusanowska, Paulina; Zielińska, Magdalena; Bernat, Katarzyna; Wojnowska-Baryła, Irena

2014-02-01

This study investigated how hydraulic retention time (HRT) and COD/N ratio affect nitrogen-converting consortia in constantly aerated granules treating high-ammonium digester supernatant. Three HRTs (10, 13, 19 h) were tested at COD/N ratios of 4.5 and 2.3. Denaturing gradient gel electrophoresis and relative real-time PCR were used to characterize the microbial communities. When changes in HRT and COD/N increased nitrogen loading, the ratio of the relative abundance of aerobic to anaerobic ammonium-oxidizers decreased. The COD/N ratio determined the species composition of the denitrifiers; however, Thiobacillus denitrificans, Pseudomonas denitrificans and Azoarcus sp. showed a high tolerance to the environmental conditions and occurred in the granules from all reactors. Denitrifier genera that support granule formation were identified, such as Pseudomonas, Shinella, and Flavobacterium. In aerated granules, nirK-possessing bacteria were more diverse than nirS-possessing bacteria. At a low COD/N ratio, N2O-reducer diversity increased because of the presence of bacteria known as aerobic denitrifiers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of copper on the abundance and diversity of ammonia oxidizers during dairy cattle manure composting.

PubMed

Yin, Yanan; Song, Wen; Gu, Jie; Zhang, Kaiyu; Qian, Xun; Zhang, Xin; Zhang, Yajun; Li, Yang; Wang, Xiaojuan

2016-12-01

This study investigated the effects of adding Cu(II) at two exposure levels (50 and 500mgkg -1 , i.e., Cu50 and Cu500 treatments, respectively) on the activity of ammonia-oxidizing microorganisms during dairy cattle manure composting. The results showed that the pH, NH 4 + -N, NO 3 - -N, and potential ammonia oxidation values were inhibited significantly by the addition of Cu(II). Furthermore, the abundances of the ammonia-oxidizing archaea (AOA) amoA gene and ammonia-oxidizing bacteria (AOB) amoA gene were determined by quantitative PCR, and their compositions were evaluated by denaturing gradient gel electrophoresis (DGGE). AOA was the dominant ammonia oxidizing microorganism, of which the abundance was much higher than AOB during composting. Cu50 and Cu500 had significant inhibitory effects on the abundance of the amoA gene. The DGGE profile and statistical analysis showed that Cu(II) changed the AOA and AOB community structure and diversity, where Nitrosomonas and Crenarchaeota dominated throughout the composting process. Copyright © 2016. Published by Elsevier Ltd.

Triclosan enriches for Dehalococcoides-like Chloroflexi in anaerobic soil at environmentally relevant concentrations.

PubMed

McNamara, Patrick J; Krzmarzick, Mark J

2013-07-01

Triclosan is an antimicrobial agent that is discharged to soils with land-applied wastewater biosolids, is persistent under anaerobic conditions, and yet its impact on anaerobic microbial communities in soils is largely unknown. We hypothesized that triclosan enriches for Dehalococcoides-like Chloroflexi because these bacteria respire organochlorides and are likely less sensitive, relative to other bacteria, to the antimicrobial effects of triclosan. Triplicate anaerobic soil microcosms were seeded with agricultural soil, which was not previously exposed to triclosan, and were amended with 1 mg kg(-1) of triclosan. Triplicate control microcosms did not receive triclosan, and the experiment was run for 618 days. The overall bacterial community (assessed by automated ribosomal intergenic spacer analysis and denaturing gradient gel electrophoresis) was not impacted by triclosan; however, the abundance of Dehalococcoides-like Chloroflexi 16S rRNA genes (determined by qPCR) increased 20-fold with triclosan amendment compared with a fivefold increase without triclosan. This work demonstrates that triclosan impacts anaerobic soil communities at environmentally relevant levels. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Microbiological study on bioremediation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) contaminated soil by agricultural waste composting.

PubMed

Chen, Yaoning; Ma, Shuang; Li, Yuanping; Yan, Ming; Zeng, Guangming; Zhang, Jiachao; Zhang, Jie; Tan, Xuebin

2016-11-01

This paper studied the degradation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in contaminated soil under composting and natural conditions, respectively. BDE-47 residue in agricultural waste-composting pile was determined during 45-day composting. The microbial communities were determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), and the relationships between the DGGE results and physico-chemical parameters were evaluated by redundancy analysis (RDA) and heatmap-clustering analysis. The results showed that the degradation rate of BDE-47 was significantly higher in agricultural waste-composting pile compared with control group, which was enhanced up to almost 15 % at the end of composting. There were different environmental factors which affected the distribution of composting bacterial and fungal communities. The bacterial community composition was more significantly affected by the addition of BDE-47 compared with other physico-chemical parameters, and BDE-47 had stronger influences on bacterial community than fungal community during the composting. Meanwhile, the most variation in distribution of fungal community was explained by pile temperature.

Arbuscular mycorrhizal fungal communities in the rhizosphere of a continuous cropping soybean system at the seedling stage.

PubMed

Cui, Jiaqi; Bai, Li; Liu, Xiaorui; Jie, Weiguang; Cai, Baiyan

Arbuscular mycorrhizae (AM) fungi play a crucial role in the growth of soybean; however, the planting system employed is thought to have an effect on AM fungal communities in the rhizosphere. This study was performed to explore the influence of continuous soybean cropping on the diversity of Arbuscular mycorrhizal (AM) fungi, and to identify the dominant AM fungus during the seedling stage. Three soybean cultivars were planted under two and three years continuous cropping, respectively. The diversity of AM fungi in the rhizosphere soil at the seedling stage was subsequently analyzed using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). The results showed that an increase in cropping years improved the colonization rate of AM in all three soybean cultivars. Moreover, the dominant species were found to be Funneliformis mosseae and Glomus species. The results of cluster analysis further confirmed that the number of years of continuous cropping significantly affected the composition of rhizospheric AM fungal communities in different soybean cultivars. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Supplemental feeding of a gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, alleviates spontaneous atopic dermatitis and modulates intestinal microbiota in NC/nga mice.

PubMed

Kaikiri, Hiroko; Miyamoto, Junki; Kawakami, Takahiro; Park, Si-Bum; Kitamura, Nahoko; Kishino, Shigenobu; Yonejima, Yasunori; Hisa, Keiko; Watanabe, Jun; Ogita, Tasuku; Ogawa, Jun; Tanabe, Soichi; Suzuki, Takuya

2017-12-01

The present study investigated the antiallergic and anti-inflammatory effects of 10-hydroxy-cis-12-octadecenoic acid (HYA), a novel gut microbial metabolite of linoleic acid, in NC/Nga mice, a model of atopic dermatitis (AD). Feeding HYA decreased the plasma immunoglobulin E level and skin infiltration of mast cells with a concomitant decrease in dermatitis score. HYA feeding decreased TNF-α and increased claudin-1, a tight junction protein, levels in the mouse skin. Cytokine expression levels in the skin and intestinal Peyer's patches cells suggested that HYA improved the Th1/Th2 balance in mice. Immunoglobulin A concentration in the feces of the HYA-fed mice was approximately four times higher than that in the control mice. Finally, denaturing gradient gel electrophoresis of the PCR-amplified 16 S rRNA gene of fecal microbes indicated the modification of microbiota by HYA. Taken together, the alterations in the intestinal microbiota might be, at least in part, associated with the antiallergic effect of HYA.

Inhibitory effects of soybean oligosaccharides and water-soluble soybean fibre on formation of putrefactive compounds from soy protein by gut microbiota.

PubMed

Nakata, Toru; Kyoui, Daisuke; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi

2017-04-01

Soybeans are part of the traditional food consumed in Asia countries. In this study, we investigated inhibitory effects of soybean oligosaccharides and water-soluble soybean fibre (Soyafibe) on putrefactive compounds from soy protein by gut microbiota in rats. Caecal microbial fermentation products and microbiota in rats fed 20% soy protein (SP-1) and whole soybean flour (SFL: protein content was 20%) diets were determined. The caecal environment in rats fed 20% soy protein without dietary fibre (SP-2) or with 2% Soyafibe (SFB) was also determined. Compared to SP-1 and SP-2 group, low indole content with high lactic acid was shown in SFL and SFB group, respectively. Using the 16S rRNA genes polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. Prevotella, Gram-negative anaerobic rods, were detected as dominant in both SFL and SFB groups. Our findings indicated that fermentable polysaccharides in soybeans have inhibitory effects on the formation of putrefactive compounds generated from soy protein by the microbiota. Copyright © 2017 Elsevier B.V. All rights reserved.

Establishment of intestinal microbiota with focus on yeasts of unweaned and weaned piglets kept under different farm conditions.

PubMed

Urubschurov, Vladimir; Janczyk, Pawel; Souffrant, Wolfgang-Bernhard; Freyer, Gertraude; Zeyner, Annette

2011-09-01

This study aimed to characterize the intestinal yeasts in weaning piglets and to establish their possible relationships with main bacterial groups. German Landrace piglets were weaned (WP, n=32) at 28 days of age or kept with the dams until day 39 without creep feed (UP, n=32). The experiment was performed at an experimental and a commercial farm (CF). Faeces were collected from the piglets, sows and pen floors on days 28, 33 and 39 for isolation of DNA and cultivation for enumeration of yeasts, enterobacteria, enterococci and lactobacilli. Fragments of the D1 domain of 26S rRNA gene were amplified and separated by denaturing gradient gel electrophoresis (DGGE). No yeasts could be cultured from water and feed samples. No or only low numbers of yeasts were detected among all UP. In WP at CF, yeasts correlated with lactobacilli (r=0.456; P=0.009) and enterobacteria (r=-0.407; P=0.021). Kazachstania slooffiae dominated among the cultured yeasts. It was the only yeast species detected by PCR-DGGE. Yeasts, especially K. slooffiae, established in the porcine gastrointestinal tract after consumption of grain-based feed and may interrelate with the intestinal microbiota. The study provides data indicating importance of K. slooffiae for the development of balanced porcine gut microbiota. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Dynamics of Bacterial Communities in Two Unpolluted Soils after Spiking with Phenanthrene: Soil Type Specific and Common Responders

PubMed Central

Ding, Guo-Chun; Heuer, Holger; Smalla, Kornelia

2012-01-01

Considering their key role for ecosystem processes, it is important to understand the response of microbial communities in unpolluted soils to pollution with polycyclic aromatic hydrocarbons (PAH). Phenanthrene, a model compound for PAH, was spiked to a Cambisol and a Luvisol soil. Total community DNA from phenanthrene-spiked and control soils collected on days 0, 21, and 63 were analyzed based on PCR-amplified 16S rRNA gene fragments. Denaturing gradient gel electrophoresis (DGGE) fingerprints of bacterial communities increasingly deviated with time between spiked and control soils. In taxon specific DGGE, significant responses of Alphaproteobacteria and Actinobacteria became only detectable after 63 days, while significant effects on Betaproteobacteria were detectable in both soils after 21 days. Comparison of the taxonomic distribution of bacteria in spiked and control soils on day 63 as revealed by pyrosequencing indicated soil type specific negative effects of phenanthrene on several taxa, many of them belonging to the Gamma-, Beta-, or Deltaproteobacteria. Bacterial richness and evenness decreased in spiked soils. Despite the significant differences in the bacterial community structure between both soils on day 0, similar genera increased in relative abundance after PAH spiking, especially Sphingomonas and Polaromonas. However, this did not result in an increased overall similarity of the bacterial communities in both soils. PMID:22934091

The effect of a non-denaturing detergent and a guanidinium-based inactivation agent on the viability of Ebola virus in mock clinical serum samples.

PubMed

Burton, J E; Easterbrook, L; Pitman, J; Anderson, D; Roddy, S; Bailey, D; Vipond, R; Bruce, C B; Roberts, A D

2017-12-01

The 2014 Ebola outbreak in West Africa required the rapid testing of clinical material for the presence of potentially high titre Ebola virus (EBOV). Safe, fast and effective methods for the inactivation of such clinical samples are required so that rapid diagnostic tests including downstream analysis by RT-qPCR or nucleotide sequencing can be carried out. One of the most commonly used guanidinium - based denaturing agents, AVL (Qiagen) has been shown to fully inactivate EBOV once ethanol is added, however this is not compatible with the use of automated nucleic acid extraction systems. Additional inactivation agents need to be identified that can be used in automated systems. A candidate inactivation agent is Triton X-100, a non-denaturing detergent that is frequently used in clinical nucleic acid extraction procedures and has previously been used for inactivation of EBOV. In this study the effect of 0.1% and 1.0% Triton X-100 (final concentration 0.08% and 0.8% respectively) alone and in combination with AVL on the viability of EBOV (10 6 TCID 50 /ml) spiked into commercially available pooled negative human serum was tested. The presence of viable EBOV in the treated samples was assessed by carrying out three serial passages of the samples in Vero E6 cells (37°C, 5% CO 2 , 1 week for each passage). At the end of each passage the cells were observed for evidence of cytopathic effect and samples were taken for rRT-PCR analysis for the presence of EBOV RNA. Before cell culture cytotoxic components of AVL and Triton X-100 were removed from the samples using size exclusion spin column technology or a hydrophobic adsorbent resin. The results of this study showed that EBOV spiked into human serum was not fully inactivated when treated with either 0.1% (v/v) Triton X-100 for 10 mins or 1.0% (v/v) Triton X-100 for 20 mins (final concentrations 0.08% and 0.8% Triton X-100 respectively). AVL alone also did not consistently provide complete inactivation. Samples treated with both AVL and 0.1% Triton X-100 for 10 or 20 mins were shown to be completely inactivated. This treatment is compatible with downstream analysis by RT-qPCR and next generation sequencing. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Effects of Agronomic Treatments on Structure and Function of Ammonia-Oxidizing Communities

PubMed Central

Phillips, Carol J.; Harris, Dave; Dollhopf, Sherry L.; Gross, Katherine L.; Prosser, James I.; Paul, Eldor A.

2000-01-01

The aim of this study was to determine the effects of different agricultural treatments and plant communities on the diversity of ammonia oxidizer populations in soil. Denaturing gradient gel electrophoresis (DGGE), coupled with specific oligonucleotide probing, was used to analyze 16S rRNA genes of ammonia oxidizers belonging to the β subgroup of the division Proteobacteria by use of DNA extracted from cultivated, successional, and native deciduous forest soils. Community profiles of the different soil types were compared with nitrification rates and most-probable-number (MPN) counts. Despite significant variation in measured nitrification rates among communities, there were no differences in the DGGE banding profiles of DNAs extracted from these soils. DGGE profiles of DNA extracted from samples of MPN incubations, cultivated at a range of ammonia concentrations, showed the presence of bands not amplified from directly extracted DNA. Nitrosomonas-like bands were seen in the MPN DNA but were not detected in the DNA extracted directly from soils. These bands were detected in some samples taken from MPN incubations carried out with medium containing 1,000 μg of NH4+-N ml−1, to the exclusion of bands detected in the native DNA. Cell concentrations of ammonia oxidizers determined by MPN counts were between 10- and 100-fold lower than those determined by competitive PCR (cPCR). Although no differences were seen in ammonia oxidizer MPN counts from the different soil treatments, cPCR revealed higher numbers in fertilized soils. The use of a combination of traditional and molecular methods to investigate the activities and compositions of ammonia oxidizers in soil demonstrates differences in fine-scale compositions among treatments that may be associated with changes in population size and function. PMID:11097922

Histamine development and bacterial diversity in microbially-challenged tonggol (Thunnus tonggol) under temperature abuse during canning manufacture.

PubMed

Hongpattarakere, Tipparat; Buntin, Nirunya; Nuylert, Aem

2016-01-01

Histamine formation and bacteriological changes caused by temperature abuse commonly occurring in the manufacturing process of standard canned tuna was assessed in microbiologically challenged tonggol (Thunnus tonggol). The in situ challenge was performed by water-soaking at 26-28 °C for 7 h to ensure the multiplication and active phase of fish microflora. Right after pre-cooking to back-bone temperature (BBT) of 50-52 °C, histamine dropped to 5.17 ± 2.71 ppm, and slowly reached 6.84 ± 1.69 ppm at 16 h abuse. On the contrary, histamine was reduced to 2.87 ± 1.23 ppm and eventually reached 5.01 ± 1.32 ppm at 24 h abuse in the pre-cooked fish previously frozen. The numbers of total aerobic bacteria, Enterobactericeae, psychrotroph, histamine forming bacteria (HFB) and diversity of fish microflora were revealed by cultural and nested PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) techniques. Interestingly, frozen storage effectively halted histamine formation in raw fish throughout 16 h abuse despite the presence of HFB. These included the prolific strains of Morganella morganii, Proteus penneri, Proteus mirabilin, Citrobacter spp. The nested PCR-DGGE profile confirmed the presence of M. morganii and Citrobacter spp. in raw fish. These prolific strains were hardly observed in the precooked fish previously frozen. Frozen storage did not only promote even histamine distribution throughout fish muscle but also enhanced histamine loss during thawing and pre-cooking. Therefore, pre-cooking and frozen storage were proven to be the effective combined hurdles not only to reduce but also prolong histamine formation of the challenged toggol throughout 24 h of temperature abuse during canning process.

Analysis of Inter-Individual Bacterial Variation in Gut of Cicada Meimuna mongolica (Hemiptera: Cicadidae).

PubMed

Zhou, Wenting; Nan, Xiaoning; Zheng, Zhou; Wei, Cong; He, Hong

2015-01-01

Intestinal bacterial community plays a crucial role in the nutrition, development, survival, and reproduction of insects. When compared with other insects with piercing-sucking mouthparts, the habitats of cicada nymphs and adults are totally different. However, little is known about the differences in the gut bacterial communities in the nymphs and adults within any cicada species. The diversity of bacteria in the gut of nymphs and adults of both genders of Meimuna mongolica (Distant) was studied using the denaturing gradient gel electrophoresis (DGGE) method. Few inter-individual variations among gut microbiota were observed, suggesting that M. mongolica typically harbors a limited and consistent suite of bacterial species. Bacteria in the genera Pseudomonas and Enterobacter were the predominant components of the gut microflora of M. mongolica at all life stages. Bacteria of Pantoea, Streptococcus, and Uruburuella were also widespread in the cicada samples but at relatively lower concentrations. The relative stability and similarity of the PCR-DGGE patterns indicate that all individuals of this cicada species harbor a characteristic bacterial community which is independent from developmental stages and genders. Related endosymbionts that could be harbored in bacteromes of cicadas were not detected in any gut samples, which could be related to the cicada species and the distribution of these endosymbionts in the cicada cavity, or due to some of the possible limitations of PCR-DGGE community profiling. It is worthwhile to further address if related cicada endosymbiont clades distribute in the alimentary canals and other internal organs through diagnostic PCR using group-specific primer sets. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Changes in Bacterial Diversity Associated with Epithelial Tissue in the Beef Cow Rumen during the Transition to a High-Grain Diet ▿

PubMed Central

Chen, Yanhong; Penner, Gregory B.; Li, Meiju; Oba, Masahito; Guan, Le Luo

2011-01-01

Our understanding of the ruminal epithelial tissue-associated bacterial (defined as epimural bacteria in this study) community is limited. In this study, we aimed to determine whether diet influences the diversity of the epimural bacterial community in the bovine rumen. Twenty-four beef heifers were randomly assigned to either a rapid grain adaptation (RGA) treatment (n = 18) in which the heifers were allowed to adapt from a diet containing 97% hay to a diet containing 8% hay over 29 days or to the control group (n = 6), which was fed 97% hay. Rumen papillae were collected when the heifers were fed 97%, 25%, and 8% hay diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis were used to characterize rumen epimural bacterial diversity and to estimate the total epimural bacterial population (copy numbers of the 16S rRNA gene). The epimural bacterial diversity from RGA heifers changed (P = 0.01) in response to the rapid dietary transition, whereas it was not affected in control heifers. A total of 88 PCR-DGGE bands were detected, and 44 were identified from phyla including Firmicutes, Bacteroidetes, and Proteobacteria. The bacteria Treponema sp., Ruminobacter sp., and Lachnospiraceae sp. were detected only when heifers were fed 25% and 8% hay diets, suggesting the presence of these bacteria is the result of adaptation to the high-grain diets. In addition, the total estimated population of rumen epimural bacteria was positively correlated with molar proportions of acetate, isobutyrate, and isovalerate, suggesting that they may play a role in volatile fatty acid metabolism in the rumen. PMID:21705529

Novel Firmicutes Group Implicated in the Dechlorination of Two Chlorinated Xanthones, Analogues of Natural Organochlorines

PubMed Central

Krzmarzick, Mark J.; Miller, Hanna R.; Yan, Tao

2014-01-01

Although the abundance and diversity of natural organochlorines are well established, much is still unknown about the degradation of these compounds. Triplicate microcosms were used to determine whether, and which, bacterial communities could dechlorinate two chlorinated xanthones (2,7-dichloroxanthone and 5,7-dichloro-1,3-dihydroxylxanthone), analogues of a diverse class of natural organochlorines. According to quantitative-PCR (qPCR) results, several known dechlorinating genera were either not present or not enriched during dechlorination of the xanthones. Denaturing gradient gel electrophoresis, however, indicated that several Firmicutes were enriched in the dechlorinating cultures compared to triplicate controls amended with nonchlorinated xanthones. One such group, herein referred to as the Gopher group, was further studied with a novel qPCR method that confirmed enrichment of Gopher group 16S rRNA genes in the dechlorinating cultures. The enrichment of the Gopher group was again tested with two new sets of triplicate microcosms. Enrichment was observed during chlorinated xanthone dechlorination in one set of these triplicate microcosms. In the other set, two microcosms showed clear enrichment while a third did not. The Gopher group is a previously unidentified group of Firmicutes, distinct from but related to the Dehalobacter and Desulfitobacterium genera; this group also contains clones from at least four unique cultures capable of dechlorinating anthropogenic organochlorines that have been previously described in the literature. This study suggests that natural chlorinated xanthones may be effective biostimulants to enhance the remediation of pollutants and highlights the idea that novel genera of dechlorinators likely exist and may be active in bioremediation and the natural cycling of chlorine. PMID:24296507

Variations in the patterns of soil organic carbon mineralization and microbial communities in response to exogenous application of rice straw and calcium carbonate.

PubMed

Feng, Shuzhen; Huang, Yuan; Ge, Yunhui; Su, Yirong; Xu, Xinwen; Wang, Yongdong; He, Xunyang

2016-11-15

The addition of exogenous inorganic carbon (CaCO3) and organic carbon has an important influence on soil organic carbon (SOC) mineralization in karst soil, but the microbial mechanisms underlying the SOC priming effect are poorly understood. We conducted a 100-day incubation experiment involving four treatments of the calcareous soil in southwestern China's karst region: control, (14)C-labeled rice straw addition, (14)C-labeled CaCO3 addition, and a combination of (14)C-labeled rice straw and CaCO3. Changes in soil microbial communities were characterized using denaturing gradient gel electrophoresis with polymerase chain reaction (PCR-DGGE) and real-time quantitative PCR (q-PCR). Both (14)C-rice straw and Ca(14)CO3 addition stimulated SOC mineralization, suggesting that organic and inorganic C affected SOC stability. Addition of straw alone had no significant effect on bacterial diversity; however, when the straw was added in combination with calcium carbonate, it had an inhibitory effect on bacterial and fungal diversity. At the beginning of the experimental period, exogenous additives increased bacterial abundance, although at the end of the 100-day incubation bacterial community abundance had gradually declined. Incubation time, exogenous input, and their interaction significantly affected SOC mineralization (in terms of priming and the cumulative amount of mineralization), microbial biomass carbon (MBC), and microbial community abundance and diversity. Moreover, the key factors influencing SOC mineralization were MBC, bacterial diversity, and soil pH. Overall, these findings support the view that inorganic C is involved in soil C turnover with the participation of soil microbial communities, promoting soil C cycling in the karst region. Copyright © 2016 Elsevier B.V. All rights reserved.

Microbial Dynamics during Aerobic Exposure of Corn Silage Stored under Oxygen Barrier or Polyethylene Films▿

PubMed Central

Dolci, Paola; Tabacco, Ernesto; Cocolin, Luca; Borreani, Giorgio

2011-01-01

The aims of this study were to compare the effects of sealing forage corn with a new oxygen barrier film with those obtained by using a conventional polyethylene film. This comparison was made during both ensilage and subsequent exposure of silage to air and included chemical, microbiological, and molecular (DNA and RNA) assessments. The forage was inoculated with a mixture of Lactobacillus buchneri, Lactobacillus plantarum, and Enterococcus faecium and ensiled in polyethylene (PE) and oxygen barrier (OB) plastic bags. The oxygen permeability of the PE and OB films was 1,480 and 70 cm3 m−2 per 24 h at 23°C, respectively. The silages were sampled after 110 days of ensilage and after 2, 5, 7, 9, and 14 days of air exposure and analyzed for fermentation characteristics, conventional microbial enumeration, and bacterial and fungal community fingerprinting via PCR-denaturing gradient gel electrophoresis (DGGE) and reverse transcription (RT)-PCR-DGGE. The yeast counts in the PE and OB silages were 3.12 and 1.17 log10 CFU g−1, respectively, with corresponding aerobic stabilities of 65 and 152 h. Acetobacter pasteurianus was present at both the DNA and RNA levels in the PE silage samples after 2 days of air exposure, whereas it was found only after 7 days in the OB silages. RT-PCR-DGGE revealed the activity of Aspergillus fumigatus in the PE samples from the day 7 of air exposure, whereas it appeared only after 14 days in the OB silages. It has been shown that the use of an oxygen barrier film can ensure a longer shelf life of silage after aerobic exposure. PMID:21821764

Evaluation of different primers for PCR-DGGE analysis of cheese-associated enterococci.

PubMed

Lorbeg, Petra Mohar; Majhenic, Andreja Canzek; Rogelj, Irena

2009-08-01

Enterococci represent an important part of bacterial microbiota in different types of artisanal cheeses, made from either raw or pasteurized milk. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) of ribosomal DNA is currently one of the most frequently used fingerprinting method to study diversity and dynamics of microbial communities and also a tool for microbial identification. Among several primer pairs for DGGE analysis published so far, six primer pairs amplifying different variable regions of 16S rDNA were selected and applied in our DGGE analysis of 12 species belonging to genus Enterococcus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactoccocus lactis). When DGGE procedures were optimized, the same set of primers was used for DGGE analysis of five cheese samples. Our study demonstrates that the use of different primer pairs generate significant differences in DGGE analysis of enterococcal population, consequently, appropriate primers regarding the purpose of analysis can be selected. For differentiation and identification of pure enterococcal isolates, primer pair P1V1/P2V1 showed the most promising results since all 12 enterococcal isolates gave distinctive DGGE fingerprints, but with multiple bands patterns; therefore, these primers do not seem to be appropriate for identification of enterococcal species in mixed cultures. Use of primer pairs HDA1/HDA2 and V3f/V3r amplifying V3 region showed better potential for detection and identification of enterococci in mixed communities, but since some bacterial species showed the same fingerprint, for clear identification combination of DGGE and some other method (e.g. species specific PCR) or combined DGGE analysis using two primer pairs generating distinctive results should be used.

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.

PubMed

Wong, Grace; Wong, Isaac; Chan, Kamfai; Hsieh, Yicheng; Wong, Season

2015-01-01

Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3 °C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths. We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.

Mineralogical Control on Microbial Diversity in a Weathered Granite?

NASA Astrophysics Data System (ADS)

Gleeson, D.; Clipson, N.; McDermott, F.

