Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.

PubMed

Flenker, Katie S; Burghardt, Elliot L; Dutta, Nirmal; Burns, William J; Grover, Julia M; Kenkel, Elizabeth J; Weaver, Tyler M; Mills, James; Kim, Hyeon; Huang, Lingyan; Owczarzy, Richard; Musselman, Catherine A; Behlke, Mark A; Ford, Bradley; McNamara, James O

2017-06-07

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

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.

Two Novel Real-Time Reverse Transcriptase PCR Assays for Rapid Detection of Bacterial Contamination in Platelet Concentrates

PubMed Central

Dreier, Jens; Störmer, Melanie; Kleesiek, Knut

2004-01-01

The incidence of platelet bacterial contamination is approximately 1 per 2,000 units and has been acknowledged as the most frequent infectious risk from transfusion. In preliminary studies, the sterility of platelet concentrates (PCs) was tested with an automated bacterial blood culturing system and molecular genetic assays. Two real-time reverse transcriptase PCR (RT-PCR) assays performed in a LightCycler instrument were developed and compared regarding specificity and sensitivity by the use of different templates to detect the majority of the clinically important bacterial species in platelets. Primers and probes specific for the conserved regions of the eubacterial 23S rRNA gene or the groEL gene (encoding the 60-kDa heat shock protein Hsp60) were designed. During the development of the 23S rRNA RT-PCR, problems caused by the contamination of reagents with bacterial DNA were noted. Treatment with 8-methoxypsoralen and UV irradiation reduced the level of contaminating DNA. The sensitivity of the assays was greatly influenced by the enzyme system which was used. With rTth DNA polymerase in a one-enzyme system, we detected 500 CFU of Escherichia coli or Staphylococcus epidermidis/ml. With a two-enzyme system consisting of Moloney murine leukemia virus RT and Taq DNA polymerase, we detected 16 CFU/ml. With groEL mRNA as the target of RT-PCR under optimized conditions, we detected 125 CFU of E. coli/ml, and no problems with false-positive results caused by reagent contamination or a cross-reaction with human nucleic acids were found. Furthermore, the use of mRNA as an indicator of viability was demonstrated. Here we report the application of novel real-time RT-PCR assays for the detection of bacterial contamination of PCs that are appropriate for transfusion services. PMID:15472337

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.

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

Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection.

PubMed

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-10-01

Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacterial colonies in infected host cells (Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy, Ernstsen et al., 2017 [1]). The infected cells were imaged with a 10× objective and number of intracellular bacterial colonies, their size distribution and the number of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method.

Method and Apparatus for Detecting and Quantifying Bacterial Spores on a Surface

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2017-01-01

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: a matrix including lanthanide ions is provided on the surface containing the bacterial spores; functionalized aromatic molecules are released from the bacterial spores on the surface; a complex of the lanthanide ion and the aromatic molecule is formed on the surface; the complex of the lanthanide ion and the aromatic molecule is excited to generate a characteristic luminescence of the complex on the surface; and the bacterial spores exhibiting the luminescence of the complex on the surface are detected and quantified.

Method and apparatus for detecting and quantifying bacterial spores on a surface

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2009-01-01

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: a matrix including lanthanide ions is provided on the surface containing the bacterial spores; functionalized aromatic molecules are released from the bacterial spores on the surface; a complex of the lanthanide ion and the aromatic molecule is formed on the surface; the complex of the lanthanide ion and the aromatic molecule is excited to generate a characteristic luminescence of the complex on the surface; and the bacterial spores exhibiting the luminescence of the complex on the surface are detected and quantified.

Towards in vivo bacterial detection in human lung(Conference Presentation)

NASA Astrophysics Data System (ADS)

Choudhary, Tushar R.; Bradley, Mark; Duncan, Rory R.; Dhaliwal, Kevin

2017-04-01

Antibiotic resistance is a serious global concern. One way to tackle this problem is to develop new and sensitive approaches to diagnose bacterial infections and prevent unnecessary antibiotic use. With recent developments in optical molecular imaging, we are one step closer to in situ rapid detection of bacterial infections. We present here bespoke fluorescent probes for bacterial detection in ex vivo human lung tissue using fluorescence lifetime imaging microscopy (FLIM). Two in-house synthesised bespoke probes were used in this study to detect and differentiate between Gram positive and Gram negative bacterial strain using their fluorescence lifetime in the ex vivo human lung tissue. The average fluorescence lifetime of Gram positive probe (n=12) was 2.40 ± 0.25 ns and Gram negative (n=12) was 6.73 ± 0.49 ns. The human lung tissue (n=12) average fluorescence lifetime value was found to be 3.43 ± 0.19 ns. Furthermore we were also able to distinguish between dead or alive bacteria in ex vivo lung tissue based on difference in their lifetime. We have developped Fibre-FLIM methods to enable clinical translation within the Proteus Project (www.proteus.ac.uk).

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.

Fluorescence spectroscopy for rapid detection and classification of bacterial pathogens.

PubMed

Sohn, Miryeong; Himmelsbach, David S; Barton, Franklin E; Fedorka-Cray, Paula J

2009-11-01

This study deals with the rapid detection and differentiation of Escherichia coli, Salmonella, and Campylobacter, which are the most commonly identified commensal and pathogenic bacteria in foods, using fluorescence spectroscopy and multivariate analysis. Each bacterial sample cultured under controlled conditions was diluted in physiologic saline for analysis. Fluorescence spectra were collected over a range of 200-700 nm with 0.5 nm intervals on the PerkinElmer Fluorescence Spectrometer. The synchronous scan technique was employed to find the optimum excitation (lambda(ex)) and emission (lambda(em)) wavelengths for individual bacteria with the wavelength interval (Deltalambda) being varied from 10 to 200 nm. The synchronous spectra and two-dimensional plots showed two maximum lambda(ex) values at 225 nm and 280 nm and one maximum lambda(em) at 335-345 nm (lambda(em) = lambda(ex) + Deltalambda), which correspond to the lambda(ex) = 225 nm, Deltalambda = 110-120 nm, and lambda(ex) = 280 nm, Deltalambda = 60-65 nm. For all three bacterial genera, the same synchronous scan results were obtained. The emission spectra from the three bacteria groups were very similar, creating difficulty in classification. However, the application of principal component analysis (PCA) to the fluorescence spectra resulted in successful classification of the bacteria by their genus as well as determining their concentration. The detection limit was approximately 10(3)-10(4) cells/mL for each bacterial sample. These results demonstrated that fluorescence spectroscopy, when coupled with PCA processing, has the potential to detect and to classify bacterial pathogens in liquids. The methodology is rapid (>10 min), inexpensive, and requires minimal sample preparation compared to standard analytical methods for bacterial detection.

Normalization of test and evaluation of biothreat detection systems: overcoming microbial air content fluctuations by using a standardized reagent bacterial mixture.

PubMed

Berchebru, Laurent; Rameil, Pascal; Gaudin, Jean-Christophe; Gausson, Sabrina; Larigauderie, Guilhem; Pujol, Céline; Morel, Yannick; Ramisse, Vincent

2014-10-01

Test and evaluation of engineered biothreat agent detection systems ("biodetectors") are a challenging task for government agencies and industries involved in biosecurity and biodefense programs. In addition to user friendly features, biodetectors need to perform both highly sensitive and specific detection, and must not produce excessive false alerts. In fact, the atmosphere displays a number of variables such as airborne bacterial content that can interfere with the detection process, thus impeding comparative tests when carried out at different times or places. To overcome these bacterial air content fluctuations, a standardized reagent bacterial mixture (SRBM), consisting in a collection of selected cultivable environmental species that are prevalent in temperate climate bioaerosols, was designed to generate a stable, reproducible, and easy to use surrogate of bioaerosol sample. The rationale, design, and production process are reported. The results showed that 8.59; CI 95%: 8.46-8.72 log cfu distributed into vials underwent a 0.95; CI 95%: 0.65-1.26 log viability decay after dehydration and subsequent reconstitution, thus advantageously mimicking a natural bioaerosol sample which is typically composed of cultivable and uncultivable particles. Dehydrated SRBM was stable for more than 12months at 4°C and allowed the reconstitution of a dead/live cells aqueous suspension that is stable for 96h at +4°C, according to plate counts. Specific detection of a simulating biothreat agent (e.g. Bacillus atrophaeus) by immuno-magnetic or PCR assays did not display any significant loss of sensitivity, false negative or positive results in the presence of SRBM. This work provides guidance on testing and evaluating detection devices, and may contribute to the establishment of suitable standards and normalized procedures. Copyright © 2014 Elsevier B.V. All rights reserved.

Method and apparatus for detecting and quantifying bacterial spores on a surface

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2009-01-01

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: bacterial spores are transferred from a place of origin to a test surface, the test surface comprises lanthanide ions. Aromatic molecules are released from the bacterial spores; a complex of the lanthanide ions and aromatic molecules is formed on the test surface, the complex is excited to generate a characteristic luminescence on the test surface; the luminescence on the test surface is detected and quantified.

Method and Apparatus for Detecting and Quantifying Bacterial Spores on a Surface

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2016-01-01

A method and an apparatus for detecting and quantifying bacterial spores on a surface. In accordance with the method: bacterial spores are transferred from a place of origin to a test surface, the test surface comprises lanthanide ions. Aromatic molecules are released from the bacterial spores; a complex of the lanthanide ions and aromatic molecules is formed on the test surface, the complex is excited to generate a characteristic luminescence on the test surface; the luminescence on the test surface is detected and quantified.

Point detection of bacterial and viral pathogens using oral samples

NASA Astrophysics Data System (ADS)

Malamud, Daniel

2008-04-01

Oral samples, including saliva, offer an attractive alternative to serum or urine for diagnostic testing. This is particularly true for point-of-use detection systems. The various types of oral samples that have been reported in the literature are presented here along with the wide variety of analytes that have been measured in saliva and other oral samples. The paper focuses on utilizing point-detection of infectious disease agents, and presents work from our group on a rapid test for multiple bacterial and viral pathogens by monitoring a series of targets. It is thus possible in a single oral sample to identify multiple pathogens based on specific antigens, nucleic acids, and host antibodies to those pathogens. The value of such a technology for detecting agents of bioterrorism at remote sites is discussed.

Molecular analysis of bacterial communities and detection of potential pathogens in a recirculating aquaculture system for Scophthalmus maximus and Solea senegalensis.

PubMed

Martins, Patrícia; Cleary, Daniel F R; Pires, Ana C C; Rodrigues, Ana Maria; Quintino, Victor; Calado, Ricardo; Gomes, Newton C M

2013-01-01

The present study combined a DGGE and barcoded 16S rRNA pyrosequencing approach to assess bacterial composition in the water of a recirculating aquaculture system (RAS) with a shallow raceway system (SRS) for turbot (Scophthalmus maximus) and sole (Solea senegalensis). Barcoded pyrosequencing results were also used to determine the potential pathogen load in the RAS studied. Samples were collected from the water supply pipeline (Sup), fish production tanks (Pro), sedimentation filter (Sed), biofilter tank (Bio), and protein skimmer (Ozo; also used as an ozone reaction chamber) of twin RAS operating in parallel (one for each fish species). Our results revealed pronounced differences in bacterial community composition between turbot and sole RAS, suggesting that in the systems studied there is a strong species-specific effect on water bacterial communities. Proteobacteria was the most abundant phylum in the water supply and all RAS compartments. Other important taxonomic groups included the phylum Bacteriodetes. The saltwater supplied displayed a markedly lower richness and appeared to have very little influence on bacterial composition. The following potentially pathogenic species were detected: Photobacterium damselae in turbot (all compartments), Tenacibaculum discolor in turbot and sole (all compartments), Tenacibaculum soleae in turbot (all compartments) and sole (Pro, Sed and Bio), and Serratia marcescens in turbot (Sup, Sed, Bio and Ozo) and sole (only Sed) RAS. Despite the presence of these pathogens, no symptomatic fish were observed. Although we were able to identify potential pathogens, this approach should be employed with caution when monitoring aquaculture systems, as the required phylogenetic resolution for reliable identification of pathogens may not always be possible to achieve when employing 16S rRNA gene fragments.

Molecular Analysis of Bacterial Communities and Detection of Potential Pathogens in a Recirculating Aquaculture System for Scophthalmus maximus and Solea senegalensis

PubMed Central

Martins, Patrícia; Cleary, Daniel F. R.; Pires, Ana C. C.; Rodrigues, Ana Maria; Quintino, Victor; Calado, Ricardo; Gomes, Newton C. M.

2013-01-01

The present study combined a DGGE and barcoded 16S rRNA pyrosequencing approach to assess bacterial composition in the water of a recirculating aquaculture system (RAS) with a shallow raceway system (SRS) for turbot (Scophthalmus maximus) and sole (Solea senegalensis). Barcoded pyrosequencing results were also used to determine the potential pathogen load in the RAS studied. Samples were collected from the water supply pipeline (Sup), fish production tanks (Pro), sedimentation filter (Sed), biofilter tank (Bio), and protein skimmer (Ozo; also used as an ozone reaction chamber) of twin RAS operating in parallel (one for each fish species). Our results revealed pronounced differences in bacterial community composition between turbot and sole RAS, suggesting that in the systems studied there is a strong species-specific effect on water bacterial communities. Proteobacteria was the most abundant phylum in the water supply and all RAS compartments. Other important taxonomic groups included the phylum Bacteriodetes. The saltwater supplied displayed a markedly lower richness and appeared to have very little influence on bacterial composition. The following potentially pathogenic species were detected: Photobacterium damselae in turbot (all compartments), Tenacibaculum discolor in turbot and sole (all compartments), Tenacibaculum soleae in turbot (all compartments) and sole (Pro, Sed and Bio), and Serratia marcescens in turbot (Sup, Sed, Bio and Ozo) and sole (only Sed) RAS. Despite the presence of these pathogens, no symptomatic fish were observed. Although we were able to identify potential pathogens, this approach should be employed with caution when monitoring aquaculture systems, as the required phylogenetic resolution for reliable identification of pathogens may not always be possible to achieve when employing 16S rRNA gene fragments. PMID:24278329

Study of bacterial meningitis in children below 5 years with comparative evaluation of gram staining, culture and bacterial antigen detection.

PubMed

Yadhav Ml, Kala

2014-04-01

Bacterial meningitis is one of the most serious infections seen in infants and children, which is associated with acute complications and chronic morbidity. Infections of Central Nervous System (CNS) still dominate the scene of childhood neurological disorders in most of the developing tropical countries. To isolate, identify and determine the antibiotic susceptibility patterns of pathogens associated with bacterial meningitis. We also aimed to comparatively evaluate of Gram staining, culture and bacterial antigen detection in cerebrospinal fluid samples. Present comparative study included 100 CSF samples of children below the age of 5 years, who were clinically suspected meningitis cases. The samples were subjected to Gram staining, culture and Latex agglutination test (LAT). The organisms isolated in the study were characterized and antibiotic susceptibility test was done according to standard guidelines. It was done by using Gaussian test. Of the 100 cases, 24 were diagnosed as Acute bacterial meningitis (ABM) cases by. Gram staining, culture and latex agglutination test. 21 (87.5%) cases were culture positive, with 2 cases being positive for polymicrobial isolates. Gram staining was positive in 17 (70.53%) cases and LAT was positive in 18 (33.33%) cases. Streptococcus pneumoniae was the predominant organism which was isolated and it was sensitive to antibiotics. In the present study, male to female ratio was 1.27:1, which showed a male preponderance. With the combination of Gram staining, culture, and LAT, 100% sensitivity and specificity can be achieved (p < 0.001). Gram staining and LAT can detect 85% of cases of ABM. Bacterial meningitis is a medical emergency and making an early diagnosis and providing treatment early are life saving and they reduce chronic morbidity.

Detection of Spiroplasma and Wolbachia in the bacterial gonad community of Chorthippus parallelus.

PubMed

Martínez-Rodríguez, P; Hernández-Pérez, M; Bella, J L

2013-07-01

We have recently detected the endosymbiont Wolbachia in multiple individuals and populations of the grasshopper Chorthippus parallelus (Orthoptera: acrididae). This bacterium induces reproductive anomalies, including cytoplasmic incompatibility. Such incompatibilities may help explain the maintenance of two distinct subspecies of this grasshopper, C. parallelus parallelus and C. parallelus erythropus, which are involved in a Pyrenean hybrid zone that has been extensively studied for the past 20 years, becoming a model system for the study of genetic divergence and speciation. To evaluate whether Wolbachia is the sole bacterial infection that might induce reproductive anomalies, the gonadal bacterial community of individuals from 13 distinct populations of C. parallelus was determined by denaturing gradient gel electrophoresis analysis of bacterial 16S rRNA gene fragments and sequencing. The study revealed low bacterial diversity in the gonads: a persistent bacterial trio consistent with Spiroplasma sp. and the two previously described supergroups of Wolbachia (B and F) dominated the gonad microbiota. A further evaluation of the composition of the gonad bacterial communities was carried out by whole cell hybridization. Our results confirm previous studies of the cytological distribution of Wolbachia in C. parallelus gonads and show a homogeneous infection by Spiroplasma. Spiroplasma and Wolbachia cooccurred in some individuals, but there was no significant association of Spiroplasma with a grasshopper's sex or with Wolbachia infection, although subtle trends might be detected with a larger sample size. This information, together with previous experimental crosses of this grasshopper, suggests that Spiroplasma is unlikely to contribute to sex-specific reproductive anomalies; instead, they implicate Wolbachia as the agent of the observed anomalies in C. parallelus.

Comparison of enterovirus detection in cerebrospinal fluid with Bacterial Meningitis Score in children

PubMed Central

Pires, Frederico Ribeiro; Franco, Andréia Christine Bonotto Farias; Gilio, Alfredo Elias; Troster, Eduardo Juan

2017-01-01

ABSTRACT Objective To measure the role of enterovirus detection in cerebrospinal fluid compared with the Bacterial Meningitis Score in children with meningitis. Methods A retrospective cohort based on analysis of medical records of pediatric patients diagnosed as meningitis, seen at a private and tertiary hospital in São Paulo, Brazil, between 2011 and 2014. Excluded were patients with critical illness, purpura, ventricular shunt or recent neurosurgery, immunosuppression, concomitant bacterial infection requiring parenteral antibiotic therapy, and those who received antibiotics 72 hours before lumbar puncture. Results The study included 503 patients. Sixty-four patients were excluded and 94 were not submitted to all tests for analysis. Of the remaining 345 patients, 7 were in the Bacterial Meningitis Group and 338 in the Aseptic Meningitis Group. There was no statistical difference between the groups. In the Bacterial Meningitis Score analysis, of the 338 patients with possible aseptic meningitis (negative cultures), 121 of them had one or more points in the Bacterial Meningitis Score, with sensitivity of 100%, specificity of 64.2%, and negative predictive value of 100%. Of the 121 patients with positive Bacterial Meningitis Score, 71% (86 patients) had a positive enterovirus detection in cerebrospinal fluid. Conclusion Enterovirus detection in cerebrospinal fluid was effective to differentiate bacterial from viral meningitis. When the test was analyzed together with the Bacterial Meningitis Score, specificity was higher when compared to Bacterial Meningitis Score alone. PMID:28767914

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.

Thin-layer chromatographic technique for rapid detection of bacterial phospholipases.

PubMed

Legakis, N J; Papavassiliou, J

1975-11-01

Silica gel thin-layer chromatography was employed to detect lecithinase activity induced from bacterial resting cell preparations induced from bacterial resting cell preparations incubated at 37 C for 4 h in the presence of purified egg yolk lecithin. Bacillus subtilis, Bacillus cereus, Serratia marcescens, and Pseudomonas aeruginosa hydrolyzed lecithin with the formation of free fatty acids as the sole lipid-soluble product. In none of the Escherichia coli and Citrobacter freundii strains tested could lecithinase activity be detected. Four among eight strains of Enterobacter aerogenes and one among 12 strains of Proteus tested produced negligible amounts of free fatty acid.

Increased detection of mastitis pathogens by real-time PCR compared to bacterial culture.

PubMed

Keane, O M; Budd, K E; Flynn, J; McCoy, F

2013-09-21

Rapid and accurate identification of mastitis pathogens is important for disease control. Bacterial culture and isolate identification is considered the gold standard in mastitis diagnosis but is time consuming and results in many culture-negative samples. Identification of mastitis pathogens by PCR has been proposed as a fast and sensitive alternative to bacterial culture. The results of bacterial culture and PCR for the identification of the aetiological agent of clinical mastitis were compared. The pathogen identified by traditional culture methods was also detected by PCR in 98 per cent of cases indicating good agreement between the positive results of bacterial culture and PCR. A mastitis pathogen could not be recovered from approximately 30 per cent of samples by bacterial culture, however, an aetiological agent was identified by PCR in 79 per cent of these samples. Therefore, a mastitis pathogen was detected in significantly more milk samples by PCR than by bacterial culture (92 per cent and 70 per cent, respectively) although the clinical relevance of PCR-positive culture-negative results remains controversial. A mixed infection of two or more mastitis pathogens was also detected more commonly by PCR. Culture-negative samples due to undetected Staphylococcus aureus infections were rare. The use of PCR technology may assist in rapid mastitis diagnosis, however, accurate interpretation of PCR results in the absence of bacterial culture remains problematic.

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system.

PubMed

Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

2016-11-14

Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.

Digital microbiology: detection and classification of unknown bacterial pathogens using a label-free laser light scatter-sensing system

NASA Astrophysics Data System (ADS)

Rajwa, Bartek; Dundar, M. Murat; Akova, Ferit; Patsekin, Valery; Bae, Euiwon; Tang, Yanjie; Dietz, J. Eric; Hirleman, E. Daniel; Robinson, J. Paul; Bhunia, Arun K.

2011-06-01

The majority of tools for pathogen sensing and recognition are based on physiological or genetic properties of microorganisms. However, there is enormous interest in devising label-free and reagentless biosensors that would operate utilizing the biophysical signatures of samples without the need for labeling and reporting biochemistry. Optical biosensors are closest to realizing this goal and vibrational spectroscopies are examples of well-established optical label-free biosensing techniques. A recently introduced forward-scatter phenotyping (FSP) also belongs to the broad class of optical sensors. However, in contrast to spectroscopies, the remarkable specificity of FSP derives from the morphological information that bacterial material encodes on a coherent optical wavefront passing through the colony. The system collects elastically scattered light patterns that, given a constant environment, are unique to each bacterial species and/or serovar. Both FSP technology and spectroscopies rely on statistical machine learning to perform recognition and classification. However, the commonly used methods utilize either simplistic unsupervised learning or traditional supervised techniques that assume completeness of training libraries. This restrictive assumption is known to be false for real-life conditions, resulting in unsatisfactory levels of accuracy, and consequently limited overall performance for biodetection and classification tasks. The presented work demonstrates preliminary studies on the use of FSP system to classify selected serotypes of non-O157 Shiga toxin-producing E. coli in a nonexhaustive framework, that is, without full knowledge about all the possible classes that can be encountered. Our study uses a Bayesian approach to learning with a nonexhaustive training dataset to allow for the automated and distributed detection of unknown bacterial classes.

Fluorescence quencher improves SCANSYSTEM for rapid bacterial detection.

PubMed

Schmidt, M; Hourfar, M K; Wahl, A; Nicol, S-B; Montag, T; Roth, W K; Seifried, E

2006-05-01

The optimized scansystem could detect contaminated platelet products within 24 h. However, the system's sensitivity was reduced by a high fluorescence background even in sterile samples, which led to the necessity of a well-trained staff for confirmation of microscope results. A new protocol of the optimized scansystem with the addition of a fluorescence quencher was evaluated. Pool platelet concentrates contaminated with five transfusion-relevant bacterial strains were tested in a blind study. In conjunction with new analysis software, the new quenching dye was able to reduce significantly unspecific background fluorescence. Sensitivity was best for Bacillus cereus and Escherichia coli (3 CFU/ml). The application of a fluorescence quencher enables automated discrimination of positive and negative test results in 60% of all analysed samples.

Bioluminescence-based system for rapid detection of natural transformation.

PubMed

Santala, Ville; Karp, Matti; Santala, Suvi

2016-07-01

Horizontal gene transfer plays a significant role in bacterial evolution and has major clinical importance. Thus, it is vital to understand the mechanisms and kinetics of genetic transformations. Natural transformation is the driving mechanism for horizontal gene transfer in diverse genera of bacteria. Our study introduces a simple and rapid method for the investigation of natural transformation. This highly sensitive system allows the detection of a transformation event directly from a bacterial population without any separation step or selection of cells. The system is based on the bacterial luciferase operon from Photorhabdus luminescens The studied molecular tools consist of the functional modules luxCDE and luxAB, which involve a replicative plasmid and an integrative gene cassette. A well-established host for bacterial genetic investigations, Acinetobacter baylyi ADP1, is used as the model bacterium. We show that natural transformation followed by homologous recombination or plasmid recircularization can be readily detected in both actively growing and static biofilm-like cultures, including very rare transformation events. The system allows the detection of natural transformation within 1 h of introducing sample DNA into the culture. The introduced method provides a convenient means to study the kinetics of natural transformation under variable conditions and perturbations. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Role of Honey in Topical and Systemic Bacterial Infections.

