Detection of bacterial contamination and DNA quantification in stored blood units in 2 veterinary hospital blood banks.

PubMed

Stefanetti, Valentina; Miglio, Arianna; Cappelli, Katia; Capomaccio, Stefano; Sgariglia, Elisa; Marenzoni, Maria L; Antognoni, Maria T; Coletti, Mauro; Mangili, Vittorio; Passamonti, Fabrizio

2016-09-01

Blood transfusions in veterinary medicine have become increasingly more common and are now an integral part of lifesaving and advanced treatment in small and large animals. Important risks associated with transfusion of blood products include the transmission of various infectious diseases. Several guidelines suggest what infectious agents to screen for in canine and feline transfusion medicine. However, while the risk of bacterial contamination of blood products during storage and administration has not been documented in veterinary medicine, it has emerged as a cause of morbidity and mortality in human transfusion medicine. Clinical experience shows that the majority of blood component bacterial contaminations are caused by only a few species. Unlike other types of bacteria, psychrotolerant species like Pseudomonas spp. and Serratia spp. can proliferate during the storage of blood units at 4°C from a very low titer at the time of blood collection to a clinically significant level (> 10(5) CFU/mL) causing clinical sepsis resulting from red blood cell concentrate transfusions in human medicine. The purpose of this report was to describe the detection and quantification procedures applied in 4 cases of bacterial contamination of canine and feline blood units, which suggest the need for further investigations to optimize patients' safety in veterinary transfusion medicine. © 2016 American Society for Veterinary Clinical Pathology.

Airborne bacterial contaminations in typical Chinese wet market with live poultry trade.

PubMed

Gao, Xin-Lei; Shao, Ming-Fei; Luo, Yi; Dong, Yu-Fang; Ouyang, Feng; Dong, Wen-Yi; Li, Ji

2016-12-01

Chinese wet markets with live poultry trade have been considered as major sources of pathogen dissemination, and sites for horizontal transfer of bacterial and viral pathogens. In this study, the pathogenic bacteria and antibiotic resistant genes (ARGs) in air samples collected at a typical Chinese wet market had been analysis and quantified. Corynebacterium minutissimum and other pathogenic bacteria accounted for 0.81-8.02% of the whole microbial community in different air samples. The four ARGs quantified in this study showed a comparable relative concentration (copies/ng_DNA) with municipal wastewater. Poultry manures were demonstrated to be important microbial contamination source in wet market, which was supported by both microbial composition based source tracking and the quantification of airborne microbial density. A series of Firmicutes and Bacteroidetes indicators of poultry area contamination were successfully screened, which will be useful for the more convenient monitoring of airborne poultry area contamination. Our results indicate bioaerosols acted as important route for the transmissions of pathogens and ARGs. Continued surveillance of airborne microbial contamination is required in poultry trade wet market. Urban live poultry markets are important sources of pathogen dissemination, and sites for horizontal transfer of viral and bacterial pathogens. In the present field-study, pathogenic bacteria and antibiotic resistance genes were focused to provide quantitative information on the levels of microbial contaminations at the indoor air of wet markets. Results demonstrated that poultry manures were important microbial contamination source in wet market, and in the meanwhile bioaerosols were identified as important route for the transmissions of microbial contaminants. A series of Firmicutes and Bacteroidetes indicators of poultry area contamination were successfully screened, which will be useful for the more convenient monitoring of airborne poultry

Perforation and bacterial contamination of microscope covers in lumbar spinal decompressive surgery.

PubMed

Osterhoff, Georg; Spirig, José; Klasen, Jürgen; Kuster, Stefan P; Zinkernagel, Annelies S; Sax, Hugo; Min, Kan

2014-01-01

To determine the integrity of microscope covers and bacterial contamination at the end of lumbar spinal decompressive surgery. A prospective study of 25 consecutive lumbar spinal decompressions with the use of a surgical microscope was performed. For detection of perforations, the microscope covers were filled with water at the end of surgery and the presence of water leakage in 3 zones (objective, ocular and control panel) was examined. For detection of bacterial contamination, swabs were taken from the covers at the same locations before and after surgery. Among the 25 covers, 1 (4%) perforation was observed and no association between perforation and bacterial contamination was seen; 3 (4%) of 75 smears from the 25 covers showed post-operative bacterial contamination, i.e. 2 in the ocular zone and 1 in the optical zone, without a cover perforation. The incidence of microscope cover perforation was very low and was not shown to be associated with bacterial contamination. External sources of bacterial contamination seem to outweigh the problem of contamination due to failure of cover integrity. © 2014 S. Karger AG, Basel.

Perforation and Bacterial Contamination of Microscope Covers in Lumbar Spinal Decompressive Surgery

PubMed Central

Osterhoff, Georg; Spirig, José; Klasen, Jürgen; Kuster, Stefan P.; Zinkernagel, Annelies S.; Sax, Hugo; Min, Kan

2014-01-01

Objective To determine the integrity of microscope covers and bacterial contamination at the end of lumbar spinal decompressive surgery. Materials and Methods A prospective study of 25 consecutive lumbar spinal decompressions with the use of a surgical microscope was performed. For detection of perforations, the microscope covers were filled with water at the end of surgery and the presence of water leakage in 3 zones (objective, ocular and control panel) was examined. For detection of bacterial contamination, swabs were taken from the covers at the same locations before and after surgery. Results Among the 25 covers, 1 (4%) perforation was observed and no association between perforation and bacterial contamination was seen; 3 (4%) of 75 smears from the 25 covers showed post-operative bacterial contamination, i.e. 2 in the ocular zone and 1 in the optical zone, without a cover perforation. Conclusions The incidence of microscope cover perforation was very low and was not shown to be associated with bacterial contamination. External sources of bacterial contamination seem to outweigh the problem of contamination due to failure of cover integrity. PMID:24903448

Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation

USDA-ARS?s Scientific Manuscript database

Bacterial contaminants can inhibit ethanol production in biofuel fermentations, and even result in stuck fermentations. Contaminants may persist in production facilities by forming recalcitrant biofilms. A two-year longitudinal study was conducted of bacterial contaminants from a Midwestern dry grin...

An assessment of air as a source of DNA contamination encountered when performing PCR.

PubMed

Witt, Nina; Rodger, Gillian; Vandesompele, Jo; Benes, Vladimir; Zumla, Alimuddin; Rook, Graham A; Huggett, Jim F

2009-12-01

Sensitive molecular methods, such as the PCR, can detect low-level contamination, and careful technique is required to reduce the impact of contaminants. Yet, some assays that are designed to detect high copy-number target sequences appear to be impossible to perform without contamination, and frequently, personnel or laboratory environment are held responsible as the source. This complicates diagnostic and research analysis when using molecular methods. To investigate the air specifically as a source of contamination, which might occur during PCR setup, we exposed tubes of water to the air of a laboratory and clean hood for up to 24 h. To increase the chances of contamination, we also investigated a busy open-plan office in the same way. All of the experiments showed the presence of human and rodent DNA contamination. However, there was no accumulation of the contamination in any of the environments investigated, suggesting that the air was not the source of contamination. Even the air from a busy open-plan office was a poor source of contamination for all of the DNA sequences investigated (human, bacterial, fungal, and rodent). This demonstrates that the personnel and immediate laboratory environment are not necessarily to blame for the observed contamination.

An Assessment of Air As a Source of DNA Contamination Encountered When Performing PCR

PubMed Central

Witt, Nina; Rodger, Gillian; Vandesompele, Jo; Benes, Vladimir; Zumla, Alimuddin; Rook, Graham A.; Huggett, Jim F.

2009-01-01

Sensitive molecular methods, such as the PCR, can detect low-level contamination, and careful technique is required to reduce the impact of contaminants. Yet, some assays that are designed to detect high copy-number target sequences appear to be impossible to perform without contamination, and frequently, personnel or laboratory environment are held responsible as the source. This complicates diagnostic and research analysis when using molecular methods. To investigate the air specifically as a source of contamination, which might occur during PCR setup, we exposed tubes of water to the air of a laboratory and clean hood for up to 24 h. To increase the chances of contamination, we also investigated a busy open-plan office in the same way. All of the experiments showed the presence of human and rodent DNA contamination. However, there was no accumulation of the contamination in any of the environments investigated, suggesting that the air was not the source of contamination. Even the air from a busy open-plan office was a poor source of contamination for all of the DNA sequences investigated (human, bacterial, fungal, and rodent). This demonstrates that the personnel and immediate laboratory environment are not necessarily to blame for the observed contamination. PMID:19949694

Aspergillus DNA contamination in blood collection tubes.

PubMed

Harrison, Elizabeth; Stalhberger, Thomas; Whelan, Ruth; Sugrue, Michele; Wingard, John R; Alexander, Barbara D; Follett, Sarah A; Bowyer, Paul; Denning, David W

2010-08-01

Fungal polymerase chain reaction (PCR)-based diagnostic methods are at risk for contamination. Sample collection containers were investigated for fungal DNA contamination using real-time PCR assays. Up to 18% of blood collection tubes were contaminated with fungal DNA, probably Aspergillus fumigatus. Lower proportions of contamination in other vessels were observed. Copyright 2010 Elsevier Inc. All rights reserved.

Bacterial community changes in response to oil contamination and perennial crop cultivation.

PubMed

Yan, Lijuan; Penttinen, Petri; Mikkonen, Anu; Lindström, Kristina

2018-05-01

We investigated bacterial community dynamics in response to used motor oil contamination and perennial crop cultivation by 16S rRNA gene amplicon sequencing in a 4-year field study. Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes were the major bacterial phyla, and Rhodococcus was the most abundant genus. Initially, oil contamination decreased the overall bacterial diversity. Actinobacteria, Betaproteobacteria, and Gammaproteobacteria were sensitive to oil contamination, exhibiting clear succession with time. However, bacterial communities changed over time, regardless of oil contamination and crop cultivation. The abundance difference of most OTUs between oil-contaminated and non-contaminated plots remained the same in later sampling years after the initial abundance difference induced by oil spike. The abundances of three oil-favored actinobacteria (Lysinimonas, Microbacteriaceae, and Marmoricola) and one betaproteobacterium (Aquabacterium) changed in different manner over time in oil-contaminated and non-contaminated soil. We propose that these taxa are potential bio-indicators for monitoring recovery from motor oil contamination in boreal soil. The effect of crop cultivation on bacterial communities became significant only after the crops achieved stable growth, likely associated with plant material decomposition by Bacteroidetes, Armatimonadetes and Fibrobacteres.

Procedures involving lipid media for detection of bacterial contamination in breweries.

PubMed

Van Vuuren, H J; Louw, H A; Loos, M A; Meisel, R

1977-02-01

The liquid equivalent of universal beer agar, designated universal beer liquid medium, and its beer-free equivalent, universal liquid medium (UL), were equally effective in demonstrating bacterial contamination in 120 of 200 samples from different stages of commercial brewing process. Growth of the contaminants after 3 days was consistently more luxuriant in the UL medium. A yeast-water substrate medium failed to reveal many contaminants detected with UL in 392 samples from three breweries and revealed only a few not detected with UL. The use of UL and a lactose-peptone medium, with microscope examination of the media for bacterial growth, permitted detection of 93% of the known contaminants compared to 87%, detected with UL alone; this combination or universal beer liquid medium plus lactose-peptone medium can therefore be recommended for the detection of bacterial contaminants in brewery samples. Bacterial contamination of pitching yeasts appeared to be a particular problem in the breweries investigated.

Bacterial DNA indicated as an important inducer of fish cathelicidins.

PubMed

Maier, Valerie Helene; Schmitt, Clemens Nikolaus Zeno; Gudmundsdottir, Sigridur; Gudmundsson, Gudmundur Hrafn

2008-04-01

Cathelicidins are antimicrobial peptides indicated as important in the control of the natural microflora as well as in the fight against bacterial invasion in mammals. Little is known about cathelicidins in fish and here the Chinook salmon (Oncorhynchus tshawytscha) embryo cell line (CHSE-214) was used as a model system to study the expression of cathelicidins due to fish pathogenic bacteria. The cDNA of cathelicidin from CHSE-214 cells (csCath) was cloned and shown to be closely related to gene 2 of both rainbow trout and Atlantic salmon. The deducted amino acid sequence showed highest sequence identity to rtCath2 with 95% and 72% for the cathelin and the antibacterial part, respectively. Cathelicidin gene expression was studied and various Gram positive and Gram negative bacteria caused the upregulation of the gene (csCath). Bacterial DNA and protein were shown important for the induction of cathelicidin expression in these cells. LPS (Escherichia coli) also causes the upregulation of cathelicidins, but digestion of the LPS with DNase I before incubation of the cells, totally abolished the upregulation of cathelicidin and suggests DNA contamination in the LPS to be the trigger for this effect. These results could explain the limited responsiveness of fish cells towards pure LPS and confirm previous suggestions that fish cells are less sensitive to LPS than mammalian cells.

Detection of bacterial DNA by PCR in dogs with stifle pathology.

PubMed

Bhandal, Jitender; Hayashi, Kei; Kim, Sun-Young; Klein, Martha; Wong, Alice; Toupadakis, Chrisoula A; Muir, Peter; Yellowley, Clare E

2013-10-01

To determine presence of bacterial DNA in canine stifles with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL) compared to normal canine stifles (control). Prospective clinical study. Dogs (n = 44). Dogs of varying age, breed, sex, and weight residing in California were assessed for stifle pathology (CCLR, MPL, or normal control). Synovial fluid of all stifles was assessed for the presence of bacterial DNA using broad-ranging 16S rRNA primers and PCR. Bacterial DNA was detected in normal control stifles and those with CCLR and MPL. There were no statistical differences in the copy numbers of bacterial DNA in the stifle synovial fluid among groups (P > .05); however, synovial fluid specimens from dogs with stifle pathology (CCLR and MPL combined) tended to have higher copy numbers of bacterial DNA than those from controls (P = .06). There was no significant difference in the number of bacterial DNA between the CCLR and MPL groups (P = .57). The copy numbers of bacterial DNA had a weak positive significant correlation with the duration of lameness in CCLR group (P < .05). Increased detection of bacterial DNA in the stifle synovial fluid may indicate joint pathology but not be directly linked to a specific joint disease. © Copyright 2013 by The American College of Veterinary Surgeons.

Glove and gown effects on intraoperative bacterial contamination.

PubMed

Ward, William G; Cooper, Joshua M; Lippert, Dylan; Kablawi, Rawan O; Neiberg, Rebecca H; Sherertz, Robert J

2014-03-01

Experiments were performed to determine the risk of bacterial contamination associated with changing outer gloves and using disposable spunlace paper versus reusable cloth gowns. Despite decades of research, there remains a lack of consensus regarding certain aspects of optimal aseptic technique including outer glove exchange while double-gloving and surgical gown type selection. In an initial glove study, 102 surgical team members were randomized to exchange or retain outer gloves 1 hour into clean orthopedic procedures; cultures were obtained 15 minutes later from the palm of the surgeon's dominant gloved hand and from the surgical gown sleeve. Surgical gown type selection was recorded. A laboratory strike-through study investigating bacterial transmission through cloth and paper gowns was performed with coagulase-negative staphylococci. In a follow-up glove study, 251 surgical team members, all wearing paper gowns, were randomized as in the first glove study. Glove study 1 revealed 4-fold higher levels of baseline bacterial contamination (31% vs 7%) on the sleeve of surgical team members wearing cloth gowns than those using paper gowns [odds ratio (95% confidence interval): 4.64 (1.72-12.53); P = 0.0016]. The bacterial strike-through study revealed that 26 of 27 cloth gowns allowed bacterial transmission through the material compared with 0 of 27 paper gowns (P < 0.001). In glove study 2, surgeons retaining outer gloves 1 hour into the case had a subsequent positive glove contamination rate of 23% compared with 13% among surgeons exchanging their original outer glove [odds ratio (95% confidence interval): 1.97 (1.02-3.80); P = 0.0419]. Paper gowns demonstrated less bacterial transmission in the laboratory and lower rates of contamination in the operating room. Disposable paper gowns are recommended for all surgical cases, especially those involving implants, because of the heightened risk of infection. Outer glove exchange just before handling implant materials

High level bacterial contamination of secondary school students' mobile phones.

PubMed

Kõljalg, Siiri; Mändar, Rando; Sõber, Tiina; Rööp, Tiiu; Mändar, Reet

2017-06-01

While contamination of mobile phones in the hospital has been found to be common in several studies, little information about bacterial abundance on phones used in the community is available. Our aim was to quantitatively determine the bacterial contamination of secondary school students' mobile phones. Altogether 27 mobile phones were studied. The contact plate method and microbial identification using MALDI-TOF mass spectrometer were used for culture studies. Quantitative PCR reaction for detection of universal 16S rRNA, Enterococcus faecalis 16S rRNA and Escherichia coli allantoin permease were performed, and the presence of tetracycline ( tet A, tet B, tet M), erythromycin ( erm B) and sulphonamide ( sul 1) resistance genes was assessed. We found a high median bacterial count on secondary school students' mobile phones (10.5 CFU/cm 2 ) and a median of 17,032 bacterial 16S rRNA gene copies per phone. Potentially pathogenic microbes ( Staphylococcus aureus , Acinetobacter spp. , Pseudomonas spp., Bacillus cereus and Neisseria flavescens ) were found among dominant microbes more often on phones with higher percentage of E. faecalis in total bacterial 16S rRNA. No differences in contamination level or dominating bacterial species between phone owner's gender and between phone types (touch screen/keypad) were found. No antibiotic resistance genes were detected on mobile phone surfaces. Quantitative study methods revealed high level bacterial contamination of secondary school students' mobile phones.

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

PubMed Central

Hashemipetroudi, Seyyed Hamidreza; Nematzadeh, Ghorbanali; Ahmadian, Gholamreza; Yamchi, Ahad; Kuhlmann, Markus

2018-01-01

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species. Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species PMID:29443017

Evaluation of Bacterial Contamination of Clipper Blades in Small Animal Private Practice.

PubMed

Mount, Rebecca; Schick, Anthea E; Lewis, Thomas P; Newton, Heide M

2016-01-01

Nosocomial infections are a growing concern in veterinary hospitals, and identifying fomites is imperative to reducing the risk of pathogen transmission. In veterinary medicine, shaving of hair is necessary prior to many procedures. Contaminated clipper blades have been cited as potential fomites involved in the transmission of pathogens in veterinary and human medicine. The primary goal of this study was to evaluate bacterial contamination of clipper blades in veterinary practices. A secondary goal was to assess whether there was an association between bacterial contamination of clipper blades and clipper blade cleaning solutions, clipper blade cleaning protocols, clipper blade storage, and type of practice. Sixty clipper blades from 60 different practices were cultured. Information regarding blade cleaning solutions, protocols, and storage was collected from each practice. Fifty-one percent (31/60) of clipper blades sampled were contaminated with bacteria. Category of cleaning solutions had a significant association with bacterial contamination (P < 0.02). Cleaning frequency (P = 0.55), storage location (P = 0.26), and practice type (P = 0.06) had no significant association with bacterial contamination. This study documented bacterial contamination of clipper blades in veterinary practices, and clipper blades should be considered potential fomites.

High level bacterial contamination of secondary school students’ mobile phones

PubMed Central

Kõljalg, Siiri; Mändar, Rando; Sõber, Tiina; Rööp, Tiiu; Mändar, Reet

2017-01-01

Introduction While contamination of mobile phones in the hospital has been found to be common in several studies, little information about bacterial abundance on phones used in the community is available. Our aim was to quantitatively determine the bacterial contamination of secondary school students’ mobile phones. Methods Altogether 27 mobile phones were studied. The contact plate method and microbial identification using MALDI-TOF mass spectrometer were used for culture studies. Quantitative PCR reaction for detection of universal 16S rRNA, Enterococcus faecalis 16S rRNA and Escherichia coli allantoin permease were performed, and the presence of tetracycline (tetA, tetB, tetM), erythromycin (ermB) and sulphonamide (sul1) resistance genes was assessed. Results We found a high median bacterial count on secondary school students’ mobile phones (10.5 CFU/cm2) and a median of 17,032 bacterial 16S rRNA gene copies per phone. Potentially pathogenic microbes (Staphylococcus aureus, Acinetobacter spp., Pseudomonas spp., Bacillus cereus and Neisseria flavescens) were found among dominant microbes more often on phones with higher percentage of E. faecalis in total bacterial 16S rRNA. No differences in contamination level or dominating bacterial species between phone owner’s gender and between phone types (touch screen/keypad) were found. No antibiotic resistance genes were detected on mobile phone surfaces. Conclusion Quantitative study methods revealed high level bacterial contamination of secondary school students’ mobile phones. PMID:28626737

Influence of Calcium in Extracellular DNA Mediated Bacterial Aggregation and Biofilm Formation

PubMed Central

Koop, Leena; Wong, Yie Kuan; Ahmed, Safia; Siddiqui, Khawar Sohail; Manefield, Mike

2014-01-01

Calcium (Ca2+) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca2+ to eDNA thereby mediating bacterial aggregation and biofilm formation. PMID:24651318

Modeling bacterial contamination of fuel ethanol fermentation.

PubMed

Bischoff, Kenneth M; Liu, Siqing; Leathers, Timothy D; Worthington, Ronald E; Rich, Joseph O

2009-05-01

The emergence of antibiotic-resistant bacteria may limit the effectiveness of antibiotics to treat bacterial contamination in fuel ethanol plants, and therefore, new antibacterial intervention methods and tools to test their application are needed. Using shake-flask cultures of Saccharomyces cerevisiae grown on saccharified corn mash and strains of lactic acid bacteria isolated from a dry-grind ethanol facility, a simple model to simulate bacterial contamination and infection was developed. Challenging the model with 10(8) CFU/mL Lactobacillus fermentum decreased ethanol yield by 27% and increased residual glucose from 6.2 to 45.5 g/L. The magnitude of the effect was proportional to the initial bacterial load, with 10(5) CFU/mL L. fermentum still producing an 8% decrease in ethanol and a 3.2-fold increase in residual glucose. Infection was also dependent on the bacterial species used to challenge the fermentation, as neither L. delbrueckii ATCC 4797 nor L. amylovorus 0315-7B produced a significant decrease in ethanol when inoculated at a density of 10(8) CFU/mL. In the shake-flask model, treatment with 2 microg/mL virginiamycin mitigated the infection when challenged with a susceptible strain of L. fermentum (MIC for virginiamycin < or =2 ppm), but treatment was ineffective at treating infection by a resistant strain of L. fermentum (MIC = 16 ppm). The model may find application in developing new antibacterial agents and management practices for use in controlling contamination in the fuel ethanol industry. Copyright 2008 Wiley Periodicals, Inc.

Fluress, fluorescein and benoxinate: recovery from bacterial contamination.

PubMed

Yolton, D P; German, C J

1980-05-01

The ability to recover from bacterial contaminations with Staphylococcus auresus and Pseudomonas aeruginosa was determined for three optometric DPAs: Fluress, fluorescein and benoxinate. Results show that Fluress recovers from contamination more rapidly than benoxinate or fluorescein. This ability to recover from contamination and the relative ease of use of Fluress may make it the DPA choice for a number of optometric procedures including applanation tonometry.

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification.

PubMed

Zeng, Yan; Wan, Yi; Zhang, Dun; Qi, Peng

2015-01-01

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3'-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

Bacterial contamination, bacterial profile and antimicrobial susceptibility pattern of isolates from stethoscopes at Jimma University Specialized Hospital.

PubMed

Shiferaw, Teklu; Beyene, Getenet; Kassa, Tesfaye; Sewunet, Tsegaye

2013-12-13

Hospital acquired infections are recognized as critical public health problems. Infections are frequently caused by organisms residing in healthcare environment, including contaminated medical equipment like Stethoscopes. To determine bacterial contamination, bacterial profile and anti-microbial susceptibility pattern of the isolates from stethoscopes at Jimma University Specialized Hospital. Cross-sectional study conducted from May to September 2011 at Jimma University Specialized Hospital. One hundred seventy-six stethoscopes owned by Health Care Workers (HCWs) and Medical students were randomly selected and studied. Self-administered structured questionnaire was used to collect socio-demographic data. Specimen was collected using moisten sterile cotton swab and 1 ml normal saline was used to transport the specimen, all laboratory investigations were done following standard microbiological techniques, at Microbiology Laboratory, Jimma University. SPSS windows version 16 used for data analysis and P <0.05 was considered statistically significant. A total, of 151 (85.8%) stethoscopes were contaminated. A total of 256 bacterial strains and a mean of 1.44×104 CFUs/diaphragm of stethoscopes was isolated. Of the 256 isolates, 133 (52%) were potential pathogens like S. aureus, Klebsiella spp., Citrobacter spp., Salmonella spp., Proteus spp., Enterobacter spp., P. aeruginosa and E. coli. All strains were resistant to multiple classes of antibiotics (two to eight classes of antibiotics). Disinfection practice was poor. Disinfection practice was found to be associated with bacterial contamination of stethoscopes (P < 0.05). High contamination rate 100 (90.9%) was observed among stethoscopes that had never been disinfected; while the least contamination 29 (72.2%) was found on those disinfected a week or less before the survey. Bacterial contamination of the stethoscope was significant. The isolates were potential pathogens and resistant to multiple classes of

The bacterial contamination rate of glucose meter test strips in the hospital setting

PubMed Central

Al-Rubeaan, Khalid A.; Saeb, Amr T. M.; AlNaqeb, Dhekra M.; AlQumaidi, Hamed M.; AlMogbel, Turki A.

2016-01-01

Objectives: To assess the rate of bacterial contamination of the multi-use vial and single-use packed glucose meter strips, and to identify the type and frequency of various bacterial contamination in different hospital wards. Methods: This prospective observational study was conducted by a team from the Strategic Center for Diabetes Research in 7 general hospitals in the Central region of Saudi Arabia during the period from August to September 2014 to assess the bacterial contamination rate of the unused strips. A total of 10,447 strips were cultured using proper agar media and incubated both aerobically and anaerobically. Results: The total bacterial contamination rate for the multi-use vials glucose strips was 31.7%, while single-use packed strips were not contaminated at all. Ministry of Health hospitals had the highest contamination rates compared with other hospitals. Critical, obstetric, and surgical wards had the highest bacterial isolates number, where most were in the risk group 3 according to the National Institute of Health guidelines. Staphylococcus species were the most common bacteria found. Conclusion: Glucose meter strips should be recognized as a source of bacterial contamination that could be behind serious hospital acquired infections. The hospital infection control team should adopt proper measures to implement protocols for glucose meter cleaning and glucose strips handling. PMID:27570855

Microbial Air Quality and Bacterial Surface Contamination in Ambulances During Patient Services

PubMed Central

Luksamijarulkul, Pipat; Pipitsangjan, Sirikun

2015-01-01

Objectives We sought to assess microbial air quality and bacterial surface contamination on medical instruments and the surrounding areas among 30 ambulance runs during service. Methods We performed a cross-sectional study of 106 air samples collected from 30 ambulances before patient services and 212 air samples collected during patient services to assess the bacterial and fungal counts at the two time points. Additionally, 226 surface swab samples were collected from medical instrument surfaces and the surrounding areas before and after ambulance runs. Groups or genus of isolated bacteria and fungi were preliminarily identified by Gram’s stain and lactophenol cotton blue. Data were analyzed using descriptive statistics, t-test, and Pearson’s correlation coefficient with a p-value of less than 0.050 considered significant. Results The mean and standard deviation of bacterial and fungal counts at the start of ambulance runs were 318±485cfu/m3 and 522±581cfu/m3, respectively. Bacterial counts during patient services were 468±607cfu/m3 and fungal counts were 656±612cfu/m3. Mean bacterial and fungal counts during patient services were significantly higher than those at the start of ambulance runs, p=0.005 and p=0.030, respectively. For surface contamination, the overall bacterial counts before and after patient services were 0.8±0.7cfu/cm2 and 1.3±1.1cfu/cm2, respectively (p<0.001). The predominant isolated bacteria and fungi were Staphylococcus spp. and Aspergillus spp., respectively. Additionally, there was a significantly positive correlation between bacterial (r=0.3, p<0.010) and fungal counts (r=0.2, p=0.020) in air samples and bacterial counts on medical instruments and allocated areas. Conclusions This study revealed high microbial contamination (bacterial and fungal) in ambulance air during services and higher bacterial contamination on medical instrument surfaces and allocated areas after ambulance services compared to the start of ambulance runs

Microbial air quality and bacterial surface contamination in ambulances during patient services.

PubMed

Luksamijarulkul, Pipat; Pipitsangjan, Sirikun

2015-03-01

We sought to assess microbial air quality and bacterial surface contamination on medical instruments and the surrounding areas among 30 ambulance runs during service. We performed a cross-sectional study of 106 air samples collected from 30 ambulances before patient services and 212 air samples collected during patient services to assess the bacterial and fungal counts at the two time points. Additionally, 226 surface swab samples were collected from medical instrument surfaces and the surrounding areas before and after ambulance runs. Groups or genus of isolated bacteria and fungi were preliminarily identified by Gram's stain and lactophenol cotton blue. Data were analyzed using descriptive statistics, t-test, and Pearson's correlation coefficient with a p-value of less than 0.050 considered significant. The mean and standard deviation of bacterial and fungal counts at the start of ambulance runs were 318±485cfu/m(3) and 522±581cfu/m(3), respectively. Bacterial counts during patient services were 468±607cfu/m(3) and fungal counts were 656±612cfu/m(3). Mean bacterial and fungal counts during patient services were significantly higher than those at the start of ambulance runs, p=0.005 and p=0.030, respectively. For surface contamination, the overall bacterial counts before and after patient services were 0.8±0.7cfu/cm(2) and 1.3±1.1cfu/cm(2), respectively (p<0.001). The predominant isolated bacteria and fungi were Staphylococcus spp. and Aspergillus spp., respectively. Additionally, there was a significantly positive correlation between bacterial (r=0.3, p<0.010) and fungal counts (r=0.2, p=0.020) in air samples and bacterial counts on medical instruments and allocated areas. This study revealed high microbial contamination (bacterial and fungal) in ambulance air during services and higher bacterial contamination on medical instrument surfaces and allocated areas after ambulance services compared to the start of ambulance runs. Additionally, bacterial and

Analysis of Salmonella sp bacterial contamination on Vannamei Shrimp using binary logit model approach

NASA Astrophysics Data System (ADS)

Oktaviana, P. P.; Fithriasari, K.

2018-04-01

Mostly Indonesian citizen consume vannamei shrimp as their food. Vannamei shrimp also is one of Indonesian exports comodities mainstay. Vannamei shrimp in the ponds and markets could be contaminated by Salmonella sp bacteria. This bacteria will endanger human health. Salmonella sp bacterial contamination on vannamei shrimp could be affected by many factors. This study is intended to identify what factors that supposedly influence the Salmonella sp bacterial contamination on vannamei shrimp. The researchers used the testing result of Salmonella sp bacterial contamination on vannamei shrimp as response variable. This response variable has two categories: 0 = if testing result indicate that there is no Salmonella sp on vannamei shrimp; 1 = if testing result indicate that there is Salmonella sp on vannamei shrimp. There are four factors that supposedly influence the Salmonella sp bacterial contamination on vannamei shrimp, which are the testing result of Salmonella sp bacterial contamination on farmer hand swab; the subdistrict of vannamei shrimp ponds; the fish processing unit supplied by; and the pond are in hectare. This four factors used as predictor variables. The analysis used is Binary Logit Model Approach according to the response variable that has two categories. The analysis result indicates that the factors or predictor variables which is significantly affect the Salmonella sp bacterial contamination on vannamei shrimp are the testing result of Salmonella sp bacterial contamination on farmer hand swab and the subdistrict of vannamei shrimp ponds.

Conventional and nonconventional strategies for controlling bacterial contamination in fuel ethanol fermentations.

PubMed

Ceccato-Antonini, Sandra Regina

2018-05-25

Ethanol bio-production in Brazil has some unique characteristics that inevitably lead to bacterial contamination, which results in the production of organic acids and biofilms and flocculation that impair the fermentation yield by affecting yeast viability and diverting sugars to metabolites other than ethanol. The ethanol-producing units commonly give an acid treatment to the cells after each fermentative cycle to decrease the bacterial number, which is not always effective. An alternative strategy must be employed to avoid bacterial multiplication but must be compatible with economic, health and environmental aspects. This review analyzes the issue of bacterial contamination in sugarcane-based fuel ethanol fermentation, and the potential strategies that may be utilized to control bacterial growth besides acid treatment and antibiotics. We have emphasized the efficiency and suitability of chemical products other than acids and those derived from natural sources in industrial conditions. In addition, we have also presented bacteriocins, bacteriophages, and beneficial bacteria as non-conventional antimicrobial agents to mitigate bacterial contamination in the bioethanol industry.

ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'.

PubMed Central

Tian, Wenzhi; Chua, Kevin; Strober, Warren; Chu, Charles C.

2002-01-01

ILG1. CONCLUSIONS: In the course of our studies using cDNA RDA, we have isolated and identified ILG1, a likely active site-specific recombinase and new member of the bacteriophage P4 family of integrases. This family of integrases is implicated in the horizontal DNA transfer of pathogenic genes between bacterial species, such as those found in pathogenic strains of E. coli, Shigella, Yersinia, and Vibrio cholera. Using ILG1 as a marker of our laboratory E. coli strain TOP10F', our evidence suggests that contaminating bacterial DNA in our subtraction experiment is due to this laboratory bacterial strain, which colonized exposed surfaces of the laboratory worker. Thus, identification of differentially expressed genes between normal and diseased states could be dramatically improved by using extra precaution to prevent bacterial contamination of samples. PMID:12393938

An investigation into the bacterial contamination of goniolenses in use in clinical practice.

PubMed

Grundon, Rachael; Scurrell, Emma; Mould, John; Hayton-Lee, Emma; Heinrich, Christine

2017-12-18

Objectives To report the incidence and evaluate the clinical significance of goniolens bacterial contamination in clinical use in dogs with three different usage protocols and one with an added cleaning protocol. Animals Studied and Methods Three groups of twenty dogs undergoing gonioscopy at a private practice in the UK had the goniolenses swabbed for bacteriology culture and identification prior to placement on the cornea. Three protocols of lens use, with 2 different types of goniolens, were studied. One protocol was then repeated with 21 dogs with a lens cleaning protocol prior to storage. Results Low levels of bacterial contamination were found in all 3 initial groups (10-15%). No correlation was found between usage protocol used and rate of contamination and no correlation was found between length of storage between use and contamination. All bacteria cultured were considered naturally occurring commensals for the canine eye and environment. The group with a cleaning protocol had a 4.7% contamination rate. This was not statistically different from the non-cleaning groups. Conclusions The rate of bacterial contamination of goniolenses in clinical practice is low and the bacterial contaminants consist of commensal bacteria, unlikely to be of detriment to the eye. Minimal contamination of the goniolenses was found and this did not appear to be of clinical significance. The introduction of a simple cleaning protocol did not produce a statistically significant reduction in bacterial contamination. © 2017 American College of Veterinary Ophthalmologists.

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

PubMed

Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

2018-05-17

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Contamination during criminal investigation: Detecting police contamination and secondary DNA transfer from evidence bags.

PubMed

Fonneløp, Ane Elida; Johannessen, Helen; Egeland, Thore; Gill, Peter

2016-07-01

As the profiling systems used in forensic analyses have become more sensitive in recent years, the risk of detecting a contamination in a DNA sample has increased proportionally. This requires more stringent work protocols and awareness to minimize the chance of contamination. Although there is high consciousness on contamination and best practice procedures in forensic labs, the same requirements are not always applied by the police. In this study we have investigated the risk of contamination from police staff. Environmental DNA was monitored by performing wipe tests (sampling of hot spots) at two large police units (scenes of crime departments). Additionally, the DNA profiles of the scenes of crime officers were compared to casework samples that their own unit had investigated in the period of 2009-2015. Furthermore, a pilot study to assess whether DNA from the outside package of an exhibit could be transferred to a DNA sample was carried out. Environmental DNA was detected in various samples from hot spots. Furthermore, 16 incidences of previously undetected police-staff contamination were found in casework that had been submitted between 2009 and 2015. In 6 cases the police officers with a matching DNA profile reported that they had not been involved with the case. We have demonstrated that DNA from the outside package can be transferred to an exhibit during examination. This experience demonstrates that when implementing the new multiplex systems, it is important to ensure that 'best practice' procedures are upgraded, and appropriate training is provided in order to ensure that police are aware of the increased contamination risks. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Bacterial contamination, bacterial profile and antimicrobial susceptibility pattern of isolates from stethoscopes at Jimma University Specialized Hospital

PubMed Central

2013-01-01

Introduction Hospital acquired infections are recognized as critical public health problems. Infections are frequently caused by organisms residing in healthcare environment, including contaminated medical equipment like Stethoscopes. Objective To determine bacterial contamination, bacterial profile and anti-microbial susceptibility pattern of the isolates from stethoscopes at Jimma University Specialized Hospital. Methodology Cross-sectional study conducted from May to September 2011 at Jimma University Specialized Hospital. One hundred seventy-six stethoscopes owned by Health Care Workers (HCWs) and Medical students were randomly selected and studied. Self-administered structured questionnaire was used to collect socio-demographic data. Specimen was collected using moisten sterile cotton swab and 1 ml normal saline was used to transport the specimen, all laboratory investigations were done following standard microbiological techniques, at Microbiology Laboratory, Jimma University. SPSS windows version 16 used for data analysis and P <0.05 was considered statistically significant. Result: A total, of 151 (85.8%) stethoscopes were contaminated. A total of 256 bacterial strains and a mean of 1.44×104 CFUs/diaphragm of stethoscopes was isolated. Of the 256 isolates, 133 (52%) were potential pathogens like S. aureus, Klebsiella spp., Citrobacter spp., Salmonella spp., Proteus spp., Enterobacter spp., P. aeruginosa and E. coli. All strains were resistant to multiple classes of antibiotics (two to eight classes of antibiotics). Disinfection practice was poor. Disinfection practice was found to be associated with bacterial contamination of stethoscopes (P < 0.05). High contamination rate 100 (90.9%) was observed among stethoscopes that had never been disinfected; while the least contamination 29 (72.2%) was found on those disinfected a week or less before the survey. Conclusion Bacterial contamination of the stethoscope was significant. The isolates were potential

High bacterial contamination rate of electrocautery tips during total hip and knee arthroplasty.

PubMed

Abdelaziz, Hussein; Zahar, Akos; Lausmann, Christian; Gehrke, Thorsten; Fickenscher, Helmut; Suero, Eduardo M; Gebauer, Matthias; Citak, Mustafa

2018-04-01

The aim of the study was to quantify the bacterial contamination rate of electrocautery tips during primary total joint replacement (TJR), as well as during aseptic and septic revision TJR. A total of 150 electrocautery tips were collected between April and July 2017. TJR surgeries were divided into three groups: (1) primary, (2) aseptic and (3) septic revisions. In each group, a total of 50 electrocautery tips were collected. A monopolar electrocautery with a reusable stainless-steel blade tip was used in all cases. The rate of bacterial contamination was determined for all groups. Correlation of exposure time and type of surgery was analyzed. The overall bacterial contamination rate was 14.7% (95% CI 9.4 to 21.4%). The highest contamination rate occurred in the septic revision group (30.0%; 95% CI 17.9 to 44.6%), followed by the primary cases group (10.0%; 95% CI 3.3 to 21.8%) and the aseptic revision group (4.0%; 95% CI 0.5 to 13.7%). Exposure time did not affect the bacterial contamination rate. In 12 out of 15 (80%) contaminations identified in the septic group, we found the same causative microorganism of the prosthetic joint infection on the electrocautery tip. The bacterial contamination of the electrocautery tips is relatively high, especially during septic hip revision arthroplasty. Electrocautery tips should be changed after debridement of infected tissue.

Risk factors for bacterial contamination during boar semen collection.

PubMed

Goldberg, Ana Maria G; Argenti, Laura E; Faccin, Jamil E; Linck, Lídia; Santi, Mônica; Bernardi, Mari Lourdes; Cardoso, Marisa R I; Wentz, Ivo; Bortolozzo, Fernando P

2013-10-01

The aim of this study was to evaluate the influence of multiple factors on bacterial contamination in 213 ejaculates from four boar studs. Semen contamination by aerobic mesophiles increased in ejaculates where the preputial fluid flowed into the collection container, collection glove was dirty, preputial hair was long (>1.0 cm), the collection lasted >7 min and boars were older than 18 months. An increase in coliforms occurred when preputial fluid dripped into the collection container, collections lasted >7 min or when penis escaped during collection. Semen contamination increased when two or more factors related to hygiene (poor hygiene of the boar, dirty preputial ostium, large preputial diverticulum, long preputial hair, dirty gloves, preputial liquid trickling from the hand of the technician into the semen container and penis escaping) were present. A vigilant protocol of collection must be followed to minimize bacterial contamination, especially avoiding dripping of preputial liquid into the semen container. Copyright © 2013 Elsevier Ltd. All rights reserved.

IDENTIFYING POTENTIAL SOURCES OF BACKGROUND CONTAMINATION IN RT-PCR

EPA Science Inventory

Extraction of nucleic acids from low biomass samples, such as drinking water, is particularly sensitive to potential background contamination because the contaminating material is minimally diluted by the sample. The presence of bacterial DNA in Taq DNA polymerase is wel...

Analysis of bacterial populations in the environment using two-dimensional gel electrophoresis of genomic DNA and complementary DNA.

PubMed

Liu, Guo-Hua; Nakamura, Tatsuo; Amemiya, Takashi; Rajendran, Narasimmalu; Itoh, Kiminori

2011-01-01

Two-dimensional gel electrophoresis (2-DGE) mapping of genomic DNA and complementary DNA (cDNA) amplicons was attempted to analyze total and active bacterial populations within soil and activated sludge samples. Distinct differences in the number and species of bacterial populations and those that were metabolically active at the time of sampling were visually observed especially for the soil community. Statistical analyses and sequencing based on the 2-DGE data further revealed the relationships between total and active bacterial populations within each community. This high-resolution technique would be useful for obtaining a better understanding of bacterial population structures in the environment.

Bacterial community dynamics during bioremediation of diesel oil-contaminated Antarctic soil.

PubMed

Vázquez, S; Nogales, B; Ruberto, L; Hernández, E; Christie-Oleza, J; Lo Balbo, A; Bosch, R; Lalucat, J; Mac Cormack, W

2009-05-01

The effect of nutrient and inocula amendment in a bioremediation field trial using a nutrient-poor Antarctic soil chronically contaminated with hydrocarbons was tested. The analysis of the effects that the treatments caused in bacterial numbers and hydrocarbon removal was combined with the elucidation of the changes occurring on the bacterial community, by 16S rDNA-based terminal restriction fragment length polymorphism (T-RFLP) typing, and the detection of some of the genes involved in the catabolism of hydrocarbons. All treatments caused a significant increase in the number of bacteria able to grow on hydrocarbons and a significant decrease in the soil hydrocarbon content, as compared to the control. However, there were no significant differences between treatments. Comparison of the soil T-RFLP profiles indicated that there were changes in the structure and composition of bacterial communities during the bioremediation trial, although the communities in treated plots were highly similar irrespective of the treatment applied, and they had a similar temporal dynamics. These results showed that nutrient addition was the main factor contributing to the outcome of the bioremediation experiment. This was supported by the lack of evidence of the establishment of inoculated consortia in soils, since their characteristic electrophoretic peaks were only detectable in soil profiles at the beginning of the experiment. Genetic potential for naphthalene degradation, evidenced by detection of nahAc gene, was observed in all soil plots including the control. In treated plots, an increase in the detection of catechol degradation genes (nahH and catA) and in a key gene of denitrification (nosZ) was observed as well. These results indicate that treatments favored the degradation of aromatic hydrocarbons and probably stimulated denitrification, at least transiently. This mesocosm study shows that recovery of chronically contaminated Antarctic soils can be successfully accelerated

[Bacterial contamination of stethoscopes in hospital].

PubMed

Davullu, Sevcan; Burger, Sandrine; Kessler, Brigitte; Meunier, Olivier

2015-01-01

Although many publications on the subject are available, only few campaigns for stethoscopes cleaning and disinfection are conducted. Stethoscope is a "not critical" reusable medical device and should benefit from a cleaning disinfection "low level" after each use. We studied the bacterial contamination of stethoscopes in our hospital and measured the role of bacterial reservoir and/or vector stethoscope could play. Mean microbial load was estimated at 1.550 CFU per stethoscope. Results obtained in our hospital and those of the literature, led us to propose a simple and efficient cleaning method. We believe motivate everyone to change their habits and carry out disinfection more frequent their very handling stethoscope.

Evaluation of Bacterial Contamination as an Indicator of Viral Contamination in a Sedimentary Aquifer in Uruguay.

PubMed

Gamazo, P; Victoria, M; Schijven, J F; Alvareda, E; Tort, L F L; Ramos, J; Burutaran, L; Olivera, M; Lizasoain, A; Sapriza, G; Castells, M; Colina, R

2018-03-21

In Uruguay, groundwater is frequently used for agricultural activities, as well as for human consumption in urban and rural areas. As in many countries worldwide, drinking water microbiological quality is evaluated only according to bacteriological standards and virological analyses are not mentioned in the legislation. In this work, the incidence of human viral (Rotavirus A, Norovirus GII, and human Adenovirus) and bacterial (total and thermotolerant coliform and Pseudomonas aeruginosa) contamination in groundwater in the Salto district, Uruguay, as well as the possible correlation between these groups of microorganisms, was studied. From a total of 134 groundwater samples, 42 (32.1%) were positive for Rotavirus, only 1 (0.7%) for both Rotavirus and Adenovirus, and 96 (72.6%) samples were positive for bacterial indicators. Results also show that Rotavirus presence was not associated with changes in chemical composition of the aquifer water. Bacteriological indicators were not adequate to predict the presence of viruses in individual groundwater samples (well scale), but a deeper spatial-temporal analysis showed that they are promising candidates to assess the viral contamination degree at aquifer scale, since from the number of wells with bacterial contamination the number of wells with viral contamination could be estimated.

Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation.

PubMed

Rich, Joseph O; Leathers, Timothy D; Bischoff, Kenneth M; Anderson, Amber M; Nunnally, Melinda S

2015-11-01

Bacterial contaminants can inhibit ethanol production in biofuel fermentations, and even result in stuck fermentations. Contaminants may persist in production facilities by forming recalcitrant biofilms. A two-year longitudinal study was conducted of bacterial contaminants from a Midwestern dry grind corn fuel ethanol facility. Among eight sites sampled in the facility, the combined liquefaction stream and yeast propagation tank were consistently contaminated, leading to contamination of early fermentation tanks. Among 768 contaminants isolated, 92% were identified as Lactobacillus sp., with the most abundant species being Lactobacillus plantarum, Lactobacillus casei, Lactobacillus mucosae, and Lactobacillus fermentum. Seven percent of total isolates showed the ability to form biofilms in pure cultures, and 22% showed the capacity to significantly inhibit ethanol production. However, these traits were not correlated. Ethanol inhibition appeared to be related to acetic acid production by contaminants, particularly by obligately heterofermentative species such as L. fermentum and L. mucosae. Published by Elsevier Ltd.

Bacterial hand contamination and transfer after use of contaminated bulk-soap-refillable dispensers.

PubMed

Zapka, Carrie A; Campbell, Esther J; Maxwell, Sheri L; Gerba, Charles P; Dolan, Michael J; Arbogast, James W; Macinga, David R

2011-05-01

Bulk-soap-refillable dispensers are prone to extrinsic bacterial contamination, and recent studies demonstrated that approximately one in four dispensers in public restrooms are contaminated. The purpose of this study was to quantify bacterial hand contamination and transfer after use of contaminated soap under controlled laboratory and in-use conditions in a community setting. Under laboratory conditions using liquid soap experimentally contaminated with 7.51 log(10) CFU/ml of Serratia marcescens, an average of 5.28 log(10) CFU remained on each hand after washing, and 2.23 log(10) CFU was transferred to an agar surface. In an elementary-school-based field study, Gram-negative bacteria on the hands of students and staff increased by 1.42 log(10) CFU per hand (26-fold) after washing with soap from contaminated bulk-soap-refillable dispensers. In contrast, washing with soap from dispensers with sealed refills significantly reduced bacteria on hands by 0.30 log(10) CFU per hand (2-fold). Additionally, the mean number of Gram-negative bacteria transferred to surfaces after washing with soap from dispensers with sealed-soap refills (0.06 log(10) CFU) was significantly lower than the mean number after washing with contaminated bulk-soap-refillable dispensers (0.74 log(10) CFU; P < 0.01). Finally, significantly higher levels of Gram-negative bacteria were recovered from students (2.82 log(10) CFU per hand) than were recovered from staff (2.22 log(10) CFU per hand) after washing with contaminated bulk soap (P < 0.01). These results demonstrate that washing with contaminated soap from bulk-soap-refillable dispensers can increase the number of opportunistic pathogens on the hands and may play a role in the transmission of bacteria in public settings.

Factors affecting use of ballistics gelatin in laboratory studies of bacterial contamination in projectile wounds.

PubMed

Evans, Jessica J; Bost, Aaron; Muci-Küchler, Karim H; DeVeaux, Linda C

2018-05-25

Ballistics gelatin is a common tissue surrogate used in bacterial contamination models for projectile wounds. Although these studies have demonstrated that bacteria are transferred from the surface of the gelatin to the wound track by a projectile, quantifiable results have been inconsistent and not repeatable in successive tests. In this study, five areas of a typical contamination model in which bacterial recovery or survival are affected were identified for optimization. The first was a contaminated "skin" surrogate, where the novel use of vacuum filtration of a bacterial culture and buffer onto filter paper was employed. The other possibly problematic areas of the bacterial distribution model included the determination of bacterial survival when the contamination model is dried, survival in solid and molten gelatin, and the effect of high-intensity lights used for recording high-speed video. Vacuum filtration of bacteria and buffer resulted in a consistent bacterial distribution and recovery. The use of phosphate buffer M9 (pH 7) aided in neutralizing the ballistics gelatin and improving bacterial survival in solid gelatin. Additionally, the use of high-intensity lights to record high-speed video and the use of a 42 ° C water bath to melt the gelatin were found to be bactericidal for gram-positive and gram-negative bacteria. Multiple areas of a typical contamination model in which bacterial survival may be impeded were identified, and methods were proposed to improve survival in each area. These methods may be used to optimize the results of bacterial contamination models for medical applications, such as understanding the progression of infection in penetrating wounds and to identify possible sources of contamination for forensic purposes.

Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

PubMed Central

Campeau, J. L.; Salim, S. Y.; Albert, E. J.; Hotte, N.

2012-01-01

Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes. PMID:22615241

Physical therapy clinic therapeutic ultrasound equipment as a source for bacterial contamination.

PubMed

Spratt, Henry G; Levine, David; Tillman, Larry

2014-10-01

A procedure commonly used in physical therapy (PT) clinics is therapeutic ultrasound (US). This equipment and associated gel comes in contact with patient skin, potentially serving as a reservoir for bacteria. In this study, we sampled US heads, gel bottle tips and gel from nine outpatient PT clinics in Southeastern Tennessee. Samples were collected using sterile swabs. At the microbiology laboratory, these swabs were used to inoculate mannitol salt agar and CHROM-MRSA agar (for Staphylococcal species) and tryptic soy broth to determine non-specific bacterial contamination. US heads, gel bottle tips and gel had variable levels of contamination. Tips of gel bottles had the highest contamination, with 52.7% positive for non-specific bacterial contamination and 3.6% positive for methicillin-resistant Staphylococcus aureus (MRSA). Contamination of gel by non-specific bacteria was found in 14.5% of bottles sampled. US heads (35.5% of those sampled) had non-specific bacterial contamination, with no MRSA detected. Disinfecting US heads after initial swabbing resulted in removal of 90.9% of non-specific contamination. Gel storage at temperatures below 40 °C was found to encourage the growth of mesophilic bacteria. This study demonstrates the need for better cleaning and storage protocols for US heads and gel bottles in PT clinics.

Effect of DNA extraction and sample preservation method on rumen bacterial population.