2003-12-01

Mineral transformation reactions and the behaviour of metals in rock and soils are affected not only by physicochemical parameters but also by biological factors, particularly by microbial activity. Microbes inhabit a wide range of niches in surface and subsurface environments, with mineral-microbe interactions being generally poorly understood. The focus of this study is to elucidate the role of microbial activity in the weathering of common silicate minerals in granitic rocks. A site in the Wicklow Mountains (Ireland) has been identified that consists of an outcrop surface of Caledonian (ca. 400 million years old) pegmatitic granite from which large intact crystals of variably weathered muscovite, plagioclase, K-feldspar and quartz were sampled, together with whole-rock granite. Culture-based microbial approaches have been widely used to profile microbial communities, particularly from copiotrophic environments, but it is now well established that for oligotrophic environments such as those that would be expected on weathering faces, perhaps less than 1% of microbial diversity can be profiled by cultural means. A number of culture-independent molecular based approaches have been developed to profile microbial diversity and community structure. These rely on successfully isolating environmental DNA from a given environment, followed by the use of the polymerase chain reaction (PCR) to amplify the typically small quantities of extracted DNA. Amplified DNA can then be analysed using cloning based approaches as well as community fingerprinting systems such as denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (TRFLP) and ribosomal intergenic spacer analysis (RISA). Community DNA was extracted and the intergenic spacer region (ITS) between small (16S) and large (23S) bacterial subunit rRNA genes was amplified. RISA fragments were then electrophoresed on a non-denaturing polyacrylamide gel. Banding patterns suggest that the bacterial population in whole rock, which contained approximately 30 separated bands (indicative of the number of bacterial ribotypes), is greater than muscovite (20), K-feldspar (15), and plagioclase feldspar (12) with quartz exhibiting the lowest number (6). These bands were excised from the gel for sequencing, allowing identification of the major populations. An automated approach was also used to assess similarity of bacterial communities present on each sample type, and this allowed for a statistical evaluation of bacterial diversity. Petrographic studies were carried out to assess mineral alteration effects. Scanning electron microscopy (SEM) was used to visualise in-situ bacterial cells.

Chip-based sequencing nucleic acids

DOEpatents

Beer, Neil Reginald

2014-08-26

A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

High-Resolution Melting-Curve Analysis of Ligation-Mediated Real-Time PCR for Rapid Evaluation of an Epidemiological Outbreak of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli ▿

PubMed Central

Woksepp, Hanna; Jernberg, Cecilia; Tärnberg, Maria; Ryberg, Anna; Brolund, Alma; Nordvall, Michaela; Olsson-Liljequist, Barbro; Wisell, Karin Tegmark; Monstein, Hans-Jürg; Nilsson, Lennart E.; Schön, Thomas

2011-01-01

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE. PMID:21956981

High-resolution melting-curve analysis of ligation-mediated real-time PCR for rapid evaluation of an epidemiological outbreak of extended-spectrum-beta-lactamase-producing Escherichia coli.

PubMed

Woksepp, Hanna; Jernberg, Cecilia; Tärnberg, Maria; Ryberg, Anna; Brolund, Alma; Nordvall, Michaela; Olsson-Liljequist, Barbro; Wisell, Karin Tegmark; Monstein, Hans-Jürg; Nilsson, Lennart E; Schön, Thomas

2011-12-01

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.

Analysis of PCR Thermocycling by Rayleigh-Bénard Convection

NASA Astrophysics Data System (ADS)

Sharma, Ruchi; Ugaz, Victor

2004-03-01

In previous studies, we demonstrated a novel device employing the circulatory flow field established by Rayleigh-Bénard convection to perform amplification of a 295 base target region from a human genomic DNA template inside a 35 uL cylindrical cavity using the polymerase chain reaction (PCR) [Krishnan, Ugaz & Burns, Science, Vol. 298, 2002, p. 793]. This design eliminates the need for dynamic external temperature control required in conventional thermocyclers that repeatedly heat and cool static sample volumes to denaturation, annealing, and extension temperatures. In this paper, we extend these studies by demonstrating the design and operation of a multiwell convective flow device capable of achieving amplification of a 191 base pair fragment associated with membrane channel proteins M1 and M2 of the influenza-A virus in as little as 15 minutes with performance comparable to a conventional thermocycler. We also study the effect of initial template concentration and observe no degradation in performance over four orders of magnitude of initial template loading dilution, consistent with conventional thermocycler results. These results illustrate the ability of convective flow PCR systems to achieve performance equal to or exceeding conventional thermocycling hardware, and demonstrate their suitability for use in rapid biodetection assays.

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

PubMed Central

Hoefel, Daniel; Monis, Paul T.; Grooby, Warwick L.; Andrews, Stuart; Saint, Christopher P.

2005-01-01

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 105 bacteria ml−1. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 103 active AOB ml−1 were detected in the membrane-intact fraction, and 1.40 × 104 active AOB ml−1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml−1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event. PMID:16269672

Analysis of oral microbiota in children with dental caries by PCR-DGGE and barcoded pyrosequencing.

PubMed

Ling, Zongxin; Kong, Jianming; Jia, Peng; Wei, Chaochun; Wang, Yuezhu; Pan, Zhiwen; Huang, Wujing; Li, Lanjuan; Chen, Hui; Xiang, Charlie

2010-10-01

Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.

A simple nucleic acid hybridization/latex agglutination assay for the rapid detection of polymerase chain reaction amplicons.

PubMed

Vollenhofer-Schrumpf, Sabine; Buresch, Ronald; Schinkinger, Manfred

2007-03-01

We have developed a new method for the detection of nucleic acid hybridization, based on a simple latex agglutination test that can be evaluated by the unaided eye. Nucleic acid, e.g., a polymerase chain reaction (PCR) product, is denatured and incubated with polystyrene beads carrying covalently bound complementary oligonucleotide sequences. Hybridization of the nucleic acids leads to aggregation of the latex particles, thereby verifying the presence of target sequence. The test is performed at room temperature, and results are available within 10 min. As a proof of principle, the hybridization/latex agglutination assay was applied to the detection of purified PCR fragments either specific for Salmonella spp. or a synthetic sequence, and to the detection of Salmonella enterica in artificially contaminated chicken samples. A few nanograms of purified PCR fragments were detectable. In artificially contaminated chicken samples, 3 colony-forming units (cfu)/25 g were detected in one of three replicates, and 30 cfu/25 g were detected in both of two replicates when samples for PCR were taken directly from primary enrichment, demonstrating the practical applicability of this test system. Even multiplex detection might be achievable. This novel kind of assay could be useful for a range of applications where hybridization of nucleic acids, e.g., PCR fragments, is to be detected.

The use of denaturing high-pressure liquid chromatography for the detection of mutations in thiopurine methyltransferase.

PubMed

Hall, A G; Hamilton, P; Minto, L; Coulthard, S A

2001-01-30

The level of expression of the enzyme thiopurine methyltransferase (TPMT) is an important determinant of the metabolism of drugs used both in the treatment of acute leukaemia (6-mercaptopurine and 6-thioguanine) and as an immunosuppressant in patients with autoimmune diseases or following organ transplantation (azathioprine). Studies of enzyme activity in red blood cells have shown that TPMT expression displays genetic polymorphism with 11% of individuals having intermediate and one in 300 undetectable levels. Patients with biallelic mutations and undetectable enzyme activity suffer life-threatening myelosuppression when treated with conventional doses of these drugs. Patients with intermediate activity have an increased risk of drug-associated toxicity. In the Caucasian populations studied to date, intermediate activity is associated with mutations at two sites of the TPMT gene, G460A and A719G (designated TPMT*3A), in 80% of cases. Detection of these mutations has, to date, been based on the analysis of restriction digests of PCR products. In order to simplify this process we have investigated the ability of denaturing high pressure liquid chromatography (DHPLC) to detect the A719G mutation. DHPLC of PCR products from 15 known heterozygotes (TPMT*3A/TPMT*1) and 18 known homozygotes (TPMT*1/TPMT*1) gave a clear pattern difference between the groups and 100% concordance with the results of restriction digests. These results suggest DHPLC represents a valuable technique for accurate and rapid detection of pharmacologically important mutations in the TPMT gene.

Effect of diesel leakage in circulating cooling water system on preponderant bacteria diversity and bactericidal effect of biocides.

PubMed

Zhong, Huiyun; Liu, Fang; Lu, Jinjin; Yang, Wei; Zhao, Chaocheng

2015-01-01

Petroleum products leakage results in adverse effect on the normal operation of a circulating cooling water system. However, relatively little research has been done to explore the effect of petroleum products leakage on circulating cooling water quality and biofilm preponderant bacteria diversity. Also, normal biocides application modes cannot fulfil the need for biofilm control. In this study, diesel oil was used as the experimental subject representing leaking petroleum products; the effect of diesel addition on biofilm preponderant bacteria diversity and the bactericidal effect of chlorine dioxide and tetradecyl dimethyl benzyl ammonium chloride (1427) was investigated. Bacterial community structures were examined by PCR-denaturing gradient gel electrophoresis and PCR cloning of 16S rDNA genes. Except for 100 mg/L diesel, increasing diesel concentration enhanced the biofilm detachment ratio compared with the control test. The microstructure of biofilm samples with 0, 300 and 900 mg/L diesel addition was observed. The species of preponderant bacteria in the biofilm sample with 300 mg/L diesel addition were more and the bacterial distribution was more uniform than those in the biofilm sample with 900 mg/L diesel addition. With ClO2 and 1427 addition, chemical oxygen demand increased, lipid phosphorus and bacterial count first decreased and then remained stable, and the bactericidal ratio first increased and then remained stable. Diesel addition variation has more obvious effect on ClO2 than 1427.

Plant-fed versus chemicals-fed rhizobacteria of Lucerne: Plant-only teabags culture media not only increase culturability of rhizobacteria but also recover a previously uncultured Lysobacter sp., Novosphingobium sp. and Pedobacter sp.

PubMed Central

Hegazi, Nabil A.; Sarhan, Mohamed S.; Fayez, Mohamed; Patz, Sascha; Murphy, Brian R.; Ruppel, Silke

2017-01-01

In an effort to axenically culture the previously uncultivable populations of the rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags culture media to mimic the nutritional matrix available in the rhizosphere. Here, we show that culture media prepared from Lucerne powder teabags substantially increased the cultivability of Lucerne rhizobacteria compared with a standard nutrient agar, where we found that the cultivable populations significantly increased by up to 60% of the total bacterial numbers as estimated by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of 16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct composition and separation of bacterial populations recovered on the plant-only teabags culture media than those developed on a standard nutrient agar. Further, the new plant medium gave preference to the micro-symbiont Sinorhizobium meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The present study may encourage other researchers to consider moving from the well-established standard culture media to the challenging new plant-only culture media. Such a move may reveal previously hidden members of rhizobacteria, and help to further explore their potential environmental impacts. PMID:28686606

Application of aerobic composting system for space agriculture

NASA Astrophysics Data System (ADS)

Oshima, Tairo; Yoshii, Takahiro; Moriya, Toshiyuki; Yamashita, Masamichi

Composting is a classical technique to decompose organic wastes such as animal bodies, straw, paper, raw sludge, and so on. Compared with burning of wastes, the composting method has many advantages. It is an inexpensive and safer method because of its self-heating without spending extra energy resources. It does not emit toxic pollutants such as dioxin, NOx , and SOx . The composting products can be used as organic fertilizers for agricultural production. Composting is a promising way for digesting organic wastes safely on spaceships or manned exploration on extraterrestrial planets. We have developed a small scale high-temperature composter in order to examine its feasobility to operate food waste disposing facility and fertilizer production in space. This composter has a heated reaction vessel containing compost soil (seed bacteria) provided by a compost factory. To determine the optimal condition for its operation, we analyzed the effect of temperature on metabolic activity (CO2 production rate), and water content. The dynamics of microbial community was studied by polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE). Water content was maintained to a range between 27% and 40% by continuously adding water. The highest CO2 emission was observed at around 70° C. PCR-DGGE analysis shows that the bacterial community of the compost soil is dramatically changed by changing reaction temperature. We will discuss the application of the composter in space in order to establish the closed recycling loop of bio-elements in space agriculture.

Difference of microbial community stressed in artificial pit muds for Luzhou-flavour liquor brewing revealed by multiphase culture-independent technology.

PubMed

Zhang, L; Zhou, R; Niu, M; Zheng, J; Wu, C

2015-11-01

Artificial pit muds (APMs) is produced by peats, aged pit muds, yellow and black clays etc. and is one of essential factors for Luzhou-flavour liquor production. The microbial community of APMs significantly influence the quality of Luzhou-flavour liquor. The aim of this study was to investigate the differences in bacterial, archaeal and fungal community of APMs, starters and materials. Multiphase culture-independent technology were employed in this study, including nested PCR-denaturing gradient gel electrophoresis (nested PCR-DGGE), phospholipid fatty acid (PLFA), phospholipid ether lipids (PLEL) and fluorescence in situ hybridization (FISH) analysis. Results suggested that the microbial diversity significantly changed under environmental stress and different culture patterns during APMs cultivation. The dominant bacteria in APMs mainly fell into Clostridiales, Lactobacillales, Bacteroidales and Rhizobiales, Archaea affiliated with Methanomicrobiales and Methanosarcinales, and fungi belonged to Saccharomycetales and Eurotiales. Furthermore, the microbial community structures of APMs cultured by ground pile pattern were more similar with that of aged pit muds, meanwhile, the relative bands intensities of microbes, which are the main contributors for liquor brewing, increased with the culture times. Not only the niche selection and biogeochemical properties of APMs, but also the mutual collaboration and constraint between different microbes may result in enriching different liquor-brewing microbes into APMs. APM cultivation technology was necessary to promote enriching functional liquor-brewing microbes into APMs. These results may facilitate understanding the microbial succession during APMs manufacture. © 2015 The Society for Applied Microbiology.

The Freshwater Sponge Ephydatia fluviatilis Harbours Diverse Pseudomonas Species (Gammaproteobacteria, Pseudomonadales) with Broad-Spectrum Antimicrobial Activity

PubMed Central

Keller-Costa, Tina; Jousset, Alexandre; van Overbeek, Leo; van Elsas, Jan Dirk; Costa, Rodrigo

2014-01-01

Bacteria are believed to play an important role in the fitness and biochemistry of sponges (Porifera). Pseudomonas species (Gammaproteobacteria, Pseudomonadales) are capable of colonizing a broad range of eukaryotic hosts, but knowledge of their diversity and function in freshwater invertebrates is rudimentary. We assessed the diversity, structure and antimicrobial activities of Pseudomonas spp. in the freshwater sponge Ephydatia fluviatilis. Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints of the global regulator gene gacA revealed distinct structures between sponge-associated and free-living Pseudomonas communities, unveiling previously unsuspected diversity of these assemblages in freshwater. Community structures varied across E. fluviatilis specimens, yet specific gacA phylotypes could be detected by PCR-DGGE in almost all sponge individuals sampled over two consecutive years. By means of whole-genome fingerprinting, 39 distinct genotypes were found within 90 fluorescent Pseudomonas isolates retrieved from E. fluviatilis. High frequency of in vitro antibacterial (49%), antiprotozoan (35%) and anti-oomycetal (32%) activities was found among these isolates, contrasting less-pronounced basidiomycetal (17%) and ascomycetal (8%) antagonism. Culture extracts of highly predation-resistant isolates rapidly caused complete immobility or lysis of cells of the protozoan Colpoda steinii. Isolates tentatively identified as P. jessenii, P. protegens and P. oryzihabitans showed conspicuous inhibitory traits and correspondence with dominant sponge-associated phylotypes registered by cultivation-independent analysis. Our findings suggest that E. fluviatilis hosts both transient and persistent Pseudomonas symbionts displaying antimicrobial activities of potential ecological and biotechnological value. PMID:24533086

Maternal consumption of fructo-oligosaccharide diminishes the severity of skin inflammation in offspring of NC/Nga mice.

PubMed

Fujiwara, Reiko; Takemura, Naoki; Watanabe, Jun; Sonoyama, Kei

2010-02-01

Strategies to manipulate the gut microbiota in infancy have been considered to prevent the development of allergic diseases later in life. We aimed to elucidate the effects of maternal dietary supplementation with a prebiotic oligosaccharide on gut microbiota and spontaneously developing atopic dermatitis-like skin lesions in the offspring of NC/Nga mice. Female NC/Nga mice were fed diets either with or without fructo-oligosaccharide supplementation during pregnancy and lactation. After weaning, offspring were fed the diets supplemented with or without fructo-oligosaccharide for 11 weeks in an air-uncontrolled conventional room. Changes in gut microbiota were assessed by denaturing gradient gel electrophoresis of the PCR-amplified 16S rRNA gene. Skin lesions were evaluated by a clinical score and scratching behaviour. Serum antibody levels were measured by ELISA, and expression levels of cytokines and chemokines in lesional tissue were evaluated by quantitative RT-PCR. Maternal supplementation with fructo-oligosaccharide modulated the gut microbiota in sucklings. Although maternal supplementation with fructo-oligosaccharide suppressed the increase in clinical skin severity score and scratching behaviour in offspring, dietary fructo-oligosaccharide after weaning was less effective. The diminution of skin lesions was accompanied by lower serum concentrations of total IgG1 and lower expression levels of TNF-alpha in the lesional tissue. These data suggest that maternal consumption of fructo-oligosaccharide diminishes the severity of atopic dermatitis-like skin lesions in the offspring of NC/Nga mice.

p-Coumaric Acid Influenced Cucumber Rhizosphere Soil Microbial Communities and the Growth of Fusarium oxysporum f.sp. cucumerinum Owen

PubMed Central

Zhou, Xingang; Wu, Fengzhi

2012-01-01

Background Autotoxicity of cucumber root exudates or decaying residues may be the cause of the soil sickness of cucumber. However, how autotoxins affect soil microbial communities is not yet fully understood. Methodology/Principal Findings The aims of this study were to study the effects of an artificially applied autotoxin of cucumber, p-coumaric acid, on cucumber seedling growth, rhizosphere soil microbial communities, and Fusarium oxysporum f.sp. cucumerinum Owen (a soil-borne pathogen of cucumber) growth. Abundance, structure and composition of rhizosphere bacterial and fungal communities were analyzed with real-time PCR, PCR-denaturing gradient gel electrophoresis (DGGE) and clone library methods. Soil dehydrogenase activity and microbial biomass C (MBC) were determined to indicate the activity and size of the soil microflora. Results showed that p-coumaric acid (0.1–1.0 µmol/g soil) decreased cucumber leaf area, and increased soil dehydrogenase activity, MBC and rhizosphere bacterial and fungal community abundances. p-Coumaric acid also changed the structure and composition of rhizosphere bacterial and fungal communities, with increases in the relative abundances of bacterial taxa Firmicutes, Betaproteobacteria, Gammaproteobacteria and fungal taxa Sordariomycete, Zygomycota, and decreases in the relative abundances of bacterial taxa Bacteroidetes, Deltaproteobacteria, Planctomycetes, Verrucomicrobia and fungal taxon Pezizomycete. In addition, p-coumaric acid increased Fusarium oxysporum population densities in soil. Conclusions/Significance These results indicate that p-coumaric acid may play a role in the autotoxicity of cucumber via influencing soil microbial communities. PMID:23118972

p-Coumaric acid influenced cucumber rhizosphere soil microbial communities and the growth of Fusarium oxysporum f.sp. cucumerinum Owen.

PubMed

Zhou, Xingang; Wu, Fengzhi

2012-01-01

Autotoxicity of cucumber root exudates or decaying residues may be the cause of the soil sickness of cucumber. However, how autotoxins affect soil microbial communities is not yet fully understood. The aims of this study were to study the effects of an artificially applied autotoxin of cucumber, p-coumaric acid, on cucumber seedling growth, rhizosphere soil microbial communities, and Fusarium oxysporum f.sp. cucumerinum Owen (a soil-borne pathogen of cucumber) growth. Abundance, structure and composition of rhizosphere bacterial and fungal communities were analyzed with real-time PCR, PCR-denaturing gradient gel electrophoresis (DGGE) and clone library methods. Soil dehydrogenase activity and microbial biomass C (MBC) were determined to indicate the activity and size of the soil microflora. Results showed that p-coumaric acid (0.1-1.0 µmol/g soil) decreased cucumber leaf area, and increased soil dehydrogenase activity, MBC and rhizosphere bacterial and fungal community abundances. p-Coumaric acid also changed the structure and composition of rhizosphere bacterial and fungal communities, with increases in the relative abundances of bacterial taxa Firmicutes, Betaproteobacteria, Gammaproteobacteria and fungal taxa Sordariomycete, Zygomycota, and decreases in the relative abundances of bacterial taxa Bacteroidetes, Deltaproteobacteria, Planctomycetes, Verrucomicrobia and fungal taxon Pezizomycete. In addition, p-coumaric acid increased Fusarium oxysporum population densities in soil. These results indicate that p-coumaric acid may play a role in the autotoxicity of cucumber via influencing soil microbial communities.

Molecular comparison of the sampling efficiency of four types of airborne bacterial samplers.

PubMed

Li, Kejun

2011-11-15

In the present study, indoor and outdoor air samples were collected using four types of air samplers often used for airborne bacterial sampling. These air samplers included two solid impactors (BioStage and RCS), one liquid impinger (BioSampler), and one filter sampler with two kinds of filters (a gelatin and a cellulose acetate filter). The collected air samples were further processed to analyze the diversity and abundance of culturable bacteria and total bacteria through standard culture techniques, denaturing gradient gel electrophoresis (DGGE) fingerprinting and quantitative polymerase chain reaction (qPCR) analysis. The DGGE analysis indicated that the air samples collected using the BioStage and RCS samplers have higher culturable bacterial diversity, whereas the samples collected using the BioSampler and the cellulose acetate filter sampler have higher total bacterial diversity. To obtain more information on the sampled bacteria, some gel bands were excised and sequenced. In terms of sampling efficiency, results from the qPCR tests indicated that the collected total bacterial concentration was higher in samples collected using the BioSampler and the cellulose acetate filter sampler. In conclusion, the sampling bias and efficiency of four kinds of air sampling systems were compared in the present study and the two solid impactors were concluded to be comparatively efficient for culturable bacterial sampling, whereas the liquid impactor and the cellulose acetate filter sampler were efficient for total bacterial sampling. Copyright © 2011 Elsevier B.V. All rights reserved.

Lactobacillus paracasei A survives gastrointestinal passage and affects the fecal microbiota of healthy infants.

PubMed

Marzotto, Marta; Maffeis, Claudio; Paternoster, Thomas; Ferrario, Rossano; Rizzotti, Lucia; Pellegrino, Maristella; Dellaglio, Franco; Torriani, Sandra

2006-11-01

This study focuses on the potentiality of a putative probiotic strain, Lactobacillus paracasei A, to survive gastrointestinal (GI) passage and modulate the resident microbiota of healthy infants. In a placebo-controlled study, 26 children aged 12-24 months received 100 g/day of either fermented milk containing strain A or pasteurized yogurt for four weeks. Fecal samples were analyzed before starting the administration, after 1, 3 and 4 weeks of consumption and after washout. The fate of strain A was followed by means of a newly developed PCR targeting a strain-specific genomic marker. The composition and dynamics of fecal microbial communities during the study were analyzed by culturing on selective media and by the PCR-denaturing gradient gel electrophoresis (DGGE) technique using universal and group-specific (Lactobacillus and Bifidobacterium) primers. The variation in enzymatic activities in infant feces during probiotic consumption was also analyzed. Strain A survived in fecal samples in most (92%) of the infants examined after 1 week of consumption, and temporarily dominated the intestinal Lactobacillus community. The administration of L. paracasei A led to a significant increment in the Lactobacillus population, while a moderate effect upon the main bacterial groups in the GI ecosystem was observed. Strain A also affected the diversity of the Lactobacillus and Bifidobacterium populations. The fecal bacterial structure of 1 - 2-year-old infants seems to combine neonate and adult-like features. The microbiota of these subjects promptly responded to probiotic consumption, later restoring the endogenous equilibrium.

Microscopic, chemical, and molecular-biological investigation of the decayed medieval stained window glasses of two Catalonian churches

PubMed Central

Piñar, Guadalupe; Garcia-Valles, Maite; Gimeno-Torrente, Domingo; Fernandez-Turiel, Jose Luis; Ettenauer, Jörg; Sterflinger, Katja

2013-01-01

We investigated the decayed historical church window glasses of two Catalonian churches, both under Mediterranean climate. Glass surfaces were studied by scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and X-ray diffraction (XRD). Their chemical composition was determined by wavelength-dispersive spectrometry (WDS) microprobe analysis. The biodiversity was investigated by molecular methods: DNA extraction from glass, amplification by PCR targeting the16S rRNA and ITS regions, and fingerprint analyses by denaturing gradient gel electrophoresis (DGGE). Clone libraries containing either PCR fragments of the bacterial 16S rDNA or the fungal ITS regions were screened by DGGE. Clone inserts were sequenced and compared with the EMBL database. Similarity values ranged from 89 to 100% to known bacteria and fungi. Biological activity in both sites was evidenced in the form of orange patinas, bio-pitting, and mineral precipitation. Analyses revealed complex bacterial communities consisting of members of the phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Fungi showed less diversity than bacteria, and species of the genera Cladosporium and Phoma were dominant. The detected Actinobacteria and fungi may be responsible for the observed bio-pitting phenomenon. Moreover, some of the detected bacteria are known for their mineral precipitation capabilities. Sequence results also showed similarities with bacteria commonly found on deteriorated stone monuments, supporting the idea that medieval stained glass biodeterioration in the Mediterranean area shows a pattern comparable to that on stone. PMID:24092957

Shifts of microbial communities of wheat (Triticum aestivum L.) cultivation in a closed artificial ecosystem.

PubMed

Qin, Youcai; Fu, Yuming; Dong, Chen; Jia, Nannan; Liu, Hong

2016-05-01

The microbial communities of plant ecosystems are in relation to plant growing environment, but the alteration in biodiversity of rhizosphere and phyllosphere microbial communities in closed and controlled environments is unknown. The purpose of this study is to analyze the change regularity of microbial communities with wheat plants dependent-cultivated in a closed artificial ecosystem. The microbial community structures in closed-environment treatment plants were investigated by a culture-dependent approach, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), and Illumina Miseq high-throughput sequencing. The results indicated that the number of microbes decreased along with time, and the magnitude of bacteria, fungi, and actinomycetes were 10(7)-10(8), 10(5), and 10(3)-10(4) CFU/g (dry weight), respectively. The analysis of PCR-DGGE and Illumina Miseq revealed that the wheat leaf surface and near-root substrate had different microbial communities at different periods of wheat ecosystem development and showed that the relative highest diversity of microbial communities appeared at late and middle periods of the plant ecosystem, respectively. The results also indicated that the wheat leaf and substrate had different microbial community compositions, and the wheat substrate had higher richness of microbial community than the leaf. Flavobacterium, Pseudomonas, Paenibacillus, Enterobacter, Penicillium, Rhodotorula, Acremonium, and Alternaria were dominant in the wheat leaf samples, and Pedobacter, Flavobacterium, Halomonas, Marinobacter, Salinimicrobium, Lysobacter, Pseudomonas, Halobacillus, Xanthomonas, Acremonium, Monographella, and Penicillium were dominant populations in the wheat near-root substrate samples.

Grassland management regimens reduce small-scale heterogeneity and species diversity of beta-proteobacterial ammonia pxidizer populations.