PubMed

Hussain, Muhammad Barkaat

2018-01-01

The development of bacterial resistance to antibiotics has made it more difficult and expensive to treat infections. Honey is getting worldwide attention as a topical therapeutic agent for wound infections and potential future candidate for systemic infections. The purpose of this review was to summarise different antibacterial bio-active compounds in honey, their synergistic interaction and their clinical implications in topical and systemic infections. In addition, contemporary testing methods for evaluating peroxide and non-peroxide antibacterial activity of honey were also critically appraised. MEDLINE, EMBASE, Cochrane Library, Pub Med, reference lists and databases were used to review the literature. Honey contains several unique antibacterial components. These components are believed to act on diverse bacterial targets, are broad spectrum, operate synergistically, prevent biofilm formation, and decrease production of virulence factors. Moreover, honey has the ability to block bacterial communication (quorum sensing), and therefore, it is unlikely that bacteria develop resistance against honey. Bacterial resistance against honey has not been documented so far. Unlike conventional antibiotics, honey only targets pathogenic bacteria without disturbing the growth of normal gastrointestinal flora when taken orally. It also contains prebiotics, probiotics, and zinc and enhances the growth of beneficial gut flora. The presence of such plethora of antibacterial properties in one product makes it a promising candidate not only in wound infections but also in systemic and particularly for gastrointestinal infections. Agar diffusion assay, being used for evaluating antibacterial activity of honey, is not the most appropriate and sensitive assay as it only detects non-peroxide activity when present at a higher level. Therefore, there is a need to develop more sensitive techniques that may be capable of detecting and evaluating different important components in honey as

Near-infrared surface-enhanced-Raman-scattering (SERS) mediated detection of single optically trapped bacterial spores

NASA Astrophysics Data System (ADS)

Alexander, Troy A.; Pellegrino, Paul M.; Gillespie, James B.

2003-08-01

A novel methodology has been developed for the investigation of bacterial spores. Specifically, this method has been used to probe the spore coat composition of two different Bacillus stearothermophilus variants. This technique may be useful in many applications; most notably, development of novel detection schemes toward potentially harmful bacteria. This method would also be useful as an ancillary environmental monitoring system where sterility is of importance (i.e., food preparation areas as well as invasive and minimally invasive medical applications). This unique detection scheme is based on the near-infrared (NIR) Surface-Enhanced-Raman-Scattering (SERS) from single, optically trapped, bacterial spores. The SERS spectra of bacterial spores in aqueous media have been measured using SERS substrates based on ~60-nm diameter gold colloids bound to 3-Aminopropyltriethoxysilane derivatized glass. The light from a 787-nm laser diode was used to trap/manipulate as well as simultaneously excite the SERS of an individual bacterial spore. The collected SERS spectra were examined for uniqueness and the applicability of this technique for the strain discrimination of Bacillus stearothermophilus spores. Comparison of normal Raman and SERS spectra reveal not only an enhancement of the normal Raman spectral features but also the appearance of spectral features absent in the normal Raman spectrum.

Detection of Bacterial Meningitis Pathogens by PCR-Mass Spectrometry in Cerebrospinal Fluid.

PubMed

Jing-Zi, Piao; Zheng-Xin, He; Wei-Jun, Chen; Yong-Qiang, Jiang

2018-06-01

Acute bacterial meningitis remains a life-threatening infectious disease with considerable morbidity and mortality. DNA-based detection methods are an urgent requisite for meningitis-causing bacterial pathogens for the prevention of outbreaks and control of infections. We proposed a novel PCR-mass spectrometry (PCR-Mass) assay for the simultaneous detection of four meningitis-causing agents, Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae, and Mycobacterium tuberculosis in the present study. A total of 138 cerebrospinal fluid (CSF) samples (including 56 CSF culture positive, 44 CSF culture negative, and 38 CSF control) were enrolled and analyzed by PCR/Mass. Results were compared to real-time PCR detection. These four targeting pathogens could be discriminated without cross-reaction by the accurate detection of the corresponding extension products with different masses. The limits of detection were 102 copies/reaction for S. pneumoniae, H. influenzae, and N. meningitidis and 103 for M. tuberculosis. The evaluation of the culture-positive CSF specimens from the meningitis patients provided an overall agreement rate of 85.7% with PCR-Mass and real-time PCR. The PCR-Mass was also able to detect the targeting pathogens from culture-negative CSF specimens from meningitis patients receiving early antibiotic treatment. PCR-Mass could be used for the molecular detection of bacterial meningitis and tuberculosis, especially when early antibiotic treatment has been administered to the suspected patients.

Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

PubMed

Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

2016-10-01

Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Simulation of Graphene Field-Effect Transistor Biosensors for Bacterial Detection.

PubMed

Wu, Guangfu; Meyyappan, Meyya; Lai, King Wai Chiu

2018-05-25

Foodborne illness is correlated with the existence of infectious pathogens such as bacteria in food and drinking water. Probe-modified graphene field effect transistors (G-FETs) have been shown to be suitable for Escherichia coli ( E. coli ) detection. Here, the G-FETs for bacterial detection are modeled and simulated with COMSOL Multiphysics to understand the operation of the biosensors. The motion of E. coli cells in electrolyte and the surface charge of graphene induced by E. coli are systematically investigated. The comparison between the simulation and experimental data proves the sensing probe size to be a key parameter affecting the surface charge of graphene induced by bacteria. Finally, the relationship among the change in source-drain current (∆ I ds ), graphene-bacteria distance and bacterial concentration is established. The shorter graphene-bacteria distance and higher bacterial concentration give rise to better sensing performance (larger ∆ I ds ) of the G-FETs biosensors. The simulation here could serve as a guideline for the design and optimization of G-FET biosensors for various applications.

Real-time polymerase chain reaction in transfusion medicine: applications for detection of bacterial contamination in blood products.

PubMed

Dreier, Jens; Störmer, Melanie; Kleesiek, Knut

2007-07-01

Bacterial contamination of blood components, particularly of platelet concentrates (PCs), represents the greatest infectious risk in blood transfusion. Although the incidence of platelet bacterial contamination is approximately 1 per 2,000 U, the urgent need for a method for the routine screening of PCs to improve safety for patients had not been considered for a long time. Besides the culturing systems, which will remain the criterion standard, rapid methods for sterility screening will play a more important role in transfusion medicine in the future. In particular, nucleic acid amplification techniques (NATs) are powerful potential tools for bacterial screening assays. The combination of excellent sensitivity and specificity, reduced contamination risk, ease of performance, and speed has made real-time polymerase chain reaction (PCR) technology an appealing alternative to conventional culture-based testing methods. When using real-time PCR for the detection of bacterial contamination, several points have to be considered. The main focus is the choice of the target gene; the assay format; the nucleic acid extraction method, depending on the sample type; and the evaluation of an ideal sampling strategy. However, several factors such as the availability of bacterial-derived nucleic acid amplification reagents, the impracticability, and the cost have limited the use of NATs until now. Attempts to reduce the presence of contaminating nucleic acids from reagents in real-time PCR have been described, but none of these approaches have proven to be very effective or to lower the sensitivity of the assay. Recently, a number of broad-range NAT assays targeting the 16S ribosomal DNA or 23S ribosomal RNA for the detection of bacteria based on real-time technology have been reported. This review will give a short survey of current approaches to and the limitations of the application of real-time PCR for bacterial detection in blood components, with emphasis on the bacterial

Characterization of bacterial coliform occurrences in different zones of a drinking water distribution system.

PubMed

Blanch, A R; Galofré, B; Lucena, F; Terradillos, A; Vilanova, X; Ribas, F

2007-03-01

To compare the bacterial coliforms detected from occurrences in three zones of a water distribution system supplied by two separate water sources. Conventional and standardized protocols for identifying enterobacterial populations were applied. Additional tests to confirm isolates were included. Analyses of diversity and population similarity were performed using the Phene Plate System, a miniaturized biochemical phenotyping method. Isolates were identified by the API 20E system in tandem with biochemical phenotyping. A total of 16 576 samples were taken from the water distribution system, with 1416 isolates analysed. A low number of coliform occurrences were observed (2%). Escherichia coli was not detected in either water origin or in Zone 2 samples; however, in Zones 1 and 3 a low number of cases of E. coli were recorded. The percentages of E. coli depended on the identification criteria. Eight biochemical profiles for coliform populations were defined according to the results of the confirmative tests. There was a high diversity among these populations in the three zones studied, although no significant variations in their composition (associated with occurrences in the different zones) were observed. Klebsiella oxytoca was the most commonly detected species irrespective of zone, although seven other enterobacterial genera were also found. Analysis of the enzymatic activity of beta-glucuronidase or application of the criteria established in the norm ISO 9308-1, in tandem with thermotolerance was needed to evaluate the occurrence of E. coli in the distribution systems. Detected occurrences of bacterial coliforms could be associated with re-growth patterns for specific sampling points in the distribution system. Seasonal differences, independent of the studied zones, were observed. Biochemical phenotyping of bacterial coliforms was shown to be a useful method on the characterization of occurrences in water distribution systems.

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

PubMed Central

Li, Yiyan; Yang, Xing; Zhao, Weian

2018-01-01

Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies. PMID:28850804

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing.

PubMed

Li, Yiyan; Yang, Xing; Zhao, Weian

2017-12-01

Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies.

Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform.

PubMed

Kaushik, Aniruddha M; Hsieh, Kuangwen; Chen, Liben; Shin, Dong Jin; Liao, Joseph C; Wang, Tza-Huei

2017-11-15

There remains an urgent need for rapid diagnostic methods that can evaluate antibiotic resistance for pathogenic bacteria in order to deliver targeted antibiotic treatments. Toward this end, we present a rapid and integrated single-cell biosensing platform, termed dropFAST, for bacterial growth detection and antimicrobial susceptibility assessment. DropFAST utilizes a rapid resazurin-based fluorescent growth assay coupled with stochastic confinement of bacteria in 20 pL droplets to detect signal from growing bacteria after 1h incubation, equivalent to 2-3 bacterial replications. Full integration of droplet generation, incubation, and detection into a single, uninterrupted stream also renders this platform uniquely suitable for in-line bacterial phenotypic growth assessment. To illustrate the concept of rapid digital antimicrobial susceptibility assessment, we employ the dropFAST platform to evaluate the antibacterial effect of gentamicin on E. coli growth. Copyright © 2017 Elsevier B.V. All rights reserved.

Impedimetric biosensor based on cell-mediated bioimprinted films for bacterial detection.

PubMed

Qi, Peng; Wan, Yi; Zhang, Dun

2013-01-15

This work presents the synthesis of bacteria-mediated bioimprinted films for selective bacterial detection. Marine pathogen sulfate-reducing bacteria (SRB) were chosen as the template bacteria. Chitosan (CS) doped with reduced graphene sheets (RGSs) was electrodeposited on an indium tin oxide electrode, and the resulting RGSs-CS hybrid film served as a platform for bacterial attachment. The electrodeposition conditions were optimized to obtain RGSs-CS hybrid films with excellent electrochemical performance. A layer of nonconductive CS film was deposited to embed the pathogen, and acetone was used to wash away the bacterial templates. Electrochemical impedance spectroscopy was performed to characterize the stepwise modification process and monitor the SRB population. Faradic impedance measurements revealed that the charge transfer resistance (R(ct)) increased with increased SRB concentration. A linear relationship between ΔR(ct) and the logarithm of SRB concentration was obtained within the concentration range of 1.0×10(4)cfum L(-1) to 1.0×10(8)cfum L(-1). The impedimetric sensor showed good selectivity towards SRB based on size and shape. Hence, selectivity for bacterial detection can be improved if the bioimprinting technique is combined with other bio-recognition elements. Copyright © 2012 Elsevier B.V. All rights reserved.

Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system

PubMed Central

Atilano, Magda Luciana; Pereira, Pedro Matos; Vaz, Filipa; Catalão, Maria João; Reed, Patricia; Grilo, Inês Ramos; Sobral, Rita Gonçalves; Ligoxygakis, Petros; Pinho, Mariana Gomes; Filipe, Sérgio Raposo

2014-01-01

Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host. DOI: http://dx.doi.org/10.7554/eLife.02277.001 PMID:24692449

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.

PubMed

Tizzano, Marco; Gulbransen, Brian D; Vandenbeuch, Aurelie; Clapp, Tod R; Herman, Jake P; Sibhatu, Hiruy M; Churchill, Mair E A; Silver, Wayne L; Kinnamon, Sue C; Finger, Thomas E

2010-02-16

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl-homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca(2+). Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either G alpha-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl-homoserine lactones serve as quorum-sensing molecules for gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms.

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

PubMed Central

Tizzano, Marco; Gulbransen, Brian D.; Vandenbeuch, Aurelie; Clapp, Tod R.; Herman, Jake P.; Sibhatu, Hiruy M.; Churchill, Mair E. A.; Silver, Wayne L.; Kinnamon, Sue C.; Finger, Thomas E.

2010-01-01

The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca2+. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms. PMID:20133764

The bacterial sealing capacity of morse taper implant-abutment systems in vitro.

PubMed

Ranieri, Rogerio; Ferreira, Andreia; Souza, Emmanuel; Arcoverde, Joao; Dametto, Fabio; Gade-Neto, Cicero; Seabra, Flavio; Sarmento, Carlos

2015-05-01

The use of Morse taper systems in dental implantology has been associated widely with a more precise adaptation between implants and their respective abutments. This may lead to an increase in the stability of the implant system and may also prevent microbial invasion through the implant-abutment interface. The aim of this study was to investigate in vitro the ability of four commercially available Morse taper system units to impede bacterial penetration through their implant-abutment interfaces. Abutments were screwed onto the implants, and the units were subsequently immersed in Streptococcus sanguinis bacterial broth (1 × 10(8) colony forming units/mL) for 48 hours. The units were examined by scanning electron microscopy (SEM) under three conditions: 1) with the implant-abutment components assembled as units to investigate for both the existence of microgaps and the presence of bacteria; 2) with the implants and abutments separated for examination of internal surfaces; and 3) with the implant-abutment components again assembled as units to measure any microgaps detected. The mean size of the microgaps in each unit was determined by measuring, under SEM, their width in four equidistant points. Microgaps were detected in all units with no significant differences in dimension (Kruskal-Wallis test, P >0.05). Within all units, the presence of bacteria was also observed. The seals provided by the interfaces of the commercially available Morse taper implant-abutment units tested were not sufficiently small to shield the implant from bacterial penetration.

Histo-FISH protocol to detect bacterial compositions and biofilms formation in vivo.

PubMed

Madar, M; Slizova, M; Czerwinski, J; Hrckova, G; Mudronova, D; Gancarcikova, S; Popper, M; Pistl, J; Soltys, J; Nemcova, R

2015-01-01

The study of biofilm function in vivo in various niches of the gastrointestinal tract (GIT) is rather limited. It is more frequently used in in vitro approaches, as an alternative to the studies focused on formation mechanisms and function of biofilms, which do not represent the actual in vivo complexity of microbial structures. Additionally, in vitro tests can sometimes lead to unreliable results. The goal of this study was to develop a simple approach to detect bacterial populations, particularly Lactobacillus and Bifidobacterium in biofilms, in vivo by the fluorescent in situ hybridisation (FISH) method. We standardised a new Histo-FISH method based on specific fluorochrome labelling probes which are able to detect Lactobacillus spp. and Bifidobacterium spp. within biofilms on the mucosal surface of the GIT embedded in paraffin in histological slices. This method is also suitable for visualisation of bacterial populations in the GIT internal content. Depending on the labelling probes, the Histo-FISH method has the potential to detect other probiotic strains or pathogenic bacteria. This original approach permits us to analyse bacterial colonisation processes as well as biofilm formation in stomach and caecum of BALB/c and germ-free mice.

Detection of Only Viable Bacterial Spores Using a Live/Dead Indicator in Mixed Populations

NASA Technical Reports Server (NTRS)

Behar, Alberto E.; Stam, Christina N.; Smiley, Ronald

2013-01-01

This method uses a photoaffinity label that recognizes DNA and can be used to distinguish populations of bacterial cells from bacterial spores without the use of heat shocking during conventional culture, and live from dead bacterial spores using molecular-based methods. Biological validation of commercial sterility using traditional and alternative technologies remains challenging. Recovery of viable spores is cumbersome, as the process requires substantial incubation time, and the extended time to results limits the ability to quickly evaluate the efficacy of existing technologies. Nucleic acid amplification approaches such as PCR (polymerase chain reaction) have shown promise for improving time to detection for a wide range of applications. Recent real-time PCR methods are particularly promising, as these methods can be made at least semi-quantitative by correspondence to a standard curve. Nonetheless, PCR-based methods are rarely used for process validation, largely because the DNA from dead bacterial cells is highly stable and hence, DNA-based amplification methods fail to discriminate between live and inactivated microorganisms. Currently, no published method has been shown to effectively distinguish between live and dead bacterial spores. This technology uses a DNA binding photoaffinity label that can be used to distinguish between live and dead bacterial spores with detection limits ranging from 109 to 102 spores/mL. An environmental sample suspected of containing a mixture of live and dead vegetative cells and bacterial endospores is treated with a photoaffinity label. This step will eliminate any vegetative cells (live or dead) and dead endospores present in the sample. To further determine the bacterial spore viability, DNA is extracted from the spores and total population is quantified by real-time PCR. The current NASA standard assay takes 72 hours for results. Part of this procedure requires a heat shock step at 80 degC for 15 minutes before the

Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

PubMed

Alcaine, S D; Tilton, L; Serrano, M A C; Wang, M; Vachet, R W; Nugen, S R

2015-10-01

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

Determinants of bacterial communities in Canadian agroforestry systems.

PubMed

Banerjee, Samiran; Baah-Acheamfour, Mark; Carlyle, Cameron N; Bissett, Andrew; Richardson, Alan E; Siddique, Tariq; Bork, Edward W; Chang, Scott X

2016-06-01

Land-use change is one of the most important factors influencing soil microbial communities, which play a pivotal role in most biogeochemical and ecological processes. Using agroforestry systems as a model, this study examined the effects of land uses and edaphic properties on bacterial communities in three agroforestry types covering a 270 km soil-climate gradient in Alberta, Canada. Our results demonstrate that land-use patterns exert stronger effects on soil bacterial communities than soil zones in these agroforestry systems. Plots with trees in agroforestry systems promoted greater bacterial abundance and to some extent species richness, which was associated with more nutrient-rich soil resources. While Acidobacteria, Actinobacteria and Alphaproteobacteria were the dominant bacterial phyla and subphyla across land uses, Arthrobacter, Acidobacteria_Gp16, Burkholderia, Rhodanobacter and Rhizobium were the keystone taxa in these agroforestry systems. Soil pH and carbon contents emerged as the major determinants of bacterial community characteristics. We found non-random co-occurrence and modular patterns of soil bacterial communities, and these patterns were controlled by edaphic factors and not their taxonomy. Overall, this study highlights the drivers and co-occurrence patterns of soil microbial communities in agroforestry systems. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Rapid, portable, multiplexed detection of bacterial pathogens directly from clinical sample matrices

DOE PAGES

Phaneuf, Christopher R.; Mangadu, Betty Lou Bosano; Piccini, Matthew E.; ...

2016-09-23

Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. Furthermore, this platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated inmore » diarrheal and enteric diseases in less than 20 min.« less

Rapid, portable, multiplexed detection of bacterial pathogens directly from clinical sample matrices

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

Phaneuf, Christopher R.; Mangadu, Betty Lou Bosano; Piccini, Matthew E.

Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. Furthermore, this platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 µL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated inmore » diarrheal and enteric diseases in less than 20 min.« less

Detection of bacterial infection with a fiber optic microendoscope

NASA Astrophysics Data System (ADS)

Mufti, Nooman; Kong, Ying; Cirillo, Jeffrey D.; Maitland, Kristen C.

2011-07-01

We present the use of fiber optic microendoscopy to image bacterial infection in the skin and lungs using an animal model. The contact probe microendoscope we have constructed has a 4 μm resolution, a 750 μm field of view, and a 1 mm outer diameter. Well resolved regions of bacterial infection were imaged for subcutaneous inocula of 106 to 101 CFU and intra-tracheal inocula of 108 to 106 CFU. Results reveal a linear relationship between average fluorescence and CFU, suggesting potential for using this device for quantitative analysis. Detection limits of 104 CFU for skin samples and 107 CFU for lung tissue were determined. In addition, bacteria were also qualitatively visible in lung tissue down to 106 CFU. Confocal imaging was used to confirm the presence of bacteria in tissue samples.

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

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

Pinzon, NM; Aukema, KG; Gralnick, JA

A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone productionmore » as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76: 1212-1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76: 3850-3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high

Duplex recombinase polymerase amplification assays incorporating competitive internal controls for bacterial meningitis detection.

PubMed

Higgins, Owen; Clancy, Eoin; Forrest, Matthew S; Piepenburg, Olaf; Cormican, Martin; Boo, Teck Wee; O'Sullivan, Nicola; McGuinness, Claire; Cafferty, Deirdre; Cunney, Robert; Smith, Terry J

2018-04-01

Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries. Copyright © 2018 Elsevier Inc. All rights reserved.

Alignment-free detection of horizontal gene transfer between closely related bacterial genomes.

PubMed

Domazet-Lošo, Mirjana; Haubold, Bernhard

2011-09-01

Bacterial epidemics are often caused by strains that have acquired their increased virulence through horizontal gene transfer. Due to this association with disease, the detection of horizontal gene transfer continues to receive attention from microbiologists and bioinformaticians alike. Most software for detecting transfer events is based on alignments of sets of genes or of entire genomes. But despite great advances in the design of algorithms and computer programs, genome alignment remains computationally challenging. We have therefore developed an alignment-free algorithm for rapidly detecting horizontal gene transfer between closely related bacterial genomes. Our implementation of this algorithm is called alfy for "ALignment Free local homologY" and is freely available from http://guanine.evolbio.mpg.de/alfy/. In this comment we demonstrate the application of alfy to the genomes of Staphylococcus aureus. We also argue that-contrary to popular belief and in spite of increasing computer speed-algorithmic optimization is becoming more, not less, important if genome data continues to accumulate at the present rate.

Simplified Protocol for Carba NP Test for Enhanced Detection of Carbapenemase Producers Directly from Bacterial Cultures

PubMed Central

Pasteran, Fernando; Tijet, Nathalie; Melano, Roberto G.

2015-01-01

We compared carbapenemase detection among 266 Gram-negative bacilli (161 carbapenemase producers) using the Carba NP tests issued by the CLSI (CNPt-CLSI) and a novel protocol (CNPt-direct) designed for carbapenemase detection direct from bacterial cultures (instead of bacterial extracts required by the CLSI tests). The specificities were comparable (100%), but the CNPt-direct was more sensitive (98% versus 84%). The CNPt-direct was easier to perform due to the direct use of colonies and offered a more robust detection of carbapenemase producers. PMID:26424841

Simplified Protocol for Carba NP Test for Enhanced Detection of Carbapenemase Producers Directly from Bacterial Cultures.

PubMed

Pasteran, Fernando; Tijet, Nathalie; Melano, Roberto G; Corso, Alejandra

2015-12-01

We compared carbapenemase detection among 266 Gram-negative bacilli (161 carbapenemase producers) using the Carba NP tests issued by the CLSI (CNPt-CLSI) and a novel protocol (CNPt-direct) designed for carbapenemase detection direct from bacterial cultures (instead of bacterial extracts required by the CLSI tests). The specificities were comparable (100%), but the CNPt-direct was more sensitive (98% versus 84%). The CNPt-direct was easier to perform due to the direct use of colonies and offered a more robust detection of carbapenemase producers. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Real-time detection of antibiotic activity by measuring nanometer-scale bacterial deformation

NASA Astrophysics Data System (ADS)

Iriya, Rafael; Syal, Karan; Jing, Wenwen; Mo, Manni; Yu, Hui; Haydel, Shelley E.; Wang, Shaopeng; Tao, Nongjian

2017-12-01

Diagnosing antibiotic-resistant bacteria currently requires sensitive detection of phenotypic changes associated with antibiotic action on bacteria. Here, we present an optical imaging-based approach to quantify bacterial membrane deformation as a phenotypic feature in real-time with a nanometer scale (˜9 nm) detection limit. Using this approach, we found two types of antibiotic-induced membrane deformations in different bacterial strains: polymyxin B induced relatively uniform spatial deformation of Escherichia coli O157:H7 cells leading to change in cellular volume and ampicillin-induced localized spatial deformation leading to the formation of bulges or protrusions on uropathogenic E. coli CFT073 cells. We anticipate that the approach will contribute to understanding of antibiotic phenotypic effects on bacteria with a potential for applications in rapid antibiotic susceptibility testing.

Bacterial community changes in an industrial algae production system.