PubMed

Fliegerova, Katerina; Tapio, Ilma; Bonin, Aurelie; Mrazek, Jakub; Callegari, Maria Luisa; Bani, Paolo; Bayat, Alireza; Vilkki, Johanna; Kopečný, Jan; Shingfield, Kevin J; Boyer, Frederic; Coissac, Eric; Taberlet, Pierre; Wallace, R John

2014-10-01

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluating bacterial pathogen DNA preservation in museum osteological collections

PubMed Central

Barnes, Ian; Thomas, Mark G

2005-01-01

Reports of bacterial pathogen DNA sequences obtained from archaeological bone specimens raise the possibility of greatly improving our understanding of the history of infectious diseases. However, the survival of pathogen DNA over long time periods is poorly characterized, and scepticism remains about the reliability of these data. In order to explore the survival of bacterial pathogen DNA in bone specimens, we analysed samples from 59 eighteenth and twentieth century individuals known to have been infected with either Mycobacterium tuberculosis or Treponema pallidum. No reproducible evidence of surviving pathogen DNA was obtained, despite the use of extraction and PCR-amplification methods determined to be highly sensitive. These data suggest that previous studies need to be interpreted with caution, and we propose that a much greater emphasis is placed on understanding how pathogen DNA survives in archaeological material, and how its presence can be properly verified and used. PMID:16608682

Correcting for Sample Contamination in Genotype Calling of DNA Sequence Data

PubMed Central

Flickinger, Matthew; Jun, Goo; Abecasis, Gonçalo R.; Boehnke, Michael; Kang, Hyun Min

2015-01-01

DNA sample contamination is a frequent problem in DNA sequencing studies and can result in genotyping errors and reduced power for association testing. We recently described methods to identify within-species DNA sample contamination based on sequencing read data, showed that our methods can reliably detect and estimate contamination levels as low as 1%, and suggested strategies to identify and remove contaminated samples from sequencing studies. Here we propose methods to model contamination during genotype calling as an alternative to removal of contaminated samples from further analyses. We compare our contamination-adjusted calls to calls that ignore contamination and to calls based on uncontaminated data. We demonstrate that, for moderate contamination levels (5%–20%), contamination-adjusted calls eliminate 48%–77% of the genotyping errors. For lower levels of contamination, our contamination correction methods produce genotypes nearly as accurate as those based on uncontaminated data. Our contamination correction methods are useful generally, but are particularly helpful for sample contamination levels from 2% to 20%. PMID:26235984

Bacterial Hand Contamination and Transfer after Use of Contaminated Bulk-Soap-Refillable Dispensers▿†

PubMed Central

Zapka, Carrie A.; Campbell, Esther J.; Maxwell, Sheri L.; Gerba, Charles P.; Dolan, Michael J.; Arbogast, James W.; Macinga, David R.

2011-01-01

Bulk-soap-refillable dispensers are prone to extrinsic bacterial contamination, and recent studies demonstrated that approximately one in four dispensers in public restrooms are contaminated. The purpose of this study was to quantify bacterial hand contamination and transfer after use of contaminated soap under controlled laboratory and in-use conditions in a community setting. Under laboratory conditions using liquid soap experimentally contaminated with 7.51 log10 CFU/ml of Serratia marcescens, an average of 5.28 log10 CFU remained on each hand after washing, and 2.23 log10 CFU was transferred to an agar surface. In an elementary-school-based field study, Gram-negative bacteria on the hands of students and staff increased by 1.42 log10 CFU per hand (26-fold) after washing with soap from contaminated bulk-soap-refillable dispensers. In contrast, washing with soap from dispensers with sealed refills significantly reduced bacteria on hands by 0.30 log10 CFU per hand (2-fold). Additionally, the mean number of Gram-negative bacteria transferred to surfaces after washing with soap from dispensers with sealed-soap refills (0.06 log10 CFU) was significantly lower than the mean number after washing with contaminated bulk-soap-refillable dispensers (0.74 log10 CFU; P < 0.01). Finally, significantly higher levels of Gram-negative bacteria were recovered from students (2.82 log10 CFU per hand) than were recovered from staff (2.22 log10 CFU per hand) after washing with contaminated bulk soap (P < 0.01). These results demonstrate that washing with contaminated soap from bulk-soap-refillable dispensers can increase the number of opportunistic pathogens on the hands and may play a role in the transmission of bacteria in public settings. PMID:21421792

Survey of Intraoperative Bacterial Contamination in Dogs Undergoing Elective Orthopedic Surgery.

PubMed

Andrade, Natalia; Schmiedt, Chad W; Cornell, Karen; Radlinsky, MaryAnn G; Heidingsfelder, Lauren; Clarke, Kevin; Hurley, David J; Hinson, Whitney D

2016-02-01

To investigate the frequency, source, and risk factors of intraoperative (IO) surgeon and patient bacterial contamination during clean orthopedic surgeries, and to investigate the relationship between IO contamination and surgical site infection (SSI) in dogs. Prospective clinical study. Client-owned dogs undergoing stifle surgery (n = 100). IO cultures were taken in each case from surgical foot wrap, peri-incisional skin, surgical gloves, and the surgical team's hands. The environment (operating room [OR] lights, computers, scrub sink faucet, anesthesia gurney, and radiology table) was sampled every 5 months. Bacteria were identified and the contamination of each case was categorized. All gloves from the surgical team were collected and tested for perforations using a water infusion test. Cases were followed for at least 8 weeks to determine the presence or absence of SSI. Perioperative variables were evaluated for association with IO contamination and SSI. Bacterial isolates were yielded from 81% of procedures from 1 or more sources; 58% had positive hand cultures, 46% had positive glove cultures, 23% had positive patient skin cultures, and 12% had positive foot wrap cultures. Staphylococcus spp. was the most commonly recovered bacteria. There was no apparent association between IO contamination and SSI. The highest level of environmental contamination was associated with the scrub sink faucet, followed by the radiology table, anesthesia gurney, and OR computers. The IO glove perforation rate was 18%. Clean orthopedic procedures commonly had clinically insignificant bacterial contamination. In our study, bacteria responsible for SSI did not appear to colonize the patient in the OR. © Copyright 2016 by The American College of Veterinary Surgeons.

Human Contamination in Public Genome Assemblies.

PubMed

Kryukov, Kirill; Imanishi, Tadashi

2016-01-01

Contamination in genome assembly can lead to wrong or confusing results when using such genome as reference in sequence comparison. Although bacterial contamination is well known, the problem of human-originated contamination received little attention. In this study we surveyed 45,735 available genome assemblies for evidence of human contamination. We used lineage specificity to distinguish between contamination and conservation. We found that 154 genome assemblies contain fragments that with high confidence originate as contamination from human DNA. Majority of contaminating human sequences were present in the reference human genome assembly for over a decade. We recommend that existing contaminated genomes should be revised to remove contaminated sequence, and that new assemblies should be thoroughly checked for presence of human DNA before submitting them to public databases.

Estimation of lactic acid bacterial cell number by DNA quantification.

PubMed

Ishii, Masaki; Matsumoto, Yasuhiko; Sekimizu, Kazuhisa

2018-01-01

Lactic acid bacteria are provided by fermented foods, beverages, medicines, and supplements. Because the beneficial effects of medicines and supplements containing functional lactic acid bacteria are related to the bacterial cell number, it is important to establish a simple method for estimating the total number of lactic acid bacterial cells in the products for quality control. Almost all of the lactic acid bacteria in the products are dead, however, making it difficult to estimate the total number of lactic acid bacterial cells in the products using a standard colony-counting method. Here we estimated the total lactic acid bacterial cell number in samples containing dead bacteria by quantifying the DNA. The number of viable Enterococcus faecalis 0831-07 cells decreased to less than 1 × 10 -8 by 15 min of heat treatment at 80°C. The amount of extracted DNA from heat-treated cells was 78% that of non-heated cells. The number of viable Lactobacillus paraplantarum 11-1 cells decreased to 1 × 10 -4 after 4 days culture. The amount of extracted DNA of the long-cultured cells, however, was maintained at 97%. These results suggest that cell number of lactic acid bacteria killed by heat-treatment or long-term culture can be estimated by DNA quantification.

Exogenous bacterial contamination of donor blood.

PubMed

Rock, G; Westwood, J C

1977-05-07

The Canadian Red Cross blood transfusion service has followed a set protocol for phlebotomy and collection of a unit of blood. Recent requirements for automated testing have necessitated that a second tube of blood be obtained from the blood line following collection of the unit. Evaluation of the techniques used, however, has indicated the possibility of bacterial contamination from the skin of donors, from insertion of the needle through an unsterile rubber stopper, and through backflow from a nonsterile vacuum tube. To test these possibilities swabs were taken from skin and stoppers of vacuum tubes. Further, vacuum tubes were deliberately contaminated with Escherichia coli. The normal sampling procedure, which involves stripping the donor line to refill and mix the blood, was then followed. This resulted in contamination of the segments and even the blood bag. These findings led to modification of the standard bleeding technique, whereby stripping was eliminated and sterile vacuum tubes were to be used at all times.

Plasma Bacterial and Mitochondrial DNA Distinguish Bacterial Sepsis from Sterile SIRS and Quantify Inflammatory Tissue Injury in Nonhuman Primates

PubMed Central

Sursal, Tolga; Stearns-Kurosawa, Deborah J; Itagaki, Kiyoshi; Oh, Sun-Young; Sun, Shiqin; Kurosawa, Shinichiro; Hauser, Carl J

2012-01-01

Systemic inflammatory response syndrome (SIRS) is a fundamental host response common to bacterial infection and sterile tissue injury. SIRS can cause organ dysfunction and death but its mechanisms are incompletely understood. Moreover, SIRS can progress to organ failure or death despite being sterile or after control of the inciting infection. Biomarkers discriminating between sepsis, sterile SIRS and post-infective SIRS would therefore help direct care. Circulating mitochondrial DNA (mtDNA) is a damage-associated molecular pattern (DAMP) reflecting cellular injury. Circulating bacterial 16S-DNA (bDNA) is a pathogen-associated pattern (PAMP) reflecting ongoing infection. We developed qPCR assays to quantify these markers and predicted their plasma levels might help distinguish sterile injury from infection. To study these events in primates we assayed banked serum from papio baboons that had undergone a brief challenge of intravenous Bacillus anthracis deltaSterne (modified to remove toxins) followed by antibiotics (anthrax) that causes organ failure and death. To investigate the progression of sepsis to “severe” sepsis and death we studied animals where anthrax was pretreated with drotrecogin alfa (aPC), which attenuates sepsis in baboons. We also contrasted lethal anthrax bacteremia against non-lethal E.coli bacteremia and against sterile tissue injury from Shiga-like toxin-1 (Stx1). bDNA and mtDNA levels in timed samples were correlated with blood culture results and assays of organ function. Sterile injury by Stx1 increased mtDNA but bDNA was undetectable: consistent with the absence of infection. The bacterial challenges caused parallel early bDNA and mtDNA increases, but bDNA detected pathogens even after bacteria were undetectable by culture. Sub-lethal E.coli challenge only caused transient rises in mtDNA consistent with a self-limited injury. In lethal anthrax challenge (n=4) bDNA increased transiently but mtDNA levels remained elevated until death

Cd and proton adsorption onto bacterial consortia grown from industrial wastes and contaminated geologic settings.

PubMed

Borrok, David M; Fein, Jeremy B; Kulpa, Charles F

2004-11-01

To model the effects of bacterial metal adsorption in contaminated environments, results from metal adsorption experiments involving individual pure stains of bacteria must be extrapolated to systems in which potentially dozens of bacterial species are present. This extrapolation may be made easier because bacterial consortia from natural environments appear to exhibit similar metal binding properties. However, bacteria that thrive in highly perturbed contaminated environments may exhibit significantly different adsorptive behavior. Here we measure proton and Cd adsorption onto a range of bacterial consortia grown from heavily contaminated industrial wastes, groundwater, and soils. We model the results using a discrete site surface complexation approach to determine binding constants and site densities for each consortium. The results demonstrate that bacterial consortia from different contaminated environments exhibit a range of total site densities (approximately a 3-fold difference) and Cd-binding constants (approximately a 10-fold difference). These ranges for Cd binding constants may be small enough to suggest that bacteria-metal adsorption in contaminated environments can be described using relatively few "averaged" bacteria-metal binding constants (in conjunction with the necessary binding constants for competing surfaces and ligands). However, if additional precision is necessary, modeling parameters must be developed separately for each contaminated environment of interest.

Natural bacterial communities serve as quantitative geochemical biosensors.

PubMed

Smith, Mark B; Rocha, Andrea M; Smillie, Chris S; Olesen, Scott W; Paradis, Charles; Wu, Liyou; Campbell, James H; Fortney, Julian L; Mehlhorn, Tonia L; Lowe, Kenneth A; Earles, Jennifer E; Phillips, Jana; Techtmann, Steve M; Joyner, Dominique C; Elias, Dwayne A; Bailey, Kathryn L; Hurt, Richard A; Preheim, Sarah P; Sanders, Matthew C; Yang, Joy; Mueller, Marcella A; Brooks, Scott; Watson, David B; Zhang, Ping; He, Zhili; Dubinsky, Eric A; Adams, Paul D; Arkin, Adam P; Fields, Matthew W; Zhou, Jizhong; Alm, Eric J; Hazen, Terry C

2015-05-12

Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts. Copyright © 2015 Smith et al.

Natural bacterial communities serve as quantitative geochemical biosensors

DOE PAGES

Smith, Mark B.; Rocha, Andrea M.; Smillie, Chris S.; ...

2015-05-12

Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination,more » even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.« less

Bacterial Contamination of Hospital Bed-Control Handsets in a Surgical Setting: A Potential Marker of Contamination of the Healthcare Environment

PubMed Central

Brady, RRW; Kalima, P; Damani, NN; Wilson, RG; Dunlop, MG

2007-01-01

INTRODUCTION Patients undergoing colorectal surgical resections have a high incidence of surgical site infection (SSI). Many patient-specific risk factors have been recognised in association with SSI in such patients, but environmental contamination is increasingly recognised as a contributor to hospital-acquired infection (HAI). This study set out to describe the bacterial contamination of the patient environment, using hospital bed-control handsets, as they are frequently handled by both staff and patients and represent a marker of environmental contamination. PATIENTS AND METHODS On two unannounced sampling events, 1 week apart, 140 bacteriological assessments were made of 70 hospital bed control handsets within a specialist colorectal surgical unit. RESULTS Of the handsets examined, 67 (95.7%) demonstrated at least one bacterial species (52.9% grew 1, 30% grew 2 and 12.9% grew 3 or more bacterial species). Of these, 29 (41.4%) bed-control handsets grew bacteria known to cause nosocomial infection, including 22 (31.4%) handsets which grew Enterococcus spp., 9 (12.9%) which grew MRSA, 2 (2.9%) which grew MSSA, 2 (2.9%) which grew coliforms, and 1 (1.4%) handset which grew anaerobes. At 1-week follow-up, 31 bed-control handsets showed evidence of contamination by the same bacterial species. CONCLUSIONS This study revealed high levels of bacteria known to cause HAI, contaminating hospital bed-control handsets in a surgical setting. Further study is now required to confirm whether hospital environmental contamination is causally involved in SSI. PMID:17959000

BACTERIAL CONTAMINATION OF STETHOSCOPES

PubMed Central

Bukharie, Huda A.; Al-Zahrani, Hussain; Rubaish, Abdullah M.; Abdulmohsen, Mohammed F.

2004-01-01

Background: A stethoscope, an essential tool of the medical profession, can become a source of nosocomial infection. Objective: To determine the frequency of bacterial contamination of stethoscopes as well as the practices used for cleaning them. Methods: Cultures were taken from 100 stethoscopes used by different medical personnel in different hospital services. The stethoscopes were collected while the staff filled in a questionnaire. Results: Thirty (30%) out of the 100 stethoscopes surveyed were contaminated with microorganisms. The majority of organisms isolated were gram-positive bacteria (gram positive bacilli 12%, gram-negative bacteria 9%, gram-positive cocci 9%). None of the stethoscopes grew methicillin-resistant staphylococcus aureus. Overall, 21% of the health personnel cleaned their stethoscopes daily, 47% weekly, and 32% yearly. None of the health care workers cleaned their stethoscopes after every patient. Nurses cleaned their stethoscopes more often than physicians and medical students. Conclusion: Stethoscopes may be important in the spread of infectious agents. Their regular disinfection after use on each patient should be considered, particularly in such areas of the hospital, as the critical care units, and oncology units which house many patients with antibiotic-resistant organisms. PMID:23012043

Apheresis technology correlates with bacterial contamination of platelets and reported septic transfusion reactions.

PubMed

Eder, Anne F; Dy, Beth A; DeMerse, Barbara; Wagner, Stephen J; Stramer, Susan L; O'Neill, E Mary; Herron, Ross M

2017-12-01

Apheresis technology to collect platelet (PLT) components differs among devices. We evaluated the relationship of the plateletpheresis device with bacterial contamination and reported septic transfusion reactions. Plateletpheresis was performed using Amicus (Fenwal, a Fresenius Kabi Company) or Trima (Trima Accel, TerumoBCT) from 2010 to 2014. All donations used inlet-line sample diversion and were tested by quality control (QC; Day 1) aerobic culture. Rates of bacterial contamination and septic reactions to PLTs were calculated for both devices. During the 5-year study period, plateletpheresis collections using Amicus and Trima devices totaled 1,486,888 and 671,955 donations, respectively. The rate of confirmed-positive bacterial cultures of apheresis PLT donations was significantly higher with Amicus than with Trima (252 vs. 112 per 10 6 donations [odds ratio {OR}, 2.3; 95% confidence interval {CI}, 1.8-2.9]). Septic transfusion reactions were caused by 30 apheresis PLT units from 25 contaminated Amicus procedures and three apheresis PLT units from three contaminated Trima procedures. The overall rate of septic reactions was significantly higher with apheresis PLT components collected with Amicus than with Trima (16.8 vs. 4.5 per 10 6 donations [OR, 3.8; 95% CI, 1.1-12.5]). All apheresis PLT components implicated in septic transfusion reactions had negative QC culture results incubated through Day 5 (i.e., false negatives). Apheresis technology affects bacterial contamination of plateletpheresis collections. The device-specific, higher rate of confirmed-positive bacterial culture results also correlated with a significantly higher rate of reported septic transfusion reactions to apheresis PLTs. © 2017 AABB.

Bacterial Contamination into Ready-to-Eat Foods Sold in Middle Thailand.

PubMed

Ananchaipattana, Chiraporn; Bari, Md Latiful; Inatsu, Yasuhiro

2016-01-01

　Bacterial contamination in ready-to-eat (RTE) foods prepared and sold by small businesses in middle Thailand was surveyed. The 135 samples were randomly purchased from open markets and supermarkets in Bangkok and Pathum Thani provinces during May 2013 to February 2015. The results indicated that the rate of contamination in cooked pork, chicken meat and cooked aquatic items were 13/39 (33%), 18/45 (40%) and 14/57 (25%), respectively and the rate of bacterial contamination of collected samples from open market and supermarket were 26/67 (39%) and 19/68 (27%), respectively. Therefore, no statistically significant difference of contamination rate between two kinds of market or among three categories of food. The most contaminated pathogenic bacteria was Staphylococcus aureus 27/135 (20%) while that of Salmonella spp. was the lowest 5/135 (4%) in each categories of collected food samples. Implementation of suitable hygienic practices in the small food businesses are thought to be required to reduce the risk of foodborne illnesses caused by the consumption of RTE foods sold in middle Thailand.

Prevalence of bacterial contamination in platelet concentrates at the National Center of Blood Transfusion (Mexico).

PubMed

Ibáñez-Cervantes, G; Bello-López, J M; Fernández-Sánchez, V; Domínguez-Mendoza, C A; Acevedo-Alfaro, L I

2017-06-01

Most common bacterial sepsis associated with transfusion is caused by contaminated Platelet Concentrates (PC). The screening of PC to detect bacterial contamination is obligatory in Mexico, and it is carried out in quality control programs. In Mexico, the identification and molecular characterization of bacterial contaminants to detect contamination sources have not been implemented due to high costs; however, it is an actual current need. One hundred PC were randomly selected and microbiologically analyzed. This sample size corresponds to 1% of the PC obtained by the National Center of Blood Transfusion (NCBT) in Mexico City according to the Official Mexican Standard NOM-253-SSA1-2012. Additionally, molecular biology tests were implemented in order to identify the possible contamination sources. Nine of the 100 PC analyzed (9%) showed bacterial contamination; analysis of the nucleotide sequences revealed the presence of characteristic microbiota from donor skin and soil. Diverse clonal relationship between the strains was identified in Staphylococcus epidermidis. Detection of contaminants associated with environmental and skin flora, shows the need to implement measures in the process of disinfecting skin at the site of phlebotomy and cleaning each of the areas involved in blood collection. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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.

PARAMETERS OF TREATED STAINLESS STEEL SURFACES IMPORTANT FOR RESISTANCE TO BACTERIAL CONTAMINATION

EPA Science Inventory

Use of materials that are resistant to bacterial contamination could enhance food safety during processing. Common finishing treatments of stainless steel surfaces used for components of poultry processing equipment were tested for resistance to bacterial attachment. Surface char...

Electromagnetic signals are produced by aqueous nanostructures derived from bacterial DNA sequences.

PubMed

Montagnier, Luc; Aïssa, Jamal; Ferris, Stéphane; Montagnier, Jean-Luc; Lavallée, Claude

2009-06-01

A novel property of DNA is described: the capacity of some bacterial DNA sequences to induce electromagnetic waves at high aqueous dilutions. It appears to be a resonance phenomenon triggered by the ambient electromagnetic background of very low frequency waves. The genomic DNA of most pathogenic bacteria contains sequences which are able to generate such signals. This opens the way to the development of highly sensitive detection system for chronic bacterial infections in human and animal diseases.

UV irradiation and autoclave treatment for elimination of contaminating DNA from laboratory consumables.

PubMed

Gefrides, Lisa A; Powell, Mark C; Donley, Michael A; Kahn, Roger

2010-02-01

Laboratories employ various approaches to ensure that their consumables are free of DNA contamination. They may purchase pre-treated consumables, perform quality control checks prior to casework, and use in-house profile databases for contamination detection. It is better to prevent contamination prior to DNA typing than identify it after samples are processed. To this end, laboratories may UV irradiate or autoclave consumables prior to use but treatment procedures are typically based on killing microorganisms and not on the elimination of DNA. We report a systematic study of UV and autoclave treatments on the persistence of DNA from saliva. This study was undertaken to determine the best decontamination strategy for the removal of DNA from laboratory consumables. We have identified autoclave and UV irradiation procedures that can eliminate nanogram quantities of contaminating DNA contained within cellular material. Autoclaving is more effective than UV irradiation because it can eliminate short fragments of contaminating DNA more effectively. Lengthy autoclave or UV irradiation treatments are required. Depending on bulb power, a UV crosslinker may take a minimum of 2h to achieve an effective dose for elimination of nanogram quantities of contaminating DNA (>7250mJ/cm(2)). Similarly autoclaving may also take 2h to eliminate similar quantities of contaminating DNA. For this study, we used dried saliva stains to determine the effective dose. Dried saliva stains were chosen because purified DNA as well as fresh saliva are less difficult to eradicate than dried stains and also because consumable contamination is more likely to be in the form of a collection of dry cells.

Bacterial community profile of contaminated soils in a typical antimony mining site.

PubMed

Wang, Ningning; Zhang, Suhuan; He, Mengchang

2018-01-01

The soils around the world's largest antimony mine have been contaminated by high concentrations of Sb and As, which might influence microbial diversity in the surrounding soils. The ecological effects of bioavailable Sb and As on the composition and diversity of microbial community in soils remain unknown. In this study, the relative abundance, taxonomic diversity and composition of bacterial community in soils from a typical Sb mine area, and the relationship between the bacterial community and bioavailable concentrations as well as environmental factors have been investigated comprehensively using high-throughput sequencing (HTS) and diffusive gradients in thin films (DGT). The results indicated that Proteobacteria, Acidobacteria, Chloroflexi, Bacteroidetes, Actinobacteria, Gemmatimonadetes, and Cyanobacteria were the dominant bacterial populations at phylum level in all soil samples, accounting for more than 80% of the bacteria sequenced. The abundance and diversity of bacterial community vary along a metal contamination gradient. Redundancy discriminate analysis (RDA) revealed that 74.74% of bacterial community variation in the contaminated soils was explained by six environmental factors (pH, Sb DGT , As DGT , potential ecological risk index (RI), TC, TN), among which pH, Sb DGT , and As DGT were dominant factors influencing the composition and diversity of bacteria. This study contributes to our understanding of microbial diversity in a local ecosystem and introduces the option of studying bioavailable Sb and As using DGT.

Plasma bacterial and mitochondrial DNA distinguish bacterial sepsis from sterile systemic inflammatory response syndrome and quantify inflammatory tissue injury in nonhuman primates.

PubMed

Sursal, Tolga; Stearns-Kurosawa, Deborah J; Itagaki, Kiyoshi; Oh, Sun-Young; Sun, Shiqin; Kurosawa, Shinichiro; Hauser, Carl J

2013-01-01

Systemic inflammatory response syndrome (SIRS) is a fundamental host response common to bacterial infection and sterile tissue injury. Systemic inflammatory response syndrome can cause organ dysfunction and death, but its mechanisms are incompletely understood. Moreover, SIRS can progress to organ failure or death despite being sterile or after control of the inciting infection. Biomarkers discriminating between sepsis, sterile SIRS, and postinfective SIRS would therefore help direct care. Circulating mitochondrial DNA (mtDNA) is a damage-associated molecular pattern reflecting cellular injury. Circulating bacterial 16S DNA (bDNA) is a pathogen-associated pattern (PAMP) reflecting ongoing infection. We developed quantitative polymerase chain reaction assays to quantify these markers, and predicting their plasma levels might help distinguish sterile injury from infection. To study these events in primates, we assayed banked serum from Papio baboons that had undergone a brief challenge of intravenous Bacillus anthracis delta Sterne (modified to remove toxins) followed by antibiotics (anthrax) that causes organ failure and death. To investigate the progression of sepsis to "severe" sepsis and death, we studied animals where anthrax was pretreated with drotrecogin alfa (activated protein C), which attenuates sepsis in baboons. We also contrasted lethal anthrax bacteremia against nonlethal E. coli bacteremia and against sterile tissue injury from Shiga-like toxin 1. Bacterial DNA and mtDNA levels in timed samples were correlated with blood culture results and assays of organ function. Sterile injury by Shiga-like toxin 1 increased mtDNA, but bDNA was undetectable: consistent with the absence of infection. The bacterial challenges caused parallel early bDNA and mtDNA increases, but bDNA detected pathogens even after bacteria were undetectable by culture. Sublethal E. coli challenge only caused transient rises in mtDNA consistent with a self-limited injury. In lethal

Bacterial Contamination of Anaesthetic and Vasopressor Drugs in the Operating Theatres

PubMed Central

Rueangchira-Urai, Rongrong; Rujirojindakul, Panthila; Geater, Alan Frederick; McNeil, Edward

2017-01-01

Objective The aim of this study was to determine the incidence of bacterial and fungal contamination in anaesthetic and vasopressor drugs before and after use in operating theatres. Methods A cross-sectional study was conducted in the operating theatres of a university hospital. We collected 945 samples of three different drugs, namely, propofol, vecuronium and ephedrine, from 20 operating rooms and refrigerators where the unused drugs were stored. Each drug was divided into two groups, the pre-use group and the post-use group. The pre-use drugs were cultured before the patient received the drug. The post-use drugs were cultured after the patient had received the drug or after the drugs had been transferred to other syringes. The culture results were reported as either positive or negative. Results Out of the 945 drug samples, 26 (2.8%, 95% confidence interval=1.8%–4.0%) gave a positive culture. Of the 317 propofol samples, 20 (6.3%) were found to have bacterial contamination, 11 in the pre-use group and 9 in the post-use group. Of the 318 ephedrine samples, 6 (1.9%) were found to be positive on culture, one in the pre-use group and five in the post-use group. Vecuronium gave no positive cultures. All organisms were non-pathogenic, and no fungal contamination was found. Conclusion The incidence of bacterial contamination in anaesthetic and vasopressor drugs was 2.8%. Anaesthetic teams must be aware of contamination issues in anaesthetic drugs that have been prepared for later use and, in order to reduce the risk of contamination, they must improve the methods of administering drugs to patients. PMID:28377840

Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal

PubMed Central

Skoglund, Pontus; Northoff, Bernd H.; Shunkov, Michael V.; Derevianko, Anatoli P.; Pääbo, Svante; Krause, Johannes; Jakobsson, Mattias

2014-01-01

One of the main impediments for obtaining DNA sequences from ancient human skeletons is the presence of contaminating modern human DNA molecules in many fossil samples and laboratory reagents. However, DNA fragments isolated from ancient specimens show a characteristic DNA damage pattern caused by miscoding lesions that differs from present day DNA sequences. Here, we develop a framework for evaluating the likelihood of a sequence originating from a model with postmortem degradation—summarized in a postmortem degradation score—which allows the identification of DNA fragments that are unlikely to originate from present day sources. We apply this approach to a contaminated Neandertal specimen from Okladnikov Cave in Siberia to isolate its endogenous DNA from modern human contaminants and show that the reconstructed mitochondrial genome sequence is more closely related to the variation of Western Neandertals than what was discernible from previous analyses. Our method opens up the potential for genomic analysis of contaminated fossil material. PMID:24469802

Potential risk for bacterial contamination in conventional reused ventilator systems and disposable closed ventilator-suction systems.

PubMed

Li, Ya-Chi; Lin, Hui-Ling; Liao, Fang-Chun; Wang, Sing-Siang; Chang, Hsiu-Chu; Hsu, Hung-Fu; Chen, Sue-Hsien; Wan, Gwo-Hwa

2018-01-01

Few studies have investigated the difference in bacterial contamination between conventional reused ventilator systems and disposable closed ventilator-suction systems. The aim of this study was to investigate the bacterial contamination rates of the reused and disposable ventilator systems, and the association between system disconnection and bacterial contamination of ventilator systems. The enrolled intubated and mechanically ventilated patients used a conventional reused ventilator system and a disposable closed ventilator-suction system, respectively, for a week; specimens were then collected from the ventilator circuit systems to evaluate human and environmental bacterial contamination. The sputum specimens from patients were also analyzed in this study. The detection rate of bacteria in the conventional reused ventilator system was substantially higher than that in the disposable ventilator system. The inspiratory and expiratory limbs of the disposable closed ventilator-suction system had higher bacterial concentrations than the conventional reused ventilator system. The bacterial concentration in the heated humidifier of the reused ventilator system was significantly higher than that in the disposable ventilator system. Positive associations existed among the bacterial concentrations at different locations in the reused and disposable ventilator systems, respectively. The predominant bacteria identified in the reused and disposable ventilator systems included Acinetobacter spp., Bacillus cereus, Elizabethkingia spp., Pseudomonas spp., and Stenotrophomonas (Xan) maltophilia. Both the reused and disposable ventilator systems had high bacterial contamination rates after one week of use. Disconnection of the ventilator systems should be avoided during system operation to decrease the risks of environmental pollution and human exposure, especially for the disposable ventilator system. ClinicalTrials.gov PRS / NCT03359148.

Bacterial Contaminants of Poultry Meat: Sources, Species, and Dynamics

PubMed Central

Rouger, Amélie; Zagorec, Monique

2017-01-01

With the constant increase in poultry meat consumption worldwide and the large variety of poultry meat products and consumer demand, ensuring the microbial safety of poultry carcasses and cuts is essential. In the present review, we address the bacterial contamination of poultry meat from the slaughtering steps to the use-by-date of the products. The different contamination sources are identified. The contaminants occurring in poultry meat cuts and their behavior toward sanitizing treatments or various storage conditions are discussed. A list of the main pathogenic bacteria of concern for the consumer and those responsible for spoilage and waste of poultry meat is established. PMID:28841156

Chemically synthesized silver nanoparticles as cell lysis agent for bacterial genomic DNA isolation

NASA Astrophysics Data System (ADS)

Goswami, Gunajit; Boruah, Himangshu; Gautom, Trishnamoni; Jyoti Hazarika, Dibya; Barooah, Madhumita; Boro, Robin Chandra

2017-12-01

Silver nanoparticles (AgNPs) have seen a recent spurt of use in varied fields of science. In this paper, we showed a novel application of AgNP as a promising microbial cell-lysis agent for genomic DNA isolation. We utilized chemically synthesized AgNPs for lysing bacterial cells to isolate their genomic DNA. The AgNPs efficiently lysed bacterial cells to yield good quality DNA that could be subsequently used for several molecular biology works.

Potential risk for bacterial contamination in conventional reused ventilator systems and disposable closed ventilator-suction systems

PubMed Central

Li, Ya-Chi; Lin, Hui-Ling; Liao, Fang-Chun; Wang, Sing-Siang; Chang, Hsiu-Chu; Hsu, Hung-Fu; Chen, Sue-Hsien

2018-01-01

Background Few studies have investigated the difference in bacterial contamination between conventional reused ventilator systems and disposable closed ventilator-suction systems. The aim of this study was to investigate the bacterial contamination rates of the reused and disposable ventilator systems, and the association between system disconnection and bacterial contamination of ventilator systems. Methods The enrolled intubated and mechanically ventilated patients used a conventional reused ventilator system and a disposable closed ventilator-suction system, respectively, for a week; specimens were then collected from the ventilator circuit systems to evaluate human and environmental bacterial contamination. The sputum specimens from patients were also analyzed in this study. Results The detection rate of bacteria in the conventional reused ventilator system was substantially higher than that in the disposable ventilator system. The inspiratory and expiratory limbs of the disposable closed ventilator-suction system had higher bacterial concentrations than the conventional reused ventilator system. The bacterial concentration in the heated humidifier of the reused ventilator system was significantly higher than that in the disposable ventilator system. Positive associations existed among the bacterial concentrations at different locations in the reused and disposable ventilator systems, respectively. The predominant bacteria identified in the reused and disposable ventilator systems included Acinetobacter spp., Bacillus cereus, Elizabethkingia spp., Pseudomonas spp., and Stenotrophomonas (Xan) maltophilia. Conclusions Both the reused and disposable ventilator systems had high bacterial contamination rates after one week of use. Disconnection of the ventilator systems should be avoided during system operation to decrease the risks of environmental pollution and human exposure, especially for the disposable ventilator system. Trial registration Clinical

A pilot study to assess the bacterial contaminants in hookah pipes in a community setting.

PubMed

Martinasek, M; Rivera, Z; Ferrer, A; Freundt, E

2018-05-01

Hookah smoking among young adults remains a public health threat. Increasing research has uncovered the deleterious effects of hookah smoking, including both acute and chronic health conditions. Due to the current lack of regulation, hookah bars/lounges lack protocols for equipment sanitation. To examine evidence of bacterial contamination in hookah pipes due to a lack of sanitation regulations. For this field/laboratory study, 10 hookah bars/lounges were studied. Isolated bacteria were characterized and identified by species using 16S ribosomal RNA gene sequencing. At the 10 hookah bars sampled, the mouthpiece had the highest bacterial prevalence and diversity. Some of the bacterial isolates were found to be antibiotic-resistant. Ten of the isolated bacteria were Gram-positive and two were identified as Gram-negative. Levels of bacterial contamination vary widely from one hookah bar to the next, and reflect a lack of industry standards for cleaning these devices. Bacterial contamination of hookah pipes may represent a fomite for transmission of infectious diseases. Our results warrant future surveillance of hookahs to monitor for potential human pathogens.

Significance of treated agrowaste residue and autochthonous inoculates (Arbuscular mycorrhizal fungi and Bacillus cereus) on bacterial community structure and phytoextraction to remediate soils contaminated with heavy metals.

PubMed

Azcón, Rosario; Medina, Almudena; Roldán, Antonio; Biró, Borbála; Vivas, Astrid

2009-04-01

In this study, we analyzed the impact of treatments such as Aspergillus niger-treated sugar beet waste (SB), PO4(3-) fertilization and autochthonous inoculants [arbuscular mycorrhizal (AM) fungi and Bacillus cereus], on the bacterial community structure in a soils contaminated with heavy metals as well as, the effectiveness on plant growth (Trifolium repens). The inoculation with AM fungi in SB amended soil, increased plant growth similarly to PO4(3-) addition, and both treatments matched in P acquisition but bacterial biodiversity estimated by denaturing gradient gel electrophoresis of amplified 16S rDNA sequences, was more stimulated by the presence of the AM fungus than by PO4(3-) fertilization. The SB amendment plus AM inoculation increased the microbial diversity by 233% and also changed (by 215%) the structure of the bacterial community. The microbial inoculants and amendment used favoured plant growth and the phytoextraction process and concomitantly modified bacterial community in the rhizosphere; thus they can be used for remediation. Therefore, the understanding of such microbial ecological aspects is important for phytoremediation and the recovery of contaminated soils.

Dynamic Effects of Biochar on the Bacterial Community Structure in Soil Contaminated with Polycyclic Aromatic Hydrocarbons.

PubMed

Song, Yang; Bian, Yongrong; Wang, Fang; Xu, Min; Ni, Ni; Yang, Xinglun; Gu, Chenggang; Jiang, Xin

2017-08-16

Amending soil with biochar is an effective soil remediation strategy for organic contaminants. This study investigated the dynamic effects of wheat straw biochar on the bacterial community structure during remediation by high-throughput sequencing. The wheat straw biochar amended into the soil significantly reduced the bioavailability and toxicity of polycyclic aromatic hydrocarbons (PAHs). Biochar amendment helped to maintain the bacterial diversity in the PAH-contaminated soil. The relationship between the immobilization of PAHs and the soil bacterial diversity fit a quadratic model. Before week 12 of the incubation, the incubation time was the main factor contributing to the changes in the soil bacterial community structure. However, biochar greatly affected the bacterial community structure after 12 weeks of amendment, and the effects were dependent upon the biochar type. Amendment with biochar mainly facilitated the growth of rare bacterial genera (relative abundance of 0.01-1%) in the studied soil. Therefore, the application of wheat straw biochar into PAH-contaminated soil can reduce the environmental risks of PAHs and benefit the soil microbial ecology.

Effects of predation and dispersal on bacterial abundance and contaminant biodegradation.

PubMed

Otto, Sally; Harms, Hauke; Wick, Lukas Y

2017-02-01

Research into the biodegradation of soil contaminants has rarely addressed the consequences of predator-prey interactions. Here, we investigated the joint effect of predation and dispersal networks on contaminant degradation by linking spatial abundances of degrader (Pseudomonas fluorescens LP6a) and predator (Bdellovibrio bacteriovorus) bacteria to the degradation of the major soil contaminant phenanthrene (PHE). We used a laboratory microcosm with a PHE passive dosing system and a glass fiber network to facilitate bacterial dispersal. Different predator-to-prey ratios and spatial arrangements of prey and predator inoculation were used to study predation pressure effects on PHE degradation. We observed that predation resulted in (i) enhanced PHE-degradation at low predator counts (PC) compared to controls lacking predation, (ii) reduced PHE-degradation at elevated PC relative to low PC, and (iii) significant effects of the spatial arrangement of prey and predator inoculation on PHE degradation. Our data suggest that predation facilitated by dispersal networks (such as fungal mycelia) may support the build-up of an effective bacterial biomass and, hence, contaminant biodegradation in heterogeneous systems such as soil. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Risk factors for contact lens bacterial contamination during continuous wear.

PubMed

Szczotka-Flynn, Loretta B; Bajaksouzian, Saralee; Jacobs, Michael R; Rimm, Alfred

2009-11-01

Microbial contamination of contact lenses is associated with corneal infection and inflammation. This study determined the microbiological, clinical, and demographic factors that are associated with bacterial contamination of a silicone hydrogel contact lens when worn for continuous wear (CW). Two hundred five healthy subjects were enrolled in the Longitudinal Analysis of Silicone Hydrogel Contact Lens Study and were fitted with lotrafilcon A lenses for monthly CW and followed for 1 year. Lenses were aseptically removed after 1 week and 4 months of wear and cultured using an agar sandwich technique. Lids and conjunctiva were routinely cultured at baseline and after 1 week and 4 months of CW. Lenses and ocular sites were considered to have substantial microbial bioburden when they harbored pathogenic organisms or high levels of commensal organisms. Univariate and multivariate logistic regression analyses were conducted to examine whether substantial conjunctival or lid bioburden, subject demographics, lens-wearing history, symptoms, and biomicroscopic signs were associated with lens bioburden. About one third (32.4%) of subjects had substantial bacterial bioburden in either eye across multiple visits. Over half (53.2%) and about one tenth (11.7%) of subjects had substantial lid and conjunctival bioburden, respectively, and 11.2% discontinued because of discomfort. The adjusted odds ratios (and 95% confidence intervals) for presence of substantial lens bioburden were 2.49 (1.17-5.30), 4.24 (1.45-12.40), and 4.11 (1.17-14.46) for substantial lid bioburden, substantial conjunctival bioburden, and lens discomfort, respectively. Bacterial contamination of silicone hydrogel contact lenses is common during CW. Substantial lens bioburden is associated with discomfort precluding successful CW. The presence of substantial lid and conjunctival bioburden is associated with a 2.5-fold and more than fourfold greater risk of substantial lens bioburden and is likely the major route

Bacterial community and arsenic functional genes diversity in arsenic contaminated soils from different geographic locations

PubMed Central

Gu, Yunfu; D. Van Nostrand, Joy; Wu, Liyou; He, Zhili; Qin, Yujia; Zhao, Fang-Jie; Zhou, Jizhong

2017-01-01

To understand how soil microbial communities and arsenic (As) functional genes respond to soil arsenic (As) contamination, five soils contaminated with As at different levels were collected from diverse geographic locations, incubated for 54 days under flooded conditions, and examined by both MiSeq sequencing of 16S rRNA gene amplicons and functional gene microarray (GeoChip 4.0). The results showed that both bacterial community structure and As functional gene structure differed among geographical locations. The diversity of As functional genes correlated positively with the diversity of 16S rRNA genes (P< 0.05). Higher diversities of As functional genes and 16S rRNA genes were observed in the soils with higher available As. Soil pH, phosphate-extractable As, and amorphous Fe content were the most important factors in shaping the bacterial community structure and As transformation functional genes. Geographic location was also important in controlling both the bacterial community and As transformation functional potential. These findings provide insights into the variation of As transformation functional genes in soils contaminated with different levels of As at different geographic locations, and the impact of environmental As contamination on the soil bacterial community. PMID:28475654

Elimination and molecular identification of endophytic bacterial contaminants during in vitro propagation of Bambusa balcooa.

PubMed

Ray, Syandan Sinha; Ali, Md Nasim; Mukherjee, Shibasis; Chatterjee, Gautam; Banerjee, Maitreyi

2017-02-01

Bambusa balcooa is an economically important, multipurpose bamboo species, decidedly used in construction industry. Availability of natural bamboo is depleting very rapidly due to accelerated deforestation and its unrestrained use. The large number and timely supply of saplings are the need of the hour for the restoration of bamboo stands. Micropropagation, being the potent alternative for season independent rapid regeneration, is restricted in bamboo because of endophytic contamination. An in vitro attempt has been taken to overcome the endophytic contamination by using broad spectrum antibiotics as surface sterilant as well as a media component. Ampicillin sodium salt (5 mg/ml for 30 min) as a surface sterilant was found as the best treatment for high bud breaking (80%) coupled with high branching and low contamination (20%) but it was found ineffective to control the contamination during multiplication stage. Then, two endophytes were isolated and minimum inhibitory concentration was determined through antibiotic susceptibility test for successful eradication at multiplication stage. Finally, contamination free cultures were obtained when streptocycline (100 μg/ml) and gentamicin sulphate (75 μg/ml) were added into the medium. The two isolated endophytes, BB1 and BB2, were identified through 16S rDNA techniques and NCBI-BLAST algorithm with 99% sequence similarity with those of Janibacter sp. (KX423734) and Serratia marcescens strain (KX423735). To our knowledge, this is the first report for B. balcooa where antibiotics were used as surface sterilant as well as medium component, to control endophytic bacterial contaminants, followed by their identification.

Detecting and Estimating Contamination of Human DNA Samples in Sequencing and Array-Based Genotype Data

PubMed Central

Jun, Goo; Flickinger, Matthew; Hetrick, Kurt N.; Romm, Jane M.; Doheny, Kimberly F.; Abecasis, Gonçalo R.; Boehnke, Michael; Kang, Hyun Min

2012-01-01

DNA sample contamination is a serious problem in DNA sequencing studies and may result in systematic genotype misclassification and false positive associations. Although methods exist to detect and filter out cross-species contamination, few methods to detect within-species sample contamination are available. In this paper, we describe methods to identify within-species DNA sample contamination based on (1) a combination of sequencing reads and array-based genotype data, (2) sequence reads alone, and (3) array-based genotype data alone. Analysis of sequencing reads allows contamination detection after sequence data is generated but prior to variant calling; analysis of array-based genotype data allows contamination detection prior to generation of costly sequence data. Through a combination of analysis of in silico and experimentally contaminated samples, we show that our methods can reliably detect and estimate levels of contamination as low as 1%. We evaluate the impact of DNA contamination on genotype accuracy and propose effective strategies to screen for and prevent DNA contamination in sequencing studies. PMID:23103226

Corneal erosions, bacterial contamination of contact lenses, and microbial keratitis.

PubMed

Willcox, Mark D P; Naduvilath, Thomas J; Vaddavalli, Pravin K; Holden, Brien A; Ozkan, Jerome; Zhu, Hua

2010-11-01

To estimate the rate of corneal erosion coupled with gram-negative bacterial contamination of contact lenses and compare this with the rate of microbial keratitis (MK) with contact lenses. The rate of corneal erosion and contact lens contamination by gram-negative bacteria were calculated from several prospective trials. These rates were used to calculate the theoretical rate of corneal erosion happening at the same time as wearing a contact lens contaminated with gram-negative bacteria. This theoretical rate was then compared with the rates of MK reported in various epidemiological and clinical trials. Corneal erosions were more frequent during extended wear (0.6-2.6% of visits) compared with daily wear (0.01-0.05% of visits). No corneal erosions were observed for lenses worn on a daily disposable basis. Contamination rates for lenses worn on a daily disposable basis were the lowest (2.4%), whereas they were the highest for low Dk lenses worn on an extended wear basis (7.1%). The estimated rate of corneal erosions occurring at the same time as wearing lenses contaminated with gram-negative bacteria was the lowest during daily wear of low Dk lenses (1.56/10,000 [95% CI: 0.23-10.57]) and the highest during extended wear of high Dk lenses (38.55/10,000 [95% CI: 24.77-60.04]). These rates were similar in magnitude to the rates reported for MK of different hydrogel lenses worn on differing wear schedules. The coincidence of corneal erosions during lens wear with gram-negative bacterial contamination of lenses may account for the relative incidence of MK during lens wear with different lens materials and modes of use.

Bacterial Contamination of Multi-dose Eye Drops at Ophthalmology Department, University of Gondar, Northwest Ethiopia.

PubMed

Tsegaw, Asegedech; Tsegaw, Asamere; Abula, Tefera; Assefa, Yared

2017-01-01

Ophthalmic solutions used for both diagnostic and therapeutic purposes were found to be contaminated with bacteria pathogens and caused serious ocular infections such as keratitis and endophthalmitis. The objective was to assess the magnitude and pattern of bacterial contamination of multi-dose ophthalmic medications and investigate the drug susceptibility pattern of the isolates in the Department of Ophthalmology at Gondar University Teaching Hospital. A total of 100 ophthalmic medications in-use by patients and eye-care workers have been taken and cultured for potential bacterial contamination in the Microbiology Department after 1 week and >1 week of use. The dropper tip and the residual eye medications were examined for contamination. The contaminating bacteria were identified using a standard procedure and drug susceptibility testing to selected antimicrobial agents was done. Eleven ophthalmic medications were contaminated by different bacterial species with a prevalence of 11%. Multi-use and longer duration of use of eye medications were associated with higher rate of contamination. The contamination level ranges from 0% for antibiotics, 20% for local anesthetics, and 40% for povidone iodine. Among bacteria identified, Staphylococcus aureus and coagulase-negative Staphylococcus species were resistant to methicillin while others were sensitive to the antibiotics tested. The prevalence of contamination was low, but methicillin-resistant Staphylococcus was a potential risk. It is recommended that the Department of Ophthalmology should design set of rules about duration of use and safe handling of ophthalmic medications by the staff and patients.

Contamination of sequence databases with adaptor sequences

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

Yoshikawa, Takeo; Sanders, A.R.; Detera-Wadleigh, S.D.

Because of the exponential increase in the amount of DNA sequences being added to the public databases on a daily basis, it has become imperative to identify sources of contamination rapidly. Previously, contaminations of sequence databases have been reported to alert the scientific community to the problem. These contaminations can be divided into two categories. The first category comprises host sequences that have been difficult for submitters to manage or control. Examples include anomalous sequences derived from Escherichia coli, which are inserted into the chromosomes (and plasmids) of the bacterial hosts. Insertion sequences are highly mobile and are capable ofmore » transposing themselves into plasmids during cloning manipulation. Another example of the first category is the infection with yeast genomic DNA or with bacterial DNA of some commercially available cDNA libraries from Clontech. The second category of database contamination is due to the inadvertent inclusion of nonhost sequences. This category includes incorporation of cloning-vector sequences and multicloning sites in the database submission. M13-derived artifacts have been common, since M13-based vectors have been widely used for subcloning DNA fragments. Recognizing this problem, the National Center for Biotechnology Information (NCBI) started to screen, in April 1994, all sequences directly submitted to GenBank, against a set of vector data retrieved from GenBank by use of key-word searches, such as {open_quotes}vector.{close_quotes} In this report, we present evidence for another sequence artifact that is widespread but that, to our knowledge, has not yet been reported. 11 refs., 1 tab.« less

Activation of the immune system by bacterial CpG-DNA

PubMed Central

Häcker, Georg; Redecke, Vanessa; Häcker, Hans

2002-01-01

The past decade has seen a remarkable process of refocusing in immunology. Cells of the innate immune system, especially macrophages and dendritic cells, have been at the centre of this process. These cells had been regarded by some scientists as non-specific, sometimes perhaps even confined to the menial job of serving T cells by scavenging antigen and presenting it to the sophisticated adaptive immune system. Only over the last few years has it become unequivocally clear that cells of the innate immunity hold, by variation of context and mode of antigen presentation, the power of shaping an adaptive immune response. The innate immune response, in turn, is to a significant degree the result of stimulation by so-called pathogen-associated molecular patterns (PAMPs). One compound with high stimulatory potential for the innate immune system is bacterial DNA. Here we will review recent evidence that bacterial DNA should be ranked with other PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid. We will further review our present knowledge of DNA recognition and DNA-dependent signal transduction in cells of the immune system. PMID:11918685

Elasticity-mediated nematiclike bacterial organization in model extracellular DNA matrix.

PubMed

Smalyukh, Ivan I; Butler, John; Shrout, Joshua D; Parsek, Matthew R; Wong, Gerard C L

2008-09-01

DNA is a common extracellular matrix component of bacterial biofilms. We find that bacteria can spontaneously order in a matrix of aligned concentrated DNA, in which rod-shaped cells of Pseudomonas aeruginosa follow the orientation of extended DNA chains. The alignment of bacteria is ensured by elasticity and liquid crystalline properties of the DNA matrix. These findings show how behavior of planktonic bacteria may be modified in extracellular polymeric substances of biofilms and illustrate the potential of using complex fluids to manipulate embedded nanosized and microsized active particles.

Plant secondary metabolite-induced shifts in bacterial community structure and degradative ability in contaminated soil.

PubMed

Uhlik, Ondrej; Musilova, Lucie; Ridl, Jakub; Hroudova, Miluse; Vlcek, Cestmir; Koubek, Jiri; Holeckova, Marcela; Mackova, Martina; Macek, Tomas

2013-10-01

The aim of the study was to investigate how selected natural compounds (naringin, caffeic acid, and limonene) induce shifts in both bacterial community structure and degradative activity in long-term polychlorinated biphenyl (PCB)-contaminated soil and how these changes correlate with changes in chlorobiphenyl degradation capacity. In order to address this issue, we have integrated analytical methods of determining PCB degradation with pyrosequencing of 16S rRNA gene tag-encoded amplicons and DNA-stable isotope probing (SIP). Our model system was set in laboratory microcosms with PCB-contaminated soil, which was enriched for 8 weeks with the suspensions of flavonoid naringin, terpene limonene, and phenolic caffeic acid. Our results show that application of selected plant secondary metabolites resulted in bacterial community structure far different from the control one (no natural compound amendment). The community in soil treated with caffeic acid is almost solely represented by Proteobacteria, Acidobacteria, and Verrucomicrobia (together over 99 %). Treatment with naringin resulted in an enrichment of Firmicutes to the exclusion of Acidobacteria and Verrucomicrobia. SIP was applied in order to identify populations actively participating in 4-chlorobiphenyl catabolism. We observed that naringin and limonene in soil foster mainly populations of Hydrogenophaga spp., caffeic acid Burkholderia spp. and Pseudoxanthomonas spp. None of these populations were detected among 4-chlorobiphenyl utilizers in non-amended soil. Similarly, the degradation of individual PCB congeners was influenced by the addition of different plant compounds. Residual content of PCBs was lowest after treating the soil with naringin. Addition of caffeic acid resulted in comparable decrease of total PCBs with non-amended soil; however, higher substituted congeners were more degraded after caffeic acid treatment compared to all other treatments. Finally, it appears that plant secondary metabolites

Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

NASA Astrophysics Data System (ADS)

Chen, K.

2017-01-01

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

PubMed

Christen, Matthias; Deutsch, Samuel; Christen, Beat

2015-08-21

Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

[Bacterial contamination of the indoor air in a transplant unit].