PubMed

Webster, Gordon; Embley, T Martin; Prosser, James I

2002-01-01

The impact of soil management practices on ammonia oxidizer diversity and spatial heterogeneity was determined in improved (addition of N fertilizer), unimproved (no additions), and semi-improved (intermediate management) grassland pastures at the Sourhope Research Station in Scotland. Ammonia oxidizer diversity within each grassland soil was assessed by PCR amplification of microbial community DNA with both ammonia oxidizer-specific, 16S rRNA gene (rDNA) and functional, amoA, gene primers. PCR products were analysed by denaturing gradient gel electrophoresis, phylogenetic analysis of partial 16S rDNA and amoA sequences, and hybridization with ammonia oxidizer-specific oligonucleotide probes. Ammonia oxidizer populations in unimproved soils were more diverse than those in improved soils and were dominated by organisms representing Nitrosospira clusters 1 and 3 and Nitrosomonas cluster 7 (closely related phylogenetically to Nitrosomonas europaea). Improved soils were only dominated by Nitrosospira cluster 3 and Nitrosomonas cluster 7. These differences were also reflected in functional gene (amoA) diversity, with amoA gene sequences of both Nitrosomonas and Nitrosospira species detected. Replicate 0.5-g samples of unimproved soil demonstrated significant spatial heterogeneity in 16S rDNA-defined ammonia oxidizer clusters, which was reflected in heterogeneity in ammonium concentration and pH. Heterogeneity in soil characteristics and ammonia oxidizer diversity were lower in improved soils. The results therefore demonstrate significant effects of soil management on diversity and heterogeneity of ammonia oxidizer populations that are related to similar changes in relevant soil characteristics.

Characterization of eubacterial and archaeal community diversity in the pit mud of Chinese Luzhou-flavor liquor by nested PCR-DGGE.

PubMed

Ding, Xiao-Fei; Wu, Chong-De; Zhang, Li-Qiang; Zheng, Jia; Zhou, Rong-Qing

2014-02-01

The aim of this study was to investigate and compare the microbial community structures of eubacteria and archaea in the pit mud of Chinese Luzhou-flavor liquor from the wall (C(w)) and bottom (C(b)) of cellar through nested PCR-denaturing gradient gel electrophoresis (DGGE). The Shannon-Wiener index (H) calculated from the DGGE profiles showed that the community diversities of eubacteria and archaea in samples from C(b) were almost higher than that from C(w). In addition, cluster analysis of the DGGE profiles revealed that some differences were found in the microbial community structure in samples from different locations. The closely relative microorganisms of all eubacterial 16S rRNA gene sequences fell into four phyla (Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria), including 12 genera and 2 uncultured eubacteria. Moreover, 37.1% eubacteria were affiliated with Clostridium. Particularly, genus Acinetobacter was absent in all samples from C(b) but present in all samples from C(w). The closely relative microorganisms of all archaeal 16S rRNA gene sequences fell into four genera, which included Methanobrevibacter, Methanoculleus, Methanobacterium and Methanosaeta, while the dominant archaea in samples from C(w) and C(b) were similar. Results presented in this study provide further understanding of the spatial differences in microbial community structure in the pit mud, and is of great importance for the production and quality improvement of Luzhou-flavor liquor.

Circulating polymerase chain reaction chips utilizing multiple-membrane activation

NASA Astrophysics Data System (ADS)

Wang, Chih-Hao; Chen, Yi-Yu; Liao, Chia-Sheng; Hsieh, Tsung-Min; Luo, Ching-Hsing; Wu, Jiunn-Jong; Lee, Huei-Huang; Lee, Gwo-Bin

2007-02-01

This paper reports a new micromachined, circulating, polymerase chain reaction (PCR) chip for nucleic acid amplification. The PCR chip is comprised of a microthermal control module and a polydimethylsiloxane (PDMS)-based microfluidic control module. The microthermal control modules are formed with three individual heating and temperature-sensing sections, each modulating a specific set temperature for denaturation, annealing and extension processes, respectively. Micro-pneumatic valves and multiple-membrane activations are used to form the microfluidic control module to transport sample fluids through three reaction regions. Compared with other PCR chips, the new chip is more compact in size, requires less time for heating and cooling processes, and has the capability to randomly adjust time ratios and cycle numbers depending on the PCR process. Experimental results showed that detection genes for two pathogens, Streptococcus pyogenes (S. pyogenes, 777 bps) and Streptococcus pneumoniae (S. pneumoniae, 273 bps), can be successfully amplified using the new circulating PCR chip. The minimum number of thermal cycles to amplify the DNA-based S. pyogenes for slab gel electrophoresis is 20 cycles with an initial concentration of 42.5 pg µl-1. Experimental data also revealed that a high reproducibility up to 98% could be achieved if the initial template concentration of the S. pyogenes was higher than 4 pg µl-1. The preliminary results of the current paper were presented at the 19th IEEE International Conference on Micro Electro Mechanical Systems (IEEE MEMS 2006), Istanbul, Turkey, 22-26 January, 2006.

Sustained high-throughput polymerase chain reaction diagnostics during the European epidemic of Bluetongue virus serotype 8.

PubMed

van Rijn, Piet A; Heutink, René G; Boonstra, Jan; Kramps, Hans A; van Gennip, René G P

2012-05-01

A real-time reverse transcription polymerase chain reaction assay (PCR test) based on genome segment 10 of Bluetongue virus (BTV) was developed. The PCR test consists of robotized viral RNA isolation from blood samples and an all-in-one method including initial denaturation of genomic double-stranded RNA, reverse transcription polymerase chain reaction (RT-PCR), and real-time detection and analysis. Reference strains of the 24 recognized BTV serotypes, isolates from different years, and geographic origins were detected. Other orbiviruses such as African horse sickness virus, Epizootic hemorrhagic disease virus, and Equine encephalosis virus were not detected. Experimentally infected animals were PCR positive from 2 days postinoculation, which was earlier than fever, other clinical signs, or seroconversion. The diagnostic sensitivity and specificity were very close to or even 100%. The PCR test played a key role in the detection of BTV serotype 8 in August 2006 in The Netherlands. The outbreak in a completely naive ruminant population allowed for further evaluation of the PCR test with field samples. In 2006, the correlation between enzyme-linked immunosorbent assay and PCR results was estimated to be 95%. In the following years, the PCR test was used for diagnosis of diseased animals, for testing of healthy animals for trade purposes, and for detection of BTV RNA in different species of the insect vector, Culicoides. In the autumn of 2008, BTV serotype 6 unexpectedly emerged in northwest Europe and was also detected with the PCR test developed in the current study. The performance in routine use over 5 years has been recorded and evaluated.

Plasma protein denaturation with graded heat exposure.

PubMed

Vazquez, R; Larson, D F

2013-11-01

During cardiopulmonary bypass (CPB), perfusion at tepid temperatures (33-35 °C) is recommended to avoid high temperature cerebral hyperthermia during and after the operation. However, the ideal temperature for uncomplicated adult cardiac surgery is an unsettled question. Typically, the heat exchanger maximum temperature is monitored between 40-42 °C to prevent denaturation of plasma proteins, but studies have not been performed to make these conclusions. Therefore, our hypothesis was to determine the temperature in which blood plasma protein degradation occurs after 2 hours of heat exposure. As a result, blood plasma proteins were exposed to heat in the 37-50 °C range for 2 hours. Plasma protein samples were loaded onto an 8-12% gradient gel for SDS-PAGE and low molecular weight plasma protein degradation was detected with graded heat exposure. Protein degradation was first detected between 43-45 °C of heat exposure. This study supports the practice of monitoring the heat exchanger between 40-42 °C to prevent denaturation of plasma proteins.

Denaturing gradient gel electrophoresis-polymerase chain reaction comparison of chitosan effects on anaerobic cultures of broiler cecal bacteria and Salmonella Typhimurium

USDA-ARS?s Scientific Manuscript database

Salmonella colonization and product contamination are major poultry industry problems. Alternatives to traditional antibiotics against Salmonella offer the potential to lessen the development of resistance to antibiotics of importance to human health. The chitin derivative chitosan has drawn substa...

Rapid detection of common Chinese glucose-6-phosphate dehydrogenase (G6PD) mutations by denaturing gradient gel electrophoresis (DGGE).

PubMed

Lam, V M; Huang, W; Lam, S T; Yeung, C Y; Johnson, P H

1996-03-01

We describe here the use of denaturing gradient gel electrophoresis (DGGE) to detect the most common Chinese glucose-6-phosphate dehydrogenase (G6PD) variants, which are the single point mutations: G-->T at nt 1376, G-->A at 1388 both in exon 12 and A-->G at nt 95 in exon 02. In each case, the mutant allele resolves well from the normal allele(s). The distinct heteroduplex bands are characteristic of a particular genotype suggesting that this feature is very useful for identifying all heterozygous carriers for this and other X-linked diseases. When the analysis is extended to other exons, DGGE scans the gene and coupled with direct sequencing, it leads to the identification of new G6PD variation(s). With this approach, we identified a mutation in exon 9 which had not been reported in Hong Kong. Since DGGE can rapidly screen many unknown samples in one gel, this approach could be used to diagnose these G6PD mutations and to identify the at-risk for counselling.

The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer

PubMed Central

Mancini, Irene; Santucci, Claudio; Sestini, Roberta; Simi, Lisa; Pratesi, Nicola; Cianchi, Fabio; Valanzano, Rosa; Pinzani, Pamela; Orlando, Claudio

2010-01-01

Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30–50% of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAFV600E detection. Traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer, resulting in false negative diagnoses. In this study, we designed a protocol to detect mutations of KRAS and BRAFV600E in 117 sporadic CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM). Using traditional PCR and direct sequencing, we found KRAS mutations in 47 (40%) patients and BRAFV600E in 10 (8.5%). The use of COLD-PCR in apparently wild-type samples allowed us to identify 15 newly mutated CRCs (10 for KRAS and 5 for BRAFV600E), raising the percentage of mutated CRCs to 48.7% for KRAS and to 12.8% for BRAFV600E. Therefore, COLD-PCR combined with HRM permits the correct identification of less represented mutations in CRC and better selection of patients eligible for targeted therapies, without requiring expensive and time-consuming procedures. PMID:20616366

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

PubMed Central

Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

2014-01-01

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detectiona)

PubMed Central

Li, Kan-Chien; Ding, Shih-Torng; Lin, En-Chung; Wang, Lon (Alex); Lu, Yen-Wen

2014-01-01

A continuous-flow microchip with a temperature gradient in microchannels was utilized to demonstrate spatial melting analysis on microbeads for clinical Single Nucleotide Polymorphisms (SNPs) genotyping on animal genomic DNA. The chip had embedded heaters and thermometers, which created a rapid and yet stable temperature gradient between 60 °C and 85 °C in a short distance as the detection region. The microbeads, which served as mobile supports carrying the target DNA and fluorescent dye, were transported across the temperature gradient. As the surrounding temperature increased, the fluorescence signals of the microbeads decayed with this relationship being acquired as the melting curve. Fast DNA denaturation, as a result of the improved heat transfer and thermal stability due to scaling, was also confirmed. Further, each individual microbead could potentially bear different sequences and pass through the detection region, one by one, for a series of melting analysis, with multiplex, high-throughput capability being possible. A prototype was tested with target DNA samples in different genotypes (i.e., wild and mutant types) with a SNP location from Landrace sows. The melting temperatures were obtained and compared to the ones using a traditional tube-based approach. The results showed similar levels of SNP discrimination, validating our proposed technique for scanning homozygotes and heterozygotes to distinguish single base changes for disease research, drug development, medical diagnostics, agriculture, and animal production. PMID:25553186

Analysis of Bacterial Community Structure in Sulfurous-Oil-Containing Soils and Detection of Species Carrying Dibenzothiophene Desulfurization (dsz) Genes

PubMed Central

Duarte, Gabriela Frois; Rosado, Alexandre Soares; Seldin, Lucy; de Araujo, Welington; van Elsas, Jan Dirk

2001-01-01

The selective effects of sulfur-containing hydrocarbons, with respect to changes in bacterial community structure and selection of desulfurizing organisms and genes, were studied in soil. Samples taken from a polluted field soil (A) along a concentration gradient of sulfurous oil and from soil microcosms treated with dibenzothiophene (DBT)-containing petroleum (FSL soil) were analyzed. Analyses included plate counts of total bacteria and of DBT utilizers, molecular community profiling via soil DNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC.Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used. Generally, counts of DBT degraders were 10- to 100-fold lower than the total culturable counts. However, PCR-DGGE showed that the numbers of bands detected in the molecular community profiles decreased with increasing oil content of the soil. Analysis of the sequences of three prominent bands of the profiles generated with the highly polluted soil samples suggested that the underlying organisms were related to Actinomyces sp., Arthrobacter sp., and a bacterium of uncertain affiliation. dszA, dszB, and dszC genes were present in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis. Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities. The molecular community profiles were largely stable over time in the untreated soil, whereas they showed a progressive change over time following treatment with DBT-containing petroleum. Direct PCR assessment revealed the presence of dszB-related signals in the untreated FSL soil and the apparent selection of dszA- and dszC-related sequences by the petroleum-DBT treatment. PCR-DGGE applied to sequential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to 10 distinct bands. Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonas sp., Stenotrophomonas maltophilia, and Rhodococcus erythropolis. Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selected in the enrichment cultures. Evidence for the presence of dszB in 12 strains was obtained, whereas dszA and dszC genes were found in only 7 and 6 strains, respectively. Most of the strains carrying dszA or dszC were classified as R. erythropolis related, and all revealed the capacity to desulfurize DBT. A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R. erythropolis strain IGTS8) dszA sequence and a large degree of internal conservation. The 37 sequences recovered were grouped in three clusters. One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second group of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar. The first two groups contained sequences obtained from both soil types and enrichment cultures (including isolates), but the last consisted of sequences obtained directly from the polluted A soil. PMID:11229891

Impact of Substratum Surface on Microbial Community Structure and Treatment Performance in Biological Aerated Filters

PubMed Central

Kim, Lavane; Pagaling, Eulyn; Zuo, Yi Y.

2014-01-01

The impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected, Burkholderiales and Rhodocyclales of the Betaproteobacteria class were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes. PMID:24141134

[Application study of droplet digital PCR to detect maternal cell contamination in prenatal diagnosis].

PubMed

Geng, J; Liu, C; Zhou, X C; Ma, J; Du, L; Lu, J; Zhou, W N; Hu, T T; Lyu, L J; Yin, A H

2017-02-25

Objective: To develop a new method based on droplet digital PCR (DD-PCR) for detection and quantification of maternal cell contamination in prenatal diagnosis. Methods: Invasive prenatal samples from 40 couples of β(IVS-Ⅱ-654)/β(N) thalassemia gene carriers who accepted prenatal diagnosis in Affiliated Women and Children's Hospital of Guangzhou Medical University from October 2015 to December 2016 were analyzed retrospectively. Specific primers and probes were designed. The concentration gradient were 50%, 25%, 12.5%, 6.25%, 3.125%, 1.562 5%. There were 40 groups of prenatal diagnostic samples. Comparing DD-PCR with quantitative fluorescent-PCR (QF-PCR) based on the short tandem repeats for assement of the sensitivity and accuracy of maternal cell contamination, respectively. Results: DD-PCR could quantify the maternal cell contamination as low as 1.562 5%. The result was proportional to the dilution titers. In the 40 prenatal samples, 6 cases (15%, 6/40) of maternal cell contamination were detected by DD-PCR, while the QF-PCR based on short tandem repeat showed 3 cases (7.5%, 3/40) with maternal cell contamination, DD-PCR was more accurate ( P= 0.002) . Conclusion: DD-PCR is a precise and sensitive method in the detection of maternal cell contamintation. It could be useful in clinical application.

A temperature control method for shortening thermal cycling time to achieve rapid polymerase chain reaction (PCR) in a disposable polymer microfluidic device

NASA Astrophysics Data System (ADS)

Bu, Minqiang; Perch-Nielsen, Ivan R.; Sørensen, Karen S.; Skov, Julia; Sun, Yi; Duong Bang, Dang; Pedersen, Michael E.; Hansen, Mikkel F.; Wolff, Anders

2013-07-01

We present a temperature control method capable of effectively shortening the thermal cycling time of polymerase chain reaction (PCR) in a disposable polymer microfluidic device with an external heater and a temperature sensor. The method employs optimized temperature overshooting and undershooting steps to achieve a rapid ramping between the temperature steps for DNA denaturation, annealing and extension. The temperature dynamics within the microfluidic PCR chamber was characterized and the overshooting and undershooting parameters were optimized using the temperature-dependent fluorescence signal from Rhodamine B. The method was validated with the PCR amplification of mecA gene (162 bp) from methicillin-resistant Staphylococcus aureus bacterium (MRSA), where the time for 30 cycles was reduced from 50 min (without over- and undershooting) to 20 min.

Fermentation properties of isomaltooligosaccharides are affected by human fecal enterotypes.

PubMed

Wu, Qinqin; Pi, Xiong'e; Liu, Wei; Chen, Huahai; Yin, Yeshi; Yu, Hongwei D; Wang, Xin; Zhu, Liying

2017-12-01

Isomaltooligosaccharides (IMOs) are enzymatically synthesized oligosaccharides that have potential prebiotic effects. Five IMO substrates with 2-16° of polymerization (DP) were studied for their fermentation capacities using human microbiomes in an in vitro batch fermentation model. Eleven fecal slurries belonging to three enterotypes, including the Bacteroides-, Prevotella- and Mixed-type, exhibited different degradation rates for long chain IMOs (DP 7 to 16). In contrast, the degradation rates for short chain IMOs (DP 2 to 6) were not affected by enterotypes. Both 16S rRNA gene sequencing and quantitative PCR demonstrated that, after fermentation, the Bifidobacterium growth with IMOs was primarily detected in the Bacteroides- and Mixed-type (non-Prevotella-type), and to a lesser degree in the Prevotella-type. Interestingly, the Prevotella-type microbiome had higher levels of propionic acid and butyric acid production than non-Prevotella-type microbiome after IMOs fermentation. Moreover, principal coordinate analysis (PCoA) of both denaturing gradient gel electrophoresis (DGGE) profiling and 16S rRNA sequencing data demonstrated that the microbiome community compositions were separately clustered based on IMO chain length, suggesting significant impact of DP on the bacterial community structure. The current results clearly demonstrated that the IMO chain length could modulate the structure and composition of the human colonic microbiome. Different responses to short and long chain IMOs were observed from three human enterotypes, indicating that IMOs may be used as therapeutic substrates for directly altering human colonic bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Microscopic and molecular studies of the diversity of free-living protozoa in meat-cutting plants.

PubMed

Vaerewijck, Mario J M; Sabbe, Koen; Baré, Julie; Houf, Kurt

2008-09-01

The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini, and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.

Influence of the diet on the microbial diversity of faecal and gastrointestinal contents in gilthead sea bream (Sparus aurata) and intestinal contents in goldfish (Carassius auratus).

PubMed

Silva, Flávia Cristina de Paula; Nicoli, Jacques Robert; Zambonino-Infante, José Luiz; Kaushik, Sadasivam; Gatesoupe, François-Joël

2011-11-01

Fish intestinal microbiota changes with the diet and this effect is of particular interest considering the increasing substitution of fish meal by plant protein sources. The objective of this work was to study the effects of partial substitution of fish meal with lupin and rapeseed meals on gut microbiota of the gilthead sea bream (Sparus aurata) and in goldfish (Carassius auratus). Faecal, gastrointestinal and intestinal contents were characterized using culture-based and molecular methods. Vibrionaceae was high in faeces and in the intestine of sea bream, while a more diverse microbiota was retrieved from the stomach, where Bacillales and Flavobacteriaceae appeared to be influenced by the diet. PCR-denaturing gradient gel electrophoresis profiles revealed a high diversity of the microbiota transiting in the sea bream digestive tract, with a shift between gastric and intestinal communities, especially in the group fed with lupin meal. The goldfish was different, with a predominance of Aeromonas spp., Shewanella putrefaciens and Staphylococcus spp. among the aerotolerant-cultivable bacteria. The culture-independent methods revealed the presence of anaerobes like Cetobacterium somerae, and that of Vibrio spp., likely in a viable, but noncultivable state. There was a trend towards decreasing diversity in goldfish microbiota with the partial substitution by lupin, which seemed to inhibit some taxa. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

[Analysis of community composition in dental plaque of elder people with root caries].

PubMed

Ma, Shan-fen; Liang, Jing-ping; Jiang, Yun-tao; Zhu, Cai-lian

2011-10-01

To analyze the community in dental plaque of elder people with root caries. Total DNAs were extracted from the root caries dental plaques of nine elders over 60 years of age. Polymerase chaid reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial composition, DGGE bands were excised from the gels for sequencing and identification. The dominant genus in root caries dental plaque of elder people were: Acinetobacte [0.9% (1/114)], Actinobaculum [1.8% (2/114)], Actinomyces [15.8% (18/114)], Aggregatibacter [0.9% (1/114)], Capnocytophaga [14.0% (16/114)], Corynebacterium [0.9% (1/114)], Haemophilus [0.9% (1/114)], Mobiluncus [0.9% (1/114)], Naxibacter [0.9% (1/114)], Neisseriaceae [10.5% (12/114)], Porphyromonas [0.9% (1/114)], Prevotella [12.3% (14/114)], Selenomonas [6.1% (7/114)], Staphylococcus [1.8% (2/114)], Oralis streptococcus [6.1% (7/114)], Mutans streptococcu [7.9% (9/114)], Tannerella [0.9% (1/114)], Treponema [1.8% (2/114)], Veillonella [10.5% (12/114)] and two uncultured unknown genus [1.8% (2/114)]. Uncultred genotypes accounted for 19.30% of the total. Gram-positive bacteria genotype accounted for 31.6% (36/114), and Gram-negative bacteria genotype accounted for 66.7% (76/114). There were many bacteria genotypes in root caries dental plaque in the elderly, which were widely distributed. Gram-negative bacteria accounted for the majority. Genotype-specific pathogenic bacteria were not found.

Bacterial consortia at different wine fermentation phases of two typical Central European grape varieties: Blaufränkisch (Frankovka modrá) and Grüner Veltliner (Veltlínske zelené).

PubMed

Godálová, Zuzana; Kraková, Lucia; Puškárová, Andrea; Bučková, Mária; Kuchta, Tomáš; Piknová, Ľubica; Pangallo, Domenico

2016-01-18

This is the first study focused to bacterial diversity and dynamic during the vinification of two important Central Europe grape vines: Blaufränkisch and Grüner Veltliner. The investigation strategy included culture-dependent and culture-independent approaches. Four different agar media were utilized for the isolation of various bacteria occurring in several fermentation stages. The isolates were clustered by fluorescent-ITS PCR and, one or more representatives of each cluster, were identified by 16 rRNA gene sequencing. The culture-independent approach, based on 16S rRNA gene amplification, combined the denaturing gradient gel electrophoresis (DGGE) method and the construction of bacterial clone library for each wine fermentation step. A complex bacterial community was identified, comprising different lactic acid bacteria and acetic acid bacteria, such as Leuconostoc spp., Lactobacillus spp. and Gluconobacter spp. Other OTUs and bacterial isolates embraced the Actinobacteria, Bacilli, Alpha-, Beta- and Gamma-proteobacteria classes. Different taxa already detected by recent studies, such as Sphingomonas, Variovorax, Pantoea, Enterobacter and Tatumella, were detected confirming the continuous occurrence of these kinds of bacteria in wine environment. Moreover, novel genera (Amycolatopsis, Hydrogenophilus, Snodgrassella, Telluria, Gilliamella, Lelliottia, and Lonsdale quercina) never detected before were recognized, too. The role of these, until now anonymous, bacteria during vinification deserves investigation, which could open a new research field in wine technology. Copyright © 2015 Elsevier B.V. All rights reserved.

Soil fungal community shift evaluation as a potential cadaver decomposition indicator.

PubMed

Chimutsa, Monica; Olakanye, Ayodeji O; Thompson, Tim J U; Ralebitso-Senior, T Komang

2015-12-01

Fungi metabolise organic matter in situ and so alter both the bio-/physico-chemical properties and microbial community structure of the ecosystem. In particular, they are responsible reportedly for specific stages of decomposition. Therefore, this study aimed to extend previous bacteria-based forensic ecogenomics research by investigating soil fungal community and cadaver decomposition interactions in microcosms with garden soil (20 kg, fresh weight) and domestic pig (Sus scrofa domesticus) carcass (5 kg, leg). Soil samples were collected at depths of 0-10 cm, 10-20 cm and 20-30 cm on days 3, 28 and 77 in the absence (control -Pg) and presence (experimental +Pg) of Sus scrofa domesticus and used for total DNA extraction and nested polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) profiling of the 18S rRNA gene. The Shannon-Wiener (H') community diversity indices were 1.25±0.21 and 1.49±0.30 for the control and experimental microcosms, respectively, while comparable Simpson species dominance (S) values were 0.65±0.109 and 0.75±0.015. Generally, and in contrast to parallel studies of the bacterial 16S rRNA and 16S rDNA profiles, statistical analysis (t-test) of the 18S dynamics showed no mathematically significant shifts in fungal community diversity (H'; p=0.142) and dominance (S; p=0.392) during carcass decomposition, necessitating further investigations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Ras mutations are rare in solitary cold and toxic thyroid nodules.

PubMed

Krohn, K; Reske, A; Ackermann, F; Müller, A; Paschke, R

2001-08-01

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

The effect of alum addition on microbial communities in poultry litter.

PubMed

Rothrock, M J; Cook, K L; Warren, J G; Sistani, K

2008-08-01

Alum [Al(2)(SO(4))(3).14H(2)O] is a common poultry litter amendment used to decrease water-soluble phosphorus or reduce ammonia volatilization, or both. Although the physiochemical effects of alum addition have been well researched, little attention has been given to the poultry litter microbial communities. The goal of this study was to use molecular biological methods [denaturing gradient gel electrophoresis (DGGE), community cloning, and quantitative real-time PCR] to characterize general, group-specific and pathogenic microbial communities in alum (10% wt/wt) and non-alum-treated litter. According to quantitative real-time PCR analyses, alum addition to the poultry litter resulted in significant reductions in both Campylobacter jejuni and Escherichia coli concentrations by the end of the first month of the experiment (3 log and 2 log, respectively). The concentrations of Salmonella spp. were below detection (<5 x 10(3) cell.g(-1) of litter) for the entire experiment. The DGGE analyses revealed significant reductions in the Clostridium/Eubacterium and low %GC gram-positive groups in the alum-treated litters by the end of the first month, with no bands detectable for either group after 8 wk of incubation. Conversely, minimal effects of alum addition were observed in the Actinomycetes community. The most significant shift in the microbial community (based on DGGE analyses) occurred in the fungal population, with a large increase in diversity and abundance within 1 mo of alum addition (1 dominant band on d 0 to 9 dominant bands at 4 wk). Specifically, the incidence of Aspergillus spp. increased from 0 to 50% of the sequences in fungal clone libraries (n = 80) over the course of the experiment. This suggests that the addition of alum to poultry litter potentially shifts the microbial populations from bacterially dominated to dominated by fungi. The ramifications of this shift in dominance are still unknown, and future work will be aimed at characterizing these fungi and elucidating their role in the acidified litter environment.