PubMed

Fulbright, Scott P; Robbins-Pianka, Adam; Berg-Lyons, Donna; Knight, Rob; Reardon, Kenneth F; Chisholm, Stephen T

2018-04-01

While microalgae are a promising feedstock for production of fuels and other chemicals, a challenge for the algal bioproducts industry is obtaining consistent, robust algae growth. Algal cultures include complex bacterial communities and can be difficult to manage because specific bacteria can promote or reduce algae growth. To overcome bacterial contamination, algae growers may use closed photobioreactors designed to reduce the number of contaminant organisms. Even with closed systems, bacteria are known to enter and cohabitate, but little is known about these communities. Therefore, the richness, structure, and composition of bacterial communities were characterized in closed photobioreactor cultivations of Nannochloropsis salina in F/2 medium at different scales, across nine months spanning late summer-early spring, and during a sequence of serially inoculated cultivations. Using 16S rRNA sequence data from 275 samples, bacterial communities in small, medium, and large cultures were shown to be significantly different. Larger systems contained richer bacterial communities compared to smaller systems. Relationships between bacterial communities and algae growth were complex. On one hand, blooms of a specific bacterial type were observed in three abnormal, poorly performing replicate cultivations, while on the other, notable changes in the bacterial community structures were observed in a series of serial large-scale batch cultivations that had similar growth rates. Bacteria common to the majority of samples were identified, including a single OTU within the class Saprospirae that was found in all samples. This study contributes important information for crop protection in algae systems, and demonstrates the complex ecosystems that need to be understood for consistent, successful industrial algae cultivation. This is the first study to profile bacterial communities during the scale-up process of industrial algae systems.

Rapid Bacterial Detection via an All-Electronic CMOS Biosensor

PubMed Central

Nikkhoo, Nasim; Cumby, Nichole; Gulak, P. Glenn; Maxwell, Karen L.

2016-01-01

The timely and accurate diagnosis of infectious diseases is one of the greatest challenges currently facing modern medicine. The development of innovative techniques for the rapid and accurate identification of bacterial pathogens in point-of-care facilities using low-cost, portable instruments is essential. We have developed a novel all-electronic biosensor that is able to identify bacteria in less than ten minutes. This technology exploits bacteriocins, protein toxins naturally produced by bacteria, as the selective biological detection element. The bacteriocins are integrated with an array of potassium-selective sensors in Complementary Metal Oxide Semiconductor technology to provide an inexpensive bacterial biosensor. An electronic platform connects the CMOS sensor to a computer for processing and real-time visualization. We have used this technology to successfully identify both Gram-positive and Gram-negative bacteria commonly found in human infections. PMID:27618185

Tape Cassette Bacteria Detection System

NASA Technical Reports Server (NTRS)

1973-01-01

The design, fabrication, and testing of an automatic bacteria detection system with a zero-g capability and based on the filter-capsule approach is described. This system is intended for monitoring the sterility of regenerated water in a spacecraft. The principle of detection is based on measuring the increase in chemiluminescence produced by the action of bacterial porphyrins (i.e., catalase, cytochromes, etc.) on a luminol-hydrogen peroxide mixture. Since viable as well as nonviable organisms initiate this luminescence, viable organisms are detected by comparing the signal of an incubated water sample with an unincubated control. Higher signals for the former indicate the presence of viable organisms. System features include disposable sealed sterile capsules, each containing a filter membrane, for processing discrete water samples and a tape transport for moving these capsules through a processing sequence which involves sample concentration, nutrient addition, incubation, a 4 Molar Urea wash and reaction with luminol-hydrogen peroxide in front of a photomultiplier tube. Liquids are introduced by means of a syringe needle which pierces a rubber septum contained in the wall of the capsule. Detection thresholds obtained with this unit towards E. coli and S. marcescens assuming a 400 ml water sample are indicated.

Bacterial Signaling to the Nervous System through Toxins and Metabolites.

PubMed

Yang, Nicole J; Chiu, Isaac M

2017-03-10

Mammalian hosts interface intimately with commensal and pathogenic bacteria. It is increasingly clear that molecular interactions between the nervous system and microbes contribute to health and disease. Both commensal and pathogenic bacteria are capable of producing molecules that act on neurons and affect essential aspects of host physiology. Here we highlight several classes of physiologically important molecular interactions that occur between bacteria and the nervous system. First, clostridial neurotoxins block neurotransmission to or from neurons by targeting the SNARE complex, causing the characteristic paralyses of botulism and tetanus during bacterial infection. Second, peripheral sensory neurons-olfactory chemosensory neurons and nociceptor sensory neurons-detect bacterial toxins, formyl peptides, and lipopolysaccharides through distinct molecular mechanisms to elicit smell and pain. Bacteria also damage the central nervous system through toxins that target the brain during infection. Finally, the gut microbiota produces molecules that act on enteric neurons to influence gastrointestinal motility, and metabolites that stimulate the "gut-brain axis" to alter neural circuits, autonomic function, and higher-order brain function and behavior. Furthering the mechanistic and molecular understanding of how bacteria affect the nervous system may uncover potential strategies for modulating neural function and treating neurological diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacteriophages for detection and control of bacterial pathogens in food and food-processing environment.

PubMed

Brovko, Lubov Y; Anany, Hany; Griffiths, Mansel W

2012-01-01

This chapter presents recent advances in bacteriophage research and their application in the area of food safety. Section 1 describes general facts on phage biology that are relevant to their application for control and detection of bacterial pathogens in food and environmental samples. Section 2 summarizes the recently acquired data on application of bacteriophages to control growth of bacterial pathogens and spoilage organisms in food and food-processing environment. Section 3 deals with application of bacteriophages for detection and identification of bacterial pathogens. Advantages of bacteriophage-based methods are presented and their shortcomings are discussed. The chapter is intended for food scientist and food product developers, and people in food inspection and health agencies with the ultimate goal to attract their attention to the new developing technology that has a tremendous potential in providing means for producing wholesome and safe food. Copyright © 2012 Elsevier Inc. All rights reserved.

Engineering Bacterial Thiosulfate and Tetrathionate Sensors for Detecting Gut Inflammation

DTIC Science & Technology

2017-04-03

Article Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation Kristina N-M Daeffler1 , Jeffrey D Galley2, Ravi U...interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we...understood. Genetically engineered sensor bacteria have untapped potential as tools for analyzing gut pathways. Bacteria have evolved sensors of a large

Real-time detection of antibiotic activity by measuring nanometer-scale bacterial deformation.

PubMed

Iriya, Rafael; Syal, Karan; Jing, Wenwen; Mo, Manni; Yu, Hui; Haydel, Shelley E; Wang, Shaopeng; Tao, Nongjian

2017-12-01

Diagnosing antibiotic-resistant bacteria currently requires sensitive detection of phenotypic changes associated with antibiotic action on bacteria. Here, we present an optical imaging-based approach to quantify bacterial membrane deformation as a phenotypic feature in real-time with a nanometer scale (∼9  nm) detection limit. Using this approach, we found two types of antibiotic-induced membrane deformations in different bacterial strains: polymyxin B induced relatively uniform spatial deformation of Escherichia coli O157:H7 cells leading to change in cellular volume and ampicillin-induced localized spatial deformation leading to the formation of bulges or protrusions on uropathogenic E. coli CFT073 cells. We anticipate that the approach will contribute to understanding of antibiotic phenotypic effects on bacteria with a potential for applications in rapid antibiotic susceptibility testing. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

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.

Multimodal imaging system for dental caries detection

NASA Astrophysics Data System (ADS)

Liang, Rongguang; Wong, Victor; Marcus, Michael; Burns, Peter; McLaughlin, Paul

2007-02-01

Dental caries is a disease in which minerals of the tooth are dissolved by surrounding bacterial plaques. A caries process present for some time may result in a caries lesion. However, if it is detected early enough, the dentist and dental professionals can implement measures to reverse and control caries. Several optical, nonionized methods have been investigated and used to detect dental caries in early stages. However, there is not a method that can singly detect the caries process with both high sensitivity and high specificity. In this paper, we present a multimodal imaging system that combines visible reflectance, fluorescence, and Optical Coherence Tomography (OCT) imaging. This imaging system is designed to obtain one or more two-dimensional images of the tooth (reflectance and fluorescence images) and a three-dimensional OCT image providing depth and size information of the caries. The combination of two- and three-dimensional images of the tooth has the potential for highly sensitive and specific detection of dental caries.

Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy.

PubMed

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-08-01

To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analyzing size and number of intracellular bacterial colonies in infected tissue culture cells. Cells are seeded in 48-well plates and infected with a GFP-expressing bacterial pathogen. Following gentamicin treatment to remove extracellular pathogens, cells are fixed and cell nuclei stained. This is followed by automated microscopy and subsequent semi-automated spot detection to determine the number of intracellular bacterial colonies, their size distribution, and the average number per host cell. Multiple 48-well plates can be processed sequentially and the procedure can be completed in one working day. As a model we quantified intracellular bacterial colonies formed by uropathogenic Escherichia coli (UPEC) during infection of human kidney cells (HKC-8). Urinary tract infections caused by UPEC are among the most common bacterial infectious diseases in humans. UPEC can colonize tissues of the urinary tract and is responsible for acute, chronic, and recurrent infections. In the bladder, UPEC can form intracellular quiescent reservoirs, thought to be responsible for recurrent infections. In the kidney, UPEC can colonize renal epithelial cells and pass to the blood stream, either via epithelial cell disruption or transcellular passage, to cause sepsis. Intracellular colonies are known to be clonal, originating from single invading UPEC. In our experimental setup, we found UPEC CFT073 intracellular bacterial colonies to be heterogeneous in size and present in nearly one third of the HKC-8 cells. This high-throughput experimental format substantially reduces experimental time and enables fast screening of the intracellular bacterial load and cellular distribution of multiple

An integrated microfluidic analysis microsystems with bacterial capture enrichment and in-situ impedance detection

NASA Astrophysics Data System (ADS)

Liu, Hai-Tao; Wen, Zhi-Yu; Xu, Yi; Shang, Zheng-Guo; Peng, Jin-Lan; Tian, Peng

2017-09-01

In this paper, an integrated microfluidic analysis microsystems with bacterial capture enrichment and in-situ impedance detection was purposed based on microfluidic chips dielectrophoresis technique and electrochemical impedance detection principle. The microsystems include microfluidic chip, main control module, and drive and control module, and signal detection and processing modulet and result display unit. The main control module produce the work sequence of impedance detection system parts and achieve data communication functions, the drive and control circuit generate AC signal which amplitude and frequency adjustable, and it was applied on the foodborne pathogens impedance analysis microsystems to realize the capture enrichment and impedance detection. The signal detection and processing circuit translate the current signal into impendence of bacteria, and transfer to computer, the last detection result is displayed on the computer. The experiment sample was prepared by adding Escherichia coli standard sample into chicken sample solution, and the samples were tested on the dielectrophoresis chip capture enrichment and in-situ impedance detection microsystems with micro-array electrode microfluidic chips. The experiments show that the Escherichia coli detection limit of microsystems is 5 × 104 CFU/mL and the detection time is within 6 min in the optimization of voltage detection 10 V and detection frequency 500 KHz operating conditions. The integrated microfluidic analysis microsystems laid the solid foundation for rapid real-time in-situ detection of bacteria.

Gibbs motif sampling: detection of bacterial outer membrane protein repeats.

PubMed Central

Neuwald, A. F.; Liu, J. S.; Lawrence, C. E.

1995-01-01

The detection and alignment of locally conserved regions (motifs) in multiple sequences can provide insight into protein structure, function, and evolution. A new Gibbs sampling algorithm is described that detects motif-encoding regions in sequences and optimally partitions them into distinct motif models; this is illustrated using a set of immunoglobulin fold proteins. When applied to sequences sharing a single motif, the sampler can be used to classify motif regions into related submodels, as is illustrated using helix-turn-helix DNA-binding proteins. Other statistically based procedures are described for searching a database for sequences matching motifs found by the sampler. When applied to a set of 32 very distantly related bacterial integral outer membrane proteins, the sampler revealed that they share a subtle, repetitive motif. Although BLAST (Altschul SF et al., 1990, J Mol Biol 215:403-410) fails to detect significant pairwise similarity between any of the sequences, the repeats present in these outer membrane proteins, taken as a whole, are highly significant (based on a generally applicable statistical test for motifs described here). Analysis of bacterial porins with known trimeric beta-barrel structure and related proteins reveals a similar repetitive motif corresponding to alternating membrane-spanning beta-strands. These beta-strands occur on the membrane interface (as opposed to the trimeric interface) of the beta-barrel. The broad conservation and structural location of these repeats suggests that they play important functional roles. PMID:8520488

Bacterial toxin-antitoxin systems: more than selfish entities?

PubMed

Van Melderen, Laurence; Saavedra De Bast, Manuel

2009-03-01

Bacterial toxin-antitoxin (TA) systems are diverse and widespread in the prokaryotic kingdom. They are composed of closely linked genes encoding a stable toxin that can harm the host cell and its cognate labile antitoxin, which protects the host from the toxin's deleterious effect. TA systems are thought to invade bacterial genomes through horizontal gene transfer. Some TA systems might behave as selfish elements and favour their own maintenance at the expense of their host. As a consequence, they may contribute to the maintenance of plasmids or genomic islands, such as super-integrons, by post-segregational killing of the cell that loses these genes and so suffers the stable toxin's destructive effect. The function of the chromosomally encoded TA systems is less clear and still open to debate. This Review discusses current hypotheses regarding the biological roles of these evolutionarily successful small operons. We consider the various selective forces that could drive the maintenance of TA systems in bacterial genomes.

Bacterial Toxin–Antitoxin Systems: More Than Selfish Entities?

PubMed Central

Van Melderen, Laurence; Saavedra De Bast, Manuel

2009-01-01

Bacterial toxin–antitoxin (TA) systems are diverse and widespread in the prokaryotic kingdom. They are composed of closely linked genes encoding a stable toxin that can harm the host cell and its cognate labile antitoxin, which protects the host from the toxin's deleterious effect. TA systems are thought to invade bacterial genomes through horizontal gene transfer. Some TA systems might behave as selfish elements and favour their own maintenance at the expense of their host. As a consequence, they may contribute to the maintenance of plasmids or genomic islands, such as super-integrons, by post-segregational killing of the cell that loses these genes and so suffers the stable toxin's destructive effect. The function of the chromosomally encoded TA systems is less clear and still open to debate. This Review discusses current hypotheses regarding the biological roles of these evolutionarily successful small operons. We consider the various selective forces that could drive the maintenance of TA systems in bacterial genomes. PMID:19325885

Translating bacterial detection by DNAzymes into a litmus test.

PubMed

Tram, Kha; Kanda, Pushpinder; Salena, Bruno J; Huan, Shuangyan; Li, Yingfu

2014-11-17

Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front-line preventative care, particularly in resource-limited regions. Thus, there is a significant need to develop low-cost and easy-to-use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pH value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lectin functionalized ZnO nanoarrays as a 3D nano-biointerface for bacterial detection.

PubMed

Zheng, Laibao; Wan, Yi; Qi, Peng; Sun, Yan; Zhang, Dun; Yu, Liangmin

2017-05-15

The detection of pathogenic bacteria is essential in various fields, such as food safety, water environmental analysis, or clinical diagnosis. Although rapid and selective techniques have been achieved based on the fast and specific binding of recognitions elements and target, the sensitive detection of bacterial pathogens was limited by their low targets-binding efficiency. The three-dimensional (3D) nano-biointerface, compared with the two-dimensional (2D) flat substrate, has a much higher binding capacity, which can offer more reactive sites to bind with bacterial targets, resulting in a great improvement of detection sensitivity. Herein, a lectin functionalized ZnO nanorod (ZnO-NR) array has been fabricated and employed as a 3D nano-biointerface for Escherichia coli (E. coli) capture and detection by multivalent binding of concanavalin A (ConA) with polysaccharides on the cellular surface of E. coli. The 3D lectin functionalized ZnO-NR array-based assay shows reasonable detection limit and efficiently expanded linear range (1.0×10 3 to 1.0×10 7 cfumL -1 ) for pathogen detection. The platform has a potential for further applications and provides an excellent sensitivity approach for detection of pathogenic bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Diagnostic utility and cost-effectiveness of reflex bacterial culture for the detection of urinary tract infection in dogs with low urine specific gravity.

PubMed

Tivapasi, Musavenga T; Hodges, Joanne; Byrne, Barbara A; Christopher, Mary M

2009-09-01

Urinary tract infections (UTIs) may be subclinical or difficult to detect in dilute urine as sediment abnormalities may not be observed. In our laboratory, bacterial culture is automatically performed (reflex culture) on samples with urine specific gravity (USG)< or =1.013 to increase the likelihood of detecting infection. The value of routine culture of dilute urine, however, has not been fully assessed. The purpose of this retrospective study was to evaluate the frequency of positive bacterial cultures and analyze the diagnostic utility and cost-effectiveness of culture compared with routine sediment examination for detecting UTI in dilute urine specimens from dogs. Urinalysis and concurrent aerobic bacterial culture results were obtained from the electronic medical record system at the University of California-Davis Veterinary Medical Teaching Hospital for samples with USG< or =1.013 analyzed from July 1998 through January 2005. Urine collection method, presence of leukocytes and bacteria, bacterial culture results, and clinical diagnosis were recorded. Cost-effectiveness of reflex culture, based on low USG as the sole criterion, was evaluated. Of 1264 urine specimens, 106 (8.4%) had positive bacterial cultures. Using culture as the gold standard, sediment evaluation had a diagnostic sensitivity of 58.5% and specificity of 98.3% (diagnostic accuracy 94.9%). An additional cost of $60 per patient was incurred, leading to average annual costs of $11,668 for reflex bacterial cultures of all samples with low USG, regardless of collection method. Within our study population, 10 urine samples needed to be cultured for each true positive result. The sensitivity of urine sediment evaluation is low for UTI in dilute urine samples; however, reflex bacterial culture does not appear to be cost-effective in dogs with USG< or =1.013 in the absence of active urine sediment or high clinical suspicion for UTI.

Evaluation of the Seeplex® Meningitis ACE Detection kit for the detection of 12 common bacterial and viral pathogens of acute meningitis.

PubMed

Shin, So Youn; Kwon, Kye Chul; Park, Jong Woo; Kim, Ji Myung; Shin, So Young; Koo, Sun Hoe

2012-01-01

Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. The lower detection limits ranged from 10(1) copies/µL to 5×10(1) copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens.

Evaluation of the Seeplex® Meningitis ACE Detection Kit for the Detection of 12 Common Bacterial and Viral Pathogens of Acute Meningitis

PubMed Central

Shin, So Youn; Kwon, Kye Chul; Park, Jong Woo; Kim, Ji Myung; Shin, So Young

2012-01-01

Background Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. Methods Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. Results The lower detection limits ranged from 101 copies/µL to 5×101 copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. Conclusions The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens. PMID:22259778

Insight into the effects of different cropping systems on soil bacterial community and tobacco bacterial wilt rate.

PubMed

Niu, Jiaojiao; Chao, Jin; Xiao, Yunhua; Chen, Wu; Zhang, Chao; Liu, Xueduan; Rang, Zhongwen; Yin, Huaqun; Dai, Linjian

2017-01-01

Rotation is an effective strategy to control crop disease and improve plant health. However, the effects of crop rotation on soil bacterial community composition and structure, and crop health remain unclear. In this study, using 16S rRNA gene sequencing, we explored the soil bacterial communities under four different cropping systems, continuous tobacco cropping (control group), tobacco-maize rotation, tobacco-lily rotation, and tobacco-turnip rotation. Results of detrended correspondence analysis and dissimilarity tests showed that soil bacterial community composition and structure changed significantly among the four groups, such that Acidobacteria and Actinobacteria were more abundant in the maize rotation group (16.6 and 11.5%, respectively) than in the control (8.5 and 7.1%, respectively). Compared with the control group (57.78%), maize and lily were effective rotation crops in controlling tobacco bacterial wilt (about 23.54 and 48.67%). On the other hand, tobacco bacterial wilt rate was increased in the turnip rotation (59.62%) relative to the control. Further study revealed that the abundances of several bacterial populations were directly correlated with tobacco bacterial wilt. For example, Acidobacteria and Actinobacteria were significantly negatively correlated to the tobacco bacterial wilt rate, so they may be probiotic bacteria. Canonical correspondence analysis showed that soil pH and calcium content were key factors in determining soil bacterial communities. In conclusion, our study revealed the composition and structure of bacterial communities under four different cropping systems and may unveil molecular mechanisms for the interactions between soil microorganisms and crop health. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis.

PubMed

Huy, Nguyen Tien; Hang, Le Thi Thuy; Boamah, Daniel; Lan, Nguyen Thi Phuong; Van Thanh, Phan; Watanabe, Kiwao; Huong, Vu Thi Thu; Kikuchi, Mihoko; Ariyoshi, Koya; Morita, Kouichi; Hirayama, Kenji

2012-12-01

Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Comparison of enterovirus detection in cerebrospinal fluid with Bacterial Meningitis Score in children.

PubMed

Pires, Frederico Ribeiro; Franco, Andréia Christine Bonotto Farias; Gilio, Alfredo Elias; Troster, Eduardo Juan

2017-01-01

To measure the role of enterovirus detection in cerebrospinal fluid compared with the Bacterial Meningitis Score in children with meningitis. A retrospective cohort based on analysis of medical records of pediatric patients diagnosed as meningitis, seen at a private and tertiary hospital in São Paulo, Brazil, between 2011 and 2014. Excluded were patients with critical illness, purpura, ventricular shunt or recent neurosurgery, immunosuppression, concomitant bacterial infection requiring parenteral antibiotic therapy, and those who received antibiotics 72 hours before lumbar puncture. The study included 503 patients. Sixty-four patients were excluded and 94 were not submitted to all tests for analysis. Of the remaining 345 patients, 7 were in the Bacterial Meningitis Group and 338 in the Aseptic Meningitis Group. There was no statistical difference between the groups. In the Bacterial Meningitis Score analysis, of the 338 patients with possible aseptic meningitis (negative cultures), 121 of them had one or more points in the Bacterial Meningitis Score, with sensitivity of 100%, specificity of 64.2%, and negative predictive value of 100%. Of the 121 patients with positive Bacterial Meningitis Score, 71% (86 patients) had a positive enterovirus detection in cerebrospinal fluid. Enterovirus detection in cerebrospinal fluid was effective to differentiate bacterial from viral meningitis. When the test was analyzed together with the Bacterial Meningitis Score, specificity was higher when compared to Bacterial Meningitis Score alone. Avaliar o papel da pesquisa de enterovírus no líquido cefalorraquidiano em comparação com o Escore de Meningite Bacteriana em crianças com meningite. Coorte retrospectiva, realizada pela análise de prontuários, incluindo pacientes pediátricos, com diagnóstico de meningite e atendidos em um hospital privado e terciário, localizado em São Paulo, entre 2011 e 2014. Foram excluídos os pacientes com doença crítica, púrpura, deriva

Vision Marker-Based In Situ Examination of Bacterial Growth in Liquid Culture Media.

PubMed

Kim, Kyukwang; Choi, Duckyu; Lim, Hwijoon; Kim, Hyeongkeun; Jeon, Jessie S

2016-12-18

The detection of bacterial growth in liquid media is an essential process in determining antibiotic susceptibility or the level of bacterial presence for clinical or research purposes. We have developed a system, which enables simplified and automated detection using a camera and a striped pattern marker. The quantification of bacterial growth is possible as the bacterial growth in the culturing vessel blurs the marker image, which is placed on the back of the vessel, and the blurring results in a decrease in the high-frequency spectrum region of the marker image. The experiment results show that the FFT (fast Fourier transform)-based growth detection method is robust to the variations in the type of bacterial carrier and vessels ranging from the culture tubes to the microfluidic devices. Moreover, the automated incubator and image acquisition system are developed to be used as a comprehensive in situ detection system. We expect that this result can be applied in the automation of biological experiments, such as the Antibiotics Susceptibility Test or toxicity measurement. Furthermore, the simple framework of the proposed growth measurement method may be further utilized as an effective and convenient method for building point-of-care devices for developing countries.

Luciferase-Zinc-Finger System for the Rapid Detection of Pathogenic Bacteria.

PubMed

Shi, Chu; Xu, Qing; Ge, Yue; Jiang, Ling; Huang, He

2017-08-09

Rapid and reliable detection of pathogenic bacteria is crucial for food safety control. Here, we present a novel luciferase-zinc finger system for the detection of pathogens that offers rapid and specific profiling. The system, which uses a zinc-finger protein domain to probe zinc finger recognition sites, was designed to bind the amplified conserved regions of 16S rDNA, and the obtained products were detected using a modified luciferase. The luciferase-zinc finger system not only maintained luciferase activity but also allowed the specific detection of different bacterial species, with a sensitivity as low as 10 copies and a linear range from 10 to 10 4 copies per microliter of the specific PCR product. Moreover, the system is robust and rapid, enabling the simultaneous detection of 6 species of bacteria in artificially contaminated samples with excellent accuracy. Thus, we envision that our luciferase-zinc finger system will have far-reaching applications.