PubMed

Matoušková, Ivanka; Holý, Ondřej

2013-12-01

For one year (August 2010 to July 2011), microbial contamination of the indoor air in the Transplant Unit of the Haemato-Oncology Clinic, Olomouc University Hospital was monitored monthly. Twenty sampling sites were singled out and a total of 240 indoor air samples were collected. An MAS-100 air sampler (Merck, GER) was used, air flow rate of 100 liters per minute, 1 minute. The measured values of indoor air temperature were stable. The relative air humidity ranged from 17% to 68%. The highest average value of microbial air contamination was found in the "staff entry room" (1170 CFU/m3). The lowest microbial air contamination (150-250 CFU/m3) was measured in the patient isolation units. The most frequently isolated bacterial strains were coagulase-negative staphylococci (94.3%), followed by Micrococcus spp. (67%) and Bacillus subtilis (11%). It can be assumed that the -source of these airborne bacterial strains are both patients and medical staff. They are classified as -opportunistic pathogens and as such can cause hospital infections among haemato-oncology patients.

Evaluation of Two Matrices for Long-Term, Ambient Storage of Bacterial DNA.

PubMed

Miernyk, Karen M; DeByle, Carolynn K; Rudolph, Karen M

2017-12-01

Culture-independent molecular analyses allow researchers to identify diverse microorganisms. This approach requires microbiological DNA repositories. The standard for DNA storage is liquid nitrogen or ultralow freezers. These use large amounts of space, are costly to operate, and could fail. Room temperature DNA storage is a viable alternative. In this study, we investigated storage of bacterial DNA using two ambient storage matrices, Biomatrica DNAstable ® Plus and GenTegra ® DNA. We created crude and clean DNA extracts from five Streptococcus pneumoniae isolates. Extracts were stored at -30°C (our usual DNA storage temperature), 25°C (within the range of temperatures recommended for the products), and 50°C (to simulate longer storage time). Samples were stored at -30°C with no product and dried at 25°C and 50°C with no product, in Biomatrica DNAstable Plus or GenTegra DNA. We analyzed the samples after 0, 1, 2, 4, 8, 16, 32, and 64 weeks using the Nanodrop 1000 to determine the amount of DNA in each aliquot and by real-time PCR for the S. pneumoniae genes lytA and psaA. Using a 50°C storage temperature, we simulated 362 weeks of 25°C storage. The average amount of DNA in aliquots stored with a stabilizing matrix was 103%-116% of the original amount added to the tubes. This is similar to samples stored at -30°C (average 102%-121%). With one exception, samples stored with a stabilizing matrix had no change in lytA or psaA cycle threshold (Ct) value over time (Ct range ≤2.9), similar to samples stored at -30°C (Ct range ≤3.0). Samples stored at 25°C with no stabilizing matrix had Ct ranges of 2.2-5.1. DNAstable Plus and GenTegra DNA can protect dried bacterial DNA samples stored at room temperature with similar effectiveness as at -30°C. It is not effective to store bacterial DNA at room temperature without a stabilizing matrix.

Bacterial DNA induces pulmonary damage via TLR-9 through cross-talk with neutrophils.

PubMed

Itagaki, Kiyoshi; Adibnia, Yasaman; Sun, Shiqin; Zhao, Cong; Sursal, Tolga; Chen, Yu; Junger, Wolfgang; Hauser, Carl J

2011-12-01

Bacterial DNA (bDNA) contains hypomethylated "CpG" repeats that can be recognized by Toll-like receptor 9 (TLR-9) as a pathogen-associated molecular pattern. The ability of bDNA to initiate lung injury via TLR-9 has been inferred on the basis of studies using artificial CpG DNA. But the role of authentic bDNA in lung injury is still unknown. Moreover, the mechanisms by which CpG DNA species can lead to pulmonary injury are unknown, although neutrophils (PMNs) are thought to play a key role in the genesis of septic acute lung injury. We evaluated the effects of bDNA on PMN-endothelial cell (EC) interactions thought critical for initiation of acute lung injury. Using a biocapacitance system to monitor real-time changes in endothelial permeability, we demonstrate here that bDNA causes EC permeability in a dose-dependent manner uniquely in the presence of PMNs. These permeability changes are inhibited by chloroquine, suggesting TLR-9 dependency. When PMNs were preincubated with bDNA and applied to ECs or when bDNA was applied to ECs without PMNs, no permeability changes were detected. To study the underlying mechanisms, we evaluated the effects of bDNA on PMN-EC adherence. Bacterial DNA significantly increased PMN adherence to ECs in association with upregulated adhesion molecules in both cell types. Taken together, our results strongly support the conclusion that bDNA can initiate lung injury by stimulating PMN-EC adhesive interactions predisposing to endothelial permeability. Bacterial DNA stimulation of TLR-9 appears to promote enhanced gene expression of adhesion molecules in both cell types. This leads to PMN-EC cross-talk, which is required for injury to occur.

Metal contamination disturbs biochemical and microbial properties of calcareous agricultural soils of the Mediterranean area.

PubMed

de Santiago-Martín, Ana; Cheviron, Natalie; Quintana, Jose R; González, Concepción; Lafuente, Antonio L; Mougin, Christian

2013-04-01

Mediterranean climate characteristics and carbonate are key factors governing soil heavy-metal accumulation, and low organic matter (OM) content could limit the ability of microbial populations to cope with resulting stress. We studied the effects of metal contamination on a combination of biological parameters in soils having these characteristics. With this aim, soils were spiked with a mixture of cadmium, copper, lead, and zinc, at the two limit values proposed by current European legislation, and incubated for ≤12 months. Then we measured biochemical (phosphatase, urease, β-galactosidase, arylsulfatase, and dehydrogenase activities) and microbial (fungal and bacterial DNA concentration by quantitative polymerase chain reaction) parameters. All of the enzyme activities were strongly affected by metal contamination and showed the following inhibition sequence: phosphatase (30-64 %) < arylsulfatase (38-97 %) ≤ urease (1-100 %) ≤ β-galactosidase (30-100 %) < dehydrogenase (69-100 %). The high variability among soils was attributed to the different proportion of fine mineral fraction, OM, crystalline iron oxides, and divalent cations in soil solution. The decrease of fungal DNA concentration in metal-spiked soils was negligible, whereas the decrease of bacterial DNA was ~1-54 % at the lowest level and 2-69 % at the highest level of contamination. The lowest bacterial DNA decrease occurred in soils with the highest OM, clay, and carbonate contents. Finally, regarding the strong inhibition of the biological parameters measured and the alteration of the fungal/bacterial DNA ratio, we provide strong evidence that disturbance on the system, even within the limiting values of contamination proposed by the current European Directive, could alter key soil processes. These limiting values should be established according to soil characteristics and/or revised when contamination is produced by a mixture of heavy metals.

Human β-defensin 3 increases the TLR9-dependent response to bacterial DNA.

PubMed

McGlasson, Sarah L; Semple, Fiona; MacPherson, Heather; Gray, Mohini; Davidson, Donald J; Dorin, Julia R

2017-04-01

Human β-defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self-DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt-3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self-DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9-dependent response. In contrast to hBD3, lipofection of self-DNA enhances inflammatory signaling, but the response is predominantly TLR9-independent. Together, these data show that hBD3 has a role in the innate immune-mediated response to pathogen DNA, increasing inflammatory signaling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy-number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA-based vaccines. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mutagenic cost of ribonucleotides in bacterial DNA

PubMed Central

Schroeder, Jeremy W.; Randall, Justin R.; Hirst, William G.; O’Donnell, Michael E.; Simmons, Lyle A.

2017-01-01

Replicative DNA polymerases misincorporate ribonucleoside triphosphates (rNTPs) into DNA approximately once every 2,000 base pairs synthesized. Ribonucleotide excision repair (RER) removes ribonucleoside monophosphates (rNMPs) from genomic DNA, replacing the error with the appropriate deoxyribonucleoside triphosphate (dNTP). Ribonucleotides represent a major threat to genome integrity with the potential to cause strand breaks. Furthermore, it has been shown in the bacterium Bacillus subtilis that loss of RER increases spontaneous mutagenesis. Despite the high rNTP error rate and the effect on genome integrity, the mechanism underlying mutagenesis in RER-deficient bacterial cells remains unknown. We performed mutation accumulation lines and genome-wide mutational profiling of B. subtilis lacking RNase HII, the enzyme that incises at single rNMP residues initiating RER. We show that loss of RER in B. subtilis causes strand- and sequence-context–dependent GC → AT transitions. Using purified proteins, we show that the replicative polymerase DnaE is mutagenic within the sequence context identified in RER-deficient cells. We also found that DnaE does not perform strand displacement synthesis. Given the use of nucleotide excision repair (NER) as a backup pathway for RER in RNase HII-deficient cells and the known mutagenic profile of DnaE, we propose that misincorporated ribonucleotides are removed by NER followed by error-prone resynthesis with DnaE. PMID:29078353

Bacterial Degraders of Coexisting Dichloromethane, Benzene, and Toluene, Identified by Stable-Isotope Probing.

PubMed

Yoshikawa, Miho; Zhang, Ming; Kurisu, Futoshi; Toyota, Koki

2017-01-01

Most bioremediation studies on volatile organic compounds (VOCs) have focused on a single contaminant or its derived compounds and degraders have been identified under single contaminant conditions. Bioremediation of multiple contaminants remains a challenging issue. To identify a bacterial consortium that degrades multiple VOCs (dichloromethane (DCM), benzene, and toluene), we applied DNA-stable isotope probing. For individual tests, we combined a 13 C-labeled VOC with other two unlabeled VOCs, and prepared three unlabeled VOCs as a reference. Over 11 days, DNA was periodically extracted from the consortia, and the bacterial community was evaluated by next-generation sequencing of bacterial 16S rRNA gene amplicons. Density gradient fractions of the DNA extracts were amplified by universal bacterial primers for the 16S rRNA gene sequences, and the amplicons were analyzed by terminal restriction fragment length polymorphism (T-RFLP) using restriction enzymes: Hha I and Msp I. The T-RFLP fragments were identified by 16S rRNA gene cloning and sequencing. Under all test conditions, the consortia were dominated by Rhodanobacter , Bradyrhizobium / Afipia , Rhizobium , and Hyphomicrobium . DNA derived from Hyphomicrobium and Propioniferax shifted toward heavier fractions under the condition added with 13 C-DCM and 13 C-benzene, respectively, compared with the reference, but no shifts were induced by 13 C-toluene addition. This implies that Hyphomicrobium and Propioniferax were the main DCM and benzene degraders, respectively, under the coexisting condition. The known benzene degrader Pseudomonas sp. was present but not actively involved in the degradation.

Risk Factors for Contact Lens Bacterial Contamination During Continuous Wear

PubMed Central

Szczotka-Flynn, Loretta B.; Bajaksouzian, Saralee; Jacobs, Michael R.; Rimm, Alfred

2009-01-01

Purpose Microbial contamination of contact lenses is associated with corneal infection and inflammation. This study determined which microbiological, clinical and demographic factors are associated with bacterial contamination of a silicone hydrogel contact lens when worn for continuous wear. Methods Two hundred five normal subjects were enrolled in the Longitudinal Analysis of Silicone Hydrogel (LASH) Contact Lens Study and were fitted with lotrafilcon A lenses for monthly continuous wear and followed for 1 year. Lenses were aseptically removed after 1 week and 4 months of wear and cultured using an agar sandwich technique. Lids and conjunctiva were routinely cultured at baseline, and after 1 week and 4 months of continuous wear. Lenses and ocular sites were considered to have substantial microbial bioburden when they harbored pathogenic organisms or high levels of commensal organisms. Univariate and multivariate logistic regression analyses were conducted to examine whether substantial conjunctival or lid bioburden, subject demographics, lens wearing history, symptoms, and biomicroscopic signs were associated with lens bioburden. Results About one-third (32.4%) of subjects had substantial bacterial bioburden in either eye across multiple visits. Over half (53.2%) and about one-tenth (11.7%) of subjects had substantial lid and conjunctival bioburden, respectively, and 11.2% discontinued due to discomfort. The adjusted odds ratios (and 95% confidence intervals) for presence of substantial lens bioburden were 2.49 (1.17–5.30), 4.24 (1.45–12.40), and 4.11 (1.17–14.46) for substantial lid bioburden, substantial conjunctival bioburden, and lens discomfort, respectively. Conclusions Bacterial contamination of silicone hydrogel contact lenses is common during continuous wear. Substantial lens bioburden is associated with discomfort precluding successful continuous wear. The presence of substantial lid and conjunctival bioburden are associated with a 2.5 fold and

On-farm and postharvest processing sources of bacterial contamination to melon rinds.

PubMed

Gagliardi, J V; Millner, P D; Lester, G; Ingram, D

2003-01-01

Multistate and international foodborne illness outbreaks, particularly involving cantaloupe and often involving rare Salmonella spp., have increased dramatically over the past 13 years. This study assessed the sources and extent of melon rind contamination in production fields and at processing and packing facilities. In the spring of 1999, cantaloupe (Cucumis melo L. [reticulatus group] cv. Cruiser) sampled from two sites in the Rio Grande River Valley showed that postharvest-processed melon rinds often had greater plate counts of bacterial contaminants than field-fresh melons. Cantaloupe in the field had 2.5 to 3.5 log CFU g(-1) rind total coliforms by aerobic plate counts, whereas washed melons had 4.0 to 5.0 log CFU g(-1). In the fall of 1999, coliforms on honeydew melons (C. melo [inodorous group] cv. Honey Brew) ranged from 2.6 to 3.7 log CFU g(-1) after processing, and total and fecal coliforms and enterococci never fell below 2.5 log CFU g(-1). A hydrocooler at another site contaminated cantaloupe rinds with up to 3.4 log CFU g(-1) total and fecal enterococci; a secondary rinse with chlorinated water incompletely removed these bacteria. Sources of coliforms and enterococci were at high levels in melon production soils, especially in furrows that were flood irrigated, in standing water at one field, and in irrigation water at both sites. At one processing facility, wash water pumped from the Rio Grande River may not have been sufficiently disinfected prior to use. Because soil, irrigation water, and process water were potential sources of bacterial contamination, monitoring and management on-farm and at processing and packing facilities should focus on water quality as an important control point for growers and packers to reduce bacterial contamination on melon rinds.

Adequate Hand Washing and Glove Use Are Necessary To Reduce Cross-Contamination from Hands with High Bacterial Loads.

PubMed

Robinson, Andrew L; Lee, Hyun Jung; Kwon, Junehee; Todd, Ewen; Rodriguez, Fernando Perez; Ryu, Dojin

2016-02-01

Hand washing and glove use are the main methods for reducing bacterial cross-contamination from hands to ready-to-eat food in a food service setting. However, bacterial transfer from hands to gloves is poorly understood, as is the effect of different durations of soap rubbing on bacterial reduction. To assess bacterial transfer from hands to gloves and to compare bacterial transfer rates to food after different soap washing times and glove use, participants' hands were artificially contaminated with Enterobacter aerogenes B199A at ∼9 log CFU. Different soap rubbing times (0, 3, and 20 s), glove use, and tomato dicing activities followed. The bacterial counts in diced tomatoes and on participants' hands and gloves were then analyzed. Different soap rubbing times did not significantly change the amount of bacteria recovered from participants' hands. Dicing tomatoes with bare hands after 20 s of soap rubbing transferred significantly less bacteria (P < 0.01) to tomatoes than did dicing with bare hands after 0 s of soap rubbing. Wearing gloves while dicing greatly reduced the incidence of contaminated tomato samples compared with dicing with bare hands. Increasing soap washing time decreased the incidence of bacteria recovered from outside glove surfaces (P < 0.05). These results highlight that both glove use and adequate hand washing are necessary to reduce bacterial cross-contamination in food service environments.

Bacterial community structure and abundances of antibiotic resistance genes in heavy metals contaminated agricultural soil.

PubMed

Zhang, Fengli; Zhao, Xiaoxue; Li, Qingbo; Liu, Jia; Ding, Jizhe; Wu, Huiying; Zhao, Zongsheng; Ba, Yue; Cheng, Xuemin; Cui, Liuxin; Li, Hongping; Zhu, Jingyuan

2018-04-01

Soil contamination with heavy metals is a worldwide problem especially in China. The interrelation of soil bacterial community structure, antibiotic resistance genes, and heavy metal contamination in soil is still unclear. Here, seven agricultural areas (G1-G7) with heavy metal contamination were sampled with different distances (741 to 2556 m) to the factory. Denaturing gradient gel electrophoresis (DGGE) and Shannon index were used to analyze bacterial community diversity. Real-time fluorescence quantitative PCR was used to detect the relative abundance of ARGs sul1, sul2, tetA, tetM, tetW, one mobile genetic elements (MGE) inti1. Results showed that all samples were polluted by Cadmium (Cd), and some of them were polluted by lead (Pb), mercury (Hg), arsenic (As), copper (Cu), and zinc (Zn). DGGE showed that the most abundant bacterial species were found in G7 with the lightest heavy metal contamination. The results of the principal component analysis and clustering analysis both showed that G7 could not be classified with other samples. The relative abundance of sul1 was correlated with Cu, Zn concentration. Gene sul2 are positively related with total phosphorus, and tetM was associated with organic matter. Total gene abundances and relative abundance of inti1 both correlated with organic matter. Redundancy analysis showed that Zn and sul2 were significantly related with bacterial community structure. Together, our results indicate a complex linkage between soil heavy metal concentration, bacterial community composition, and some global disseminated ARG abundance.

Bacterial contamination of boar semen affects the litter size.

PubMed

Maroto Martín, Luis O; Muñoz, Eduardo Cruz; De Cupere, Françoise; Van Driessche, Edilbert; Echemendia-Blanco, Dannele; Rodríguez, José M Machado; Beeckmans, Sonia

2010-07-01

One hundred and fifteen semen samples were collected from 115 different boars from two farms in Cuba. The boars belonged to five different breeds. Evaluation of the semen sample characteristics (volume, pH, colour, smell, motility of sperm cells) revealed that they meet international standards. The samples were also tested for the presence of agglutinated sperm cells and for bacterial contamination. Seventy five percent of the ejaculates were contaminated with at least one type of bacteria and E. coli was by far the major contaminant, being present in 79% of the contaminated semen samples (n=68). Other contaminating bacteria belonged to the genera Proteus (n=31), Serratia (n=31), Enterobacter (n=24), Klebsiella (n=12), Staphylococcus (n=10), Streptococcus (n=8) and Pseudomonas (n=7). Only in one sample anaerobic bacteria were detected. Pearson's analysis of the data revealed that there is a positive correlation between the presence of E. coli and sperm agglutination, and a negative correlation between sperm agglutination and litter size. One-way ANOVA and post hoc Tukey analysis of 378 litters showed that the litter size is significantly reduced when semen is used that is contaminated with spermagglutinating E. coli above a threshold value of 3.5x10(3)CFU/ml. Copyright 2010 Elsevier B.V. All rights reserved.

Field investigations of bacterial contaminants and their effects on extended porcine semen.

PubMed

Althouse, G C; Kuster, C E; Clark, S G; Weisiger, R M

2000-03-15

Field investigations (n=23) were made over a 3-yr period at North American boar studs and farms in which the primary complaint was sperm agglutination in association with decreased sperm longevity of extended semen, and increased regular returns to estrus and/or vaginal discharges across parity. Microscopic examination of extended semen from these units revealed depressed gross motility (usually <30%), sperm agglutination, and sperm cell death occurring within 2 d of semen collection and processing regardless of the semen extender used. The extended semen exhibited a high number of induced acrosome abnormalities (>20%). Sample pH was acidic (5.7 to 6.4) in 93% of the submitted samples. Aerobic culture yielded a variety of bacteria from different genera. A single bacterial contaminant was obtained from 66% of the submitted samples (n=37 doses); 34% contained 2 or more different bacterial genera. The most frequently isolated contaminant bacteria from porcine extended semen were Alcaligenes xylosoxydans (n=3), Burkholderia cepacia (n=6), Enterobacter cloacae (n=6), Escherichia coli (n=6), Serratia marcescens (n=5), and Stenotrophomonas [Xanthomonas] maltophilia (n=6); these 6 bacteria accounted for 71% of all contaminated samples, and were spermicidal when re-inoculated and incubated in fresh, high quality extended semen. All contaminant bacteria were found to be resistant to the aminoglycoside gentamicin, a common preservative antibiotic used in commercial porcine semen extenders. Eleven genera were spermicidal in conjunction with an acidic environment, while 2 strains (E. coli, S. maltophilia) were spermicidal without this characteristic acidic environment. Bacteria originated from multiple sources at the stud/farm, and were of animal and nonanimal origin. A minimum contamination technique (MCT) protocol was developed to standardize hygiene and sanitation. This protocol focused on MCT's during boar preparation, semen collection, semen processing and laboratory

Adsorption of bacterial plasmids in pure mineral mixtures

NASA Astrophysics Data System (ADS)

Zhang, L.; Cochran, J. P.; Seaman, J. C.; Parrott, B.

2017-12-01

Microorganisms play an important role in controlling the fate and transport of subsurface contaminants through the direct degradation of organic contaminants to the control of chemical redox conditions that impact the speciation and partitioning of inorganic contaminants. Genes that control these processes, including the relative tolerance associated with direct exposure to toxic contaminants, are found within the bacteria's chromosomal DNA and also within distinct, circular DNA elements called plasmids. Plasmids are mobile genetic elements that can be exchanged with other bacterial species through horizontal gene transfer (HGT). The frequency of HGT in soil is influenced by several factors, with the physicochemical characteristics of soil possibly being a primary factor. Thus, the objective for our research was to determine the movement and persistence of bacterial plasmids within soil. Our current study focuses on batch sorption experiments designed to evaluate the partitioning of bacterial plasmids in idealized mineral mixtures that represent the clay mineralogy of highly weathered soils of the Southeastern US. Specifically, we compared plasmid adsorption among pure goethite, kaolinite, and a mixture of goethite and kaolinite. We also determined the adsorption of plasmids on the above minerals over increasing pH (3 to 10). Our results show that adsorption decreased in the following order: goethite > kaolinite > mixture of goethite and kaolinite. We also found that plasmids adsorption was higher at lower pH levels, with pH 3 having the adsorption maximum. However, at pH 3, DNA denaturing may have occurred, leading to aggregation or precipitation of plasmids on the mineral surfaces. Our study was the first steps in determining the influence of soil properties on plasmid adsorption. Our future goals are to determine the adsorption in other pure minerals and in natural soils.

MULTIPLE IMAGING TECHNIQUES DEMONSTRATE THE MANIPULATION OF SURFACES TO REDUCE BACTERIAL CONTAMINATION

EPA Science Inventory

Surface imaging techniques were combined to determine appropriate manipulation of technologically important surfaces for commercial applications. Stainless steel surfaces were engineered to reduce bacterial contamination, biofilm formation, and corrosion during product processing...

Bacterial contamination of amniotic membrane in a tissue bank from Iran.

PubMed

Aghayan, Hamid Reza; Goodarzi, Parisa; Baradaran-Rafii, Alireza; Larijani, Bagher; Moradabadi, Leila; Rahim, Fakher; Arjmand, Babak

2013-09-01

Human Amniotic Membrane (AM) transplantation can promote tissue healing and reduce inflammation, tissue scarring and neovascularization. Homa Peyvand Tamin (HPT) tissue bank has focused on manufacturing human cell and tissue based products including AM. The purpose of this study is to evaluate and identify bacterial contamination of AMs that is produced by HPT for several ophthalmic applications. From July 2006 to April 2011, 122 placentas from cesarean sections were retrieved by HPT after obtaining informed consent from the donors. Besides testing donor's blood sample for viral markers, microbiological evaluation was performed pre and post processing. During tissue processing, decontamination was performed by an antibiotic cocktail including; Gentamicin, Ceftriaxone and Cloxacillin. Of 271 cesarean section AM donors who were screened as potential donors, 122 were accepted for processing and assessed for microbiological contamination. Donors' age were between 21 and 41 years (Mean = 27.61 ± 0.24). More than 92% of mothers were in their first or second gravidity with full term pregnancies. The most prevalent organisms were Staphylococci species (72.53%). After processing, contamination rates markedly decreased by 84.62% (p value = 0.013). According to our results, most of bacterial contaminations were related to donation process and the contamination pattern suggests procurement team as a source. Therefore we recommend that regular training programs should be implemented by tissue banks for procurement staff. These programs should focus on improved donor screening and proper aseptic technique for tissue retrieval. We also suggest that tissue banks should periodically check the rate and types of tissue contaminations. These data help them to find system faults and to update processing methods.

Swabbing of waiting room magazines reveals only low levels of bacterial contamination

PubMed Central

Charnock, Colin

2005-01-01

Previous studies have shown that toys in waiting rooms of general practice surgeries can be contaminated with potentially pathogenic bacteria. The question was raised as to whether magazines might also be sources of contamination. Swabbing of the front page of 15 magazines from 11 general practice surgeries, followed by analysis for total and specific bacteria, revealed low levels of contamination. Among targeted groups of pathogens only two colonies of Staphylococcus aureus were detected. Magazines do not seem to be potentially important vectors of bacterial transfer in the setting examined. PMID:15667764

Bacterial contamination hypothesis: a new concept in endometriosis.

PubMed

Khan, Khaleque N; Fujishita, Akira; Hiraki, Koichi; Kitajima, Michio; Nakashima, Masahiro; Fushiki, Shinji; Kitawaki, Jo

2018-04-01

Endometriosis is a multifactorial disease that mainly affects women of reproductive age. The exact pathogenesis of this disease is still debatable. The role of bacterial endotoxin (lipopolysaccharide, LPS) and Toll-like receptor 4 (TLR4) in endometriosis were investigated and the possible source of endotoxin in the pelvic environment was examined. The limulus amoebocyte lysate test was used to measure the endotoxin levels in the menstrual fluid and peritoneal fluid and their potential role in the growth of endometriosis was investigated. Menstrual blood and endometrial samples were cultured for the presence of microbes. The effect of gonadotrophin-releasing hormone agonist (GnRHa) treatment on intrauterine microbial colonization (IUMC) and the occurrence of endometritis was investigated. Lipopolysaccharide regulates the pro-inflammatory response in the pelvis and growth of endometriosis via the LPS/TLR4 cascade. The menstrual blood was highly contaminated with Escherichea coli and the endometrial samples were colonized with other microbes. A cross-talk between inflammation and ovarian steroids or the stress reaction also was observed in the pelvis. Treatment with GnRHa further worsens intrauterine microbial colonization, with the consequent occurrence of endometritis in women with endometriosis. For the first time, a new concept called the "bacterial contamination hypothesis" is proposed in endometriosis. This study's findings of IUMC in women with endometriosis could hold new therapeutic potential in addition to the conventional estrogen-suppressing agent.

Identification and elimination of bacterial contamination during in vitro propagation of Guadua angustifolia Kunth.

PubMed

Nadha, Harleen Kaur; Salwan, Richa; Kasana, Ramesh Chand; Anand, Manju; Sood, Anil

2012-04-01

Guadua angustifolia Kunth is a very important bamboo species with significant utility in pharmaceutical, paper, charcoal, and construction industries. Microbial contamination is a major problem encountered during establishment of in vitro cultures of Guadua. This study has been designed to analyze the identity of contaminating bacteria and to develop the strategy to eliminate them during micropropagation of Guadua. We isolated and consequently analyzed partial sequence analysis of the 16S rRNA gene to identify two contaminating bacteria as (1) Pantoea agglomerans and (2) Pantoea ananatis. In addition, we also- performed antibiotic sensitivity testing on these bacterial isolates. We identified kanamycin and streptomycin sulfate as potentially useful antibiotics in eliminating the contaminating bacteria. We grew shoots on multiplication medium containing BAP (2 mg/l) and adenine sulfate (10 mg/l) supplemented with kanamycin (10 μg/ml) for 10 days and transferred them to fresh medium without antibiotics and found that bacterial growth was inhibited. Moreover, we observed intensive formation of high-quality shoots. Streptomycin sulfate also inhibited bacterial growth but at higher concentration. We also demonstrated that shoots grown in streptomycin sulfate tended to be shorter and had yellow leaves. Thus, we have developed a novel strategy to identify and inhibit intriguing microbial contaminations of (1) Pantoea agglomerans and (2) Pantoea ananatis during establishment of in vitro cultures of Guadua. This would improve in vitro establishment of an important bamboo, Guadua angustifolia Kunth for large scale propagation.

Bacterial contamination along implant-abutment interface in external and internal-hex dental implants

PubMed Central

de Oliveira, Greison Rabelo; Olate, Sergio; Pozzer, Leandro; Cavalieri-Pereira, Lucas; Rodrigues-Chessa, Jaime G; Albergaría-Barbosa, José Ricardo

2014-01-01

The aim of this research was to evaluate bacterial contamination along the implant-abutment interface in relation to the size of the interface. 80 brand name implants were used, 40 internal-hex and 40 external-hex. The implants were handled in a sterile atmosphere inside a box, where they were inoculated with 0.3 μl of the Streptococcus sanguis ATCC10556 bacterium in the interior and the abutment was immediately installed with a torque of 30 Ncm for the external-hex and 20 Ncm for the internal-hex; the system was included in an Eppendorf control for 30 seconds and then placed in an Eppendorf control for 30 days. The implants were removed and assessed under a scanning electron microscope while the Eppendorf controls were bred in blood agar to analyze the colonies formed. The data were analyzed using the Chi-squared, Kruskal-Wallis and Mann-Whitney tests, considering a value of p<0.05 to obtain statistical significance. Five implants were excluded due to probable external contamination. Microspaces of up to 86.8 μm were observed in the external-hex implants and up to 53.9 μm in the internal-hex implants with no significant differences between the different systems being observed (p>0.05). The contamination observed was produced mainly in the external-hex implants and statistically significant differences were observed between the different hex systems from the same company. No significant differences were observed between interface size and bacterial contamination. Within our limitations, there was no relation between the size of the implant-abutment interface and bacterial contamination with Streptococcus sanguis ATCC10556. PMID:24753751

STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

PubMed Central

Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

2009-01-01

Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

Comparison of commercially-available preservatives for maintaining the integrity of bacterial DNA in human milk.

PubMed

Lackey, Kimberly A; Williams, Janet E; Price, William J; Carrothers, Janae M; Brooker, Sarah L; Shafii, Bahman; McGuire, Mark A; McGuire, Michelle K

2017-10-01

Inhibiting changes to bacteria in human milk between sample collection and analysis is necessary for unbiased characterization of the milk microbiome. Although cold storage is considered optimal, alternative preservation is sometimes necessary. The objective of this study was to compare the effectiveness of several commercially-available preservatives with regard to maintaining bacterial DNA in human milk for delayed microbiome analysis. Specifically, we compared Life Technologies' RNAlater® stabilization solution, Biomatrica's DNAgard® Saliva, Advanced Instruments' Broad Spectrum Microtabs II™, and Norgen Biotek Corporation's Milk DNA Preservation and Isolation Kit. Aliquots of 8 pools of human milk were treated with each preservative. DNA was extracted immediately and at 1, 2, 4, and 6wk, during which time milk was held at 37°C. The V1-V3 region of the bacterial 16S rRNA gene was amplified and sequenced. Changes in bacterial community structure and diversity over time were evaluated. Comparable to other studies, the most abundant genera were Streptococcus (33.3%), Staphylococcus (14.0%), Dyella (6.3%), Pseudomonas (3.0%), Veillonella (2.5%), Hafnia (2.0%), Prevotella (1.7%), Rhodococcus (1.6%), and Granulicatella (1.4%). Overall, use of Norgen's Milk DNA Preservation and Isolation Kit best maintained the consistency of the bacterial community structure. Total DNA, diversity, and evenness metrics were also highest in samples preserved with this method. When collecting human milk for bacterial community analysis in field conditions where cold storage is not available, our results suggest that Norgen's Milk DNA Preservation and Isolation Kit may be a useful method, at least for a period of 2weeks. Copyright © 2017 Elsevier B.V. All rights reserved.

Terahertz spectroscopy for the isothermal detection of bacterial DNA by magnetic bead-based rolling circle amplification.

PubMed

Yang, Xiang; Yang, Ke; Zhao, Xiang; Lin, Zhongquan; Liu, Zhiyong; Luo, Sha; Zhang, Yang; Wang, Yunxia; Fu, Weiling

2017-12-04

The demand for rapid and sensitive bacterial detection is continuously increasing due to the significant requirements of various applications. In this study, a terahertz (THz) biosensor based on rolling circle amplification (RCA) was developed for the isothermal detection of bacterial DNA. The synthetic bacterium-specific sequence of 16S rDNA hybridized with a padlock probe (PLP) that contains a sequence fully complementary to the target sequence at the 5' and 3' ends. The linear PLP was circularized by ligation to form a circular PLP upon recognition of the target sequence; then the capture probe (CP) immobilized on magnetic beads (MBs) acted as a primer to initialize RCA. As DNA molecules are much less absorptive than water molecules in the THz range, the RCA products on the surface of the MBs cause a significant decrease in THz absorption, which can be sensitively probed by THz spectroscopy. Our results showed that 0.12 fmol of synthetic bacterial DNA and 0.05 ng μL -1 of genomic DNA could be effectively detected using this assay. In addition, the specificity of this strategy was demonstrated by its low signal response to interfering bacteria. The proposed strategy not only represents a new method for the isothermal detection of the target bacterial DNA but also provides a general methodology for sensitive and specific DNA biosensing using THz spectroscopy.

Wautersia: The Contingency Water Container Bacterial Contamination Investigation

NASA Technical Reports Server (NTRS)

Shkedi, Brienne; Labuda, Laura; Bruce, Rebekah

2009-01-01

The Orbiter delivers water to the International Space Station (ISS) in Contingency Water Containers (CWCs) on each flight to the ISS. These CWCs are routinely sampled during each mission to verify the quality of the delivered water. Of the 5 samples returned on STS118/ 13A.1 in August 2007, two exhibited microbial growth exceeding potable water acceptability limits and historical data by orders of magnitude . The microbe was identified as Wautersia species and an investigation was launched to find the source of the contamination. Since then, samples collected on subsequent flights indicated additional CWCs had the same bacteria, as well as several on-orbit systems. An investigation was launched to try to find and address the source of the bacterial contamination. This paper will discuss how Wautersia was found, what Wautersia is, the investigation, and resolution.

Bacterial and fungal DNA extraction from blood samples: automated protocols.

PubMed

Lorenz, Michael G; Disqué, Claudia; Mühl, Helge

2015-01-01

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA.

Changes in the Bacterial Community Structure of Remediated Anthracene-Contaminated Soils

PubMed Central

Delgado-Balbuena, Laura; Bello-López, Juan M.; Navarro-Noya, Yendi E.; Rodríguez-Valentín, Analine; Luna-Guido, Marco L.; Dendooven, Luc

2016-01-01

Mixing soil or adding earthworms (Eisenia fetida (Savigny, 1826)) accelerated the removal of anthracene, a polycyclic aromatic hydrocarbon, from a pasture and an arable soil, while a non-ionic surfactant (Surfynol® 485) inhibited the removal of the contaminant compared to the untreated soil. It was unclear if the treatments affected the soil bacterial community and consequently the removal of anthracene. Therefore, the bacterial community structure was monitored by means of 454 pyrosequencing of the 16S rRNA gene in the pasture and arable soil mixed weekly, amended with Surfynol® 485, E. fetida or organic material that served as food for the earthworms for 56 days. In both soils, the removal of anthracene was in the order: mixing soil weekly (100%) > earthworms applied (92%) > organic material applied (77%) > untreated soil (57%) > surfactant applied (34%) after 56 days. There was no clear link between removal of anthracene from soil and changes in the bacterial community structure. On the one hand, application of earthworms removed most of the contaminant from the arable soil and had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of the Acidobacteria, Chloroflexi and Gemmatimonadetes, and an increase in that of the Proteobacteria compared to the unamended soil. Mixing the soil weekly removed all anthracene from the arable soil, but had little or no effect on the bacterial community structure. On the other hand, application of the surfactant inhibited the removal of anthracene from the arable soil compared to the untreated soil, but had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of Cytophagia (Bacteroidetes), Chloroflexi, Gemmatimonadetes and Planctomycetes and an increase in that of the Flavobacteria (Bacteroidetes) and Proteobacteria. Additionally, the removal of anthracene was similar in the different treatments of both the arable and pasture soil, but the

Differentiation of some species of Neisseriaceae and other bacterial groups by DNA-DNA hybridization.

PubMed

Tønjum, T; Bukholm, G; Bøvre, K

1989-05-01

DNA-DNA hybridization using total genomic DNA probes may represent a way of differentiating between miscellaneous bacterial species. This was studied with type and reference strains of 20 species in Moraxella, Kingella, and other selected Gram-negative groups. Both radioactive and biotin labelling were employed. Most of the species examined were easily distinguished, such as Moraxella (Branhamella) catarrhalis, M.(B.) ovis, M. atlantae, M. phenylpyruvica, M. osloensis, Neisseria elongata, N. meningitidis, Kingella kingae, K. indologenes, K. dentrificans, Oligella urethralis, Eikenella corrodens, Cardiobacterium hominis, Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Gardnerella vaginalis, and DF-2. This reflected the extent of the genetic distances between them as a basis for identification by hybridization. There was some clustering in the Moraxella group. Especially the closely related Moraxella nonliquefaciens, M. lacunata and M. bovis showed strong hybridization affinities. This leads to potential problems in distinguishing these three species from each other by DNA-DNA hybridization with total genomic probes alone.

Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.

PubMed

Villequey, Camille; Kong, Xu-Dong; Heinis, Christian

2017-11-01

Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Joint Estimation of Contamination, Error and Demography for Nuclear DNA from Ancient Humans

PubMed Central

Slatkin, Montgomery

2016-01-01

When sequencing an ancient DNA sample from a hominin fossil, DNA from present-day humans involved in excavation and extraction will be sequenced along with the endogenous material. This type of contamination is problematic for downstream analyses as it will introduce a bias towards the population of the contaminating individual(s). Quantifying the extent of contamination is a crucial step as it allows researchers to account for possible biases that may arise in downstream genetic analyses. Here, we present an MCMC algorithm to co-estimate the contamination rate, sequencing error rate and demographic parameters—including drift times and admixture rates—for an ancient nuclear genome obtained from human remains, when the putative contaminating DNA comes from present-day humans. We assume we have a large panel representing the putative contaminant population (e.g. European, East Asian or African). The method is implemented in a C++ program called ‘Demographic Inference with Contamination and Error’ (DICE). We applied it to simulations and genome data from ancient Neanderthals and modern humans. With reasonable levels of genome sequence coverage (>3X), we find we can recover accurate estimates of all these parameters, even when the contamination rate is as high as 50%. PMID:27049965

Bacterial contamination of fresh traumatic wounds at Trauma Center, Siriraj Hospital, Bangkok, Thailand.

PubMed

Sirijatuphat, Rujipas; Siritongtaworn, Preecha; Sripojtham, Vipaporn; Boonyasiri, Adhiratha; Thamlikitkul, Visanu

2014-03-01

Fresh traumatic wound is a common health problem in patients attending Trauma Center at Siriraj Hospital in Bangkok, Thailand. Antibiotic prophylaxis was given to nearly 90% of such patients. A contributing factor to a high prevalence of antibiotic prophylaxis is a lack of data on bacterial contamination at fresh traumatic wounds in Thai patients. To determine prevalence and characters of bacterial contamination, and incidence of wound infection in adult patients with fresh traumatic wounds attending Siriraj Trauma Center. Prospective study was conducted in 330 adult patients with fresh traumatic wounds from March 2012 and September 2012. All patients received wound care and antibiotic prophylaxis according to the judgment of their responsible physicians. A wound swab culture was taken from all patients. The patients were either called by telephone or asked to have follow-up visits in order to determine incidence of wound infection. The infected patient received regular care. Sixty-three percent and 8% of the patients had lacerated wounds and bite wounds, respectively. Ninety-one percent of them received antibiotics of which dicloxacillin and co-amoxiclav accounted for 80.3% and 11.4%, respectively. Wound swab cultures revealed that potential pathogenic bacteria i.e. S. aureus, streptococci, Enterobacteriaceae, Aeromonas spp., Acinetobacter spp. and non-fermentative gram-negative rods (NF GNR) were recovered from 7% of wounds. Incidence of wound infection was 1.2%, and all infected wounds were found in patients who had a contaminated wound and received antibiotic prophylaxis. Bacterial contaminations of infected patients were NF GNR, E. cloacae, and mixed organisms. All wound infections were successfully treated with appropriate wound care. More than 90% of adult patients with fresh traumatic wound at Siriraj Trauma Center received prophylactic antibiotics. Less than 10% of these wounds were contaminated with potentially pathogenic bacteria. Incidence of wound

Bacterial contaminants from frozen puff pastry production process and their growth inhibition by antimicrobial substances from lactic acid bacteria.

PubMed

Rumjuankiat, Kittaporn; Keawsompong, Suttipun; Nitisinprasert, Sunee

2017-05-01

Seventy-five bacterial contaminants which still persisted to cleaning system from three puff pastry production lines (dough forming, layer and filling forming, and shock freezing) were identified using 16S rDNA as seven genera of Bacillus , Corynebacterium , Dermacoccus , Enterobacter , Klebsiella, Pseudomonas , and Staphylococcus with detection frequencies of 24.00, 2.66, 1.33, 37.33, 1.33, 2.66, and 30.66, respectively. Seventeen species were discovered while only 11 species Bacillus cereus, B. subtilis, B. pumilus, Corynebacterium striatum , Dermacoccus barathri , Enterobacter asburiae, Staphylococcus kloosii, S. haemolyticus, S. hominis, S. warneri , and S. aureus were detected at the end of production. Based on their abundance, the highest abundance of E. asburiae could be used as a biomarker for product quality. While a low abundance of the mesophile pathogen C. striatum , which causes respiratory and nervous infection and appeared only at the shock freezing step was firstly reported for its detection in bakery product. Six antimicrobial substances (AMSs) from lactic acid bacteria, FF1-4, FF1-7, PFUR-242, PFUR-255, PP-174, and nisin A were tested for their inhibition activities against the contaminants. The three most effective were FF1-7, PP-174, and nisin A exhibiting wide inhibition spectra of 88.00%, 85.33%, and 86.66%, respectively. The potential of a disinfectant solution containing 800 AU/ml of PP-174 and nisin A against the most resistant strains of Enterobacter , Staphylococcus , Bacillus and Klebsiella was determined on artificially contaminated conveyor belt coupons at 0, 4, 8, 12, and 16 hr. The survival levels of the test strains were below 1 log CFU/coupon at 0 hr. The results suggested that a combined solution of PP-174 and nisin A may be beneficial as a sanitizer to inhibit bacterial contaminants in the frozen puff pastry industry.

The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence.

PubMed

Kasu, Mohaimin; Shires, Karen

2015-07-01

The production of full DNA profiles from biological evidence found in soil has a high failure rate due largely to the inhibitory substance humic acid (HA). Abundant in various natural soils, HA co-extracts with DNA during extraction and inhibits DNA profiling by binding to the molecular components of the genotyping assay. To successfully utilize traces of soil contaminated evidence, such as that found at many murder and rape crime scenes in South Africa, a reliable HA removal extraction system would often be selected based on previous validation studies. However, for many standard forensic DNA extraction systems, peer-reviewed publications detailing the efficacy on soil evidence is either lacking or is incomplete. Consequently, these sample types are often not collected or fail to yield suitable DNA material due to the use of unsuitable methodology. The aim of this study was to validate the common forensic DNA collection and extraction systems used in South Africa, namely DNA IQ, FTA elute and Nucleosave for processing blood and saliva contaminated with HA. A forensic appropriate volume of biological evidence was spiked with HA (0, 0.5, 1.5 and 2.5 mg/ml) and processed through each extraction protocol for the evaluation of HA removal using QPCR and STR-genotyping. The DNA IQ magnetic bead system effectively removed HA from highly contaminated blood and saliva, and generated consistently acceptable STR profiles from both artificially spiked samples and crude soil samples. This system is highly recommended for use on soil-contaminated evidence over the cellulose card-based systems currently being preferentially used for DNA sample collection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

PubMed

Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

2014-11-01

DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

Absence of bacterial DNA in culture-negative urine from cats with and without lower urinary tract disease.

PubMed

Lund, Heidi Sjetne; Skogtun, Gaute; Sørum, Henning; Eggertsdóttir, Anna Vigdís

2015-10-01

A diagnosis of bacterial cystitis commonly relies on a positive microbiological culture demonstrating the presence of a significant number of colony-forming units/ml urine, as urine within the upper urinary tract, bladder and proximal urethra generally is considered sterile. Recent studies from human and veterinary medicine indicate the presence of non-culturable bacteria in culture-negative urine samples. The aim of the present study was to determine the occurrence of bacterial DNA in culture-negative urine samples from cats with signs of feline lower urinary tract disease (FLUTD) and healthy control cats by 16S ribosomal DNA PCR and subsequent sequencing. The study sample included 38 culture-negative urine samples from cats with FLUTD and 43 culture-negative samples from control cats. Eight culture-positive urine samples from cats with FLUTD were included as external positive controls in addition to negative reaction controls. Of possible methodological limitations, degradation of DNA due to storage, the use of non-sedimented urine for DNA isolation and lack of internal positive reaction controls should be mentioned. The positive controls were recognised, but occurrence of bacterial DNA in culture-negative urine from cats with or without signs of lower urinary tract disease was not demonstrated. However, considering the possible methodological limitations, the presence of bacterial DNA in the urine of culture-negative FLUTD cats cannot be excluded based on the present results alone. Therefore, a prospective study reducing the possibility of degradation of DNA due to storage, in combination with modifications enhancing the chance of detecting even lower levels of bacterial DNA in culture-negative samples, seems warranted. © ISFM and AAFP 2014.

Improving soil bacterial taxa–area relationships assessment using DNA meta-barcoding

PubMed Central

Terrat, S; Dequiedt, S; Horrigue, W; Lelievre, M; Cruaud, C; Saby, N P A; Jolivet, C; Arrouays, D; Maron, P-A; Ranjard, L; Chemidlin Prévost-Bouré, N

2015-01-01

The evaluation of the taxa–area relationship (TAR) with molecular fingerprinting data demonstrated the spatial structuration of soil microorganisms and provided insights into the processes shaping their diversity. The increasing use of massive sequencing technologies in biodiversity investigations has now raised the question of the advantages of such technologies over the fingerprinting approach for elucidation of the determinism of soil microbial community assembly in broad-scale biogeographic studies. Our objectives in this study were to compare DNA fingerprinting and meta-barcoding approaches for evaluating soil bacterial TAR and the determinism of soil bacterial community assembly on a broad scale. This comparison was performed on 392 soil samples from four French geographic regions with different levels of environmental heterogeneity. Both molecular approaches demonstrated a TAR with a significant slope but, because of its more sensitive description of soil bacterial community richness, meta-barcoding provided significantly higher and more accurate estimates of turnover rates. Both approaches were useful in evidencing the processes shaping bacterial diversity variations on a broad scale. When different taxonomic resolutions were considered for meta-barcoding data, they significantly influenced the estimation of turnover rates but not the relative importance of each component process. Altogether, DNA meta-barcoding provides a more accurate evaluation of the TAR and may lead to re-examination of the processes shaping soil bacterial community assembly. This should provide new insights into soil microbial ecology in the context of sustainable use of soil resources. PMID:25293875

Comparison of different methods for isolation of bacterial DNA from retail oyster tissues

USDA-ARS?s Scientific Manuscript database

Oysters are filter-feeders that bio-accumulate bacteria in water while feeding. To evaluate the bacterial genomic DNA extracted from retail oyster tissues, including the gills and digestive glands, four isolation methods were used. Genomic DNA extraction was performed using the Allmag™ Blood Genomic...

Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons.

PubMed Central

Cheetham, M E; Brion, J P; Anderton, B H

1992-01-01

The bacterial heat-shock protein DnaJ has been implicated in protein folding and protein complex dissociation. The DnaJ protein interacts with the prokaryotic analogue of Hsp70, DnaK, and accelerates the rate of ATP hydrolysis by DnaK. Several yeast homologues of DnaJ, with different proposed subcellular localizations and functions, have recently been isolated and are the only eukaryotic forms of DnaJ so far described. We have isolated cDNAs corresponding to two alternatively spliced transcripts of a novel human gene, HSJ1, which show sequence similarity to the bacterial DnaJ protein and the yeast homologues. The cDNA clones were isolated from a human brain-frontal-cortex expression library screened with a polyclonal antiserum raised to paired-helical-filament (PHF) proteins isolated from extracts of the brains of patients suffering from Alzheimer's disease. The similarity between the predicted human protein sequences and the bacterial and yeast proteins is highest at the N-termini, this region also shows a limited similarity to viral T-antigens and is a possible common motif involved in the interaction with DnaK/Hsp70. Northern-blot analysis has shown that human brain contains higher levels of mRNA for the DnaJ homologue than other tissues examined, and hybridization studies with riboprobes in situ show a restricted pattern of expression of the mRNA within the brain, with neuronal layers giving the strongest signal. These findings suggest that the DnaJ-DnaK (Hsp70) interaction is general to eukaryotes and, indeed, to higher organisms. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1599432

Human β‐defensin 3 increases the TLR9‐dependent response to bacterial DNA

PubMed Central

McGlasson, Sarah L.; Semple, Fiona; MacPherson, Heather; Gray, Mohini; Davidson, Donald J.

2017-01-01

Human β‐defensin 3 (hBD3) is a cationic antimicrobial peptide with potent bactericidal activity in vitro. HBD3 is produced in response to pathogen challenge and can modulate immune responses. The amplified recognition of self‐DNA by human plasmacytoid dendritic cells has been previously reported, but we show here that hBD3 preferentially enhances the response to bacterial DNA in mouse Flt‐3 induced dendritic cells (FLDCs) and in human peripheral blood mononuclear cells. We show the effect is mediated through TLR9 and although hBD3 significantly increases the cellular uptake of both E. coli and self‐DNA in mouse FLDCs, only the response to bacterial DNA is enhanced. Liposome transfection also increases uptake of bacterial DNA and amplifies the TLR9‐dependent response. In contrast to hBD3, lipofection of self‐DNA enhances inflammatory signaling, but the response is predominantly TLR9‐independent. Together, these data show that hBD3 has a role in the innate immune‐mediated response to pathogen DNA, increasing inflammatory signaling and promoting activation of the adaptive immune system via antigen presenting cells including dendritic cells. Therefore, our data identify an additional immunomodulatory role for this copy‐number variable defensin, of relevance to host defence against infection and indicate a potential for the inclusion of HBD3 in pathogen DNA‐based vaccines. PMID:28102569

DNA barcoding detects contamination and substitution in North American herbal products

PubMed Central

2013-01-01

Background Herbal products available to consumers in the marketplace may be contaminated or substituted with alternative plant species and fillers that are not listed on the labels. According to the World Health Organization, the adulteration of herbal products is a threat to consumer safety. Our research aimed to investigate herbal product integrity and authenticity with the goal of protecting consumers from health risks associated with product substitution and contamination. Methods We used DNA barcoding to conduct a blind test of the authenticity for (i) 44 herbal products representing 12 companies and 30 different species of herbs, and (ii) 50 leaf samples collected from 42 herbal species. Our laboratory also assembled the first standard reference material (SRM) herbal barcode library from 100 herbal species of known provenance that were used to identify the unknown herbal products and leaf samples. Results We recovered DNA barcodes from most herbal products (91%) and all leaf samples (100%), with 95% species resolution using a tiered approach (rbcL + ITS2). Most (59%) of the products tested contained DNA barcodes from plant species not listed on the labels. Although we were able to authenticate almost half (48%) of the products, one-third of these also contained contaminants and or fillers not listed on the label. Product substitution occurred in 30/44 of the products tested and only 2/12 companies had products without any substitution, contamination or fillers. Some of the contaminants we found pose serious health risks to consumers. Conclusions Most of the herbal products tested were of poor quality, including considerable product substitution, contamination and use of fillers. These activities dilute the effectiveness of otherwise useful remedies, lowering the perceived value of all related products because of a lack of consumer confidence in them. We suggest that the herbal industry should embrace DNA barcoding for authenticating herbal products through

Mitochondria and mitochondrial DNA as relevant targets for environmental contaminants.

PubMed

Roubicek, Deborah A; Souza-Pinto, Nadja C de

2017-11-01

The mitochondrial DNA (mtDNA) is a closed circular molecule that encodes, in humans, 13 polypeptides components of the oxidative phosphorylation complexes. Integrity of the mitochondrial genome is essential for mitochondrial function and cellular homeostasis, and mutations and deletions in the mtDNA lead to oxidative stress, mitochondrial dysfunction and cell death. In vitro and in situ studies suggest that when exposed to certain genotoxins, mtDNA accumulates more damage than nuclear DNA, likely owing to its organization and localization in the mitochondrial matrix, which tends to accumulate lipophilic, positively charged molecules. In that regard, several relevant environmental and occupational contaminants have physical-chemical characteristics that indicate that they might accumulate in mitochondria and target mtDNA. Nonetheless, very little is known so far about mtDNA damage and mitochondrial dysfunction due to environmental exposure, either in model organisms or in humans. In this article, we discuss some of the characteristics of mtDNA which render it a potentially relevant target for damage by environmental contaminants, as well as possible functional consequences of damage/mutation accumulation. In addition, we review the data available in the literature focusing on mitochondrial effects of the most common classes of environmental pollutants. From that, we conclude that several lines of experimental evidence support the idea that mitochondria and mtDNA are susceptible and biologically relevant targets for pollutants, and more studies, including mechanistic ones, are needed to shed more light into the contribution of mitochondrial dysfunction to the environmental and human health effects of chemical exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

Assessment of the bacterial contamination of hand air dryer in washrooms.