Bacillus pumilus ES4: candidate plant growth-promoting bacterium to enhance establishment of plants in mine tailings

PubMed Central

de-Bashan, Luz E.; Hernandez, Juan-Pablo; Bashan, Yoav; Maier, Raina

2014-01-01

Three plant growth-promoting bacteria (PGPB; Bacillus pumilus ES4, B. pumilus RIZO1, and Azospirillum brasilense Cd) were tested for their ability to enhance plant growth and development of the native Sonoran Desert shrub quailbush (Atriplex lentiformis) and for their effect on the native bacterial community in moderately acidic, high-metal content (AHMT) and in neutral, low metal content natural tailings (NLMT) in controlled greenhouse experiments. Inoculation of quailbush with all three PGPB significantly enhanced plant growth parameters, such as germination, root length, dry weight of shoots and roots, and root/shoot ratio in both types of tailings. The effect of inoculation on the indigenous bacterial community by the most successful PGPB Bacillus pumilus ES4 was evaluated by denaturating gradient gel electrophoresis (PCR-DGGE) fingerprinting and root colonization was followed by specific fluorescent in situ hybridization (FISH). Inoculation with this strain significantly changed the bacterial community over a period of 60 days. FISH analysis showed that the preferred site of colonization was the root tips and root elongation area. This study shows that inoculation of native perennial plants with PGPB can be used for developing technologies for phytostabilizing mine tailings. PMID:25009362

Studies into the prevalence of Mycoplasma species in small ruminants in Benue State, North-central Nigeria.

PubMed

Akwuobu, Chinedu A; Ayling, Roger D; Chah, Kennedy Foinkfu; Oboegbulem, Stephen I

2014-08-01

The indicative prevalence of respiratory Mycoplasma species in small ruminants (SR) was determined in North-central Nigeria. Nasal swabs from 172 sheep and 336 goats from the Northeast, Northwest and South Senatorial Districts of Benue State were examined. Initial Mycoplasma isolation used Mycoplasma culture techniques followed by digitonin sensitivity testing. Species identification was done using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Overall, Mycoplasma organisms were isolated from 131 (25.8 %) of the 508 SR examined. Prevalence rates of 18.1 and 29.8 % were recorded for sheep and goats, respectively. A total of 135 isolates of Mycoplasma belonging to three different species were identified: Mycoplasma ovipneumoniae (127), Mycoplasma arginini (7) and Mycoplasma mycoides subspecies capri (1). More than one Mycoplasma species were detected in four (3.1 %) of the 131 confirmed Mycoplasma positive cultures. Mycoplasma was isolated from 16.2 and 29.1 % of animals with and without respiratory signs, respectively. The high isolation rate of mycoplasmas in apparently healthy and clinically sick sheep and goats in this study indicates a carrier status in these SR which may constitute a serious problem in disease control.

Suppression of Listeria monocytogenes by the Native Micro-Flora in Teewurst Sausage.

PubMed

Austin-Watson, Clytrice; Grant, Ar'Quette; Brice, Michline

2013-10-21

Modern consumers are interested in the use of non-chemical methods to control pathogens when heat sterilization is not an option. Such is the case with teewurst sausage, a raw spreadable sausage and a popular German commodity. Although Listeria was not found in teewurst, the optimal microbial growing conditions of teewurst coupled with the ubiquity of L. monocytogenes in nature, makes the possibility of contamination of products very possible. This pilot study was conducted to examine teewurst's native micro-flora's ability to suppress the outgrowth of L. monocytogenes at 10 °C using standard plate counts and PCR-DGGE. Traditional plating methods showed L. monocytogenes growth significantly decreased when in competition with the teewurst's native micro-flora ( p < 0.05). The native micro-flora of the teewurst suppressed the overall growth of L. monocytogenes by an average of two logs, under these conditions. Denaturing Gradient Gel Electrophoresis (DGGE) amplicons with unique banding patterns were extracted from DGGE gel for identification. Brochothrix thermosphacta and Lactobacillus curvatus were identified as a part of the teewurst's native micro-flora. Although the native micro-flora did not decrease L. monocytogenes to below limits of detection, it was enough of a decrease to warrant further investigation.

Molecular detection of Lactobacillus species in the neovagina of male-to-female transsexual women

PubMed Central

Petricevic, Ljubomir; Kaufmann, Ulrike; Domig, Konrad J.; Kraler, Manuel; Marschalek, Julian; Kneifel, Wolfgang; Kiss, Herbert

2014-01-01

There is a general opinion that penile skin lined neovagina of transsexual women is not able to support the growth of lactobacilli. This study was undertaken to prove if lactobacilli strains could survive in neovagina and to characterise the most dominant Lactobacillus species. Sixty three male-to-female transsexual women without abnormal vaginal discharge, clinical signs of infection were recruited on an ongoing basis from among transsexual outpatients in an academic research institution and tertiary care centre. Neovaginal smears were taken for molecular Lactobacillus spp. profiling by denaturing gradient gel electrophoresis (PCR–DGGE). Lactobacillus species were detected from 47/63 transsexual women (75%). The 279 Lactobacillus signals detected by PCR-DGGE technique belonged to 13 different species. Lactobacilli of the L. delbrueckii group (L. gasseri, L. crispatus, L. johnsonii, L. iners, L. jensenii) were predominant. More than 90% of women harboured a combination of two or more neovaginal Lactobacillus species. In this study we report the frequent occurrence of lactobacilli from neovagina of transsexual women. Both, frequency and composition were similar to the normal lactic acid bacterial microflora in both women of reproductive age and postmenopausal women. PMID:24434849

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains.

PubMed

Hamet, Maria Fernanda; Londero, Alejandra; Medrano, Micaela; Vercammen, Elisabeth; Van Hoorde, Koenraad; Garrote, Graciela L; Huys, Geert; Vandamme, Peter; Abraham, Analía G

2013-12-01

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)5-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization and stability of lactobacilli and yeast microbiota in kefir grains.

PubMed

Vardjan, T; Mohar Lorbeg, P; Rogelj, I; Čanžek Majhenič, A

2013-05-01

Characterization and stability of lactobacilli and yeasts from kefir grains using culture-dependent and culture-independent methods were investigated in this study. Culture-dependent analysis, followed by sequencing of 16S ribosomal DNA for bacteria and 26S rRNA gene for yeasts, revealed 3 different species of lactobacilli and yeasts, respectively. The most frequently isolated bacterial species were Lactobacillus kefiranofaciens ssp. kefirgranum, Lb. parakefiri, and Lb. kefiri, whereas yeasts belonged to Kluyveromyces marxianus, Kazachstania exigua, and Rhodosporidium kratochvilovae. This study is the first to report on the presence of R. kratochvilovae in kefir grains. On the other hand, PCR-denaturing gradient gel electrophoresis in the culture-independent method showed that the dominant microorganisms were Lb. kefiranofaciens ssp. kefirgranum, Kl. marxianus and Ka. exigua, but did not reveal bands corresponding to Lb. parakefiri, Lb. kefiri, or R. kratochvilovae. Our results support the necessity of combining more techniques for detailed and reliable study of microbial communities in kefir grains. Another interesting finding confirmed that the detected dominant microbiota of kefir grains is very stable and did not change over experimental time. This finding is important to ensure consistent product quality. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Operational and biological analyses of branched water-adjustment and combined treatment of wastewater from a chemical industrial park.

PubMed

Xu, Ming; Cao, Jiashun; Li, Chao; Tu, Yong; Wu, Haisuo; Liu, Weijing

2018-01-01

The combined biological processes of branched water-adjustment, chemical precipitation, hydrolysis acidification, secondary sedimentation, Anoxic/Oxic and activated carbon treatment were used for chemical industrial wastewater treatment in the Taihu Lake Basin. Full-scale treatment resulted in effluent chemical oxygen demand, total nitrogen, NH 3 -N and total phosphorus of 35.1, 5.20, 3.10 and 0.15 mg/L, respectively, with a total removal efficiency of 91.1%, 67.1%, 70.5% and 89.3%, respectively. In this process, short-circuited organic carbon from brewery wastewater was beneficial for denitrification and second-sulfate reduction. The concentration of effluent fluoride was 6.22 mg/L, which also met the primary standard. Gas Chromatography-Mass Spectrometry analysis revealed that many types of refractory compounds were present in the inflow. Microbial community analysis performed in the summer by PCR-denaturing gradient gel electrophoresis and MiSeq demonstrated that certain special functional bacteria, such as denitrificans, phosphorus-accumulating bacteria, sulfate- and perhafnate-reducing bacteria, aromatic compound-degrading bacteria and organic fluoride-degrading bacteria, present in the bio-tanks were responsible for the acceptable specific biological pollutant reduction achieved.

Microbial community of cyanobacteria mats in the intertidal zone of oil-polluted coast of Saudi Arabia.

PubMed

Al-Thukair, A A; Abed, R M M; Mohamed, L

2007-02-01

Cyanobacterial mats are found at various locations along the coast of the Eastern Province of Saudi Arabia. Those mats were affected by severe oil pollution following 1991 oil spill. In this study, samples from Abu Ali Island were collected at three selected sampling sites across the intertidal zone (Lower, Middle, and Upper) in order to understand the effect of extreme environmental conditions of high salinity, temperature and desiccation on distribution of cyanobacteria along the oil polluted intertidal zone. Our investigation of composition of cyanobacteria and diatoms was carried out using light microscopy, and Denaturant Gradient Gel Electrophoresis (DGGE) technique. Light microscopy identification revealed dominant cyanobacteria to be affiliated with genera Phormidium, Microcoleus, and Schizothrix, and to a lesser extent with Oscillatoria, Halothece, and various diatom species. The analysis of DGGE of PCR-amplified 16S rRNA fragments showed that the diversity of cyanobacteria decreases as we proceed from the lower to the upper intertidal zone. Accordingly, the tidal regime, salinity, elevated ambient air temperature, and desiccation periods have a great influence on the distribution of cyanobacterial community in the oil polluted intertidal zone of Abu Ali Island.

DGGE and multivariate analysis of a yeast community in spontaneous cocoa fermentation process.

PubMed

Ferreira, A C R; Marques, E L S; Dias, J C T; Rezende, R P

2015-12-28

Cocoa bean is the main raw material used in the production of chocolate. In southern Bahia, Brazil, cocoa farming and processing is an important economic activity. The fermentation of cocoa is the processing stage that yields important chocolate flavor precursors and complex microbial involvement is essential for this process. In this study, PCR-denaturing gradient gel electrophoreses (DGGE) was used to investigate the diversity of yeasts present during the spontaneous fermentation of cocoa in southern Bahia. The DGGE analysis revealed a richness of 8 to 13 distinct bands of varied intensities among the samples; and samples taken at 24, 36, and 48 h into the fermentation process were found to group with 70% similarity and showed the greatest diversity of bands. Hierarchical clustering showed that all samples had common operational taxonomic units (OTUs) and the highest number of OTUs was found in the 48 h sample. Variations in pH and temperature observed within the fermenting mass over time possibly had direct effects on the composition of the existing microbial community. The findings reported here indicate that a heterogeneous yeast community is involved in the complex cocoa fermentation process, which is known to involve a succession of specialized microorganisms.

Houttuynia cordata Facilitates Metformin on Ameliorating Insulin Resistance Associated with Gut Microbiota Alteration in OLETF Rats.

PubMed

Wang, Jing-Hua; Bose, Shambhunath; Lim, Soo-Kyoung; Ansari, AbuZar; Chin, Young-Won; Choi, Han Seok; Kim, Hojun

2017-09-22

Metformin and Houttuynia cordata are representative anti-diabetic therapeutics in western and oriental medicine, respectively. The current study examined the synergistic anti-diabetic effect of Houttuynia cordata extraction (HCE) and metformin combination in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Fecal microbiota were analyzed by denaturing gradient gel electrophoresis (DGGE) and real-time PCR. Combining HCE + metformin resulted in significantly ameliorated glucose tolerance (oral glucose tolerance test (OGTT))-the same as metformin alone. Particularly, results of the insulin tolerance test (ITT) showed that combining HCE + metformin dramatically improved insulin sensitivity as compared to metformin treatment alone. Both fecal and serum endotoxin, as well as cytokines (tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6)) were significantly ameliorated by HCE + metformin compared to metformin alone. Meanwhile, the activation of AMPK (adenosine monophosphate-activated protein kinase) by metformin was distinctly enhanced by HCE. Both of HCE and metformin evidently changed the gut microbiota composition, causing the alteration of bacterial metabolite, like short-chain fatty acids. H. cordata , together with metformin, exerts intensive sensibilization to insulin; the corresponding mechanisms are associated with alleviation of endotoxemia via regulation of gut microbiota, particularly Roseburia , Akkermansia , and Gram-negative bacterium.

Houttuynia cordata Facilitates Metformin on Ameliorating Insulin Resistance Associated with Gut Microbiota Alteration in OLETF Rats

PubMed Central

Bose, Shambhunath; Lim, Soo-Kyoung; Ansari, AbuZar; Chin, Young-Won; Choi, Han Seok; Kim, Hojun

2017-01-01

Metformin and Houttuynia cordata are representative anti-diabetic therapeutics in western and oriental medicine, respectively. The current study examined the synergistic anti-diabetic effect of Houttuynia cordata extraction (HCE) and metformin combination in Otsuka Long–Evans Tokushima Fatty (OLETF) rats. Fecal microbiota were analyzed by denaturing gradient gel electrophoresis (DGGE) and real-time PCR. Combining HCE + metformin resulted in significantly ameliorated glucose tolerance (oral glucose tolerance test (OGTT))—the same as metformin alone. Particularly, results of the insulin tolerance test (ITT) showed that combining HCE + metformin dramatically improved insulin sensitivity as compared to metformin treatment alone. Both fecal and serum endotoxin, as well as cytokines (tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6)) were significantly ameliorated by HCE + metformin compared to metformin alone. Meanwhile, the activation of AMPK (adenosine monophosphate-activated protein kinase) by metformin was distinctly enhanced by HCE. Both of HCE and metformin evidently changed the gut microbiota composition, causing the alteration of bacterial metabolite, like short-chain fatty acids. H. cordata, together with metformin, exerts intensive sensibilization to insulin; the corresponding mechanisms are associated with alleviation of endotoxemia via regulation of gut microbiota, particularly Roseburia, Akkermansia, and Gram-negative bacterium. PMID:28937612

Bacterial microbiota profile in gills of modified atmosphere-packaged oysters stored at 4 °C.

PubMed

Chen, Huibin; Wang, Meiying; Lin, Xiangzhi; Shi, Caihua; Liu, Zhiyu

2017-02-01

As filter-feeding bivalves, oysters can accumulate microorganisms into their gills, causing spoilage and potential safety issues. This study aims to investigate the changes in the gill microbiota of oysters packed under air and modified atmospheres (MAs, 50% CO 2 : 50% N 2 , 70% CO 2 : 30% O 2 , and 50% CO 2 : 50% O 2 ) during storage at 4 °C. The diversity of bacterial microbiota in oyster gills was profiled through polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis on the 16S rRNA gene V3 region to describe the variation during the entire storage period. The DGGE profile revealed high bacterial diversity in the air- and MA-packaged oyster gills, and the spoilage bacterial microbiota varied in the MA-packaged oyster gills. Results indicated that CO 2 :O 2 (70%:30%) was suitable for oyster MA packaging and that high bacterial loads in oyster gills need to be considered during storage. In addition, Lactobacillus and Lactococcus species were found to grow dominantly in fresh oyster gills under MA packaging, which supports the potential application of MA packaging for oyster storage. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dietary supplementation with sorbitol results in selective enrichment of lactobacilli in rat intestine.

PubMed

Sarmiento-Rubiano, Luz Adriana; Zúñiga, Manuel; Pérez-Martínez, Gaspar; Yebra, María Jesús

2007-01-01

A potential prebiotic action has been ascribed to sorbitol, but in vivo evidence of this remains scarce. In the present work, the effect of sorbitol was compared to that of fructo-oligosaccharides (FOS) in a rat model. Microbiota changes, particularly in lactobacilli, were analyzed on fecal, colonic and cecal samples. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons using universal primers showed that FOS and sorbitol diets exerted a strong influence upon gut microbiota patterns. When Lactobacillus group-specific primers were used, DGGE profiles revealed five DNA bands that belonged to Lactobacillus johnsonii, Lactobacillus sp. AD102, Lactobacillus intestinalis, Lactobacillus murinus and Lactobacillus reuteri. Although these species are present in all dietary groups, quantification by real-time PCR showed that sorbitol and FOS intake increased L. reuteri cell numbers, and sorbitol also contributed to maintaining the levels of Lactobacillus sp. AD102. Analysis of organic acid concentrations showed that sorbitol intake significantly increased colonic and cecal butyrate levels. Hence, sorbitol, which is widely used as a low-calorie sweetener, has the capacity, in our animal model, to modify gut microbiota activity in such a way as to possibly contribute to healthy colonic mucosa.

Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil

PubMed Central

Oved, Tamar; Shaviv, Avi; Goldrath, Tal; Mandelbaum, Raphi T.; Minz, Dror

2001-01-01

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the α-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein. PMID:11472914

Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR.

PubMed

Gentilini, Fabio; Turba, Maria E

2014-01-01

A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations. Copyright © 2014 Elsevier B.V. All rights reserved.

The "COLD-PCR approach" for early and cost-effective detection of tyrosine kinase inhibitor resistance mutations in EGFR-positive non-small cell lung cancer.

PubMed

Mairinger, Fabian D; Vollbrecht, Claudia; Streubel, Anna; Roth, Andreas; Landt, Olfert; Walter, Henry F R; Kollmeier, Jens; Mairinger, Thomas

2014-01-01

Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients' exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies. Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing. COLD-PCR-based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible. COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings

PubMed Central

Wong, Grace; Wong, Isaac; Chan, Kamfai; Hsieh, Yicheng; Wong, Season

2015-01-01

Background Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3°C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses. Methodology/Principal Findings In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the “archaic” method of hand-transferring PCR tubes between water baths. Conclusions/Significance We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings. PMID:26146999

Molecular diversity of the rumen microbiome of Norwegian reindeer on natural summer pasture.

PubMed

Sundset, Monica A; Edwards, Joan E; Cheng, Yan Fen; Senosiain, Roberto S; Fraile, Maria N; Northwood, Korinne S; Praesteng, Kirsti E; Glad, Trine; Mathiesen, Svein D; Wright, André-Denis G

2009-02-01

The molecular diversity of the rumen microbiome was investigated in five semi-domesticated adult female Norwegian reindeer (Rangifer tarandus tarandus) grazing on natural summer pastures on the coast of northern Norway (71.00 degrees N, 25.30 degrees E). Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using quantitative real-time polymerase chain reaction (PCR), were 3.17x10(9), 5.17x10(11) and 4.02x10(7), respectively. Molecular diversity of rumen methanogens was revealed using a 16S rRNA gene library (54 clones) constructed using pooled PCR products from the whole rumen contents of the five individual reindeer. Based upon a similarity criterion of <97%, a total of 19 distinct operational taxonomic units (OTUs) were identified, nine of which are potential new species. The 16S rRNA sequences generated from the reindeer rumen exhibited a high degree of sequence similarity to methanogens affiliated with the families Methanobacteriaceae (14 OTUs) and Methanosarcinaceae (one OTU). Four of the OTUs detected belonged to a group of uncultivated archaea previously found in domestic ruminants and thought to be dominant in the rumen together with Methanobrevibacter spp. Denaturing gradient gel electrophoresis profiling of the rumen bacterial 16S rRNA gene and the protozoal 18S rRNA gene indicated a high degree of animal variation, although some bands were common to all individuals. Automated ribosomal intergenic spacer analysis (ARISA) profiling of the ruminal Neocallimastigales population indicated that the reindeer are likely to contain more than one type of anaerobic fungus. The ARISA profile from one animal was distinct from the other four. This is the first molecular investigation of the ruminal methanogenic archaea in reindeer, revealing higher numbers than expected based on methane emission data available. Also, many of the reindeer archaeal 16S rRNA gene sequences were similar to those reported in domesticated ruminants in Australia, Canada, China, New Zealand and Venezuela, supporting previous findings that there seems to be no host type or geographical effect on the methanogenic archaea community structure in ruminants.

Microbial Community Composition Associated with Maotai Liquor Fermentation.

PubMed

Wang, Qiang; Zhang, Hongxun; Liu, Xiu

2016-06-01

The solid-state fermentation state of Chinese Maotai liquor involves the interaction of several complex microbial communities leading to the generation of the most complex liquor fermentation system in the world and contributes to the unique flavor and aroma of the liquor. In this study, total DNA was extracted from 3 fermented grain samples (FG1, FG2, and FG3) and 12 environmental samples, including Daqu (DA1, DA2, DA3, and DA4), cellar mud (CS1, CS2, and CS3), soil (SL1 and SL2), air (A1 and A2), and sorghum (SH), and the 16S and 18S rRNA genes were amplified. The distribution of typical microorganisms in the samples was analyzed using nested PCR-denaturing gradient gel electrophoresis, while quantitative PCR amplification of 16S rRNA and internal transcribed spacer genes was performed to estimate the microbial abundance present in each sample. The results indicated that Daqu was the primary source of bacteria, followed by the air, soil, and sorghum samples, while the majority of the fungi responsible for Maotai liquor fermentation were from Daqu and sorghum. Highest bacterial concentrations were found in fermented grains, followed by Daqu and sorghum, while the highest fungal concentrations were found in Daqu, followed by sorghum and an air sample from outside the liquor production area. The findings of this study may provide information regarding the mechanisms responsible for flavor development in Maotai liquor, and may be used to further optimize the traditional art of making liquor. © 2016 Institute of Food Technologists®

Microbiological and meteorological analysis of two Australian dust storms in April 2009.

PubMed

Lim, Natalie; Munday, Chris I; Allison, Gwen E; O'Loingsigh, Tadhg; De Deckker, Patrick; Tapper, Nigel J

2011-12-15

Dust is an important source of bioaerosols including bacteria. In this study, the microbiology and meteorology of specific dust storms in Australia were investigated. The samples were collected from two dust events in April 2009 that were characterised by intense cold fronts that entrained dust from the highly erodible and drought-stricken Mallee and Riverina regions of Victoria and central NSW. In the first storm, the dust travelled eastward over Canberra and Sydney, and in the second storm, the dust travelled east/southeastward over Canberra and Melbourne. Rain fell on both cities during the second dust storm. Dust and rain samples were collected, cultured, and the composition compared using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Multiple bands were evident on DGGE indicative of a diverse microflora, and identification of several bands confirmed the presence of multiple genera and species representing three phyla. Numerous bands represented Bacillus species, and these were present in multiple dust samples collected from both Canberra and Melbourne. Interestingly, the microflora present in rain samples collected in Canberra during the second dust storm was quite different and the DGGE banding patterns from these samples clustered separately to most dust samples collected at the same time. Identification of several DGGE bands and PCR products from these rain samples indicated the presence of Pseudomonas species. These results indicate that Australian dust and rain have a diverse microflora and highlights the contribution of dust events to the distribution of microbes in the environment. Copyright © 2011 Elsevier B.V. All rights reserved.

Microbial communities in air and wine of a winery at two consecutive vintages.

PubMed

Pérez-Martín, Fátima; Seseña, Susana; Fernández-González, Mónica; Arévalo, María; Palop, María Llanos

2014-11-03

The aim of this study was to assess, both quantitatively and qualitatively, the populations of lactic acid bacteria (LAB) and yeasts in air and wine of a winery, in order to evaluate the possible exchange of microorganisms between them. Samples were taken in a winery located in Castilla-La Mancha (Spain) during the winemaking period of two consecutive vintages (2011 and 2012). The microbial composition was determined by using both a culture-dependent method and a culture-independent method, PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In addition, genetic characterization of isolates from plates was carried out. A high diversity of species was detected in air and wine samples from both vintages. Leuconostoc mesenteroides was the predominant lactic acid bacteria in air from both vintages while Oenococcus oeni was the predominant in wine. Saccharomyces cerevisiae was the most frequently isolated yeast in both air and wine. Typing of O. oeni and S. cerevisiae isolates from air and wine samples showed the presence of coincident genotypes in both samples, that would confirm the exchange of microorganisms between the two environments, air and wine, and furthermore some of these genotypes were also found at samples taken at different vintages, indicating that they would remain in the winery. The results display the influence of the activity taking place in the winery and the moment of fermentation of the wines in tanks, on the microorganisms present in the air and the role of the air for the dispersal of microorganisms within the winery. Copyright © 2014 Elsevier B.V. All rights reserved.

Differences between bacterial communities in the gut of a soil-feeding termite (Cubitermes niokoloensis) and its mounds.

PubMed

Fall, Saliou; Hamelin, Jérôme; Ndiaye, Farma; Assigbetse, Komi; Aragno, Michel; Chotte, Jean Luc; Brauman, Alain

2007-08-01

In tropical ecosystems, termite mound soils constitute an important soil compartment covering around 10% of African soils. Previous studies have shown (S. Fall, S. Nazaret, J. L. Chotte, and A. Brauman, Microb. Ecol. 28:191-199, 2004) that the bacterial genetic structure of the mounds of soil-feeding termites (Cubitermes niokoloensis) is different from that of their surrounding soil. The aim of this study was to characterize the specificity of bacterial communities within mounds with respect to the digestive and soil origins of the mound. We have compared the bacterial community structures of a termite mound, termite gut sections, and surrounding soil using PCR-denaturing gradient gel electrophoresis (DGGE) analysis and cloning and sequencing of PCR-amplified 16S rRNA gene fragments. DGGE analysis revealed a drastic difference between the genetic structures of the bacterial communities of the termite gut and the mound. Analysis of 266 clones, including 54 from excised bands, revealed a high level of diversity in each biota investigated. The soil-feeding termite mound was dominated by the Actinobacteria phylum, whereas the Firmicutes and Proteobacteria phyla dominate the gut sections of termites and the surrounding soil, respectively. Phylogenetic analyses revealed a distinct clustering of Actinobacteria phylotypes between the mound and the surrounding soil. The Actinobacteria clones of the termite mound were diverse, distributed among 10 distinct families, and like those in the termite gut environment lightly dominated by the Nocardioidaceae family. Our findings confirmed that the soil-feeding termite mound (C. niokoloensis) represents a specific bacterial habitat in the tropics.

Characterization of sulfur oxidizing bacteria related to biogenic sulfuric acid corrosion in sludge digesters.

PubMed

Huber, Bettina; Herzog, Bastian; Drewes, Jörg E; Koch, Konrad; Müller, Elisabeth

2016-07-18

Biogenic sulfuric acid (BSA) corrosion damages sewerage and wastewater treatment facilities but is not well investigated in sludge digesters. Sulfur/sulfide oxidizing bacteria (SOB) oxidize sulfur compounds to sulfuric acid, inducing BSA corrosion. To obtain more information on BSA corrosion in sludge digesters, microbial communities from six different, BSA-damaged, digesters were analyzed using culture dependent methods and subsequent polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). BSA production was determined in laboratory scale systems with mixed and pure cultures, and in-situ with concrete specimens from the digester headspace and sludge zones. The SOB Acidithiobacillus thiooxidans, Thiomonas intermedia, and Thiomonas perometabolis were cultivated and compared to PCR-DGGE results, revealing the presence of additional acidophilic and neutrophilic SOB. Sulfate concentrations of 10-87 mmol/L after 6-21 days of incubation (final pH 1.0-2.0) in mixed cultures, and up to 433 mmol/L after 42 days (final pH <1.0) in pure A. thiooxidans cultures showed huge sulfuric acid production potentials. Additionally, elevated sulfate concentrations in the corroded concrete of the digester headspace in contrast to the concrete of the sludge zone indicated biological sulfur/sulfide oxidation. The presence of SOB and confirmation of their sulfuric acid production under laboratory conditions reveal that these organisms might contribute to BSA corrosion within sludge digesters. Elevated sulfate concentrations on the corroded concrete wall in the digester headspace (compared to the sludge zone) further indicate biological sulfur/sulfide oxidation in-situ. For the first time, SOB presence and activity is directly relatable to BSA corrosion in sludge digesters.