Gold Nanorod-based Photo-PCR System for One-Step, Rapid Detection of Bacteria

PubMed Central

Kim, Jinjoo; Kim, Hansol; Park, Ji Ho; Jon, Sangyong

2017-01-01

The polymerase chain reaction (PCR) has been an essential tool for diagnosis of infectious diseases, but conventional PCR still has some limitations with respect to applications to point-of-care (POC) diagnostic systems that require rapid detection and miniaturization. Here we report a light-based PCR method, termed as photo-PCR, which enables rapid detection of bacteria in a single step. In the photo-PCR system, poly(enthylene glycol)-modified gold nanorods (PEG-GNRs), used as a heat generator, are added into the PCR mixture, which is subsequently periodically irradiated with a 808-nm laser to create thermal cycling. Photo-PCR was able to significantly reduce overall thermal cycling time by integrating bacterial cell lysis and DNA amplification into a single step. Furthermore, when combined with KAPA2G fast polymerase and cooling system, the entire process of bacterial genomic DNA extraction and amplification was further shortened, highlighting the potential of photo-PCR for use in a portable, POC diagnostic system. PMID:29071186

Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR.

PubMed

Wagner, Karoline; Springer, Burkard; Imkamp, Frank; Opota, Onya; Greub, Gilbert; Keller, Peter M

2018-04-01

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was

Detection of human bacterial pathogens in ticks collected from Louisiana black bears (Ursus americanus luteolus)

PubMed Central

Leydet, Brian F.; Liang, Fang-Ting

2013-01-01

There are 4 major human-biting tick species in the northeastern United States, which include: Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, and Ixodes scapularis. The black bear is a large mammal that has been shown to be parasitized by all the aforementioned ticks. We investigated the bacterial infections in ticks collected from Louisiana black bears (Ursus americanus subspecies luteolus). Eighty-six ticks were collected from 17 black bears in Louisiana from June 2010 to March 2011. All 4 common human-biting tick species were represented. Each tick was subjected to polymerase chain reaction (PCR) targeting select bacterial pathogens and symbionts. Bacterial DNA was detected in 62% of ticks (n=53). Rickettsia parkeri, the causative agent of an emerging spotted fever group rickettsiosis, was identified in 66% of A. maculatum, 28% of D. variabilis, and 11% of I. scapularis. The Lyme disease bacterium, Borrelia burgdorferi, was detected in 2 I. scapularis, while one Am. americanum was positive for Borrelia bissettii, a putative human pathogen. The rickettsial endosymbionts Candidatus Rickettsia andeanae, rickettsial endosymbiont of I. scapularis, and Rickettsia amblyommii were detected in their common tick hosts at 21%, 39%, and 60%, respectively. All ticks were PCR-negative for Anaplasma phagocytophilum, Ehrlichia spp., and Babesia microti. This is the first reported detection of R. parkeri in vector ticks in Louisiana; we also report the novel association of R. parkeri with I. scapularis. Detection of both R. parkeri and Bo. burgdorferi in their respective vectors in Louisiana demands further investigation to determine potential for human exposure to these pathogens. PMID:23415850

Evolvable social agents for bacterial systems modeling.

PubMed

Paton, Ray; Gregory, Richard; Vlachos, Costas; Saunders, Jon; Wu, Henry

2004-09-01

We present two approaches to the individual-based modeling (IbM) of bacterial ecologies and evolution using computational tools. The IbM approach is introduced, and its important complementary role to biosystems modeling is discussed. A fine-grained model of bacterial evolution is then presented that is based on networks of interactivity between computational objects representing genes and proteins. This is followed by a coarser grained agent-based model, which is designed to explore the evolvability of adaptive behavioral strategies in artificial bacteria represented by learning classifier systems. The structure and implementation of the two proposed individual-based bacterial models are discussed, and some results from simulation experiments are presented, illustrating their adaptive properties.

Comparative Genomic Analyses of the Bacterial Phosphotransferase System

PubMed Central

Barabote, Ravi D.; Saier, Milton H.

2005-01-01

We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer. PMID:16339738

Detection and inhibition of bacterial cell-cell communication.

PubMed

Rice, Scott A; McDougald, Diane; Givskov, Michael; Kjelleberg, Staffan

2008-01-01

Bacteria communicate with other members of their community through the secretion and perception of small chemical cues or signals. The recognition of a signal normally leads to the expression of a large suite of genes, which in some bacteria are involved in the regulation of virulence factors, and as a result, these signaling compounds are key regulatory factors in many disease processes. Thus, it is of interest when studying pathogens to understand the mechanisms used to control the expression of virulence genes so that strategies might be devised for the control of those pathogens. Clearly, the ability to interfere with this process of signaling represents a novel approach for the treatment of bacterial infections. There is a broad range of compounds that bacteria can use for signaling purposes, including fatty acids, peptides, N-acylated homoserine lactones, and the signals collectively called autoinducer 2 (AI-2). This chapter will focus on the latter two signaling systems as they are present in a range of medically relevant bacteria, and here we describe assays for determining whether an organism produces a particular signal and assays that can be used to identify inhibitors of the signaling cascade. Lastly, the signal detection and inhibition assays will be directly linked to the expression of virulence factors of specific pathogens.

Bacterial communities in the collection and chlorinated distribution sections of a drinking water system in Budapest, Hungary.

PubMed

Homonnay, Zalán G; Török, György; Makk, Judit; Brumbauer, Anikó; Major, Eva; Márialigeti, Károly; Tóth, Erika

2014-07-01

Bacterial communities of a bank-filtered drinking water system were investigated by aerobic cultivation and clone library analysis. Moreover, bacterial communities were compared using sequence-aided terminal restriction fragment length polymorphism (T-RFLP) fingerprinting at ten characteristic points located at both the collecting and the distributing part of the water supply system. Chemical characteristics of the samples were similar, except for the presence of chlorine residuals in the distribution system and increased total iron concentration in two of the samples. Assimilable organic carbon (AOC) concentration increased within the collection system, it was reduced by chlorination and it increased again in the distribution system. Neither fecal indicators nor pathogens were detected by standard cultivation techniques. Chlorination reduced bacterial diversity and heterotrophic plate counts. Community structures were found to be significantly different before and after chlorination: the diverse communities in wells and the collection system were dominated by chemolithotrophic (e.g., Gallionella and Nitrospira) and oligocarbophilic-heterotrophic bacteria (e.g., Sphingomonas, Sphingopyxis, and Bradyrhizobium). After chlorination in the distribution system, the most characteristic bacterium was related to the facultative methylotrophic Methylocella spp. Communities changed within the distribution system too, Mycobacterium spp. or Sphingopyxis spp. became predominant in certain samples. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System

PubMed Central

Prest, E. I.; Weissbrodt, D. G.; Hammes, F.; van Loosdrecht, M. C. M.; Vrouwenvelder, J. S.

2016-01-01

Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (<1–3.6 ng L-1), which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson’s correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously

Long-Term Bacterial Dynamics in a Full-Scale Drinking Water Distribution System.

PubMed

Prest, E I; Weissbrodt, D G; Hammes, F; van Loosdrecht, M C M; Vrouwenvelder, J S

2016-01-01

Large seasonal variations in microbial drinking water quality can occur in distribution networks, but are often not taken into account when evaluating results from short-term water sampling campaigns. Temporal dynamics in bacterial community characteristics were investigated during a two-year drinking water monitoring campaign in a full-scale distribution system operating without detectable disinfectant residual. A total of 368 water samples were collected on a biweekly basis at the water treatment plant (WTP) effluent and at one fixed location in the drinking water distribution network (NET). The samples were analysed for heterotrophic plate counts (HPC), Aeromonas plate counts, adenosine-tri-phosphate (ATP) concentrations, and flow cytometric (FCM) total and intact cell counts (TCC, ICC), water temperature, pH, conductivity, total organic carbon (TOC) and assimilable organic carbon (AOC). Multivariate analysis of the large dataset was performed to explore correlative trends between microbial and environmental parameters. The WTP effluent displayed considerable seasonal variations in TCC (from 90 × 103 cells mL-1 in winter time up to 455 × 103 cells mL-1 in summer time) and in bacterial ATP concentrations (<1-3.6 ng L-1), which were congruent with water temperature variations. These fluctuations were not detected with HPC and Aeromonas counts. The water in the network was predominantly influenced by the characteristics of the WTP effluent. The increase in ICC between the WTP effluent and the network sampling location was small (34 × 103 cells mL-1 on average) compared to seasonal fluctuations in ICC in the WTP effluent. Interestingly, the extent of bacterial growth in the NET was inversely correlated to AOC concentrations in the WTP effluent (Pearson's correlation factor r = -0.35), and positively correlated with water temperature (r = 0.49). Collecting a large dataset at high frequency over a two year period enabled the characterization of previously

Direct fluorescent antibody technique for the detection of bacterial kidney disease in paraffin-embedded tissues

USGS Publications Warehouse

Ochiai, T.; Yasutake, W.T.; Gould, R.W.

1985-01-01

The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.

Novel aptamer-linked nanoconjugate approach for detection of waterborne bacterial pathogens: an update

NASA Astrophysics Data System (ADS)

Singh, Gulshan; Manohar, Murli; Adegoke, Anthony Ayodeji; Stenström, Thor Axel; Shanker, Rishi

2017-01-01

The lack of microbiologically safe water in underdeveloped nations is the prime cause of infectious disease outbreaks. The need for the specific identification and detection of microorganisms encourages the development of advanced, rapid, sensitive and highly specific methods for the monitoring of pathogens and management of potential risk to human health. The rapid molecular assays based on detection of specific molecular signatures offer advantages over conventional methods in terms of specificity and sensitivity but require complex instrumentation and skilled personnel. Nanotechnology is an emerging area and provides a robust approach for the identification of pathogenic microorganism utilizing the peculiar properties of nanomaterials, i.e. small size (1-100 nm) and large surface area. This emerging technology promises to fulfill the urgent need of a novel strategy to enhance the bacterial identification and quantitation in the environment. In this context, the peculiar properties of gold nanoparticles, their plasmonic shifts, and changes in magnetic properties have been utilized for the simple and cost-effective detection of bacterial nucleic acids, antigens and toxins with quite improved sensitivity. One of the promising leads to develop an advance detection method might be the coupling of nucleic acid aptamers (capable of interacting specifically with bacteria, protozoa, and viruses) with nanomaterials. Such aptamer-nano conjugate can be used for the specific recognition of infectious agents in different environmental matrices. This review summarizes the application of nanotechnology in the area of pathogen detection and discusses the prospects of coupling nucleic acid aptamers with nanoparticles for the specific detection of targeted pathogens.

Evaluation of the 3D BacT/ALERT automated culture system for the detection of microbial contamination of platelet concentrates.

PubMed

McDonald, C P; Rogers, A; Cox, M; Smith, R; Roy, A; Robbins, S; Hartley, S; Barbara, J A J; Rothenberg, S; Stutzman, L; Widders, G

2002-10-01

Bacterial transmission remains the major component of morbidity and mortality associated with transfusion-transmitted infections. Platelet concentrates are the most common cause of bacterial transmission. The BacT/ALERT 3D automated blood culture system has the potential to screen platelet concentrates for the presence of bacteria. Evaluation of this system was performed by spiking day 2 apheresis platelet units with individual bacterial isolates at final concentrations of 10 and 100 colony-forming units (cfu) mL-1. Fifteen organisms were used which had been cited in platelet transmission and monitoring studies. BacT/ALERT times to detection were compared with thioglycollate broth cultures, and the performance of five types of BacT/ALERT culture bottles was evaluated. Sampling was performed immediately after the inoculation of the units, and 10 replicates were performed per organism concentration for each of the five types of BacT/ALERT bottles. The mean times for the detection of these 15 organisms by BacT/ALERT, with the exception of Propionibacterium acnes, ranged from 9.1 to 48.1 h (all 10 replicates were positive). In comparison, the time range found using thioglycollate was 12.0-32.3 h (all 10 replicates were positive). P. acnes' BacT/ALERT mean detection times ranged from 89.0 to 177.6 h compared with 75.6-86.4 h for the thioglycollate broth. BacT/ALERT, with the exception of P. acnes, which has dubious clinical significance, gave equivalent or shorter detection times when compared with the thioglycollate broth system. The BacT/ALERT system detected a range of organisms at levels of 10 and 100 cfu mL-1. This study validates the BacT/ALERT microbial detection system for screening platelets. Currently, the system is the only practically viable option available for routinely screening platelet concentrates to prevent bacterial transmission.

Bacterial detection using bacteriophages and gold nanorods by following time-dependent changes in Raman spectral signals.

PubMed

Moghtader, Farzaneh; Tomak, Aysel; Zareie, Hadi M; Piskin, Erhan

2018-03-27

This study attemps to develop bacterial detection strategies using bacteriophages and gold nanorods (GNRs) by Raman spectral analysis. Escherichia coli was selected as the target and its specific phage was used as the bioprobe. Target bacteria and phages were propagated/purified by traditional techniques. GNRs were synthesized by using hexadecyltrimethyl ammonium bromide (CTAB) as stabilizer. A two-step detection strategy was applied: Firstly, the target bacteria were interacted with GNRs in suspensions, and then they were dropped onto silica substrates for detection. It was possible to obtain clear surface-enchanced Raman spectroscopy (SERS) peaks of the target bacteria, even without using phages. In the second step, the phage nanoemulsions were droped onto the bacterial-GNRs complexes on those surfaces and time-dependent changes in the Raman spectra were monitored at different time intervals upto 40 min. These results demonstrated that how one can apply phages with plasmonic nanoparticles for detection of pathogenic bacteria very effectively in a quite simple test.

Electrical response of culture media during bacterial growth on a paper-based device

NASA Astrophysics Data System (ADS)

Srimongkon, Tithimanan; Buerkle, Marius; Nakamura, Akira; Enomae, Toshiharu; Ushijima, Hirobumi; Fukuda, Nobuko

2017-05-01

In this work, we evaluated the feasibility of a paper-based bacterial detection system. The paper served as a substrate for the measurement electrodes and the culture medium. Using a printing technique, we patterned gold electrodes onto the paper substrate and applied Luria broth (LB) agar gel as a culture medium on top of the electrodes. As the first step towards the development of a bacterial detection system, we determined changes in the surface potential during bacterial growth and monitored these changes over 24 h. This allowed us to correlate changes in the surface potential with the different growth phases of the bacteria.

Single-shot detection of bacterial endospores via coherent Raman spectroscopy.

PubMed

Pestov, Dmitry; Wang, Xi; Ariunbold, Gombojav O; Murawski, Robert K; Sautenkov, Vladimir A; Dogariu, Arthur; Sokolov, Alexei V; Scully, Marlan O

2008-01-15

Recent advances in coherent Raman spectroscopy hold exciting promise for many potential applications. For example, a technique, mitigating the nonresonant four-wave-mixing noise while maximizing the Raman-resonant signal, has been developed and applied to the problem of real-time detection of bacterial endospores. After a brief review of the technique essentials, we show how extensions of our earlier experimental work [Pestov D, et al. (2007) Science 316:265-268] yield single-shot identification of a small sample of Bacillus subtilis endospores (approximately 10(4) spores). The results convey the utility of the technique and its potential for "on-the-fly" detection of biohazards, such as Bacillus anthracis. The application of optimized coherent anti-Stokes Raman scattering scheme to problems requiring chemical specificity and short signal acquisition times is demonstrated.

Evaluation of vaginal pH for detection of bacterial vaginosis

PubMed Central

Hemalatha, R.; Ramalaxmi, Baru Anantha; Swetha, Eluru; Balakrishna, N.; Mastromarino, Paola

2013-01-01

Background & objectives: Bacterial vaginosis (BV) is highly prevalent among women in reproductive age group. Little information exists on routine vaginal pH measurement in women with BV. We undertook this study to assess the utility of vaginal pH determination for initial evaluation of bacterial vaginosis. Methods: In this cross-sectional study vaginal swabs were collected from women with complaints of white discharge, back ache and pain abdomen attending a government hospital and a community health clinic, and subjected to vaginal pH determination, Gram stain, wet mount and whiff test. Nugent score and Amsel criteria were used for BV confirmation. Results: Of the 270 women included in the analysis, 154 had BV based on Nugents’ score. The mean vaginal pH in women with BV measured by pH strips and pH glove was 5 and 4.9, respectively. The vaginal pH was significantly higher in women with BV. Vaginal discharge was prevalent in 84.8 per cent women, however, only 56.8 per cent of these actually had BV by Nugent score (NS). Presence of clue cells and positive whiff test were significant for BV. Vaginal pH >4.5 by pH strips and pH Glove had a sensitivity of 72 and 79 per cent and specificity of 60 and 53 per cent, respectively to detect BV. Among the combination criteria, clue cells and glove pH >4.5 had highest sensitivity and specificity to detect BV. Interpretation & conclusions: Vaginal pH determination is relatively sensitive, but less specific in detecting women with BV. Inclusion of whiff test along with pH test reduced the sensitivity, but improved specificity. Both, the pH strip and pH glove are equally suitable for screening women with BV on outpatient basis. PMID:24135180

Dynamic laser speckle to detect motile bacterial response of Pseudomonas aeruginosa

NASA Astrophysics Data System (ADS)

Sendra, H.; Murialdo, S.; Passoni, L.

2007-11-01

This proposal deals with the technique for detection of motile response of Pseudomonas aeruginosa using dynamic laser speckle or biospeckle as an alternative method. The study of bacterial displacement plays an essential role in biocatalysts processes and biodegradation. Hence, some biodegrading enzymes are benign catalytic that could be used for the production of industrially useful compounds as well as in wastewater treatments. This work presents an experimental set up and a computational process using frame sequences of dynamic laser speckle as a novel application. The objective was the detection of different levels of motility in bacteria. The encouraging results were achieved through a direct and non invasive observation method of the phenomenon.

Disposable bioluminescence-based biosensor for detection of bacterial count in food.

PubMed

Luo, Jinping; Liu, Xiaohong; Tian, Qing; Yue, Weiwei; Zeng, Jing; Chen, Guangquan; Cai, Xinxia

2009-11-01

A biosensor for rapid detection of bacterial count based on adenosine 5'-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin-luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10(3) to 10(8) colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n=22) within 5min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.

Membrane Lipids as Indicators for Viable Bacterial Communities Inhabiting Petroleum Systems.

PubMed

Gruner, Andrea; Mangelsdorf, Kai; Vieth-Hillebrand, Andrea; Horsfield, Brian; van der Kraan, Geert M; Köhler, Thomas; Janka, Christoph; Morris, Brandon E L; Wilkes, Heinz

2017-08-01

Microbial activity in petroleum reservoirs has been implicated in a suite of detrimental effects including deterioration of petroleum quality, increases in oil sulfur content, biofouling of steel pipelines and other infrastructures, and well plugging. Here, we present a biogeochemical approach, using phospholipid fatty acids (PLFAs), for detecting viable bacteria in petroleum systems. Variations within the bacterial community along water flow paths (producing well, topside facilities, and injection well) can be elucidated in the field using the same technique, as shown here within oil production plants in the Molasse Basin of Upper Austria. The abundance of PLFAs is compared to total cellular numbers, as detected by qPCR of the 16S rDNA gene, to give an overall comparison between the resolutions of both methods in a true field setting. Additionally, the influence of biocide applications on lipid- and DNA-based quantification was investigated. The first oil field, Trattnach, showed significant PLFA abundances and cell numbers within the reservoir and topside facilities. In contrast, the second field (Engenfeld) showed very low PLFA levels overall, likely due to continuous treatment of the topside facilities with a glutaraldehyde-based antimicrobial. In comparison, Trattnach is dosed once per week in a batch fashion. Changes within PLFA compositions across the flow path, throughout the petroleum production plants, point to cellular adaptation within the system and may be linked to shifts in the dominance of certain bacterial types in oil reservoirs versus topside facilities. Overall, PLFA-based monitoring provides a useful tool to assess the abundance and high-level taxonomic diversity of viable microbial populations in oil production wells, topside infrastructure, pipelines, and other related facilities.

Comparison of two suspension arrays for simultaneous detection of five biothreat bacterial in powder samples.

PubMed

Yang, Yu; Wang, Jing; Wen, Haiyan; Liu, Hengchuan

2012-01-01

We have developed novel Bio-Plex assays for simultaneous detection of Bacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis, and Burkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identify Bacillus anthracis, Yersinia pestis, and Brucella spp. at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detect Bacillus anthracis sterne spore and Yersinia pestis EV76 from mimic "write powder" samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

Adenosine Monophosphate-Based Detection of Bacterial Spores

NASA Technical Reports Server (NTRS)

Kern, Roger G.; Chen, Fei; Venkateswaran, Kasthuri; Hattori, Nori; Suzuki, Shigeya

2009-01-01

A method of rapid detection of bacterial spores is based on the discovery that a heat shock consisting of exposure to a temperature of 100 C for 10 minutes causes the complete release of adenosine monophosphate (AMP) from the spores. This method could be an alternative to the method described in the immediately preceding article. Unlike that method and related prior methods, the present method does not involve germination and cultivation; this feature is an important advantage because in cases in which the spores are those of pathogens, delays involved in germination and cultivation could increase risks of infection. Also, in comparison with other prior methods that do not involve germination, the present method affords greater sensitivity. At present, the method is embodied in a laboratory procedure, though it would be desirable to implement the method by means of a miniaturized apparatus in order to make it convenient and economical enough to encourage widespread use.

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.

Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion.

PubMed

Fällman, Erik; Schedin, Staffan; Jass, Jana; Andersson, Magnus; Uhlin, Bernt Eric; Axner, Ove

2004-06-15

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle's displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads.

Recognition of bacterial plant pathogens: local, systemic and transgenerational immunity.

PubMed

Henry, Elizabeth; Yadeta, Koste A; Coaker, Gitta

2013-09-01

Bacterial pathogens can cause multiple plant diseases and plants rely on their innate immune system to recognize and actively respond to these microbes. The plant innate immune system comprises extracellular pattern recognition receptors that recognize conserved microbial patterns and intracellular nucleotide binding leucine-rich repeat (NLR) proteins that recognize specific bacterial effectors delivered into host cells. Plants lack the adaptive immune branch present in animals, but still afford flexibility to pathogen attack through systemic and transgenerational resistance. Here, we focus on current research in plant immune responses against bacterial pathogens. Recent studies shed light onto the activation and inactivation of pattern recognition receptors and systemic acquired resistance. New research has also uncovered additional layers of complexity surrounding NLR immune receptor activation, cooperation and sub-cellular localizations. Taken together, these recent advances bring us closer to understanding the web of molecular interactions responsible for coordinating defense responses and ultimately resistance. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Bacterial uptake of antibiotics in model unsaturated systems

NASA Astrophysics Data System (ADS)

Zhang, W.; Chen, Z.; Zhang, Y.; Zhao, Z.; Wang, G.; Gao, Y.; Boyd, S. A.; Zhu, D.; Li, H.

2016-12-01

Anthropogenic antibiotics are ubiquitously present in the environment due to large uses in human medicine and animal agriculture, and are causing unintended consequence to human and ecosystem health. Bacterial uptake of antibiotics could exert selection pressure on antibiotic resistance development among bacteria population. Therefore, understanding environmental factors controlling bioavailability of antibiotics to bacteria is critical to better assessing exposure risks and developing mitigation strategies. Nonetheless, conventional bioavailability assays are often performed in water-saturated systems that do not represent unsaturated soils where most bacteria live, therefore neglecting soil water as a controlling factor in determining the extent of antibiotic bacterial uptake. Therefore, we propose to study bacterial uptake of antibiotics in model unsaturated systems using GFP-tagged Escherichia coli bioreporter for tetracyclines. Our preliminary studies demonstrated the important role of water content (or water matric potential) in determining the bioavailability of antibiotics, and complex interactions of water potential, tetracycline diffusion, and E. coli growth. Therefore, unsaturated processes are important for understanding antibiotic resistance development and developing mitigation strategies.

Bacterial wetwood detection in Fagus grandifolia and Prunus serotina sapwood using a conducting polymer electronic-nose device

Treesearch

A.D. Wilson

2014-01-01

New electronic gas-detection methods were developed and tested for the diagnosis of bacterial wetwood disease in Fagus grandifolia (American beech) and Prunus serotina (black cherry) using a Conducting Polymer (CP)-type electronic nose (e-nose), the Aromascan A32S, based on detection of headspace...

Comparison of individual and pooled sampling methods for detecting bacterial pathogens of fish

USGS Publications Warehouse

Mumford, Sonia; Patterson, Chris; Evered, J.; Brunson, Ray; Levine, J.; Winton, J.

2005-01-01

Examination of finfish populations for viral and bacterial pathogens is an important component of fish disease control programs worldwide. Two methods are commonly used for collecting tissue samples for bacteriological culture, the currently accepted standards for detection of bacterial fish pathogens. The method specified in the Office International des Epizooties Manual of Diagnostic Tests for Aquatic Animals permits combining renal and splenic tissues from as many as 5 fish into pooled samples. The American Fisheries Society (AFS) Blue Book/US Fish and Wildlife Service (USFWS) Inspection Manual specifies the use of a bacteriological loop for collecting samples from the kidney of individual fish. An alternative would be to more fully utilize the pooled samples taken for virology. If implemented, this approach would provide substantial savings in labor and materials. To compare the relative performance of the AFS/USFWS method and this alternative approach, cultures of Yersinia ruckeri were used to establish low-level infections in groups of rainbow trout (Oncorhynchus mykiss) that were sampled by both methods. Yersinia ruckeri was cultured from 22 of 37 groups by at least 1 method. The loop method yielded 18 positive groups, with 1 group positive in the loop samples but negative in the pooled samples. The pooled samples produced 21 positive groups, with 4 groups positive in the pooled samples but negative in the loop samples. There was statistically significant agreement (Spearman coefficient 0.80, P < 0.001) in the relative ability of the 2 sampling methods to permit detection of low-level bacterial infections of rainbow trout.