PubMed

Alharbi, Sulaiman Ali; Salmen, Saleh Hussein; Chinnathambi, Arunachalam; Alharbi, Naiyf S; Zayed, M E; Al-Johny, Bassam O; Wainwright, Milton

2016-03-01

The present study was carried out, using standard techniques, to identify and count the bacterial contamination of hand air dryers, used in washrooms. Bacteria were isolated from the air flow, outlet nozzle of warm air dryers in fifteen air dryers used in these washrooms. Bacteria were found to be relatively numerous in the air flows. Bacterially contaminated air was found to be emitted whenever a warm air dryer was running, even when not being used for hand drying. Our investigation shows that Staphylococcus haemolyticus, Micrococcus luteus, Pseudomonas alcaligenes, Bacillus cereus and Brevundimonad diminuta/vesicularis were emitted from all of the dryers sampled, with 95% showing evidence of the presence of the potential pathogen S. haemolyticus. It is concluded that hot air dryers can deposit pathogenic bacteria onto the hands and body of users. Bacteria are distributed into the general environment whenever dryers are running and could be inhaled by users and none-users alike. The results provide an evidence base for the development and enhancement of hygienic hand drying practices.

Assessment of the bacterial contamination of hand air dryer in washrooms

PubMed Central

Alharbi, Sulaiman Ali; Salmen, Saleh Hussein; Chinnathambi, Arunachalam; Alharbi, Naiyf S.; Zayed, M.E.; Al-Johny, Bassam O.; Wainwright, Milton

2015-01-01

The present study was carried out, using standard techniques, to identify and count the bacterial contamination of hand air dryers, used in washrooms. Bacteria were isolated from the air flow, outlet nozzle of warm air dryers in fifteen air dryers used in these washrooms. Bacteria were found to be relatively numerous in the air flows. Bacterially contaminated air was found to be emitted whenever a warm air dryer was running, even when not being used for hand drying. Our investigation shows that Staphylococcus haemolyticus, Micrococcus luteus, Pseudomonas alcaligenes, Bacillus cereus and Brevundimonad diminuta/vesicularis were emitted from all of the dryers sampled, with 95% showing evidence of the presence of the potential pathogen S. haemolyticus. It is concluded that hot air dryers can deposit pathogenic bacteria onto the hands and body of users. Bacteria are distributed into the general environment whenever dryers are running and could be inhaled by users and none-users alike. The results provide an evidence base for the development and enhancement of hygienic hand drying practices. PMID:26981009

Large-scale preparation of plasmid DNA.

PubMed

Heilig, J S; Elbing, K L; Brent, R

2001-05-01

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

Reducing bacterial contamination in an Orthopedic Theatre ventilated by natural ventilation, in a Developing Country.

PubMed

Thomas, Stephanie; Palmer, Rish; Phillipo, Edward; Chipungu, Geoffrey

2016-05-31

All surgical procedures have the potential for infection and some of the main sources are contamination from airborne particles, theatre personnel and the theatre environment.  There is strong evidence that the use of ultra-clean air flow systems in orthopedic operating theatres reduces the incidence of deep sepsis after surgery. In the developing world however, this is often an unrealistic solution. The aim of this study was to establish baseline levels of contamination in a working orthopedic theatre, at the Queen Elizabeth Central Hospital, Blantyre, Malawi. To feedback results to the theatre team, promote infection prevention discussion and work with the team to implement workable and realistic goals to improve the intra-operative environment. Samples were collected from theatre equipment available at the time of surgery, from theatre water and theatre air using passive air sampling techniques. Samples were immediately transferred to the Central Microbiology Laboratory for culture on basic culture media. Bacterial contamination of theatre equipment, intra-operative theatre air and water was detected. Results were discussed with the theatre and infection prevention team who were receptive to feedback with regards to infection prevention strategies and keen to develop simple measures which could be put in place to change practice. In this setting, we suggest that implementing workable and realistic goals such as, establishing baseline rates of bacterial contamination and introduction of strict protocols for asepsis and theatre etiquette, may reduce bacterial contamination rates and subsequent intra-operative infection in the absence of expensive engineering solutions.

Bioremediation and reclamation of soil contaminated with petroleum oil hydrocarbons by exogenously seeded bacterial consortium: a pilot-scale study.

PubMed

Mukherjee, Ashis K; Bordoloi, Naba K

2011-03-01

Spillage of petroleum hydrocarbons causes significant environmental pollution. Bioremediation is an effective process to remediate petroleum oil contaminant from the ecosystem. The aim of the present study was to reclaim a petroleum oil-contaminated soil which was unsuitable for the cultivation of crop plants by using petroleum oil hydrocarbon-degrading microbial consortium. Bacterial consortium consisting of Bacillus subtilis DM-04 and Pseudomonas aeruginosa M and NM strains were seeded to 20% (v/w) petroleum oil-contaminated soil, and bioremediation experiment was carried out for 180 days under laboratory condition. The kinetics of hydrocarbon degradation was analyzed using biochemical and gas chromatographic (GC) techniques. The ecotoxicity of the elutriates obtained from petroleum oil-contaminated soil before and post-treatment with microbial consortium was tested on germination and growth of Bengal gram (Cicer aretinum) and green gram (Phaseolus mungo) seeds. Bacterial consortium showed a significant reduction in total petroleum hydrocarbon level in contaminated soil (76% degradation) as compared to the control soil (3.6% degradation) 180 days post-inoculation. The GC analysis confirmed that bacterial consortium was more effective in degrading the alkane fraction compared to aromatic fraction of crude petroleum oil hydrocarbons in soil. The nitrogen, sulfur, and oxygen compounds fraction was least degraded. The reclaimed soil supported the germination and growth of crop plants (C. aretinum and P. mungo). In contrast, seeds could not be germinated in petroleum oil-contaminated soil. The present study reinforces the application of bacterial consortium rather than individual bacterium for the effective bioremediation and reclamation of soil contaminated with petroleum oil.

Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes.

PubMed

Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

2015-09-24

Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs) were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA) indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future.

Natural Bacterial Communities Serve as Quantitative Geochemical Biosensors

PubMed Central

Smith, Mark B.; Rocha, Andrea M.; Smillie, Chris S.; Olesen, Scott W.; Paradis, Charles; Wu, Liyou; Campbell, James H.; Fortney, Julian L.; Mehlhorn, Tonia L.; Lowe, Kenneth A.; Earles, Jennifer E.; Phillips, Jana; Joyner, Dominique C.; Elias, Dwayne A.; Bailey, Kathryn L.; Hurt, Richard A.; Preheim, Sarah P.; Sanders, Matthew C.; Yang, Joy; Mueller, Marcella A.; Brooks, Scott; Watson, David B.; Zhang, Ping; He, Zhili; Dubinsky, Eric A.; Adams, Paul D.; Arkin, Adam P.; Fields, Matthew W.; Zhou, Jizhong; Alm, Eric J.

2015-01-01

ABSTRACT Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. PMID:25968645

Evaluating the efficacy of bioremediating a diesel-contaminated soil using ecotoxicological and bacterial community indices.

PubMed

Khudur, Leadin Salah; Shahsavari, Esmaeil; Miranda, Ana F; Morrison, Paul D; Nugegoda, Dayanthi; Ball, Andrew S

2015-10-01

Diesel represents a common environmental contaminant as a result of operation, storage, and transportation accidents. The bioremediation of diesel in a contaminated soil is seen as an environmentally safe approach to treat contaminated land. The effectiveness of the remediation process is usually assessed by the degradation of the total petroleum hydrocarbon (TPH) concentration, without considering ecotoxicological effects. The aim of this study was to assess the efficacy of two bioremediation strategies in terms of reduction in TPH concentration together with ecotoxicity indices and changes in the bacterial diversity assessed using PCR-denaturing gradient gel electrophoresis (DGGE). The biostimulation strategy resulted in a 90 % reduction in the TPH concentration versus 78 % reduction from the natural attenuation strategy over 12 weeks incubation in a laboratory mesocosm-containing diesel-contaminated soil. In contrast, the reduction in the ecotoxicity resulting from the natural attenuation treatment using the Microtox and earthworm toxicity assays was more than double the reduction resulting from the biostimulation treatment (45 and 20 % reduction, respectively). The biostimulated treatment involved the addition of nitrogen and phosphorus in order to stimulate the microorganisms by creating an optimal C:N:P molar ratio. An increased concentration of ammonium and phosphate was detected in the biostimulated soil compared with the naturally attenuated samples before and after the remediation process. Furthermore, through PCR-DGGE, significant changes in the bacterial community were observed as a consequence of adding the nutrients together with the diesel (biostimulation), resulting in the formation of distinctly different bacterial communities in the soil subjected to the two strategies used in this study. These findings indicate the suitability of both bioremediation approaches in treating hydrocarbon-contaminated soil, particularly biostimulation. Although

Food safety in raw milk production: risk factors associated to bacterial DNA contamination.

PubMed

Cerva, Cristine; Bremm, Carolina; Reis, Emily Marques dos; Bezerra, André Vinícius Andrade; Loiko, Márcia Regina; Cruz, Cláudio Estêvão Farias da; Cenci, Alexander; Mayer, Fabiana Quoos

2014-06-01

While human illness from milkborne pathogens may be linked to contamination of the product after pasteurization or improper pasteurization, such diseases are usually associated with consumption of raw milk or its by-products. Molecular biology tools were applied to investigate contamination by Listeria monocytogenes, Salmonella spp., some pathogenic strains of Escherichia coli, and Campylobacter jejuni in 548 raw milk samples from 125 dairy farms established in two regions from southern Brazil. Moreover, 15 variables were evaluated for their association with raw milk contamination levels, and the risk factors were determined by multiple regression analysis. Salmonella spp. were more frequently detected, followed by pathogenic E. coli. There was difference in contamination index between the regions, in which risk factors such as temporary cattle confinement, low milk production, low milking machine cleaning frequency, and milk storage area without tile walls were identified. The risk factors were specific to each region studied. Nevertheless, the data can be used to improve milk quality of dairy farms/herds with similar management practices.

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

PubMed Central

Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

2011-01-01

Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA

Bacterial contamination of ultrasound probes in different radiological institutions before and after specific hygiene training: do we have a general hygienical problem?

PubMed

Sartoretti, Thomas; Sartoretti, Elisabeth; Bucher, Candid; Doert, Aleksis; Binkert, Christoph; Hergan, Klaus; Meissnitzer, Matthias; Froehlich, Johannes; Kolokythas, Orpheus; Matoori, Simon; Orasch, Christina; Kos, Sebastian; Sartoretti-Schefer, Sabine; Gutzeit, Andreas

2017-10-01

Aim was to investigate hygienic conditions of ultrasound probes before and after hygiene training in radiology institutions in comparison to bacterial contamination in public places. In three radiology departments, bacterial contamination was evaluated using baseline agar plates for cultures taken from 36 ultrasound probes. Afterwards teams were trained by a hygiene service centre and 36 ultrasound probes were routinely disinfected with regular disinfecting wipes and then evaluated. In comparison, bacterial contamination in public places (bus poles, n = 11; toilet seats, n = 10) were analysed. Plates were routinely incubated and the number of colony forming units (CFU) analysed. Cultures taken from the probes showed a median of 53 CFU before and 0 CFU after training (p < 0.001). Cultures taken from public places showed a median of 4 CFU from toilets and 28 from bus poles and had lower bacterial load in comparison to ultrasound probes before training (p = 0.055, toilets; p = 0.772, bus poles), without statistical significance. Bacterial contamination of ultrasound probes prior to hygiene training proved to be high and showed higher bacterial load than toilets seats or bus poles. Radiologists should be aware that the lack of hygiene in the field of ultrasound diagnostics puts patients at risk of healthcare-associated infections. • Hospital-associated infections are a problem for patient care. • Hygiene training of staff prevents bacterial contamination of ultrasound probes. • Disinfection of ultrasound probes is an easy method to protect patients.

Influence of hang time and location on bacterial contamination of intravenous bags in a veterinary emergency and critical care setting.

PubMed

Guillaumin, Julien; Olp, Nichole M; Magnusson, Karissa D; Butler, Amy L; Daniels, Joshua B

2017-09-01

To assess the rate of bacterial contamination of fluid and ports in intravenous bags in a veterinary emergency room (ER) and intensive care unit (ICU). Experimental model. Ninety intravenous fluid bags of lactated balanced-electrolytes solution (1 L) hung in a university hospital. Bags were hung in 2 different locations in the ER (sink and bins) and one location in the ICU (sink) for 11 days. Bags were punctured 3 times daily with a sterile needle to simulate clinical use. Injection ports were swabbed and 50 mL of fluid were collected in duplicates on days 0, 2, 4, 7, and 10. Aerobic bacterial cultures were performed on the fluid and injection port. Contamination was defined as bacterial growth of a similar phenotype across 2 consecutive times. Increase in the fluid contamination rate from day 0 was tested using an exact binomial test. Port contamination rate between locations was tested using Fisher's exact test. Combined bacterial growth on injection ports reached a mean (95% confidence interval) of 8.1 (0.005-16.2) cfu/port on day 10. The combined port contamination was 3.3%, 11.1%, 17.8%, and 31.1% on days 0, 2, 4, and 7, respectively. Port contamination was similar between ER and ICU. However, port contamination was higher in the sink versus the bins area (38.3% vs 16.7%, P = 0.032). No fluid bag was contaminated at days 0 and 2. The contamination rate of fluid bag was 1.1% and 4.4% on days 4 and 7, respectively. All bags with contaminated fluid were in the ER (6.7%, 95% exact binomial confidence interval 1.9-16.2%). Injection port contamination reached 31.1% on day 7. Contamination was more likely when the bags were hung next to a sink. In our model of bag puncture, fluid contamination occurred between days 2 and 4. © Veterinary Emergency and Critical Care Society 2017.

Health Threats from Contamination of Spices Commercialized in Romania: Risks of Fungal and Bacterial Infections.

PubMed

Man, Adrian; Mare, Anca; Toma, Felicia; Curticăpean, Augustin; Santacroce, Luigi

2016-01-01

The study of fungal contamination in food and mycotoxicoses is a priority today, both internationally and nationally. The purpose of this study is to have a general view over the quality of the most common spices that are sold in Romanian markets, by assessing the degree of fungal, bacterial and mycotoxin contamination in pepper and chili powders. We tested four types of spices: white pepper, black pepper, sweet and hot chili powders from 12 different distributing companies, summing a total of 35 sample types. The fungal and bacterial load was assessed by Standard Plate Count, while the mycotoxin content by High-performance liquid chromatography. Environmental conditions (humidity, pH) and the selling price for each product were also followed. Fungi were observed in 72.7% of black pepper samples, 33.3% in white pepper, 30% in sweet chili and 25% in hot chili products. The most common isolated fungus was Aspergillus spp., while Rhizopus, Mucor, Fusarium, Penicillium, Absidia species were found, in smaller percentage. Four producers (44.4%) presented fungal contamination of over 10^3 CFU/g and two producers (22.2%) presented no fungal contamination in their products. Bacterial contamination was found in 85.7% of the tested products, consisting mostly in Bacillus spp. Aflatoxin B1 was present in all the tested products, mostly in black pepper (mean value 126.3 ng/g); Ochratoxin A was present in sweet chili (mean value 328 ng/g) and Zearalenone in hot chili (mean value 604 ng/g) and sweet chili (mean value 382 ng/g). All spices presented either fungal contamination, mycotoxin contamination, or both. The high humidity and the high pH of spices represent favorable conditions for fungal growth. The selling price was partly related to the physic-chemical conditions and microbiological quality of the spices. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Prevalence of antibacterial resistant bacterial contaminants from mobile phones of hospital inpatients

PubMed Central

Vinod Kumar, B.; Hobani, Yahya Hasan; Abdulhaq, Ahmed; Jerah, Ahmed Ali; Hakami, Othman M.; Eltigani, Magdeldin; Bidwai, Anil K.

2014-01-01

Mobile phones contaminated with bacteria may act as fomites. Antibiotic resistant bacterial contamination of mobile phones of inpatients was studied. One hundred and six samples were collected from mobile phones of patients admitted in various hospitals in Jazan province of Saudi Arabia. Eighty-nine (83.9%) out of 106 mobile phones were found to be contaminated with bacteria. Fifty-two (49.0%) coagulase-negative Staphylococcus, 12 (11.3%) Staphylococcus aureus, 7 (6.6%) Enterobacter cloacae, 3 (2.83%) Pseudomonas stutzeri, 3 (2.83%) Sphingomonas paucimobilis, 2 (1.8%) Enterococcus faecalis and 10 (9.4%) aerobic spore bearers were isolated. All the isolated bacteria were found to be resistant to various antibiotics. Hence, regular disinfection of mobile phones of hospital inpatients is advised. PMID:25292217

Prevalence of antibacterial resistant bacterial contaminants from mobile phones of hospital inpatients.

PubMed

Kumar, B Vinod; Hobani, Yahya Hasan; Abdulhaq, Ahmed; Jerah, Ahmed Ali; Hakami, Othman M; Eltigani, Magdeldin; Bidwai, Anil K

2014-01-01

Mobile phones contaminated with bacteria may act as fomites. Antibiotic resistant bacterial contamination of mobile phones of inpatients was studied. One hundred and six samples were collected from mobile phones of patients admitted in various hospitals in Jazan province of Saudi Arabia. Eighty-nine (83.9%) out of 106 mobile phones were found to be contaminated with bacteria. Fifty-two (49.0%) coagulase-negative Staphylococcus, 12 (11.3%) Staphylococcus aureus, 7 (6.6%) Enterobacter cloacae, 3 (2.83%) Pseudomonas stutzeri, 3 (2.83%) Sphingomonas paucimobilis, 2 (1.8%) Enterococcus faecalis and 10 (9.4%) aerobic spore bearers were isolated. All the isolated bacteria were found to be resistant to various antibiotics. Hence, regular disinfection of mobile phones of hospital inpatients is advised.

Identification of bacterial contaminants from calcium carbonate filler production lines and an evaluation of biocide based decontamination procedures.

PubMed

Odić, Duško; Prah, Jana; Avguštin, Gorazd

2017-04-01

The aim of this study was to analyze the bacterial community in the production line of a calcium carbonate filler production company and to investigate possible causes for bacterial presence. Throughout 2012, 24 carbonate slurry and six groundwater samples were analyzed. Pseudomonas and Microbacterium were the most frequent contaminants in the slurry, whereas Pseudomonas and Brevundimonas dominated the groundwater samples. Of the 43 different bacterial strains isolated, only five were found both in the slurry and the groundwater, indicating that the latter was not a major source of contamination. The efficacy of 54 commercial biocidal formulations was tested against an artificial bacterial consortium composed of selected slurry isolates. A formulation containing 7.5-15% (v v -1 ) bronopol and 1.0-2.5% (v v -1 ) [chloroisothiazolinone (CIT) + methylisothiazolinone (MIT)] exhibited the highest efficacy. Of the possible causes for bacterial presence, sporogenesis and biocide adsorption to carbonate particles were found to be less probable compared to bacterial adsorption to particles, and the acquisition of resistance to biocides.

Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

NASA Astrophysics Data System (ADS)

Novianty, E.; Kartikasari, L. R.; Lee, J. H.; Cahyadi, M.

2017-04-01

Meat based food products have a big opportunity to mix and adulterated with other meats. Muslim communities are prohibited to consume pork-containing product or other pig derivatives in food. Therefore, the high sensitivity, fast, cheap and accurate approach is needed to detect pig contamination in raw meat and meat-processed product such as meatball. The aim of this study was to identify pork contamination in meatball using genetic marker of mitochondrial DNA cytochrome b gene by duplex-PCR. Samples were prepared and designed by following the proportions 0, 1, 5, 10, 25% of pork in meatballs, respectively. The DNA genome was extracted from meatballs and polymerase chain reaction (PCR) was performed using species specific primer to isolate mt-DNA cytochrome b gene. The results showed that the DNA genome was successfully isolated from pork, beef, and contaminated meatballs. Furthermore, 2% agarose gels was able to visualize of duplex-PCR to identify pork contamination in meatballs up to very small proportion (1%). It can be concluded that duplex-PCR of mt-DNA cytochrome b gene was very sensitive to identify pork contamination in meatball with the presence of specific 398 bp DNA band.

Bacterial contamination of stethoscopes used by health workers: public health implications.

PubMed

Uneke, Chigozie J; Ogbonna, Annayo; Oyibo, Patrick G; Onu, Christian M

2010-08-04

This study was designed to assess both the potential for bacterial transmission by stethoscopes used by health-care workers in Nigeria and the implications for patient safety and control of hospital-acquired infections. A structured questionnaire was administered to health workers and the surface of the diaphragm of their stethoscopes swabbed for bacteriological analysis using standard techniques. Of the 107 stethoscopes surveyed, 84 (79%) were contaminated with bacteria; 59 (81%) of the contaminated stethoscopes belonged to physicians and 25 (74%) were from other health workers. Isolates included Staphylococcus aureus (54%), Pseudomonas aeruginosa (19%), Enterococcus faecalis (14%), and Escherichia coli (13%). All stethoscopes that had never been cleaned were contaminated while lower levels of contamination were found on those cleaned one week or less before the survey (chi(2) = 22.4, P < .05). Contamination was significantly higher on stethoscopes cleaned with only water (100%) compared to those cleaned with alcohol (49%) (chi(2) = 30.17, P < .05). Significantly fewer (9%) stethoscopes from health workers who washed their hands after seeing each patient were contaminated when compared with the instruments (86%) of those who did not practice hand washing (chi(2) = 23.79, P < .05). E. coli showed the highest antibiotic resistance, while S. aureus showed the highest antibiotic susceptibility. Strict adherence to stethoscope disinfection practices by health workers can minimize cross-contamination and ensure improved patient safety in hospital environments.

DNA-sensing inflammasomes: regulation of bacterial host defense and the gut microbiota.

PubMed

Man, Si Ming; Karki, Rajendra; Kanneganti, Thirumala-Devi

2016-06-01

DNA sensors are formidable immune guardians of the host. At least 14 cytoplasmic DNA sensors have been identified in recent years, each with specialized roles in driving inflammation and/or cell death. Of these, AIM2 is a sensor of dsDNA, and forms an inflammasome complex to activate the cysteine protease caspase-1, mediates the release of the proinflammatory cytokines IL-1β and IL-18, and induces pyroptosis. The inflammasome sensor NLRP3 can also respond to DNA in the forms of oxidized mitochondrial DNA and the DNA derivative RNA:DNA hybrids produced by bacteria, whereas the putative inflammasome sensor IFI16 responds to viral DNA in the nucleus. Although inflammasomes provoke inflammation for anti-microbial host defense, they must also maintain homeostasis with commensal microbiota. Here, we outline recent advances highlighting the complex relationship between DNA-sensing inflammasomes, bacterial host defense and the gut microbiota. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Linkage between bacterial and fungal rhizosphere communities in hydrocarbon-contaminated soils is related to plant phylogeny

PubMed Central

Bell, Terrence H; El-Din Hassan, Saad; Lauron-Moreau, Aurélien; Al-Otaibi, Fahad; Hijri, Mohamed; Yergeau, Etienne; St-Arnaud, Marc

2014-01-01

Phytoremediation is an attractive alternative to excavating and chemically treating contaminated soils. Certain plants can directly bioremediate by sequestering and/or transforming pollutants, but plants may also enhance bioremediation by promoting contaminant-degrading microorganisms in soils. In this study, we used high-throughput sequencing of bacterial 16S rRNA genes and the fungal internal transcribed spacer (ITS) region to compare the community composition of 66 soil samples from the rhizosphere of planted willows (Salix spp.) and six unplanted control samples at the site of a former petrochemical plant. The Bray–Curtis distance between bacterial communities across willow cultivars was significantly correlated with the distance between fungal communities in uncontaminated and moderately contaminated soils but not in highly contaminated (HC) soils (>2000 mg kg−1 hydrocarbons). The mean dissimilarity between fungal, but not bacterial, communities from the rhizosphere of different cultivars increased substantially in the HC blocks. This divergence was partly related to high fungal sensitivity to hydrocarbon contaminants, as demonstrated by reduced Shannon diversity, but also to a stronger influence of willows on fungal communities. Abundance of the fungal class Pezizomycetes in HC soils was directly related to willow phylogeny, with Pezizomycetes dominating the rhizosphere of a monophyletic cluster of cultivars, while remaining in low relative abundance in other soils. This has implications for plant selection in phytoremediation, as fungal associations may affect the health of introduced plants and the success of co-inoculated microbial strains. An integrated understanding of the relationships between fungi, bacteria and plants will enable the design of treatments that specifically promote effective bioremediating communities. PMID:23985744

Use of on-section immunolabeling and cryosubstitution for studies of bacterial DNA distribution.

PubMed Central

Hobot, J A; Bjornsti, M A; Kellenberger, E

1987-01-01

Escherichia coli cells were very rapidly frozen and substituted at a low temperature with 3% glutaraldehyde in acetone. Infiltration and embedding with Lowicryl K4M were carried out at -35 degrees C. This procedure resulted in good structural preservation of both the nucleoid morphology and its DNA plasm, such that immunolabeling with the protein-A gold technique could be carried out. With antibodies specific for either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), it was shown that dsDNA was present throughout the nucleoid but that ssDNA was located on the nucleoid periphery. Chloramphenicol-treated cells, in which protein synthesis but not DNA replication is stopped, produced a characteristic ringlike nucleoid shape and had both dsDNA and ssDNA present throughout the annular section of the DNA plasm. The relationship between metabolically active DNA and overall bacterial genome organization is discussed. Images PMID:3553155

Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis).

PubMed

Nilsen, Frances M; Parrott, Benjamin B; Bowden, John A; Kassim, Brittany L; Somerville, Stephen E; Bryan, Teresa A; Bryan, Colleen E; Lange, Ted R; Delaney, J Patrick; Brunell, Arnold M; Long, Stephen E; Guillette, Louis J

2016-03-01

Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida's north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different "treatments" of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics. Published by Elsevier B.V.

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.

Dynamic changes of bacterial community under bioremediation with Sphingobium sp. LY-6 in buprofezin-contaminated soil.

PubMed

Liu, Yuan; Hou, Qianqian; Liu, Wanru; Meng, Yawen; Wang, Guangli

2015-08-01

Buprofezin is a commonly used chemical with satisfactory biological activity against sucking insect pests, but its disposal can cause serious environmental problems. To study the feasibility of remedying contamination by buprofezin, microcosm experiments were carried out to study the effects of various concentrations of buprofezin and Sphingobium sp. LY-6 on soil bacterial communities in soils collected from vegetable fields. In this experiment, the results showed that buprofezin was effectively degraded by Sphingobium sp. LY-6 in incubation soils. Comparing to non-incubated soils, the cumulative degradation ratio of buprofezin was significantly increased, up to the extent of 85 and 51%, in the initial concentration of 10 and 100 mg kg(-1). The abundance and community structure of the bacterial communities were analysed by real-time PCR (qPCR) and terminal-restriction fragment length polymorphism (T-RFLP). The findings suggest that buprofezin had a negative effect on soil bacterial community, and decreases in bacterial abundance were observed in the later part of the incubation period. The bacterial community structure and diversity shifted significantly at each sampling time. In conclusion, the buprofezin-degrading strain LY-6 played a major role in the bioremediation of the buprofezin-contaminated soil and influenced the dynamics and structure of the bacterial community, demonstrating the great potential of exogenous microorganisms for soil remediation.

High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

PubMed

Inoue, Daisuke; Hinoura, Takuji; Suzuki, Noriko; Pang, Junqin; Malla, Rabin; Shrestha, Sadhana; Chapagain, Saroj Kumar; Matsuzawa, Hiroaki; Nakamura, Takashi; Tanaka, Yasuhiro; Ike, Michihiko; Nishida, Kei; Sei, Kazunari

2015-01-01

Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1-37 pathogen species/groups, including 1-27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

Synergistic Processing of Biphenyl and Benzoate: Carbon Flow Through the Bacterial Community in Polychlorinated-Biphenyl-Contaminated Soil

NASA Astrophysics Data System (ADS)

Leewis, Mary-Cathrine; Uhlik, Ondrej; Leigh, Mary Beth

2016-02-01

Aerobic mineralization of PCBs, which are toxic and persistent organic pollutants, involves the upper (biphenyl, BP) and lower (benzoate, BZ) degradation pathways. The activity of different members of the soil microbial community in performing one or both pathways, and their synergistic interactions during PCB biodegradation, are not well understood. This study investigates BP and BZ biodegradation and subsequent carbon flow through the microbial community in PCB-contaminated soil. DNA stable isotope probing (SIP) was used to identify the bacterial guilds involved in utilizing 13C-biphenyl (unchlorinated analogue of PCBs) and/or 13C-benzoate (product/intermediate of BP degradation and analogue of chlorobenzoates). By performing SIP with two substrates in parallel, we reveal microbes performing the upper (BP) and/or lower (BZ) degradation pathways, and heterotrophic bacteria involved indirectly in processing carbon derived from these substrates (i.e. through crossfeeding). Substrate mineralization rates and shifts in relative abundance of labeled taxa suggest that BP and BZ biotransformations were performed by microorganisms with different growth strategies: BZ-associated bacteria were fast growing, potentially copiotrophic organisms, while microbes that transform BP were oligotrophic, slower growing, organisms. Our findings provide novel insight into the functional interactions of soil bacteria active in processing biphenyl and related aromatic compounds in soil, revealing how carbon flows through a bacterial community.

The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

NASA Astrophysics Data System (ADS)

Roos, C.; Santos, J. N.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

2013-07-01

Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm-2) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms.

Effects of ultrasonic pretreatment on quantity and composition of bacterial DNA recovered from granular activated carbon used for drinking water treatment.

PubMed

Kim, Tae Gwan; Kim, Sun-Hye; Cho, Kyung-Suk

2014-01-01

Effects of ultrasonic pretreatment on bacterial DNA recovery from granular activated carbon (GAC) were investigated. GAC (Calgon F400), biologically activated, was sampled from an actual drinking water plant. Different ultrasonic energy densities (0-400 J·cm(-3)) were applied with agitation (250 rpm for 30 min), and recovered bacterial DNA was quantified using quantitative PCR. Energy density was linearly correlated with the concentration of carbon fines produced from GAC during ultrasonication. Ultrasonication alone had no effect on DNA recovery at ≤60 J·cm(-3), but a strongly adverse effect at >67 J·cm(-3) due to the produced carbon fines. Agitation along with ultrasonication strongly enhanced the bacterial DNA recovery when ≤40 J·cm(-3) was applied, although it did not affect the production of carbon fines. Ribosomal tag pyrosequencing was used to compare recovered bacterial communities (0, 20 and 30 J·cm(-3) with or without agitation). Ultrasonication allowed for obtaining a more diverse and richer bacterial community from GAC, compared with the control. Agitation did not show a positive effect on community organization (richness and diversity). Consistently, canonical correspondence analysis indicated that the energy density was associated with the relative abundances of particular bacterial members (P < 0.05), while agitation did not. Correspondence analysis revealed that the recovered bacterial communities were grouped according to the applied energy densities. In conclusion, ultrasonication and agitation influence the recovered DNA in quality and quantity, respectively, and carbon fines as a by-product by ultrasonication interfere with the DNA recovery.

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

PubMed

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-09-18

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

Reducing DNA context dependence in bacterial promoters

PubMed Central

Carr, Swati B.; Densmore, Douglas M.

2017-01-01

Variation in the DNA sequence upstream of bacterial promoters is known to affect the expression levels of the products they regulate, sometimes dramatically. While neutral synthetic insulator sequences have been found to buffer promoters from upstream DNA context, there are no established methods for designing effective insulator sequences with predictable effects on expression levels. We address this problem with Degenerate Insulation Screening (DIS), a novel method based on a randomized 36-nucleotide insulator library and a simple, high-throughput, flow-cytometry-based screen that randomly samples from a library of 436 potential insulated promoters. The results of this screen can then be compared against a reference uninsulated device to select a set of insulated promoters providing a precise level of expression. We verify this method by insulating the constitutive, inducible, and repressible promotors of a four transcriptional-unit inverter (NOT-gate) circuit, finding both that order dependence is largely eliminated by insulation and that circuit performance is also significantly improved, with a 5.8-fold mean improvement in on/off ratio. PMID:28422998

Assessment of bacterial superficial contamination in classical or ritually slaughtered cattle using metagenetics and microbiological analysis.

PubMed

Korsak, N; Taminiau, B; Hupperts, C; Delhalle, L; Nezer, C; Delcenserie, V; Daube, G

2017-04-17

The aim of this study was to investigate the influence of the slaughter technique (Halal vs Classical slaughter) on the superficial contamination of cattle carcasses, by using traditional microbiological procedures and 16S rDNA metagenetics. The purpose was also to investigate the neck area to identify bacteria originating from the digestive or the respiratory tract. Twenty bovine carcasses (10 from each group) were swabbed at the slaughterhouse, where both slaughtering methods are practiced. Two swabbing areas were chosen: one "legal" zone of 1600cm 2 (composed of zones from rump, flank, brisket and forelimb) and locally on the neck area (200cm 2 ). Samples were submitted to classical microbiology for aerobic Total Viable Counts (TVC) at 30°C and Enterobacteriaceae counts, while metagenetic analysis was performed on the same samples. The classical microbiological results revealed no significant differences between both slaughtering practices; with values between 3.95 and 4.87log CFU/100cm 2 and 0.49 and 1.94log CFU/100cm 2 , for TVC and Enterobacteriaceae respectively. Analysis of pyrosequencing data showed that differences in the bacterial population abundance between slaughtering methods were mainly observed in the "legal" swabbing zone compared to the neck area. Bacterial genera belonging to the Actinobacteria phylum were more abundant in the "legal" swabbing zone in "Halal" samples, while Brevibacterium and Corynebacterium were encountered more in "Halal" samples, in all swabbing areas. This was also the case for Firmicutes bacterial populations (families of Aerococcaceae, Planococcaceae). Except for Planococcoceae, the analysis of Operational Taxonomic Unit (OTU) abundances of bacteria from the digestive or respiratory tract revealed no differences between groups. In conclusion, the slaughtering method does not influence the superficial microbiological pattern in terms of specific microbiological markers of the digestive or respiratory tract. However

Comparison of bacterial DNA profiles of footwear insoles and soles of feet for the forensic discrimination of footwear owners.

PubMed

Goga, Haruhisa

2012-09-01

It is crucial to identify the owner of unattended footwear left at a crime scene. However, retrieving enough DNA for DNA profiling from the owner's foot skin (plantar skin) cells from inside the footwear is often unsuccessful. This is sometimes because footwear that is used on a daily basis contains an abundance of bacteria that degrade DNA. Further, numerous other factors related to the inside of the shoe, such as high humidity and temperature, can encourage bacterial growth inside the footwear and enhance DNA degradation. This project sought to determine if bacteria from inside footwear could be used for footwear trace evidence. The plantar skins and insoles of shoes of volunteers were swabbed for bacteria, and their bacterial community profiles were compared using bacterial 16S rRNA terminal restriction fragment length polymorphism analysis. Sufficient bacteria were recovered from both footwear insoles and the plantar skins of the volunteers. The profiling identified that each volunteer's plantar skins harbored unique bacterial communities, as did the individuals' footwear insoles. In most cases, a significant similarity in the bacterial community was identified for the matched foot/insole swabs from each volunteer, as compared with those profiles from different volunteers. These observations indicate the probability to discriminate the owner of footwear by comparing the microbial DNA fingerprint from inside footwear with that of the skin from the soles of the feet of the suspected owner. This novel strategy will offer auxiliary forensic footwear evidence for human DNA identification, although further investigations into this technique are required.

Human Mitochondrial DNA and Endogenous Bacterial Surrogates for Risk Assessment of Graywater Reuse

EPA Science Inventory

Previous graywater risk assessment studies have focused on fecal contamination, yet the low density of fecal indicators may not provide the most useful approach to assess pathogen removal during graywater treatment. In this study, we employed high throughput bacterial sequencing ...

Bacterial community composition characterization of a lead-contaminated Microcoleus sp. consortium.

PubMed

Giloteaux, Ludovic; Solé, Antoni; Esteve, Isabel; Duran, Robert

2011-08-01

A Microcoleus sp. consortium, obtained from the Ebro delta microbial mat, was maintained under different conditions including uncontaminated, lead-contaminated, and acidic conditions. Terminal restriction fragment length polymorphism and 16S rRNA gene library analyses were performed in order to determine the effect of lead and culture conditions on the Microcoleus sp. consortium. The bacterial composition inside the consortium revealed low diversity and the presence of specific terminal-restriction fragments under lead conditions. 16S rRNA gene library analyses showed that members of the consortium were affiliated to the Alpha, Beta, and Gammaproteobacteria and Cyanobacteria. Sequences closely related to Achromobacter spp., Alcaligenes faecalis, and Thiobacillus species were exclusively found under lead conditions while sequences related to Geitlerinema sp., a cyanobacterium belonging to the Oscillatoriales, were not found in presence of lead. This result showed a strong lead selection of the bacterial members present in the Microcoleus sp. consortium. Several of the 16S rRNA sequences were affiliated to nitrogen-fixing microorganisms including members of the Rhizobiaceae and the Sphingomonadaceae. Additionally, confocal laser scanning microscopy and scanning and transmission electron microscopy showed that under lead-contaminated condition Microcoleus sp. cells were grouped and the number of electrodense intracytoplasmic inclusions was increased.

Near-infrared spectroscopy for the detection and quantification of bacterial contaminations in pharmaceutical products.

PubMed

Quintelas, Cristina; Mesquita, Daniela P; Lopes, João A; Ferreira, Eugénio C; Sousa, Clara

2015-08-15

Accurate detection and quantification of microbiological contaminations remains an issue mainly due the lack of rapid and precise analytical techniques. Standard methods are expensive and time-consuming being associated to high economic losses and public health threats. In the context of pharmaceutical industry, the development of fast analytical techniques able to overcome these limitations is crucial and spectroscopic techniques might constitute a reliable alternative. In this work we proved the ability of Fourier transform near infrared spectroscopy (FT-NIRS) to detect and quantify bacteria (Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, Salmonella enterica, Staphylococcus epidermidis) from 10 to 10(8) CFUs/mL in sterile saline solutions (NaCl 0.9%). Partial least squares discriminant analysis (PLSDA) models showed that FT-NIRS was able to discriminate between sterile and contaminated solutions for all bacteria as well as to identify the contaminant bacteria. Partial least squares (PLS) models allowed bacterial quantification with limits of detection ranging from 5.1 to 9 CFU/mL for E. coli and B. subtilis, respectively. This methodology was successfully validated in three pharmaceutical preparations (contact lens solution, cough syrup and topic anti-inflammatory solution) proving that this technique possess a high potential to be routinely used for the detection and quantification of bacterial contaminations. Copyright © 2015 Elsevier B.V. All rights reserved.

Inexpensive metagenomic DNA extraction protocol with high quality from marine sediments contaminated by petroleum hydrocarbons.

PubMed

García-Bautista, I; Toledano-Thompson, T; Dantán-González, E; González-Montilla, J; Valdez-Ojeda, R

2017-09-21

Marine environments are a reservoir of relevant information on dangerous contaminants such as hydrocarbons, as well as microbial communities with probable degradation skills. However, to access microbial diversity, it is necessary to obtain high-quality DNA. An inexpensive, reliable, and effective metagenomic DNA (mgDNA) extraction protocol from marine sediments contaminated with petroleum hydrocarbons was established in this study from modifications to Zhou's protocol. The optimization included pretreatment of sediment with saline solutions for the removal of contaminants, a second precipitation and enzymatic degradation of RNA, followed by purification of mgDNA extracted by electroelution. The results obtained indicated that the modifications applied to 12 sediments with total petroleum hydrocarbon (TPH) concentrations from 22.6-174.3 (µg/g dry sediment) yielded 20.3-321.3 ng/µL mgDNA with A 260 /A 280 and A 260 /A 230 ratios of 1.75 ± 0.08 and 1.19 ± 0.22, respectively. The 16S rRNA amplification confirmed the purity of the mgDNA. The suitability of this mgDNA extraction protocol lies in the fact that all chemical solutions utilized are common in all molecular biology laboratories, and the use of dialysis membrane does not require any sophisticated or expensive equipment, only an electrophoretic chamber.

The action of the bacterial toxin, microcin B17, on DNA gyrase.

PubMed

Parks, William M; Bottrill, Andrew R; Pierrat, Olivier A; Durrant, Marcus C; Maxwell, Anthony

2007-04-01

Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.

Potential bacterial contamination of eyedrops used for tonometry.

PubMed

Palmberg, R; Gutierrez, Y S; Miller, D; Feuer, W J; Anderson, D R

1994-05-15

We compared the potential for bacterial contamination of a proparacaine hydrochloride solution preserved with benzalkonium chloride, which is used with fluorescein paper for tonometry, to that of a fluorescein-benoxinate hydrochloride combination solution preserved with chlorobutanol. We contaminated bottles of each solution with Pseudomonas aeruginosa or Staphylococcus aureus (10(7) organisms per milliliter of eyedrop solution). From the fluorescein-benoxinate hydrochloride solution, Staphylococcus organisms were cultured in declining numbers over time, with a half-life of nine seconds, and no Staphylococcus organisms were recovered after five minutes. Pseudomonas organisms disappeared from this solution within 15 seconds. Neither species was viable after 15 seconds in the proparacaine hydrochloride solution. Additionally, we cultured 12 bottles of fluorescein-benoxinate hydrochloride and 15 bottles of proparacaine hydrochloride that had been in use in the clinic for one month. No organisms were recovered from a drop dispensed from any bottle or from the tips, caps, rims, or solution from within any bottle, except that five colonies of Staphylococcus grew from the external rim of one bottle. We conclude that both solutions sterilize themselves rapidly and effectively. Either may be used safely for tonometry.

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

PubMed

Maye, Peter; Stover, Mary Louise; Liu, Yaling; Rowe, David W; Gong, Shiaochin; Lichtler, Alexander C

2009-03-13

Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

Generation of Nanobodies against SlyD and development of tools to eliminate this bacterial contaminant from recombinant proteins.

PubMed

Hu, Yaozhong; Romão, Ema; Vertommen, Didier; Vincke, Cécile; Morales-Yánez, Francisco; Gutiérrez, Carlos; Liu, Changxiao; Muyldermans, Serge

2017-09-01

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis-trans isomerase (SlyD) from E. coli. This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. Copyright © 2017 Elsevier Inc. All rights reserved.

IDENTIFICATION OF BACTERIAL DNA MARKERS FOR THE DETECTION OF HUMAN FECAL POLLUTION IN WATER

EPA Science Inventory

We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human a...

Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

DOE PAGES

Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; ...

2015-04-30

The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy numbermore » of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.« less

Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

PubMed Central

Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Taş, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

2015-01-01

The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below −10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

DNA-labeled micro- and nanoparticles: a new approach to study contaminant transport in the subsurface

NASA Astrophysics Data System (ADS)

McNew, C.; Wang, C.; Kocis, T. N.; Murphy, N. P.; Dahlke, H. E.

2017-12-01

Though our understanding of contaminant behavior in the subsurface has improved, our ability to measure and predict complex contaminant transport pathways at hillslope to watershed scales is still lacking. By utilizing bio-molecular nanotechnology developed for nano-medicines and drug delivery, we are able to produce DNA-labeled micro- and nanoparticles for use in a myriad of environmental systems. Control of the fabrication procedure allows us to produce particles of custom size, charge, and surface functionality to mimic the transport properties of the particulate contaminant or colloid of interest. The use of custom sequenced DNA allows for the fabrication of an enormous number of unique particle labels (approximately 1.61 x 1060 unique sequences) and the ability to discern between varied spatial and temporal applications, or the transport effect of varied particle size, charge, or surface properties. To date, this technology has been utilized to study contaminant transport from lab to field scales, including surface and open channel flow applications, transport in porous media, soil retention, and even subglacial flow pathways. Here, we present the technology for production and detection of the DNA-labeled particles along with the results from a current hillslope study at the Sierra Foothills Research and Extension Center (SFREC). This field study utilizes spatial and temporal variations in DNA-labeled particle applications to identify subsurface pollutant transport pathways through the four distinct soil horizons present at the SFREC site. Results from this and previous studies highlight the tremendous potential of the DNA-labeled particle technology for studying contaminant transport through the subsurface.

Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities

PubMed Central

Biswas, Kristi; Taylor, Michael W.; Gear, Kim

2017-01-01

The application of high-throughput, next-generation sequencing technologies has greatly improved our understanding of the human oral microbiome. While deciphering this diverse microbial community using such approaches is more accurate than traditional culture-based methods, experimental bias introduced during critical steps such as DNA extraction may compromise the results obtained. Here, we systematically evaluate four commonly used microbial DNA extraction methods (MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Mini Kit, Zymo Bacterial/Fungal DNA Mini PrepTM, phenol:chloroform-based DNA isolation) based on the following criteria: DNA quality and yield, and microbial community structure based on Illumina amplicon sequencing of the V3–V4 region of the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) 1 region of fungi. Our results indicate that DNA quality and yield varied significantly with DNA extraction method. Representation of bacterial genera in plaque and saliva samples did not significantly differ across DNA extraction methods and DNA extraction method showed no effect on the recovery of fungal genera from plaque. By contrast, fungal diversity from saliva was affected by DNA extraction method, suggesting that not all protocols are suitable to study the salivary mycobiome. PMID:28099455

Denitrifying Bacteria from the Genus Rhodanobacter Dominate Bacterial Communities in the Highly Contaminated Subsurface of a Nuclear Legacy Waste Site

PubMed Central

Green, Stefan J.; Prakash, Om; Jasrotia, Puja; Overholt, Will A.; Cardenas, Erick; Hubbard, Daniela; Tiedje, James M.; Watson, David B.; Schadt, Christopher W.; Brooks, Scott C.

2012-01-01

The effect of long-term mixed-waste contamination, particularly uranium and nitrate, on the microbial community in the terrestrial subsurface was investigated at the field scale at the Oak Ridge Integrated Field Research Challenge (ORIFRC) site in Oak Ridge, TN. The abundance, community composition, and distribution of groundwater microorganisms were examined across the site during two seasonal sampling events. At representative locations, subsurface sediment was also examined from two boreholes, one sampled from the most heavily contaminated area of the site and another from an area with low contamination. A suite of DNA- and RNA-based molecular tools were employed for community characterization, including quantitative PCR of rRNA and nitrite reductase genes, community composition fingerprinting analysis, and high-throughput pyrotag sequencing of rRNA genes. The results demonstrate that pH is a major driver of the subsurface microbial community structure and that denitrifying bacteria from the genus Rhodanobacter (class Gammaproteobacteria) dominate at low pH. The relative abundance of bacteria from this genus was positively correlated with lower-pH conditions, and these bacteria were abundant and active in the most highly contaminated areas. Other factors, such as the concentration of nitrogen species, oxygen level, and sampling season, did not appear to strongly influence the distribution of Rhodanobacter bacteria. The results indicate that these organisms are acid-tolerant denitrifiers, well suited to the acidic, nitrate-rich subsurface conditions, and pH is confirmed as a dominant driver of bacterial community structure in this contaminated subsurface environment. PMID:22179233

Pyrosequencing analysis of bacterial diversity in soils contaminated long-term with PAHs and heavy metals: Implications to bioremediation.

PubMed

Kuppusamy, Saranya; Thavamani, Palanisami; Megharaj, Mallavarapu; Venkateswarlu, Kadiyala; Lee, Yong Bok; Naidu, Ravi

2016-11-05

Diversity, distribution and composition of bacterial community of soils contaminated long-term with both polycyclic aromatic hydrocarbons (PAHs) and heavy metals were explored for the first time following 454 pyrosequencing. Strikingly, the complete picture of the Gram positive (+ve) and Gram negative (-ve) bacterial profile obtained in our study illustrates novel postulates that include: (1) Metal-tolerant and PAH-degrading Gram -ves belonging to the class Alphaproteobacteria persist relatively more in the real contaminated sites compared to Gram +ves, (2) Gram +ves are not always resistant to heavy metal toxicity, (3) Stenotrophomonas followed by Burkholderia and Pseudomonas are the dominant genera of PAH degraders with high metabolic activity in long-term contaminated soils, (4) Actinobacteria is the predominant group among the Gram +ves in soils contaminated with high molecular weight PAHs that co-exist with toxic heavy metals like Pb, Cu and Zn, (5) Microbial communities are nutrient-driven in natural environments and (6) Catabolically potential Gram +/-ves with diverse applicability to remediate the real contaminated sites evolve eventually in the historically-polluted soils. Thus, the most promising indigenous Gram +/-ve strains from the long-term contaminated sites with increased catabolic potential, enzymatic activity and metal tolerance need to be harnessed for mixed contaminant cleanups. Copyright © 2016 Elsevier B.V. All rights reserved.

Three-step Channel Conformational Changes Common to DNA Packaging Motors of Bacterial Viruses T3, T4, SPP1, and Phi29

PubMed Central

Wang, Shaoying; Ji, Zhouxiang; Yan, Erfu; Haque, Farzin; Guo, Peixuan

2016-01-01

The DNA packaging motor of dsDNA bacterial viruses contains a head-tail connector with a channel for genome to enter during assembly and to exit during host infection. The DNA packaging motor of bacterial virus phi29 was recently reported to use the “One-way Revolution” mechanism for DNA packaging. This raises a question of how dsDNA is ejected during infection if the channel acts as a one-way inward valve. Here we report a three step conformational change of the portal channel that is common among DNA translocation motors of bacterial viruses T3, T4, SPP1, and phi29. The channels of these motors exercise three discrete steps of gating, as revealed by electrophysiological assays. It is proposed that the three step channel conformational changes occur during DNA entry process, resulting in a structural transition in preparation of DNA movement in the reverse direction during ejection. PMID:27181501

Development of an Efficient Bacterial Consortium for the Potential Remediation of Hydrocarbons from Contaminated Sites

PubMed Central

Patowary, Kaustuvmani; Patowary, Rupshikha; Kalita, Mohan C.; Deka, Suresh

2016-01-01

The intrinsic biodegradability of hydrocarbons and the distribution of proficient degrading microorganisms in the environment are very crucial for the implementation of bioremediation practices. Among others, one of the most favorable methods that can enhance the effectiveness of bioremediation of hydrocarbon-contaminated environment is the application of biosurfactant producing microbes. In the present study, the biodegradation capacities of native bacterial consortia toward total petroleum hydrocarbons (TPH) with special emphasis to poly aromatic hydrocarbons were determined. The purpose of the study was to isolate TPH degrading bacterial strains from various petroleum contaminated soil of Assam, India and develop a robust bacterial consortium for bioremediation of crude oil of this native land. From a total of 23 bacterial isolates obtained from three different hydrocarbons contaminated samples five isolates, namely KS2, PG1, PG5, R1, and R2 were selected as efficient crude oil degraders with respect to their growth on crude oil enriched samples. Isolates KS2, PG1, and R2 are biosurfactant producers and PG5, R1 are non-producers. Fourteen different consortia were designed involving both biosurfactant producing and non-producing isolates. Consortium 10, which comprises two Bacillus strains namely, Bacillus pumilus KS2 and B. cereus R2 (identified by 16s rRNA sequencing) has shown the best result in the desired degradation of crude oil. The consortium showed degradation up to 84.15% of TPH after 5 weeks of incubation, as revealed from gravimetric analysis. FTIR (Fourier transform infrared) and GCMS (Gas chromatography-mass spectrometer) analyses were correlated with gravimetric data which reveals that the consortium has removed a wide range of petroleum hydrocarbons in comparison with abiotic control including different aliphatic and aromatic hydrocarbons. PMID:27471499

Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation.

PubMed

Feng, Jinsong; Ma, Lina; Nie, Jiatong; Konkel, Michael E; Lu, Xiaonan

2018-03-01

Campylobacter jejuni is a microaerophilic bacterium and is believed to persist in a biofilm to antagonize environmental stress. This study investigated the influence of environmental conditions on the formation of C. jejuni biofilm. We report an extracellular DNA (eDNA)-mediated mechanism of biofilm formation in response to aerobic and starvation stress. The eDNA was determined to represent a major form of constitutional material of C. jejuni biofilms and to be closely associated with bacterial lysis. Deletion mutation of the stress response genes spoT and recA enhanced the aerobic influence by stimulating lysis and increasing eDNA release. Flagella were also involved in biofilm formation but mainly contributed to attachment rather than induction of lysis. The addition of genomic DNA from either Campylobacter or Salmonella resulted in a concentration-dependent stimulation effect on biofilm formation, but the effect was not due to forming a precoating DNA layer. Enzymatic degradation of DNA by DNase I disrupted C. jejuni biofilm. In a dual-species biofilm, eDNA allocated Campylobacter and Salmonella at distinct spatial locations that protect Campylobacter from oxygen stress. Our findings demonstrated an essential role and multiple functions of eDNA in biofilm formation of C. jejuni , including facilitating initial attachment, establishing and maintaining biofilm, and allocating bacterial cells. IMPORTANCE Campylobacter jejuni is a major cause of foodborne illness worldwide. In the natural environment, the growth of C. jejuni is greatly inhibited by various forms of environmental stress, such as aerobic stress and starvation stress. Biofilm formation can facilitate the distribution of C. jejuni by enabling the survival of this fragile microorganism under unfavorable conditions. However, the mechanism of C. jejuni biofilm formation in response to environmental stress has been investigated only partially. The significance of our research is in identifying extracellular

Air suctioning during colon biopsy forceps removal reduces bacterial air contamination in the endoscopy suite.