Influence of red wine polyphenols and ethanol on the gut microbiota ecology and biochemical biomarkers.

PubMed

Queipo-Ortuño, María Isabel; Boto-Ordóñez, María; Murri, Mora; Gomez-Zumaquero, Juan Miguel; Clemente-Postigo, Mercedes; Estruch, Ramon; Cardona Diaz, Fernando; Andrés-Lacueva, Cristina; Tinahones, Francisco J

2012-06-01

Few studies have investigated the effect of dietary polyphenols on the complex human gut microbiota, and they focused mainly on single polyphenol molecules and select bacterial populations. The objective was to evaluate the effect of a moderate intake of red wine polyphenols on select gut microbial groups implicated in host health benefits. Ten healthy male volunteers underwent a randomized, crossover, controlled intervention study. After a washout period, all of the subjects received red wine, the equivalent amount of de-alcoholized red wine, or gin for 20 d each. Total fecal DNA was submitted to polymerase chain reaction(PCR)-denaturing gradient gel electrophoresis and real-time quantitative PCR to monitor and quantify changes in fecal microbiota. Several biochemical markers were measured. The dominant bacterial composition did not remain constant over the different intake periods. Compared with baseline, the daily consumption of red wine polyphenol for 4 wk significantly increased the number of Enterococcus, Prevotella, Bacteroides, Bifidobacterium, Bacteroides uniformis, Eggerthella lenta, and Blautia coccoides-Eubacterium rectale groups (P < 0.05). In parallel, systolic and diastolic blood pressures and triglyceride, total cholesterol, HDL cholesterol, and C-reactive protein concentrations decreased significantly (P < 0.05). Moreover, changes in cholesterol and C-reactive protein concentrations were linked to changes in the bifidobacteria number. This study showed that red wine consumption can significantly modulate the growth of select gut microbiota in humans, which suggests possible prebiotic benefits associated with the inclusion of red wine polyphenols in the diet. This trial was registered at controlled-trials.com as ISRCTN88720134.

Bacaba beverage produced by Umutina Brazilian Amerindians: Microbiological and chemical characterization

PubMed Central

Puerari, Cláudia; Magalhães-Guedes, Karina Teixeira; Schwan, Rosane Freitas

2015-01-01

Bacaba chicha is a beverage prepared by the indigenous Umutina people from the bacaba fruit (Oenocarpus bacaba), a purple berry that is rich in fat and carbohydrates, as well as a source of phenolic compounds. In this study, samples of bacaba chicha beverage were collected, and the microbial community was assessed using culture-dependent and -independent techniques. The nutritional composition and metabolite profiles were analyzed, and species belonging to lactic acid bacteria (LAB) and yeasts were detected. The LAB group detected by culture-dependent analysis included Enterococcus hormaechei and Leuconostoc lactis. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) detected additional Propionibacterium avidum, Acetobacter spp., and uncultured bacteria. Pichia caribbica and Pichia guilliermondii were detected in a culture-dependent method, and Pichia caribbica was confirmed by PCR-DGGE analysis. The pH value of the beverage was 6.2. The nutritional composition was as follows: 16.47 ± 0.73 g 100 mL-1 dry matter, 2.2 ± 0.0 g 100 mL-1 fat, 3.36 ± 0.44 g 100 mL-1 protein, and 10.87 ± 0.26 g 100 mL-1 carbohydrate. The metabolites detected were 2.69 g L-1 succinic acid, 0.9 g L-1 acetic acid, 0.49 g L-1 citric acid, 0.52 g L-1 ethanol, and 0.4 g L-1 glycerol. This is the first study to identify microbial diversity in bacaba chicha spontaneous fermentation. This study is also the starting step in the immaterial record of this Brazilian indigenous beverage prepared from bacaba fruit. PMID:26691483

Bacaba beverage produced by Umutina Brazilian Amerindians: Microbiological and chemical characterization.

PubMed

Puerari, Cláudia; Magalhães-Guedes, Karina Teixeira; Schwan, Rosane Freitas

2015-01-01

Bacaba chicha is a beverage prepared by the indigenous Umutina people from the bacaba fruit (Oenocarpus bacaba), a purple berry that is rich in fat and carbohydrates, as well as a source of phenolic compounds. In this study, samples of bacaba chicha beverage were collected, and the microbial community was assessed using culture-dependent and -independent techniques. The nutritional composition and metabolite profiles were analyzed, and species belonging to lactic acid bacteria (LAB) and yeasts were detected. The LAB group detected by culture-dependent analysis included Enterococcus hormaechei and Leuconostoc lactis. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) detected additional Propionibacterium avidum, Acetobacter spp., and uncultured bacteria. Pichia caribbica and Pichia guilliermondii were detected in a culture-dependent method, and Pichia caribbica was confirmed by PCR-DGGE analysis. The pH value of the beverage was 6.2. The nutritional composition was as follows: 16.47 ± 0.73 g 100 mL-1 dry matter, 2.2 ± 0.0 g 100 mL-1 fat, 3.36 ± 0.44 g 100 mL-1 protein, and 10.87 ± 0.26 g 100 mL-1 carbohydrate. The metabolites detected were 2.69 g L-1 succinic acid, 0.9 g L-1 acetic acid, 0.49 g L-1 citric acid, 0.52 g L-1 ethanol, and 0.4 g L-1 glycerol. This is the first study to identify microbial diversity in bacaba chicha spontaneous fermentation. This study is also the starting step in the immaterial record of this Brazilian indigenous beverage prepared from bacaba fruit.

Effects of Crude Oil, Dispersant, and Oil-Dispersant Mixtures on Human Fecal Microbiota in an In Vitro Culture System

PubMed Central

Kim, Jong Nam; Kim, Bong-Soo; Kim, Seong-Jae; Cerniglia, Carl E.

2012-01-01

ABSTRACT The Deepwater Horizon oil spill of 2010 raised concerns that dispersant and dispersed oil, as well as crude oil itself, could contaminate shellfish and seafood habitats with hazardous residues that had potential implications for human health and the ecosystem. However, little is known about the effects of crude oil and dispersant on the human fecal microbiota. The aim of this research was to evaluate the potential effects of Deepwater Horizon crude oil, Corexit 9500 dispersant, and their combination on human fecal microbial communities, using an in vitro culture test system. Fecal specimens from healthy adult volunteers were made into suspensions, which were then treated with oil, dispersant, or oil-dispersant mixtures under anaerobic conditions in an in vitro culture test system. Perturbations of the microbial community, compared to untreated control cultures, were assessed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and pyrosequencing methods. DGGE and pyrosequencing analysis showed that oil-dispersant mixtures reduced the diversity of fecal microbiota from all individuals. Real-time PCR results indicated that the copy numbers of 16S rRNA genes in cultures treated with dispersed oil or oil alone were significantly lower than those in control incubations. The abundance of the Bacteroidetes decreased in crude oil-treated and dispersed-oil-treated cultures, while the Proteobacteria increased in cultures treated with dispersed oil. In conclusion, the human fecal microbiota was affected differently by oil and dispersed oil, and the influence of dispersed oil was significantly greater than that of either oil or dispersant alone compared to control cultures. PMID:23093387

Rumen protozoa and methanogenesis: not a simple cause-effect relationship.

PubMed

Morgavi, Diego P; Martin, Cécile; Jouany, Jean-Pierre; Ranilla, Maria José

2012-02-01

Understanding the interactions between hydrogen producers and consumers in the rumen ecosystem is important for ruminant production and methane mitigation. The present study explored the relationships between rumen protozoa, methanogens and fermentation characteristics. A total of six donor sheep harbouring (F, faunated) or not (D, defaunated) protozoa in their rumens (D animals were kept without protozoa for a period of a few months (D - ) or for more than 2 years (D+)) were used in in vitro and in vivo experiments. In vitro the absence of protozoa decreased NH3 and butyrate production and had no effect on methane. In contrast, the liquid-associated bacterial and methanogens fraction of D+ inocula produced more methane than D -  and F inoculum (P < 0·05). In vivo fermentation parameters of donor animals showed the same trend on NH3 and butyrate and showed that D+ animals were high methane emitters, while D -  were the lowest ( - 35 %). The concentration of dissolved dihydrogen measured after feeding followed the opposite trend. Methane emissions did not correlate with the relative abundance of methanogens in the rumen measured by quantitative PCR, but there was a trend for higher methanogens concentration in the solid-associated population of D+ animals compared with D -  animals. In contrast, PCR-denaturing gradient gel electrophoresis profiles of methanogens' methyl coenzyme-M reductase A gene showed a clear clustering in liquid-associated fractions for all three groups of donors but fewer differences in solid-associated fractions. These results show that the absence of protozoa may affect differently the methanogen community and methane emissions in wethers.

Microbial community and treatment ability investigation in AOAO process for the optoelectronic wastewater treatment using PCR-DGGE biotechnology.

PubMed

Chen, Hsi-Jien; Lin, Yi-Zi; Fanjiang, Jen-Mao; Fan, Chihhao

2013-04-01

This study aimed to explore the microbial community variation and treatment ability of a full-scale anoxic-aerobic-anoxic-aerobic (AOAO) process used for optoelectronic wastewater treatment. The sludge samples in the biological treatment units were collected and subsequently subjected to polymerase chain reaction (PCR) amplification and denaturing gradient gel electrophoresis identification and the wastewater components such as BOD5 and NH3-N were evaluated during the processes. The group specific primers selected were targeting at the kingdom Bacteria, the Acidobacterium, the α-proteobacteria, the β-proteobacteria ammonia oxidizers, Actinobacteria and methyllotrophs, and the 16S rDNA clone libraries were established. Ten different clones were obtained using the Bacteria primers and eight different clones were obtained using the β-proteobacteria ammonia oxidizer primers. Over 95 % of BOD5 and 90 % of NH3-N were removed from the system. The microbial community analysis showed that the Janthinobacterium sp. An8 and Nitrosospira sp. were the dominant species throughout the AOAO process. Across the whole clone library, six clones showed closely related to Janthinobacterium sp. and these species seemed to be the dominant species with more than 50 % occupancy of the total population. Nitrosospira sp. was the predominant species within the β-proteobacteria and occupied more than 30 % of the total population in the system. These two strains were the novel species specific to the AOAO process for optoelectronic treatment, and they were found strongly related to the system capability of removing aquatic contaminants by inspecting the wastewater concentration variation across the system.

Community structure of ammonia-oxidizing bacteria under long-term application of mineral fertilizer and organic manure in a sandy loam soil.

PubMed

Chu, Haiyan; Fujii, Takeshi; Morimoto, Sho; Lin, Xiangui; Yagi, Kazuyuki; Hu, Junli; Zhang, Jiabao

2007-01-01

The effects of mineral fertilizer (NPK) and organic manure on the community structure of soil ammonia-oxidizing bacteria (AOB) was investigated in a long-term (16-year) fertilizer experiment. The experiment included seven treatments: organic manure, half organic manure N plus half fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and the control (without fertilization). N fertilization greatly increased soil nitrification potential, and mineral N fertilizer had a greater impact than organic manure, while N deficiency treatment (PK) had no significant effect. AOB community structure was analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the amoA gene, which encodes the alpha subunit of ammonia monooxygenase. DGGE profiles showed that the AOB community was more diverse in N-fertilized treatments than in the PK-fertilized treatment or the control, while one dominant band observed in the control could not be detected in any of the fertilized treatments. Phylogenetic analysis showed that the DGGE bands derived from N-fertilized treatments belonged to Nitrosospira cluster 3, indicating that N fertilization resulted in the dominance of Nitrosospira cluster 3 in soil. These results demonstrate that long-term application of N fertilizers could result in increased soil nitrification potential and the AOB community shifts in soil. Our results also showed the different effects of mineral fertilizer N versus organic manure N; the effects of P and K on the soil AOB community; and the importance of balanced fertilization with N, P, and K in promoting nitrification functions in arable soils.

Community Structure of Ammonia-Oxidizing Bacteria under Long-Term Application of Mineral Fertilizer and Organic Manure in a Sandy Loam Soil▿

PubMed Central

Chu, Haiyan; Fujii, Takeshi; Morimoto, Sho; Lin, Xiangui; Yagi, Kazuyuki; Hu, Junli; Zhang, Jiabao

2007-01-01

The effects of mineral fertilizer (NPK) and organic manure on the community structure of soil ammonia-oxidizing bacteria (AOB) was investigated in a long-term (16-year) fertilizer experiment. The experiment included seven treatments: organic manure, half organic manure N plus half fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and the control (without fertilization). N fertilization greatly increased soil nitrification potential, and mineral N fertilizer had a greater impact than organic manure, while N deficiency treatment (PK) had no significant effect. AOB community structure was analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the amoA gene, which encodes the α subunit of ammonia monooxygenase. DGGE profiles showed that the AOB community was more diverse in N-fertilized treatments than in the PK-fertilized treatment or the control, while one dominant band observed in the control could not be detected in any of the fertilized treatments. Phylogenetic analysis showed that the DGGE bands derived from N-fertilized treatments belonged to Nitrosospira cluster 3, indicating that N fertilization resulted in the dominance of Nitrosospira cluster 3 in soil. These results demonstrate that long-term application of N fertilizers could result in increased soil nitrification potential and the AOB community shifts in soil. Our results also showed the different effects of mineral fertilizer N versus organic manure N; the effects of P and K on the soil AOB community; and the importance of balanced fertilization with N, P, and K in promoting nitrification functions in arable soils. PMID:17098920

Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica.

PubMed

Pan, Qi; Wang, Feng; Zhang, Yang; Cai, Minghong; He, Jianfeng; Yang, Haizhen

2013-08-01

Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station, Fildes Peninsula, King George Island, Antarctica, using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The classes alpha-, beta-, and gamma-Proteobacteria, as well as the phylum Actinobacteria, were found to be the dominant bacteria in the soils around the Great Wall Station. Although the selected samples were not contaminated by oil, a relationship between soil parameters, microbial biodiversity, and human impact was still seen. Sample sites in human impacted areas showed lower bacterial biodiversity (average H' = 2.65) when compared to non-impacted sites (average H' = 3.05). There was no statistically significant correlation between soil bacterial diversity and total organic carbon (TOC), total nitrogen, or total phosphorus contents of the soil. Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters. In conclusion, microbial biodiversity and community characteristics within relatively small scales (1.5 km) were determined as a function of local environment parameters and anthropogenic impact.

Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

PubMed Central

Alexander, Crispin G.; Wanner, Randy; Johnson, Christopher M.; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

2014-01-01

Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein–ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide–PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes. PMID:25262836

Selection of stable scFv antibodies by phage display.

PubMed

Brockmann, Eeva-Christine

2012-01-01

ScFv fragments are popular recombinant antibody formats but often suffer from limited stability. Phage display is a powerful tool in antibody engineering and applicable also for stability selection. ScFv variants with improved stability can be selected from large randomly mutated phage displayed libraries with a specific antigen after the unstable variants have been inactivated by heat or GdmCl. Irreversible scFv denaturation, which is a prerequisite for efficient selection, is achieved by combining denaturation with reduction of the intradomain disulfide bonds. Repeated selection cycles of increasing stringency result in enrichment of stabilized scFv fragments. Procedures for constructing a randomly mutated scFv library by error-prone PCR and phage display selection for enrichment of stable scFv antibodies from the library are described here.

Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

PubMed

Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

2015-02-03

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

Molecular diversity and tools for deciphering the methanogen community structure and diversity in freshwater sediments.

PubMed

Chaudhary, Prem Prashant; Brablcová, Lenka; Buriánková, Iva; Rulík, Martin

2013-09-01

Methanogenic archaeal communities existing in freshwater sediments are responsible for approximately 50 % of the total global emission of methane. This process contributes significantly to global warming and, hence, necessitates interventional control measures to limit its emission. Unfortunately, the diversity and functional interactions of methanogenic populations occurring in these habitats are yet to be fully characterized. Considering several disadvantages of conventional culture-based methodologies, in recent years, impetus is given to molecular biology approaches to determine the community structure of freshwater sedimentary methanogenic archaea. 16S rRNA and methyl coenzyme M reductase (mcrA) gene-based cloning techniques are the first choice for this purpose. In addition, electrophoresis-based (denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, and terminal restriction fragment length polymorphism) and quantitative real-time polymerase chain reaction techniques have also found extensive applications. These techniques are highly sensitive, rapid, and reliable as compared to traditional culture-dependent approaches. Molecular diversity studies revealed the dominance of the orders Methanomicrobiales and Methanosarcinales of methanogens in freshwater sediments. The present review discusses in detail the status of the diversity of methanogens and the molecular approaches applied in this area of research.

Automated detection system of single nucleotide polymorphisms using two kinds of functional magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Liu, Hongna; Li, Song; Wang, Zhifei; Li, Zhiyang; Deng, Yan; Wang, Hua; Shi, Zhiyang; He, Nongyue

2008-11-01

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome wide codominant SNPs identification. Therefore, large-scale codominant SNPs identification, especially for those associated with complex diseases, has induced the need for completely high-throughput and automated SNP genotyping method. Herein, we present an automated detection system of SNPs based on two kinds of functional magnetic nanoparticles (MNPs) and dual-color hybridization. The amido-modified MNPs (NH 2-MNPs) modified with APTES were used for DNA extraction from whole blood directly by electrostatic reaction, and followed by PCR, was successfully performed. Furthermore, biotinylated PCR products were captured on the streptavidin-coated MNPs (SA-MNPs) and interrogated by hybridization with a pair of dual-color probes to determine SNP, then the genotype of each sample can be simultaneously identified by scanning the microarray printed with the denatured fluorescent probes. This system provided a rapid, sensitive and highly versatile automated procedure that will greatly facilitate the analysis of different known SNPs in human genome.

Genotypic distribution of a specialist model microorganism, Methanosaeta, along an estuarine gradient: does metabolic restriction limit niche differentiation potential?

PubMed

Carbonero, Franck; Oakley, Brian B; Hawkins, Robert J; Purdy, Kevin J

2012-05-01

A reductionist ecological approach of using a model genus was adopted in order to understand how microbial community structure is driven by metabolic properties. The distribution along an estuarine gradient of the highly specialised genus Methanosaeta was investigated and compared to the previously determined distribution of the more metabolically flexible Desulfobulbus. Methanosaeta genotypic distribution along the Colne estuary (Essex, UK) was determined by DNA- and RNA-based denaturing gradient gel electrophoresis and 16S rRNA gene sequence analyses. Methanosaeta distribution was monotonic, with a consistently diverse community and no apparent niche partitioning either in DNA or RNA analyses. This distribution pattern contrasts markedly with the previously described niche partitioning and sympatric differentiation of the model generalist, Desulfobulbus. To explain this difference, it is hypothesised that Methanosaeta's strict metabolic needs limit its adaptation potential, thus populations do not partition into spatially distinct groups and so do not appear to be constrained by gross environmental factors such as salinity. Thus, at least for these two model genera, it appears that metabolic flexibility may be an important factor in spatial distribution and this may be applicable to other microbes.

Microorganisms with Novel Dissimilatory (Bi)Sulfite Reductase Genes Are Widespread and Part of the Core Microbiota in Low-Sulfate Peatlands ▿ †

PubMed Central

Steger, Doris; Wentrup, Cecilia; Braunegger, Christina; Deevong, Pinsurang; Hofer, Manuel; Richter, Andreas; Baranyi, Christian; Pester, Michael; Wagner, Michael; Loy, Alexander

2011-01-01

Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented “core” members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions. PMID:21169452

Phylogenetically and Spatially Close Marine Sponges Harbour Divergent Bacterial Communities

PubMed Central

Hardoim, Cristiane C. P.; Esteves, Ana I. S.; Pires, Francisco R.; Gonçalves, Jorge M. S.; Cox, Cymon J.; Xavier, Joana R.; Costa, Rodrigo

2012-01-01

Recent studies have unravelled the diversity of sponge-associated bacteria that may play essential roles in sponge health and metabolism. Nevertheless, our understanding of this microbiota remains limited to a few host species found in restricted geographical localities, and the extent to which the sponge host determines the composition of its own microbiome remains a matter of debate. We address bacterial abundance and diversity of two temperate marine sponges belonging to the Irciniidae family - Sarcotragus spinosulus and Ircinia variabilis – in the Northeast Atlantic. Epifluorescence microscopy revealed that S. spinosulus hosted significantly more prokaryotic cells than I. variabilis and that prokaryotic abundance in both species was about 4 orders of magnitude higher than in seawater. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of S. spinosulus and I. variabilis differed markedly from each other – with higher number of ribotypes observed in S. spinosulus – and from those of seawater. Four PCR-DGGE bands, two specific to S. spinosulus, one specific to I. variabilis, and one present in both sponge species, affiliated with an uncultured sponge-specific phylogenetic cluster in the order Acidimicrobiales (Actinobacteria). Two PCR-DGGE bands present exclusively in S. spinosulus fingerprints affiliated with one sponge-specific phylogenetic cluster in the phylum Chloroflexi and with sponge-derived sequences in the order Chromatiales (Gammaproteobacteria), respectively. One Alphaproteobacteria band specific to S. spinosulus was placed in an uncultured sponge-specific phylogenetic cluster with a close relationship to the genus Rhodovulum. Our results confirm the hypothesized host-specific composition of bacterial communities between phylogenetically and spatially close sponge species in the Irciniidae family, with S. spinosulus displaying higher bacterial community diversity and distinctiveness than I. variabilis. These findings suggest a pivotal host-driven effect on the shape of the marine sponge microbiome, bearing implications to our current understanding of the distribution of microbial genetic resources in the marine realm. PMID:23300853

Evidence of Campylobacter jejuni reduction in broilers with early synbiotic administration.

PubMed

Baffoni, Loredana; Gaggìa, Francesca; Garofolo, Giuliano; Di Serafino, Gabriella; Buglione, Enrico; Di Giannatale, Elisabetta; Di Gioia, Diana

2017-06-19

C. jejuni is considered a food safety concern to both public health authorities and consumers since it is the leading bacterial cause of food-borne gastroenteritis in humans. A high incidence of C. jejuni in broiler flocks is often correlated to pathogen recovery in retail poultry meat, which is the main source of human infection. In this work broiler chickens were fed with a synbiotic product mixed with conventional feed using two different administration strategies. The synbiotic was formulated with the microencapsulated probiotic Bifidobacterium longum PCB133 and a xylo-oligosaccharide (XOS). 1-day old chicks were infected with C. jejuni strain M1 (10 5 cells) and the synbiotic mixture was then administered starting from the first and the 14th day of chicken life (for animal groups GrpC and GrpB respectively). The goal of this study was to monitor C. jejuni load at caecum level at different sampling time by real-time PCR, identifying the best administration strategy. The microbiological analysis of the caecal content also considered the quantification of Campylobacter spp., Bifidobacterium spp. and B. longum. The supplemented synbiotic was more successful in reducing C. jejuni and Campylobacter spp. when administered lifelong, compared to the shorter supplementation (GrpB). Bifidobacterium spp. quantification did not show significant differences among treatments and B. longum PCB133 was detected in both supplemented groups evidencing the successful colonization of the strain. Moreover, the samples of the control group (GrpA) and GrpC were analysed with PCR-denaturing gradient gel electrophoresis (PCR-DGGE) to compare the caecal microbial community profiles at the beginning and at the end of the trial. Pattern analysis evidenced the strong influence of the early synbiotic supplementation, although a physiological change in the microbial community, occurring during growth, could be observed. Experimental results demonstrate that the synbiotic approach at farm level can be an effective strategy, combined with biosecurity measures, to improve the safety of poultry meat. Copyright © 2017 Elsevier B.V. All rights reserved.

15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

PubMed Central

Alexandrescu, A. T.; Rathgeb-Szabo, K.; Rumpel, K.; Jahnke, W.; Schulthess, T.; Kammerer, R. A.

1998-01-01

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding. PMID:9521116

Product differentiation during continuous-flow thermal gradient PCR.

PubMed

Crews, Niel; Wittwer, Carl; Palais, Robert; Gale, Bruce

2008-06-01

A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.

Immuno-PCR for one step detection of H5N1 avian influenza virus and Newcastle disease virus using magnetic gold particles as carriers.

PubMed

Deng, MingJun; Long, Ling; Xiao, XiZhi; Wu, ZhenXing; Zhang, FengJuan; Zhang, YanMing; Zheng, XiaoLong; Xin, XueQian; Wang, Qun; Wu, DongLai

2011-06-15

Detecting avian influenza virus (AIV) and Newcastle disease virus (NDV) at low concentrations from tracheal and cloacal swabs of avian influenza- and Newcastle disease-infected poultry was carried out using a highly sensitive immunological-polymerase chain reaction (immuno-PCR) method. Magnetic gold particles were pre-coated with a capture antibody, either a monoclonal anti-AIV/H5 or monoclonal anti-NDV/F and viruses serially diluted ten-fold from 10(2) to 10(-5)EID(50)/ml. A biotinylated detection antibody bound to the viral antigen was then linked via a streptavidin bridge to biotinylated reporter DNA. After extensive washing, reporter DNA was released by denaturation, transferred to PCR tubes, amplified, electrophoresed and visualized. An optimized immuno-PCR method was able to detect as little as 10(-4)EID(50)/ml AIV and NDV. To further evaluate the specificity and the clinical application of this IPCR assay for AIV H5N1 and NDV, the tracheal swab specimens, taken from chickens which were infected with H5N1/AIV, H9N2/AIV, H7N2/AIV, NDV, IBDV, IBV/H(120), were detected by IPCR. Our data demonstrated that this monoclonal antibody-based immuno-PCR method provides a platform capable of rapid screening of clinical samples for trace levels of AIV H5 and NDV in one step. Copyright © 2011 Elsevier B.V. All rights reserved.

Characterisation of prototype Nurmi cultures using culture-based microbiological techniques and PCR-DGGE.