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

Detection of bacterial endospores by means of ultrafast coherent Raman spectroscopy

NASA Astrophysics Data System (ADS)

Pestov, Dmitry Sergeyevich

This work is devoted to formulation and development of a laser spectroscopic technique for rapid detection of biohazards, such as Bacillus anthracis spores. Coherent anti-Stokes Raman scattering (CARS) is used as an underlying process for active retrieval of species-specific characteristics of an analyte. Vibrational modes of constituent molecules are Raman-excited by a pair of ultrashort, femtosecond laser pulses, and then probed through inelastic scattering of a third, time-delayed laser field. We first employ the already known time-resolved CARS technique. We apply it to the spectroscopy of easy-to-handle methanol-water mixtures, and then continue building our expertise on solutions of dipicolinic acid (DPA) and its salts, which happen to be marker molecules for bacterial spores. Various acquisition schemes are evaluated, and the preference is given to multi-channel frequency-resolved detection, when the whole CARS spectrum is recorded as a function of the probe pulse delay. We demonstrate a simple detection algorithm that manages to differentiate DPA solution from common interferents. We investigate experimentally the advantages and disadvantages of near-resonant probing of the excited molecular coherence, and finally observe the indicative backscattered CARS signal from DPA and NaDPA powders. The possibility of selective Raman excitation via pulse shaping of the preparation pulses is also demonstrated. The analysis of time-resolved CARS experiments on powders and B. subtilis spores, a harmless surrogate for B. anthracis, facilitates the formulation of a new approach, where we take full advantage of the multi-channel frequency-resolved acquisition and spectrally discriminate the Raman-resonant CARS signal from the background due to other instantaneous four-wave mixing (FWM) processes. Using narrowband probing, we decrease the magnitude of the nonresonant FWM, which is further suppressed by the timing of the laser pulses. The devised technique, referred to as

Chemical polyglycosylation and nanolitre detection enables single-molecule recapitulation of bacterial sugar export

NASA Astrophysics Data System (ADS)

Kong, Lingbing; Almond, Andrew; Bayley, Hagan; Davis, Benjamin G.

2016-05-01

The outermost protective layer of both Gram-positive and Gram-negative bacteria is composed of bacterial capsular polysaccharides. Insights into the interactions between the capsular polysaccharide and its transporter and the mechanism of sugar export would not only increase our understanding of this key process, but would also help in the design of novel therapeutics to block capsular polysaccharide export. Here, we report a nanolitre detection system that makes use of the bilayer interface between two droplets, and we use this system to study single-molecule recapitulation of sugar export. A synthetic strategy of polyglycosylation based on tetrasaccharide monomers enables ready synthetic access to extended fragments of K30 oligosaccharides and polysaccharides. Examination of the interactions between the Escherichia coli sugar transporter Wza and very small amounts of fragments of the K30 capsular polysaccharide substrate reveal the translocation of smaller but not larger fragments. We also observe capture events that occur only on the intracellular side of Wza, which would complement coordinated feeding by adjunct biosynthetic machinery.

Direct molecular testing to assess the incidence of meningococcal and other bacterial causes of meningitis among persons reported with unspecified bacterial meningitis.

PubMed

Ramautar, Arianne E; Halse, Tanya A; Arakaki, Lola; Antwi, Mike; Del Rosso, Paula; Dorsinville, Marie; Nazarian, Elizabeth; Steiner-Sichel, Linda; Lee, Lillian; Dickinson, Michelle; Wroblewski, Danielle; Dumas, Nellie; Musser, Kimberlee; Isaac, Beth; Rakeman, Jennifer; Weiss, Don

2015-11-01

Confirmed and probable cases of invasive Neisseria meningitidis (Nm) infection are reportable in New York City. We conducted a study to identify Nm among culture-negative reports of bacterial and viral meningitis. During the study period, 262 reports of suspected meningitis were eligible. Cerebrospinal fluid (CSF) specimens from 138 patients were obtained for testing. No Nm cases were detected. Results from real-time polymerase chain reaction and 16S on CSF specimens were concordant with hospital microbiology findings in 80%; however, other pathogenic organisms were detected in 14 culture-negative specimens. New York City's surveillance system appears to be effective at capturing cases of Nm meningitis. Nucleic acid testing is useful for detecting the presence of bacterial DNA when antibiotic therapy precedes lumbar puncture or bacterial cultures are negative. It remains unanswered whether culture-negative cases of Nm bacteremia are being missed by reportable disease surveillance. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Cassette bacteria detection system. [for monitoring the sterility of regenerated water in spacecraft

NASA Technical Reports Server (NTRS)

1974-01-01

The design, fabrication, and testing of an automatic bacteria detection system, with a zero-g capability, based on the filter-capable approach, and intended for monitoring the sterility of regenerated water in spacecraft is discussed. The principle of detection is based on measuring the increase in chemiluminescence produced by the action of bacterial porphyrins on a luminol-hydrogen peroxide mixture. Viable organisms are detected by comparing the signal of an incubated water sample with an unincubated control. High signals for the incubated water sample indicate the presence of viable organisms.

Rapid and direct detection of Invivo kinetics of pathogenic bacterial infection from mouse blood and urine.

PubMed

Gopal, Judy; Lee, Chia-Hsun; Wu, Hui-Fen

2012-06-06

This study demonstrates the first use of matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) to trace the Invivo infection kinetics of the well known deadly pathogen Staphylococcus aureus in Swiss albino mice. The growth curve of the bacteria from the point of injection (200μL of bacterial suspension (10(8)cfu/mL)) into the mouse blood till mortality (death) was periodically analyzed using the plate counting method and MALDI-MS. Bacterial counts of 10(3)cfu/mL were observed in the log phase of the growth curve in the blood and 10(2)cfu/mL were observed in the urine samples. Death occurred in the log phase of the growth curve, where the bacterial counts showed steady increase. In other cases, the bacteria counts started decreasing after 48h and by 96h the bacteria got totally eliminated from the mouse and these mice survived. Direct MALDI-MS was not feasible for tracking the bacteria in the infected blood. However, ionic liquid 1-Butyl-3-methylimidazolium tetrafluoroborate was successful in enabling bacterial detection amidst the strong blood peaks. But, in the case of the urine analysis, it was observed that direct MALDI-MS was adequate to enable detection. The results obtained prove the efficacy of MALDI-MS for analyzing pathogenic bacteria in clinical samples. This article is part of a Special Issue entitled: Proteomics: The clinical link. Copyright © 2011 Elsevier B.V. All rights reserved.

Simultaneous amplification of two bacterial genes: more reliable method of Helicobacter pylori detection in microbial rich dental plaque samples.

PubMed

Chaudhry, Saima; Idrees, Muhammad; Izhar, Mateen; Butt, Arshad Kamal; Khan, Ayyaz Ali

2011-01-01

Polymerase Chain reaction (PCR) assay is considered superior to other methods for detection of Helicobacter pylori (H. pylori) in oral cavity; however, it also has limitations when sample under study is microbial rich dental plaque. The type of gene targeted and number of primers used for bacterial detection in dental plaque samples can have a significant effect on the results obtained as there are a number of closely related bacterial species residing in plaque biofilm. Also due to high recombination rate of H. pylori some of the genes might be down regulated or absent. The present study was conducted to determine the frequency of H. pylori colonization of dental plaque by simultaneously amplifying two genes of the bacterium. One hundred dental plaque specimens were collected from dyspeptic patients before their upper gastrointestinal endoscopy and presence of H. pylori was determined through PCR assay using primers targeting two different genes of the bacterium. Eighty-nine of the 100 samples were included in final analysis. With simultaneous amplification of two bacterial genes 51.6% of the dental plaque samples were positive for H. pylori while this prevalence increased to 73% when only one gene amplification was used for bacterial identification. Detection of H. pylori in dental plaque samples is more reliable when two genes of the bacterium are simultaneously amplified as compared to one gene amplification only.

Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems

PubMed Central

Shaw, Jennifer L. A.; Weyrich, Laura S.; Sawade, Emma; Drikas, Mary; Cooper, Alan J.

2015-01-01

Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. PMID:26162884

Bacterial DNA in water and dialysate: detection and significance for patient outcomes.

PubMed

Handelman, Garry J; Megdal, Peter A; Handelman, Samuel K

2009-01-01

The fluid used for hemodialysis may contain DNA fragments from bacteria, which could be harmful for patient outcomes. DNA fragments from bacteria, containing the nonmethylated CpG motif, can trigger inflammation through the monocyte and lymphocyte Toll-like receptor 9, and these DNA fragments have been observed in dialysate. The fragments may transfer across the dialyzer into the patient's bloodstream during hemodialysis treatment. During hemodiafiltration, the fragments would be introduced directly into the bloodstream. The DNA fragments may arise from biofilm in the pipes of the water system, from growth of bacteria in the water, or as contaminants in the bicarbonate and salt mixture used for preparation of dialysate. Current filtration methods, such as Diasafe filters, are not able to remove these fragments. It would be prudent to seek to reduce or eliminate these contaminants. However, the cost and effort of decreasing bacterial DNA content may ultimately require substantial facility improvements; we therefore need to fund research studies to determine if modifications to reduce bacterial DNA content are clinically warranted. This research will require methods to accurately determine the species of bacteria that contribute the DNA, since this information will allow the source to be established as biofilm, bicarbonate mixtures, or other problems in the dialysis system such as bacterial growth or leakage during water preparation. In this review, the evidence for bacterial DNA fragments will be examined and suggestions for further studies will be described.

Comparison of direct-plating and broth-enrichment culture methods for detection of potential bacterial pathogens in respiratory secretions.

PubMed

Kaur, Ravinder; Wischmeyer, Jareth; Morris, Matthew; Pichichero, Michael E

2017-11-01

We compared the recovery of potential respiratory bacterial pathogens and normal flora from nasopharyngeal specimens collected from children during health and at the onset of acute otitis media (AOM) by selective direct-plating and overnight broth-enrichment. Overall, 3442 nasal wash (NW) samples collected from young children were analysed from a 10-year prospective study. NWs were cultured by (1) direct-plating to TSAII/5 % sheep blood agar and chocolate agar plates and (2) overnight broth-enrichment in BacT/ALERT SA-broth followed by plating. Standard microbiology techniques were applied to identify three dominant respiratory bacterial pathogens: Streptococcus pneumoniae (Spn), Haemophilus influenzae (Hflu) and Moraxella catarrhalis (Mcat) as well as two common nasal flora, Staphylococcus aureus (SA) and alpha-haemolytic Streptococci (AHS).Results/Key findings. Direct-plating of NW resulted in isolation of Spn from 37.8 %, Hflu from 13.6 % and Mcat from 33.2 % of samples. In comparison, overnight broth-enrichment isolated fewer Spn (30.1 %), Hflu (6.2 %) and Mcat (16.2 %) (P<0.001-0.0001). Broth-enrichment resulted in significant increased isolation of SA (6.0 %) and AHS (30.1 %) (P<0.0001). Competition between bacterial species in broth when both species were detected by direct-plating was assessed, and it was found that SA and AHS out-competed other species during broth-enrichment when samples were collected from healthy children but not during AOM. In middle ear fluids (MEF) at the onset of AOM, broth-enrichment resulted in higher recovery of Spn (+10.4 %, P<0.001), Hflu (+4.4 %, P=0.39) and Mcat (+13.5 %, <0.001). Broth-enrichment significantly reduces the accurate detection of bacterial respiratory pathogens and increases identification of SA and AHS in NW. Broth-enrichment improves detection of bacterial respiratory pathogens in MEF samples.

The Yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.

PubMed

Siggers, Keri A; Lesser, Cammie F

2008-07-17

Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors.

Extraction of Total Nucleic Acids From Ticks for the Detection of Bacterial and Viral Pathogens

PubMed Central

Crowder, Chris D.; Rounds, Megan A.; Phillipson, Curtis A.; Picuri, John M.; Matthews, Heather E.; Halverson, Justina; Schutzer, Steven E.; Ecker, David J.; Eshoo, Mark W.

2010-01-01

Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot. PMID:20180313

Piezoelectric immunochip coated with thin films of bacterial cellulose nanocrystals for dengue detection.

PubMed

Pirich, Cleverton Luiz; de Freitas, Rilton Alves; Torresi, Roberto Manuel; Picheth, Guilherme Fadel; Sierakowski, Maria Rita

2017-06-15

Low-cost piezoelectric devices, such as simple frequency monitoring quartz crystal microbalance (QCM) devices, have good clinical utility as fast diagnostic tools for the detection of several diseases. However, unspecific antigen recognition, poor molecular probe adsorption and the need for sample dilution are still common drawbacks that hinder their use in routine diagnosis. In this work, piezoelectric sensors were previously coated with thin films of bacterial cellulose nanocrystals (CN) to provide a more sensitive and adapted interface for the attachment of monoclonal immunoglobulin G (IgGNS1) and to favor specific detection of non-structural protein 1 (NS1) of dengue fever. The assembly of the immunochip surface was analyzed by atomic force microscopy (AFM) and the NS1 detection was followed by quartz crystal microbalance with (QCM-D) and without energy dissipation monitoring (QCM). The CN surface was able to immobilize 2.30±0.5mgm -2 of IgGNS1, as confirmed by AFM topography and phase images along with QCM-D. The system was able to detect the NS1 protein in serum with only 10-fold dilution in the range of 0.01-10µgmL -1 by both QCM and QCM-D. The limits of detection of the two devices were 0.1μgmL -1 for QCM-D and 0.32μgmL -1 for QCM. As a result, QCM-D and QCM apparatuses can be used to follow NS1 recognition and have good potential for more sensitive, fast and/or less expensive diagnostic assays for dengue. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of bacterial communities of conventional and A-stage activated sludge systems

PubMed Central

Gonzalez-Martinez, Alejandro; Rodriguez-Sanchez, Alejandro; Lotti, Tommaso; Garcia-Ruiz, Maria-Jesus; Osorio, Francisco; Gonzalez-Lopez, Jesus; van Loosdrecht, Mark C. M.

2016-01-01

The bacterial community structure of 10 different wastewater treatment systems and their influents has been investigated through pyrosequencing, yielding a total of 283486 reads. These bioreactors had different technological configurations: conventional activated sludge (CAS) systems and very highly loaded A-stage systems. A-stage processes are proposed as the first step in an energy producing municipal wastewater treatment process. Pyrosequencing analysis indicated that bacterial community structure of all influents was similar. Also the bacterial community of all CAS bioreactors was similar. Bacterial community structure of A-stage bioreactors showed a more case-specific pattern. A core of genera was consistently found for all influents, all CAS bioreactors and all A-stage bioreactors, respectively, showing that different geographical locations in The Netherlands and Spain did not affect the functional bacterial communities in these technologies. The ecological roles of these bacteria were discussed. Influents and A-stage bioreactors shared several core genera, while none of these were shared with CAS bioreactors communities. This difference is thought to reside in the different operational conditions of the two technologies. This study shows that bacterial community structure of CAS and A-stage bioreactors are mostly driven by solids retention time (SRT) and hydraulic retention time (HRT), as suggested by multivariate redundancy analysis. PMID:26728449

Diagnostic Accuracy of FebriDx: A Rapid Test to Detect Immune Responses to Viral and Bacterial Upper Respiratory Infections.

PubMed

Self, Wesley H; Rosen, Jeffrey; Sharp, Stephan C; Filbin, Michael R; Hou, Peter C; Parekh, Amisha D; Kurz, Michael C; Shapiro, Nathan I

2017-10-07

C-reactive protein (CRP) and myxovirus resistance protein A (MxA) are associated with bacterial and viral infections, respectively. We conducted a prospective, multicenter, cross-sectional study of adults and children with febrile upper respiratory tract infections (URIs) to evaluate the diagnostic accuracy of a rapid CRP/MxA immunoassay to identify clinically significant bacterial infection with host response and acute pathogenic viral infection. The reference standard for classifying URI etiology was an algorithm that included throat bacterial culture, upper respiratory PCR for viral and atypical pathogens, procalcitonin, white blood cell count, and bandemia. The algorithm also allowed for physician override. Among 205 patients, 25 (12.2%) were classified as bacterial, 53 (25.9%) as viral, and 127 (62.0%) negative by the reference standard. For bacterial detection, agreement between FebriDx and the reference standard was 91.7%, with FebriDx having a sensitivity of 80% (95% CI: 59-93%), specificity of 93% (89-97%), positive predictive value (PPV) of 63% (45-79%), and a negative predictive value (NPV) of 97% (94-99%). For viral detection, agreement was 84%, with a sensitivity of 87% (75-95%), specificity of 83% (76-89%), PPV of 64% (63-75%), and NPV of 95% (90-98%). FebriDx may help to identify clinically significant immune responses associated with bacterial and viral URIs that are more likely to require clinical management or therapeutic intervention, and has potential to assist with antibiotic stewardship.

rmpM genosensor for detection of human brain bacterial meningitis in cerebrospinal fluid.

PubMed

Dash, Sandip Kumar; Sharma, Minakshi; Khare, Shashi; Kumar, Ashok

2013-09-01

Human brain bacterial meningitis is a life-threatening disease caused mainly by Neisseria meningitidis, lead to damage of the outer membrane covering (meninges) of brain or even death. The usual methods of diagnosis are either time-consuming or have some limitations. The specific rmpM (reduction-modifiable protein M) virulent gene based genosensor is more sensitive, specific, and can detect N. meningitidis directly from the patient cerebrospinal fluid in 30 min including 1-min response time. 5'-Thiol-labeled single-stranded DNA (ssDNA) probe was immobilized onto screen-printed gold electrode (SPGE) and hybridized with denatured (95 °C) single-stranded genomic DNA (ssG-DNA) for 10 min at 25 °C. The electrochemical response was measured by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance using redox indicators. The sensitivity of the genosensor was 9.5087 (μA/cm(2))/ng with DPV and limit of detection was 3 ng/6 μL ssG-DNA. The immobilization of the ssDNA probe and hybridization with ssG-DNA from N. meningitidis was characterized by atomic force microscopy and Fourier transform infrared spectroscopy. The rmpM genosensor was stable for 6 months at 4 °C with 10 % loss in initial DPV current. The advantage of rmpM genosensor is to detect bacterial meningitis simultaneously in multiple patients using SPGE array during an outbreak of the disease.

Differential resistance of drinking water bacterial populations to monochloramine disinfection.

PubMed

Chiao, Tzu-Hsin; Clancy, Tara M; Pinto, Ameet; Xi, Chuanwu; Raskin, Lutgarde

2014-04-01

The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations.

The Effect of Antibiotic Exposure and Specimen Volume on the Detection of Bacterial Pathogens in Children With Pneumonia.

PubMed

Driscoll, Amanda J; Deloria Knoll, Maria; Hammitt, Laura L; Baggett, Henry C; Brooks, W Abdullah; Feikin, Daniel R; Kotloff, Karen L; Levine, Orin S; Madhi, Shabir A; O'Brien, Katherine L; Scott, J Anthony G; Thea, Donald M; Howie, Stephen R C; Adrian, Peter V; Ahmed, Dilruba; DeLuca, Andrea N; Ebruke, Bernard E; Gitahi, Caroline; Higdon, Melissa M; Kaewpan, Anek; Karani, Angela; Karron, Ruth A; Mazumder, Razib; McLellan, Jessica; Moore, David P; Mwananyanda, Lawrence; Park, Daniel E; Prosperi, Christine; Rhodes, Julia; Saifullah, Md; Seidenberg, Phil; Sow, Samba O; Tamboura, Boubou; Zeger, Scott L; Murdoch, David R

2017-06-15

Antibiotic exposure and specimen volume are known to affect pathogen detection by culture. Here we assess their effects on bacterial pathogen detection by both culture and polymerase chain reaction (PCR) in children. PERCH (Pneumonia Etiology Research for Child Health) is a case-control study of pneumonia in children aged 1-59 months investigating pathogens in blood, nasopharyngeal/oropharyngeal (NP/OP) swabs, and induced sputum by culture and PCR. Antibiotic exposure was ascertained by serum bioassay, and for cases, by a record of antibiotic treatment prior to specimen collection. Inoculated blood culture bottles were weighed to estimate volume. Antibiotic exposure ranged by specimen type from 43.5% to 81.7% in 4223 cases and was detected in 2.3% of 4863 controls. Antibiotics were associated with a 45% reduction in blood culture yield and approximately 20% reduction in yield from induced sputum culture. Reduction in yield of Streptococcus pneumoniae from NP culture was approximately 30% in cases and approximately 32% in controls. Several bacteria had significant but marginal reductions (by 5%-7%) in detection by PCR in NP/OP swabs from both cases and controls, with the exception of S. pneumoniae in exposed controls, which was detected 25% less frequently compared to nonexposed controls. Bacterial detection in induced sputum by PCR decreased 7% for exposed compared to nonexposed cases. For every additional 1 mL of blood culture specimen collected, microbial yield increased 0.51% (95% confidence interval, 0.47%-0.54%), from 2% when volume was ≤1 mL to approximately 6% for ≥3 mL. Antibiotic exposure and blood culture volume affect detection of bacterial pathogens in children with pneumonia and should be accounted for in studies of etiology and in clinical management. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Pathogen espionage: multiple bacterial adrenergic sensors eavesdrop on host communication systems.

PubMed

Karavolos, Michail H; Winzer, Klaus; Williams, Paul; Khan, C M Anjam

2013-02-01

The interactions between bacterial pathogens and their eukaryotic hosts are vital in determining the outcome of infections. Bacterial pathogens employ molecular sensors to detect and facilitate adaptation to changes in their niche. The sensing of these extracellular signals enables the pathogen to navigate within mammalian hosts. Intercellular bacterial communication is facilitated by the production and sensing of autoinducer (AI) molecules via quorum sensing. More recently, AI-3 and the host neuroendocrine (NE) hormones adrenaline and noradrenaline were reported to display cross-talk for the activation of the same signalling pathways. Remarkably, there is increasing evidence to suggest that enteric bacteria sense and respond to the host NE stress hormones adrenaline and noradrenaline to modulate virulence. These responses can be inhibited by α and β-adrenergic receptor antagonists implying a bacterial receptor-based sensing and signalling cascade. In Escherichia coli O157:H7 and Salmonella, QseC has been proposed as the adrenergic receptor. Strikingly, there is an increasing body of evidence that not all the bacterial adrenergic responses require signalling through QseC. Here we provide additional hypotheses to reconcile these observations implicating the existence of alternative adrenergic receptors including BasS, QseE and CpxA and their associated signalling cascades with major roles in interkingdom communication. © 2012 Blackwell Publishing Ltd.

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.

Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform

PubMed Central

Renner, Lars D.; Zan, Jindong; Hu, Linda I.; Martinez, Manuel; Resto, Pedro J.; Siegel, Adam C.; Torres, Clint; Hall, Sara B.; Slezak, Tom R.

2016-01-01

ABSTRACT An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying

Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform.

PubMed

Renner, Lars D; Zan, Jindong; Hu, Linda I; Martinez, Manuel; Resto, Pedro J; Siegel, Adam C; Torres, Clint; Hall, Sara B; Slezak, Tom R; Nguyen, Tuan H; Weibel, Douglas B

2017-02-15

An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity

Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems.

PubMed

Shaw, Jennifer L A; Monis, Paul; Weyrich, Laura S; Sawade, Emma; Drikas, Mary; Cooper, Alan J

2015-09-01

Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Community acquired bacterial pneumonia: aetiology, laboratory detection and antibiotic susceptibility pattern.

PubMed

Akter, Sonia; Shamsuzzaman, S M; Jahan, Ferdush

2014-08-01

This cross sectional study was conducted to identify the common bacterial causes of community acquired pneumonia (CAP) from sputum and blood by culture and polymerase chain reaction (PCR) and to evaluate the effectiveness of these tests. A total of 105 sputum and blood samples were collected from patients with pneumonia on clinical suspicion. Common causative bacterial agents of pneumonia were detected by Gram staining, cultures, biochemical tests and PCR. Among 55 sputum culture positive cases, a majority (61.82%) of the patients were in the age group between 21-50 years and the ratio between male and female was 2.5:1. Most (61.90%) of the cases were from the lower socio-economic group. Out of 105 samples, 23 (37.12%) were positive by Gram stain, 29 (27.62%) yielded growth in culture media and 37 (35.24%) were positive by PCR for Streptococcus pneumoniae and Haemophilus influenzae. Streptococcus pneumoniae was the most common aetiological agent (19.05%) followed by Klebsiella pneumoniae (13.33%), Haemophilus influenzae (8.57%) and Pseudomonas aeruginosa (5.71%). Multiplex PCR is a useful technique for rapid diagnosis of bacterial causes of pneumonia directly from sputum and blood. Considering culture as a gold standard, the sensitivity of PCR was 96.55% and specificity was 88.15%. More than 80% of Streptococcus pneumoniae isolates were found to be sensitive to ampicillin, amoxycillinclavulanate, and ceftriaxone. Susceptibilities to other antimicrobials ranged from 65% for azithromycin to 70% for levofloxacin. On the other hand, the Gram negative organisms were more sensitive to meropenem, ceftriaxone, amoxycillin-clavulanate and amikacin.