PubMed

Vavricka, S R; Tutuian, R; Imhof, A; Wildi, S; Gubler, C; Fruehauf, H; Ruef, C; Schoepfer, A M; Fried, M

2010-09-01

Bacterial contamination of endoscopy suites is of concern; however studies evaluating bacterial aerosols are lacking. We aimed to determine the effectiveness of air suctioning during removal of biopsy forceps in reducing bacterial air contamination. This was a prospective single-blinded trial involving 50 patients who were undergoing elective nontherapeutic colonoscopy. During colonoscopy, endoscopists removed the biopsy forceps first without and then with suctioning following contact with the sigmoid mucosa. A total of 50 L of air was collected continuously for 30 seconds at 30-cm distance from the biopsy channel valve of the colonoscope, with time starting at forceps removal. Airborne bacteria were collected by an impactor air sampler (MAS-100). Standard Petri dishes with CNA blood agar were used to culture Gram-positive bacteria. Main outcome measure was the bacterial load in endoscopy room air. At the beginning and end of the daily colonoscopy program, the median (and interquartile [IQR] range) bioaerosol burden was 4 colony forming units (CFU)/m (3) (IQR 3 - 6) and 16 CFU/m (3) (IQR 13 - 18), respectively. Air suctioning during removal of the biopsy forceps reduced the bioaerosol burden from a median of 14 CFU/m (3) (IQR 11 - 29) to a median of 7 CFU/m (3) (IQR 4 - 16) ( P = 0.0001). Predominantly enterococci were identified on the agar plates. The bacterial aerosol burden during handling of biopsy forceps can be reduced by applying air suction while removing the forceps. This simple method may reduce transmission of infectious agents during gastrointestinal endoscopies. Copyright Georg Thieme Verlag KG Stuttgart . New York.

Low-energy plasma immersion ion implantation to induce DNA transfer into bacterial E. coli

NASA Astrophysics Data System (ADS)

Sangwijit, K.; Yu, L. D.; Sarapirom, S.; Pitakrattananukool, S.; Anuntalabhochai, S.

2015-12-01

Plasma immersion ion implantation (PIII) at low energy was for the first time applied as a novel biotechnology to induce DNA transfer into bacterial cells. Argon or nitrogen PIII at low bias voltages of 2.5, 5 and 10 kV and fluences ranging from 1 × 1012 to 1 × 1017 ions/cm2 treated cells of Escherichia coli (E. coli). Subsequently, DNA transfer was operated by mixing the PIII-treated cells with DNA. Successes in PIII-induced DNA transfer were demonstrated by marker gene expressions. The induction of DNA transfer was ion-energy, fluence and DNA-size dependent. The DNA transferred in the cells was confirmed functioning. Mechanisms of the PIII-induced DNA transfer were investigated and discussed in terms of the E. coli cell envelope anatomy. Compared with conventional ion-beam-induced DNA transfer, PIII-induced DNA transfer was simpler with lower cost but higher efficiency.

A 28,000 Years Old Cro-Magnon mtDNA Sequence Differs from All Potentially Contaminating Modern Sequences

PubMed Central

Caramelli, David; Milani, Lucio; Vai, Stefania; Modi, Alessandra; Pecchioli, Elena; Girardi, Matteo; Pilli, Elena; Lari, Martina; Lippi, Barbara; Ronchitelli, Annamaria; Mallegni, Francesco; Casoli, Antonella; Bertorelle, Giorgio; Barbujani, Guido

2008-01-01

Background DNA sequences from ancient speciments may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal) and early modern (Cro-Magnoid) Europeans. Methodology/Principal Findings We typed the mitochondrial DNA (mtDNA) hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23) and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences. Conclusions/Significance: The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of early modern Europeans. PMID:18628960

[Predictive factors of contamination in a blood culture with bacterial growth in an Emergency Department].

PubMed

Hernández-Bou, S; Trenchs Sainz de la Maza, V; Esquivel Ojeda, J N; Gené Giralt, A; Luaces Cubells, C

2015-06-01

The aim of this study is to identify predictive factors of bacterial contamination in positive blood cultures (BC) collected in an emergency department. A prospective, observational and analytical study was conducted on febrile children aged on to 36 months, who had no risk factors of bacterial infection, and had a BC collected in the Emergency Department between November 2011 and October 2013 in which bacterial growth was detected. The potential BC contamination predicting factors analysed were: maximum temperature, time to positivity, initial Gram stain result, white blood cell count, absolute neutrophil count, band count, and C-reactive protein (CRP). Bacteria grew in 169 BC. Thirty (17.8%) were finally considered true positives and 139 (82.2%) false positives. All potential BC contamination predicting factors analysed, except maximum temperature, showed significant differences between true positives and false positives. CRP value, time to positivity, and initial Gram stain result are the best predictors of false positives in BC. The positive predictive values of a CRP value≤30mg/L, BC time to positivity≥16h, and initial Gram stain suggestive of a contaminant in predicting a FP, are 95.1, 96.9 and 97.5%, respectively. When all 3 conditions are applied, their positive predictive value is 100%. Four (8.3%) patients with a false positive BC and discharged to home were revaluated in the Emergency Department. The majority of BC obtained in the Emergency Department that showed positive were finally considered false positives. Initial Gram stain, time to positivity, and CRP results are valuable diagnostic tests in distinguishing between true positives and false positives in BC. The early detection of false positives will allow minimising their negative consequences. Copyright © 2014 Asociación Española de Pediatría. Published by Elsevier España, S.L.U. All rights reserved.

Mechanism of foreign DNA selection in a bacterial adaptive immune system

PubMed Central

Sashital, Dipali G.; Wiedenheft, Blake; Doudna, Jennifer A.

2012-01-01

Summary In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary “non-self” DNA sequences for destruction, while avoiding binding to “self” sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for non-self target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved 3-base pair motif that is required for non-self target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade. PMID:22521690

Calibration of optimal use parameters for an ultraviolet light-emitting diode in eliminating bacterial contamination on needleless connectors.

PubMed

Hutchens, M P; Drennan, S L; Cambronne, E D

2015-06-01

Needleless connectors may develop bacterial contamination and cause central-line-associated bloodstream infections (CLABSI) despite rigorous application of best-practice. Ultraviolet (UV) light-emitting diodes (LED) are an emerging, increasingly affordable disinfection technology. We tested the hypothesis that a low-power UV LED could reliably eliminate bacteria on needleless central-line ports in a laboratory model of central-line contamination. Needleless central-line connectors were inoculated with Staphylococcus aureus. A 285 nm UV LED was used in calibrated fashion to expose contaminated connectors. Ports were directly applied to agar plates and flushed with sterile saline, allowing assessment of bacterial survival on the port surface and in simulated usage flow-through fluid. UV applied to needleless central-line connectors was highly lethal at 0·5 cm distance at all tested exposure times. At distances >1·5 cm both simulated flow-through and port surface cultures demonstrated significant bacterial growth following UV exposure. Logarithmic-phase S. aureus subcultures were highly susceptible to UV induction/maintenance dosing. Low-power UV LED doses at fixed time and distance from needleless central-line connector ports reduced cultivable S. aureus from >10(6) CFU to below detectable levels in this laboratory simulation of central-line port contamination. Low-power UV LEDs may represent a feasible alternative to current best-practice in connector decontamination. © 2015 The Society for Applied Microbiology.

EDTA addition enhances bacterial respiration activities and hydrocarbon degradation in bioaugmented and non-bioaugmented oil-contaminated desert soils.

PubMed

Al Kharusi, Samiha; Abed, Raeid M M; Dobretsov, Sergey

2016-03-01

The low number and activity of hydrocarbon-degrading bacteria and the low solubility and availability of hydrocarbons hamper bioremediation of oil-contaminated soils in arid deserts, thus bioremediation treatments that circumvent these limitations are required. We tested the effect of Ethylenediaminetetraacetic acid (EDTA) addition, at different concentrations (i.e. 0.1, 1 and 10 mM), on bacterial respiration and biodegradation of Arabian light oil in bioaugmented (i.e. with the addition of exogenous alkane-degrading consortium) and non-bioaugmented oil-contaminated desert soils. Post-treatment shifts in the soils' bacterial community structure were monitored using MiSeq sequencing. Bacterial respiration, indicated by the amount of evolved CO2, was highest at 10 mM EDTA in bioaugmented and non-bioaugmented soils, reaching an amount of 2.2 ± 0.08 and 1.6 ± 0.02 mg-CO2 g(-1) after 14 days of incubation, respectively. GC-MS revealed that 91.5% of the C14-C30 alkanes were degraded after 42 days when 10 mM EDTA and the bacterial consortium were added together. MiSeq sequencing showed that 78-91% of retrieved sequences in the original soil belonged to Deinococci, Alphaproteobacteria, Gammaproteobacteia and Bacilli. The same bacterial classes were detected in the 10 mM EDTA-treated soils, however with slight differences in their relative abundances. In the bioaugmented soils, only Alcanivorax sp. MH3 and Parvibaculum sp. MH21 from the exogenous bacterial consortium could survive until the end of the experiment. We conclude that the addition of EDTA at appropriate concentrations could facilitate biodegradation processes by increasing hydrocarbon availability to microbes. The addition of exogenous oil-degrading bacteria along with EDTA could serve as an ideal solution for the decontamination of oil-contaminated desert soils. Copyright © 2016 Elsevier Ltd. All rights reserved.

Powerful bacterial killing by buckwheat honeys is concentration-dependent, involves complete DNA degradation and requires hydrogen peroxide.

PubMed

Brudzynski, Katrina; Abubaker, Kamal; Wang, Tony

2012-01-01

Exposure of bacterial cells to honey inhibits their growth and may cause cell death. Our previous studies showed a cause-effect relationship between hydroxyl radical generated from honey hydrogen peroxide and growth arrest. Here we explored the role of hydroxyl radicals as inducers of bacterial cells death. The bactericidal effect of ·OH on antibiotic-resistant clinical isolates of MRSA and VRE and standard bacterial strains of E. coli and B. subtiles was examined using a broth microdilution assay supplemented with 3'-(p-aminophenyl) fluorescein (APF) as the ·OH trap, followed by colony enumeration. Bactericidal activities of eight honeys (six varieties of buckwheat, blueberry and manuka honeys) were analyzed. The MBC/MIC ratio ≤4 and the killing curves indicated that honeys exhibited powerful, concentration-dependent bactericidal effect. The extent of killing depended on the ratio of honey concentration to bacterial load, indicating that honey dose was critical for its bactericidal efficacy. The killing rate and potency varied between honeys and ranged from over a 6-log(10) to 4-log(10) CFU/ml reduction of viable cells, equivalent to complete bacterial eradication. The maximal killing was associated with the extensive degradation of bacterial DNA. Honey concentration at which DNA degradation occurred correlated with cell death observed in the concentration-dependent cell-kill on agar plates. There was no quantitative relationship between the ·OH generation by honey and bactericidal effect. At the MBC, where there was no surviving cells and no DNA was visible on agarose gels, the ·OH levels were on average 2-3x lower than at Minimum Inhibitory Concentration (MICs) (p < 0.0001). Pre-treatment of honey with catalase, abolished the bactericidal effect. This raised possibilities that either the abrupt killing prevented accumulation of ·OH (dead cells did not generate ·OH) or that DNA degradation and killing is the actual footprint of ·OH action. In conclusion

Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

PubMed Central

SOLANKY, DIPESH; HAYDEL, SHELLEY E.

2012-01-01

This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

Non-Enzymatic Detection of Bacterial Genomic DNA Using the Bio-Barcode Assay

PubMed Central

Hill, Haley D.; Vega, Rafael A.; Mirkin, Chad A.

2011-01-01

The detection of bacterial genomic DNA through a non-enzymatic nanomaterials based amplification method, the bio-barcode assay, is reported. The assay utilizes oligonucleotide functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double stranded DNA. These blockers bind to specific regions of the target DNA upon cooling, and prevent the duplex DNA from re-hybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (barcodes) act as amplification surrogates. The barcodes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 femtomolar, and this is the first demonstration of a barcode type assay for the detection of double stranded, genomic DNA. PMID:17927207

The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

ERIC Educational Resources Information Center

Falconer, A. C.; Hayes, L. J.

1986-01-01

Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

PubMed

Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

2014-05-01

Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

Metal resistant plants and phytoremediation of environmental contamination

DOEpatents

Meagher, Richard B.; Li, Yujing; Dhankher, Om P.

2010-04-20

The present disclosure provides a method of producing transgenic plants which are resistant to at least one metal ion by transforming the plant with a recombinant DNA comprising a nucleic acid encoding a bacterial arsenic reductase under the control of a plant expressible promoter, and a nucleic acid encoding a nucleotide sequence encoding a phytochelatin biosynthetic enzyme under the control of a plant expressible promoter. The invention also relates a method of phytoremediation of a contaminated site by growing in the site a transgenic plant expressing a nucleic acid encoding a bacterial arsenate reductase and a nucleic acid encoding a phytochelatin biosynthetic enzyme.

Surface tailored organobentonite enhances bacterial proliferation and phenanthrene biodegradation under cadmium co-contamination.

PubMed

Mandal, Asit; Biswas, Bhabananda; Sarkar, Binoy; Patra, Ashok K; Naidu, Ravi

2016-04-15

Co-contamination of soil and water with polycyclic aromatic hydrocarbon (PAH) and heavy metals makes biodegradation of the former extremely challenging. Modified clay-modulated microbial degradation provides a novel insight in addressing this issue. This study was conducted to evaluate the growth and phenanthrene degradation performance of Mycobacterium gilvum VF1 in the presence of a palmitic acid (PA)-grafted Arquad® 2HT-75-based organobentonite in cadmium (Cd)-phenanthrene co-contaminated water. The PA-grafted organobentonite (ABP) adsorbed a slightly greater quantity of Cd than bentonite at up to 30mgL(-1) metal concentration, but its highly negative surface charge imparted by carboxylic groups indicated the potential of being a significantly superior adsorbent of Cd at higher metal concentrations. In systems co-contained with Cd (5 and 10mgL(-1)), the Arquad® 2HT-75-modified bentonite (AB) and PA-grafted organobentonite (ABP) resulted in a significantly higher (72-78%) degradation of phenanthrene than bentonite (62%) by the bacterium. The growth and proliferation of bacteria were supported by ABP which not only eliminated Cd toxicity through adsorption but also created a congenial microenvironment for bacterial survival. The macromolecules produced during ABP-bacteria interaction could form a stable clay-bacterial cluster by overcoming the electrostatic repulsion among individual components. Findings of this study provide new insights for designing clay modulated PAH bioremediation technologies in mixed-contaminated water and soil. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of lauric acid-potassium hydroxide concentration on bacterial contamination of spray washed broiler carcasses

USDA-ARS?s Scientific Manuscript database

A series of experiments were conducted to examine reductions in bacterial contamination of broiler carcasses washed in a spray cabinet with various concentrations of lauric acid (LA)-potassium hydroxide (KOH) solutions. Fifty eviscerated carcasses and 5 ceca were obtained from the processing line of...

VUV absorption spectroscopy of bacterial spores and DNA components

NASA Astrophysics Data System (ADS)

Fiebrandt, Marcel; Lackmann, Jan-Wilm; Raguse, Marina; Moeller, Ralf; Awakowicz, Peter; Stapelmann, Katharina

2017-01-01

Low-pressure plasmas can be used to inactivate bacterial spores and sterilize goods for medical and pharmaceutical applications. A crucial factor are damages induced by UV and VUV radiation emitted by the plasma. To analyze inactivation processes and protection strategies of spores, absorption spectra of two B. subtilis strains are measured. The results indicate, that the inner and outer coat of the spore significantly contribute to the absorption of UV-C and also of the VUV, protecting the spore against radiation based damages. As the sample preparation can significantly influence the absorption spectra due to salt residues, the cleaning procedure and sample deposition is tested for its reproducibility by measuring DNA oligomers and pUC18 plasmid DNA. The measurements are compared and discussed with results from the literature, showing a strong decrease of the salt content enabling the detection of absorption structures in the samples.

Bacterial Contamination of Boar Semen and its Relationship to Sperm Quality Preserved in Commercial Extender Containing Gentamicin Sulfate.

PubMed

Gączarzewicz, D; Udała, J; Piasecka, M; Błaszczyk, B; Stankiewicz, T

2016-09-01

This study was designed to determine the degree and type of bacterial contamination in boar semen (79 ejaculates from Large White and Landrace boars) and its consequences for sperm quality during storage (27 extended semen samples, 16°C for five days) under practical conditions of artificial insemination (AI). The results revealed the presence of aerobic bacteria in 99% of the ejaculates (from 80 to 370 ×106 colony-forming units/mL). Most of the ejaculates contained two or three bacterial contaminants, while the Staphylococcus, Streptococcus, and Pseudomonas bacterial genera were most frequently isolated. Also detected were Enterobacter spp., Bacillus spp., Proteus spp., Escherichia coli, P. fluorescens, and P. aeruginosa. In general, the growth of certain bacterial types isolated prior to semen processing (Enterobacter spp., E. coli, P. fluorescens, and P. aeruginosa) was not discovered on different days of storage, but fluctuations (with a tendency towards increases) were found in the frequencies of Bacillus spp., Pseudomonas spp., and Staphylococcus spp. isolates up to the end of storage. Semen preserved for five days exhibited decreases in sperm motility and increases in the average number of total aerobic bacteria; this was associated with sperm agglutination, plasma membrane disruption, and acrosome damage. We inferred that, due to the different degrees and types of bacterial contaminants in the boar ejaculates, the inhibitory activity of some antimicrobial agents used in swine extenders (such as gentamicin sulfate) may be limited. Because such agents can contribute to the overgrowth of certain aerobic bacteria and a reduction in the quality of stored semen, procedures with high standards of hygiene and microbiological control should be used when processing boar semen.

DNA DAMAGE AND EXTERNAL LESIONS IN BROWN BULLHEAD FROM CONTAMINATED HABITATS

EPA Science Inventory

The single cell gel electrophoresis ("Comet") assay was used to compare levels of DNA damage in brown bullheads (Ameiurus nebulosus) collected from three known contaminated locations, the Cuyahoga River, Ashtabula River, and Ashumet Pond (Cape Cod), with brown bullheads collected...

DNA DAMAGE AND EXTERNAL LESIONS IN BROWN BULLHEADS FROM CONTAMINATED HABITATS

EPA Science Inventory

The Comet assay was used to compare levels of DNA damage in brown bullheads (Ameiurus nebulosus) collected from three known contaminated locations, the Cuyahoga River, Ashtabula River, and Ashumet Pond (Cape Cod), with brown bullheads collected from three paired reference sites, ...

Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota

PubMed Central

Gill, Christina; Blow, Frances; Darby, Alistair C.

2016-01-01

Background Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. Results After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. Conclusions An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures

Evaluation of Lysis Methods for the Extraction of Bacterial DNA for Analysis of the Vaginal Microbiota.

PubMed

Gill, Christina; van de Wijgert, Janneke H H M; Blow, Frances; Darby, Alistair C

2016-01-01

Recent studies on the vaginal microbiota have employed molecular techniques such as 16S rRNA gene sequencing to describe the bacterial community as a whole. These techniques require the lysis of bacterial cells to release DNA before purification and PCR amplification of the 16S rRNA gene. Currently, methods for the lysis of bacterial cells are not standardised and there is potential for introducing bias into the results if some bacterial species are lysed less efficiently than others. This study aimed to compare the results of vaginal microbiota profiling using four different pretreatment methods for the lysis of bacterial samples (30 min of lysis with lysozyme, 16 hours of lysis with lysozyme, 60 min of lysis with a mixture of lysozyme, mutanolysin and lysostaphin and 30 min of lysis with lysozyme followed by bead beating) prior to chemical and enzyme-based DNA extraction with a commercial kit. After extraction, DNA yield did not significantly differ between methods with the exception of lysis with lysozyme combined with bead beating which produced significantly lower yields when compared to lysis with the enzyme cocktail or 30 min lysis with lysozyme only. However, this did not result in a statistically significant difference in the observed alpha diversity of samples. The beta diversity (Bray-Curtis dissimilarity) between different lysis methods was statistically significantly different, but this difference was small compared to differences between samples, and did not affect the grouping of samples with similar vaginal bacterial community structure by hierarchical clustering. An understanding of how laboratory methods affect the results of microbiota studies is vital in order to accurately interpret the results and make valid comparisons between studies. Our results indicate that the choice of lysis method does not prevent the detection of effects relating to the type of vaginal bacterial community one of the main outcome measures of epidemiological studies

MOLECULAR EVALUATION OF CHANGES IN PLANKTONIC BACTERIAL POPULATIONS RESULTING FROM EQUINE FECAL CONTAMINATION IN A SUB-WATERSHED

EPA Science Inventory

Considerable emphasis has been placed on developing watershed-based strategies with the potential to reduce non-point-source fecal contamination. Molecular methods applied used 16S-ribosomal-deoxyribonucleic-acid (rDNA) to try to determine sources of fecal contamination. Objectiv...

Phylogenetic and Functional Diversity of Total (DNA) and Expressed (RNA) Bacterial Communities in Urban Green Infrastructure Bioswale Soils.

PubMed

Gill, Aman S; Lee, Angela; McGuire, Krista L

2017-08-15

New York City (NYC) is pioneering green infrastructure with the use of bioswales and other engineered soil-based habitats to provide stormwater infiltration and other ecosystem functions. In addition to avoiding the environmental and financial costs of expanding traditional built infrastructure, green infrastructure is thought to generate cobenefits in the form of diverse ecological processes performed by its plant and microbial communities. Yet, although plant communities in these habitats are closely managed, we lack basic knowledge about how engineered ecosystems impact the distribution and functioning of soil bacteria. We sequenced amplicons of the 16S ribosomal subunit, as well as seven genes associated with functional pathways, generated from both total (DNA-based) and expressed (RNA) soil communities in the Bronx, NYC, NY, in order to test whether bioswale soils host characteristic bacterial communities with evidence for enriched microbial functioning, compared to nonengineered soils in park lawns and tree pits. Bioswales had distinct, phylogenetically diverse bacterial communities, including taxa associated with nutrient cycling and metabolism of hydrocarbons and other pollutants. Bioswale soils also had a significantly greater diversity of genes involved in several functional pathways, including carbon fixation ( cbbL-R [ cbbL gene, red-like subunit] and apsA ), nitrogen cycling ( noxZ and amoA ), and contaminant degradation ( bphA ); conversely, no functional genes were significantly more abundant in nonengineered soils. These results provide preliminary evidence that urban land management can shape the diversity and activity of soil communities, with positive consequences for genetic resources underlying valuable ecological functions, including biogeochemical cycling and degradation of common urban pollutants. IMPORTANCE Management of urban soil biodiversity by favoring taxa associated with decontamination or other microbial metabolic processes is a

Phylogenetic and Functional Diversity of Total (DNA) and Expressed (RNA) Bacterial Communities in Urban Green Infrastructure Bioswale Soils

PubMed Central

Lee, Angela; McGuire, Krista L.

2017-01-01

ABSTRACT New York City (NYC) is pioneering green infrastructure with the use of bioswales and other engineered soil-based habitats to provide stormwater infiltration and other ecosystem functions. In addition to avoiding the environmental and financial costs of expanding traditional built infrastructure, green infrastructure is thought to generate cobenefits in the form of diverse ecological processes performed by its plant and microbial communities. Yet, although plant communities in these habitats are closely managed, we lack basic knowledge about how engineered ecosystems impact the distribution and functioning of soil bacteria. We sequenced amplicons of the 16S ribosomal subunit, as well as seven genes associated with functional pathways, generated from both total (DNA-based) and expressed (RNA) soil communities in the Bronx, NYC, NY, in order to test whether bioswale soils host characteristic bacterial communities with evidence for enriched microbial functioning, compared to nonengineered soils in park lawns and tree pits. Bioswales had distinct, phylogenetically diverse bacterial communities, including taxa associated with nutrient cycling and metabolism of hydrocarbons and other pollutants. Bioswale soils also had a significantly greater diversity of genes involved in several functional pathways, including carbon fixation (cbbL-R [cbbL gene, red-like subunit] and apsA), nitrogen cycling (noxZ and amoA), and contaminant degradation (bphA); conversely, no functional genes were significantly more abundant in nonengineered soils. These results provide preliminary evidence that urban land management can shape the diversity and activity of soil communities, with positive consequences for genetic resources underlying valuable ecological functions, including biogeochemical cycling and degradation of common urban pollutants. IMPORTANCE Management of urban soil biodiversity by favoring taxa associated with decontamination or other microbial metabolic processes is a

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives.

PubMed

Collin, Frédéric; Karkare, Shantanu; Maxwell, Anthony

2011-11-01

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme.

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

PubMed Central

Rothrock, Michael J.; Hiett, Kelli L.; Gamble, John; Caudill, Andrew C.; Cicconi-Hogan, Kellie M.; Caporaso, J. Gregory

2014-01-01

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. PMID:25548939

Parvovirus B19V DNA contamination in Chinese plasma and plasma derivatives

PubMed Central

2012-01-01

Background To ensure the safety of plasma derivatives, screening for human parvovirus B19V genomic DNA in donated plasma using a pooling strategy is performed in some countries. We investigated the prevalence of B19V DNA and anti-B19V antibodies in Chinese plasma pools, plasma derivatives and plasma donations to evaluate the risk posed by B19V. Methods Using a Q-PCR assay developed in-house, we tested for B19V genomic DNA in 142 plasma pools collected between January 2009 and June 2011 from two Chinese blood products manufacturers. Plasma derivatives collected between 1993–1995 (10 batches of albumin, 155 batches of intravenous immunoglobulin, IVIG) and 2009–2011 (50 batches of albumin, 54 batches of IVIG, 35 batches of factor VIII, 7 batches of fibrinogen, and 17 batches of prothrombin complex concentrate, PCC) were also tested for B19V contamination. In addition, B19V genome prevalence in minipools(including 90 individual donations) of 49680 individual plasma samples collected between August 2011 and March 2012 by a single Chinese manufacturer was investigated. IgM/IgG was also investigated in plasma pools/derivatives and in minipools with B19V-DNA titers above 1x104 and 1x106 geq/mL using B19 ELISA IgM/IgG assay(Virion-Serion, Würzburg, Germany), respectively. Results B19V-DNA was detected in 54.2% of plasma pools from two Chinese blood product manufacturers; among recently produced blood products, B19V was detected in 21/54 IVIG samples, 19/35 factor VIII samples, 6/7 fibrinogen samples, and 12/17 PCC samples, but not in albumin samples. The levels of B19V-DNA in these samples varied from 102-107 geq/mL. In samples with >104 geq/mL genome DNA, B19V-specific IgG was also found in all corresponding plasma pools and IVIG, whereas none was detected in the majority of other plasma derivatives. Screening of plasma donations indicated that most minipools were contaminated with B19V-DNA (102-108 geq/mL) and one donation had 1.09 × 1010 geq/mL B19V genomic DNA

Assessing genetic structure and diversity of airborne bacterial communities by DNA fingerprinting and 16S rDNA clone library

NASA Astrophysics Data System (ADS)

Maron, Pierre-Alain; Lejon, David P. H.; Carvalho, Esmeralda; Bizet, Karine; Lemanceau, Philippe; Ranjard, Lionel; Mougel, Christophe

The density, genetic structure and diversity of airborne bacterial communities were assessed in the outdoor atmosphere. Two air samples were collected on the same location (north of France) at two dates (March 2003 (sample1) and May 2003 (sample 2)). Molecular culture -independent methods were used to characterise airborne bacterial communities regardless of the cell culturability. The automated-ribosomal intergenic spacer analysis (A-RISA) was performed to characterise the community structure in each sample. For both sampling dates, complex A-RISA patterns were observed suggesting a highly diverse community structure, comparable to those found in soil, water or sediment environments. Furthermore, differences in the genetic structure of airborne bacterial communities were observed between samples 1 and 2 suggesting an important variability in time. A clone library of 16S rDNA directly amplified from air DNA of sample 1 was constructed and sequenced to analyse the community composition and diversity. The Proteobacteria group had the greatest representation (60%), with bacteria belonging to the different subdivisions α- (19%), β-(21%), γ-(12%) and δ-(8%). Firmicute and Actinobacteria were also well represented with 14% and 12%, respectively. Most of the identified bacteria are known to be commonly associated with soil or plant environments suggesting that the atmosphere is mainly colonised transiently by microorganisms from local sources, depending on air fluxes.

Impact of roadside ditch dredging on bacterial communities and biological contamination of a tidal creek

NASA Astrophysics Data System (ADS)

Jones, Chance E.; Barkovskii, Andrei L.

2017-03-01

Tidal creek networks form the primary hydrologic link between estuaries and land-based activities on barrier islands. A possible impact from the excavation of drainage ditch systems on bacterial communities and biological contamination was studied in the water column and sediments of headwater, mid-stream, and mouth sites of the intertidal Oakdale Creek on Sapelo Island, GA. Community analysis was performed using the MiSeq Illumina platform and revealed that dredging was the cause of a significant rise in Proteobacteria, especially γ-proteobacteria. Targeted biological contaminants included fecal indicator bacteria, Enterococcus spp. (Entero-1), pathogens, Shigella spp. (ipaH), and Salmonella spp (invA), virulence associated genes (VG's) of pathogenic E. coli (eaeA, hlyD, stx1, stx2, and set1B), integrons (intI1, intI2), and tetracycline resistance genes (TRGs). Incidence and gene concentrations of Shigella spp., eaeA and set1B, and of TRGs increased 3-20 folds after the onset of dredging, and followed the dredging schedule. Principal Component Analysis suggested possible common carriers for Shigella spp., some TRGs, and the pathogenic E. coli eaeA gene. At the site of dredging, all of the above contaminants were detected at high concentrations. We concluded that excavation of roadside ditches caused significant changes in bacterial composition and a rise in incidence and concentrations of biological contaminants in the creek. The authors suggest a different approach for the maintenance of this material be explored.

The degree of bacterial contamination while performing spine surgery.

PubMed

Ahn, Dong Ki; Park, Hoon Seok; Kim, Tae Woo; Yang, Jong Hwa; Boo, Kyung Hwan; Kim, In Ja; Lee, Hye Jin

2013-03-01

Prospective experimental study. To evaluate bacterial contamination during surgery. The participants of surgery and ventilation system have been known as the most significant sources of contamination. Two pairs of air culture blood agar plate for G(+) bacteria and MacConkey agar plate for G(-) bacteria were placed at 3 different locations in a conventional operation room: in the surgical field, under the airflow of local air conditioner, and pathway to door while performing spine surgeries. One pair of culture plates was retrieved after one hour and the other pair was retrieved after 3 hours. The cultured bacteria were identified and number of colonies was counted. There was no G(-) bacteria identified. G(+) bacteria grew on all 90 air culture blood agar plates. The colony count of one hour group was 14.5±5.4 in the surgical field, 11.3±6.6 under the local air conditioner, and 13.1±8.7 at the pathway to the door. There was no difference among the 3 locations. The colony count of 3 hours group was 46.4±19.5, 30.3±12.9, and 39.7±15.2, respectively. It was more at the surgical field than under the air conditioner (p=0.03). The number of colonies of one hour group was 13.0±7.0 and 3 hours group was 38.8±17.1. There was positive correlation between the time and the number of colonies (r=0.76, p=0.000). Conventional operation room was contaminated by G(+) bacteria. The degree of contamination was most high at the surgical field. The number of bacteria increased right proportionally to the time.

The Degree of Bacterial Contamination While Performing Spine Surgery

PubMed Central

Ahn, Dong Ki; Park, Hoon Seok; Yang, Jong Hwa; Boo, Kyung Hwan; Kim, In Ja; Lee, Hye Jin

2013-01-01

Study Design Prospective experimental study. Purpose To evaluate bacterial contamination during surgery. Overview of Literature The participants of surgery and ventilation system have been known as the most significant sources of contamination. Methods Two pairs of air culture blood agar plate for G(+) bacteria and MacConkey agar plate for G(-) bacteria were placed at 3 different locations in a conventional operation room: in the surgical field, under the airflow of local air conditioner, and pathway to door while performing spine surgeries. One pair of culture plates was retrieved after one hour and the other pair was retrieved after 3 hours. The cultured bacteria were identified and number of colonies was counted. Results There was no G(-) bacteria identified. G(+) bacteria grew on all 90 air culture blood agar plates. The colony count of one hour group was 14.5±5.4 in the surgical field, 11.3±6.6 under the local air conditioner, and 13.1±8.7 at the pathway to the door. There was no difference among the 3 locations. The colony count of 3 hours group was 46.4±19.5, 30.3±12.9, and 39.7±15.2, respectively. It was more at the surgical field than under the air conditioner (p=0.03). The number of colonies of one hour group was 13.0±7.0 and 3 hours group was 38.8±17.1. There was positive correlation between the time and the number of colonies (r=0.76, p=0.000). Conclusions Conventional operation room was contaminated by G(+) bacteria. The degree of contamination was most high at the surgical field. The number of bacteria increased right proportionally to the time. PMID:23508998

Mobile laminar air flow screen for additional operating room ventilation: reduction of intraoperative bacterial contamination during total knee arthroplasty.

PubMed

Sossai, D; Dagnino, G; Sanguineti, F; Franchin, F

2011-12-01

Surgical site infections are important complications in orthopedic surgery. A mobile laminar air flow (LAF) screen could represent a useful addition to an operating room (OR) with conventional turbulent air ventilation (12.5 air changes/h), as it could decrease the bacterial count near the operating field. The purpose of this study was to evaluate LAF efficacy at reducing bacterial contamination in the surgical area during 34 total knee arthroplasties (TKAs). The additional unit was used in 17 operations; the LAF was positioned beside the operating table between two of the surgeons, with the air flow directed towards the surgical area (wound). The whole team wore conventional OR clothing and the correct hygiene procedures and rituals were used. Bacterial air contamination (CFU/m(3)) was evaluated in the wound area in 17 operations with the LAF unit and 17 without the LAF unit. The LAF unit reduced the mean bacterial count in the wound area from 23.5 CFU/m(3) without the LAF to 3.5 CFU/m(3) with the LAF (P < 0.0001), which is below the suggested limit for an OR with ultraclean laminar ventilation. There were no significant differences in the mean bacterial count in the instrument table area: 28.6 CFU/m(3) were recorded with the LAF (N = 6) unit and 30.8 CFU/m(3) (N = 6) without the LAF unit (P = 0.631). During six operations with LAF and six without LAF, particle counts were performed and the number of 0.5 μm particles was analyzed. The particle counts decreased significantly when the LAF unit was used (P = 0.003). When a mobile LAF unit was added to the standard OR ventilation, bacterial contamination of the wound area significantly decreased to below the accepted level for an ultraclean OR, preventing SSI infections.

Diversity of ribosomal 16S DNA- and RNA-based bacterial community in an office building drinking water system.

PubMed

Inkinen, J; Jayaprakash, B; Santo Domingo, J W; Keinänen-Toivola, M M; Ryu, H; Pitkänen, T

2016-06-01

Next-generation sequencing of 16S ribosomal RNA genes (rDNA) and ribosomal RNA (rRNA) was used to characterize water and biofilm microbiome collected from a drinking water distribution system of an office building after its first year of operation. The total bacterial community (rDNA) and active bacterial members (rRNA) sequencing databases were generated by Illumina MiSeq PE250 platform. As estimated by Chao1 index, species richness in cold water system was lower (180-260) in biofilms (Sphingomonas spp., Methylobacterium spp., Limnohabitans spp., Rhizobiales order) than in waters (250-580), (also Methylotenera spp.) (P = 0·005, n = 20). Similarly species richness (Chao1) was slightly higher (210-580) in rDNA libraries compared to rRNA libraries (150-400; P = 0·054, n = 24). Active Mycobacterium spp. was found in cross-linked polyethylene (PEX), but not in corresponding copper pipeline biofilm. Nonpathogenic Legionella spp. was found in rDNA libraries but not in rRNA libraries. Microbial communities differed between water and biofilms, between cold and hot water systems, locations in the building and between water rRNA and rDNA libraries, as shown by clear clusters in principal component analysis (PcoA). By using the rRNA method, we found that not all bacterial community members were active (e.g. Legionella spp.), whereas other members showed increased activity in some locations; for example, Pseudomonas spp. in hot water circulations' biofilm and order Rhizobiales and Limnohabitans spp. in stagnated locations' water and biofilm. rRNA-based methods may be better than rDNA-based methods for evaluating human health implications as rRNA methods can be used to describe the active bacterial fraction. This study indicates that copper as a pipeline material might have an adverse impact on the occurrence of Mycobacterium spp. The activity of Legionella spp. maybe questionable when detected solely by using DNA-based methods. © 2016 The Society for Applied

Analysis of Bacterial and Fungal Nucleic Acid in Canine Sterile Granulomatous and Pyogranulomatous Dermatitis and Panniculitis.

PubMed

Rosa, Fabio B; Older, Caitlin E; Meason-Smith, Courtney; Suchodolski, Jan S; Lingsweiler, Sonia; Mansell, Joanne E; Hoffmann, Aline Rodrigues

2018-01-01

Next generation sequencing (NGS) studies are revealing a diverse microbiota on the skin of dogs. The skin microbiota of canine sterile granulomatous and pyogranulomatous dermatitis (SGPD) has yet to be investigated using NGS techniques. NGS targeting the 16S rRNA and ITS-1 region of bacterial and fungal DNA, respectively, were used to investigate if bacterial and fungal DNA were associated with skin lesions in cases of canine SGPD. The study included 20 formalin-fixed paraffin-embedded (FFPE) skin samples and 12 fresh samples from SGPD-affected dogs, and 10 FFPE and 10 fresh samples from healthy dogs. DNA was extracted from deep dermis and panniculus, and microbial DNA was amplified using primers targeting the bacterial 16S rRNA V1-V3 and fungal ITS-1 regions. The amplified DNA was utilized for NGS on an Illumina MiSeq instrument. The sequences were processed using QIIME. No differences in fungal or bacterial alpha diversity were observed between the SGPD and control samples. Beta diversity analysis demonstrated differences in the bacterial communities between SGPD and control, but not in the fungal communities. Compared to controls, the family Erysipelotrichaceae and genus Staphylococcus were significantly more abundant in the SGPD FFPE samples, and genus Corynebacterium were more abundant in fresh samples. The bacteria found to be more abundant in SGPD are common inhabitants of skin surfaces, and likely secondary contaminants in SGPD cases. This study provides additional evidence that SGPD lesions are likely sterile.

Etiologic Diagnosis of Lower Respiratory Tract Bacterial Infections Using Sputum Samples and Quantitative Loop-Mediated Isothermal Amplification

PubMed Central

Peng, Peichao; Cheng, Xiaoxing; Wang, Guoqing; Qian, Minping; Gao, Huafang; Han, Bei; Chen, Yusheng; Hu, Yinghui; Geng, Rong; Hu, Chengping; Zhang, Wei; Yang, Jingping; Wan, Huanying; Yu, Qin; Wei, Liping; Li, Jiashu; Tian, Guizhen; Wang, Qiuyue; Hu, Ke; Wang, Siqin; Wang, Ruiqin; Du, Juan; He, Bei; Ma, Jianjun; Zhong, Xiaoning; Mu, Lan; Cai, Shaoxi; Zhu, Xiangdong; Xing, Wanli; Yu, Jun; Deng, Minghua; Gao, Zhancheng

2012-01-01

Etiologic diagnoses of lower respiratory tract infections (LRTI) have been relying primarily on bacterial cultures that often fail to return useful results in time. Although DNA-based assays are more sensitive than bacterial cultures in detecting pathogens, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. Here we report a nationwide cohort study on 2986 suspected LRTI patients across P. R. China. We compared the performance of a DNA-based assay qLAMP (quantitative Loop-mediated isothermal AMPlification) with that of standard bacterial cultures in detecting a panel of eight common respiratory bacterial pathogens from sputum samples. Our qLAMP assay detects the panel of pathogens in 1047(69.28%) patients from 1533 qualified patients at the end. We found that the bacterial titer quantified based on qLAMP is a predictor of probability that the bacterium in the sample can be detected in culture assay. The relatedness of the two assays fits a logistic regression curve. We used a piecewise linear function to define breakpoints where latent pathogen abruptly change its competitive relationship with others in the panel. These breakpoints, where pathogens start to propagate abnormally, are used as cutoffs to eliminate the influence of contaminations from normal flora. With help of the cutoffs derived from statistical analysis, we are able to identify causative pathogens in 750 (48.92%) patients from qualified patients. In conclusion, qLAMP is a reliable method in quantifying bacterial titer. Despite the fact that there are always latent bacteria contaminated in sputum samples, we can identify causative pathogens based on cutoffs derived from statistical analysis of competitive relationship. Trial Registration ClinicalTrials.gov NCT00567827 PMID:22719933

Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins

PubMed Central

1991-01-01

The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one- fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1. PMID:1714460

Heavy tailed bacterial motor switching statistics define macroscopic transport properties during upstream contamination by E. coli

NASA Astrophysics Data System (ADS)

Figueroa-Morales, N.; Rivera, A.; Altshuler, E.; Darnige, T.; Douarche, C.; Soto, R.; Lindner, A.; Clément, E.

The motility of E. Coli bacteria is described as a run and tumble process. Changes of direction correspond to a switch in the flagellar motor rotation. The run time distribution is described as an exponential decay of characteristic time close to 1s. Remarkably, it has been demonstrated that the generic response for the distribution of run times is not exponential, but a heavy tailed power law decay, which is at odds with the motility findings. We investigate the consequences of the motor statistics in the macroscopic bacterial transport. During upstream contamination processes in very confined channels, we have identified very long contamination tongues. Using a stochastic model considering bacterial dwelling times on the surfaces related to the run times, we are able to reproduce qualitatively and quantitatively the evolution of the contamination profiles when considering the power law run time distribution. However, the model fails to reproduce the qualitative dynamics when the classical exponential run and tumble distribution is considered. Moreover, we have corroborated the existence of a power law run time distribution by means of 3D Lagrangian tracking. We then argue that the macroscopic transport of bacteria is essentially determined by the motor rotation statistics.

DNA damage and external lesions in brown bullheads (Ameiurus nebulosus) from contaminated habitats

USGS Publications Warehouse

Yang, X.; Meier, J.; Chang, L.; Rowan, M.; Baumann, P.C.

2006-01-01

The Comet assay was used to compare levels of DNA damage in brown bullheads (Ameiurus nebulosus) collected from three known contaminated locations, the Cuyahoga River (OH, USA), Ashtabula River (OH, USA; both tributaries to Lake Erie, USA), and Ashumet Pond (Cape Cod, MA, USA), with brown bullheads collected from three paired reference sites, Old Woman Creek (OH, USA), Conneaut River (OH, USA; both tributaries to Lake Erie), and Great Herring Pond (mainland MA, USA), respectively. Blood was sampled from each fish, and the Comet assay was conducted on erythrocytes. The assay results demonstrate that fish from the three contaminated sites each suffered higher DNA damage compared with fish from their respective reference sites. The results also show that the genetic damage was associated with the occurrence of external lesions and deformities in fish. The Comet assay is sufficiently sensitive to detect exposure of natural fish populations to environmental levels of genotoxic contaminants. ?? 2006 SETAC.

Bacterial contamination of stethoscope chest pieces and the effect of daily cleaning.

PubMed

Fujita, H; Hansen, B; Hanel, R

2013-01-01

Stethoscopes are a potential source of nosocomial infection for hospitalized humans, a phenomenon not previously studied in companion animals. To determine if daily cleaning of stethoscope chest pieces reduces bacterial contamination between cleanings. Client-owned dogs and cats. Prospective observational study. In phase 1, bacterial cultures were obtained from the chest pieces of 10 participant stethoscopes once weekly for 3 weeks. In phase 2, stethoscopes were cleaned daily and 2 culture samples were obtained once weekly, immediately before and after cleaning with 70% isopropyl alcohol, for 3 weeks. Daily cleaning eliminated bacteria immediately after each cleaning (P = .004), but did not reduce the rate of positive cultures obtained before cleaning in phase 2. Cultures were positive for 20/30 (67%) samples during phase 1 and 18/30 (60%) obtained before daily cleaning during phase 2. Recovered organisms included normal skin flora, agents of opportunistic infections, and potential pathogens. The only genus that was repeatedly recovered from the same stethoscope for 2 or more consecutive weeks was Bacillus sp. Daily cleaning was highly effective at removing bacteria, but provided no reduction in precleaning contamination. Cleaning stethoscopes after use on dogs or cats infected with pathogenic bacteria and before use on immunocompromised animals should be considered. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Viral and bacterial contamination in a sedimentary aquifer in Uruguay: evaluation of coliforms as regional indicators of viral contamination.

NASA Astrophysics Data System (ADS)

Gamazo, Pablo; Colina, Rodney; Victoria, Matias; Alvareda, Elena; Burutatran, Luciana; Ramos, Julian; Olivera, María; Soler, Joan

2015-04-01

In many areas of Uruguay groundwater is the only source of water for human consumption and for industrial-agricultural economic activities. Traditionally considered as a safe source, groundwater is commonly used without any treatment. The Uruguayan law requires bacteriological (fecal) analysis for most water uses, but virological analyses are not mentioned in the legislation. In the Salto district, where groundwater is used for human consumption and for agricultural activities, bacterial contamination has been detected in several wells but no viruses analysis have been performed. The Republic University (UDELAR), with the support of the National Agency for Research and Innovation (ANII), is studying the incidence of virus and fecal bacteria in groundwater on an intensive agriculture area of the Salto district. An initial screening campaign of 44 wells was performed in which, besides total and fecal coliforms, rotavirus and adenovirus were detected. A subgroup of the screening wells (15) where selected for bimonthly sampling during a year. In accordance with literature results, single well data analysis shows that coliform and viral contamination can be considered as independent variables. However, when spatial data is integrated, coliform and viral contamination show linear correlation. In this work we present the survey results, we analyse the temporal incidence of variables like precipitation, temperature and chemical composition in well contamination and we discuss the value of coliforms as global indicator of viral contamination for the Salto aquifer.

Bacterial community composition in different sediments from the Eastern Mediterranean Sea: a comparison of four 16S ribosomal DNA clone libraries.

PubMed

Polymenakou, Paraskevi N; Bertilsson, Stefan; Tselepides, Anastasios; Stephanou, Euripides G

2005-10-01

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.

Biphenyl-Metabolizing Microbial Community and a Functional Operon Revealed in E-Waste-Contaminated Soil.

PubMed

Jiang, Longfei; Luo, Chunling; Zhang, Dayi; Song, Mengke; Sun, Yingtao; Zhang, Gan

2018-05-18

Primitive electronic waste (e-waste) recycling activities release massive amounts of persistent organic pollutants (POPs) and heavy metals into surrounding soils, posing a major threat to the ecosystem and human health. Microbes capable of metabolizing POPs play important roles in POPs remediation in soils, but their phylotypes and functions remain unclear. Polychlorinated biphenyls (PCBs), one of the main pollutants in e-waste contaminated soils, have drawn increasing attention due to their high persistence, toxicity, and bioaccumulation. In the present study, we employed the culture-independent method of DNA stable-isotope probing to identify active biphenyl and PCB degraders in e-waste-contaminated soil. A total of 19 rare operational taxonomic units and three dominant bacterial genera ( Ralstonia, Cupriavidus, and uncultured bacterium DA101) were enriched in the 13 C heavy DNA fraction, confirming their functions in PCBs metabolism. Additionally, a 13.8 kb bph operon was amplified, containing a bphA gene labeled by 13 C that was concentrated in the heavy DNA fraction. The tetranucleotide signature characteristics of the bph operon suggest that it originated from Ralstonia. The bph operon may be shared by horizontal gene transfer because it contains a transposon gene and is found in various bacterial species. This study gives us a deeper understanding of PCB-degrading mechanisms and provides a potential resource for the bioremediation of PCBs-contaminated soils.

Maternal blood contamination of collected cord blood can be identified using DNA methylation at three CpGs.

PubMed

Morin, Alexander M; Gatev, Evan; McEwen, Lisa M; MacIsaac, Julia L; Lin, David T S; Koen, Nastassja; Czamara, Darina; Räikkönen, Katri; Zar, Heather J; Koenen, Karestan; Stein, Dan J; Kobor, Michael S; Jones, Meaghan J

2017-01-01

Cord blood is a commonly used tissue in environmental, genetic, and epigenetic population studies due to its ready availability and potential to inform on a sensitive period of human development. However, the introduction of maternal blood during labor or cross-contamination during sample collection may complicate downstream analyses. After discovering maternal contamination of cord blood in a cohort study of 150 neonates using Illumina 450K DNA methylation (DNAm) data, we used a combination of linear regression and random forest machine learning to create a DNAm-based screening method. We identified a panel of DNAm sites that could discriminate between contaminated and non-contaminated samples, then designed pyrosequencing assays to pre-screen DNA prior to being assayed on an array. Maternal contamination of cord blood was initially identified by unusual X chromosome DNA methylation patterns in 17 males. We utilized our DNAm panel to detect contaminated male samples and a proportional amount of female samples in the same cohort. We validated our DNAm screening method on an additional 189 sample cohort using both pyrosequencing and DNAm arrays, as well as 9 publically available cord blood 450K data sets. The rate of contamination varied from 0 to 10% within these studies, likely related to collection specific methods. Maternal blood can contaminate cord blood during sample collection at appreciable levels across multiple studies. We have identified a panel of markers that can be used to identify this contamination, either post hoc after DNAm arrays have been completed, or in advance using a targeted technique like pyrosequencing.

Bacterial Communities in the Groundwater of Xikuangshan Antimony Mine, China

NASA Astrophysics Data System (ADS)

Wu, M.; Wang, H.; Wang, N.; Wang, M.

2017-12-01

Xikuangshan (XKS) is the biggest antimony (Sb) mine around the word, which causes serious environmental contamination due to the mining actives. To fully understand the bacterial compositions in the groundwater around the mining area in XKS and their correlation with environmental factors, groundwater samples were collected and subject to 16S rDNA high throughput sequencing. Results indicated that Proteobacteria (especially Gamma-Proteobacteria) dominated bacterial communities in high-Sb groundwater samples, whereas Bacteroidetes predominated in low-Sb groundwater. Furthermore, antimony concentration was found to be the most significant factor shaping bacterial communities (P=0.002) with an explanation of 9.16% of the variation. Other factors such as pH, contents of Mg, Ca and orthophosphate were also observed to significantly correlate with bacterial communities. This was the first report to show the important impact of Sb concentration on bacterial community structure in the groundwater in the mining area. Our results will enhance the understanding of subsurface biogeochemical processes mediated by microbes.

DNA damage in Mexican children living in high-risk contaminated scenarios.

PubMed

Jasso-Pineda, Yolanda; Díaz-Barriga, Fernando; Yáñez-Estrada, Leticia; Pérez-Vázquez, Francisco Javier; Pérez-Maldonado, Ivan Nelinho

2015-06-15

The aim of this study was to evaluate the deoxyribonucleic acid (DNA) damage (as a biomarker of biological effects) in children living in areas at high risk of contamination in Mexico using the comet assay. The alkaline comet assay was performed in order to assess DNA damage levels in blood cells of 276 children living in eleven communities in four states of Mexico. Moreover, levels of arsenic and 1-hydroxypyrene (1-OHP) in urine and lead and total DDT [sum of 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT)] in blood were quantified. We found urinary 1-OHP levels between DNA damage (comet assay), the most important finding in our study was that children exposed to a chemical mixture [high levels of exposure to polycyclic aromatic hydrocarbons (PAHs) and DDT were found] had the significant highest DNA damage level (p<0.05) in their blood cells (olive tail moment=7.5±3.5), when compared with DNA damage levels in children living in the other scenarios assessed in this work. Finally, significant correlations were observed between urinary arsenic levels (r=0.32, p<0.05); urinary 1-OHP levels (r=0.65, p<0.01); total DDT in blood levels (r=0.59, p<0.01) and DNA damage. In conclusion, the data indicates that children living in areas which are at high risk of contamination showed high levels of biomarkers of exposure in urine or blood. Moreover, the exposure levels contribute to DNA damage and suggest an increased health risk in studied sites at risk of great pollution. Copyright © 2015 Elsevier B.V. All rights reserved.

Seasonal Liza aurata tissue-specific DNA integrity in a multi-contaminated coastal lagoon (Ria de Aveiro, Portugal).