PubMed

Waters, Sinéad M; Murphy, Richard A; Power, Ronan F G

2006-08-01

Undefined Nurmi-type cultures (NTCs) have been used successfully to prevent salmonella colonisation in poultry for decades. Such cultures are derived from the caecal contents of specific-pathogen-free birds and are administered via drinking water or spray application onto eggs in the hatchery. These cultures consist of many non-culturable and obligately anaerobic bacteria. Due to their undefined nature it is difficult to obtain approval from regulatory agencies to use these preparations as direct fed microbials for poultry. In this study, 10 batches of prototype NTCs were produced using an identical protocol over a period of 2 years. Traditional microbiological techniques and a molecular culture-independent methodology, polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE), were applied to characterise these cultures and also to examine if the constituents of the NTCs were identical. Culture-dependent analysis of these cultures included plating on a variety of selective and semi-selective agars, examination of colony morphology, Gram-staining and a series of biochemical tests (API, BioMerieux, France). Two sets of PCR-DGGE studies were performed. These involved the amplification of universal and subsequently lactic acid bacteria (LAB)-specific hypervariable regions of a 16S rRNA gene by PCR. Resultant amplicons were subjected to DGGE. Sequence analysis was performed on subsequent bands present in resultant DGGE profiles using the Basic Local Alignment Search Tool (BLAST). Microbiological culturing techniques tended to isolate common probiotic bacterial species from the genera Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Clostridium, Escherichia, Pediococcus and Enterobacterium as well as members of the genera, Actinomyces, Bacteroides, Propionibacterium, Capnocytophaga, Proteus, and Klebsiella. Bacteroides, Enterococcus, Escherichia, Brevibacterium, Klebsiella, Lactobacillus, Clostridium, Bacillus, Eubacterium, Serratia, Citrobacter, Enterobacter, Pectobacterium and Pantoea spp. in addition to unculturable bacteria were identified as constituents of the NTCs using universal PCR-DGGE analysis. A number of the sequences detected by LAB-specific PCR-DGGE were homologous to those of a number of Lactobacillus spp., including L. fermentum, L. pontis, L. crispatus, L. salivarius, L. casei, L. suntoryeus, L. vaginalis, L. gasseri, L. aviaries, L. johnsonii, L. acidophilus, and L. mucosae in addition to a range of unculturable lactobacilli. While NTCs are successful due to their complexity, the presence of members of Lactobacillus spp. amongst other probiotic genera, in these samples possibly lends to the success of the NTC cultures as probiotics or competitive exclusion products in poultry over the decades. PCR-DGGE proved to be an effective tool in detecting non-culturable organisms present in these complex undefined cultures. In conclusion, while the culture-dependent identification methods or PCR-DGGE alone cannot comprehensively elucidate the bacterial species present in such complex cultures, their complementarity provides useful information on the identity of the constituents of NTCs and will aid in future development of defined probiotics. Moreover, for the purpose of analysing prototype NTCs during their development, PCR-DGGE overcomes the limitations associated with conventional culturing methods including their low sensitivities, inability to detect unculturable bacteria and unknown species, very slow turnabout time and poor reproducibility. This study demonstrated that PCR-DGGE is indeed more valuable in detecting predominant microbial populations between various NTCs than as an identification methodology, being more applicable as a quality control method used to analyse for batch-to-batch variation during NTC production.

Monitoring of bioaerosol inhalation risks in different environments using a six-stage Andersen sampler and the PCR-DGGE method.

PubMed

Xu, Zhenqiang; Yao, Maosheng

2013-05-01

Increasing evidences show that inhalation of indoor bioaerosols has caused numerous adverse health effects and diseases. However, the bioaerosol size distribution, composition, and concentration level, representing different inhalation risks, could vary with different living environments. The six-stage Andersen sampler is designed to simulate the sampling of different human lung regions. Here, the sampler was used in investigating the bioaerosol exposure in six different environments (student dorm, hospital, laboratory, hotel room, dining hall, and outdoor environment) in Beijing. During the sampling, the Andersen sampler was operated for 30 min for each sample, and three independent experiments were performed for each of the environments. The air samples collected onto each of the six stages of the sampler were incubated on agar plates directly at 26 °C, and the colony forming units (CFU) were manually counted and statistically corrected. In addition, the developed CFUs were washed off the agar plates and subjected to polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) for diversity analysis. Results revealed that for most environments investigated, the culturable bacterial aerosol concentrations were higher than those of culturable fungal aerosols. The culturable bacterial and fungal aerosol fractions, concentration, size distribution, and diversity were shown to vary significantly with the sampling environments. PCR-DGGE analysis indicated that different environments had different culturable bacterial aerosol compositions as revealed by distinct gel band patterns. For most environments tested, larger (>3 μm) culturable bacterial aerosols with a skewed size distribution were shown to prevail, accounting for more than 60 %, while for culturable fungal aerosols with a normal size distribution, those 2.1-4.7 μm dominated, accounting for 20-40 %. Alternaria, Cladosporium, Chaetomium, and Aspergillus were found abundant in most environments studied here. Viable microbial load per unit of particulate matter was also shown to vary significantly with the sampling environments. The results from this study suggested that different environments even with similar levels of total microbial culturable aerosol concentrations could present different inhalation risks due to different bioaerosol particle size distribution and composition. This work fills literature gaps regarding bioaerosol size and composition-based exposure risks in different human dwellings in contrast to a vast body of total bioaerosol levels.

Decrease in fungal biodiversity along an available phosphorous gradient in arable Andosol soils in Japan.

PubMed

Bao, Zhihua; Matsushita, Yuko; Morimoto, Sho; Hoshino, Yuko Takada; Suzuki, Chika; Nagaoka, Kazunari; Takenaka, Makoto; Murakami, Hiroharu; Kuroyanagi, Yukiko; Urashima, Yasufumi; Sekiguchi, Hiroyuki; Kushida, Atsuhiko; Toyota, Koki; Saito, Masanori; Tsushima, Seiya

2013-06-01

Andosols comprise one of the most important soil groups for agricultural activities in Japan because they cover about 46.5% of arable upland fields. In this soil group, available phosphorus (P) is accumulated by application of excessive fertilizer, but little is known about the influence of increasing P availability on microbial community diversity at large scales. We collected soil samples from 9 agro-geographical sites with Andosol soils across an available P gradient (2048.1-59.1 mg P2O5·kg(-1)) to examine the influence of P availability on the fungal community diversity. We used polymerase chain reaction - denaturing gradient gel electrophoresis to analyze the fungal communities based on 18S rRNA genes. Statistical analyses revealed a high negative correlation between available P and fungal diversity (H'). Fungal diversity across all sites exhibited a significant hump-shaped relationship with available P (R(2) = 0.38, P < 0.001). In addition, the composition of the fungal community was strongly correlated with the available P gradient. The ribotype F6, which was positively correlated with available P, was closely related to Mortierella. The results show that both the diversity and the composition of the fungal community were influenced by available P concentrations in Andosols, at a large scale. This represents an important step toward understanding the processes responsible for the maintenance of fungal diversity in Andosolic soils.

Current Protocols in Protein Science

PubMed Central

Huynh, Kathy

2015-01-01

The purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables the rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as a low cost, initial screen to discover new protein:ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for the small-scale, high-throughout thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. PMID:25640896

Genetic profiling of the oral microbiota associated with severe early-childhood caries.

PubMed

Li, Y; Ge, Y; Saxena, D; Caufield, P W

2007-01-01

The determination of the composition of the microbial community in the oral cavity is usually based on cultivation methods; however, nearly half of the bacteria in the saliva and the dental plaque are not cultivable. In this study, we evaluated the difference in oral microbial diversity between children with severe early-childhood caries (S-ECC) and caries-free (CF) controls by means of a cultivation-independent approach called denaturing gradient gel electrophoresis (DGGE). Pooled dental plaque samples were collected from 20 children aged 2 to 8 years. Total microbial genomic DNA was isolated from those subjects, and a portion of the 16S rRNA gene locus was PCR amplified by using universal primers. We observed that the mean species richness of the bacterial population was greater in the CF children (n = 12) (42 +/- 3.7) than in the S-ECC children (n = 8) (35 +/- 4.3); the difference was statistically significant (P = 0.005). The overall diversity of plaque samples as measured by the Shannon index was 3.5 for the S-ECC group and 3.7 for the CF group (P = 0.004). Differences in DGGE profiles were distinguished on the basis of a cluster analysis. Sequence analysis of excised DGGE bands consisted of 2.7 phylotypes, on average. After adjusting for the number of observed bands, we estimated that the S-ECC group exhibited 94.5 total phylotypes and that the CF group exhibited 113.4. These results suggest that the microbial diversity and complexity of the microbial biota in dental plaque are significantly less in S-ECC children than in CF children.

Molecular and Culture-Based Assessment of the Microbial Diversity of Diabetic Chronic Foot Wounds and Contralateral Skin Sites

PubMed Central

Oates, Angela; Bowling, Frank L.; Boulton, Andrew J. M.

2012-01-01

Wound debridement samples and contralateral (healthy) skin swabs acquired from 26 patients attending a specialist foot clinic were analyzed by differential isolation and eubacterium-specific PCR-denaturing gradient gel electrophoresis (DGGE) in conjunction with DNA sequencing. Thirteen of 26 wounds harbored pathogens according to culture analyses, with Staphylococcus aureus being the most common (13/13). Candida (1/13), pseudomonas (1/13), and streptococcus (7/13) were less prevalent. Contralateral skin was associated with comparatively low densities of bacteria, and overt pathogens were not detected. According to DGGE analyses, all wounds contained significantly greater eubacterial diversity than contralateral skin (P < 0.05), although no significant difference in total eubacterial diversity was detected between wounds from which known pathogens had been isolated and those that were putatively uninfected. DGGE amplicons with homology to Staphylococcus sp. (8/13) and S. aureus (2/13) were detected in putatively infected wound samples, while Staphylococcus sp. amplicons were detected in 11/13 noninfected wounds; S. aureus was not detected in these samples. While a majority of skin-derived DGGE consortial fingerprints could be differentiated from wound profiles through principal component analysis (PCA), a large minority could not. Furthermore, wounds from which pathogens had been isolated could not be distinguished from putatively uninfected wounds on this basis. In conclusion, while chronic wounds generally harbored greater eubacterial diversity than healthy skin, the isolation of known pathogens was not associated with qualitatively distinct consortial profiles or otherwise altered diversity. The data generated support the utility of both culture and DGGE for the microbial characterization of chronic wounds. PMID:22553231

Hydrolysis, acidification and methanogenesis during low-temperature anaerobic digestion of dilute dairy wastewater in an inverted fluidised bioreactor.

PubMed

Bialek, Katarzyna; Cysneiros, Denise; O'Flaherty, Vincent

2014-10-01

The application of low-temperature (10 °C) anaerobic digestion (LtAD) for the treatment of complex dairy-based wastewater in an inverted fluidised bed (IFB) reactor was investigated. Inadequate mixing intensity provoked poor hydrolysis of the substrate (mostly protein), which resulted in low chemical oxygen demand (COD) removal efficiency throughout the trial, averaging ~69 % at the best operational period. Overgrowth of the attached biomass to the support particles (Extendospheres) induced bed stratification by provoking agglutination of the particles and supporting their washout by sedimentation, which contributed to unstable bioprocess performance at the organic loading rates (OLRs) between 0.5 and 5 kg COD m(-3) day(-1). An applied OLR above 2 kg COD m(-3) day(-1) additionally promoted acidification and strongly influenced the microbial composition and dynamics. Hydrogenotrophic methanogens appeared to be the mostly affected group by the Extendospheres particle washout as a decrease in their abundance was observed by quantitative PCR analysis towards the end of the trial, although the specific methanogenic activity and maximum substrate utilisation rate on H2/CO2 indicated high metabolic activity and preference towards hydrogenotrophic methanogenesis of the reactor biomass at this stage. The bacterial community in the bioreactor monitored via denaturing gradient gel electrophoresis (DGGE) also suggested an influence of OLR stress on bacterial community structure and population dynamics. The data presented in this work can provide useful information in future optimisation of fluidised reactors intended for digestion of complex industrial wastewaters during LtAD.

Genetic diversity pattern of microeukaryotic communities and its relationship with the environment based on PCR-DGGE and T-RFLP techniques in Dongshan Bay, southeast China

NASA Astrophysics Data System (ADS)

Zhang, Wenjing; Mo, Yuanyuan; Yang, Jun; Zhou, Jing; Lin, Yuanshao; Isabwe, Alain; Zhang, Jian; Gao, Xiu; Yu, Zheng

2018-07-01

Microeukaryotes play important roles in aquatic ecosystems, and could act as drivers of the biological nutrient cycling processes. However, compared with prokaryotic ones, little is known about the genetic diversity pattern of their community, and the environmental factors affecting their ecological pattern, especially in marine ecosystems. In this study, we used denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) to explore the genetic diversity structure of microeukaryotic communities in Dongshan Bay, southeast China. Our results revealed that microeukaryotic diversity ranged from 31 to 48 phylotypes (on average, 42) using the DGGE approach, while from 22 to 38 phylotypes (on average, 30) based on T-RFLP method, and the Shannon-Wiener diversity (DGGE-based) was relatively higher, suggesting that DGGE displayed a slightly higher resolution than T-RFLP. Surprisingly, the DGGE showed significant horizontal difference among microeukaryotic communities, but was similar with T-RFLP analysis that had no significant influence on microeukaryotic diversity at vertical scale. Further, redundancy analysis (RDA) indicated that the DGGE-based microeukaryotic communities distribution was significantly correlated with three environmental factors (Chl-a, TP and salinity), whereas microeukaryotic community revealed by T-RFLP was affected by four environmental factors namely salinity, temperature, depth and NOX-N. Compared with RDA, BIO-ENV analysis showed that heterotrophic bacteria and NOX-N were important environmental variable influencing microeukaryotic communities in both methods. These differences may be attributed to the noisy effects caused by the relatively large number of environmental variables. Generally speaking, despite differences in beta-diversity ordination for both DGGE and T-RFLP methods, there exists some consistency in the results of both techniques in terms of microeukaryotes responses to the environmental variables. These results suggested that environmental parameters had a great effect on spatial distribution of microeukaryotic community and contributed to marine ecosystem health to be further evaluated.

Bacterial communities in pigmented biofilms formed on the sandstone bas-relief walls of the Bayon Temple, Angkor Thom, Cambodia.

PubMed

Kusumi, Asako; Li, Xianshu; Osuga, Yu; Kawashima, Arata; Gu, Ji-Dong; Nasu, Masao; Katayama, Yoko

2013-01-01

The Bayon temple in Angkor Thom, Cambodia has shown serious deterioration and is subject to the formation of various pigmented biofilms. Because biofilms are damaging the bas-reliefs, low reliefs engraved on the surface of sandstone, information about the microbial community within them is indispensable to control biofilm colonization. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of biofilm samples from the pigmented sandstone surfaces showed that the bacterial community members in the biofilms differed clearly from those in the air and had low sequence similarity to database sequences. Non-destructive sampling of biofilm revealed novel bacterial groups of predominantly Rubrobacter in salmon pink biofilm, Cyanobacteria in chrome green biofilm, Cyanobacteria and Chloroflexi in signal violet biofilm, Chloroflexi in black gray biofilm, and Deinococcus-Thermus, Cyanobacteria, and Rubrobacter in blue green biofilm. Serial peeling-off of a thick biofilm by layers with adhesive sheets revealed a stratified structure: the blue-green biofilm, around which there was serious deterioration, was very rich in Cyanobacteria near the surface and Chloroflexi in deep layer below. Nitrate ion concentrations were high in the blue-green biofilm. The characteristic distribution of bacteria at different biofilm depths provides valuable information on not only the biofilm formation process but also the sandstone weathering process in the tropics.

Influence of isolated bacterial strains on the in situ biodegradation of endosulfan and the reduction of endosulfan- contaminated soil toxicity.

PubMed

Kong, Lingfen; Zhang, Yu; Zhu, Lusheng; Wang, Jinhua; Wang, Jun; Du, Zhongkun; Zhang, Cheng

2018-09-30

The recently discovered endosulfan-degrading bacterial strains Pusillimonas sp. JW2 and Bordetella petrii NS were isolated from endosulfan-polluted water and soil environments. The optimal conditions for the growth and biodegradation activity of the strains JW2 and NS were studied in detail. In addition, the ability of the strains JW2 and NS to biodegrade endosulfan in soils during in situ bioremediation experiments was investigated. At a concentration of 2 mg of endosulfan per kilogram of soil, both JW2 and NS had positive effects on the degradation of endosulfan; JW2 degraded 100% and 91.5% of α- and β-endosulfan, respectively, and NS degraded 95.1% and 90.3% of α- and β-endosulfan, respectively. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of soil samples showed the successful colonization of JW2 and NS, and the toxicity of the soil decreased, as determined by single-cell gel electrophoresis (SCGE) assays of Eiseniafetida and micronucleus (MN) assays of Viciafaba root tip cells. Furthermore, the metabolic products of the bacterially degraded endosulfan from the in situ experiments were identified as endosulfan ether and lactone. This study provided potentially foundational backgrounds information for the remediation of endosulfan-contaminated soil. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of key micro-organisms involved in Douchi fermentation by statistical analysis and their use in an experimental fermentation.

PubMed

Chen, C; Xiang, J Y; Hu, W; Xie, Y B; Wang, T J; Cui, J W; Xu, Y; Liu, Z; Xiang, H; Xie, Q

2015-11-01

To screen and identify safe micro-organisms used during Douchi fermentation, and verify the feasibility of producing high-quality Douchi using these identified micro-organisms. PCR-denaturing gradient gel electrophoresis (DGGE) and automatic amino-acid analyser were used to investigate the microbial diversity and free amino acids (FAAs) content of 10 commercial Douchi samples. The correlations between microbial communities and FAAs were analysed by statistical analysis. Ten strains with significant positive correlation were identified. Then an experiment on Douchi fermentation by identified strains was carried out, and the nutritional composition in Douchi was analysed. Results showed that FAAs and relative content of isoflavone aglycones in verification Douchi samples were generally higher than those in commercial Douchi samples. Our study indicated that fungi, yeasts, Bacillus and lactic acid bacteria were the key players in Douchi fermentation, and with identified probiotic micro-organisms participating in fermentation, a higher quality Douchi product was produced. This is the first report to analyse and confirm the key micro-organisms during Douchi fermentation by statistical analysis. This work proves fermentation micro-organisms to be the key influencing factor of Douchi quality, and demonstrates the feasibility of fermenting Douchi using identified starter micro-organisms. © 2015 The Society for Applied Microbiology.

Distribution of oil-degrading bacteria in coastal seawater, Toyama Bay, Japan.

PubMed

Tanaka, Daisuke; Tanaka, Shunsuke; Yamashiro, Yoko; Nakamura, Shogo

2008-10-01

Oil-degrading bacteria are considered to play an important role in the biodegradation of spilled or released oil in the sea. The distribution of indigenous oil-degrading bacteria in the coastal seawater of Toyama Bay, Japan, was examined. Surface seawater samples with or without oil film in fishing port were analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of bacterial 16S rDNA. Sequence analysis revealed that several DGGE bands clearly detected only in samples with oil film corresponded to Cyanobacteria. Moreover, we cultured surface seawater samples with oil film in two different liquid culture media, a marine broth and an NSW medium; each culture contained 0.5% (w/v) C-heavy oil. Emulsification of the oil was observed at day 6 in the marine broth and day 9 in the NSW medium. Time-dependent changes of bacterial communities in those culture media were analyzed by DGGE. Interestingly, we found that Alcanivorax sp. became one of the dominant bacteria in each culture medium when emulsification of the oil began. Alcanivorax sp. is one of the well-known oil-degrading bacteria in seawater and is associated with the production of biosurfactants. These results suggest that Cyanobacteria and Alcanivorax play important roles in the bioremediation of oil-contaminated areas in Toyama Bay.

Occurrence and activity of Archaea in aerated activated sludge wastewater treatment plants.

PubMed

Gray, Neil D; Miskin, Ian P; Kornilova, Oksana; Curtis, Thomas P; Head, Ian M

2002-03-01

The occurrence, distribution and activity of archaeal populations within two aerated, activated sludge wastewater treatment systems, one treating domestic waste and the second treating mixed domestic and industrial wastewater, were investigated by denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified ribosomal RNA gene fragments and process measurements. In the plant receiving mixed industrial and domestic waste the archaeal populations found in the mixed liquor were very similar to those in the influent sewage, though a small number of DGGE bands specific to the mixed liquor were identified. In contrast, the activated sludge treating principally domestic waste harboured distinct archaeal populations associated with the mixed liquor that were not prevalent in the influent sewage. We deduce that the Archaea in the plant treating mixed wastewater were derived principally from the influent, whereas those in the plant treating solely domestic waste were actively growing in the treatment plant. Archaeal 16S rRNA gene sequences related to the Methanosarcinales, Methanomicrobiales and the Methanobacteriales were detected. Methanogenesis was measured in activated sludge samples incubated under oxic and anoxic conditions, demonstrating that the methanogens present in both activated sludge plants were active only in anoxic incubations. The relatively low rates of methanogenesis measured indicated that, although active, the methanogens play a minor role in carbon turnover in activated sludge.

Osteogenesis imperfecta type I: Molecular heterogeneity for COL1A1 null alleles of type I collagen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willing, M.C.; Deschenes, S.P.; Pitts, S.H.

Osteogenesis imperfecta (OI) type I is the mildest form of inherited brittle-bone disease. Dermal fibroblasts from most affected individuals produce about half the usual amount of type I procollagen, as a result of a COL1A1 {open_quotes}null{close_quotes} allele. Using PCR amplification of genomic DNA from affected individuals, followed by denaturing gradient gel electrophoresis (DGGE) and SSCP, we identified seven different COL1A1 gene mutations in eight unrelated families with OI type I. Three families have single nucleotide substitutions that alter 5{prime} donor splice sites; two of these unrelated families have the same mutation. One family has a point mutation, in an exon,more » that creates a premature termination codon, and four have small deletions or insertions, within exons, that create translational frameshifts and new termination codons downstream of the mutation sites. Each mutation leads to both marked reduction in steady-state levels of mRNA from the mutant allele and a quantitative decrease in type I procollagen production. Our data demonstrate that different molecular mechanisms that have the same effect on type I collagen production result in the same clinical phenotype. 58 refs., 4 figs., 1 tab.« less

Effects of different sources of physically effective fiber on rumen microbial populations.

PubMed

Shaw, C N; Kim, M; Eastridge, M L; Yu, Z

2016-03-01

Physically effective fiber is needed by dairy cattle to prevent ruminal acidosis. This study aimed to examine the effects of different sources of physically effective fiber on the populations of fibrolytic bacteria and methanogens. Five ruminally cannulated Holstein cows were each fed five diets differing in physically effective fiber sources over 15 weeks (21 days/period) in a Latin Square design: (1) 44.1% corn silage, (2) 34.0% corn silage plus 11.5% alfalfa hay, (3) 34.0% corn silage plus 5.1% wheat straw, (4) 36.1% corn silage plus 10.1% wheat straw, and (5) 34.0% corn silage plus 5.5% corn stover. The impact of the physically effective fiber sources on total bacteria and archaea were examined using denaturing gradient gel electrophoresis. Specific real-time PCR assays were used to quantify total bacteria, total archaea, the genus Butyrivibrio, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens and three uncultured rumen bacteria that were identified from adhering ruminal fractions in a previous study. No significant differences were observed among the different sources of physical effective fiber with respect to the microbial populations quantified. Any of the physically effective fiber sources may be fed to dairy cattle without negative impact on the ruminal microbial community.

[Diversity of oil-degrading bacteria isolated form the Indian Ocean sea surface].

PubMed

Wu, Changliang; Wang, Xin; Shao, Zongze

2010-09-01

In order to investigate the diversity of oil-degrading bacteria in the surface seawater across the India Ocean, and to obtain new oil-degrading bacteria. Potential oil-degrading bacteria were selected out via 1:1 mixture of diesel and crude oil as sole carbon source. Meanwhile, the community structure of 13 enrichments was analyzed by polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE). We obtained 51 unique strains of 29 genera after screening via morphological, physiological, biochemical and 16S rRNA analyses. They mainly belonged to a and gamma-Proteobacteria. The four genera Alcanivorax (accounting for 18%), Novosphingobium (10%), Marinobacter (6%) and Thalassospira (6%) were the most predominant bacteria. Ecological analyses showed that the bacteria had high diversity with Shannon-Winner index (H) of 4.57968, and distributed even with Evenness index (E) as 0.8664771. Then Further experiments revealed oil-degrading capability of 49 strains. In addition, our investigation revealed oil-degrading ability of genera Sinomonas, Knoellia and Mesoflavibacter for the first time. DGGE fingerprint patterns indicated that the genus Alcanivorax was an important oil-degrading bacteria in the surface seawater across the India Ocean. This study demonstrated a high diversity of the oil-degradation bacteria in the surface seawater of Indian Ocean, these bacteria are of potential in bioremediation of marine oil pollution.

Suppression of Listeria monocytogenes by the Native Micro-Flora in Teewurst Sausage

PubMed Central

Austin-Watson, Clytrice; Grant, Ar’Quette; Brice, Michline

2013-01-01

Modern consumers are interested in the use of non-chemical methods to control pathogens when heat sterilization is not an option. Such is the case with teewurst sausage, a raw spreadable sausage and a popular German commodity. Although Listeria was not found in teewurst, the optimal microbial growing conditions of teewurst coupled with the ubiquity of L. monocytogenes in nature, makes the possibility of contamination of products very possible. This pilot study was conducted to examine teewurst’s native micro-flora’s ability to suppress the outgrowth of L. monocytogenes at 10 °C using standard plate counts and PCR-DGGE. Traditional plating methods showed L. monocytogenes growth significantly decreased when in competition with the teewurst’s native micro-flora (p < 0.05). The native micro-flora of the teewurst suppressed the overall growth of L. monocytogenes by an average of two logs, under these conditions. Denaturing Gradient Gel Electrophoresis (DGGE) amplicons with unique banding patterns were extracted from DGGE gel for identification. Brochothrix thermosphacta and Lactobacillus curvatus were identified as a part of the teewurst’s native micro-flora. Although the native micro-flora did not decrease L. monocytogenes to below limits of detection, it was enough of a decrease to warrant further investigation. PMID:28239131

Functional Stability Of A Mixed Microbial Consortia Producing PHA From Waste Carbon Sources

DOE Office of Scientific and Technical Information (OSTI.GOV)

David N. Thompson; Erik R. Coats; William A. Smith

2006-04-01

Polyhydroxyalkanoates (PHAs), naturally-occurring biological polyesters that are microbially synthesized from a myriad of carbon sources, can be utilized as biodegradable substitutes for petroleum-derived thermoplastics. However, current PHA commercialization schemes are limited by high feedstock costs, the requirement for aseptic reactors, and high separation and purification costs. Bacteria indigenous to municipal waste streams can accumulate large quantities of PHA under environmentally controlled conditions; hence, a potentially more environmentally-effective method of production would utilize these consortia to produce PHAs from inexpensive waste carbon sources. In this study, PHA production was accomplished in sequencing batch bioreactors utilizing mixed microbial consortia from municipal activatedmore » sludge as inoculum, in cultures grown on real wastewaters. PHA production averaged 85%, 53%, and 10% of the cell dry weight from methanol-enriched pulp-and-paper mill foul condensate, fermented municipal primary solids, and biodiesel wastewater, respectively. The PHA-producing microbial consortia were examined to explore the microbial community changes that occurred during reactor operations, employing denaturing gradient gel electrophoresis (DGGE) of 16S-rDNA from PCR-amplified DNA extracts. Distinctly different communities were observed both between and within wastewaters following enrichment. More importantly, stable functions were maintained despite the differing and contrasting microbial populations.« less

Sulfate-reducing bacteria and their activities in cyanobacterial mats of Solar Lake (Sinai, Egypt)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teske, A.; Ramsing, N.B.; Habicht, K.

1998-08-01

The sulfate-reducing bacteria within the surface layer of the hypersaline cyanobacterial mat of Solar Lake (Sinai, Egypt) were investigated with combined microbiological, molecular, and biogeochemical approaches. The diurnally oxic surface layer contained between 10{sup 6} and 10{sup 7} cultivable sulfate-reducing bacteria ml{sup {minus}1} day{sup {minus}1}, both in the same range as and sometimes higher than those in anaerobic deeper mat layers. In the oxic surface layer and in the mat layers below, filamentous sulfate-reducing Desulfonema bacteria were found in variable densities of 10{sup 4} and 10{sup 6} cells ml{sup {minus}1}. A Desulfonema-related, diurnally migrating bacterium was detected with PCR andmore » denaturing gradient gel electrophoresis within and below the oxic surface layer. Facultative aerobic respiration, filamentous morphology, motility, diurnal migration, and aggregate formation were the most conspicuous adaptations of Solar Lake sulfate-reducing bacteria to the mat matrix and to diurnal oxygen stress. A comparison of sulfate reduction rates within the mat and previously published photosynthesis rates showed that CO{sub 2} from sulfate reduction in the upper 5 mm accounted for 7 to 8% of the total photosynthetic CO{sub 2} demand of the mat.« less

The microbial diversity of an industrially produced lambic beer shares members of a traditionally produced one and reveals a core microbiota for lambic beer fermentation.