Effect of semi-permeable cover system on the bacterial diversity during sewage sludge composting.

PubMed

Robledo-Mahón, Tatiana; Aranda, Elisabet; Pesciaroli, Chiara; Rodríguez-Calvo, Alfonso; Silva-Castro, Gloria Andrea; González-López, Jesús; Calvo, Concepción

2018-06-01

Sewage sludge composting is a profitable process economically viable and environmentally friendly. In despite of there are several kind of composting types, the use of combined system of semipermeable cover film and aeration air-floor is widely developed at industrial scale. However, the knowledge of the linkages between microbial communities structure, enzyme activities and physico-chemical factors under these conditions it has been poorly explored. Thus, the aim of this study was to investigate the bacterial dynamic and community structure using next generation sequencing coupled to analyses of microbial enzymatic activity and culturable dependent techniques in a full-scale real composting plant. Sewage sludge composting process was conducted using a semi-permeable Gore-tex cover, in combination with an air-insufflation system. The highest values of enzymatic activities such as dehydrogenase, protease and arylsulphatase were detected in the first 5 days of composting; suggesting that during this period of time a greater degrading activity of organic matter took place. Culturable bacteria identified were in agreement with the bacteria found by massive sequencing technologies. The greatest bacterial diversity was detected between days 15 and 30, with Actinomycetales and Bacillales being the predominant orders at the beginning and end of the process. Bacillus was the most representative genus during all the process. A strong correlation between abiotic factors as total organic content and organic matter and enzymatic activities such as dehydrogenase, alkaline phosphatase, and ß-glucosidase activity was found. Bacterial diversity was strongly influenced by the stage of the process, community-structure change was concomitant with a temperature rise, rendering favorable conditions to stimulate microbial activity and facilitate the change in the microbial community linked to the degradation process. Moreover, results obtained confirmed that the use of semipermeable

Newer systems for bacterial resistances to toxic heavy metals.

PubMed Central

Silver, S; Ji, G

1994-01-01

Bacterial plasmids contain specific genes for resistances to toxic heavy metal ions including Ag+, AsO2-, AsO4(3-), Cd2+, Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, and Zn2+. Recent progress with plasmid copper-resistance systems in Escherichia coli and Pseudomonas syringae show a system of four gene products, an inner membrane protein (PcoD), an outer membrane protein (PcoB), and two periplasmic Cu(2+)-binding proteins (PcoA and PcoC). Synthesis of this system is governed by two regulatory proteins (the membrane sensor PcoS and the soluble responder PcoR, probably a DNA-binding protein), homologous to other bacterial two-component regulatory systems. Chromosomally encoded Cu2+ P-type ATPases have recently been recognized in Enterococcus hirae and these are closely homologous to the bacterial cadmium efflux ATPase and the human copper-deficiency disease Menkes gene product. The Cd(2+)-efflux ATPase of gram-positive bacteria is a large P-type ATPase, homologous to the muscle Ca2+ ATPase and the Na+/K+ ATPases of animals. The arsenic-resistance system of gram-negative bacteria functions as an oxyanion efflux ATPase for arsenite and presumably antimonite. However, the structure of the arsenic ATPase is fundamentally different from that of P-type ATPases. The absence of the arsA gene (for the ATPase subunit) in gram-positive bacteria raises questions of energy-coupling for arsenite efflux. The ArsC protein product of the arsenic-resistance operons of both gram-positive and gram-negative bacteria is an intracellular enzyme that reduces arsenate [As(V)] to arsenite [As(III)], the substrate for the transport pump. Newly studied cation efflux systems for Cd2+, Zn2+, and Co2+ (Czc) or Co2+ and Ni2+ resistance (Cnr) lack ATPase motifs in their predicted polypeptide sequences. Therefore, not all plasmid-resistance systems that function through toxic ion efflux are ATPases. The first well-defined bacterial metallothionein was found in the cyanobacterium Synechococcus

Investigation of polymerase chain reaction assays to improve detection of bacterial involvement in bovine respiratory disease.

PubMed

Bell, Colin J; Blackburn, Paul; Elliott, Mark; Patterson, Tony I A P; Ellison, Sean; Lahuerta-Marin, Angela; Ball, Hywel J

2014-09-01

Bovine respiratory disease (BRD) causes severe economic losses to the cattle farming industry worldwide. The major bacterial organisms contributing to the BRD complex are Mannheimia haemolytica, Histophilus somni, Mycoplasma bovis, Pasteurella multocida, and Trueperella pyogenes. The postmortem detection of these organisms in pneumonic lung tissue is generally conducted using standard culture-based techniques where the presence of therapeutic antibiotics in the tissue can inhibit bacterial isolation. In the current study, conventional and real-time polymerase chain reaction (PCR) assays were used to assess the prevalence of these 5 organisms in grossly pneumonic lung samples from 150 animals submitted for postmortem examination, and the results were compared with those obtained using culture techniques. Mannheimia haemolytica was detected in 51 cases (34%) by PCR and in 33 cases (22%) by culture, H. somni was detected in 35 cases (23.3%) by PCR and in 6 cases (4%) by culture, Myc. bovis was detected in 53 cases (35.3%) by PCR and in 29 cases (19.3%) by culture, P. multocida was detected in 50 cases (33.3%) by PCR and in 31 cases (20.7%) by culture, and T. pyogenes was detected in 42 cases (28%) by PCR and in 31 cases (20.7%) by culture, with all differences being statistically significant. The PCR assays indicated positive results for 111 cases (74%) whereas 82 cases (54.6%) were culture positive. The PCR assays have demonstrated a significantly higher rate of detection of all 5 organisms in cases of pneumonia in cattle in Northern Ireland than was detected by current standard procedures. © 2014 The Author(s).

Harnessing CRISPR-Cas systems for bacterial genome editing.

PubMed

Selle, Kurt; Barrangou, Rodolphe

2015-04-01

Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

CRISPR technologies for bacterial systems: Current achievements and future directions.

PubMed

Choi, Kyeong Rok; Lee, Sang Yup

2016-11-15

Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial microbiology-metabolic engineering, synthetic biology, and other related disciplines-is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR technologies. Next, we describe CRISPR/Cas-derived technologies for bacterial strain development, including genome editing and gene expression regulation applications. Then, other CRISPR technologies possessing great potential for industrial applications are described, including typing and tracking of bacterial strains, virome identification, vaccination of bacteria, and advanced antimicrobial approaches. For each application, we note our suggestions for additional improvements as well. In the same context, replication of CRISPR/Cas-based chromosome imaging technologies developed originally in eukaryotic systems is introduced with its potential impact on studying bacterial chromosomal dynamics. Also, the current patent status of CRISPR technologies is reviewed. Finally, we provide some insights to the future of CRISPR technologies for bacterial systems by proposing complementary techniques to be developed for the use of CRISPR technologies in even wider range of applications. Copyright © 2016. Published by Elsevier Inc.

Electrically-receptive and thermally-responsive paper-based sensor chip for rapid detection of bacterial cells.

PubMed

Khan, Muhammad S; Misra, Santosh K; Dighe, Ketan; Wang, Zhen; Schwartz-Duval, Aaron S; Sar, Dinabandhu; Pan, Dipanjan

2018-07-01

Although significant technological advancements have been made in the development of analytical biosensor chips for detecting bacterial strains (E. coli, S. Mutans and B. Subtilis), critical requirements i.e. limit of detection (LOD), fast time of response, ultra-sensitivity with high reproducibility and good shelf-life with robust sensing capability have yet to be met within a single sensor chip. In order to achieve these criteria, we present an electrically-receptive thermally-responsive (ER-TR) sensor chip comprised of simple filter paper used as substrate coated with composite of poly(N-isopropylacrylamide) polymer (PNIPAm) - graphene nanoplatelet (GR) followed by evaporation of Au electrodes for capturing both Gram-positive (S. mutans and B. subtilis) and Gram-negative (E. coli) bacterial cells in real-time. Autoclave water, tap water, lake water and milk samples were tested with ER-TR chip with and without bacterial strains at varying concentration range 10 1 -10 5 cells/mL. The sensor was integrated with in-house built printed circuit board (PCB) to transmit/receive electrical signals. The interaction of E. coli, S. mutans and B. subtilis cells with fibers of PNIPAm-GR resulted in a change of electrical resistance and the readout was monitored wirelessly in real-time using MATLAB algorithm. Finally, prepared ER-TR chip exhibited the reproducibility of 85-97% with shelf-life of up to four weeks after testing with lake water sample. Copyright © 2018 Elsevier B.V. All rights reserved.

Targeting bacterial secretion systems: benefits of disarmament in the microcosm.

PubMed

Baron, Christian; Coombes, Brian

2007-03-01

Secretion systems are used by many bacterial pathogens for the delivery of virulence factors to the extracellular space or directly into host cells. They are attractive targets for the development of novel anti-virulence drugs as their inactivation would lead to pathogen attenuation or avirulence, followed by clearance of the bacteria by the immune system. This review will present the state of knowledge on the assembly and function of type II, type III and type IV secretion systems in Gram-negative bacteria focusing on insights provided by structural analyses of several key components. The suitability of transcription factors regulating the expression of secretion system components and of ATPases, lytic transglycosylases and protein assembly factors as drug targets will be discussed. Recent progress using innovative in vivo as well as in vitro screening strategies led to a first set of secretion system inhibitors with potential for further development as anti-infectives. The discovery of such inhibitors offers exciting and innovative opportunities to further develop these anti-virulence drugs into monotherapy or in combination with classical antibiotics. Bacterial growth per se would not be inhibited by such drugs so that the selection for mutations causing resistance could be reduced. Secretion system inhibitors may therefore avoid many of the problems associated with classical antibiotics and may constitute a valuable addition to our arsenal for the treatment of bacterial infections.

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

PubMed

Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

2017-06-01

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Bioinformatic Strategy for the Detection, Classification and Analysis of Bacterial Autotransporters

PubMed Central

Celik, Nermin; Webb, Chaille T.; Leyton, Denisse L.; Holt, Kathryn E.; Heinz, Eva; Gorrell, Rebecca; Kwok, Terry; Naderer, Thomas; Strugnell, Richard A.; Speed, Terence P.; Teasdale, Rohan D.; Likić, Vladimir A.; Lithgow, Trevor

2012-01-01

Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters. PMID:22905239

Bacterial diversity in water injection systems of Brazilian offshore oil platforms.

PubMed

Korenblum, Elisa; Valoni, Erika; Penna, Mônica; Seldin, Lucy

2010-01-01

Biogenic souring and microbial-influenced corrosion is a common scenario in water-flooded petroleum reservoirs. Water injection systems are continuously treated to control bacterial contamination, but some bacteria that cause souring and corrosion can persist even after different treatments have been applied. Our aim was to increase our knowledge of the bacterial communities that persist in the water injection systems of three offshore oil platforms in Brazil. To achieve this goal, we used a culture-independent molecular approach (16S ribosomal RNA gene clone libraries) to analyze seawater samples that had been subjected to different treatments. Phylogenetic analyses revealed that the bacterial communities from the different platforms were taxonomically different. A predominance of bacterial clones affiliated with Gammaproteobacteria, mostly belonging to the genus Marinobacter (60.7%), were observed in the platform A samples. Clones from platform B were mainly related to the genera Colwellia (37.9%) and Achromobacter (24.6%), whereas clones obtained from platform C were all related to unclassified bacteria. Canonical correspondence analyses showed that different treatments such as chlorination, deoxygenation, and biocide addition did not significantly influence the bacterial diversity in the platforms studied. Our results demonstrated that the injection water used in secondary oil recovery procedures contained potentially hazardous bacteria, which may ultimately cause souring and corrosion.

Detecting Lyme disease using antibody-functionalized carbon nanotubes

NASA Astrophysics Data System (ADS)

Dailey, Jennifer; Lerner, Mitchell; Goldsmith, Brett; Brisson, Dustin; Johnson, A. T. Charlie

2011-03-01

We combine antibodies for Lyme flagellar protein with carbon nanotube transistors to create an electronic sensor capable of definitive detection of Lyme disease. Over 35,000 cases of Lyme disease are reported in the United States each year, of which more than 23 percent are originally misdiagnosed. Rational design of the coupling of the biological system to the electronic system gives us a flexible sensor platform which we can apply to several biological systems. By coupling these antibodies to carbon nanotubes in particular, we allow for fast, sensitive, highly selective, electronic detection. Unlike antibody or biomarker detection, bacterial protein detection leads to positive identification of both early and late stage bacterial infections, and is easily expandable to environmental monitoring.

Lack of Accuracy of Body Temperature for Detecting Serious Bacterial Infection in Febrile Episodes.

PubMed

De, Sukanya; Williams, Gabrielle J; Teixeira-Pinto, Armando; Macaskill, Petra; McCaskill, Mary; Isaacs, David; Craig, Jonathan C

2015-09-01

Body temperature is a time-honored marker of serious bacterial infection, but there are few studies of its test performance. The aim of our study was to determine the accuracy of temperature measured on presentation to medical care for detecting serious bacterial infection. Febrile children 0-5 years of age presenting to the emergency department of a tertiary care pediatric hospital were sampled consecutively. The accuracy of the axillary temperature measured at presentation was evaluated using logistic regression models to generate receiver operating characteristic curves. Reference standard tests for serious bacterial infection were standard microbiologic/radiologic tests and clinical follow-up. Age, clinicians' impression of appearance of the child (well versus unwell) and duration of illness were assessed as possible effect modifiers. Of 15,781 illness episodes 1120 (7.1%) had serious bacterial infection. The area under the receiver operating characteristic curve for temperature was 0.60 [95% confidence intervals (CI): 0.58-0.62]. A threshold of ≥ 38°C had a sensitivity of 0.67 (95% CI: 0.64-0.70), specificity of 0.45 (95% CI: 0.44-0.46), positive likelihood ratio of 1.2 (95% CI: 1.2-1.3) and negative likelihood ratio of 0.7 (95% CI: 0.7-0.8). Age and illness duration had a small but significant effect on the accuracy of temperature increasing its "rule-in" potential. Measured temperature at presentation to hospital is not an accurate marker of serious bacterial infection in febrile children. Younger age and longer duration of illness increase the rule-in potential of temperature but without substantial overall change in its test accuracy.

Recent Developments in Antibody-Based Assays for the Detection of Bacterial Toxins

PubMed Central

Zhu, Kui; Dietrich, Richard; Didier, Andrea; Doyscher, Dominik; Märtlbauer, Erwin

2014-01-01

Considering the urgent demand for rapid and accurate determination of bacterial toxins and the recent promising developments in nanotechnology and microfluidics, this review summarizes new achievements of the past five years. Firstly, bacterial toxins will be categorized according to their antibody binding properties into low and high molecular weight compounds. Secondly, the types of antibodies and new techniques for producing antibodies are discussed, including poly- and mono-clonal antibodies, single-chain variable fragments (scFv), as well as heavy-chain and recombinant antibodies. Thirdly, the use of different nanomaterials, such as gold nanoparticles (AuNPs), magnetic nanoparticles (MNPs), quantum dots (QDs) and carbon nanomaterials (graphene and carbon nanotube), for labeling antibodies and toxins or for readout techniques will be summarized. Fourthly, microscale analysis or minimized devices, for example microfluidics or lab-on-a-chip (LOC), which have attracted increasing attention in combination with immunoassays for the robust detection or point-of-care testing (POCT), will be reviewed. Finally, some new materials and analytical strategies, which might be promising for analyzing toxins in the near future, will be shortly introduced. PMID:24732203

Impact of water quality on the bacterial populations and off-flavours in recirculating aquaculture systems.

PubMed

Auffret, Marc; Yergeau, Étienne; Pilote, Alexandre; Proulx, Émilie; Proulx, Daniel; Greer, Charles W; Vandenberg, Grant; Villemur, Richard

2013-05-01

A variety of factors affecting water quality in recirculating aquaculture systems (RAS) are associated with the occurrence of off-flavours. In this study, we report the impact of water quality on the bacterial diversity and the occurrence of the geosmin-synthesis gene (geoA) in two RAS units operated for 252 days. Unit 2 displayed a higher level of turbidity and phosphate, which affected the fresh water quality compared with unit 1. In the biofilter, nitrification is one of the major processes by which high water quality is maintained. The bacterial population observed in the unit 1 biofilter was more stable throughout the experiment, with a higher level of nitrifying bacteria compared with the unit 2 biofilter. Geosmin appeared in fish flesh after 84 days in unit 2, whereas it appeared in unit 1 after 168 days, but at a much lower level. The geoA gene was detected in both units, 28 days prior to the detection of geosmin in fish flesh. In addition, we detected sequences associated with Sorangium and Nannocystis (Myxococcales): members of these genera are known to produce geosmin. These sequences were observed at an earlier time in unit 2 and at a higher level than in unit 1. This study confirms the advantages of new molecular methods to understand the occurrence of geosmin production in RAS. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation.

PubMed

Daeffler, Kristina N-M; Galley, Jeffrey D; Sheth, Ravi U; Ortiz-Velez, Laura C; Bibb, Christopher O; Shroyer, Noah F; Britton, Robert A; Tabor, Jeffrey J

2017-04-03

There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two-component systems from marine Shewanella species, and validate them in laboratory Escherichia coli Then, we port these sensors into a gut-adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Analysis of bacterial metagenomes from the Southwestern Gulf of Mexico for pathogens detection.

PubMed

Escobedo-Hinojosa, Wendy; Pardo-López, Liliana

2017-07-31

Little is known about the diversity of bacteria in the Southwestern Gulf of Mexico. The aim of the study illustrated in this perspective was to search for the presence of bacterial pathogens in this ecosystem, using metagenomic data recently generated by the Mexican research group known as the Gulf of Mexico Research Consortium. Several genera of bacteria annotated as pathogens were detected in water and sediment marine samples. As expected, native and ubiquitous pathogenic bacteria genera such as Burkolderia, Halomonas, Pseudomonas, Shewanella and Vibrio were highly represented. Surprisingly, non-native genera of public health concern were also detected, including Borrelia, Ehrlichia, Leptospira, Mycobacterium, Mycoplasma, Salmonella, Staphylococcus, Streptococcus and Treponema. While there are no previous metagenomics studies of this environment, the potential influences of natural, anthropogenic and ecological factors on the diversity of putative pathogenic bacteria found in it are reviewed. The taxonomic annotation herein reported provides a starting point for an improved understanding of bacterial biodiversity in the Southwestern Gulf of Mexico. It also represents a useful tool in public health as it may help identify infectious diseases associated with exposure to marine water and ingestion of fish or shellfish, and thus may be useful in predicting and preventing waterborne disease outbreaks. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Applications of CRISPR/Cas System to Bacterial Metabolic Engineering.

PubMed

Cho, Suhyung; Shin, Jongoh; Cho, Byung-Kwan

2018-04-05

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli . Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics.

Species Specific Bacterial Spore Detection Using Lateral-Flow Immunoassay with DPA-Triggered Tb Luminescence

NASA Technical Reports Server (NTRS)

Ponce, Adrian

2003-01-01

A method of detecting bacterial spores incorporates (1) A method of lateral-flow immunoassay in combination with (2) A method based on the luminescence of Tb3+ ions to which molecules of dipicolinic acid (DPA) released from the spores have become bound. The present combination of lateral-flow immunoassay and DPA-triggered Tb luminescence was developed as a superior alternative to a prior lateral-flow immunoassay method in which detection involves the visual observation and/or measurement of red light scattered from colloidal gold nanoparticles. The advantage of the present combination method is that it affords both (1) High selectivity for spores of the species of bacteria that one seeks to detect (a characteristic of lateral-flow immunoassay in general) and (2) Detection sensitivity much greater (by virtue of the use of DPA-triggered Tb luminescence instead of gold nanoparticles) than that of the prior lateral-flow immunoassay method

Detection of bacterial growth by gas absorption.

PubMed

Waters, J R

1992-05-01

When 24 different aerobic organisms were grown in a shaken culture, all were found to first absorb gas from the headspace. In a rudimentary medium, such as tryptic soy broth, 16 of the 24 organisms did not produce gas following the initial gas absorption. We have developed a simple, noninvasive method for detecting both gas absorption and production in multiple culture vials. The time to positivity was compared with that obtained by the BACTEC 460 blood culture system. For nearly all of these organisms, there was no difference. For some of those organisms that did not produce gas, e.g. Staphylococcus epidermidis, Moraxella osloensis, and Neisseria meningitidis, detection by gas absorption was a few hours faster. Gas absorption appears to be a promising technique for a new automated blood culture system because of its simplicity and because medium without special additives can be used to detect organisms that do not produce gas.

The diagnostic value of CRP, IL-8, PCT, and sTREM-1 in the detection of bacterial infections in pediatric oncology patients with febrile neutropenia.

PubMed

Miedema, Karin G E; de Bont, Eveline S J M; Elferink, Rob F M Oude; van Vliet, Michel J; Nijhuis, Claudi S M Oude; Kamps, Willem A; Tissing, Wim J E

2011-10-01

In this study, we evaluated C-reactive protein (CRP), interleukin (IL)-8, procalcitonin (PCT), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as predictors for bacterial infection in febrile neutropenia, plus their usefulness in febrile neutropenia during chemotherapy-induced gastrointestinal mucositis. Plasma was obtained from pediatric oncology patients at presentation with febrile neutropenia (n = 43) and 24-48 h later (n = 17). The patients were classified as having or not having a bacterial infection. Plasma was also obtained of patients in the absence and in the presence of mucositis (n = 26). At presentation with febrile neutropenia, median IL-8 and PCT levels were significantly increased in patients with a bacterial infection, in contrast to CRP and sTREM-1. IL-8 was the most sensitive marker for the early detection of bacterial infection, in combination with clinical parameters or PCT the sensitivity reached 100%. After 24-48 h, only PCT was significantly elevated during bacterial infection. IL-8 levels were significantly increased during mucositis. Mucositis did not cause considerable changes in PCT levels. IL-8 is the most useful marker for the early detection of bacterial infections, compared with CRP, PCT, and sTREM-1. IL-8 in combination with clinical parameters or PCT might be even more useful. Gastrointestinal mucositis alone does not affect PCT levels, in contrast to IL-8 levels, and therefore, PCT might be more useful for the detection of bacterial infections during mucositis than IL-8.

Detection of bacterial contamination in platelet concentrates by a sensitive flow cytometric assay (BactiFlow): a multicentre validation study.

PubMed

Vollmer, T; Dreier, J; Schottstedt, V; Bux, J; Tapernon, K; Sibrowski, W; Kleesiek, K; Knabbe, C

2012-08-01

Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk. As a result of septic complications, particularly observed with older PCs, the shelf life of PCs has been reduced in Germany to 4 days. In this study, bacterial screening of PCs by BactiFlow (BF) flow cytometry was introduced in three German blood services to evaluate the robustness and applicability of the assay. Results were used to discuss the potential for the extension of PC shelf life to 5 days. A total of 1956 PCs were tested on days 4 or 5+ after PC production using the BF, whereas the BacT/Alert culture system served as reference method. Two PCs were confirmed positive by culture only and were identified as Propionibacterium acnes and Staphylococcus species. Two PCs were confirmed positive for Streptococcus mitis by BF and culture. Additionally, two PCs were culture-positive only in one culture bottle (aerobic: S. mitis and anaerobic: S. hominis). Retrospective analysis of bacterial growth kinetics provide the indication that corresponding bacterial titres were most likely below the BF analytical detection limit (<150 CFU mL(-1) ) and had probably no transfusion relevance. All remaining specimens were tested negative. Testing of PCs by BF was successfully implemented. The BF proved sufficient as a rapid screening method to improve PC safety. This study further provides data supporting the extension of PC shelf life to 5 days after negative BF testing on day 4. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.

A system for the rapid detection of bacterial contamination in cell-based therapeutica

NASA Astrophysics Data System (ADS)

Bolwien, Carsten; Erhardt, Christian; Sulz, Gerd; Thielecke, Hagen; Johann, Robert; Pudlas, Marieke; Mertsching, Heike; Koch, Steffen

2010-02-01

Monitoring the sterility of cell or tissue cultures is of major concern, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. Our sterility-control system is based on a Raman micro-spectrometer and is able to perform fast sterility testing on microliters of liquid samples. In conventional sterility control, samples are incubated for weeks to proliferate the contaminants to concentrations above the detection limit of conventional analysis. By contrast, our system filters particles from the liquid sample. The filter chip fabricated in microsystem technology comprises a silicon nitride membrane with millions of sub-micrometer holes to retain particles of critical sizes and is embedded in a microfluidic cell specially suited for concomitant microscopic observation. After filtration, identification is carried out on the single particle level: image processing detects possible contaminants and prepares them for Raman spectroscopic analysis. A custom-built Raman-spectrometer-attachment coupled to the commercial microscope uses 532nm or 785nm Raman excitation and records spectra up to 3400cm-1. In the final step, the recorded spectrum of a single particle is compared to an extensive library of GMP-relevant organisms, and classification is carried out based on a support vector machine.