PubMed

Oliveira, M; Maria, V L; Ahmad, I; Pacheco, M; Santos, M A

2010-10-01

In this study, the DNA integrity of golden grey mullet (Liza aurata) collected in differently contaminated sites of a coastal lagoon, Ria de Aveiro (Portugal), was assessed, over the period of 1 year, using the DNA alkaline unwinding assay, in four different tissues (gill, kidney, liver and blood) and compared to a reference site. The four tissues displayed different DNA integrity basal levels, clearly affected by seasonal factors. Gill and kidney were, respectively, the most and least sensitive tissues. All sites demonstrated the capacity to interfere with DNA integrity. The sites displaying the highest and lowest DNA damage capability were, respectively, Barra (subject to naval traffic) and Vagos (contaminated with polycyclic aromatic hydrocarbons). In terms of seasonal variability, autumn seems to be the more critical season (more DNA damage) unlike summer when no DNA damage was found in any tissue. Data recommend the continued monitoring of this aquatic system. Copyright © 2010 Elsevier Ltd. All rights reserved.

Is repetitive intraoperative splash basin use a source of bacterial contamination in total joint replacement?

PubMed

Glait, Sergio A; Schwarzkopf, Ran; Gould, Steven; Bosco, Joseph; Slover, James

2011-09-09

Splash basins are used in arthroplasty cases to wash instruments. Several studies in the literature have shown these basins being a potential source of bacterial infection. This study assesses the risk of contamination of intraoperative splash basins used to wash and store instruments. A total of 46 random clean primary arthroplasty cases (32 hips, 13 knees, and 1 unicondylar knee) were studied by taking cultures of sterile splash basins as soon as they are opened (controls) and again at wound closure after instruments and debris have come into contact with the sterile water. All cultures were taken with sterile culture swabs and sent to the laboratory for aerobic, anaerobic, and fungal culture. Outcome measured was any positive culture. A total of 92 cultures from 46 cases were tested. Only 1 (2.17%) control culture, which grew Streptococcus viridans, was positive for bacterial growth. One of 46 samples (2.17%) taken at wound closure was positive for coagulase-negative Staphylococcus. Mean time between basin opening and wound closure was 180±45 minutes. For the 1 infected sample taken at the conclusion of the case, it was 240 minutes. Previous studies show contamination rates as high as 74% for splash basins used intraoperatively. Our study contradicts the belief that splash basins are a high source of infection, with only 2.17% of basins showing contamination. Splash basins can be a potential source of contamination, but the risk is not as high as previously cited in the orthopedic literature. Copyright 2011, SLACK Incorporated.

The development and application of a molecular community profiling strategy to identify polymicrobial bacterial DNA in the whole blood of septic patients.

PubMed

Faria, M M P; Conly, J M; Surette, M G

2015-10-16

The application of molecular based diagnostics in sepsis has had limited success to date. Molecular community profiling methods have indicated that polymicrobial infections are more common than suggested by standard clinical culture. A molecular profiling approach was developed to investigate the propensity for polymicrobial infections in patients predicted to have bacterial sepsis. Disruption of blood cells with saponin and hypotonic shock enabled the recovery of microbial cells with no significant changes in microbial growth when compared to CFU/ml values immediately prior to the addition of saponin. DNA extraction included a cell-wall digestion step with both lysozyme and mutanolysin, which increased the recovery of terminal restriction fragments by 2.4 fold from diverse organisms. Efficiencies of recovery and limits of detection using Illumina sequencing of the 16S rRNA V3 region were determined for both viable cells and DNA using mock bacterial communities inoculated into whole blood. Bacteria from pre-defined communities could be recovered following lysis and removal of host cells with >97% recovery of total DNA present. Applying the molecular profiling methodology to three septic patients in the intensive care unit revealed microbial DNA from blood had consistent alignment with cultured organisms from the primary infection site providing evidence for a bloodstream infection in the absence of a clinical lab positive blood culture result in two of the three cases. In addition, the molecular profiling indicated greater diversity was present in the primary infection sample when compared to clinical diagnostic culture. A method for analyzing bacterial DNA from whole blood was developed in order to characterize the bacterial DNA profile of sepsis infections. Preliminary results indicated that sepsis infections were polymicrobial in nature with the bacterial DNA recovered suggesting a more complex etiology when compared to blood culture data.

Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

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

Kennedy, Edward M.; Cullen, Bryan R., E-mail: bryan.cullen@duke.edu

CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called singlemore » guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA

Effect of mobile laminar airflow units on airborne bacterial contamination during neurosurgical procedures.

PubMed

von Vogelsang, A-C; Förander, P; Arvidsson, M; Löwenhielm, P

2018-03-24

Surgical site infections (SSIs) after neurosurgery are potentially life-threatening and entail great costs. SSIs may occur from airborne bacteria in the operating room, and ultraclean air is desired during infection-prone cleaning procedures. Door openings and the number of persons present in the operating room affect the air quality. Mobile laminar airflow (MLAF) units, with horizontal laminar airflow, have previously been shown to reduce airborne bacterial contamination. To assess the effect of MLAF units on airborne bacterial contamination during neurosurgical procedures. In a quasi-experimental design, bacteria-carrying particles (colony-forming units: cfu) during neurosurgical procedures were measured with active air-sampling in operating rooms with conventional turbulent ventilation, and with additional MLAF units. The MLAF units were shifted between operating rooms monthly. Colony-forming unit count and bacterial species detection were conducted after incubation. Data was collected for a period of 18 months. A total of 233 samples were collected during 45 neurosurgical procedures. The use of MLAF units significantly reduced the numbers of cfu in the surgical site area (P < 0.001) and above the instrument table (P < 0.001). Logistic regression showed that the only significant predictor affecting cfu count was the use of MLAF units (odds ratio: 41.6; 95% confidence interval: 11.3-152.8; P < 0.001). The most frequently detected bacteria were coagulase-negative staphylococci. MLAF successfully reduces cfu during neurosurgery to ultraclean air levels. MLAF units are valuable when the main operating room ventilation system is unable to produce ultraclean air in infection-prone clean neurosurgery. Copyright © 2018 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Bacterial contamination of ex vivo processed PBPC products under clean room conditions.

PubMed

Ritter, Markus; Schwedler, Joachim; Beyer, Jörg; Movassaghi, Kamran; Mutters, Reinier; Neubauer, Andreas; Schwella, Nimrod

2003-11-01

Patients undergoing high-dose radio- and/or chemotherapy and autologous or allogeneic PBPC transplantation are at high risk for infections owing to profound immunosuppression. In this study, the rate of microbial contamination of ex vivo processed PBPC products was analyzed, comparing preparation under clean room conditions to standard laboratory conditions. After implementation of good manufacturing practice conditions in the two participating institutions, the microbial contamination rate of 366 PBPC harvests from 198 patients was determined under certified clean room conditions (Group A) from 2000 until 2002. To investigate influence of improved environmental conditions along with other parameters, this set of samples was compared with a historical control set of 1413 PBPC products, which have been processed ex vivo under a clean bench in a regular laboratory room and were harvested from 626 patients (Group B) from 1989 until 2000. In Group B microbial contamination was found in 74 PBPC products (5.2%) from 57 patients. In Group A microbial growth was detected in 3 leukapheresis products (0.8%) from 3 patients. After exclusion of PBPC products, which were probably contaminated before manipulation, statistical analysis showed a significant difference (chi2= 10.339; p < 0.001). These data suggest an impact of clean room conditions on the bacterial contamination rate of PBPC products. To identify confounding variables, variables like technique of leukapheresis, culture methodology, and microbial colonization of central venous catheters were taken into account. Further variables might be identified in following studies.

Evaluation of a pulsed xenon ultraviolet disinfection system to decrease bacterial contamination in operating rooms.

PubMed

El Haddad, Lynn; Ghantoji, Shashank S; Stibich, Mark; Fleming, Jason B; Segal, Cindy; Ware, Kathy M; Chemaly, Roy F

2017-10-10

Environmental cleanliness is one of the contributing factors for surgical site infections in the operating rooms (ORs). To decrease environmental contamination, pulsed xenon ultraviolet (PX-UV), an easy and safe no-touch disinfection system, is employed in several hospital environments. The positive effect of this technology on environmental decontamination has been observed in patient rooms and ORs during the end-of-day cleaning but so far, no study explored its feasibility between surgical cases in the OR. In this study, 5 high-touch surfaces in 30 ORs were sampled after manual cleaning and after PX-UV intervention mimicking between-case cleaning to avoid the disruption of the ORs' normal flow. The efficacy of a 1-min, 2-min, and 8-min cycle were tested by measuring the surfaces' contaminants by quantitative cultures using Tryptic Soy Agar contact plates. We showed that combining standard between-case manual cleaning of surfaces with a 2-min cycle of disinfection using a portable xenon pulsed ultraviolet light germicidal device eliminated at least 70% more bacterial load after manual cleaning. This study showed the proof of efficacy of a 2-min cycle of PX-UV in ORs in eliminating bacterial contaminants. This method will allow a short time for room turnover and a potential reduction of pathogen transmission to patients and possibly surgical site infections.

Sources and Pathways of Bacterial Contamination in Urban Streams and Ocean Beaches, Santa Barbara, California

NASA Astrophysics Data System (ADS)

Johnson, R. D.; Mendez, G. O.; La, J. X.; Izbicki, J. A.

2005-12-01

Streams and ocean beaches in Santa Barbara, California, occasionally have concentrations of fecal indicator bacteria that exceed public health standards for recreational water, forcing temporary beach closures. Possible sources of fecal bacteria contamination include transient human populations, animal populations, and leaking sewer lines. The purpose of this three-year study is to identify important sources of fecal bacteria affecting the urban streams and beaches and to identify important pathways of transport. Contamination may enter streams and beaches directly by surface runoff, but also may be transmitted short distances through shallow ground water. Our analysis of existing historical data shows that fecal indicator bacteria concentrations are higher in near-shore ocean water following extreme high tides. The possible role of near shore ground water in supplying contaminants to the sea will be investigated by sampling water from an array of shallow wells installed for this study between an older city sewer line and the ocean. The ground water flux to the ocean will be inferred from water levels in these wells, and further tested by radium isotope values in near shore ocean samples. Two additional well arrays will be installed to test for leakage from residential sewage hookups and measure associated exchanges between ground water, streams, and ocean. Preliminary data collected by this study show fecal indicator bacteria concentrations in urban reaches of Mission Creek and its tributaries, the principle drainage through the city, are higher during low flow periods than during periods of higher flow. Analysis of preliminary data also shows short-term temporal variations in bacterial concentrations during twenty-four hour periods. Human enterovirus has been detected in our sample from one urban-drain tributary to Mission Creek. In order to identify the origins of fecal indicator bacteria water samples from Mission Creek, its tributaries, urban drains, and

Bioremediation of PCB-contaminated shallow river sediments: The efficacy of biodegradation using individual bacterial strains and their consortia.

PubMed

Horváthová, Hana; Lászlová, Katarína; Dercová, Katarína

2018-02-01

Elimination of dangerous toxic and hydrophobic chlorinated aromatic compounds, mainly PCBs from the environment, is one of the most important aims of the environmental biotechnologies. In this work, biodegradation of an industrial mixture of PCBs (Delor 103, equivalent to Aroclor 1242) was performed using bacterial consortia composed of four bacterial strains isolated from the historically PCB-contaminated sediments and characterized as Achromobacter xylosoxidans, Stenotrophomonas maltophilia, Ochrobactrum anthropi and Rhodococcus ruber. The objective of this research was to determine the biodegradation ability of the individual strains and artificially prepared consortia composed of two or three bacterial strains mentioned above. Based on the growth parameters, six consortia were constructed and inoculated into the historically contaminated sediment samples collected in the efflux canal of Chemko Strážske plant - the former producer of the industrial mixtures of PCBs. The efficacy of the biotreatment, namely bioaugmentation, was evaluated by determination of ecotoxicity of treated and non-treated sediments. The most effective consortia were those containing the strain R. ruber. In the combination with A. xylosoxidans, the biodegradation of the sum of the indicator congeners was 85% and in the combination with S. maltophilia nearly 80%, with inocula applied in the ratio 1:1 in both cases. Consortium containing the strain R. ruber and S. maltophilia showed pronounced degradation of the highly chlorinated PCB congeners. Among the consortia composed of three bacterial strains, only that consisting of O. anthropi, R. ruber and A. xylosoxidans showed higher biodegradation (73%). All created consortia significally reduced the toxicity of the contaminated sediment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation and characterization of a hydrocarbonoclastic bacterial enrichment from total petroleum hydrocarbon contaminated sediments: potential candidates for bioaugmentation in bio-based processes.

PubMed

Di Gregorio, Simona; Siracusa, Giovanna; Becarelli, Simone; Mariotti, Lorenzo; Gentini, Alessandro; Lorenzi, Roberto

2016-06-01

Seven hydrocarbonoclastic new bacterial isolates were isolated from dredged sediments of a river estuary in Italy. The sediments were contaminated by shipyard activities since decades, mainly ascribable to the exploitation of diesel oil as the fuel for recreational and commercial navigation of watercrafts. The bacterial isolates were able to utilize diesel oil as sole carbon source. Their metabolic capacities were evaluated by GC-MS analysis, with reference to the depletion of both the normal and branched alkanes, the nC18 fatty acid methyl ester and the unresolved complex mixture of organic compounds. They were taxonomically identified as different species of Stenotrophomonas and Pseudomonas spp. by the combination of amplified ribosomal DNA restriction analysis (ARDRA) and repetitive sequence-based PCR (REP-PCR) analysis. The metabolic activities of interest were analyzed both in relation to the single bacterial strains and to the combination of the latter as a multibacterial species system. After 6 days of incubation in mineral medium with diesel oil as sole carbon source, the Stenotrophomonas sp. M1 strain depleted 43-46 % of Cn-alkane from C28 up to C30, 70 % of the nC18 fatty acid methyl ester and the 46 % of the unresolved complex mixture of organic compounds. On the other hand, the Pseudomonas sp. NM1 strain depleted the 76 % of the nC18 fatty acid methyl ester, the 50 % of the unresolved complex mixture of organic compounds. The bacterial multispecies system was able to completely deplete Cn-alkane from C28 up to C30 and to deplete the 95 % of the unresolved complex mixture of organic compounds. The isolates, either as single strains and as a bacterial multispecies system, were proposed as candidates for bioaugmentation in bio-based processes for the decontamination of dredged sediments.

Effects of the inoculant strain Sphingomonas paucimobilis 20006FA on soil bacterial community and biodegradation in phenanthrene-contaminated soil.

PubMed

Coppotelli, B M; Ibarrolaza, A; Del Panno, M T; Morelli, I S

2008-02-01

The effects of the inoculant strain Sphingomonas paucimobilis 20006FA (isolated from a phenanthrene-contaminated soil) on the dynamics and structure of microbial communities and phenanthrene elimination rate were studied in soil microcosms artificially contaminated with phenanthrene. The inoculant managed to be established from the first inoculation as it was evidenced by denaturing gradient gel electrophoresis analysis, increasing the number of cultivable heterotrophic and PAH-degrading cells and enhancing phenanthrene degradation. These effects were observed only during the inoculation period. Nevertheless, the soil biological activity (dehydrogenase activity and CO(2) production) showed a late increase. Whereas gradual and successive changes in bacterial community structures were caused by phenanthrene contamination, the inoculation provoked immediate, significant, and stable changes on soil bacterial community. In spite of the long-term establishment of the inoculated strain, at the end of the experiment, the bioaugmentation did not produce significant changes in the residual soil phenanthrene concentration and did not improve the residual effects on the microbial soil community.

Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

PubMed

Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

2015-12-01

Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival. Copyright © 2015 Elsevier Ltd. All rights reserved.

Forensic Analysis of Human DNA from Samples Contamined with Bioweapons Agents

DTIC Science & Technology

2011-10-01

Forensic analysis of human DNA from samples contaminated with bioweapons agents Jason Timbers Kathryn Wright Royal Canadian Mounted...Police Forensic Science and Identification Service Prepared By: Royal Canadian Mounted Police RCMP Forensic Science Identification Services... Royal Canadian Mounted Police Forensic Science and Identification Service Prepared By: Royal Canadian Mounted Police RCMP Forensic Science

Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

PubMed

Fuchs-Telka, Sabine; Fister, Susanne; Mester, Patrick-Julian; Wagner, Martin; Rossmanith, Peter

2017-02-01

DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr + ][Ntf 2 - ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

Isolation of PCR quality microbial community DNA from heavily contaminated environments.

PubMed

Gunawardana, Manjula; Chang, Simon; Jimenez, Abraham; Holland-Moritz, Daniel; Holland-Moritz, Hannah; La Val, Taylor P; Lund, Craig; Mullen, Madeline; Olsen, John; Sztain, Terra A; Yoo, Jennifer; Moss, John A; Baum, Marc M

2014-07-01

Asphalts, biochemically degraded oil, contain persistent, water-soluble compounds that pose a significant challenge to the isolation of PCR quality DNA. The adaptation of existing DNA purification protocols and commercial kits proved unsuccessful at overcoming this hurdle. Treatment of aqueous asphalt extracts with a polyamide resin afforded genomic microbial DNA templates that could readily be amplified by PCR. Physicochemically distinct asphalt samples from five natural oil seeps successfully generated the expected 291 bp amplicons targeting a region of the 16S rRNA gene, illustrating the robustness of the method. DNA recovery yields were in the 50-80% range depending on how the asphalt sample was seeded with exogenous DNA. The scope of the new method was expanded to include soil with high humic acid content. DNA from soil samples spiked with a range of humic acid concentrations was extracted with a commercial kit followed by treatment with the polyamide resin. The additional step significantly improved the purity of the DNA templates, especially at high humic acid concentrations, based on qPCR analysis of the bacterial 16S rRNA genes. The new method has the advantages of being inexpensive, simple, and rapid and should provide a valuable addition to protocols in the field of petroleum and soil microbiology. Copyright © 2014 Elsevier B.V. All rights reserved.

Yeast Derived LysA2 Can Control Bacterial Contamination in Ethanol Fermentation.

PubMed

Kim, Jun-Seob; Daum, M Angela; Jin, Yong-Su; Miller, Michael J

2018-05-24

Contamination of fuel-ethanol fermentations continues to be a significant problem for the corn and sugarcane-based ethanol industries. In particular, members of the Lactobacillaceae family are the primary bacteria of concern. Currently, antibiotics and acid washing are two major means of controlling contaminants. However, antibiotic use could lead to increased antibiotic resistance, and the acid wash step stresses the fermenting yeast and has limited effectiveness. Bacteriophage endolysins such as LysA2 are lytic enzymes with the potential to contribute as antimicrobials to the fuel ethanol industries. Our goal was to evaluate the potential of yeast-derived LysA2 as a means of controlling Lactobacillaceae contamination. LysA2 intracellularly produced by Pichia pastoris showed activity comparable to Escherichia coli produced LysA2. Lactic Acid Bacteria (LAB) with the A4α peptidoglycan chemotype (L-Lys-D-Asp crosslinkage) were the most sensitive to LysA2, though a few from that chemotype were insensitive. Pichia -expressed LysA2, both secreted and intracellularly produced, successfully improved ethanol productivity and yields in glucose (YPD60) and sucrose-based (sugarcane juice) ethanol fermentations in the presence of a LysA2 susceptible LAB contaminant. LysA2 secreting Sacharomyces cerevisiae did not notably improve production in sugarcane juice, but it did control bacterial contamination during fermentation in YPD60. Secretion of LysA2 by the fermenting yeast, or adding it in purified form, are promising alternative tools to control LAB contamination during ethanol fermentation. Endolysins with much broader lytic spectrums than LysA2 could supplement or replace the currently used antibiotics or the acidic wash.

Comparison of two freshwater turtle species as monitors of radionuclide and chemical contamination: DNA damage and residue analysis

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

Meyers-Schoene, L.; Shugart, L.R.; Beauchamp, J.J.

1993-08-01

Two species of turtles that occupy different ecological niches were compared for their usefulness as monitors of freshwater ecosystems where both low-level radioactive and nonradioactive contaminants are present. The pond slider (Trachemys scripta) and common snapping turtle (Chelydra serpentina) were analyzed for the presence of [sup 90]Sr, [sup 137]Cs, [sup 60]Co, and Hg, radionuclides and chemicals known to be present at the contaminated site, and single-strand breaks in liver DNA. The integrity of the DNA was examined by the alkaline unwinding assay, a technique that detects strand breaks as a biological marker of possible exposure to genotoxic agents. This measuremore » of DNA damage was significantly increased in both species of turtles at the contaminated site compared with turtles of the same species at a reference site, and shows that contaminant-exposed populations were under more severe genotoxic stress than those at the reference site. The level of strand breaks observed at the contaminated site was high and in the range reported for other aquatic species exposed to deleterious concentrations of genotoxic agents such as chemicals and ionizing radiation. Statistically significantly higher concentrations of radionuclides and Hg were detected in the turtles from the contaminated area. Mercury concentrations were significantly higher in the more carnivorous snapping turtle compared with the slider; however, both species were effective monitors of the contaminants.« less

Bacterial Contamination and Disinfection Status of Laryngoscopes Stored in Emergency Crash Carts

PubMed Central

Lee, Jung Won; Shin, Hee Bong; Lee, In Kyung

2017-01-01

Objectives To identify bacterial contamination rates of laryngoscope blades and handles stored in emergency crash carts by hospital and area according to the frequency of intubation attempts. Methods One hundred forty-eight handles and 71 blades deemed ready for patient use from two tertiary hospitals were sampled with sterile swabs using a standardized rolling technique. Samples were considered negative (not contaminated) if no colonies were present on the blood agar plate after an 18-hour incubation period. Samples were stratified by hospital and according to the frequency of intubation attempts (10 attempts per year) using the χ2-test and Fisher exact test. Results One or more species of bacteria were isolated from 4 (5.6%) handle tops, 20 (28.2%) handles with knurled surfaces, and 27 (18.2%) blades. No significant differences were found in microbial contamination levels on the handle tops and blades between the two hospitals and two areas according to the frequency of intubation attempts. However, significant differences were found between the two hospitals and two areas in the level of microbial contamination on the handles with knurled surfaces (p<0.05). Conclusions Protocols and policies must be reviewed to standardize procedures to clean and disinfect laryngoscope blades and handles; handles should be re-designed to eliminate points of contact with the blade; and single-use, one-piece laryngoscopes should be introduced. PMID:28605891

DNA-polyfluorophore Chemosensors for Environmental Remediation: Vapor-phase Identification of Petroleum Products in Contaminated Soil†

PubMed Central

Jiang, Wei; Wang, Shenliang; Yuen, Lik Hang; Kwon, Hyukin; Ono, Toshikazu

2013-01-01

Contamination of soil and groundwater by petroleum-based products is an extremely widespread and important environmental problem. Here we have tested a simple optical approach for detecting and identifying such industrial contaminants in soil samples, using a set of fluorescent DNA-based chemosensors in pattern-based sensing. We used a set of diverse industrial volatile chemicals to screen and identify a set of five short oligomeric DNA fluorophores on PEG-polystyrene microbeads that could differentiate the entire set after exposure to their vapors in air. We then tested this set of five fluorescent chemosensor compounds for their ability to respond with fluorescence changes when exposed to headgas over soil samples contaminated with one of ten different samples of crude oil, petroleum distillates, fuels, lubricants and additives. Statistical analysis of the quantitative fluorescence change data (as Δ(R,G,B) emission intensities) revealed that these five chemosensors on beads could differentiate all ten product mixtures at 1000 ppm in soil within 30 minutes. Tests of sensitivity with three of the contaminant mixtures showed that they could be detected and differentiated in amounts at least as low as one part per million in soil. The results establish that DNA-polyfluorophores may have practical utility in monitoring the extent and identity of environmental spills and leaks, while they occur and during their remediation. PMID:23878719

Identification of Fic-1 as an enzyme that inhibits bacterial DNA replication by AMPylating GyrB, promoting filament formation.

PubMed

Lu, Canhua; Nakayasu, Ernesto S; Zhang, Li-Qun; Luo, Zhao-Qing

2016-01-26

The morphology of bacterial cells is important for virulence, evasion of the host immune system, and coping with environmental stresses. The widely distributed Fic proteins (filamentation induced by cAMP) are annotated as proteins involved in cell division because of the presence of the HPFx[D/E]GN[G/K]R motif. We showed that the presence of Fic-1 from Pseudomonas fluorescens significantly reduced the yield of plasmid DNA when expressed in Escherichia coli or P. fluorescens. Fic-1 interacted with GyrB, a subunit of DNA gyrase, which is essential for bacterial DNA replication. Fic-1 catalyzed the AMPylation of GyrB at Tyr(109), a residue critical for binding ATP, and exhibited auto-AMPylation activity. Mutation of the Fic-1 auto-AMPylated site greatly reduced AMPylation activity toward itself and toward GyrB. Fic-1-dependent AMPylation of GyrB triggered the SOS response, indicative of DNA replication stress or DNA damage. Fic-1 also promoted the formation of elongated cells when the SOS response was blocked. We identified an α-inhibitor protein that we named anti-Fic-1 (AntF), encoded by a gene immediately upstream of Fic-1. AntF interacted with Fic-1, inhibited the AMPylation activity of Fic-1 for GyrB in vitro, and blocked Fic-1-mediated inhibition of DNA replication in bacteria, suggesting that Fic-1 and AntF comprise a toxin-antitoxin module. Our work establishes Fic-1 as an AMPylating enzyme that targets GyrB to inhibit DNA replication and may target other proteins to regulate bacterial morphology. Copyright © 2016, American Association for the Advancement of Science.

Bioremediation potential of a tropical soil contaminated with a mixture of crude oil and production water.

PubMed

Alvarez, Vanessa Marques; Santos, Silvia Cristina Cunha Dos Santos; Casella, Renata da Costa; Vital, Ronalt Leite; Sebastin, Gina Vasquez; Seldin, Lucy

2008-12-01

A typical tropical soil from the northeast of Brazil, where an important terrestrial oil field is located, was accidentally contaminated with a mixture of oil and saline production water. To study the bioremediation potential in this area, molecular methods based on PCR-DGGE were used to determine the diversity of the bacterial communities in bulk and in contaminated soils. Bacterial fingerprints revealed that the bacterial communities were affected by the presence of the mixture of oil and production water, and different profiles were observed when the contaminated soils were compared with the control. Halotolerant strains capable of degrading crude oil were also isolated from enrichment cultures obtained from the contaminated soil samples. Twenty-two strains showing these features were characterized genetically by amplified ribosomal DNA restriction analysis (ARDRA) and phenotypically by their colonial morphology and tolerance to high NaCl concentrations. Fifteen ARDRA groups were formed. Selected strains were analyzed by 16S rDNA sequencing, and Actinobacteria was identified as the main group found. Strains were also tested for their growth capability in the presence of different oil derivatives (hexane, dodecane, hexadecane, diesel, gasoline, toluene, naphthalene, o-xylene, and p-xylene) and different degradation profiles were observed. PCR products were obtained from 12 of the 15 ARDRA representatives when they were screened for the presence of the alkane hydroxylase gene (alkB). Members of the genera Rhodococcus and Gordonia were identified as predominant in the soil studied. These genera are usually implicated in oil degradation processes and, as such, the potential for bioremediation in this area can be considered as feasible.

DNA damage as a biomarker of genotoxic contamination in Mytilus galloprovincialis from the south coast of Portugal.

PubMed

Almeida, Catarina; Pereira, Catarina; Gomes, Tânia; Bebianno, Maria João; Cravo, Alexandra

2011-09-01

DNA damage was evaluated in the haemolymph of Mytilus galloprovincialis from nine sites along the south coast of Portugal using the comet assay. DNA damage was low, in the same range of sites considered to suffer low impact from genotoxic contaminants. Even so, differences between sites, seasons and genders were found. Highest values were in mussels from the main estuaries and the fishery harbour, reflecting higher genotoxin levels, whereas the lowest values can be used as a baseline for future work. Non-contaminant related factors (e.g. temperature and oxygen) were also shown to influence DNA damage. Between seasons, highest values were in summer related not only to the increase of tourism in this region (∼10-fold), but also to temperature. Between genders, males were found to be more sensitive. The condition index was also generally higher in summer. Lipid peroxidation, another damage biomarker, was measured in gills to assess if there is any association between the responses of both biomarkers and if they are similarly affected by the same environmental conditions. LPO like DNA damage was higher in summer. This work confirms that DNA damage is a sensitive biomarker to discriminate genotoxic contamination, even in areas considered to suffer low impact from genotoxins.

Mangrove Bacterial Diversity and the Impact of Oil Contamination Revealed by Pyrosequencing: Bacterial Proxies for Oil Pollution

PubMed Central

dos Santos, Henrique Fragoso; Cury, Juliano Carvalho; do Carmo, Flávia Lima; dos Santos, Adriana Lopes; Tiedje, James; van Elsas, Jan Dirk; Rosado, Alexandre Soares; Peixoto, Raquel Silva

2011-01-01

Background Mangroves are transitional coastal ecosystems in tropical and sub-tropical regions and represent biologically important and productive ecosystems. Despite their great ecological and economic importance, mangroves are often situated in areas of high anthropogenic influence, being exposed to pollutants, such as those released by oil spills. Methodology/Principal Findings A microcosm experiment was conducted, which simulated an oil spill in previously pristine mangrove sediment. The effect of the oil spill on the extant microbial community was studied using direct pyrosequencing. Extensive bacterial diversity was observed in the pristine mangrove sediment, even after oil contamination. The number of different OTUs only detected in contaminated samples was significantly higher than the number of OTUs only detected in non-contaminated samples. The phylum Proteobacteria, in particular the classes Gammaproteobacteria and Deltaproteobacteria, were prevalent before and after the simulated oil spill. On the other hand, the order Chromatiales and the genus Haliea decreased upon exposure to 2 and 5% oil, these are proposed as sensitive indicators of oil contamination. Three other genera, Marinobacterium, Marinobacter and Cycloclasticus increased their prevalence when confronted with oil. These groups are possible targets for the biomonitoring of the impact of oil in mangrove settings. Conclusions/Significance We suggest the use of sequences of the selected genera as proxies for oil pollution, using qPCR assessments. The quantification of these genera in distinct mangrove systems in relation to the local oil levels would permit the evaluation of the level of perturbance of mangroves, being useful in field monitoring. Considering the importance of mangroves to many other environments and the susceptibility of such areas to oil spills this manuscript will be of broad interest. PMID:21399677

Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing

PubMed Central

Eastman, Alexander W.; Yuan, Ze-Chun

2015-01-01

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects. PMID

Comprehensive analysis of DNA polymerase III α subunits and their homologs in bacterial genomes

PubMed Central

Timinskas, Kęstutis; Balvočiūtė, Monika; Timinskas, Albertas; Venclovas, Česlovas

2014-01-01

The analysis of ∼2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an ‘impaired’ version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set. PMID:24106089

APPLICATION OF RADIOISOTOPE-LABELED BACTERIA IN FOOD SANITATION STUDIES. IV. ON THE BACTERIAL CONTAMINATION AND DECONTAMINATION OF LIVING OYSTER (in Japanese)

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

Bito, T.; Takase, A.

Decontamination of the living oyster was studied by Y/sup 90/or Y/sup 91/ -labeled coliform bacteria. At low temperature, precultural temperature affected the contamination of the oyster but the uptake of bacteria by the oyster was distinctly observed below 5 deg C. In a study on elapsing change of contamination, it was determined that the whole bacterial load of the oyster depended mainly on the gill, but was not negligible on the digestive tract. The contaminating rate of the oyster was successfully measured by automatic recording of the shift of radioactivity in the contaminated cultural water with a dip-type G-M countermore » ratemeter and chart recorder, because the bacterial concentration of the closed cultural water decrease according to the bacterial uptake of the oyster. On decontamination of the oyster, it was observed that the most effective temperature existed in the region of 15 to 25 deg C, and at the upper or lower of this region the decontaminating efficiency decreased. As for the exchange rate of the culture water, the decontaminating effect on the oyster was proportional to an increase of the rate to 7.5 times per hour. However, more was of no effect. An increase in the cultured water volume was harmful for the decontamination of the oyster because of the exchanging efficiency of the water. (P.C.H.)« less

Fragmentation of contaminant and endogenous DNA in ancient samples determined by shotgun sequencing; prospects for human palaeogenomics.

PubMed

García-Garcerà, Marc; Gigli, Elena; Sanchez-Quinto, Federico; Ramirez, Oscar; Calafell, Francesc; Civit, Sergi; Lalueza-Fox, Carles

2011-01-01

Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks. METHODOLOGY/PRINCIPALS FINDINGS: To investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrón site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants. We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5' and 3' ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone.

Bacterial community structure and variation in a full-scale seawater desalination plant for drinking water production.

PubMed

Belila, A; El-Chakhtoura, J; Otaibi, N; Muyzer, G; Gonzalez-Gil, G; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S

2016-05-01

Microbial processes inevitably play a role in membrane-based desalination plants, mainly recognized as membrane biofouling. We assessed the bacterial community structure and diversity during different treatment steps in a full-scale seawater desalination plant producing 40,000 m(3)/d of drinking water. Water samples were taken over the full treatment train consisting of chlorination, spruce media and cartridge filters, de-chlorination, first and second pass reverse osmosis (RO) membranes and final chlorine dosage for drinking water distribution. The water samples were analyzed for water quality parameters (total bacterial cell number, total organic carbon, conductivity, pH, etc.) and microbial community composition by 16S rRNA gene pyrosequencing. The planktonic microbial community was dominated by Proteobacteria (48.6%) followed by Bacteroidetes (15%), Firmicutes (9.3%) and Cyanobacteria (4.9%). During the pretreatment step, the spruce media filter did not impact the bacterial community composition dominated by Proteobacteria. In contrast, the RO and final chlorination treatment steps reduced the Proteobacterial relative abundance in the produced water where Firmicutes constituted the most dominant bacterial group. Shannon and Chao1 diversity indices showed that bacterial species richness and diversity decreased during the seawater desalination process. The two-stage RO filtration strongly reduced the water conductivity (>99%), TOC concentration (98.5%) and total bacterial cell number (>99%), albeit some bacterial DNA was found in the water after RO filtration. About 0.25% of the total bacterial operational taxonomic units (OTUs) were present in all stages of the desalination plant: the seawater, the RO permeates and the chlorinated drinking water, suggesting that these bacterial strains can survive in different environments such as high/low salt concentration and with/without residual disinfectant. These bacterial strains were not caused by contamination during

Remediation of petroleum hydrocarbon-contaminated sites by DNA diagnosis-based bioslurping technology.

PubMed

Kim, Seungjin; Krajmalnik-Brown, Rosa; Kim, Jong-Oh; Chung, Jinwook

2014-11-01

The application of effective remediation technologies can benefit from adequate preliminary testing, such as in lab-scale and Pilot-scale systems. Bioremediation technologies have demonstrated tremendous potential with regards to cost, but they cannot be used for all contaminated sites due to limitations in biological activity. The purpose of this study was to develop a DNA diagnostic method that reduces the time to select contaminated sites that are good candidates for bioremediation. We applied an oligonucleotide microarray method to detect and monitor genes that lead to aliphatic and aromatic degradation. Further, the bioremediation of a contaminated site, selected based on the results of the genetic diagnostic method, was achieved successfully by applying bioslurping in field tests. This gene-based diagnostic technique is a powerful tool to evaluate the potential for bioremediation in petroleum hydrocarbon contaminated soil. Copyright © 2014 Elsevier B.V. All rights reserved.

Bacterial Diversity and Bioremediation Potential of the Highly Contaminated Marine Sediments at El-Max District (Egypt, Mediterranean Sea).

PubMed

Amer, Ranya A; Mapelli, Francesca; El Gendi, Hamada M; Barbato, Marta; Goda, Doaa A; Corsini, Anna; Cavalca, Lucia; Fusi, Marco; Borin, Sara; Daffonchio, Daniele; Abdel-Fattah, Yasser R

2015-01-01

Coastal environments worldwide are threatened by the effects of pollution, a risk particularly high in semienclosed basins like the Mediterranean Sea that is poorly studied from bioremediation potential perspective especially in the Southern coast. Here, we investigated the physical, chemical, and microbiological features of hydrocarbon and heavy metals contaminated sediments collected at El-Max bay (Egypt). Molecular and statistical approaches assessing the structure of the sediment-dwelling bacterial communities showed correlations between the composition of bacterial assemblages and the associated environmental parameters. Fifty strains were isolated on mineral media supplemented by 1% crude oil and identified as a diverse range of hydrocarbon-degrading bacteria involved in different successional stages of biodegradation. We screened the collection for biotechnological potential studying biosurfactant production, biofilm formation, and the capability to utilize different hydrocarbons. Some strains were able to grow on multiple hydrocarbons as unique carbon source and presented biosurfactant-like activities and/or capacity to form biofilm and owned genes involved in different detoxification/degradation processes. El-Max sediments represent a promising reservoir of novel bacterial strains adapted to high hydrocarbon contamination loads. The potential of the strains for exploitation for in situ intervention to combat pollution in coastal areas is discussed.

Bacterial contamination of epidural catheters: microbiological examination of 502 epidural catheters used for postoperative analgesia.

PubMed

Steffen, Peter; Seeling, Wulf; Essig, Andreas; Stiepan, Erika; Rockemann, Michael Georg

2004-03-01

To investigate the frequency of bacterial colonization of epidural catheters used for postoperative pain treatment longer than 24 hours in abdominal, thoracic, or trauma surgery patients. Retrospective study. Intermediate care facility and general ward of a university hospital. 502 patients who received epidural catheters after abdominal, thoracic, or vascular surgery at our institution from January 1996 to December 2000. Placement of an epidural catheter, which was used for postoperative pain treatment, for more than 24 hours. The puncture site dressing included saturation each day with povidone-iodine. Microbiologic monitoring of epidural catheter tips and daily examination of puncture sites with regard to signs of inflammation took place. Four times daily patients were examined to check adequacy of pain treatment and neurologic deficits. Catheter tip cultures were positive in 29 patients (5.8%). Staphylococcus epidermidis was isolated in 22 cases (76%). No case of spinal epidural abscess was observed within 6 months after epidural catheterization. The average catheterization time was 5 days (quartile range: 4 to 6 days). Meticulous management ensures a relatively low level of bacterial contamination in epidural catheters applied for postoperative pain treatment greater than 5 days. Contamination rarely leads to spinal epidural infection.

Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods.

PubMed

La Duc, Myron T; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J A; Venkateswaran, Kasthuri

2009-10-01

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.

Assembling the bacterial segrosome.

PubMed

Hayes, Finbarr; Barillà, Daniela

2006-05-01

Genome segregation in prokaryotes is a highly ordered process that integrates with DNA replication, cytokinesis and other fundamental facets of the bacterial cell cycle. The segrosome is the nucleoprotein complex that mediates DNA segregation in bacteria, its assembly and organization is best understood for plasmid partition. The recent elucidation of structures of the ParB plasmid segregation protein bound to centromeric DNA, and of the tertiary structures of other segregation proteins, are key milestones in the path to deciphering the molecular basis of bacterial DNA segregation.

Multiple reservoirs contribute to intraoperative bacterial transmission.

PubMed

Loftus, Randy W; Brown, Jeremiah R; Koff, Matthew D; Reddy, Sundara; Heard, Stephen O; Patel, Hetal M; Fernandez, Patrick G; Beach, Michael L; Corwin, Howard L; Jensen, Jens T; Kispert, David; Huysman, Bridget; Dodds, Thomas M; Ruoff, Kathryn L; Yeager, Mark P

2012-06-01

Intraoperative stopcock contamination is a frequent event associated with increased patient mortality. In the current study we examined the relative contributions of anesthesia provider hands, the patient, and the patient environment to stopcock contamination. Our secondary aims were to identify risk factors for stopcock contamination and to examine the prior association of stopcock contamination with 30-day postoperative infection and mortality. Additional microbiological analyses were completed to determine the prevalence of bacterial pathogens within intraoperative bacterial reservoirs. Pulsed-field gel electrophoresis was used to assess the contribution of reservoir bacterial pathogens to 30-day postoperative infections. In a multicenter study, stopcock transmission events were observed in 274 operating rooms, with the first and second cases of the day in each operating room studied in series to identify within- and between-case transmission events. Reservoir bacterial cultures were obtained and compared with stopcock set isolates to determine the origin of stopcock contamination. Between-case transmission was defined by the isolation of 1 or more bacterial isolates from the stopcock set of a subsequent case (case 2) that were identical to reservoir isolates from the preceding case (case 1). Within-case transmission was defined by the isolation of 1 or more bacterial isolates from a stopcock set that were identical to bacterial reservoirs from the same case. Bacterial pathogens within these reservoirs were identified, and their potential contribution to postoperative infections was evaluated. All patients were followed for 30 days postoperatively for the development of infection and all-cause mortality. Stopcock contamination was detected in 23% (126 out of 548) of cases with 14 between-case and 30 within-case transmission events confirmed. All 3 reservoirs contributed to between-case (64% environment, 14% patient, and 21% provider) and within-case (47

DNA methylation differentially regulates cytokine secretion in gingival epithelia in response to bacterial challenges.

PubMed

Drury, Jeanie L; Chung, Whasun Oh

2015-03-01

Epigenetic modifications are changes in gene expression without altering DNA sequence. We previously reported that bacteria-specific innate immune responses are regulated by epigenetic modifications. Our hypothesis is that DNA methylation affects gingival cytokine secretion in response to bacterial stimulation. Gingival epithelial cells (GECs) were treated with DNMT-1 inhibitors prior to Porphyromonas gingivalis (Pg) or Fusobacterium nucleatum (Fn) exposure. Protein secretion was assessed using ELISA. Gene expression was quantified using qRT-PCR. The ability of bacteria to invade inhibitor pretreated GECs was assessed utilizing flow cytometry. Changes were compared to unstimulated GECs. GEC upregulation of IL-6 and CXCL1 by Pg or Fn stimulation was significantly diminished by inhibitor pretreatment. Pg stimulated IL-1α secretion and inhibitor pretreatment significantly enhanced this upregulation, while Fn alone or with inhibitor pretreatment had no effect on IL-1α expression. GEC upregulation of human beta-definsin-2 in response to Pg and Fn exposure was enhanced following the inhibitor pretreatment. GEC susceptibility to bacterial invasion was unaltered. These results suggest that DNA methylation differentially affects gingival cytokine secretion in response to Pg or Fn. Our data provide basis for better understanding of how epigenetic modifications, brought on by exposure to oral bacteria, will subsequently affect host susceptibility to oral diseases. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Bacterial and Fungal Diversity of an Aged PAH- and Heavy Metal-Contaminated Soil is Affected by Plant Cover and Edaphic Parameters.

PubMed

Bourceret, Amélia; Cébron, Aurélie; Tisserant, Emilie; Poupin, Pascal; Bauda, Pascale; Beguiristain, Thierry; Leyval, Corinne

2016-04-01

Industrial wasteland soils with aged PAH and heavy metal contaminations are environments where pollutant toxicity has been maintained for decades. Although the communities may be well adapted to the presence of stressors, knowledge about microbial diversity in such soils is scarce. Soil microbial community dynamics can be driven by the presence of plants, but the impact of plant development on selection or diversification of microorganisms in these soils has not been established yet. To test these hypotheses, aged-contaminated soil samples from a field trial were collected. Plots planted with alfalfa were compared to bare soil plots, and bacterial and fungal diversity and abundance were assessed after 2 and 6 years. Using pyrosequencing of 16S rRNA gene and ITS amplicons, we showed that the bacterial community was dominated by Proteobacteria, Actinobacteria, and Bacteroidetes and was characterized by low Acidobacteria abundance, while the fungal community was mainly represented by members of the Ascomycota. The short-term toxic impact of pollutants usually reduces the microbial diversity, yet in our samples bacterial and fungal species richness and diversity was high suggesting that the community structure and diversity adapted to the contaminated soil over decades. The presence of plants induced higher bacterial and fungal diversity than in bare soil. It also increased the relative abundance of bacterial members of the Actinomycetales, Rhizobiales, and Xanthomonadales orders and of most fungal orders. Multivariate analysis showed correlations between microbial community structure and heavy metal and PAH concentrations over time, but also with edaphic parameters (C/N, pH, phosphorus, and nitrogen concentrations).

Bacteria and bacterial DNA in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis

PubMed Central

Armingohar, Zahra; Jørgensen, Jørgen J.; Kristoffersen, Anne Karin; Abesha-Belay, Emnet; Olsen, Ingar

2014-01-01

Background Several studies have reported an association between chronic periodontitis (CP) and cardiovascular diseases. Detection of periodontopathogens, including red complex bacteria (RCB), in vascular lesions has suggested these bacteria to be involved in the pathogenesis of atherosclerosis and abdominal aortic aneurysms. Objective In this study, we investigate bacteria and their DNA in vascular biopsies from patients with vascular diseases (VD; i.e. abdominal aortic aneurysms, atherosclerotic carotid, and common femoral arteries), with and without CP. Methods DNA was extracted from vascular biopsies selected from 40 VD patients: 30 with CP and 10 without CP. The V3-V5 region of the 16S rDNA (V3-V5) was polymerase chain reaction (PCR)-amplified, and the amplicons were cloned into Escherichia coli, sequenced, and classified (GenBank and the Human Oral Microbiome database). Species-specific primers were used for the detection of Porphyromonas gingivalis. In addition, 10 randomly selected vascular biopsies from the CP group were subjected to scanning electron microscopy (SEM) for visualization of bacteria. Checkerboard DNA–DNA hybridization was performed to assess the presence of RCB in 10 randomly selected subgingival plaque samples from CP patients. Results A higher load and mean diversity of bacteria were detected in vascular biopsies from VD patients with CP compared to those without CP. Enterobacteriaceae were frequently detected in vascular biopsies together with cultivable, commensal oral, and not-yet-cultured bacterial species. While 70% of the subgingival plaque samples from CP patients showed presence of RCB, only P. gingivalis was detected in one vascular biopsy. Bacterial cells were seen in all 10 vascular biopsies examined by SEM. Conclusions A higher bacterial load and more diverse colonization were detected in VD lesions of CP patients as compared to patients without CP. This indicated that a multitude of bacterial species both from the gut and the

Bioaugmentation of thiabendazole-contaminated soils from a wastewater disposal site: Factors driving the efficacy of this strategy and the diversity of the indigenous soil bacterial community.

PubMed

Papadopoulou, Evangelia S; Genitsaris, Savvas; Omirou, Michalis; Perruchon, Chiara; Stamatopoulou, Anastasia; Ioannides, Ioannis; Karpouzas, Dimitrios G

2018-02-01

The application of the fungicide thiabendazole (TBZ) in fruit packaging plants (FPP) results in the production of effluents which are often disposed in adjacent field sites. These require remediation to prevent further environmental dispersal of TBZ. We assessed the bioaugmentation potential of a newly isolated TBZ-degrading bacterial consortium in a naturally contaminated soil (NCS) exhibiting a natural gradient of TBZ levels (12000, 400, 250 and 12 mg kg -1 ). The effect of aging on bioaugmentation efficacy was comparatively tested in a soil with similar physicochemical properties and soil microbiota, which was artificially, contaminated with the same TBZ levels (ACS). The impact of bioaugmentation and TBZ on the bacterial diversity in the NCS was explored via amplicon sequencing. Bioaugmentation effectively removed TBZ from both soils at levels up to 400 mg kg -1 but failed at the highest contamination level (12000 mg kg -1 ). Dissipation of TBZ in bioaugmented samples showed a concentration-dependent pattern, while aging of TBZ had a slight effect on bioaugmentation efficiency. Bioaugmentation had no impact on the soil bacterial diversity, in contrast to TBZ contamination. Soils from the hotspots of TBZ contamination (12000 mg kg -1 ) showed a drastically lower α-diversity driven by the dominance of β- and γ-proteobacteria at the expense of all other bacterial phyla, especially Actinobacteria. Overall, bioaugmentation with specialized microbial inocula could be an effective solution for the recovery of disposal sites contaminated with persistent chemicals like TBZ. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacterial DNA detected on pathologically changed heart valves using 16S rRNA gene amplification.

PubMed

Chalupova, Miroslava; Skalova, Anna; Hajek, Tomas; Geigerova, Lenka; Kralova, Dana; Liska, Pavel; Hecova, Hana; Molacek, Jiri; Hrabak, Jaroslav

2018-05-22

Nowadays, dental diseases are one of the most common illnesses in the world. Some of them can lead to translocation of oral bacteria to the bloodstream causing intermittent bacteraemia. Therefore, a potential association between oral infection and cardiovascular diseases has been discussed in recent years as a result of adhesion of oral microbes to the heart valves. The aim of this study was to detect oral bacteria on pathologically changed heart valves not caused by infective endocarditis. In the study, patients with pathologically changed heart valves were involved. Samples of heart valves removed during heart valve replacement surgery were cut into two parts. One aliquot was cultivated aerobically and anaerobically. Bacterial DNA was extracted using Ultra-Deep Microbiome Prep (Molzym GmbH, Bremen, Germany) followed by a 16S rRNA gene PCR amplification using Mastermix 16S Complete kit (Molzym GmbH, Bremen, Germany). Positive PCR products were sequenced and the sequences were analyzed using BLAST database ( http://www.ncbi.nlm.nih/BLAST ). During the study period, 41 samples were processed. Bacterial DNA of the following bacteria was detected in 21 samples: Cutibacterium acnes (formerly Propionibacterium acnes) (n = 11; 52.38% of patients with positive bacterial DNA detection), Staphylococcus sp. (n = 9; 42.86%), Streptococcus sp. (n = 1; 4.76%), Streptococcus sanguinis (n = 4; 19.05%), Streptococcus oralis (n = 1; 4.76%), Carnobacterium sp. (n = 1; 4.76%), Bacillus sp. (n = 2; 9.52%), and Bergeyella sp. (n = 1; 4.76%). In nine samples, multiple bacteria were found. Our results showed significant appearance of bacteria on pathologically changed heart valves in patients with no symptoms of infective endocarditis.

Limiting and detecting bacterial contamination of apheresis platelets: inlet-line diversion and increased culture volume improve component safety.

PubMed

Eder, Anne F; Kennedy, Jean M; Dy, Beth A; Notari, Edward P; Skeate, Robert; Bachowski, Gary; Mair, David C; Webb, Jonathan S; Wagner, Stephen J; Dodd, Roger Y; Benjamin, Richard J

2009-08-01

Septic transfusion reactions to apheresis platelets (PLTs) continue to occur despite preventive measures. This study evaluated the effect of two operational changes designed to reduce bacterial risk: 1) introducing inlet-line sample diversion on two-arm procedures and 2) increasing the sample volume cultured from 4 to 8 mL from all donations. Aerobic culture results and septic transfusion reactions reported between December 1, 2006, and July 31, 2008 (Period 2), were compared to March 1, 2004, to May 31, 2006 (Period 1). During Period 2, a total of 781,936 apheresis PLT collections were cultured, of which 130 donations (1:6015) were confirmed positive and 9 (1:86,882) had negative culture results but were associated with 11 septic reactions. Confirmed-positive cultures from two-arm procedures decreased (27.2 to 14.7 per 105 collections; odds ratio [OR], 0.54; 95% confidence interval [CI], 0.41-0.70) in Period 2, owing to a lower rate of skin flora contamination. Detection of contamination of one-arm collections significantly increased by 54% in Period 2 (13.7 vs. 21.1 per 105 collections; OR, 1.54; 95% CI, 1.05-2.27). Fewer septic transfusion reactions occurred in Period 2, but the difference did not reach significance (1.7 vs. 1.2 per 105 donations; OR, 0.68; 95% CI, 0.30-1.53). Inlet-line diversion decreased bacterial contamination during two-arm collections by more than 46%. Concurrently, doubling the sample volume was associated with a 54% relative increase in culture sensitivity. These interventions act cooperatively to decrease bacterial risk.

Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity.

PubMed

Erttmann, Saskia F; Härtlova, Anetta; Sloniecka, Marta; Raffi, Faizal A M; Hosseinzadeh, Ava; Edgren, Tomas; Rofougaran, Reza; Resch, Ulrike; Fällman, Maria; Ek, Torben; Gekara, Nelson O

2016-07-19

The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome-dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1β (IL-1β) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of Anaerobic Aniline-Degrading Bacteria at a Contaminated Industrial Site.

PubMed

Sun, Weimin; Li, Yun; McGuinness, Lora R; Luo, Shuai; Huang, Weilin; Kerkhof, Lee J; Mack, E Erin; Häggblom, Max M; Fennell, Donna E

2015-09-15

Anaerobic aniline biodegradation was investigated under different electron-accepting conditions using contaminated canal and groundwater aquifer sediments from an industrial site. Aniline loss was observed in nitrate- and sulfate-amended microcosms and in microcosms established to promote methanogenic conditions. Lag times of 37 days (sulfate amended) to more than 100 days (methanogenic) were observed prior to activity. Time-series DNA-stable isotope probing (SIP) was used to identify bacteria that incorporated (13)C-labeled aniline in the microcosms established to promote methanogenic conditions. In microcosms from heavily contaminated aquifer sediments, a phylotype with 92.7% sequence similarity to Ignavibacterium album was identified as a dominant aniline degrader as indicated by incorporation of (13)C-aniline into its DNA. In microcosms from contaminated canal sediments, a bacterial phylotype within the family Anaerolineaceae, but without a match to any known genus, demonstrated the assimilation of (13)C-aniline. Acidovorax spp. were also identified as putative aniline degraders in both of these two treatments, indicating that these species were present and active in both the canal and aquifer sediments. There were multiple bacterial phylotypes associated with anaerobic degradation of aniline at this complex industrial site, which suggests that anaerobic transformation of aniline is an important process at the site. Furthermore, the aniline degrading phylotypes identified in the current study are not related to any known aniline-degrading bacteria. The identification of novel putative aniline degraders expands current knowledge regarding the potential fate of aniline under anaerobic conditions.