PubMed

Spitaels, Freek; Wieme, Anneleen D; Janssens, Maarten; Aerts, Maarten; Van Landschoot, Anita; De Vuyst, Luc; Vandamme, Peter

2015-08-01

The microbiota involved in lambic beer fermentations in an industrial brewery in West-Flanders, Belgium, was determined through culture-dependent and culture-independent techniques. More than 1300 bacterial and yeast isolates from 13 samples collected during a one-year fermentation process were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by sequence analysis of rRNA and various protein-encoding genes. The bacterial and yeast communities of the same samples were further analyzed using denaturing gradient gel electrophoresis of PCR-amplified V3 regions of the 16S rRNA genes and D1/D2 regions of the 26S rRNA genes, respectively. In contrast to traditional lambic beer fermentations, there was no Enterobacteriaceae phase and a larger variety of acetic acid bacteria were found in industrial lambic beer fermentations. Like in traditional lambic beer fermentations, Saccharomyces cerevisiae, Saccharomyces pastorianus, Dekkera bruxellensis and Pediococcus damnosus were the microorganisms responsible for the main fermentation and maturation phases. These microorganisms originated most probably from the wood of the casks and were considered as the core microbiota of lambic beer fermentations. Copyright © 2015 Elsevier Ltd. All rights reserved.

Searching for links in the biotic characteristics and abiotic parameters of nine different biogas plants

PubMed Central

Walter, Andreas; Knapp, Brigitte A.; Farbmacher, Theresa; Ebner, Christian; Insam, Heribert; Franke‐Whittle, Ingrid H.

2012-01-01

Summary To find links between the biotic characteristics and abiotic process parameters in anaerobic digestion systems, the microbial communities of nine full‐scale biogas plants in South Tyrol (Italy) and Vorarlberg (Austria) were investigated using molecular techniques and the physical and chemical properties were monitored. DNA from sludge samples was subjected to microarray hybridization with the ANAEROCHIP microarray and results indicated that sludge samples grouped into two main clusters, dominated either by Methanosarcina or by Methanosaeta, both aceticlastic methanogens. Hydrogenotrophic methanogens were hardly detected or if detected, gave low hybridization signals. Results obtained using denaturing gradient gel electrophoresis (DGGE) supported the findings of microarray hybridization. Real‐time PCR targeting Methanosarcina and Methanosaeta was conducted to provide quantitative data on the dominating methanogens. Correlation analysis to determine any links between the microbial communities found by microarray analysis, and the physicochemical parameters investigated was conducted. It was shown that the sludge samples dominated by the genus Methanosarcina were positively correlated with higher concentrations of acetate, whereas sludge samples dominated by representatives of the genus Methanosaeta had lower acetate concentrations. No other correlations between biotic characteristics and abiotic parameters were found. Methanogenic communities in each reactor were highly stable and resilient over the whole year. PMID:22950603

Response of soil microbial activities and microbial community structure to vanadium stress.

PubMed

Xiao, Xi-Yuan; Wang, Ming-Wei; Zhu, Hui-Wen; Guo, Zhao-Hui; Han, Xiao-Qing; Zeng, Peng

2017-08-01

High levels of vanadium (V) have long-term, hazardous impacts on soil ecosystems and biological processes. In the present study, the effects of V on soil enzymatic activities, basal respiration (BR), microbial biomass carbon (MBC), and the microbial community structure were investigated through 12-week greenhouse incubation experiments. The results showed that V content affected soil dehydrogenase activity (DHA), BR, and MBC, while urease activity (UA) was less sensitive to V stress. The average median effective concentration (EC 50 ) thresholds of V were predicted using a log-logistic dose-response model, and they were 362mgV/kg soil for BR and 417mgV/kg soil for DHA. BR and DHA were more sensitive to V addition and could be used as biological indicators for soil V pollution. According to a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, the structural diversity of the microbial community decreased for soil V contents ranged between 254 and 1104mg/kg after 1 week of incubation. As the incubation time increased, the diversity of the soil microbial community structure increased for V contents ranged between 354 and 1104mg/kg, indicating that some new V-tolerant bacterial species might have replicated under these conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

An endophytic bacterium Acinetobacter calcoaceticus Sasm3-enhanced phytoremediation of nitrate-cadmium compound polluted soil by intercropping Sedum alfredii with oilseed rape.

PubMed

Chen, Bao; Ma, Xiaoxiao; Liu, Guiqing; Xu, Xiaomeng; Pan, Fengshan; Zhang, Jie; Tian, Shengke; Feng, Ying; Yang, Xiaoe

2015-11-01

Intensive agricultural system with high input of fertilizer results in high agricultural output. However, excessive fertilization in intensive agricultural system has great potential to cause nitrate and heavy metal accumulation in soil, which is adverse to human health. The main objective of the present study was to observe the effects of intercropping and inoculation of endophytic bacterium Acinetobacter calcoaceticus Sasm3 on phytoremediation of combined contaminated soil in oilseed rape (Brassica napus L.). The results showed that with Sasm3 inoculation, the biomass of rape was increased by 10-20% for shoot, 64% for root, and 23-29% for seeds while the nitrate accumulation in rape was decreased by 14% in root and by 12% in shoot. The cadmium concentration in rape increased significantly with mono-inoculating treatment, whereas it decreased significantly after intercropping treatment. By denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR analysis, the diversity of bacterial community and the number of nirS and nirK gene copies increased significantly with inoculation or/and intercropping treatment. In conclusion, the endophytic bacterium Sasm3-inoculated intercropping system not only improved the efficiency of clearing cadmium from soil without obstructing crop production, but also improved the quality of crop.

Rubrobacter-related bacteria associated with rosy discolouration of masonry and lime wall paintings.

PubMed

Schabereiter-Gurtner, C; Piñar, G; Vybiral, D; Lubitz, W; Rölleke, S

2001-11-01

A molecular approach was chosen to analyse the correlation between bacterial colonisation and rosy discolouration of masonry and lime wall paintings of two historically important buildings in Austria and Germany. The applied molecular method included PCR amplification of genes encoding the small subunit rRNA of bacteria (16S rDNA), genetic fingerprinting by denaturing gradient gel electrophoresis (DGGE), construction of 16S rDNA clone libraries, and comparative phylogenetic sequence analyses. The bacterial community of one red-pigmented biofilm sampled in Herberstein (Austria) contained bacteria phylogenetically related to the genera Saccharopolyspora, Nocardioides, Pseudonocardia, Rubrobacter, and to a Kineococcus-like bacterium. The bacterial community of the second red-pigmented biofilm sampled in Herberstein contained bacteria related to Arthrobacter, Comamonas, and to Rubrobacter. Rubrobacter-related 16S rDNA sequences were the most abundant. In the red-pigmented biofilm sampled in Burggen (Germany), only Rubrobacter-related bacteria were identified. No Rubrobacter-related bacteria were detected in non-rosy biofilms. The majority of sequences (70%) obtained from the bacterial communities of the three investigated rosy biofilms were related to sequences of the genus Rubrobacter (red-pigmented bacteria), demonstrating a correlation between Rubrobacter-related bacteria and the phenomenon of rosy discolouration of masonry and lime wall paintings.

Community structure of denitrifying and total bacteria during nitrogen accumulation in an ammonia-loaded biofilter.

PubMed

Yasuda, T; Waki, M; Fukumoto, Y; Hanajima, D; Kuroda, K; Suzuki, K; Matsumoto, T; Uenishi, H

2017-12-01

To obtain insight into the complex behaviour of denitrifying and total bacterial groups during the nitrogen accumulation process in an ammonia-loaded biofiltration system. Denitrifying and total bacterial communities in a laboratory-scale rockwool biofilter with intermittent water recirculation were analysed by using denaturing gradient gel electrophoresis targeting nosZ and metabarcoding sequencing of the 16S rRNA gene. Gene abundance was evaluated by quantitative PCR. The nosZ number increased from 6·59 × 10 6 to 3·33 × 10 8 copies per gram dry sample over the 436 days of operation, during which nitrogen mass balance errors increased to 39%. The nosZ sequences associated with the genera Castellaniella, Hyphomicrobium and Pseudomonas were detected. Metabarcoding sequencing analysis indicated that the proportions of the genera for which at least one denitrifying strain or species possessing nosZ had been characterized corresponded well to the nitrogen loss. In addition, the genus Nitrosococcus (γ-proteobacteria) increased its relative abundance at days 317 and 436. The increased proportion of denitrifying bacteria in this ammonia-loaded biofiltration system could be related to the nitrogen loss. These results will help to clarify the complex behaviour of nitrifiers and denitrifiers within ammonia-loaded biofiltration systems. © 2017 The Society for Applied Microbiology.

Microbial community dynamics and biogas production from manure fractions in sludge bed anaerobic digestion.

PubMed

Nordgård, A S R; Bergland, W H; Bakke, R; Vadstein, O; Østgaard, K; Bakke, I

2015-12-01

To elucidate how granular sludge inoculum and particle-rich organic loading affect the structure of the microbial communities and process performance in upflow anaerobic sludge bed (UASB) reactors. We investigated four reactors run on dairy manure filtrate and four on pig manure supernatant for three months achieving similar methane yields. The reactors fed with less particle rich pig manure stabilized faster and had highest capacity. Microbial community dynamics analysed by a PCR/denaturing gradient gel electrophoresis approach showed that influent was a major determinant for the composition of the reactor communities. Comparisons of pre- and non-adapted inoculum in the reactors run on pig manure supernatant showed that the community structure of the nonadapted inoculum adapted in approximately two months. Microbiota variance partitioning analysis revealed that running time, organic loading rate and inoculum together explained 26 and 31% of the variance in bacterial and archaeal communities respectively. The microbial communities of UASBs adapted to the reactor conditions in treatment of particle rich manure fractions, obtaining high capacity, especially on pig manure supernatant. These findings provide relevant insight into the microbial community dynamics in startup and operation of sludge bed reactors for methane production from slurry fractions, a major potential source of biogas. © 2015 The Society for Applied Microbiology.

Response of ammonia-oxidizing betaproteobacteria to short-term fertilization in a salt marsh in China

NASA Astrophysics Data System (ADS)

Ma, Yuexin; Tao, Wei; Liu, Jiao; Liu, Changfa; Li, Jin; Liu, Jichen

2018-03-01

This study examines the impacts of short-term (6 months) fertilization on the community structure and abundance of ammonia-oxidizing betaproteobacteria (β-AOB) and the potential nitrification rate in sediment colonized by Suaeda heteroptera in a saltmarsh located in Shuangtai estuary, China. The sediment samples were collected from plots treated with different amounts of an N fertilizer (urea supplied at 0.1, 0.2, 0.4, and 0.8 g/kg (nitrogen content in dry sediment)), and with different forms of N fertilizers (urea, (NH4)2SO4, and NH4NO3, each supplied at 0.2 g/kg). The fertilizers were applied 1-4 times during the plant-growing season in May, July, August and September of 2013. Untreated plots were included as a control. As revealed in denaturing gradient gel electrophoresis of the 16S rRNA gene, the β-AOB community responded to both the amount and form of N. Real-time quantitative PCR indicated that both abundance and potential nitrification rate of β-AOB increased after N addition, regardless of concentration and form (except NH4NO3). These results provide evidence that short-term N application influences the sediment β-AOB community, β-AOB abundance and potential nitrification rate in a saltmarsh ecosystem.

Nutrients removal in hybrid fluidised bed bioreactors operated with aeration cycles.

PubMed

Martin, Martin; Enríquez, L López; Fernández-Polanco, M; Villaverde, S; Garcia-Encina, P A

2007-01-01

Abstract Two hybrid fluidised bed reactors filled with sepiolite and granular activated carbon (GAC) were operated with short cycled aeration for removing organic matter, total nitrogen and phosphorous, respectively. Both reactors were continuously operated with synthetic and/or industrial wastewater containing 350-500 mg COD/L, 110-130 mg NKT/L, 90-100 mg NH3-N/L and 12-15 mg P/L for 8 months. The reactor filled with sepiolite, treating only synthetic wastewater, removed COD, ammonia, total nitrogen and phosphorous up to 88, 91, 55 and 80% with a hydraulic retention time (HRT) of 10 h, respectively. These efficiencies correspond to removal rates of 0.95 kgCODm(-3)d(-1) and 0.16 kg total N m(-3)d(-1). The reactor filled with GAC was operated for 4 months with synthetic wastewater and 4 months with industrial wastewater, removing 98% of COD, 96% of ammonia, and 66% of total nitrogen, with an HRT of 13.6 h. No significant phosphorous removing activity was observed in this reactor. Microbial communities growing with both reactors were followed using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) techniques. The microbial fingerprints, i.e. DGGE profiles, indicated that biological communities in both reactors were stable along the operational period even when the operating conditions were changed.

[Bacterial diversity in sequencing batch biofilm reactor (SBBR) for landfill leachate treatment using PCR-DGGE].

PubMed

Xiao, Yong; Yang, Zhao-hui; Zeng, Guang-ming; Ma, Yan-he; Liu, You-sheng; Wang, Rong-juan; Xu, Zheng-yong

2007-05-01

For studying the bacterial diversity and the mechanism of denitrification in sequencing bath biofilm reactor (SBBR) treating landfill leachate to provide microbial evidence for technique improvements, total microbial DNA was extracted from samples which were collected from natural landfill leachate and biofilm of a SBBR that could efficiently remove NH4+ -N and COD of high concentration. 16S rDNA fragments were amplified from the total DNA successfully using a pair of universal bacterial 16S rDNA primer, GC341F and 907R, and then were used for denaturing gradient gel electrophoresis (DGGE) analysis. The bands in the gel were analyzed by statistical methods and excided from the gel for sequencing, and the sequences were used for homology analysis and then two phylogenetic trees were constructed using DNAStar software. Results indicated that the bacterial diversity of the biofilm in SBBR and the landfill leachate was abundant, and no obvious change of community structure happened during running in the biofilm, in which most bacteria came from the landfill leachate. There may be three different modes of denitrification in the reactor because several different nitrifying bacteria, denitrifying bacteria and anaerobic ammonia oxidation bacteria coexisted in it. The results provided some valuable references for studying microbiological mechanism of denitrification in SBBR.

Effect of seed sludge on characteristics and microbial community of aerobic granular sludge.

PubMed

Song, Zhiwei; Pan, Yuejun; Zhang, Kun; Ren, Nanqi; Wang, Aijie

2010-01-01

Aerobic granular sludge was cultivated by using different kinds of seed sludge in sequencing batch airlift reactor. The influence of seed sludge on physical and chemical properties of granular sludge was studied; the microbial community structure was probed by using scanning electron microscope and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that seed sludge played an important role on the formation of aerobic granules. Seed sludge taken from beer wastewater treatment plant (inoculum A) was more suitable for cultivating aerobic granules than that of sludge from municipal wastewater treatment plant (inoculum B). Cultivated with inoculum A, large amount of mature granules formed after 35 days operation, its SVI reached 32.75 mL/g, and SOUR of granular sludge was beyond 1.10 mg/(g x min). By contrast, it needed 56 days obtaining mature granules using inoculum B. DGGE profiles indicated that the dominant microbial species in mature granules were 18 and 11 OTU when inoculum A and B were respectively employed as seed sludge. The sequencing results suggested that dominant species in mature granules cultivated by inoculum A were Paracoccus sp., Devosia hwasunensi, Pseudoxanthomonas sp., while the dominant species were Lactococcus raffinolactis and Pseudomonas sp. in granules developed from inoculum B.

Kinetics of nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium.

PubMed

Lee, Mong-Chuan; Lin, Yen-Hui; Yu, Huang-Wei

2014-11-01

A mathematical model system was derived to describe the kinetics of ammonium nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium. The model incorporates diffusive mass transport and Monod kinetics. The model was solved using a combination of the orthogonal collocation method and Gear's method. A batch test was conducted to observe the nitrification of ammonium-nitrogen ([Formula: see text]-N) and the growth of nitrifying biomass. The compositions of nitrifying bacterial community in the batch kinetic test were analyzed using PCR-DGGE method. The experimental results show that the most staining intensity abundance of bands occurred on day 2.75 with the highest biomass concentration of 46.5 mg/L. Chemostat kinetic tests were performed independently to evaluate the biokinetic parameters used in the model prediction. In the column test, the removal efficiency of [Formula: see text]-N was approximately 96 % while the concentration of suspended nitrifying biomass was approximately 16 mg VSS/L and model-predicted biofilm thickness reached up to 0.21 cm in the steady state. The profiles of denaturing gradient gel electrophoresis (DGGE) of different microbial communities demonstrated that indigenous nitrifying bacteria (Nitrospira and Nitrobacter) existed and were the dominant species in the fixed biofilm process.

Bacterial population in traditional sourdough evaluated by molecular methods.

PubMed

Randazzo, C L; Heilig, H; Restuccia, C; Giudici, P; Caggia, C

2005-01-01

To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.

Isolation of a sulfide-producing bacterial consortium from cooling-tower water: Evaluation of corrosive effects on galvanized steel.

PubMed

Ilhan-Sungur, Esra; Ozuolmez, Derya; Çotuk, Ayşın; Cansever, Nurhan; Muyzer, Gerard

2017-02-01

Sulfidogenic Clostridia and sulfate reducing bacteria (SRB) often cohabit in nature. The presence of these microorganisms can cause microbially influenced corrosion (MIC) of materials in different ways. To investigate this aspect, bacteria were isolated from cooling tower water and used in corrosion tests of galvanized steel. The identity of the isolates was determined by comparative sequence analysis of PCR-amplified 16S rDNA gene fragments, separated by denaturing gradient gel electrophoresis (DGGE). This analysis showed that, in spite of the isolation process, colonies were not pure and consisted of a mixture of bacteria affiliated with Desulfosporosinus meridiei and Clostridium sp. To evaluate the corrosive effect, galvanized steel coupons were incubated with a mixed culture for 4, 8, 24, 72, 96, 168, 360 and 744 h, along with a control set in sterile culture medium only. The corrosion rate was determined by weight loss, and biofilm formation and corroded surfaces were observed by scanning electron microscopy (SEM). Although the sulfide-producing bacterial consortium led to a slight increase in the corrosion of galvanized steel coupons, when compared to the previous studies it can be said that Clostridium sp. can reduce the corrosive effect of the Desulfosporosinus sp. strain. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography

PubMed Central

Franciosa, Giovanna; Pourshaban, Manoocheher; De Luca, Alessandro; Buccino, Anna; Dallapiccola, Bruno; Aureli, Paolo

2004-01-01

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism. PMID:15240298

Comparative analysis of nitrifying bacteria in full-scale oxidation ditch and aerated nitrification biofilter by using fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE).

PubMed

Mertoglu, Bulent; Calli, Baris; Girgin, Emine; Inanc, Bulent; Ozturk, Izzet

2005-01-01

In this study, nitrification performances and composition of nitrifying populations in a full-scale oxidation ditch and a high-rate submerged media nitrification biofilter were comparatively analyzed. In addition to different reactor configurations, effects of differing operational conditions on the nitrification efficiency and bacterial diversity were also explored and evaluated thoroughly. In microbial analysis of sludge samples fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques were used complementary to each other. The extended aeration oxidation ditch subjected to the study is operated as a nitrogen and phosphorus removal system consisting of anaerobic, anoxic, and aerobic zones. The high-rate submerged media aerated filter is operated as nitrification step following the conventional activated sludge unit and the nitrified wastewater is discharged to the sea without complete nitrogen removal. In situ hybridization results have indicated that Nitrosomonas-like ammonia oxidizing and Nitrospira-related nitrite oxidizing bacteria were intensively present in vigorous flocs in nitrification biofilter while carbonaceous bacteria belong to beta subclass of Proteobacteria were considerably dominant in oxidation ditch. Low quantities of nitrifiers in oxidation ditch were also confirmed by the dissimilarity in intensive bands between two systems obtained with DGGE analysis.

Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples.

PubMed

Pires, Ana C C; Cleary, Daniel F R; Almeida, Adelaide; Cunha, Angela; Dealtry, Simone; Mendonça-Hagler, Leda C S; Smalla, Kornelia; Gomes, Newton C M

2012-08-01

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.

Denaturing Gradient Gel Electrophoresis and Barcoded Pyrosequencing Reveal Unprecedented Archaeal Diversity in Mangrove Sediment and Rhizosphere Samples

PubMed Central

Pires, Ana C. C.; Cleary, Daniel F. R.; Almeida, Adelaide; Cunha, Ângela; Dealtry, Simone; Mendonça-Hagler, Leda C. S.; Smalla, Kornelia

2012-01-01

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages. PMID:22660713

Endophytic bacterial diversity in the phyllosphere of Amazon Paullinia cupana associated with asymptomatic and symptomatic anthracnose.

PubMed

Bogas, Andréa Cristina; Ferreira, Almir José; Araújo, Welington Luiz; Astolfi-Filho, Spartaco; Kitajima, Elliot Watanabe; Lacava, Paulo Teixeira; Azevedo, João Lúcio

2015-01-01

Endophytes colonize an ecological niche similar to that of phytopathogens, which make them candidate for disease suppression. Anthracnose is a disease caused by Colletotrichum spp., a phytopathogen that can infect guarana (Paullinia cupana), an important commercial crop in the Brazilian Amazon. We investigated the diversity of endophytic bacteria inhabiting the phyllosphere of asymptomatic and symptomatic anthracnose guarana plants. The PCR-denaturation gradient gel electrophoresis (PCR-DGGE) fingerprints revealed differences in the structure of the evaluated communities. Detailed analysis of endophytic bacteria composition using culture-dependent and 16S rRNA clone libraries revealed the presence of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria phyla. Firmicutes comprised the majority of isolates in asymptomatic plants (2.40E(-4)). However, cloning and sequencing of 16S rRNA revealed differences at the genus level for Neisseria (1.4E(-4)), Haemophilus (2.1E(-3)) and Arsenophonus (3.6E(-5)) in asymptomatic plants, Aquicella (3.5E(-3)) in symptomatic anthracnose plants, and Pseudomonas (1.1E(-3)), which was mainly identified in asymptomatic plants. In cross-comparisons of the endophytic bacterial communities as a whole, symptomatic anthracnose plants contained higher diversity, as reflected in the Shannon-Weaver and Simpson indices estimation (P < 0.05). Similarly, comparisons using LIBSHUFF and heatmap analysis for the relative abundance of operational taxonomic units (OTUs) showed differences between endophytic bacterial communities. These data are in agreement with the NMSD and ANOSIM analysis of DGGE profiles. Our results suggest that anthracnose can restructure endophytic bacterial communities by selecting certain strains in the phyllosphere of P. cupana. The understanding of these interactions is important for the development of strategies of biocontrol for Colletotrichum.

Microbial diversity of a Camembert-type cheese using freeze-dried Tibetan kefir coculture as starter culture by culture-dependent and culture-independent methods.

PubMed

Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei

2014-01-01

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.

Phylogenetic analysis of Archaea in the deep-sea sediments of west Pacific Warm Pool.

PubMed

Wang, Peng; Xiao, Xiang; Wang, Fengping

2005-06-01

Archaea are known to play important roles in carbon cycling in marine sediments. The main compositions of archaeal community in five deep-sea sediment samples collected from west Pacific Warm Pool area (WP-0, WP-1, WP-2, WP-3, WP-4), and in five sediment layers (1 cm-, 3 cm-, 6 cm-, 10 cm-, 12 cm- layer) of the 12 cm sediment core of WP-0 were checked and compared by denaturing gradient gel electrophoresis and 16 S rRNA gene sequencing. It was revealed that all the deep-sea sediment samples checked contained members of non-thermophilic marine group I crenarchaeota as the predominant archaeal group. To further detect groups of archaea possibly relating with C1 metabolism, PCR amplification was carried out using primers targeting methane-oxidizing archaea. Although no methane-oxidizing archaea was detected, a group of novel archaea (named as WPA) was instead identified from all these five WP samples by clone analysis. They could be placed in the euryarchaeota kingdom, separated into two distinct groups, the main group was peripherally related with methanogens, the other group related with Thermoplasma. The vertical distributions of WPA, archaea and bacteria along the WP-0 sediment column were determined by quantitative-PCR. It was found that bacteria dominated at all depths, the numbers of bacteria were 10-10(4) times more than those of archaea. The proportion of archaea versus bacteria had a depth related increasing tendency, it was lowest at the first layer (0.01%), reached highest at the 12 cm- layer (10%). WPA only constituted a small proportion of the archaeal community (0.05% to 5%) of west Pacific Warm Pool sediment.

Description of development of rumen ecosystem by PCR assay in milk-fed, weaned and finished lambs in an intensive fattening system.

PubMed

Belanche, A; Balcells, J; de la Fuente, G; Yañez-Ruíz, D R; Fondevila, M; Calleja, L

2010-10-01

This study examined the reticulo-rumen characteristics of the microbial community and its fermentative characteristics in milk-fed, at weaning and finished lambs in a conventional fattening system. Five lambs were assigned to each of three groups: milk-fed lambs slaughtered at 30 days (T30), weaned lambs slaughtered at 45 days (T45) and 'finished lambs' slaughtered at 90 days (T90). At slaughter, rumen size, fermentation parameters (pH, volatile fatty acids and microbial enzyme activity) and protozoal counts were recorded. Quantitative PCR was used to quantify the genes encoding 16S and 18S ribosomal DNA of the rumen bacterial and protozoal populations, respectively, and the sequential colonization of the rumen by cellulolytic (Ruminococcus albus, Ruminococcus flavefaciens) and amylolytic (Prevotella ruminicola, Streptococcus bovis) bacteria, and protozoa (Entodinium sp.). Denaturing gradient gel electrophoresis was used to study the development of rumen microbiota biodiversity. Intake of solid food before weaning caused a significant increase in rumen weight (p < 0.0001) and bacterial DNA (p < 0.05) and volatile fatty acid analysis concentration (p < 0.01), whereas pH declined. In milk-fed lambs, cellulolytic bacteria were evident after 30 days. Thereafter, in the 45-day and 90-day groups, the proportions of R. flavefaciens decreased and R. albus increased. Amylolytic bacteria were present in milk-fed lambs; the proportion of P. ruminicola increased in fattening lambs and S. bovis was the least abundant species. Protozoal concentrations were irregular; milk-fed lambs had a significant number of protozoa species from Entodinium and subfamily Isotrichiidae, but they disappeared at weaning. Lamb rumen were refaunated in some individuals at 90 days (Entodinium and subfamily Diplodiniinae spp.), although individual concentrations were variable. © 2009 Blackwell Verlag GmbH.

Enrichment of Geobacter species in response to stimulation of Fe(III) reduction in sandy aquifer sediments

USGS Publications Warehouse

Snoeyenbos-West, O.L.; Nevin, K.P.; Anderson, R.T.; Lovely, D.R.