Agreement between microscopic examination and bacterial culture of bile samples for detection of bactibilia in dogs and cats with hepatobiliary disease.

PubMed

Pashmakova, Medora B; Piccione, Julie; Bishop, Micah A; Nelson, Whitney R; Lawhon, Sara D

2017-05-01

OBJECTIVE To evaluate the agreement between results of microscopic examination and bacterial culture of bile samples from dogs and cats with hepatobiliary disease for detection of bactibilia. DESIGN Cross-sectional study. ANIMALS 31 dogs and 21 cats with hepatobiliary disease for which subsequent microscopic examination and bacterial culture of bile samples was performed from 2004 through 2014. PROCEDURES Electronic medical records of included dogs and cats were reviewed to extract data regarding diagnosis, antimicrobials administered, and results of microscopic examination and bacterial culture of bile samples. Agreement between these 2 diagnostic tests was assessed by calculation of the Cohen κ value. RESULTS 17 (33%) dogs and cats had bactibilia identified by microscopic examination of bile samples, and 11 (21%) had bactibilia identified via bacterial culture. Agreement between these 2 tests was substantial (percentage agreement [positive and negative results], 85%; κ = 0.62; 95% confidence interval, 0.38 to 0.89) and improved to almost perfect when calculated for only animals that received no antimicrobials within 24 hours prior to sample collection (percentage agreement, 94%; κ = 0.84; 95% confidence interval, 0.61 to 1.00). CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that agreement between microscopic examination and bacterial culture of bile samples for detection of bactibilia is optimized when dogs and cats are not receiving antimicrobials at the time of sample collection. Concurrent bacterial culture and microscopic examination of bile samples are recommended for all cats and dogs evaluated for hepatobiliary disease.

Cone-morse implant connection system significantly reduces bacterial leakage between implant and abutment: an in vitro study.

PubMed

Baj, A; Bolzoni, A; Russillo, A; Lauritano, D; Palmieri, A; Cura, F; Silvestre, F J; Giannì, A B

2017-01-01

Osseointegrated implants are very popular dental treatments today in the world. In osseointegrated implants, the occlusal forces are transmitted from prosthesis through an abutment to a dental implant. The abutment is connected to the implant by mean of a screw. A screw is the most used mean for connecting an implant to an abutment. Frequently the screws break and are lost. There is an alternative to screw retained abutment systems: the cone-morse connection (CMC). The CMC, thanks to the absence of the abutment screw, guarantees no micro-gaps, no micro-movements, and a reduction of bacterial leakage between implant and abutment. As P. gingivalis and T. forsythia penetration might have clinical relevance, it was the purpose of this investigation to evaluate molecular leakage of these two bacteria in a new CMC implants systems (Leone Spa®, Florence, Italy). To identify the capability of the implant to protect the internal space from the external environment, the passage of genetically modified Escherichia coli across implant-abutment interface was evaluated. Four cone-morse Leone implants (Leone® Spa, Florence, Italy) were immerged in a bacterial culture for 24 h and bacteria amount was then measured inside implant-abutment interface with Real-time PCR. Bacteria were detected inside all studied implants, with a median percentage of 3% for P. gingivalis and 4% for T. forsythia. Cone-morse connection implant system has very low bacterial leakage percentage and is similar to one-piece implants.

Simultaneous Detection of 13 Key Bacterial Respiratory Pathogens by Combination of Multiplex PCR and Capillary Electrophoresis.

PubMed

Jiang, Lu Xi; Ren, Hong Yu; Zhou, Hai Jian; Zhao, Si Hong; Hou, Bo Yan; Yan, Jian Ping; Qin, Tian; Chen, Yu

2017-08-01

Lower respiratory tract infections continue to pose a significant threat to human health. It is important to accurately and rapidly detect respiratory bacteria. To compensate for the limits of current respiratory bacteria detection methods, we developed a combination of multiplex polymerase chain reaction (PCR) and capillary electrophoresis (MPCE) assay to detect thirteen bacterial pathogens responsible for lower respiratory tract infections, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Mycoplasma pneumoniae, Legionella spp., Bordetella pertussis, Mycobacterium tuberculosis complex, Corynebacterium diphtheriae, and Streptococcus pyogenes. Three multiplex PCR reactions were built, and the products were analyzed by capillary electrophoresis using the high-throughput DNA analyzer. The specificity of the MPCE assay was examined and the detection limit was evaluated using DNA samples from each bacterial strain and the simulative samples of each strain. This assay was further evaluated using 152 clinical specimens and compared with real-time PCR reactions. For this assay, three nested-multiplex-PCRs were used to detect these clinical specimens. The detection limits of the MPCE assay for the 13 pathogens were very low and ranged from 10-7 to 10-2 ng/μL. Furthermore, analysis of the 152 clinical specimens yielded a specificity ranging from 96.5%-100.0%, and a sensitivity of 100.0% for the 13 pathogens. This study revealed that the MPCE assay is a rapid, reliable, and high-throughput method with high specificity and sensitivity. This assay has great potential in the molecular epidemiological survey of respiratory pathogens. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene

PubMed Central

Tunney, Michael M.; Patrick, Sheila; Curran, Martin D.; Ramage, Gordon; Hanna, Donna; Nixon, James R.; Gorman, Sean P.; Davis, Richard I.; Anderson, Neil

1999-01-01

In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for Propionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results. PMID:10488193

Bacterial community of iron tubercles from a drinking water distribution system and its occurrence in stagnant tap water.

PubMed

Chen, Lu; Jia, Rui-Bao; Li, Li

2013-07-01

Bacteria in drinking water distribution systems can cause deterioration of the water quality, and the microbial quality of tap water is closely related to consumer health. In the present study, the potential effects of bacteria attached to cast iron pipes on tap water in a distribution system were investigated. Comparison of the bacterial community composition of pipe tubercles with that of stagnant tap water samples based on a denaturing gradient gel electrophoresis analysis of the 16S rRNA gene revealed that the communities were related. Specifically, the main bacterial members were identical to each other. The bacterial community was found to be dominated by Firmicutes, Actinobacteria, and Proteobacteria, which included Rhizobium, Pseudomonas, Lactococcus, Brevundimonas, Rheinheimera, Arthrobacter, Bacillus, and Herbaspirillum. Heterotrophic bacteria proliferation was observed during the period of stagnation, followed by a decrease of assimilable organic carbon and a slight increase of microbially available phosphorus. These findings indicated that the regrowth of bacteria might be boosted by the release of nutrients such as phosphorus from the pipe walls, as well as the decline of residual chlorine during stagnation. Inorganic contaminants at low levels, including Al, Mn, Zn, Pb, Cr, Cu, and Ni, were detected in tubercles and were concentrated in particulates from tap water following the release of iron during stagnation.

[Investigation of bacterial and viral etiology in community acquired central nervous system infections with molecular methods].

PubMed

Kahraman, Hasip; Tünger, Alper; Şenol, Şebnem; Gazi, Hörü; Avcı, Meltem; Örmen, Bahar; Türker, Nesrin; Atalay, Sabri; Köse, Şükran; Ulusoy, Sercan; Işıkgöz Taşbakan, Meltem; Sipahi, Oğuz Reşat; Yamazhan, Tansu; Gülay, Zeynep; Alp Çavuş, Sema; Pullukçu, Hüsnü

2017-07-01

In this multicenter prospective cohort study, it was aimed to evaluate the bacterial and viral etiology in community-acquired central nervous system infections by standart bacteriological culture and multiplex polymerase chain reaction (PCR) methods. Patients hospitalized with central nervous system infections between April 2012 and February 2014 were enrolled in the study. Demographic and clinical information of the patients were collected prospectively. Cerebrospinal fluid (CSF) samples of the patients were examined by standart bacteriological culture methods, bacterial multiplex PCR (Seeplex meningitis-B ACE Detection (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, Group B streptococci) and viral multiplex PCR (Seeplex meningitis-V1 ACE Detection kits herpes simplex virus-1 (HSV1), herpes simplex virus-2 (HSV2), varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein Barr virus (EBV) and human herpes virus 6 (HHV6)) (Seeplex meningitis-V2 ACE Detection kit (enteroviruses)). Patients were classified as purulent meningitis, aseptic meningitis and encephalitis according to their clinical, CSF (leukocyte level, predominant cell type, protein and glucose (blood/CSF) levels) and cranial imaging results. Patients who were infected with a pathogen other than the detection of the kit or diagnosed as chronic meningitis and other diseases during the follow up, were excluded from the study. A total of 79 patients (28 female, 51 male, aged 42.1 ± 18.5) fulfilled the study inclusion criteria. A total of 46 patients were classified in purulent meningitis group whereas 33 were in aseptic meningitis/encephalitis group. Pathogens were detected by multiplex PCR in 41 patients. CSF cultures were positive in 10 (21.7%) patients (nine S.pneumoniae, one H.influenzae) and PCR were positive for 27 (58.6%) patients in purulent meningitis group. In this group one type of bacteria were detected in 18 patients (14 S.pneumoniae, two N

Simultaneous multicolor detection system of the single-molecular microbial antigen by total internal reflection fluorescence microscopy with fluorescent nanocrystal quantum dots

NASA Astrophysics Data System (ADS)

Hoshino, Akiyoshi; Fujioka, Kouki; Yamamoto, Mayu; Manabe, Noriyoshi; Yasuhara, Masato; Suzuki, Kazuo; Yamamoto, Kenji

2005-11-01

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

Changes in bacterial composition of biofilm in a metropolitan drinking water distribution system.

PubMed

Revetta, R P; Gomez-Alvarez, V; Gerke, T L; Santo Domingo, J W; Ashbolt, N J

2016-07-01

This study examined the development of bacterial biofilms within a metropolitan distribution system. The distribution system is fed with different source water (i.e. groundwater, GW and surface water, SW) and undergoes different treatment processes in separate facilities. The biofilm community was characterized using 16S rRNA gene clone libraries and functional potential analysis, generated from total DNA extracted from coupons in biofilm annular reactors fed with onsite drinking water for up to 18 months. Differences in the bacterial community structure were observed between GW and SW. Representatives that explained the dissimilarity were associated with the classes Betaproteobacteria, Alphaproteobacteria, Actinobacteria, Gammaproteobacteria and Firmicutes. After 9 months the biofilm bacterial community from both GW and SW were dominated by Mycobacterium species. The distribution of the dominant operational taxonomic unit (OTU) (Mycobacterium) positively correlated with the drinking water distribution system (DWDS) temperature. In this study, the biofilm community structure observed between GW and SW were dissimilar, while communities from different locations receiving SW did not show significant differences. The results suggest that source water and/or the water quality shaped by their respective treatment processes may play an important role in shaping the bacterial communities in the distribution system. In addition, several bacterial groups were present in all samples, suggesting that they are an integral part of the core microbiota of this DWDS. These results provide an ecological insight into biofilm bacterial structure in chlorine-treated drinking water influenced by different water sources and their respective treatment processes. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

PubMed Central

Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

2015-01-01

The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. PMID:25628612

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens.

PubMed

Higgins, Owen; Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert; Smith, Terry J

2018-02-09

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae , Neisseria meningitidis and Haemophilus influenzae . Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae , N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology.

Biomimetic/Optical Sensors for Detecting Bacterial Species

NASA Technical Reports Server (NTRS)

Homer, Margie; Ksendzov, Alexander; Yen, Shiao-Pin; Ryan, Margaret; Lazazzera, Beth

2006-01-01

Biomimetic/optical sensors have been proposed as means of real-time detection of bacteria in liquid samples through real-time detection of compounds secreted by the bacteria. Bacterial species of interest would be identified through detection of signaling compounds unique to those species. The best-characterized examples of quorum-signaling compounds are acyl-homoserine lactones and peptides. Each compound, secreted by each bacterium of an affected species, serves as a signal to other bacteria of the same species to engage in a collective behavior when the population density of that species reaches a threshold level analogous to a quorum. A sensor according to the proposal would include a specially formulated biomimetic film, made of a molecularly imprinted polymer (MIP), that would respond optically to the signaling compound of interest. The MIP film would be integrated directly onto an opticalwaveguide- based ring resonator for optical readout. Optically, the sensor would resemble the one described in Chemical Sensors Based on Optical Ring Resonators (NPO-40601), NASA Tech Briefs, Vol. 29, No. 10 (October 2005), page 32. MIPs have been used before as molecular- recognition compounds, though not in the manner of the present proposal. Molecular imprinting is an approach to making molecularly selective cavities in a polymer matrix. These cavities function much as enzyme receptor sites: the chemical functionality and shape of a cavity in the polymer matrix cause the cavity to bind to specific molecules. An MIP matrix is made by polymerizing monomers in the presence of the compound of interest (template molecule). The polymer forms around the template. After the polymer solidifies, the template molecules are removed from the polymer matrix by decomplexing them from their binding sites and then dissolving them, leaving cavities that are matched to the template molecules in size, shape, and chemical functionality. The cavities thus become molecular-recognition sites

Development of a panel of recombinase polymerase amplification assays for detection of common bacterial urinary tract infection pathogens.

PubMed

Raja, B; Goux, H J; Marapadaga, A; Rajagopalan, S; Kourentzi, K; Willson, R C

2017-08-01

To develop and evaluate the performance of a panel of isothermal real-time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens. The panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Enterococcus faecalis. All five RPAs required reaction times of under 12 min to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross-react with high concentrations of nontarget bacterial genomic DNA. In a 50-sample retrospective clinical study, the five-RPA assay panel was found to have a specificity of 100% (95% CI, 78-100%) and a sensitivity of 89% (95% CI, 75-96%) for UTI detection. The analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established. Rapid identification of the causative pathogens of UTIs can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture's 48-72-h turnaround time. The routine and widespread use of RPA to supplement or replace culture-based methods could profoundly impact UTI management and the emergence of multidrug-resistant pathogens. © 2017 The Society for Applied Microbiology.

Ultraviolet micro-Raman spectrograph for the detection of small numbers of bacterial cells

NASA Astrophysics Data System (ADS)

Chadha, S.; Nelson, W. H.; Sperry, J. F.

1993-11-01

The construction of a practical UV micro-Raman spectrograph capable of selective excitation of bacterial cells and other microscopic samples has been described. A reflective objective is used to focus cw laser light on a sample and at the same time collect the scattered light at 180°. With the aid of a quartz lens the image produced is focused on the slits of a spectrograph equipped with a single 2400 grooves/mm grating optimized for 250 nm. Spectra were detected by means of a blue-intensified diode array detector. Resonance Raman spectra of Bacillus subtilis and Flavobacterium capsulatum excited by the 257.2 nm output of a cw laser were recorded in the 900-1800 cm-1 region. Bacterial cells were immobilized on a quartz plate by means of polylysine and were counted visually. Cooling was required to retard sample degradation. Sample sizes ranged from 1 to 50 cells with excitation times varying from 15 to 180 s. Excellent spectra have been obtained from 20 cells in 15 s using a spectrograph having only 3% throughput.

Microbial Genomics: The Expanding Universe of Bacterial Defense Systems.

PubMed

Forsberg, Kevin J; Malik, Harmit S

2018-04-23

Bacteria protect themselves against infection using multiple defensive systems that move by horizontal gene transfer and accumulate in genomic 'defense islands'. A recent study exploited these features to uncover ten novel defense systems, substantially expanding the catalog of bacterial defense systems and predicting the discovery of many more. Copyright © 2018 Elsevier Ltd. All rights reserved.

Detection of bacterial pathogens including potential new species in human head lice from Mali.

PubMed

Amanzougaghene, Nadia; Fenollar, Florence; Sangaré, Abdoul Karim; Sissoko, Mahamadou S; Doumbo, Ogobara K; Raoult, Didier; Mediannikov, Oleg

2017-01-01

In poor African countries, where no medical and biological facilities are available, the identification of potential emerging pathogens of concern at an early stage is challenging. Head lice, Pediculus humanus capitis, have a short life, feed only on human blood and do not transmit pathogens to their progeny. They are, therefore, a perfect tool for the xenodiagnosis of current or recent human infection. This study assessed the occurrence of bacterial pathogens from head lice collected in two rural villages from Mali, where a high frequency of head lice infestation had previously been reported, using molecular methods. Results show that all 600 head lice, collected from 117 individuals, belonged to clade E, specific to West Africa. Bartonella quintana, the causative agent of trench fever, was identified in three of the 600 (0.5%) head lice studied. Our study also shows, for the first time, the presence of the DNA of two pathogenic bacteria, namely Coxiella burnetii (5.1%) and Rickettsia aeschlimannii (0.6%), detected in human head lice, as well as the DNA of potential new species from the Anaplasma and Ehrlichia genera of unknown pathogenicity. The finding of several Malian head lice infected with B. quintana, C. burnetii, R. aeschlimannii, Anaplasma and Ehrlichia is alarming and highlights the need for active survey programs to define the public health consequences of the detection of these emerging bacterial pathogens in human head lice.

Lysozyme as a recognition element for monitoring of bacterial population.

PubMed

Zheng, Laibao; Wan, Yi; Yu, Liangmin; Zhang, Dun

2016-01-01

Bacterial infections remain a significant challenge in biomedicine and environment safety. Increasing worldwide demand for point-of-care techniques and increasing concern on their safe development and use, require a simple and sensitive bioanalysis for pathogen detection. However, this goal is not yet achieved. A design for fluorescein isothiocyanate-labeled lysozyme (FITC-LYZ), which provides quantitative binding information for gram-positive bacteria, Micrococcus luteus, and detects pathogen concentration, is presented. The functional lysozyme is used not only as the pathogenic detection platform, but also as a tracking reagent for microbial population in antibacterial tests. A nonlinear relationship between the system response and the logarithm of the bacterial concentration was observed in the range of 1.2×10(2)-1.2×10(5) cfu mL(-1). The system has a potential for further applications and provides a facile and simple method for detection of pathogenic bacteria. Meanwhile, the fluorescein isothiocyanate -labeled lysozyme is also employed as the tracking agent for antibacterial dynamic assay, which show a similar dynamic curve compared with UV-vis test. Copyright © 2015 Elsevier B.V. All rights reserved.

Dynamics of Immune System Gene Expression upon Bacterial Challenge and Wounding in a Social Insect (Bombus terrestris)

PubMed Central

Erler, Silvio; Popp, Mario; Lattorff, H. Michael G.

2011-01-01

The innate immune system which helps individuals to combat pathogens comprises a set of genes representing four immune system pathways (Toll, Imd, JNK and JAK/STAT). There is a lack of immune genes in social insects (e.g. honeybees) when compared to Diptera. Potentially, this might be compensated by an advanced system of social immunity (synergistic action of several individuals). The bumble bee, Bombus terrestris, is a primitively eusocial species with an annual life cycle and colonies headed by a single queen. We used this key pollinator to study the temporal dynamics of immune system gene expression in response to wounding and bacterial challenge. Antimicrobial peptides (AMP) (abaecin, defensin 1, hymenoptaecin) were strongly up-regulated by wounding and bacterial challenge, the latter showing a higher impact on the gene expression level. Sterile wounding down-regulated TEP A, an effector gene of the JAK/STAT pathway, and bacterial infection influenced genes of the Imd (relish) and JNK pathway (basket). Relish was up-regulated within the first hour after bacterial challenge, but decreased strongly afterwards. AMP expression following wounding and bacterial challenge correlates with the expression pattern of relish whereas correlated expression with dorsal was absent. Although expression of AMPs was high, continuous bacterial growth was observed throughout the experiment. Here we demonstrate for the first time the temporal dynamics of immune system gene expression in a social insect. Wounding and bacterial challenge affected the innate immune system significantly. Induction of AMP expression due to wounding might comprise a pre-adaptation to accompanying bacterial infections. Compared with solitary species this social insect exhibits reduced immune system efficiency, as bacterial growth could not be inhibited. A negative feedback loop regulating the Imd-pathway is suggested. AMPs, the end product of the Imd-pathway, inhibited the up-regulation of the

Distinct antimicrobial peptide expression determines host species-specific bacterial associations

PubMed Central

Franzenburg, Sören; Walter, Jonas; Künzel, Sven; Wang, Jun; Baines, John F.; Bosch, Thomas C. G.; Fraune, Sebastian

2013-01-01

Animals are colonized by coevolved bacterial communities, which contribute to the host’s health. This commensal microbiota is often highly specific to its host-species, inferring strong selective pressures on the associated microbes. Several factors, including diet, mucus composition, and the immune system have been proposed as putative determinants of host-associated bacterial communities. Here we report that species-specific antimicrobial peptides account for different bacterial communities associated with closely related species of the cnidarian Hydra. Gene family extensions for potent antimicrobial peptides, the arminins, were detected in four Hydra species, with each species possessing a unique composition and expression profile of arminins. For functional analysis, we inoculated arminin-deficient and control polyps with bacterial consortia characteristic for different Hydra species and compared their selective preferences by 454 pyrosequencing of the bacterial microbiota. In contrast to control polyps, arminin-deficient polyps displayed decreased potential to select for bacterial communities resembling their native microbiota. This finding indicates that species-specific antimicrobial peptides shape species-specific bacterial associations. PMID:24003149

Bacterial community of biofilms developed under different water supply conditions in a distribution system.

PubMed

Sun, Huifang; Shi, Baoyou; Bai, Yaohui; Wang, Dongsheng

2014-02-15

In order to understand the bacterial community characteristics of biofilms developed under different finished water supply histories in drinking water distribution systems (DWDS), biofilm samples on different type of iron corrosion scales in a real DWDS were collected and systematically investigated using 454 pyrosequencing of 16S rRNA gene. The richness and diversity estimators showed that biofilms formed in DWDS transporting finished groundwater (GW) had the lowest level of bacterial diversity. From phylum to genus level, the dominant bacterial groups found in the biofilms under finished surface water (SW) and GW conditions were distinct. Proteobacteria was the dominant group in all biofilm samples (in the range of 40%-97%), but was relatively higher in biofilms with GW. The relative abundance of Firmicutes in biofilms with SW (28%-35%) was significantly higher (p<0.01) than that in biofilms with GW (0.5%-2.88%). Statistical analysis (Spearman's rank) revealed that alkalinity and chemical oxygen demand (CODMn) positively correlated with the relative abundance of Proteobacteria and Firmicutes, respectively. The abundance of sequences affiliated to iron-reducing bacteria (mainly Bacillus) and iron-oxidizing bacteria (mainly Acidovorax) were relatively higher in biofilms with SW, which might contribute to the formation of much thicker or tubercle-formed corrosion scales under SW supply condition. Several potential opportunistic pathogens, such as Burkholderia fungorum, Mycobacterium neoaurum, Mycobacterium frederiksbergense were detected in the biofilms. Copyright © 2013 Elsevier B.V. All rights reserved.

Detection of mastitis pathogens by analysis of volatile bacterial metabolites.

PubMed

Hettinga, K A; van Valenberg, H J F; Lam, T J G M; van Hooijdonk, A C M

2008-10-01

The ability to detect mastitis pathogens based on their volatile metabolites was studied. Milk samples from cows with clinical mastitis, caused by Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli were collected. In addition, samples from cows without clinical mastitis and with low somatic cell count (SCC) were collected for comparison. All mastitis samples were examined by using classical microbiological methods, followed by headspace analysis for volatile metabolites. Milk from culture-negative samples contained a lower number and amount of volatile components compared with cows with clinical mastitis. Because of variability between samples within a group, comparisons between pathogens were not sufficient for classification of the samples by univariate statistics. Therefore, an artificial neural network was trained to classify the pathogen in the milk samples based on the bacterial metabolites. The trained network differentiated milk from uninfected and infected quarters very well. When comparing pathogens, Staph. aureus produced a very different pattern of volatile metabolites compared with the other samples. Samples with coagulase-negative staphylococci and E. coli had enough dissimilarity with the other pathogens, making it possible to separate these 2 pathogens from each other and from the other samples. The 2 streptococcus species did not show significant differences between each other but could be identified as a different group from the other pathogens. Five groups can thus be identified based on the volatile bacterial metabolites: Staph. aureus, coagulase-negative staphylococci, streptococci (Strep. uberis and Strep. dysgalactiae as one group), E. coli, and uninfected quarters.

Bacterial mycophagy: definition and diagnosis of a unique bacterial-fungal interaction.