Effects of root inoculation with bacteria on the growth, Cd uptake and bacterial communities associated with rape grown in Cd-contaminated soil.

PubMed

Chen, Zhao-jin; Sheng, Xia-fang; He, Lin-yan; Huang, Zhi; Zhang, Wen-hui

2013-01-15

Two metal-resistant and plant growth-promoting bacteria (Burkholderia sp. J62 and Pseudomonas thivervalensis Y-1-3-9) were evaluated for their impacts on plant growth promotion, Cd availability in soil, and Cd uptake in rape (Brassica napus) grown in different level (0, 50, and 100 mg kg(-1)) of Cd-contaminated soils. The impacts of the bacteria on the rape-associated bacterial community structures were also evaluated using denaturing gradient gel electrophoresis (DGGE) analysis of bacterial DNA extracted from the root interior and rhizosphere and bulk soil samples collected at day 60 after inoculation. Canonical correspondence analysis (CCA) was used to have a comparative analysis of DGGE profiles. Inoculation with live bacteria not only significantly increased root (ranging from 38% to 86%), stem (ranging from 27% to 65%) and leaf (ranging from 23% to 55%) dry weights and water-extractive Cd contents (ranging from 59% to 237%) in the rhizosphere soils of the rape but also significantly increased root (ranging from 10% to 61%), stem (ranging from 41% to 57%) and leaf (ranging from 46% to 68%) total Cd uptake of rape compared to the dead bacterial-inoculated controls. DGGE and sequence analyses showed that the bacteria could colonize the rhizosphere soils and root interiors of rape plants. DGGE-CCA also showed that root interior and rhizosphere and bulk soil community profiles from the live bacteria-inoculated rape were significantly different from those from the dead bacteria-inoculated rape respectively. These results suggested that the bacteria had the potential to promote the growth and Cd uptake of rape and to influence the development of the rape-associated bacterial community structures. Copyright © 2012 Elsevier B.V. All rights reserved.

Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods▿ †

PubMed Central

La Duc, Myron T.; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J. A.; Venkateswaran, Kasthuri

2009-01-01

A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments. PMID:19700540

Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

PubMed

Dembinski, Gina M; Picard, Christine J

2017-11-01

Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex ® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex ® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex ® 16HS system is used. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Bioremediation of high molecular weight polyaromatic hydrocarbons co-contaminated with metals in liquid and soil slurries by metal tolerant PAHs degrading bacterial consortium.

PubMed

Thavamani, Palanisami; Megharaj, Mallavarapu; Naidu, Ravi

2012-11-01

Bioremediation of polyaromatic hydrocarbons (PAH) contaminated soils in the presence of heavy metals have proved to be difficult and often challenging due to the ability of toxic metals to inhibit PAH degradation by bacteria. In this study, a mixed bacterial culture designated as consortium-5 was isolated from a former manufactured gas plant (MGP) site. The ability of this consortium to utilise HMW PAHs such as pyrene and BaP as a sole carbon source in the presence of toxic metal Cd was demonstrated. Furthermore, this consortium has proven to be effective in degradation of HMW PAHs even from the real long term contaminated MGP soil. Thus, the results of this study demonstrate the great potential of this consortium for field scale bioremediation of PAHs in long term mix contaminated soils such as MGP sites. To our knowledge this is the first study to isolate and characterize metal tolerant HMW PAH degrading bacterial consortium which shows great potential in bioremediation of mixed contaminated soils such as MGP.

EFFECT OF PRE-EVISCERATION, SKIN-ON CARCASS DECONTAMINATION SANITATION STRATEGIES FOR REDUCING BACTERIAL CONTAMINATION OF BEEF DURING SKINNING

USDA-ARS?s Scientific Manuscript database

The effectiveness of pre-evisceration, skin-on carcass sanitation on reducing bacterial contamination of beef carcasses was tested using 3 cattle per treatment and 3 cattle as controls at each of 3 abattoirs in southern Wisconsin. The sanitation procedure included stunning, bleeding, tying off the e...

Bacterial Diversity and Bioremediation Potential of the Highly Contaminated Marine Sediments at El-Max District (Egypt, Mediterranean Sea)

PubMed Central

Amer, Ranya A.; El Gendi, Hamada M.; Goda, Doaa A.; Corsini, Anna; Cavalca, Lucia; Fusi, Marco; Daffonchio, Daniele; Abdel-Fattah, Yasser R.

2015-01-01

Coastal environments worldwide are threatened by the effects of pollution, a risk particularly high in semienclosed basins like the Mediterranean Sea that is poorly studied from bioremediation potential perspective especially in the Southern coast. Here, we investigated the physical, chemical, and microbiological features of hydrocarbon and heavy metals contaminated sediments collected at El-Max bay (Egypt). Molecular and statistical approaches assessing the structure of the sediment-dwelling bacterial communities showed correlations between the composition of bacterial assemblages and the associated environmental parameters. Fifty strains were isolated on mineral media supplemented by 1% crude oil and identified as a diverse range of hydrocarbon-degrading bacteria involved in different successional stages of biodegradation. We screened the collection for biotechnological potential studying biosurfactant production, biofilm formation, and the capability to utilize different hydrocarbons. Some strains were able to grow on multiple hydrocarbons as unique carbon source and presented biosurfactant-like activities and/or capacity to form biofilm and owned genes involved in different detoxification/degradation processes. El-Max sediments represent a promising reservoir of novel bacterial strains adapted to high hydrocarbon contamination loads. The potential of the strains for exploitation for in situ intervention to combat pollution in coastal areas is discussed. PMID:26273661

Use of DNA Markers for Investigating Sources of Bacteria in Contaminated Ground Water: Wooster Township, Wayne County, Ohio

USGS Publications Warehouse

Dumouchelle, Denise H.

2006-01-01

In 2004, a public-health nuisance was declared by the Wayne County Board of Health in the Scenic Heights Drive-Batdorf Road area of Wooster Township, Wayne County, Ohio, because of concerns about the safety of water from local wells. Repeated sampling had detected the presence of fecal-indicator bacteria and elevated nitrate concentrations. In June 2006, the U.S. Geological Survey (USGS), in cooperation with the Ohio Environmental Protection Agency (Ohio EPA), collected and analyzed samples from some of the affected wells to help investigate the possibility of human-origin bacterial contamination. Water samples from 12 wells and 5 home sewage-treatment systems (HSTS) were collected. Bromide concentrations were determined in samples from the 12 wells. Samples from 5 of the 12 wells were analyzed for wastewater compounds. Total coliform, enterococci and Escherichia coli (E. coli) bacteria concentrations were determined for samples from 8 of the 12 wells. In addition, two microbial source-tracking tools that employ DNA markers were used on samples from several wells and a composite sample of water from five septic tanks. The DNA markers from the Enterococcus faecium species and the order Bacteroidales are associated with specific sources, either human or ruminant sources. Bromide concentrations ranged from 0.04 to 0.18 milligrams per liter (mg/L). No wastewater compounds were detected at concentrations above the reporting limits. Samples from the 12 wells also were collected by Ohio EPA and analyzed for chloride and nitrate. Chloride concentrations ranged from 12.6 to 61.6 mg/L and nitrate concentrations ranged from 2.34 to 11.9 mg/L (as N). Total coliforms and enterococci were detected in samples from 8 wells, at concentrations from 2 to 200 colony-forming units per 100 milliliters (CFU/100 mL) and 0.5 to 17 CFU/100 mL, respectively. E. coli were detected in samples from three of the eight wells, at concentrations of 1 or 2 CFU/100 mL. Tests for the human

Platelet transfusion therapy in sub-Saharan Africa: bacterial contamination, recipient characteristics and acute transfusion reactions

PubMed Central

Hume, Heather A.; Ddungu, Henry; Angom, Racheal; Baluku, Hannington; Kajumbula, Henry; Kyeyune-Byabazaire, Dorothy; Orem, Jackson; Ramirez-Arcos, Sandra; Tobian, Aaron A.R.

2017-01-01

Background Little data are available on bacterial contamination (BC) of platelet units or acute transfusion reactions to platelet transfusions (PT) in sub-Saharan Africa (SSA). Methods This prospective observational study evaluated the rate of BC of whole blood derived platelet units (WB-PU), the utility of performing Gram stains (GS) to prevent septic reactions, characteristics of patients receiving PT and the rate of acute reactions associated with PT at the Uganda Cancer Institute in Kampala, Uganda. An aliquot of each WB-PU studied was taken to perform GS and culture using the Bactec™ 9120 instrument. Study participants were monitored for reactions. Results 337 WB-PU were evaluated for BC, of which 323 units were transfused in 151 transfusion episodes to 50 patients. The frequency of BC ranged from 0.3%–2.1% (according to criteria used to define BC). The GS had high specificity (99.1%), but low sensitivity to detect units with BC. The median platelet count prior to PT was 10,900 (IQR 6,000–18,900) cells/μL. 78% of PT were given to patients with no bleeding. Acute reactions occurred in 11 transfusion episodes, involving 13 WB-PU, for a rate of 7.3% (95%CI=3.7–12.7%) per transfusion episode. All recipients of units with positive bacterial cultures were receiving antibiotics at the time of transfusion; none experienced a reaction. Conclusions The rate of BC observed in this study is lower than previously reported in SSA, but still remains a safety issue. As GS appears to be an ineffective screening tool, alternate methods should be explored to prevent transfusing bacterially-contaminated platelets in SSA. PMID:27079627

Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis.

PubMed

Escalante, Adelfo; Rodríguez, María Elena; Martínez, Alfredo; López-Munguía, Agustín; Bolívar, Francisco; Gosset, Guillermo

2004-06-15

The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.

Response of the bacterial community in oil-contaminated marine water to the addition of chemical and biological dispersants.

PubMed

Couto, Camila Rattes de Almeida; Jurelevicius, Diogo de Azevedo; Alvarez, Vanessa Marques; van Elsas, Jan Dirk; Seldin, Lucy

2016-12-15

The use of dispersants in different stages of the oil production chain and for the remediation of water and soil is a well established practice. However, the choice for a chemical or biological dispersant is still a controversial subject. Chemical surfactants that persist long in the environment may pose problems of toxicity themselves; therefore, biosurfactants are considered to constitute an environmentally friendly and effective alternative. Nevertheless, the putative effects of such agents on the microbiomes of oil-contaminated and uncontaminated marine environments have not been sufficiently evaluated. Here, we studied the effects of the surfactant Ultrasperse II ® and the surfactin (biosurfactant) produced by Bacillus sp. H2O-1 on the bacterial communities of marine water. Specifically, we used quantitative PCR and genetic fingerprint analyses to study the abundance and structure of the bacterial communities in marine water collected from two regions with contrasting climatic conditions. The addition of either chemical surfactant or biosurfactant influenced the structure and abundance of total and oil-degrading bacterial communities of oil-contaminated and uncontaminated marine waters. Remarkably, the bacterial communities responded similarly to the addition of oil and/or either the surfactant or the biosurfactant in both set of microcosms. After 30 days of incubation, the addition of surfactin enhanced the oil-degrading bacteria more than the chemical surfactant. However, no increase of hydrocarbon biodegradation values was observed, irrespective of the dispersant used. These data contribute to an increased understanding of the impact of novel dispersants on marine bacteriomes before commercial release into the environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

DNA-binding by Haemophilus influenzae and Escherichia coli YbaB, members of a widely-distributed bacterial protein family.

PubMed

Cooley, Anne E; Riley, Sean P; Kral, Keith; Miller, M Clarke; DeMoll, Edward; Fried, Michael G; Stevenson, Brian

2009-07-13

Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind. Both the H. influenzae and the E. coli YbaB proteins bound to tested DNAs. DNA-binding was not well competed with poly-dI-dC, indicating some sequence preferences for those two proteins. Analyses of binding characteristics determined that both YbaB orthologs bind as homodimers. Different DNA sequence preferences were observed between H. influenzae YbaB, E. coli YbaB and B. burgdorferi EbfC, consistent with amino acid differences in the putative DNA-binding domains of these proteins. Three distinct members of the YbaB/EbfC bacterial protein family have now been demonstrated to bind DNA. Members of this protein family are encoded by a broad range of bacteria, including many pathogenic species, and results of our studies suggest that all such proteins have DNA-binding activities. The functions of YbaB/EbfC family members in each bacterial species are as-yet unknown, but given the ubiquity of these DNA-binding proteins among Eubacteria, further investigations are warranted.

Bacterial DNA of Ocean and Land on the Surface of the International Space Station.

NASA Astrophysics Data System (ADS)

Grebennikova, Tatiana

A.V. Syroeshkin2, T.V. Grebennikova1, E.V. Shubralova3, V.A. Shuvalov3, O.S. Tsygankov4, V.B. Lapshin2 1D. I. Ivanovsky Virology Institute, Moscow, Russia 2 Academician E. K. Fedorov Institute of Applied Geophysics, Moscow, Russia 3S.P. Korolev Rocket and Space Corporation «Energia» Korolev, Russia 4Central Research Institute of Machine Building, Korolev, Russia Existence of biological molecules as markers of microorganisms in the space environment has always attracted attention of researchers. There is great attention to the search for extraterrestrial life forms [Nicholson W.L. 2009, Kawaguchi Y. et al 2013], and as well as the coping mechanisms of living organisms in the interplanetary space [Hotchin J. et al 1965, Baranov V.M. 2009, Horneck G. et al 2010]. Experiments on American and Japanese segments of the International Space Station (ISS) over the different nature of resistance during prolonged stay in space were conducted [Scalzi G et al 2012, Wassmann M. et al 2012]. As a result of these experiments confirmed the possibility of preserving the viability of organisms in an open space for a long time. Consequence, became interested in the transfer of living matter from the stratosphere to near-Earth space [Smith D.J. 2013]. We hypothesized that viable forms, or at least, intact DNA can be transferred to the orbit of the ISS with the ascending branch of the global electric circuit. Samples of cosmic dust collected from the surface of the window of the ISS during the exit of an astronaut in space. Samples (washes with material of tampons and tampons) which were in vacuo, were analyzed for the presence of bacterial DNA by nested PCR using primers specific DNA genus Mycobacterium, the DNA of the strain of the genus Bacillus anthracis and DNA encoding the bacterial 16S ribosomal RNA after transportation of the samples to Earth. The results of amplification, followed by sequencing and phylogenetic analysis showed the presence in samples of cosmic dust DNA

Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes.

PubMed

Lopez, Victor; Cauvi, David M; Arispe, Nelson; De Maio, Antonio

2016-07-01

The cellular response to stress is orchestrated by the expression of a family of proteins termed heat shock proteins (hsp) that are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis. The bulk of hsp function occurs within the cytosol and subcellular compartments. However, some hsp have also been found outside cells released by an active mechanism independent of cell death. Extracellular hsp act as signaling molecules directed at activating a systemic response to stress. The export of hsp requires the translocation from the cytosol into the extracellular milieu across the plasma membrane. We have proposed that membrane insertion is the initial step in this export process. We investigated the interaction of the major inducible hsp from mammalian (Hsp70) and bacterial (DnaK) species with liposomes. We found that mammalian Hsp70 displayed a high specificity for negatively charged phospholipids, such as phosphatidyl serine, whereas DnaK interacted with all lipids tested regardless of the charge. Both proteins were inserted into the lipid bilayer as demonstrated by resistance to acid or basic washes that was confirmed by partial protection from proteolytic cleavage. Several regions of mammalian Hsp70 were inserted into the membrane with a small portion of the N-terminus end exposed to the outer phase of the liposome. In contrast, the N-terminus end of DnaK was inserted into the membrane, exposing the C-terminus end outside the liposome. Mammalian Hsp70 was found to make high oligomeric complexes upon insertion into the membranes whereas DnaK only formed dimers within the lipid bilayer. These observations suggest that both Hsp70s interact with lipids, but mammalian Hsp70 displays a high degree of specificity and structure as compared with the bacterial form.

[Studies on the mycotic and bacterial risk of contamination and the use of nipagin in the artificial insemination of cryosperm].

PubMed

Glander, H G; Rytter, M; Schönborn, C

1984-01-01

Primary bacterial and mycological contamination was studied in random human ejaculates. After various phases of cryopreservation secondary microbiological contamination was investigated. Furthermore, semen samples of several donors were inoculated with suspension of different concentrations of yeasts and with the test bacteria Escherichia coli K12 and Staphylococcus aureus SG 511. Microbiological results were then compared before an after cryopreservation. The cryopreserving process was carried out with and without antibiotics to test the antibacterial effectiveness of antibiotics under these conditions. Moreover, we investigated the usability of a chemical preservative (Nipagin) at cryopreservation. The following results were obtained: 9.8 per cent of samples showed primary mycological contamination (1.9 per cent with Candida albicans). Cryopreservation reduced the concentration of fungi by more than 90 per cent on average. The bacteriological investigations have shown, that with one exception the semen was without pathogenetic bacteria. This situation was altered scarcely during the cryopreserving process (also by use of cryoprotective medium without antibiotics). Out of 25 ejaculates 11 had primary non-pathogenetic bacteria. Test bacteria inoculated into semen were not influenced by the addition of antibiotics to the cryoprotective medium (CPM). Nipagin prevented respectively reduced a bacterial or a mycological contamination of the CPM during a storage of 14 days dependent of nipagin concentration but reduced the motility of human spermatozoa after cryopreservation. The results suggest the conclusion to prefer a portionate storage without nipagin at a temperature of nearly + 1 degree C, thereby the microbiological contamination may be neglected.

Comparative evaluation of environmental contamination and DNA damage induced by electronic-waste in Nigeria and China.

PubMed

Alabi, Okunola A; Bakare, Adekunle A; Xu, Xijin; Li, Bin; Zhang, Yuling; Huo, Xia

2012-04-15

In the last decade, China and Nigeria have been prime destinations for the world's e-waste disposal leading to serious environmental contamination. We carried out a comparative study of the level of contamination using soils and plants from e-waste dumping and processing sites in both countries. Levels of polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) were analyzed using gas chromatography/spectrophotometry and heavy metals using atomic absorption spectrophotometry. DNA damage was assayed in human peripheral blood lymphocytes using an alkaline comet assay. Soils and plants were highly contaminated with toxic PAHs, PCBs, PBDEs, and heavy metals in both countries. Soil samples from China and plant samples from Nigeria were more contaminated. There was a positive correlation between the concentrations of organics and heavy metals in plant samples and the surrounding soils. In human lymphocytes, all tested samples induced significant (p<0.05) concentration-dependent increases in DNA damage compared with the negative control. These findings suggest that e-waste components/constituents can accumulate, in soil and surrounding vegetation, to toxic and genotoxic levels that could induce adverse health effects in exposed individuals. Copyright © 2012 Elsevier B.V. All rights reserved.

Bacterial contamination of bath-water from spinal cord lesioned patients with pressure ulcers exercising in the water.

PubMed

Biering-Sørensen, F; Schröder, A K; Wilhelmsen, M; Lomberg, B; Nielsen, H; Høiby, N

2000-02-01

To evaluate the bacterial contamination of the water from the microflora of pressure ulcers in para- and tetraplegic patients, when they were exercising in water at 36 degrees C for half an hour. Twelve spinal cord lesioned (SCL) patients with ulcers participated, and six of the 12 SCL patients with healed ulcers constituted a control group. The evaluation was performed both with and without the ulcer covered with a moisture reactive occlusive dressing, DuoDERM. Bacterial samples were taken from the bath-water before and after the exercise programmes and additionally specimens were obtained from the ulcer, the patients skin and urine, and from the skin of the physiotherapist who exercised the patient. A similar procedure was carried out in the controls. The bacteriologic analyses showed no significant difference in the contamination of the bath-water after exercising with or without DuoDERM covering the ulcers. In half of the patients DuoDERM loosened. After all exercise programmes with or without DuoDERM dressing the water was contaminated with facultative aerobic intestinal bacteria, ie E. faecalis and Enterobacteriaceae (E. coli, Klebsiella species, Proteus species, Enterobacter species). In nearly one-third of the exercise sessions the bath-water was contaminated with P. aeruginosa before starting, and after the exercise programmes one fourth of the ulcers were colonized with these pathogens. The water specimen showed the bacteria from the intestine to be much more prominent than the bacteria coming from the ulcers. Thus the pressure ulcers were of minor importance for the bath-water and ought not to prohibit patients from the potential benefits of water exercise, but chlorination of the water in the training pool seems appropriate.

Optimal vitamin D plasma levels are associated with lower bacterial DNA translocation in HIV/hepatitis c virus coinfected patients.

PubMed

García-Álvarez, Mónica; Berenguer, Juan; Jiménez-Sousa, Maria Ángeles; Vázquez-Morón, Sonia; Carrero, Ana; Gutiérrez-Rivas, Mónica; Aldámiz-Echevarría, Teresa; López, Juan Carlos; García-Broncano, Pilar; Resino, Salvador

2016-04-24

Vitamin D has been linked to the immune response modulation and the integrity of the intestinal mucosal barrier. Therefore, vitamin D might be involved in bacterial translocation related to HIV infection. Our major aim was to analyze the association between plasma levels of 25-hydroxy-vitamin D [25(OH)D] and bacterial 16S ribosomal DNA (bactDNA) in 120 HIV/hepatitis c virus (HCV) coinfected patients. Cross-sectional study. Plasma 25(OH)D levels were quantified by enzyme immunoassay. The vitamin D status was defined as deficient (<25 nmol/l), insufficient (25-74 nmol/l), and optimal (≥75 nmol/l) plasma levels. Plasma bactDNA levels were measured by quantitative real-time PCR. For bactDNA levels the cutoffs used were as follows: low [p75th). Eighteen (15%) patients had 25(OH)D deficiency, 93 (77.5%) had insufficiency and nine (7.5%) had 25(OH)D optimal values. The bactDNA levels were lower in patients with 25(OH)D at least 75 nmol/l [37 copies/μl] than in patients with 25(OH)D insufficiency [84.2 copies/μl; P = 0.042]. Conversely, low bactDNA levels (DNA levels above p25th were found only in 11.1% of them (P = 0.029). The plasma 25(OH)D not less than 75 nmol/l was associated with low bactDNA levels (bacterial translocation and inflammation in HIV/HCV coinfected patients.

No difference in portal and hepatic venous bacterial DNA in patients with cirrhosis undergoing transjugular intrahepatic portosystemic shunt insertion.

PubMed

Mortensen, Christian; Karlsen, Stine; Grønbæk, Henning; Nielsen, Dennis T; Frevert, Susanne; Clemmesen, Jens O; Møller, Søren; Jensen, Jørgen S; Bendtsen, Flemming

2013-10-01

Bacterial translocation (BT) with immune activation may lead to hemodynamical alterations and poor outcomes in patients with cirrhosis. We investigated bacterial DNA (bDNA), a marker of BT, and its relation to portal pressure and markers of inflammation in the portal and hepatic veins in patients with cirrhosis undergoing TIPS insertion. We analysed plasma for bDNA and markers of inflammation in 28 patients [median portal pressure gradient 15 (11-19) mmHg] during TIPS treatment for refractory ascites (n = 19) or acute variceal bleeding (n = 9). Advanced cirrhosis was present in the majority [Child-Pugh class (A/B/C): 1/14/13], and most often caused by alcohol (n = 21). bDNA was detectable in one or both samples in 16 of 28 patients (57%). bDNA was present in 39% of the samples from the portal vein vs 43% of the samples in the hepatic vein (P = 0.126). Antibiotics had no effect on bDNA or markers of inflammation. Markers of inflammation did not differ between the hepatic and portal veins with the exceptions of soluble urokinase plasminogen activating receptor (suPAR) and vascular endothelial growth factor (VEGF), both higher in the hepatic vein (P = 0.031 and 0.003 respectively). No transhepatic gradient of bDNA was evident, suggesting that no major hepatic elimination of bDNA occurs in advanced liver disease. bDNA, in contrast to previous reports was largely unrelated to a panel of markers of inflammation and without relation to portal pressure. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Integrated metagenomics and molecular ecological network analysis of bacterial community composition during the phytoremediation of cadmium-contaminated soils by bioenergy crops.

PubMed

Chen, Zhaojin; Zheng, Yuan; Ding, Chuanyu; Ren, Xuemin; Yuan, Jian; Sun, Feng; Li, Yuying

2017-11-01

Two energy crops (maize and soybean) were used in the remediation of cadmium-contaminated soils. These crops were used because they are fast growing, have a large biomass and are good sources for bioenergy production. The total accumulation of cadmium in maize and soybean plants was 393.01 and 263.24μg pot -1 , respectively. The rhizosphere bacterial community composition was studied by MiSeq sequencing. Phylogenetic analysis was performed using 16S rRNA gene sequences. The rhizosphere bacteria were divided into 33 major phylogenetic groups according to phyla. The dominant phylogenetic groups included Proteobacteria, Acidobacteria, Actinobacteria, Gemmatimonadetes, and Bacteroidetes. Based on principal component analysis (PCA) and unweighted pair group with arithmetic mean (UPGMA) analysis, we found that the bacterial community was influenced by cadmium addition and bioenergy cropping. Three molecular ecological networks were constructed for the unplanted, soybean- and maize-planted bacterial communities grown in 50mgkg -1 cadmium-contaminated soils. The results indicated that bioenergy cropping increased the complexity of the bacterial community network as evidenced by a higher total number of nodes, the average geodesic distance (GD), the modularity and a shorter geodesic distance. Proteobacteria and Acidobacteria were the keystone bacteria connecting different co-expressed operational taxonomic units (OTUs). The results showed that bioenergy cropping altered the topological roles of individual OTUs and keystone populations. This is the first study to reveal the effects of bioenergy cropping on microbial interactions in the phytoremediation of cadmium-contaminated soils by network reconstruction. This method can greatly enhance our understanding of the mechanisms of plant-microbe-metal interactions in metal-polluted ecosystems. Copyright © 2017 Elsevier Inc. All rights reserved.

Unique DNA-barcoded aerosol test particles for studying aerosol transport

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

Harding, Ruth N.; Hara, Christine A.; Hall, Sara B.

Data are presented for the first use of novel DNA-barcoded aerosol test particles that have been developed to track the fate of airborne contaminants in populated environments. Until DNATrax (DNA Tagged Reagents for Aerosol eXperiments) particles were developed, there was no way to rapidly validate air transport models with realistic particles in the respirable range of 1–10 μm in diameter. The DNATrax particles, developed at Lawrence Livermore National Laboratory (LLNL) and tested with the assistance of the Pentagon Force Protection Agency, are the first safe and effective materials for aerosol transport studies that are identified by DNA molecules. The usemore » of unique synthetic DNA barcodes overcomes the challenges of discerning the test material from pre-existing environmental or background contaminants (either naturally occurring or previously released). The DNATrax particle properties are demonstrated to have appropriate size range (approximately 1–4.5 μm in diameter) to accurately simulate bacterial spore transport. As a result, we describe details of the first field test of the DNATrax aerosol test particles in a large indoor facility.« less

Unique DNA-barcoded aerosol test particles for studying aerosol transport

DOE PAGES

Harding, Ruth N.; Hara, Christine A.; Hall, Sara B.; ...

2016-03-22

Data are presented for the first use of novel DNA-barcoded aerosol test particles that have been developed to track the fate of airborne contaminants in populated environments. Until DNATrax (DNA Tagged Reagents for Aerosol eXperiments) particles were developed, there was no way to rapidly validate air transport models with realistic particles in the respirable range of 1–10 μm in diameter. The DNATrax particles, developed at Lawrence Livermore National Laboratory (LLNL) and tested with the assistance of the Pentagon Force Protection Agency, are the first safe and effective materials for aerosol transport studies that are identified by DNA molecules. The usemore » of unique synthetic DNA barcodes overcomes the challenges of discerning the test material from pre-existing environmental or background contaminants (either naturally occurring or previously released). The DNATrax particle properties are demonstrated to have appropriate size range (approximately 1–4.5 μm in diameter) to accurately simulate bacterial spore transport. As a result, we describe details of the first field test of the DNATrax aerosol test particles in a large indoor facility.« less

Bacterial contamination of stored water and stored food: a potential source of diarrhoeal disease in West Africa.

PubMed Central

Mølbak, K.; Højlyng, N.; Jepsen, S.; Gaarslev, K.

1989-01-01

The food and water hygiene in two Liberian communities was studied in a house-to-house diarrhoea survey. The level of contamination with enterobacteria of drinking water stored in the households was significantly higher than at the water sources. Food hygiene standards were low, particularly in the urban slum where storage of cooked food for long periods led to bacterial multiplication at high levels. Infant foods were particularly heavily contaminated. It is concluded that when water supply programmes are planned, the presence of other risk factors for water-related diseases should be investigated. To ensure maximum health benefits, water projects should as a rule be accompanied by other interventions. PMID:2703024

Bacterial diversity in the active stage of a bioremediation system for mineral oil hydrocarbon-contaminated soils.

PubMed

Popp, Nicole; Schlömann, Michael; Mau, Margit

2006-11-01

Soils contaminated with mineral oil hydrocarbons are often cleaned in off-site bioremediation systems. In order to find out which bacteria are active during the degradation phase in such systems, the diversity of the active microflora in a degrading soil remediation system was investigated by small-subunit (SSU) rRNA analysis. Two sequential RNA extracts from one soil sample were generated by a procedure incorporating bead beating. Both extracts were analysed separately by generating individual SSU rDNA clone libraries from cDNA of the two extracts. The sequencing results showed moderate diversity. The two clone libraries were dominated by Gammaproteobacteria, especially Pseudomonas spp. Alphaproteobacteria and Betaproteobacteria were two other large groups in the clone libraries. Actinobacteria, Firmicutes, Bacteroidetes and Epsilonproteobacteria were detected in lower numbers. The obtained sequences were predominantly related to genera for which cultivated representatives have been described, but were often clustered together in the phylogenetic tree, and the sequences that were most similar were originally obtained from soils and not from pure cultures. Most of the dominant genera in the clone libraries, e.g. Pseudomonas, Acinetobacter, Sphingomonas, Acidovorax and Thiobacillus, had already been detected in (mineral oil hydrocarbon) contaminated environmental samples. The occurrence of the genera Zymomonas and Rhodoferax was novel in mineral oil hydrocarbon-contaminated soil.

Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

PubMed

Woo, P C Y; Lau, S K P; Teng, J L L; Tse, H; Yuen, K-Y

2008-10-01

In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However

DNA Electrochemistry and Electrochemical Sensors for Nucleic Acids.

PubMed

Ferapontova, Elena E

2018-06-12

Sensitive, specific, and fast analysis of nucleic acids (NAs) is strongly needed in medicine, environmental science, biodefence, and agriculture for the study of bacterial contamination of food and beverages and genetically modified organisms. Electrochemistry offers accurate, simple, inexpensive, and robust tools for the development of such analytical platforms that can successfully compete with other approaches for NA detection. Here, electrode reactions of DNA, basic principles of electrochemical NA analysis, and their relevance for practical applications are reviewed and critically discussed.

Bacterial diversity of floor drain biofilms and drain waters in a Listeria monocytogenes contaminated food processing environment.

PubMed

Dzieciol, Monika; Schornsteiner, Elisa; Muhterem-Uyar, Meryem; Stessl, Beatrix; Wagner, Martin; Schmitz-Esser, Stephan

2016-04-16

Sanitation protocols are applied on a daily basis in food processing facilities to prevent the risk of cross-contamination with spoilage organisms. Floor drain water serves along with product-associated samples (slicer dust, brine or cheese smear) as an important hygiene indicator in monitoring Listeria monocytogenes in food processing facilities. Microbial communities of floor drains are representative for each processing area and are influenced to a large degree by food residues, liquid effluents and washing water. The microbial communities of drain water are steadily changing, whereas drain biofilms provide more stable niches. Bacterial communities of four floor drains were characterized using 16S rRNA gene pyrosequencing to better understand the composition and exchange of drain water and drain biofilm communities. Furthermore, the L. monocytogenes contamination status of each floor drain was determined by applying cultivation-independent real-time PCR quantification and cultivation-dependent detection according to ISO11290-1. Pyrosequencing of 16S rRNA genes of drain water and drain biofilm bacterial communities yielded 50,611 reads, which were clustered into 641 operational taxonomic units (OTUs), affiliated to 16 phyla dominated by Proteobacteria, Firmicutes and Bacteroidetes. The most abundant OTUs represented either product- (Lactococcus lactis) or fermentation- and food spoilage-associated phylotypes (Pseudomonas mucidolens, Pseudomonas fragi, Leuconostoc citreum, and Acetobacter tropicalis). The microbial communities in DW and DB samples were distinct in each sample type and throughout the whole processing plant, indicating the presence of indigenous specific microbial communities in each processing compartment. The microbiota of drain biofilms was largely different from the microbiota of the drain water. A sampling approach based on drain water alone may thus only provide reliable information on planktonic bacterial cells but might not allow conclusions

Evaluation of the Efficacy of Disinfectant Footmats for the Reduction of Bacterial Contamination on Footwear in a Large Animal Veterinary Hospital.

PubMed

Hornig, K J; Burgess, B A; Saklou, N T; Johnson, V; Malmlov, A; Van Metre, D C; Morley, P S; Byers, S R

2016-11-01

Infection control is critical to providing high-quality patient care. Many veterinary teaching hospitals (VTHs) utilize footbaths or footmats at entrances and key control points throughout the facility to decrease trafficking of pathogenic microorganism on contaminated footwear. To compare efficacy of 4 disinfectants used in footmats for decreasing bacterial contamination of footwear in a large animal hospital. A single adult dairy cow was housed in a stall for 4 days to facilitate stall contamination with fecal material. Overboots were experimentally contaminated with organic material in a standardized manner. Each boot was randomly assigned to 1 of 5 treatments (no treatment, or exposure to 1 of 4 disinfectants: an accelerated peroxygen [AHP], a peroxygen [VIRKON], a quaternary ammonium [QUAT], and a phenolic disinfectant [PHENOLIC]) by stepping on a soaked footmat and collecting samples from boot soles. Generalized linear modeling was used to analyze differences in bacterial counts. Reductions in colony-forming units (CFUs) on treated boots ranged from no detectable reduction to 0.45 log 10 and varied by disinfectant. Percentage reductions in total bacterial counts generally were larger (albeit still modest) for AHP and QUAT disinfectants (range 37-45%) and smallest for the PHENOLIC (no detectable reduction). In general, use of disinfectant footmats was associated with significant reductions in viable bacteria on overboots-albeit with variable efficacy. Footmats may be useful adjuncts to cleaning and disinfection programs for decreasing trafficking of microorganisms throughout VTHs but should not be considered as a sole prevention method. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Bacterial diversity and reductive dehalogenase redundancy in a 1,2-dichloroethane-degrading bacterial consortium enriched from a contaminated aquifer

PubMed Central

2010-01-01

Background Bacteria possess a reservoir of metabolic functionalities ready to be exploited for multiple purposes. The use of microorganisms to clean up xenobiotics from polluted ecosystems (e.g. soil and water) represents an eco-sustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and accurate strategies for monitoring and identification of bacteria and catabolic genes involved in the degradation of xenobiotics, key processes to follow up the activities in situ. Results We report the characterization of the response of an enriched bacterial community of a 1,2-dichloroethane (1,2-DCA) contaminated aquifer to the spiking with 5 mM lactate as electron donor in microcosm studies. After 15 days of incubation, the microbial community structure was analyzed. The bacterial 16S rRNA gene clone library showed that the most represented phylogenetic group within the consortium was affiliated with the phylum Firmicutes. Among them, known degraders of chlorinated compounds were identified. A reductive dehalogenase genes clone library showed that the community held four phylogenetically-distinct catalytic enzymes, all conserving signature residues previously shown to be linked to 1,2-DCA dehalogenation. Conclusions The overall data indicate that the enriched bacterial consortium shares the metabolic functionality between different members of the microbial community and is characterized by a high functional redundancy. These are fundamental features for the maintenance of the community's functionality, especially under stress conditions and suggest the feasibility of a bioremediation treatment with a potential prompt dehalogenation and a process stability over time. PMID:20170484

[Investigation of bacterial diversity in the biological desulfurization reactor for treating high salinity wastewater by the 16S rDNA cloning method].

PubMed

Liu, Wei-Guo; Liang, Cun-Zhen; Yang, Jin-Sheng; Wang, Gui-Ping; Liu, Miao-Miao

2013-02-01

The bacterial diversity in the biological desulfurization reactor operated continuously for 1 year was studied by the 16S rDNA cloning and sequencing method. Forty clones were randomly selected and their partial 16S rDNA genes (ca. 1,400 bp) were sequenced and blasted. The results indicated that there were dominant bacterias in the biological desulfurization reactor, where 33 clones belonged to 3 different published phyla, while 1 clone belonged to unknown phylum. The dominant bacterial community in the system was Proteobacteria, which accounted for 85.3%. The bacterial community succession was as follows: the gamma-Proteobacteria(55.9%), beta-Proteobacteria(17.6%), Actinobacteridae (8.8%), delta-Proteobacteria (5.9%) , alpha-Proteobacteria(5.9%), and Sphingobacteria (2.9%). Halothiobacillus sp. ST15 and Thiobacillus sp. UAM-I were the major desulfurization strains.

Bacterial DNA-induced NK cell IFN-gamma production is dependent on macrophage secretion of IL-12.

PubMed

Chace, J H; Hooker, N A; Mildenstein, K L; Krieg, A M; Cowdery, J S

1997-08-01

Bacterial DNA (bDNA) activates B cells and macrophages and can augment inflammatory responses by inducing release of proinflammatory cytokines. We found that bDNA stimulation of mouse spleen cells induced NK cell IFN-gamma production that was dependent upon the presence of unmethylated CpG motifs, and oligonucleotides with internal CpG motifs could also induce splenocytes to secrete IFN-gamma. The bDNA-induced IFN-gamma response was strictly macrophages dependent. While splenocytes from SCID mice secreted IFN-gamma in response to bDNA, depletion of macrophages eliminated this response. Additionally, purified NK cells did not respond to bDNA; however, addition of macrophages restored the NK cell IFN-gamma response. Coculture of NK cells with preactivated macrophages further increased bDNA-induced NK cell IFN-gamma production. Anti-IL-12 or IL-10 inhibited bDNA-induced IFN-gamma response. Treatment of purified macrophages with bDNA resulted in IL-12 secretion accompanied by an increase in IL-12 p40 mRNA level. Although isolated NK cells did not make IFN-gamma in response to bDNA, NK cells costimulated with IL-12 gained the ability to respond to bDNA. These experiments show that bDNA induces macrophage IL-12 production which, in turn, stimulates NK cell IFN-gamma production. Macrophage-derived IL-12 renders NK cells responsive to bDNA permitting an even greater IFN-gamma response to bDNA.

Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis.

PubMed

Washington, Tracy A; Smith, Janet L; Grossman, Alan D

2017-10-01

DnaA is the widely conserved bacterial AAA+ ATPase that functions as both the replication initiator and a transcription factor. In many organisms, DnaA controls expression of its own gene and likely several others during growth and in response to replication stress. To evaluate the effects of DnaA on gene expression, separate from its role in replication initiation, we analyzed changes in mRNA levels in Bacillus subtilis cells with and without dnaA, using engineered strains in which dnaA is not essential. We found that dnaA was required for many of the changes in gene expression in response to replication stress. We also found that dnaA indirectly affected expression of several regulons during growth, including those controlled by the transcription factors Spo0A, AbrB, PhoP, SinR, RemA, Rok and YvrH. These effects were largely mediated by the effects of DnaA on expression of sda. DnaA activates transcription of sda, and Sda inhibits histidine protein kinases required for activation of the transcription factor Spo0A. We also found that loss of dnaA caused a decrease in the development of genetic competence. Together, our results indicate that DnaA plays an important role in modulating cell physiology, separate from its role in replication initiation. © 2017 John Wiley & Sons Ltd.

Surveillance study of bacterial contamination of the parent's cell phone in the NICU and the effectiveness of an anti-microbial gel in reducing transmission to the hands.

PubMed

Beckstrom, A C; Cleman, P E; Cassis-Ghavami, F L; Kamitsuka, M D

2013-12-01

To determine the bacterial contamination rate of the parent's cell phone and the effectiveness of anti-microbial gel in reducing transmission of bacteria from cell phone to hands. Cross-sectional study of cultures from the cell phone and hands before and after applying anti-microbial gel (n=50). All cell phones demonstrated bacterial contamination. Ninety percent had the same bacteria on the cell phone and their cleaned hands. Twenty two percent had no growth on their hands after applying anti-microbial gel after they had the same bacteria on the cell phone and hands. Ninety-two percent of parents were aware that cell phones carried bacteria, but only 38% cleaned their cell phones at least weekly. Bacterial contamination of cell phones may serve as vectors for nosocomial infection in the neonatal intensive care unit. Bacteria transmitted from cell phone to hands may not be eliminated using anti-microbial gel. Development of hand hygiene and cell phone cleaning guidelines are needed regarding bedside cell phone use.

Internal and external contamination of donor corneas before in situ excision: bacterial risk factors in 93 donors.

PubMed

Robert, Pierre-Yves; Camezind, Philippe; Drouet, Mireille; Ploy, Marie-Cécile; Adenis, Jean-Paul

2002-04-01

Most studies of corneal donor contamination concentrate on postenucleation contamination of the eyeball. The purpose of the present study was to evaluate the relative contamination of in situ excised corneal tissue and relevance to final success or rejection by recipients of the corneal grafts. Ninety-three donors underwent anterior chamber puncture (ACP) and corneal epithelium scarification (CS) before and after disinfection with 5% povidone-iodine. Following in situ excision, corneas were preserved in culture medium at +35 degrees C. Morphological and bacteriological assessment was carried out after culture, and recipients were followed up over a 2-year period. Samples taken by ACP, CS before disinfection, CS after disinfection and a culture medium sample were contaminated by bacteria in, respectively, 8 (8.6%), 23 (24.7%) 4 (4.3%) and 5 (5.4%) donors. Contamination of aqueous humor was significantly associated with age, death-to-sample time and premortem systemic infection. Contamination of epithelium significantly increased culture medium contamination. External bacteria on donor cornea are mainly skin bacteria (especially Staphylococcus) and can be partially eliminated by a povidone-iodine wash. Internal bacteria are mainly gut bacteria and may be due to perimortem bacteriemia. However, bacterial infection at the time of death appears to have no effect on the incidence of endophthalmitis in recipients and should no longer prevent use of such corneal tissue in grafts.

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

PubMed Central

Low, Serene; Raina, Jean-Baptiste; Skarshewski, Adam; Le, Xuyen H.; Butler, Margaret K.; Stocker, Roman; Seymour, Justin; Tyson, Gene W.

2016-01-01

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics. PMID:27688978

Bacterial diversity and real-time PCR based assessment of linA and linB gene distribution at hexachlorocyclohexane contaminated sites.

PubMed

Lal, Devi; Jindal, Swati; Kumari, Hansi; Jit, Simran; Nigam, Aeshna; Sharma, Pooja; Kumari, Kirti; Lal, Rup

2015-03-01

The disposal of hexachlorocyclohexane (HCH) muck has created large number of HCH dumpsites all over the world from where the harmful HCH isomers are leaking into the environment. Bacteria have evolved at such contaminated sites that have the ability to degrade HCH. Degradation of various HCH isomers in bacterial strains is mediated primarily by two genes: linA and linB which encode dehydrochlorinase and haloalkane dehalogenase respectively. In this study we explored one such highly contaminated HCH dumpsite located in Lucknow, Uttar Pradesh, India. To assess the biostimulation potential of the contaminated site, microbial diversity study and real-time PCR based quantification of lin genes was carried out. The soil samples from dumpsite and surrounding areas were found to be highly contaminated with HCH residue levels as high as 1.8 × 10(5)  mg kg(-1). The residues were detected in areas upto 13 km from the dumpsite. Sphingomonads, Chromohalobacter, and Marinobacter were the dominant genera present at the dump-site. Role of Sphingomonads in HCH degradation has been well documented. The highest copy numbers of linA and linB genes as determined using real-time PCR were 6.2 × 10(4) and 5.3 × 10(5), respectively, were found in sample from the dump site. The presence of Sphingomonads, linA, and linB genes from HCH contaminated soil indicates the presence of indigenous bacterial communities capable of HCH degradation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of the bacterial strains using Biolog plates in the contaminated soil from Riyadh community.

PubMed

Al-Dhabaan, Fahad Abdullah M; Bakhali, Ali Hassan

2017-05-01

Routine manufacture, detonation and disposal of explosives in land and groundwater have resulted in complete pollution. Explosives are xenobiotic compounds, being toxic to biological systems, and their recalcitrance leads to persistence in the environment. The methods currently used for the remediation of explosive contaminated sites are expensive and can result in the formation of toxic products. The present study aimed to investigate the bacterial strains using the Biolog plates in the soil from the Riyadh community. The microbial strains were isolated using the spread plate technique and were identified using the Biolog method. In this study we have analyzed from bacterial families of soil samples, obtained from the different sites in 5 regions at Explosive Institute. Our results conclude that Biolog MicroPlates were developed for the rapid identification of bacterial isolates by sole-carbon source utilization and can be used for the identification of bacteria. Out of five communities, only four families of bacteria indicate that the microbial community lacks significant diversity in region one from the Riyadh community in Saudi Arabia. More studies are needed to be carried out in different regions to validate our results.

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

PubMed

Valle-García, David; Griffiths, Lyra M; Dyer, Michael A; Bernstein, Emily; Recillas-Targa, Félix

2014-01-01

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

Rapid measurement and prediction of bacterial contamination in milk using an oxygen electrode.

PubMed

Numthuam, Sonthaya; Suzuki, Hiroaki; Fukuda, Junji; Phunsiri, Suthiluk; Rungchang, Saowaluk; Satake, Takaaki

2009-03-01

An oxygen electrode was used to measure oxygen consumption to determine bacterial contamination in milk. Dissolved oxygen (DO) measured at 10-35 degrees C for 2 hours provided a reasonable prediction efficiency (r > or = 0.90) of the amount of bacteria between 1.9 and 7.3 log (CFU/mL). A temperature-dependent predictive model was developed that has the same prediction accuracy like the normal predictive model. The analysis performed with and without stirring provided the same prediction efficiency, with correlation coefficient of 0.90. The measurement of DO is a simple and rapid method for the determination of bacteria in milk.

[Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA].

PubMed

Jia, Xiao-Wei; Zhang, Guo-Hui; Shi, Hai-Yan

2012-12-01

Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription. We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction. The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15. kod-ssb may in future be used to enhance DNA and cDNA amplification.

Bacterial Community Response to Petroleum Hydrocarbon Amendments in Freshwater, Marine, and Hypersaline Water-Containing Microcosms

PubMed Central

Jurelevicius, Diogo; Alvarez, Vanessa Marques; Marques, Joana Montezano; de Sousa Lima, Laryssa Ribeiro Fonseca; Dias, Felipe de Almeida

2013-01-01

Hydrocarbon-degrading bacterial communities from freshwater, marine, and hypersaline Brazilian aquatic ecosystems (with water salinities corresponding to 0.2%, 4%, and 5%, respectively) were enriched with different hydrocarbons (heptadecane, naphthalene, or crude oil). Changes within the different microcosms of bacterial communities were analyzed using cultivation approaches and molecular methods (DNA and RNA extraction, followed by genetic fingerprinting and analyses of clone libraries based on the 16S rRNA-coding gene). A redundancy analysis (RDA) of the genetic fingerprint data and a principal component analysis (PCA) of the clone libraries revealed hydrocarbon-enriched bacterial communities specific for each ecosystem studied. However, within the same ecosystem, different bacterial communities were selected according to the petroleum hydrocarbon used. In general, the results demonstrated that Acinetobacter and Cloacibacterium were the dominant genera in freshwater microcosms; the Oceanospirillales order and the Marinobacter, Pseudomonas, and Cycloclasticus genera predominated in marine microcosms; and the Oceanospirillales order and the Marinobacter genus were selected in the different hydrocarbon-containing microcosms in hypersaline water. Determination of total petroleum hydrocarbons (TPHs) in all microcosms after 32 days of incubation showed a decrease in the hydrocarbon concentration compared to that for the controls. A total of 50 (41.3%) isolates from the different hydrocarbon-contaminated microcosms were associated with the dominant operational taxonomic units (OTUs) obtained from the clone libraries, and their growth in the hydrocarbon contaminating the microcosm from which they were isolated as the sole carbon source was observed. These data provide insight into the general response of bacterial communities from freshwater, marine, and hypersaline aquatic ecosystems to petroleum hydrocarbon contamination. PMID:23872573

Effects of bacterial contamination of media on the diagnosis of Tritrichomonas foetus by culture and real-time PCR.

PubMed

Clothier, Kristin A; Villanueva, Michelle; Torain, Andrea; Hult, Cynthia; Wallace, Rachel

2015-03-15

The venereal pathogen Tritrichomonas foetus causes early embryonic death and abortion in cattle. With no approved treatment, control involves detection of infected animals and their removal from the herd. Culture is the traditional diagnostic method; standard media are formulated to support protozoal growth while suppressing competing organisms which may prevent microscopic recognition of T. foetus. Real-time PCR increases diagnostic sensitivity and specificity over culture but requires intact T. foetus DNA for detection. The purposes of this study were 1) to evaluate the effects of resident preputial bacteria that are not suppressed by antimicrobials in a commercial culture medium (InPouch™) on T. foetus detection by culture and PCR, and 2) to determine the performance of a laboratory-prepared culture medium on T. foetus detection by culture and PCR in samples with and without this bacterial contamination. A known concentration of one of three different strains of T. foetus inoculated into InPouch™ (IP) or modified Diamonds-Plastridge media (DPM) were co-incubated with a smegma culture media (CONTAM) for 24h and examined microscopically for the presence of identifiable T. foetus. PCR was performed on IP samples to determine if CONTAM also affected T. foetus DNA detection. A PCR protocol was then validated in DPM that performed similarly to the established IP PCR method. IP and DPM with CONTAM were spiked with serial dilutions that mimic field infections of one of four T. foetus strains and evaluated by real-time PCR; cycles to threshold (Ct) values and "positive" classification were compared between media. T. foetus motility and morphology as well as media pH were severely altered in IP samples with CONTAM compared to those without as well as to DPM medium with and without CONTAM (P<0.0001). PCR testing demonstrated significantly greater Ct values were for T. foetus DNA (P<0.001) in IP contaminated with smegma bacteria compared to those without. When using T

Escherichia coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ.

PubMed

Koponen, Jonna K; Turunen, Anna-Mari; Ylä-Herttuala, Seppo

2002-03-01

Real-time PCR is a powerful method for the quantification of gene expression in biological samples. This method uses TaqMan chemistry based on the 5' -exonuclease activity of the AmpliTaq Gold DNA polymerase which releases fluorescence from hybridized probes during synthesis of each new PCR product. Many gene therapy studies use lacZ, encoding Escherichia coli beta-galactosidase, as a marker gene. Our results demonstrate that E. coli DNA contamination in AmpliTaq Gold polymerase interferes with TaqMan analysis of lacZ gene expression and decreases sensitivity of the method below the level required for biodistribution and long-term gene expression studies. In biodistribution analyses the contamination can lead to false-negative results by masking low-level lacZ expression in target and ectopic tissues, and false-positive results if sufficient controls are not used. We conclude that, to get reliable TaqMan results with lacZ, adequate controls should be included in each run to rule out contamination from AmpliTaq Gold polymerase.

Bacterial Contamination of Anaesthetic and Vasopressor Drugs in the Operating Theatres: Ameliyathanelerde Anestetik ve Vazopressör İlaçların Bakteriyel Kontaminasyonu.

PubMed

Rueangchira-Urai, Rongrong; Rujirojindakul, Panthila; Geater, Alan Frederick; McNeil, Edward

2017-02-01

The aim of this study was to determine the incidence of bacterial and fungal contamination in anaesthetic and vasopressor drugs before and after use in operating theatres. A cross-sectional study was conducted in the operating theatres of a university hospital. We collected 945 samples of three different drugs, namely, propofol, vecuronium and ephedrine, from 20 operating rooms and refrigerators where the unused drugs were stored. Each drug was divided into two groups, the pre-use group and the post-use group. The pre-use drugs were cultured before the patient received the drug. The post-use drugs were cultured after the patient had received the drug or after the drugs had been transferred to other syringes. The culture results were reported as either positive or negative. Out of the 945 drug samples, 26 (2.8%, 95% confidence interval=1.8%-4.0%) gave a positive culture. Of the 317 propofol samples, 20 (6.3%) were found to have bacterial contamination, 11 in the pre-use group and 9 in the post-use group. Of the 318 ephedrine samples, 6 (1.9%) were found to be positive on culture, one in the pre-use group and five in the post-use group. Vecuronium gave no positive cultures. All organisms were non-pathogenic, and no fungal contamination was found. The incidence of bacterial contamination in anaesthetic and vasopressor drugs was 2.8%. Anaesthetic teams must be aware of contamination issues in anaesthetic drugs that have been prepared for later use and, in order to reduce the risk of contamination, they must improve the methods of administering drugs to patients.