2000-01-01

Engineered stimulation of Fe(III) has been proposed as a strategy to enhance the immobilization of radioactive and toxic metals in metal-contaminated subsurface environments. Therefore, laboratory and field studies were conducted to determine which microbial populations would respond to stimulation of Fe(III) reduction in the sediments of sandy aquifers. In laboratory studies, the addition of either various organic electron donors or electron shuttle compounds stimulated Fe(III) reduction and resulted in Geobacter sequences becoming important constituents of the Bacterial 16S rDNA sequences that could be detected with PCR amplification and denaturing gradient gel electrophoresis (DGGE). Quantification of Geobacteraceae sequences with a PCR most-probable-number technique indicated that the extent to which numbers of Geobacter increased was related to the degree of stimulation of Fe(III) reduction. Geothrix species were also enriched in some instances, but were orders of magnitude less numerous than Geobacter species. Shewanella species were not detected, even when organic compounds known to be electron donors for Shewanella species were used to stimulate Fe(III) reduction in the sediments. Geobacter species were also enriched in two field experiments in which Fe(III) reduction was stimulated with the addition of benzoate or aromatic hydrocarbons. The apparent growth of Geobacter species concurrent with increased Fe(III) reduction suggests that Geobacter species were responsible for much of the Fe(III) reduction in all of the stimulation approaches evaluated in three geographically distinct aquifers. Therefore, strategies for subsurface remediation that involve enhancing the activity of indigenous Fe(III)-reducing populations in aquifers should consider the physiological properties of Geobacter species in their treatment design.

Soil Microbial Community Responses to Additions of Organic Carbon Substrates and Heavy Metals (Pb and Cr)

PubMed Central

Nakatsu, Cindy H.; Carmosini, Nadia; Baldwin, Brett; Beasley, Federico; Kourtev, Peter; Konopka, Allan

2005-01-01

Microcosm experiments were conducted with soils contaminated with heavy metals (Pb and Cr) and aromatic hydrocarbons to determine the effects of each upon microbial community structure and function. Organic substrates were added as a driving force for change in the microbial community. Glucose represented an energy source used by a broad variety of bacteria, whereas fewer soil species were expected to use xylene. The metal amendments were chosen to inhibit the acute rate of organic mineralization by either 50% or 90%, and lower mineralization rates persisted over the entire 31-day incubation period. Significant biomass increases were abolished when metals were added in addition to organic carbon. The addition of organic carbon alone had the most significant impact on community composition and led to the proliferation of a few dominant phylotypes, as detected by PCR-denaturing gradient gel electrophoresis of bacterial 16S rRNA genes. However, the community-wide effects of heavy metal addition differed between the two carbon sources. For glucose, either Pb or Cr produced large changes and replacement with new phylotypes. In contrast, many phylotypes selected by xylene treatment were retained when either metal was added. Members of the Actinomycetales were very prevalent in microcosms with xylene and Cr(VI); gene copy numbers of biphenyl dioxygenase and phenol hydroxylase (but not other oxygenases) were elevated in these microcosms, as determined by real-time PCR. Much lower metal concentrations were needed to inhibit the catabolism of xylene than of glucose. Cr(VI) appeared to be reduced during the 31-day incubations, but in the case of glucose there was substantial microbial activity when much of the Cr(VI) remained. In the case of xylene, this was less clear. PMID:16332740

Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease.

PubMed

Di Cagno, Raffaella; Rizzello, Carlo G; Gagliardi, Francesca; Ricciuti, Patrizia; Ndagijimana, Maurice; Francavilla, Ruggiero; Guerzoni, M Elisabetta; Crecchio, Carmine; Gobbetti, Marco; De Angelis, Maria

2009-06-01

This study aimed at investigating the fecal microbiotas of children with celiac disease (CD) before (U-CD) and after (T-CD) they were fed a gluten-free diet and of healthy children (HC). Brothers or sisters of T-CD were enrolled as HC. Each group consisted of seven children. PCR-denaturing gradient gel electrophoresis (DGGE) analysis with V3 universal primers revealed a unique profile for each fecal sample. PCR-DGGE analysis with group- or genus-specific 16S rRNA gene primers showed that the Lactobacillus community of U-CD changed significantly, while the diversity of the Lactobacillus community of T-CD was quite comparable to that of HC. Compared to HC, the ratio of cultivable lactic acid bacteria and Bifidobacterium to Bacteroides and enterobacteria was lower in T-CD and even lower in U-CD. The percentages of strains identified as lactobacilli differed as follows: HC (ca. 38%) > T-CD (ca. 17%) > U-CD (ca. 10%). Lactobacillus brevis, Lactobacillus rossiae, and Lactobacillus pentosus were identified only in fecal samples from T-CD and HC. Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus gasseri were identified only in several fecal samples from HC. Compared to HC, the composition of Bifidobacterium species of T-CD varied, and it varied even more for U-CD. Forty-seven volatile organic compounds (VOCs) belonging to different chemical classes were identified using gas-chromatography mass spectrometry-solid-phase microextraction analysis. The median concentrations varied markedly for HC, T-CD, and U-CD. Overall, the r(2) values for VOC data for brothers and sisters were equal to or lower than those for unrelated HC and T-CD. This study shows the effect of CD pathology on the fecal microbiotas of children.

Microbial Diversity of a Camembert-Type Cheese Using Freeze-Dried Tibetan Kefir Coculture as Starter Culture by Culture-Dependent and Culture-Independent Methods

PubMed Central

Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei

2014-01-01

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese. PMID:25360757

Characterization of a Highly Enriched Microbial Consortium Reductively Dechlorinating 2,3-Dichlorophenol and 2,4,6-Trichlorophenol and the Corresponding cprA Genes from River Sediment.

PubMed

El-Sayed, Wael S

2016-08-26

Anaerobic reductive dechlorination of 2,3-dichlorophenol (2,3DCP) and 2,4,6-trichlorophenol (2,4,6TCP) was investigated in microcosms from River Nile sediment. A stable sediment-free anaerobic microbial consortium reductively dechlorinating 2,3DCP and 2,4,6TCP was established. Defined sediment-free cultures showing stable dechlorination were restricted to ortho chlorine when enriched with hydrogen as the electron donor, acetate as the carbon source, and either 2,3-DCP or 2,4,6-TCP as electron acceptors. When acetate, formate, or pyruvate were used as electron donors, dechlorination activity was lost. Only lactate can replace dihydrogen as an electron donor. However, the dechlorination potential was decreased after successive transfers. To reveal chlororespiring species, the microbial community structure of chlorophenol-reductive dechlorinating enrichment cultures was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Eight dominant bacteria were detected in the dechlorinating microcosms including members of the genera Citrobacter, Geobacter, Pseudomonas, Desulfitobacterium, Desulfovibrio and Clostridium. Highly enriched dechlorinating cultures were dominated by four bacterial species belonging to the genera Pseudomonas, Desulfitobacterium, and Clostridium. Desulfitobacterium represented the major fraction in DGGE profiles indicating its importance in dechlorination activity, which was further confirmed by its absence resulting in complete loss of dechlorination. Reductive dechlorination was confirmed by the stoichiometric dechlorination of 2,3DCP and 2,4,6TCP to metabolites with less chloride groups and by the detection of chlorophenol RD cprA gene fragments in dechlorinating cultures. PCR amplified cprA gene fragments were cloned and sequenced and found to cluster with the cprA/pceA type genes of Dehalobacter restrictus.

Taxonomic structure and monitoring of the dominant population of lactic acid bacteria during wheat flour sourdough type I propagation using Lactobacillus sanfranciscensis starters.

PubMed

Siragusa, Sonya; Di Cagno, Raffaella; Ercolini, Danilo; Minervini, Fabio; Gobbetti, Marco; De Angelis, Maria

2009-02-01

The structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains of Lactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g(-1). Partial sequence analysis of the 16S rRNA and recA genes and PCR-denaturating gradient gel electrophoresis analysis using the rpoB gene allowed identification of Weissella confusa, Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rossiae, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Pediococcus pentosaceus, and Lactobacillus spp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain of L. sanfranciscensis was found in all the sourdoughs. Except for L. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes of L. sanfranciscensis and with W. confusa or L. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.

Further characterization of the ABR gene in medulloblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wright-White, E.C.; Haken, M.S. von; McDonald J.D.

1994-09-01

Although brain tumors are the most common type of solid cancer in children, little is known about their etiology at the molecular genetic level. Using a panel of 20 chromosome 17p markers, we have previously determined that loss of distal chromosome 17p DNA sequences occurs in 14 of 35 specimens (40%) of medulloblastoma, one of the most common pediatric intracranial neoplasms. Analysis of these same tumors using a PCR-denaturing gradient gel electrophoresis technique has shown only two p53 gene mutations. These results suggest that a tumor suppressor gene in addition to p53 may be located on distal chromosome 17p. Consensusmore » deletion mapping of our specimens suggests that the smallest site of chromosomal loss in defined distally by marker 144-D6, the most telomeric probe as yet identified on chromosome 17p, and proximally by the ABR marker, a BCR homologous gene containing two highly polymorphic VNTR regions. We have used fluorescence in situ hybridization and pulsed-field gel electrophoresis to determine that the ABR gene lies transcriptionally oriented 5{prime} to 3{prime} at a distance of 240 kb from marker 144-D6. We have also constructed a cosmid contig map spanning 120 kb of this region. Using one of these cosmids as a probe, we have detected breakpoints in three of the tumor specimens that lie between the two VNTR regions within the ABR gene. We have subsequently designed PCR primers to cover the breakpoint region which include the ABR exons which have the strongest homology to the BCR gene (Mbcr region), and are screening our tumor specimens for mutations. These results suggest that loss of ABR gene sequences may be involved in the etiology of medulloblastoma.« less

Microbial Contents of Vacuum Cleaner Bag Dust and Emitted Bioaerosols and Their Implications for Human Exposure Indoors

PubMed Central

Veillette, Marc; Knibbs, Luke D.; Pelletier, Ariane; Charlebois, Remi; Blais Lecours, Pascale; He, Congrong; Morawska, Lidia

2013-01-01

Vacuum cleaners can release large concentrations of particles, both in their exhaust air and from resuspension of settled dust. However, the size, variability, and microbial diversity of these emissions are unknown, despite evidence to suggest they may contribute to allergic responses and infection transmission indoors. This study aimed to evaluate bioaerosol emission from various vacuum cleaners. We sampled the air in an experimental flow tunnel where vacuum cleaners were run, and their airborne emissions were sampled with closed-face cassettes. Dust samples were also collected from the dust bag. Total bacteria, total archaea, Penicillium/Aspergillus, and total Clostridium cluster 1 were quantified with specific quantitative PCR protocols, and emission rates were calculated. Clostridium botulinum and antibiotic resistance genes were detected in each sample using endpoint PCR. Bacterial diversity was also analyzed using denaturing gradient gel electrophoresis (DGGE), image analysis, and band sequencing. We demonstrated that emission of bacteria and molds (Penicillium/Aspergillus) can reach values as high as 1E5 cell equivalents/min and that those emissions are not related to each other. The bag dust bacterial and mold content was also consistent across the vacuums we assessed, reaching up to 1E7 bacterial or mold cell equivalents/g. Antibiotic resistance genes were detected in several samples. No archaea or C. botulinum was detected in any air samples. Diversity analyses showed that most bacteria are from human sources, in keeping with other recent results. These results highlight the potential capability of vacuum cleaners to disseminate appreciable quantities of molds and human-associated bacteria indoors and their role as a source of exposure to bioaerosols. PMID:23934489

Identification of Bacteria Potentially Responsible for Oxic and Anoxic Sulfide Oxidation in Biofilters of a Recirculating Mariculture System

PubMed Central

Cytryn, Eddie; van Rijn, Jaap; Schramm, Andreas; Gieseke, Armin; de Beer, Dirk; Minz, Dror

2005-01-01

Bacteria presumably involved in oxygen- or nitrate-dependent sulfide oxidation in the biofilters of a recirculating marine aquaculture system were identified using a new application of reverse transcription-PCR denaturing gradient gel electrophoresis (DGGE) analysis termed differential-transcription (DT)-DGGE. Biofilter samples were incubated in various concentrations of sulfide or thiosulfate (0 to 5 mM) with either oxygen or nitrate as the sole electron acceptor. Before and after short-term incubations (10 to 20 h), total DNA and RNA were extracted, and a 550-bp fragment of the 16S rRNA genes was PCR amplified either directly or after reverse transcription. DGGE analysis of DNA showed no significant change of the original microbial consortia upon incubation. In contrast, DGGE of cDNA revealed several phylotypes whose relative band intensities markedly increased or decreased in response to certain incubation conditions, indicating enhanced or suppressed rRNA transcription and thus implying metabolic activity under these conditions. Specifically, species of the gammaproteobacterial genus Thiomicrospira and phylotypes related to symbiotic sulfide oxidizers could be linked to oxygen-dependent sulfide oxidation, while members of the Rhodobacteraceae (genera Roseobacter, Rhodobacter, and Rhodobium) were putatively active in anoxic, nitrate-dependent sulfide oxidation. For all these organisms, the physiology of their closest cultured relatives matches their DT-DGGE-inferred function. In addition, higher band intensities following exposure to 5 mM sulfide and nitrate were observed for Thauera-, Hydrogenophaga-, and Dethiosulfovibrio-like phylotypes. For these genera, nitrate-dependent sulfide oxidation has not been documented previously and therefore DT-DGGE might indicate a higher relative tolerance to high sulfide concentrations than that of other community members. We anticipate that DT-DGGE will be of general use in tracing functionally equivalent yet phylogenetically diverse microbial populations in nature. PMID:16204531

Probiotics Can Induce Follicle Maturational Competence: The Danio rerio Case1

PubMed Central

Gioacchini, Giorgia; Giorgini, Elisabetta; Merrifield, Daniel L.; Hardiman, Gary; Borini, Andrea; Vaccari, Lisa; Carnevali, Oliana

2011-01-01

ABSTRACT In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the maturation phase. PMID:22088919

Markers and mapping revisited: finding your gene.

PubMed

Jones, Neil; Ougham, Helen; Thomas, Howard; Pasakinskiene, Izolda

2009-01-01

This paper is an update of our earlier review (Jones et al., 1997, Markers and mapping: we are all geneticists now. New Phytologist 137: 165-177), which dealt with the genetics of mapping, in terms of recombination as the basis of the procedure, and covered some of the first generation of markers, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), simple sequence repeats (SSRs) and quantitative trait loci (QTLs). In the intervening decade there have been numerous developments in marker science with many new systems becoming available, which are herein described: cleavage amplification polymorphism (CAP), sequence-specific amplification polymorphism (S-SAP), inter-simple sequence repeat (ISSR), sequence tagged site (STS), sequence characterized amplification region (SCAR), selective amplification of microsatellite polymorphic loci (SAMPL), single nucleotide polymorphism (SNP), expressed sequence tag (EST), sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), microarrays, diversity arrays technology (DArT), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and methylation-sensitive PCR. In addition there has been an explosion of knowledge and databases in the area of genomics and bioinformatics. The number of flowering plant ESTs is c. 19 million and counting, with all the opportunity that this provides for gene-hunting, while the survey of bioinformatics and computer resources points to a rapid growth point for future activities in unravelling and applying the burst of new information on plant genomes. A case study is presented on tracking down a specific gene (stay-green (SGR), a post-transcriptional senescence regulator) using the full suite of mapping tools and comparative mapping resources. We end with a brief speculation on how genome analysis may progress into the future of this highly dynamic arena of plant science.

Niche partitioning of marine group I Crenarchaeota in the euphotic and upper mesopelagic zones of the East China Sea.

PubMed

Hu, Anyi; Jiao, Nianzhi; Zhang, Rui; Yang, Zao

2011-11-01

Marine group I Crenarchaeota (MGI) represents a ubiquitous and numerically predominant microbial population in marine environments. An understanding of the spatial dynamics of MGI and its controlling mechanisms is essential for an understanding of the role of MGI in energy and element cycling in the ocean. In the present study, we investigated the diversity and abundance of MGI in the East China Sea (ECS) by analysis of crenarchaeal 16S rRNA gene, the ammonia monooxygenase gene amoA, and the biotin carboxylase gene accA. Quantitative PCR analyses revealed that these genes were higher in abundance in the mesopelagic than in the euphotic zone. In addition, the crenarchaeal amoA gene was positively correlated with the copy number of the MGI 16S rRNA gene, suggesting that most of the MGI in the ECS are nitrifiers. Furthermore, the ratios of crenarchaeal accA to amoA or to MGI 16S rRNA genes increased from the euphotic to the mesopelagic zone, suggesting that the role of MGI in carbon cycling may change from the epipelagic to the mesopelagic zones. Denaturing gradient gel electrophoretic profiling of the 16S rRNA genes revealed depth partitioning in MGI community structures. Clone libraries of the crenarchaeal amoA and accA genes showed both "shallow" and "deep" groups, and their relative abundances varied in the water column. Ecotype simulation analysis revealed that MGI in the upper ocean could diverge into special ecotypes associated with depth to adapt to the light gradient across the water column. Overall, our results showed niche partitioning of the MGI population and suggested a shift in their ecological functions between the euphotic and mesopelagic zones of the ECS.

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

PubMed

Babu, Binoy; Washburn, Brian K; Ertek, Tülin Sarigül; Miller, Steven H; Riddle, Charles B; Knox, Gary W; Ochoa-Corona, Francisco M; Olson, Jennifer; Katırcıoğlu, Yakup Zekai; Paret, Mathews L

2017-09-01

Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/μl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes. Copyright © 2017 Elsevier B.V. All rights reserved.

A disposable, self-contained PCR chip.

PubMed

Kim, Jitae; Byun, Doyoung; Mauk, Michael G; Bau, Haim H

2009-02-21

A disposable, self-contained polymerase chain reaction (PCR) chip with on-board stored, just-on-time releasable, paraffin-passivated, dry reagents is described. During both storage and sample preparation, the paraffin immobilizes and protects the stored reagents. Fluid flow through the reactor leaves the reagents undisturbed. Prior to the amplification step, the chamber is filled with target analyte suspended in water. Upon heating the PCR chamber to the DNA's denaturation temperature, the paraffin melts and moves out of the way, and the reagents are released and hydrated. To better understand the reagent release process, a scaled up model of the reactor was constructed and the paraffin migration was visualized. Experiments were carried out with a 30 microl reactor demonstrating detectable amplification (with agarose gel electrophoresis) of 10 fg ( approximately 200 copies) of lambda DNA template. The in-reactor storage and on-time release of the PCR reagents reduce the number of needed operations and significantly simplifies the flow control that would, otherwise, be needed in lab-on-chip devices.

A Disposable, Self-Contained PCR Chip

PubMed Central

Kim, Jitae; Byun, Doyoung; Mauk, Michael G.; Bau, Haim H.

2009-01-01

A disposable, self-contained polymerase chain reaction (PCR) chip with on-board stored, just on time releasable, paraffin-passivated, dry reagents is described. During both storage and sample preparation, the paraffin immobilizes and protects the stored reagents. Fluid flow through the reactor leaves the reagents undisturbed. Prior to the amplification step, the chamber is filled with target analyte suspended in water. Upon heating the PCR chamber to the DNA’s denaturation temperature, the paraffin melts and moves out of the way, and the reagents are released and hydrated. To better understand the reagent release process, a scaled up model of the reactor was constructed and the paraffin migration was visualized. Experiments were carried out with a 30 μl reactor demonstrating detectable amplification (with agarose gel electrophoresis) of 10 fg (~200 copies) of lambda DNA template. The in-reactor storage and on-time release of the PCR reagents reduce the number of needed operations and significantly simplify the flow control that would, otherwise, be needed in lab-on-chip devices. PMID:19190797

Chaperonin-based biolayer interferometry to assess the kinetic stability of metastable, aggregation-prone proteins

PubMed Central

Lea, Wendy A.; Naik, Subhashchandra; Chaudhri, Tapan; Machen, Alexandra J.; O’Neil, Pierce T.; McGinn-Straub, Wesley; Tischer, Alexander; Auton, Matthew T.; Burns, Joshua R.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.

2017-01-01

Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy to decrease disease pathologies brought on by protein folding defects or deleterious kinetic transitions. Current methods of examining ligand binding to these marginally stable native states are limited, because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, multi-domain) and metastable proteins (e.g. low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant-pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein immobilized on a BLI biosensor to increasing denaturant concentrations (urea or GnHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remain is detected by increased GroEL binding. Since this kinetic denaturant pulse is brief, the amplitude of the GroEL binding to the immobilized protein depends on the duration of exposure to denaturant, the concentration of denaturant, wash times, and the underlying protein unfolding/refolding kinetics; fixing all other parameters and plotting GroEL binding amplitude versus denaturant pulse concentration results in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein is manifested by a decreased GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules/solution conditions that can stabilize or destabilize thermally stable proteins, multi-domain proteins, oligomeric proteins, and most importantly, aggregation prone metastable proteins. PMID:27505032

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

PubMed

Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

2016-11-01

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Effects of thermally induced denaturation on technological-functional properties of whey protein isolate-based films.

PubMed

Schmid, M; Krimmel, B; Grupa, U; Noller, K

2014-09-01

This study examined how and to what extent the degree of denaturation affected the technological-functional properties of whey protein isolate (WPI)-based coatings. It was observed that denaturation affected the material properties of WPI-coated films significantly. Surface energy decreased by approximately 20% compared with native coatings. Because the surface energy of a coating should be lower than that of the substrate, this might result in enhanced wettability characteristics between WPI-based solution and substrate surface. Water vapor barrier properties increased by about 35% and oxygen barrier properties increased by approximately 33%. However, significant differences were mainly observed between coatings made of fully native WPI and ones with a degree of denaturation of 25%. Higher degrees of denaturation did not lead to further improvement of material properties. This observation offers cost-saving potential: a major share of denatured whey proteins may be replaced by fully native ones that are not exposed to energy-intensive heat treatment. Furthermore, native WPI solutions can be produced with higher dry matter content without gelatinizing. Hence, less moisture has to be removed through drying, resulting in reduced energy consumption. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere.

PubMed

Kimura, Hiroyuki; Ishibashi, Jun-Ichiro; Masuda, Harue; Kato, Kenji; Hanada, Satoshi

2007-04-01

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.

PCR amplification and genetic analysis in a microwell cell culturing chip.

PubMed

Lindström, Sara; Hammond, Maria; Brismar, Hjalmar; Andersson-Svahn, Helene; Ahmadian, Afshin

2009-12-21

We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity.

Analysis of protein stability and ligand interactions by thermal shift assay.

PubMed

Huynh, Kathy; Partch, Carrie L

2015-02-02

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

PubMed Central

Le Bourhis, Anne-Gaëlle; Saunier, Katiana; Doré, Joël; Carlier, Jean-Philippe; Chamba, Jean-François; Popoff, Michel-Robert; Tholozan, Jean-Luc

2005-01-01

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g. PMID:15640166

Rapid Detection of Haptoglobin Gene Deletion in Alkaline-Denatured Blood by Loop-Mediated Isothermal Amplification Reaction

PubMed Central

Soejima, Mikiko; Egashira, Kouichi; Kawano, Hiroyuki; Kawaguchi, Atsushi; Sagawa, Kimitaka; Koda, Yoshiro

2011-01-01

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin antibodies. Being homozygous for the haptoglobin gene deletion allele (HPdel) is the only known cause of congenital anhaptoglobinemia, and detection of HPdel before transfusion is important to prevent anaphylactic shock. In this study, we developed a loop-mediated isothermal amplification (LAMP)-based screening for HPdel. Optimal primer sets and temperature for LAMP were selected for HPdel and the 5′ region of the HP using genomic DNA as a template. Then, the effects of diluent and boiling on LAMP amplification were examined using whole blood as a template. Blood samples diluted 1:100 with 50 mmol/L NaOH without boiling gave optimal results as well as those diluted 1:2 with water followed by boiling. The results from 100 blood samples were fully concordant with those obtained by real-time PCR methods. Detection of the HPdel allele by LAMP using alkaline-denatured blood samples is rapid, simple, accurate, and cost effective, and is readily applicable in various clinical settings because this method requires only basic instruments. In addition, the simple preparation of blood samples using NaOH saves time and effort for various genetic tests. PMID:21497293

Multiplex Amplification Coupled with COLD-PCR and High Resolution Melting Enables Identification of Low-Abundance Mutations in Cancer Samples with Low DNA Content

PubMed Central

Milbury, Coren A.; Chen, Clark C.; Mamon, Harvey; Liu, Pingfang; Santagata, Sandro; Makrigiorgos, G. Mike

2011-01-01

Thorough screening of cancer-specific biomarkers, such as DNA mutations, can require large amounts of genomic material; however, the amount of genomic material obtained from some specimens (such as biopsies, fine-needle aspirations, circulating-DNA or tumor cells, and histological slides) may limit the analyses that can be performed. Furthermore, mutant alleles may be at low-abundance relative to wild-type DNA, reducing detection ability. We present a multiplex-PCR approach tailored to amplify targets of interest from small amounts of precious specimens, for extensive downstream detection of low-abundance alleles. Using 3 ng of DNA (1000 genome-equivalents), we amplified the 1 coding exons (2-11) of TP53 via multiplex-PCR. Following multiplex-PCR, we performed COLD-PCR (co-amplification of major and minor alleles at lower denaturation temperature) to enrich low-abundance variants and high resolution melting (HRM) to screen for aberrant melting profiles. Mutation-positive samples were sequenced. Evaluation of mutation-containing dilutions revealed improved sensitivities after COLD-PCR over conventional-PCR. COLD-PCR improved HRM sensitivity by approximately threefold to sixfold. Similarly, COLD-PCR improved mutation identification in sequence-chromatograms over conventional PCR. In clinical specimens, eight mutations were detected via conventional-PCR-HRM, whereas 12 were detected by COLD-PCR-HRM, yielding a 33% improvement in mutation detection. In summary, we demonstrate an efficient approach to increase screening capabilities from limited DNA material via multiplex-PCR and improve mutation detection sensitivity via COLD-PCR amplification. PMID:21354058

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls.

PubMed

Phutikanit, Nawapen; Suwimonteerabutr, Junpen; Harrison, Dion; D'Occhio, Michael; Carroll, Bernie; Techakumphu, Mongkol

2010-03-05

The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates. Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90%) were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8%) and the lowest in fibroblastic DNA (92.3%, P < or = 0.05). Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P < or = 0.05). The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic cells.

Estimating conformation content of a protein using citrate-stabilized Au nanoparticles

NASA Astrophysics Data System (ADS)

Deka, Jashmini; Paul, Anumita; Chattopadhyay, Arun

2010-08-01

Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration.Herein we report the use of the optical properties of citrate-stabilized gold nanoparticles (Au NPs) for estimation of native or denatured conformation content in a mixture of a protein in solution. The UV-vis extinction spectrum of citrate-stabilized Au NPs is known to broaden differently in the presence of native and denatured states of α-amylase, bovine serum albumin (BSA) or amyloglucosidase (AMG). On the other hand, herein we show that when a mixture of native and denatured protein was present in the medium, the broadening of the spectrum differed for different fractional content of the conformations. Also, the total area under the extinction spectrum varied linearly with the change in the mole fraction content of a state and for a constant total protein concentration. Transmission electron microscopy (TEM) measurements revealed different levels of agglomeration for different fractional contents of the native or denatured state of a protein. In addition, time-dependent denaturation of a protein could be followed using the present method. The rate constants calculated for denaturation indicated a possible fast change in conformation of a protein before complete thermal denaturation. The observations have been explained based on the changes in extinction coefficient (thereby oscillator strength) upon interaction of citrate-stabilized NPs with proteins being in different states and levels of agglomeration. Electronic supplementary information (ESI) available: Additional UV-vis and fluorescence spectra and graphs based on UV-vis studies. See DOI: 10.1039/c0nr00154f

Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting.

PubMed

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2008-04-01

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.