Potential effects of environmental contaminants on P450 aromatase activity and DNA damage in swallows from the Rio Grande and Somerville, Texas

USGS Publications Warehouse

Sitzlar, M.A.; Mora, M.A.; Fleming, J.G.W.; Bazer, F.W.; Bickham, J.W.; Matson, C.W.

2009-01-01

Cliff swallows (Petrochelidon pyrrhonota) and cave swallows (P. fulva) were sampled during the breeding season at several locations in the Rio Grande, Texas, to evaluate the potential effects of environmental contaminants on P450 aromatase activity in brain and gonads and DNA damage in blood cells. The tritiated water-release aromatase assay was used to measure aromatase activity and flow cytometry was used to measure DNA damage in nucleated blood cells. There were no significant differences in brain and gonadal aromatase activities or in estimates of DNA damage (HPCV values) among cave swallow colonies from the Lower Rio Grande Valley (LRGV) and Somerville. However, both brain and gonadal aromatase activities were significantly higher (P < 0.05) in male cliff swallows from Laredo than in those from Somerville. Also, DNA damage estimates were significantly higher (P < 0.05) in cliff swallows (males and females combined) from Laredo than in those from Somerville. Contaminants of current high use in the LRGV, such as atrazine, and some of the highly persistent organochlorines, such as toxaphene and DDE, could be potentially associated with modulation of aromatase activity in avian tissues. Previous studies have indicated possible DNA damage in cliff swallows. We did not observe any differences in aromatase activity or DNA damage in cave swallows that could be associated with contaminant exposure. Also, the differences in aromatase activity and DNA damage between male cliff swallows from Laredo and Somerville could not be explained by contaminants measured at each site in previous studies. Our study provides baseline information on brain and gonadal aromatase activity in swallows that could be useful in future studies. ?? 2008 Springer Science+Business Media, LLC.

Superstatistical model of bacterial DNA architecture

NASA Astrophysics Data System (ADS)

Bogachev, Mikhail I.; Markelov, Oleg A.; Kayumov, Airat R.; Bunde, Armin

2017-02-01

Understanding the physical principles that govern the complex DNA structural organization as well as its mechanical and thermodynamical properties is essential for the advancement in both life sciences and genetic engineering. Recently we have discovered that the complex DNA organization is explicitly reflected in the arrangement of nucleotides depicted by the universal power law tailed internucleotide interval distribution that is valid for complete genomes of various prokaryotic and eukaryotic organisms. Here we suggest a superstatistical model that represents a long DNA molecule by a series of consecutive ~150 bp DNA segments with the alternation of the local nucleotide composition between segments exhibiting long-range correlations. We show that the superstatistical model and the corresponding DNA generation algorithm explicitly reproduce the laws governing the empirical nucleotide arrangement properties of the DNA sequences for various global GC contents and optimal living temperatures. Finally, we discuss the relevance of our model in terms of the DNA mechanical properties. As an outlook, we focus on finding the DNA sequences that encode a given protein while simultaneously reproducing the nucleotide arrangement laws observed from empirical genomes, that may be of interest in the optimization of genetic engineering of long DNA molecules.

Cyclic AMP Regulates Bacterial Persistence through Repression of the Oxidative Stress Response and SOS-Dependent DNA Repair in Uropathogenic Escherichia coli.

PubMed

Molina-Quiroz, Roberto C; Silva-Valenzuela, Cecilia; Brewster, Jennifer; Castro-Nallar, Eduardo; Levy, Stuart B; Camilli, Andrew

2018-01-09

Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic Escherichia coli , as measured by survival after exposure to a β-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic E. coli IMPORTANCE Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic E. coli , the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to β-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds. Copyright © 2018 Molina-Quiroz et al.

Emerging contaminants and nutrients synergistically affect the spread of class 1 integron-integrase (intI1) and sul1 genes within stable streambed bacterial communities.

PubMed

Subirats, Jèssica; Timoner, Xisca; Sànchez-Melsió, Alexandre; Balcázar, José Luis; Acuña, Vicenç; Sabater, Sergi; Borrego, Carles M

2018-07-01

Wastewater effluents increase the nutrient load of receiving streams while introducing a myriad of anthropogenic chemical pollutants that challenge the resident aquatic (micro)biota. Disentangling the effects of both kind of stressors and their potential interaction on the dissemination of antibiotic resistance genes in bacterial communities requires highly controlled manipulative experiments. In this work, we investigated the effects of a combined regime of nutrients (at low, medium and high concentrations) and a mixture of emerging contaminants (ciprofloxacin, erythromycin, sulfamethoxazole, diclofenac, and methylparaben) on the bacterial composition, abundance and antibiotic resistance profile of biofilms grown in artificial streams. In particular, we investigated the effect of this combined stress on genes encoding resistance to ciprofloxacin (qnrS), erythromycin (ermB), sulfamethoxazole (sul1 and sul2) as well as the class 1 integron-integrase gene (intI1). Only genes conferring resistance to sulfonamides (sul1 and sul2) and intI1 gene were detected in all treatments during the study period. Besides, bacterial communities exposed to emerging contaminants showed higher copy numbers of sul1 and intI1 genes than those not exposed, whereas nutrient amendments did not affect their abundance. However, bacterial communities exposed to both emerging contaminants and a high nutrient concentration (1, 25 and 1 mg L -1 of phosphate, nitrate and ammonium, respectively) showed the highest increase on the abundance of sul1 and intI1 genes thus suggesting a factors synergistic effect of both stressors. Since none of the treatments caused a significant change on the composition of bacterial communities, the enrichment of sul1 and intI1 genes within the community was caused by their dissemination under the combined pressure exerted by nutrients and emerging contaminants. To the best of our knowledge, this is the first study demonstrating the contribution of nutrients on

Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

PubMed Central

2011-01-01

Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool. PMID:21314965

Effect of biostimulants on 2,4,6-trinitrotoluene (TNT) degradation and bacterial community composition in contaminated aquifer sediment enrichments.

PubMed

Fahrenfeld, Nicole; Zoeckler, Jeffrey; Widdowson, Mark A; Pruden, Amy

2013-04-01

2,4,6-Trinitrotoluene (TNT) is a toxic and persistent explosive compound occurring as a contaminant at numerous sites worldwide. Knowledge of the microbial dynamics driving TNT biodegradation is limited, particularly in native aquifer sediments where it poses a threat to water resources. The purpose of this study was to quantify the effect of organic amendments on anaerobic TNT biodegradation rate and pathway in an enrichment culture obtained from historically contaminated aquifer sediment and to compare the bacterial community dynamics. TNT readily biodegraded in all microcosms, with the highest biodegradation rate obtained under the lactate amended condition followed by ethanol amended and naturally occurring organic matter (extracted from site sediment) amended conditions. Although a reductive pathway of TNT degradation was observed across all conditions, denaturing gradient gel electrophoresis (DGGE) analysis revealed distinct bacterial community compositions. In all microcosms, Gram-negative γ- or β-Proteobacteria and Gram-positive Negativicutes or Clostridia were observed. A Pseudomonas sp. in particular was observed to be stimulated under all conditions. According to non-metric multidimensional scaling analysis of DGGE profiles, the microcosm communities were most similar to heavily TNT-contaminated field site sediment, relative to moderately and uncontaminated sediments, suggesting that TNT contamination itself is a major driver of microbial community structure. Overall these results provide a new line of evidence of the key bacteria driving TNT degradation in aquifer sediments and their dynamics in response to organic carbon amendment, supporting this approach as a promising technology for stimulating in situ TNT bioremediation in the subsurface.

Metal concentrations, sperm motility, and RNA/DNA ratio in two echinoderm species from a highly contaminated fjord (the Sørfjord, Norway).

PubMed

Catarino, Ana I; Cabral, Henrique N; Peeters, Kris; Pernet, Philippe; Punjabi, Usha; Dubois, Philippe

2008-07-01

The present study evaluated the effects of field metal contamination on sperm motility and the RNA/DNA ratio in echinoderms. Populations of Asterias rubens and Echinus acutus that occur naturally along a contamination gradient of sediments by cadmium, copper, lead, and zinc in a Norwegian fjord (the Sørfjord) were studied. Sperm motility, a measure of sperm quality, was quantified using a computer-assisted sperm analysis system. The RNA/DNA ratio, a measure of protein synthesis, was assessed by a one-dye (ethidium bromide)/one-enzyme (RNase), 96-well microplate fluorometric assay. Although both species accumulate metals at high concentrations, neither sperm motility parameters in A. rubens nor the RNA/DNA ratio in both species were affected. The Sørfjord is still one of the most metal-contaminated marine sites in Europe, but even so, populations of A. rubens and E. acutus are able to endure under these conditions.

What Healthcare Workers Should Know about Environmental Bacterial Contamination in the Intensive Care Unit.

PubMed

Russotto, Vincenzo; Cortegiani, Andrea; Fasciana, Teresa; Iozzo, Pasquale; Raineri, Santi Maurizio; Gregoretti, Cesare; Giammanco, Anna; Giarratano, Antonino

2017-01-01

Intensive care unit- (ICU-) acquired infections are a major health problem worldwide. Inanimate surfaces and equipment contamination may play a role in cross-transmission of pathogens and subsequent patient colonization or infection. Bacteria contaminate inanimate surfaces and equipment of the patient zone and healthcare area, generating a reservoir of potential pathogens, including multidrug resistant species. Traditional terminal cleaning methods have limitations. Indeed patients who receive a bed from prior patient carrying bacteria are exposed to an increased risk (odds ratio 2.13, 95% confidence intervals 1.62-2.81) of being colonized and potentially infected by the same bacterial species of the previous patient. Biofilm formation, even on dry surfaces, may play a role in reducing the efficacy of terminal cleaning procedures since it enables bacteria to survive in the environment for a long period and provides increased resistance to commonly used disinfectants. No-touch methods (e.g., UV-light, hydrogen peroxide vapour) are under investigation and further studies with patient-centred outcomes are needed, before considering them the standard of terminal cleaning in ICUs. Healthcare workers should be aware of the role of environmental contamination in the ICU and consider it in the broader perspective of infection control measures and stewardship initiatives.

Mechanisms of bacterial DNA replication restart

PubMed Central

Windgassen, Tricia A; Wessel, Sarah R; Bhattacharyya, Basudeb

2018-01-01

Abstract Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved ‘DNA replication restart’ pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT). PMID:29202195

Addressing the risk of bacterial contamination in platelets: a hospital economic perspective.

PubMed

Li, Justin W; Brecher, Mark E; Jacobson, Jessica L; Harm, Sarah K; Chen, Dorothy; El-Gamil, Audrey; Dobson, Al; Mintz, Paul D

2017-10-01

Bacterially contaminated platelets (PLTs) remain a serious risk. The Food and Drug Administration has issued draft guidance recommending hospitals implement secondary testing or transfuse PLTs that have been treated with pathogen reduction technology (PRT). The cost implications of these approaches are not well understood. We modeled incurred costs when hospitals acquire, process, and transfuse PLTs that are PRT treated with INTERCEPT (Cerus Corp.) or secondary tested with the PLT PGD Test (Verax Biomedical). Hospitals will spend $221.27 (30.0%) more per PRT-treated apheresis PLT unit administered compared to a Zika-tested apheresis PLT unit that is irradiated and PGD tested in hospital. This difference is reflected in PRT PLT units having: 1) a higher hospital purchase price ($100.00 additional charge compared to an untreated PLT); 2) lower therapeutic effectiveness than untreated PLTs among hematologic-oncologic patients, which contributes to additional transfusions ($96.05); or 3) fewer PLT storage days, which contributes to higher outdating cost from expired PLTs ($67.87). Only a small portion of the incremental costs for PRT-treated PLTs are offset by costs that may be avoided, including primary bacterial culture, secondary bacterial testing ($26.65), hospital irradiation ($8.50), Zika testing ($4.47), and other costs ($3.03). The significantly higher cost of PRT-treated PLTs over PGD-tested PLTs should interest stakeholders. For hospitals that outdate PLTs, savings associated with expiration extension to 7 days by adding PGD testing will likely be substantially greater than the cost of implementing PGD-testing. Our findings might usefully inform a hospital's decision to select a particular blood safety approach. © 2017 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.

Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

NASA Astrophysics Data System (ADS)

Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

2018-03-01

The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

Experimental iron-inactivated Pasteurella multocida A: 1 vaccine adjuvanted with bacterial DNA is safe and protects chickens from fowl cholera.

PubMed

Herath, Chitra; Kumar, Pankaj; Singh, Mithilesh; Kumar, Devender; Ramakrishnan, Saravanan; Goswami, Tapas Kumar; Singh, Ajit; Ram, G C

2010-03-08

Fowl cholera is a serious problem in large and small scale poultry production. The present study describes the development and testing of an inactivated whole-cell, low-cost, safe, and effective vaccine for fowl cholera based on a previous work (Vaccine 23:5590-5598). Pasteurella multocida A: 1 grown in the presence of low FeCl(3) concentrations, inactivated with higher concentrations of FeCl(3), and adjuvanted with bacterial DNA from P. multocida B: 2 containing immunostimulatory CpG motifs protect chickens with a lethal P. multocida A: 1 challenge. Chickens were immunized with two whole-cell inactivated vaccine doses at 4 weeks apart and challenged 4 weeks after booster immunization. Experimental vaccines were pure, easy injectable, and caused very little distress in chickens due to their aqueous consistency. Vaccines and bacterial DNA (bDNA) posed no safety problems when chickens were injected subcutaneously (s.c.) with a single, double, and overdose of these preparations. Immunized chickens produced systemic IgY antibodies (Ab) responses and vaccine adjuvanted with bDNA protected 100% chickens from lethal intrapertoneal (i.p.) P. multocida A: 1 challenge. This work suggests that use of bDNA as an adjuvant can improve the cost-effectiveness of inactivated veterinary vaccines for their use in developing countries. Our future studies will focus on safety and potency evaluation of experimental and current vaccines using bDNA as an adjuvant. Copyright 2010 Elsevier Ltd. All rights reserved.

Health risk assessment of heavy metals and bacterial contamination in drinking water sources: a case study of Malakand Agency, Pakistan.

PubMed

Nawab, Javed; Khan, Sardar; Ali, Sharafat; Sher, Hassan; Rahman, Ziaur; Khan, Kifayatullah; Tang, Jianfeng; Ahmad, Aziz

2016-05-01

Human beings are frequently exposed to pathogens and heavy metals through ingestion of contaminated drinking water throughout the world particularly in developing countries. The present study aimed to assess the quality of water used for drinking purposes in Malakand Agency, Pakistan. Water samples were collected from different sources (dug wells, bore wells, tube wells, springs, and hand pumps) and analyzed for different physico-chemical parameters and bacterial pathogens (fecal coliform bacteria) using standard methods, while heavy metals were analyzed using atomic absorption spectrophotometry (AAS-PEA-700). In the study area, 70 % of water sources were contaminated with F. coliform representing high bacterial contamination. The heavy metals, such as Cd (29 and 8 %), Ni (16 and 78 %), and Cr (7 %), exceeded their respective safe limits of WHO (2006) and Pak-EPA (2008), respectively, in water sources, while Pb (9 %) only exceeded from WHO safe limit. The risk assessment tools such as daily intake of metals (DIMs) and health risk indexes (HRIs) were used for health risk estimation and were observed in the order of Ni > Cr > Mn > Pb > Cd and Cd > Ni > Pb > Mn > Cr, respectively. The HRI values of heavy metals for both children and adults were <1, showing lack of potential health risk to the local inhabitants of the study area.

Patterns of benthic bacterial diversity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)

PubMed Central

Quero, Grazia Marina; Cassin, Daniele; Botter, Margherita; Perini, Laura; Luna, Gian Marco

2015-01-01

Prokaryotes in coastal sediments are fundamental players in the ecosystem functioning and regulate processes relevant in the global biogeochemical cycles. Nevertheless, knowledge on benthic microbial diversity patterns across spatial scales, or as function to anthropogenic influence, is still limited. We investigated the microbial diversity in two of the most chemically polluted sites along the coast of Italy. One site is the Po River Prodelta (Northern Adriatic Sea), which receives contaminant discharge from one of the largest rivers in Europe. The other site, the Mar Piccolo of Taranto (Ionian Sea), is a chronically polluted area due to steel production plants, oil refineries, and intense maritime traffic. We collected sediments from 30 stations along gradients of contamination, and studied prokaryotic diversity using Illumina sequencing of amplicons of a 16S rDNA gene fragment. The main sediment variables and the concentration of eleven metals, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) were measured. Chemical analyses confirmed the high contamination in both sites, with concentrations of PCBs particularly high and often exceeding the sediment guidelines. The analysis of more than 3 millions 16S rDNA sequences showed that richness decreased with higher contamination levels. Multivariate analyses showed that contaminants significantly shaped community composition. Assemblages differed significantly between the two sites, but showed wide within-site variations related with spatial gradients in the chemical contamination, and the presence of a core set of OTUs shared by the two geographically distant sites. A larger importance of PCB-degrading taxa was observed in the Mar Piccolo, suggesting their potential selection in this historically polluted site. Our results indicate that sediment contamination by multiple contaminants significantly alter benthic prokaryotic diversity in coastal areas, and suggests considering the potential

Characterization of a di-n-butyl phthalate-degrading bacterial consortium and its application in contaminated soil.

PubMed

Yang, Jing; Guo, Chuling; Liu, Shasha; Liu, Weiting; Wang, Han; Dang, Zhi; Lu, Guining

2018-04-17

Dibutyl phthalate (DBP), as a plasticizer, is widely used in China, and it is easily released into diverse environments. In this study, we have obtained a stable bacterial consortium (B1) enriched from municipal sewage treatment plant activated sludge. The obtained bacterial consortium B1 was capable of degrading DBP and was mainly composed of Pandoraea sp. and Microbacterium sp. From the initial concentrations of 35-500 mg L -1 , DBP was efficiently degraded by the consortium, with the degradation rates above 92% within 3 days. The optimal temperature for DBP degradation was 30 °C and consortium B1 could adapt to a wide range of pH (5.5-8.5). The analysis of Illumina sequencing further showed that the relative abundance of Pandoraea was increased at the beginning of the degradation, while Microbacterium was decreased. In the later stage of the degradation, the change of the relative abundance of Pandoraea and Microbacterium was opposite. Apart from DBP, consortium B1 could also utilize dimethyl phthalate (DMP), di-2-ethylhexyl phthalate (DEHP), and phthalic acid (PA) as the sole carbon. Moreover, adding B1 to DBP-contaminated soil could greatly improve the removal rate of DBP, suggesting that B1 has a great potential for the bioremediation of DBP-contaminated environments.

Cellular telephone as reservoir of bacterial contamination: myth or fact.

PubMed

Walia, Satinder S; Manchanda, Adesh; Narang, Ramandeep S; N, Anup; Singh, Balwinder; Kahlon, Sukhdeep S

2014-01-01

To assess bacterial contamination of cellular telephone of dental care personnel, and to determine factors contributing to their contamination. A descriptive, cross-sectional study was conducted, which included 300 people using a cellular telephone The study group (hundred in each group) comprised of Dental Health Care Personnel (DHCP), In-Hospital Personnel (IHP) and Out-Hospital Personnel (OHP) of a dental college cum hospital. Swab was wiped along the front and all sides of cellular handset and it was incubated in glucose broth. The swab was subplated onto growth media plates made with half Mac Conkey's agar and half blood agar and allowed to incubate for 48 hours at 37(o)C. Isolates were tested for antimicrobial susceptibility. The analysis of presence or absence of microorganisms in the DHCP, IHP and OHP group showed no pyogenic growth in 28%, 31% and 41% cases respectively, the distribution of which was not significant (p>.05). Among non potential pathogens, spore bearing gram positive bacilli were seen in 20 cases of DHCP group, 16 cases of IHP group and 17 cases of OHP group; the distribution of which was not significant (p>.05) Among potential pathogens, significant differences were observed in the distribution of growth of Enterobacter (p<.001), Pseudomonas species (p<.05), Acinetobacter bacteria (p<.05) and Methicillin-resistant Staphylococcus aureus (MRSA) bacteria (p<.001) between the participants of different groups. RESULTs of this study showed that fomites such as cellular telephones can potentially act as "Trojan horses", thus causing Hospital-Acquired Infections (HAIs) in the dental setting.

The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

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

Dong, Jinlan; George, Nicholas P.; Duckett, Katrina L.

2010-05-25

Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 {angstrom} resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeaemore » and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.« less

The T4 Phage DNA Mimic Protein Arn Inhibits the DNA Binding Activity of the Bacterial Histone-like Protein H-NS*

PubMed Central

Ho, Chun-Han; Wang, Hao-Ching; Ko, Tzu-Ping; Chang, Yuan-Chih; Wang, Andrew H.-J.

2014-01-01

The T4 phage protein Arn (Anti restriction nuclease) was identified as an inhibitor of the restriction enzyme McrBC. However, until now its molecular mechanism remained unclear. In the present study we used structural approaches to investigate biological properties of Arn. A structural analysis of Arn revealed that its shape and negative charge distribution are similar to dsDNA, suggesting that this protein could act as a DNA mimic. In a subsequent proteomic analysis, we found that the bacterial histone-like protein H-NS interacts with Arn, implying a new function. An electrophoretic mobility shift assay showed that Arn prevents H-NS from binding to the Escherichia coli hns and T4 p8.1 promoters. In vitro gene expression and electron microscopy analyses also indicated that Arn counteracts the gene-silencing effect of H-NS on a reporter gene. Because McrBC and H-NS both participate in the host defense system, our findings suggest that T4 Arn might knock down these mechanisms using its DNA mimicking properties. PMID:25118281

Interleukin-10 functions in vitro and in vivo to inhibit bacterial DNA-induced secretion of interleukin-12.

PubMed

Anitescu, M; Chace, J H; Tuetken, R; Yi, A K; Berg, D J; Krieg, A M; Cowdery, J S

1997-12-01

Bacterial DNA (bDNA) has a number of biologic properties, including the ability to induce interleukin-12 (IL-12) production by macrophages. We studied the role of the regulatory cytokine IL-10 as a potential inhibitor of bDNA-induced IL-12 production. IL-10 concentrations as low as 0.3 ng/ml profoundly inhibited bDNA-induced macrophage IL-12 production as measured by Elispot analysis of IL-12 p40-secreting cells. Additionally, we found that IL-10 inhibited bDNA-induced IL-12 secretion by the macrophage cell lines J774 and RAW 264. Preincubation of splenic adherent cells with IL-10 markedly reduced bDNA-induced transcription of IL-12 p40 mRNA. Interestingly, after 2 h of exposure, bDNA also induces transcription of IL-10 mRNA by splenic adherent cells. The importance of IL-10 in the in vivo regulation of bDNA-induced cytokine secretion was illustrated by the response of mice with disrupted IL-10 genes (IL-10 ko mice) to i.v. bDNA challenge. Compared to +/+ mice, IL-10 knockout (ko) mice exhibited increased numbers of IL-12 and interferon-gamma (IFN-gamma)-secreting cells following either single or repeated challenge with bDNA. These findings indicate that IL-10 plays a key role in regulating bDNA-induced production of inflammatory cytokines.

Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.

PubMed

Hammond, Elizabeth R; McGillivray, Brent C; Wicker, Sophie M; Peek, John C; Shelling, Andrew N; Stone, Peter; Chamley, Larry W; Cree, Lynsey M

2017-01-01

To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. Prospective embryo cohort study. Academic center and private in vitro fertilization (IVF) clinic. Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

PubMed Central

Agúndez, Leticia; González-Prieto, Coral; Machón, Cristina; Llosa, Matxalen

2012-01-01

Background Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. Methodology/Principal Findings We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. Conclusions/Significance The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells. PMID:22292089

Bacterial Contamination of Adult House Flies (Musca domestica) and Sensitivity of these Bacteria to Various Antibiotics, Captured from Hamadan City, Iran.

PubMed

Nazari, Mansour; Mehrabi, Tahereh; Hosseini, Seyed Mostafa; Alikhani, Mohammad Yousef

2017-04-01

House flies ( Musca domestica ) have been known as a mechanical vector in spreading infectious diseases such as cholera, shigellosis, salmonellosis and skin infections. To investigate the bacterial contaminations of house flies and determine the resistance of these bacteria against various antibiotics. An analytical descriptive cross- sectional study was conducted. The study was performed from July to September 2015 in Hamadan University of Medical Sciences, Hamadan, Iran. A total number of 300 house flies were collected from four places, 75 flies from each place, including two educational hospitals belonging to Hamadan University of Medical Sciences, a fruit and vegetables center, and a livestock slaughter. The body surface of house flies was washed using the physiological sterile serum and the obtained solution was centrifuged at 2000 rpm for five minutes. The identification of bacteria was carried out using the phenotypic methods. The resistance of bacteria against various antibiotics was determined using the disk diffusion approach. Data were analysed by the employment of SPSS software package version 20.0. A total number of 394 bacterial strains were isolated from 275 house flies. The most prevalent type of bacteria was Bacillus spp which was detected in 31.1% of house flies. Moreover, Staphylococcus s pp. (22.9%), Escherichia coli (11.6%) were other prevalent species, whereas, Enterococcus s pp. was the least prevalent type of bacteria in the collected house flies. In terms of resistance to antibiotics, it was identified that bacteria extracted from house flies which were collected from hospital environments were more resistant to antibiotics compared with the resistance of bacteria extracted from house flies which were collected from non hospital environments. The maximum bacterial isolation was found in houseflies from hospital No.1 environment from around the accumulation of garbage. It is a well-known fact that house flies are a source of bacterial

Unraveling a mechanism of honey antibacterial action: polyphenol/H₂O₂-induced oxidative effect on bacterial cell growth and on DNA degradation.

PubMed

Brudzynski, Katrina; Abubaker, Kamal; Miotto, Danielle

2012-07-15

Several compounds with antibacterial activities were identified in honey however, a mechanism by which they lead to bacterial growth inhibition and bacterial death remains still unknown. We recently found that honeys possess DNA degrading activity mediated by honey hydrogen peroxide and an unknown honey component(s). Here we provide evidence that active honeys (MIC90 of 6.25-12.5% v/v) possessed significantly higher levels of phenolics (p<0.02) of higher radical scavenging activities (p<0.005) than honeys of average activity. Removal of H2O2 by catalase eliminated bacteriostatic activities caused by both phenolics and H2O2 suggesting that the growth inhibition resulted from the coupling chemistry between these compounds. Both phenolics and H2O2 were involved in DNA degradation by honeys. Treatment of plasmid DNA with H2O2 alone did not affect the DNA integrity but H2O2 removal from honey by catalase prevented DNA degradation. Polyphenols extracted from honeys degraded plasmid DNA in the presence of H2O2 and Cu(II) in the Fenton-type reaction. The extent of DNA degradation was inversely related to the polyphenol concentration in this system as well as in honeys. At low content, honey polyphenols exerted pro-oxidant activity damaging to DNA. In conclusion, honey phenolics with pro-oxidant activities were necessary intermediates that conferred oxidative action of H2O2. Phenolic/H2O2-induced oxidative stress constituted the mechanism of honey bacteriostatic and DNA damaging activities. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

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.

Hamstring graft bacterial contamination during anterior cruciate ligament reconstruction: clinical and microbiological study.

PubMed

Badran, Mohamad Aboelnour; Moemen, Dalia Mohamed

2016-09-01

Autograft preparation for anterior cruciate ligament (ACL) reconstruction has a potential for graft contamination. The purpose of this study was to evaluate the possibility of bacterial contamination of hamstring autograft during preparation and when dropped onto the operating room floor and methods of graft decontamination. Sixty hamstring tendon autograft specimens were used as the test group. Excess tendon not used in the ACL procedure was divided into five segments. One segment, at the completion of preparation, was sent for culture as a control; the remaining four segments were dropped onto the floor adjacent to the surgical field for 15 seconds. One segment was cultured without undergoing any further treatment. Cultures were taken from each segment after immersion in 10 % povidone-iodine solution, 4 % chlorhexidine and bacitracin, respectively, for three minutes. Cultures of a skin swab and floor swab were taken at the same time and place that the ACL was dropped. Cultures of control graft tissue from ten patients (16.7 %) were positive for bacteria. No patient developed post-operative infection. Ninety organisms were identified, with Staphylococcus epidermidis being the most common isolate. Grafts rinsed in either bacitracin or 4 % chlorhexidine solutions were less likely to be culture positive. A high rate of contamination can be expected during autograft preparation for ACL reconstruction. Soaking the hamstring autograft in either bacitracin or 4 % chlorhexidine solution is effective for decontamination, particulary if graft is dropped on the floor.

Assessment of levels of bacterial contamination of large wild game meat in Europe.

PubMed

Membré, Jeanne-Marie; Laroche, Michel; Magras, Catherine

2011-08-01

The variations in prevalence and levels of pathogens and fecal contamination indicators in large wild game meat were studied to assess their potential impact on consumers. This analysis was based on hazard analysis, data generation and statistical analysis. A total of 2919 meat samples from three species (red deer, roe deer, wild boar) were collected at French game meat traders' facilities using two sampling protocols. Information was gathered on the types of meat cuts (forequarter or haunch; first sampling protocol) or type of retail-ready meat (stewing meat or roasting meat; second protocol), and also on the meat storage conditions (frozen or chilled), country of origin (eight countries) and shooting season (autumn, winter, spring). The samples were analyzed in both protocols for detection and enumeration of Escherichia coli, coagulase+staphylococci and Clostridium perfringens. In addition, detection and enumeration of thermotolerant coliforms and Listeria monocytogenes were performed for samples collected in the first and second protocols, respectively. The levels of bacterial contamination of the raw meat were determined by performing statistical analysis involving probabilistic techniques and Bayesian inference. C. perfringens was found in the highest numbers for the three indicators of microbial quality, hygiene and good handling, and L. monocytogenes in the lowest. Differences in contamination levels between game species and between meats distributed as chilled or frozen products were not significant. These results might be included in quantitative exposure assessments. Copyright © 2011 Elsevier Ltd. All rights reserved.

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

DOE PAGES

Rinke, Christian; Low, Serene; Woodcroft, Ben J.; ...

2016-09-22

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. For this study, we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diversemore » Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (~100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.« less

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

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

Rinke, Christian; Low, Serene; Woodcroft, Ben J.

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. For this study, we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diversemore » Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (~100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.« less

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

PubMed Central

Maruyama, Toru; Yamagishi, Keisuke; Mori, Tetsushi; Takeyama, Haruko

2015-01-01

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples. PMID:26389587

Effects of structural injure in the bile bacterial contamination after balloon transduodenal sphincteroplasty (papillary dilation) in dogs.

PubMed

Zavadinack Netto, Martin; Fagundes, Djalma José; Bandeira, César Orlando Peralta

2006-01-01

To evaluate, in dogs, the biliary sphincter subjected to dilation by hydrostatic balloon by the point of view of structural alterations of the papilla and the biochemestry and bacterial contamination of the bile. Twenty dogs were submitted to laparotomy, duodenotomy, and enlargement of the major duodenal papilla- GA(n=10) - with balloon of 8mm inflated with pressure of 0,5 atm, during 2 minutes or to the sham procedure - GB(n=10). Blood samples collected on times t(0 day), t(7 days) and t(28 days) were subjected to dosages of alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) for cholestasis evaluation. The collected material from the gall bladder at the same times were registered and numbered to be submitted to culture in BHI, blood agar (rich, non-selective element) and Mac Conkey (selective element for Gram-negative bacillus. On the 28th day three fragments of the papilla were tranversally cut by the choledoc axis 3mm from the duodenal papilla and the cuts, stained with hematoxylin-eosin and Masson's tricome, were evaluated according to their inflammatory reaction. The GGT and ALP averages on the three periods in the groups A and B did not show significant differences, not being characterizes the cholestasis. The bacterian contamination was significantly higher in GA (2,19) than in GB (1,96); the contamination was lower in the initial time compared with 7 and 28 days (t0bacterial contamination of the gall bladder, not associated with cholestasis. The morphologic lesions are more intense in the late phase, not associated with an eventual papilla esthenosis.

Bacterial contamination of fabric and metal-bead identity card lanyards: a cross-sectional study.

PubMed

Pepper, Thomas; Hicks, Georgina; Glass, Stephen; Philpott-Howard, John

2014-01-01

In healthcare, fabric or metal-bead lanyards are universally used for carrying identity cards. However there is little information on microbial contamination with potential pathogens that may readily re-contaminate disinfected hands. We examined 108 lanyards from hospital staff. Most grew skin flora but 7/108 (6%) had potentially pathogenic bacteria: four grew methicillin-susceptible Staphylococcus aureus, and four grew probable fecal flora: 3 Clostridium perfringens and 1 Clostridium bifermentans (one lanyard grew both S. aureus and C. bifermentans). Unused (control) lanyards had little or no such contamination. The median duration of lanyard wear was 12 months (interquartile range 3-36 months). 17/108 (16%) of the lanyards had reportedly undergone decontamination including wiping with alcohol, chlorhexidine or chlorine dioxide; and washing with soap and water or by washing machine. Metal-bead lanyards had significantly lower median bacterial counts than those from fabric lanyards (1 vs. 4 CFU/cm(2); Mann-Whitney U=300.5; P<0.001). 12/32 (38%) of the metal-bead lanyards grew no bacteria, compared with 2/76 (3%) of fabric lanyards. We recommend that an effective decontamination regimen be instituted by those who use fabric lanyards, or that fabric lanyards be discarded altogether in preference for metal-bead lanyards or clip-on identity cards. Copyright © 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Comparison of DNA extraction methods used to detect bacterial and yeast DNA from spiked whole blood by real-time PCR.

PubMed

Dalla-Costa, Libera M; Morello, Luis G; Conte, Danieli; Pereira, Luciane A; Palmeiro, Jussara K; Ambrosio, Altair; Cardozo, Dayane; Krieger, Marco A; Raboni, Sonia M

2017-09-01

Sepsis is the leading cause of death in intensive care units (ICUs) worldwide and its diagnosis remains a challenge. Blood culturing is the gold standard technique for blood stream infection (BSI) identification. Molecular tests to detect pathogens in whole blood enable early use of antimicrobials and affect clinical outcomes. Here, using real-time PCR, we evaluated DNA extraction using seven manual and three automated commercially available systems with whole blood samples artificially contaminated with Escherichia coli, Staphylococcus aureus, and Candida albicans, microorganisms commonly associated with BSI. Overall, the commercial kits evaluated presented several technical limitations including long turnaround time and low DNA yield and purity. The performance of the kits was comparable for detection of high microorganism loads (10 6 CFU/mL). However, the detection of lower concentrations was variable, despite the addition of pre-processing treatment to kits without such steps. Of the evaluated kits, the UMD-Universal CE IVD kit generated a higher quantity of DNA with greater nucleic acid purity and afforded the detection of the lowest microbial load in the samples. The inclusion of pre-processing steps with the kit seems to be critical for the detection of microorganism DNA directly from whole blood. In conclusion, future application of molecular techniques will require overcoming major challenges such as the detection of low levels of microorganism nucleic acids amidst the large quantity of human DNA present in samples or differences in the cellular structures of etiological agents that can also prevent high-quality DNA yields. Copyright © 2017 Elsevier B.V. All rights reserved.

Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size

PubMed Central

2014-01-01

Background Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days. Results Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left

eDNA-based bioassessment of coastal sediments impacted by an oil spill.

PubMed

Xie, Yuwei; Zhang, Xiaowei; Yang, Jianghua; Kim, Seonjin; Hong, Seongjin; Giesy, John P; Yim, Un Hyuk; Shim, Won Joon; Yu, Hongxia; Khim, Jong Seong

2018-07-01

Oil spills offshore can cause long-term ecological effects on coastal marine ecosystems. Despite their important ecological roles in the cycling of energy and nutrients in food webs, effects on bacteria, protists or arthropods are often neglected. Environmental DNA (eDNA) metabarcoding was applied to characterize changes in the structure of micro- and macro-biota communities of surface sediments over a 7-year period since the occurrence of Hebei Spirit oil spill on December 7, 2007. Alterations in diversities and structures of micro- and macro-biota were observed in the contaminated area where concentrations of polycyclic aromatic hydrocarbons were greater. Successions of bacterial, protists and metazoan communities revealed long-term ecological effects of residual oil. Residual oil dominated the largest cluster of the community-environment association network. Presence of bacterial families (Aerococcaceae and Carnobacteriaceae) and the protozoan family (Platyophryidae) might have conferred sensitivity of communities to oil pollution. Hydrocarbon-degrading bacterial families (Anaerolinaceae, Desulfobacteraceae, Helicobacteraceae and Piscirickettsiaceae) and algal family (Araphid pennate) were resistant to adverse effects of spilt oil. The protistan family (Subulatomonas) and arthropod families (Folsomia, Sarcophagidae Opomyzoidea, and Anomura) appeared to be positively associated with residual oil pollution. eDNA metabarcoding can provide a powerful tool for assessing effects of anthropogenic pollution, such as oil spills on sediment communities and its long-term trends in coastal marine environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pyrosequence analysis of bacterial communities in aerobic bioreactors treating polycyclic aromatic hydrocarbon-contaminated soil

PubMed Central

Richardson, Stephen D.; Aitken, Michael D.

2011-01-01

Two aerobic, lab-scale, slurry-phase bioreactors were used to examine the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil and the associated bacterial communities. The two bioreactors were operated under semi-continuous (draw-and-fill) conditions at a residence time of 35 days, but one was fed weekly and the other monthly. Most of the quantified PAHs, including high-molecular-weight compounds, were removed to a greater extent in the weekly-fed bioreactor, which achieved total PAH removal of 76%. Molecular analyses, including pyrosequencing of 16S rRNA genes, revealed significant shifts in the soil bacterial communities after introduction to the bioreactors and differences in the abundance and types of bacteria in each of the bioreactors. The weekly-fed bioreactor displayed a more stable bacterial community with gradual changes over time, whereas the monthly-fed bioreactor community was less consistent and may have been more strongly influenced by the influx of untreated soil during feeding. Phylogenetic groups containing known PAH-degrading bacteria previously identified through stable-isotope probing of the untreated soil were differentially affected by bioreactor conditions. Sequences from members of the Acidovorax and Sphingomonas genera, as well as the uncultivated ‘‘Pyrene Group 2’’ were abundant in the bioreactors. However, the relative abundances of sequences from the Pseudomonas, Sphingobium, and Pseudoxanthomonas genera, as well as from a group of unclassified anthracene degraders, were much lower in the bioreactors compared to the untreated soil. PMID:21369833

Cellular Telephone as Reservoir of Bacterial Contamination: Myth or Fact

PubMed Central

Walia, Satinder S.; Manchanda, Adesh; Narang, Ramandeep S.; N., Anup; Singh, Balwinder; Kahlon, Sukhdeep S.

2014-01-01

Objective: To assess bacterial contamination of cellular telephone of dental care personnel, and to determine factors contributing to their contamination. Materials and Methods: A descriptive, cross-sectional study was conducted, which included 300 people using a cellular telephone The study group (hundred in each group) comprised of Dental Health Care Personnel (DHCP), In-Hospital Personnel (IHP) and Out-Hospital Personnel (OHP) of a dental college cum hospital. Swab was wiped along the front and all sides of cellular handset and it was incubated in glucose broth. The swab was subplated onto growth media plates made with half Mac Conkey’s agar and half blood agar and allowed to incubate for 48 hours at 37oC. Isolates were tested for antimicrobial susceptibility. Result: The analysis of presence or absence of microorganisms in the DHCP, IHP and OHP group showed no pyogenic growth in 28%, 31% and 41% cases respectively, the distribution of which was not significant (p>.05). Among non potential pathogens, spore bearing gram positive bacilli were seen in 20 cases of DHCP group, 16 cases of IHP group and 17 cases of OHP group; the distribution of which was not significant (p>.05) Among potential pathogens, significant differences were observed in the distribution of growth of Enterobacter (p<.001), Pseudomonas species (p<.05), Acinetobacter bacteria (p<.05) and Methicillin-resistant Staphylococcus aureus (MRSA) bacteria (p<.001) between the participants of different groups. Conclusion: Results of this study showed that fomites such as cellular telephones can potentially act as “Trojan horses”, thus causing Hospital-Acquired Infections (HAIs) in the dental setting. PMID:24596722

Bacterial Degradation of Aromatic Compounds

PubMed Central

Seo, Jong-Su; Keum, Young-Soo; Li, Qing X.

2009-01-01

Aromatic compounds are among the most prevalent and persistent pollutants in the environment. Petroleum-contaminated soil and sediment commonly contain a mixture of polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatics. Aromatics derived from industrial activities often have functional groups such as alkyls, halogens and nitro groups. Biodegradation is a major mechanism of removal of organic pollutants from a contaminated site. This review focuses on bacterial degradation pathways of selected aromatic compounds. Catabolic pathways of naphthalene, fluorene, phenanthrene, fluoranthene, pyrene, and benzo[a]pyrene are described in detail. Bacterial catabolism of the heterocycles dibenzofuran, carbazole, dibenzothiophene, and dibenzodioxin is discussed. Bacterial catabolism of alkylated PAHs is summarized, followed by a brief discussion of proteomics and metabolomics as powerful tools for elucidation of biodegradation mechanisms. PMID:19440284

Bacterial Contamination on Household Toys and Association with Water, Sanitation and Hygiene Conditions in Honduras

PubMed Central

Stauber, Christine E.; Walters, Adam; de Aceituno, Anna M. Fabiszewski; Sobsey, Mark D.

2013-01-01

There is growing evidence that household water treatment interventions improve microbiological water quality and reduce diarrheal disease risk. Few studies have examined, however, the impact of water treatment interventions on household-level hygiene and sanitation. This study examined the association of four water and sanitation conditions (access to latrines, improved sanitation, improved water and the plastic biosand filter) on the levels of total coliforms and E. coli on existing and introduced toys during an on-going randomized controlled trial of the plastic biosand filter (plastic BSF). The following conditions were associated with decreased bacterial contamination on children’s toys: access to a latrine, access to improved sanitation and access to the plastic BSF. Overall, compared to existing toys, introduced toys had significantly lower levels of both E. coli and total coliforms. Results suggest that levels of fecal indicator bacteria contamination on children’s toys may be associated with access to improved water and sanitation conditions in the home. In addition, the fecal indicator bacteria levels on toys probably vary with duration in the household. Additional information on how these toys become contaminated is needed to determine the usefulness of toys as indicators or sentinels of water, sanitation and hygiene conditions, behaviors and risks. PMID:23598302

Bacterial contamination on household toys and association with water, sanitation and hygiene conditions in Honduras.

PubMed

Stauber, Christine E; Walters, Adam; Fabiszewski de Aceituno, Anna M; Sobsey, Mark D

2013-04-18

There is growing evidence that household water treatment interventions improve microbiological water quality and reduce diarrheal disease risk. Few studies have examined, however, the impact of water treatment interventions on household-level hygiene and sanitation. This study examined the association of four water and sanitation conditions (access to latrines, improved sanitation, improved water and the plastic biosand filter) on the levels of total coliforms and E. coli on existing and introduced toys during an on-going randomized controlled trial of the plastic biosand filter (plastic BSF). The following conditions were associated with decreased bacterial contamination on children's toys: access to a latrine, access to improved sanitation and access to the plastic BSF. Overall, compared to existing toys, introduced toys had significantly lower levels of both E. coli and total coliforms. Results suggest that levels of fecal indicator bacteria contamination on children's toys may be associated with access to improved water and sanitation conditions in the home. In addition, the fecal indicator bacteria levels on toys probably vary with duration in the household. Additional information on how these toys become contaminated is needed to determine the usefulness of toys as indicators or sentinels of water, sanitation and hygiene conditions, behaviors and risks.

A Novel Strategy to Predict Carcinogenicity of Antiparasitics Based on a Combination of DNA Lesions and Bacterial Mutagenicity Tests

PubMed Central

Liu, Qianying; Lei, Zhixin; Zhu, Feng; Ihsan, Awais; Wang, Xu; Yuan, Zonghui

2017-01-01

Genotoxicity and carcinogenicity testing of pharmaceuticals prior to commercialization is requested by regulatory agencies. The bacterial mutagenicity test was considered having the highest accuracy of carcinogenic prediction. However, some evidences suggest that it always results in false-positive responses when the bacterial mutagenicity test is used to predict carcinogenicity. Along with major changes made to the International Committee on Harmonization guidance on genotoxicity testing [S2 (R1)], the old data (especially the cytotgenetic data) may not meet current guidelines. This review provides a compendium of retrievable results of genotoxicity and animal carcinogenicity of 136 antiparasitics. Neither genotoxicity nor carcinogenicity data is available for 84 (61.8%), while 52 (38.2%) have been evaluated in at least one genotoxicity or carcinogenicity study, and only 20 (14.7%) in both genotoxicity and carcinogenicity studies. Among 33 antiparasitics with at least one old result in in vitro genotoxicity, 15 (45.5%) are in agreement with the current ICH S2 (R1) guidance for data acceptance. Compared with other genotoxicity assays, the DNA lesions can significantly increase the accuracy of prediction of carcinogenicity. Together, a combination of DNA lesion and bacterial tests is a more accurate way to predict carcinogenicity. PMID:29170735

Amplification and sequence analysis of partial bacterial 16S ribosomal RNA gene in gallbladder bile from patients with primary biliary cirrhosis.

PubMed

Hiramatsu, K; Harada, K; Tsuneyama, K; Sasaki, M; Fujita, S; Hashimoto, T; Kaneko, S; Kobayashi, K; Nakanuma, Y

2000-07-01

The etiopathogenesis of bile duct lesion in primary biliary cirrhosis is unknown, though the participation of bacteria and/or their components and products is suspected. In this study, we tried to detect and identify bacteria in the bile of patients with primary biliary cirrhosis by polymerase chain reaction using universal bacterial primers of the 16S ribosomal RNA gene. Gallbladder bile samples from 15 patients with primary biliary cirrhosis, 5 with primary sclerosing cholangitis, 5 with hepatitis C virus-related liver cirrhosis, 11 with cholecystolithiasis, and from 12 normal adult gallbladders were used. In addition to the culture study, partial bacterial 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) taking advantage of universal primers that can amplify the gene of almost all bacterial species, and the amplicons were cloned and sequenced. Sequence homology with specific bacterial species was analyzed by database research. Bacterial contamination at every step of the bile sampling, DNA extraction and PCR study was avoided. Furthermore, to confirm whether bacterial DNA is detectable in liver explants, the same analysis was performed using 10 liver explants of patients with primary biliary cirrhosis. In primary biliary cirrhosis, 75% (p<0.0001) of 100 clones were identified as so-called gram-positive cocci while these cocci were positive in only 5% in cholecystolithiasis (p<0.0001). In cholecystolithiasis gram-negative rods were predominant instead. One bacterial species detected in a normal adult was not related to those detected in primary biliary cirrhosis and cholecystolithiasis patients. No bacterial DNA was detected by PCR amplification in 10 liver explants of patients with primary biliary cirrhosis. The present results raise several possible roles of gram-positive bacteria in bile in the etiopathogenesis of primary biliary cirrhosis. However, these results could also reflect an epiphenomenon due to decreased bile flow in the

Purification of microalgae from bacterial contamination using a disposable inertia-based microfluidic device

NASA Astrophysics Data System (ADS)

Godino, Neus; Jorde, Felix; Lawlor, Daryl; Jaeger, Magnus; Duschl, Claus

2015-08-01

Microalgae are a promising source of bioactive ingredients for the food, pharmaceutical and cosmetic industries. Every microalgae research group or production facility is facing one major problem regarding the potential contamination of the algal cell with bacteria. Prior to the storage of the microalgae in strain collections or to cultivation in bioreactors, it is necessary to carry out laborious purification procedures to separate the microalgae from the undesired bacterial cells. In this work, we present a disposable microfluidic cartridge for the high-throughput purification of microalgae samples based on inertial microfluidics. Some of the most relevant microalgae strains have a larger size than the relatively small, few micron bacterial cells, so making them distinguishable by size. The inertial microfluidic cartridge was fabricated with inexpensive materials, like pressure sensitive adhesive (PSA) and thin plastic layers, which were patterned using a simple cutting plotter. In spite of fabrication restrictions and the intrinsic difficulties of biological samples, the separation of microalgae from bacteria reached values in excess of 99%, previously only achieved using conventional high-end and high cost lithography methods. Moreover, due to the simple and high-throughput characteristic of the separation, it is possible to concatenate serial purification to exponentially decrease the absolute amount of bacteria in the final purified sample.

The bacterial segrosome: a dynamic nucleoprotein machine for DNA trafficking and segregation.

PubMed

Hayes, Finbarr; Barillà, Daniela

2006-02-01

The genomes of unicellular and multicellular organisms must be partitioned equitably in coordination with cytokinesis to ensure faithful transmission of duplicated genetic material to daughter cells. Bacteria use sophisticated molecular mechanisms to guarantee accurate segregation of both plasmids and chromosomes at cell division. Plasmid segregation is most commonly mediated by a Walker-type ATPase and one of many DNA-binding proteins that assemble on a cis-acting centromere to form a nucleoprotein complex (the segrosome) that mediates intracellular plasmid transport. Bacterial chromosome segregation involves a multipartite strategy in which several discrete protein complexes potentially participate. Shedding light on the basis of genome segregation in bacteria could indicate new strategies aimed at combating pathogenic and antibiotic-resistant bacteria.

Rehosting of Bacterial Chaperones for High-Quality Protein Production▿

PubMed Central

Martínez-Alonso, Mónica; Toledo-Rubio, Verónica; Noad, Rob; Unzueta, Ugutz; Ferrer-Miralles, Neus; Roy, Polly; Villaverde, Antonio

2009-01-01

Coproduction of DnaK/DnaJ in Escherichia coli enhances solubility but promotes proteolytic degradation of their substrates, minimizing the yield of unstable polypeptides. Higher eukaryotes have orthologs of DnaK/DnaJ but lack the linked bacterial proteolytic system. By coexpression of DnaK and DnaJ in insect cells with inherently misfolding-prone recombinant proteins, we demonstrate simultaneous improvement of soluble protein yield and quality and proteolytic stability. Thus, undesired side effects of bacterial folding modulators can be avoided by appropriate rehosting in heterologous cell expression systems. PMID:19820142

Dimensionless Analysis Applied to Bacterial Chemotaxis towards NAPL Contaminants

NASA Astrophysics Data System (ADS)

Wang, X.; GAO, B.; Zhong, W.; Kihaule, K. S.; Ford, R.

2017-12-01

The use of chemotactic bacteria in bioremediation may improve the efficiency and decrease the cost of restoration, which means it has the potential to address environmental problems caused by oil spills. However, most previous studies were focused at the laboratory-scale and there lacks a formalism that can use these laboratory-scale results as input to evaluate the relative importance of chemotaxis at the field scale. In this study, a dimensionless equation is formulated to solve this problem. First, the main influential factors were extracted based on previous researches in environmental bioremediation and then five sets of dimensionless numbers were obtained according to Buckingham theory. After collecting basic parameter values and supplementary calculations to determine the concentration gradient of the chemoattractant, all dimensionless numbers were calculated and categorized into two types, those that were sensitive to chemotaxis or those to groundwater velocity. The bacteria ratio (BR), defined as the ratio of maximum bacteria concentration to its original value, was correlated with a combination of dimensionless numbers to yield, BR=cP1-0.085P20.329P30.1P4-0.098. For a bacterial ratio greater than one, the bioremediation strategy based on chemotaxis is expected to be effective, and chemotactic bacteria are expected to accumulate around NAPL contaminant sources efficiently.

Contamination of Hospital Water Supplies in Gilan, Iran, with Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa

PubMed Central

Ahmadi Jalali Moghadam, Masoumeh; Asfaram Meshginshahr, Sajad

2015-01-01

This study is designed to determine the contamination degree of hospital water supplies with Pseudomonas aeruginosa, Legionella pneumophila, and E. coli in Gilan, Iran. Samples were collected directly into sterile containers and concentrated by centrifuge. Half part of any sample transferred to yeast extract broth and the second part transferred to Trypticase Soy Broth and incubated for 3 days. DNA was extracted by using commercial kit. Four rounds of PCR were performed as follows: multiplex PCR for detecting Pseudomonas aeruginosa, Integron 1, and Metallo-β-lactamases gene; PCR for detecting Legionella pneumophila and mip gene separately; PCR for detecting E. coli; and another PCR for detecting whole bacterial presence. Contamination rates of cold, warm, and incubator water samples with P. aeruginosa, were 16.6%, 37.5%, and 6.8% consequently. Degrees of contamination with L. pneumophila were 3.3%, 9.3%, and 10.9% and with E. coli were zero, 6.2%, and zero. Total bacterial contamination of cold, warm, and incubator water samples was 93.3%, 84.4%, and 89.0% consequently. Metallo-β-lactamases gene was found in 20.0% of all samples. Contamination degree with P. aeruginosa was considerable and with L. pneumophila was moderate. Metallo-β-lactamases gene was found frequently indicating widespread multiple drug resistance bacteria. We suggest using new decontamination method based on nanotechnology. PMID:26448745

Risk of bacterial cross infection associated with inspiration through flow-based spirometers.