PubMed

Leveau, Johan H J; Preston, Gail M

2008-01-01

This review analyses the phenomenon of bacterial mycophagy, which we define as a set of phenotypic behaviours that enable bacteria to obtain nutrients from living fungi and thus allow the conversion of fungal into bacterial biomass. We recognize three types of bacterial strategies to derive nutrition from fungi: necrotrophy, extracellular biotrophy and endocellular biotrophy. Each is characterized by a set of uniquely sequential and differently overlapping interactions with the fungal target. We offer a detailed analysis of the nature of these interactions, as well as a comprehensive overview of methodologies for assessing and quantifying their individual contributions to the mycophagy phenotype. Furthermore, we discuss future prospects for the study and exploitation of bacterial mycophagy, including the need for appropriate tools to detect bacterial mycophagy in situ in order to be able to understand, predict and possibly manipulate the way in which mycophagous bacteria affect fungal activity, turnover, and community structure in soils and other ecosystems.

Rapid onsite detection of bacterial spores of biothreat importance by paper-based colorimetric method using erbium-pyrocatechol violet complex.

PubMed

Shivakiran, M S; Venkataramana, M; Lakshmana Rao, P V

2016-01-01

Dipicolinic acid (DPA) is an important chemical marker for the detection of bacterial spores. In this study, complexes of lanthanide series elements such as erbium, europium, neodymium, and terbium were prepared with pyrocatechol violet and effectively immobilized the pyrocatechol violet (PV)-metal complex on a filter paper using polyvinyl alcohol. These filter paper strips were employed for the onsite detection of bacterial spores. The test filter papers were evaluated quantitatively with different concentrations of DPA and spores of various bacteria. Among the four lanthanide ions, erbium displayed better sensitivity than the other ions. The limit of detection of this test for DPA was 60 μM and 5 × 10(6) spores. The effect of other non-spore-forming bacteria and interfering chemicals on the test strips was also evaluated. The non-spore-forming bacteria did not have considerable effect on the test strip whereas chemicals such as EDTA had significant effects on the test results. The present test is rapid and robust, capable of providing timely results for better judgement to save resources on unnecessary decontamination procedures during false alarms.

Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

PubMed Central

2010-01-01

Background Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic

Rapid pathogen detection with bacterial-assembled magnetic mesoporous silica.

PubMed

Lee, Soo Youn; Lee, Jiho; Lee, Hye Sun; Chang, Jeong Ho

2014-03-15

We report rapid and accurate pathogen detection by coupling with high efficiency magnetic separation of pathogen by Ni(2+)-heterogeneous magnetic mesoporous silica (Ni-HMMS) and real time-polymerase chain reaction (RT-PCR) technique. Ni-HMMS was developed with a significant incorporation of Fe particles within the silica mesopores by programmed thermal hydrogen reaction and functionalized with Ni(2+) ion on the surface by the wet impregnation process. High abundant Ni(2+) ions on the Ni-HMMS surface were able to assemble with cell wall component protein NikA (nickel-binding membrane protein), which contains several pathogenic bacteria including Escherichia coli O157:H7. NikA protein expression experiment showed the outstanding separation rate of the nikA gene-overexpressed E. coli (pSY-Nik) when comparing with wild-type E. coli (44.5 ± 13%) or not over-expressed E. coli (pSY-Nik) (53.2 ± 2.7%). Moreover, Ni-HMMS showed lower obstacle effect by large reaction volume (10 mL) than spherical core/shell-type silica magnetic nanoparticles functionalized with Ni(2+) (ca. 40 nm-diameters). Finally, the Ni-HMMS was successfully assessed to separate pathogenic E. coli O157:H7 and applied to direct and rapid RT-PCR to quantitative detection at ultralow concentration (1 Log10 cfu mL(-1)) in the real samples (milk and Staphylococcus aureus culture broth) without bacterial amplification and DNA extraction step. © 2013 Elsevier B.V. All rights reserved.

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

PubMed Central

Clancy, Eoin; Cormican, Martin; Boo, Teck Wee; Cunney, Robert

2018-01-01

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. PMID:29425124

Viable bacterial population and persistence of foodborne pathogens on the pear carpoplane.

PubMed

Duvenage, Francois J; Duvenage, Stacey; Du Plessis, Erika M; Volschenk, Quinton; Korsten, Lise

2017-03-01

Knowledge on the culturable bacteria and foodborne pathogen presence on pears is important for understanding the impact of postharvest practices on food safety assurance. Pear fruit bacteria were investigated from the point of harvest, following chlorine drenching and after controlled atmosphere (CA) storage to assess the impact on natural bacterial populations and potential foodborne pathogens. Salmonella spp. and Listeria monocytogenes were detected on freshly harvested fruit in season one. During season one, chemical drenching and CA storage did not have a significant effect on the bacterial load of orchard pears, except for two farms where the populations were lower 'after CA storage'. During season two, bacterial populations of orchard pears from three of the four farms increased significantly following drenching; however, the bacterial load decreased 'after CA storage'. Bacteria isolated following enumeration included Enterobacteriaceae, Microbacteriaceae, Pseudomonadaceae and Bacillaceae, with richness decreasing 'after drench' and 'after CA storage'. Salmonella spp. and L. monocytogenes were not detected after postharvest practices. Postharvest practices resulted in decreased bacterial species richness. Understanding how postharvest practices have an impact on the viable bacterial populations of pear fruit will contribute to the development of crop-specific management systems for food safety assurance. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

C-reactive Protein Versus Neutrophil/lymphocyte Ratio in Differentiating Bacterial and Non-bacterial Pneumonia in Children.

PubMed

Gauchan, E; Adhikari, S

2016-09-01

Pneumonia is a leading cause of childhood mortality in a low resource country. Simple laboratory markers can help differentiate between bacterial and non-bacterial pneumonias for appropriate management. In children aged one to 60 months with features of lower respiratory infection, C-reactive protein (CRP) and neutrophil-lymphocyte ratio (NLR) were used to differentiate between bacterial and non-bacterial pneumonias. The cutoff values for detecting bacterial pneumonias were evaluated by statistical tools. Bacterial pneumonia was diagnosed in 285 (43.6%) children out of 654 studied. At a cut-off value of 36 mg/L CRP was predictive of bacterial pneumonias with sensitivity and specificity of 61.8% and 91.3% respectively while the sensitivity and specificity for predicting bacterial pneumonia using NLR was 45.6% and 64% respectively with 1.28 used as a cut-off. Our study shows that CRP is superior to NLR in differentiating bacterial from non-bacterial pneumonias in children.

Pyrosequencing analysis for characterization of bacterial diversity in a soil as affected by integrated livestock-cotton production systems

USDA-ARS?s Scientific Manuscript database

Impacts of integrated livestock-crop production systems compared to specialized systems on soil bacterial diversity have not been well documented. We used a bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) method to evaluate bacterial diversity of a clay loam soil (Fine, mixed, thermic To...

Rapid detection of bacterial endotoxins in ophthalmic viscosurgical device materials by direct analysis in real time mass spectrometry.

PubMed

Li, Hongli; Hitchins, Victoria M; Wickramasekara, Samanthi

2016-11-02

Bacterial endotoxins are lipopolysaccharides bound to the bacterial cell wall and released when bacteria rupture or disintegrate. Possible contamination of endotoxin in ophthalmic devices can cause a painful eye inflammation or result in toxic anterior segment syndrome after cataract surgery. Measurement of bacterial endotoxin in medical device materials is difficult since endotoxin binds with polymer matrix and some of the materials are very viscous and non-water soluble, where traditional enzyme-based Limulus amebocyte lysate (LAL) assay cannot be applied. Here we propose a rapid and high throughput ambient ionization mass spectrometric (MS) method using direct analysis in real time (DART) for the evaluation of endotoxin contamination in medical device materials. Large and structurally complex endotoxin instantaneously breaks down into low-mass characteristic fragment ions using DART and is detected by MS in both positive and negative ion modes. This method enables the identification and separation of endotoxin from medical materials with a detection limit of 0.03 ng mL -1 endotoxins in aqueous solution. Ophthalmic viscosurgical device materials including sodium hyaluronate (NaHA), non-water soluble perfluoro-n-octane (PFO) and silicone oil (SO) were spiked with different known concentrations of endotoxin and analyzed by DART MS, where the presence of endotoxin was successfully detected and featured small mass fragment ions were generated for NaHA, PFO and SO as well. Current findings showed the feasibility of measuring endotoxin contamination in medical device materials using DART-MS, which can lead to a one-step analysis of endotoxins in different matrices, avoiding any potential contamination during sample pre-treatment steps. Published by Elsevier B.V.

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

Detection of bacterial pathogens including potential new species in human head lice from Mali

PubMed Central

Amanzougaghene, Nadia; Fenollar, Florence; Sangaré, Abdoul Karim; Sissoko, Mahamadou S.; Doumbo, Ogobara K.; Raoult, Didier

2017-01-01

In poor African countries, where no medical and biological facilities are available, the identification of potential emerging pathogens of concern at an early stage is challenging. Head lice, Pediculus humanus capitis, have a short life, feed only on human blood and do not transmit pathogens to their progeny. They are, therefore, a perfect tool for the xenodiagnosis of current or recent human infection. This study assessed the occurrence of bacterial pathogens from head lice collected in two rural villages from Mali, where a high frequency of head lice infestation had previously been reported, using molecular methods. Results show that all 600 head lice, collected from 117 individuals, belonged to clade E, specific to West Africa. Bartonella quintana, the causative agent of trench fever, was identified in three of the 600 (0.5%) head lice studied. Our study also shows, for the first time, the presence of the DNA of two pathogenic bacteria, namely Coxiella burnetii (5.1%) and Rickettsia aeschlimannii (0.6%), detected in human head lice, as well as the DNA of potential new species from the Anaplasma and Ehrlichia genera of unknown pathogenicity. The finding of several Malian head lice infected with B. quintana, C. burnetii, R. aeschlimannii, Anaplasma and Ehrlichia is alarming and highlights the need for active survey programs to define the public health consequences of the detection of these emerging bacterial pathogens in human head lice. PMID:28931077

Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections

PubMed Central

Gadsby, N.J.; McHugh, M.P.; Russell, C.D.; Mark, H.; Conway Morris, A.; Laurenson, I.F.; Hill, A.T.; Templeton, K.E.

2015-01-01

The frequent lack of a positive and timely microbiological diagnosis in patients with lower respiratory tract infection (LRTI) is an important obstacle to antimicrobial stewardship. Patients are typically prescribed broad-spectrum empirical antibiotics while microbiology results are awaited, but, because these are often slow, negative, or inconclusive, de-escalation to narrow-spectrum agents rarely occurs in clinical practice. The aim of this study was to develop and evaluate two multiplex real-time PCR assays for the sensitive detection and accurate quantification of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. We found that all eight bacterial targets could be reliably quantified from sputum specimens down to a concentration of 100 CFUs/reaction (8333 CFUs/mL). Furthermore, all 249 positive control isolates were correctly detected with our assay, demonstrating effectiveness on both reference strains and local clinical isolates. The specificity was 98% on a panel of nearly 100 negative control isolates. Bacterial load was quantified accurately when three bacterial targets were present in mixtures of varying concentrations, mimicking likely clinical scenarios in LRTI. Concordance with culture was 100% for culture-positive sputum specimens, and 90% for bronchoalveolar lavage fluid specimens, and additional culture-negative bacterial infections were detected and quantified. In conclusion, a quantitative molecular test for eight key bacterial causes of LRTI has the potential to provide a more sensitive decision-making tool, closer to the time-point of patient admission than current standard methods. This should facilitate de-escalation from broad-spectrum to narrow-spectrum antibiotics, substantially improving patient management and supporting efforts to curtail inappropriate antibiotic use. PMID:25980353

Inhibition of bacterial oxidation of ferrous iron by lead nitrate in sulfate-rich systems

USGS Publications Warehouse

Wang, Hongmei; Gong, Linfeng; Cravotta,, Charles A.; Yang, Xiaofen; Tuovinen, Olli H.; Dong, Hailiang; Fu, Xiang

2013-01-01

Inhibition of bacterial oxidation of ferrous iron (Fe(II)) by Pb(NO3)2 was investigated with a mixed culture of Acidithiobacillus ferrooxidans. The culture was incubated at 30 °C in ferrous-sulfate medium amended with 0–24.2 mM Pb(II) added as Pb(NO3)2. Anglesite (PbSO4) precipitated immediately upon Pb addition and was the only solid phase detected in the abiotic controls. Both anglesite and jarosite (KFe3(SO4)2(OH)6) were detected in inoculated cultures. Precipitation of anglesite maintained dissolved Pb concentrations at 16.9–17.6 μM regardless of the concentrations of Pb(NO3)2 added. Fe(II) oxidation was suppressed by 24.2 mM Pb(NO3)2 addition even when anglesite was removed before inoculation. Experiments with 0–48 mM KNO3 demonstrated that bacterial Fe(II) oxidation decreased as nitrate concentration increased. Therefore, inhibition of Fe(II) oxidation at 24.2 mM Pb(NO3)2 addition resulted from nitrate toxicity instead of Pb addition. Geochemical modeling that considered the initial precipitation of anglesite to equilibrium followed by progressive oxidation of Fe(II) and the precipitation of jarosite and an amorphous iron hydroxide phase, without allowing plumbojarosite to precipitate were consistent with the experimental time-series data on Fe(II) oxidation under biotic conditions. Anglesite precipitation in mine tailings and other sulfate-rich systems maintains dissolved Pb concentrations below the toxicity threshold of A. ferrooxidans.

Inhibition of bacterial oxidation of ferrous iron by lead nitrate in sulfate-rich systems.

PubMed

Wang, Hongmei; Gong, Linfeng; Cravotta, Charles A; Yang, Xiaofen; Tuovinen, Olli H; Dong, Hailiang; Fu, Xiang

2013-01-15

Inhibition of bacterial oxidation of ferrous iron (Fe(II)) by Pb(NO(3))(2) was investigated with a mixed culture of Acidithiobacillus ferrooxidans. The culture was incubated at 30 °C in ferrous-sulfate medium amended with 0-24.2 mM Pb(II) added as Pb(NO(3))(2). Anglesite (PbSO(4)) precipitated immediately upon Pb addition and was the only solid phase detected in the abiotic controls. Both anglesite and jarosite (KFe(3)(SO(4))(2)(OH)(6)) were detected in inoculated cultures. Precipitation of anglesite maintained dissolved Pb concentrations at 16.9-17.6 μM regardless of the concentrations of Pb(NO(3))(2) added. Fe(II) oxidation was suppressed by 24.2 mM Pb(NO(3))(2) addition even when anglesite was removed before inoculation. Experiments with 0-48 mM KNO(3) demonstrated that bacterial Fe(II) oxidation decreased as nitrate concentration increased. Therefore, inhibition of Fe(II) oxidation at 24.2 mM Pb(NO(3))(2) addition resulted from nitrate toxicity instead of Pb addition. Geochemical modeling that considered the initial precipitation of anglesite to equilibrium followed by progressive oxidation of Fe(II) and the precipitation of jarosite and an amorphous iron hydroxide phase, without allowing plumbojarosite to precipitate were consistent with the experimental time-series data on Fe(II) oxidation under biotic conditions. Anglesite precipitation in mine tailings and other sulfate-rich systems maintains dissolved Pb concentrations below the toxicity threshold of A. ferrooxidans. Copyright © 2012 Elsevier B.V. All rights reserved.

Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae.

PubMed

Wang, Xin; Mair, Raydel; Hatcher, Cynthia; Theodore, M Jordan; Edmond, Karen; Wu, Henry M; Harcourt, Brian H; Carvalho, Maria da Gloria S; Pimenta, Fabiana; Nymadawa, Pagbajab; Altantsetseg, Dorjpurev; Kirsch, Mariah; Satola, Sarah W; Cohn, Amanda; Messonnier, Nancy E; Mayer, Leonard W

2011-04-01

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction. Published by Elsevier GmbH.

Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid.

PubMed

Wisselink, Henk J; Cornelissen, Jan B W J; van der Wal, Fimme J; Kooi, Engbert A; Koene, Miriam G; Bossers, Alex; Smid, Bregtje; de Bree, Freddy M; Antonis, Adriaan F G

2017-07-13

Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents. The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10 -1 to 2 × 10 0  CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni. It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

Detection of bacterial-reactive natural IgM antibodies in desert bighorn sheep populations

PubMed Central

Palmer, Amy L.; Zielke, Ryszard A.; Sikora, Aleksandra E.; Beechler, Brianna R.; Jolles, Anna E.; Epps, Clinton W.

2017-01-01

Ecoimmunology is a burgeoning field of ecology which studies immune responses in wildlife by utilizing general immune assays such as the detection of natural antibody. Unlike adaptive antibodies, natural antibodies are important in innate immune responses and often recognized conserved epitopes present in pathogens. Here, we describe a procedure for measuring natural antibodies reactive to bacterial antigens that may be applicable to a variety of organisms. IgM from desert bighorn sheep plasma samples was tested for reactivity to outer membrane proteins from Vibrio coralliilyticus, a marine bacterium to which sheep would have not been exposed. Immunoblotting demonstrated bighorn sheep IgM could bind to a variety of bacterial cell envelope proteins while ELISA analysis allowed for rapid determination of natural antibody levels in hundreds of individual animals. Natural antibody levels were correlated with the ability of plasma to kill laboratory strains of E. coli bacteria. Finally, we demonstrate that natural antibody levels varied in two distinct populations of desert bighorn sheep. These data demonstrate a novel and specific measure of natural antibody function and show that this varies in ecologically relevant ways. PMID:28662203

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

PubMed Central

Pinzon, Neissa M.; Aukema, Kelly G.; Gralnick, Jeffrey A.; Wackett, Lawrence P.

2011-01-01

ABSTRACT A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. PMID:21712420

Real-time bacterial microcolony counting using on-chip microscopy

NASA Astrophysics Data System (ADS)

Jung, Jae Hee; Lee, Jung Eun

2016-02-01

Observing microbial colonies is the standard method for determining the microbe titer and investigating the behaviors of microbes. Here, we report an automated, real-time bacterial microcolony-counting system implemented on a wide field-of-view (FOV), on-chip microscopy platform, termed ePetri. Using sub-pixel sweeping microscopy (SPSM) with a super-resolution algorithm, this system offers the ability to dynamically track individual bacterial microcolonies over a wide FOV of 5.7 mm × 4.3 mm without requiring a moving stage or lens. As a demonstration, we obtained high-resolution time-series images of S. epidermidis at 20-min intervals. We implemented an image-processing algorithm to analyze the spatiotemporal distribution of microcolonies, the development of which could be observed from a single bacterial cell. Test bacterial colonies with a minimum diameter of 20 μm could be enumerated within 6 h. We showed that our approach not only provides results that are comparable to conventional colony-counting assays but also can be used to monitor the dynamics of colony formation and growth. This microcolony-counting system using on-chip microscopy represents a new platform that substantially reduces the detection time for bacterial colony counting. It uses chip-scale image acquisition and is a simple and compact solution for the automation of colony-counting assays and microbe behavior analysis with applications in antibacterial drug discovery.

Real-time bacterial microcolony counting using on-chip microscopy

PubMed Central

Jung, Jae Hee; Lee, Jung Eun

2016-01-01

Observing microbial colonies is the standard method for determining the microbe titer and investigating the behaviors of microbes. Here, we report an automated, real-time bacterial microcolony-counting system implemented on a wide field-of-view (FOV), on-chip microscopy platform, termed ePetri. Using sub-pixel sweeping microscopy (SPSM) with a super-resolution algorithm, this system offers the ability to dynamically track individual bacterial microcolonies over a wide FOV of 5.7 mm × 4.3 mm without requiring a moving stage or lens. As a demonstration, we obtained high-resolution time-series images of S. epidermidis at 20-min intervals. We implemented an image-processing algorithm to analyze the spatiotemporal distribution of microcolonies, the development of which could be observed from a single bacterial cell. Test bacterial colonies with a minimum diameter of 20 μm could be enumerated within 6 h. We showed that our approach not only provides results that are comparable to conventional colony-counting assays but also can be used to monitor the dynamics of colony formation and growth. This microcolony-counting system using on-chip microscopy represents a new platform that substantially reduces the detection time for bacterial colony counting. It uses chip-scale image acquisition and is a simple and compact solution for the automation of colony-counting assays and microbe behavior analysis with applications in antibacterial drug discovery. PMID:26902822

Demonstration of bacterial biofilms in culture-negative silicone stent and jones tube.

PubMed

Parsa, Kami; Schaudinn, Christoph; Gorur, Amita; Sedghizadeh, Parish P; Johnson, Thomas; Tse, David T; Costerton, John W

2010-01-01

To demonstrate the presence of bacterial biofilms on a dacryocystorhinostomy silicone stent and a Jones tube. One dacryocystorhinostomy silicone stent and one Jones tube were removed from 2 patients who presented with an infection of their respective nasolacrimal system. Cultures were obtained, and the implants were processed for scanning electron microscopy and confocal laser scanning microscopy, advanced microscopic methods that are applicable for detection of uncultivable biofilm organisms. Routine bacterial cultures revealed no growth, but bacterial biofilms on outer and inner surfaces of both implants were confirmed by advanced microscopic techniques. To the authors' knowledge, this is the first article that documents the presence of biofilms on a Crawford stent or a Jones tube on patients who presented with infections involving the nasolacrimal system. Although initial cultures revealed absence of any bacterial growth, confocal laser scanning microscopy and scanning electron microscopy documented bacterial colonization. Clinicians should consider the role of biofilms and the limitation of our standard culturing techniques while treating patients with device- or implant-related infections.

Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis.

PubMed

Maruyama, Kohei; Takeyama, Haruko; Nemoto, Etsuo; Tanaka, Tsuyoshi; Yoda, Kiyoshi; Matsunaga, Tadashi

2004-09-20

Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs. Copyright 2004 Wiley Periodicals, Inc.

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

PubMed

Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

2012-01-01

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

Detection and validation of QTL affecting bacterial cold water disease resistance in rainbow trout using restriction-site associated DNA sequencing

USDA-ARS?s Scientific Manuscript database

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. Using microsatellites genome scan we have previously detected significant and suggestive QTL with major effects on the phenotypic variation of survival following challenge with Flavobacterium psychrophilum...

Nanoparticle Approaches against Bacterial Infections

PubMed Central

Gao, Weiwei; Thamphiwatana, Soracha; Angsantikul, Pavimol; Zhang, Liangfang

2014-01-01

Despite the wide success of antibiotics, the treatment of bacterial infection still faces significant challenges, particularly the emergence of antibiotic resistance. As a result, nanoparticle drug delivery platforms including liposomes, polymeric nanoparticles, dendrimers, and various inorganic nanoparticles have been increasingly exploited to enhance the therapeutic effectiveness of existing antibiotics. This review focuses on areas where nanoparticle approaches hold significant potential to advance the treatment of bacterial infection. These areas include targeted antibiotic delivery, environmentally responsive antibiotic delivery, combinatorial antibiotic delivery, nanoparticle-enabled antibacterial vaccination, and nanoparticle-based bacterial detection. In each area we highlight the innovative antimicrobial nanoparticle platforms and review their progress made against bacterial infections. PMID:25044325

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.

Bacterial diversity of bacteriomes and organs of reproductive, digestive and excretory systems in two cicada species (Hemiptera: Cicadidae)

PubMed Central

Zheng, Zhou; Wang, Dandan; He, Hong

2017-01-01

Cicadas form intimate symbioses with bacteria to obtain nutrients that are scarce in the xylem fluid they feed on. The obligate symbionts in cicadas are purportedly confined to specialized bacteriomes, but knowledge of bacterial communities associated with cicadas is limited. Bacterial communities in the bacteriomes and organs of reproductive, digestive and excretory systems of two cicada species (Platypleura kaempferi and Meimuna mongolica) were investigated using different methods, and the bacterial diversity and distribution patterns of dominant bacteria in different tissues were compared. Within each species, the bacterial communities of testes are significantly different from those of bacteriomes and ovaries. The dominant endosymbiont Candidatus Sulcia muelleri is found not only in the bacteriomes and reproductive organs, but also in the “filter chamber + conical segment” of both species. The transmission mode of this endosymbiont in the alimentary canal and its effect on physiological processes merits further study. A novel bacterium of Rhizobiales, showing ~80% similarity to Candidatus Hodgkinia cicadicola, is dominant in the bacteriomes and ovaries of P. kaempferi. Given that the genome of H. cicadicola exhibits rapid sequence evolution, it is possible that this novel bacterium is a related endosymbiont with beneficial trophic functions similar to that of H. cicadicola in some other cicadas. Failure to detect H. cicadicola in M. mongolica suggests that it has been subsequently replaced by another bacterium, a yeast or gut microbiota which compensates for the loss of H. cicadicola. The distribution of this novel Rhizobiales species in other cicadas and its identification require further investigation to help establish the definition of the bacterial genus Candidatus Hodgkinia and to provide more information on sequence divergence of related endosymbionts of cicadas. Our results highlight the complex bacterial communities of cicadas, and are informative

Bacterial diversity of bacteriomes and organs of reproductive, digestive and excretory systems in two cicada species (Hemiptera: Cicadidae).

PubMed

Zheng, Zhou; Wang, Dandan; He, Hong; Wei, Cong

2017-01-01

Cicadas form intimate symbioses with bacteria to obtain nutrients that are scarce in the xylem fluid they feed on. The obligate symbionts in cicadas are purportedly confined to specialized bacteriomes, but knowledge of bacterial communities associated with cicadas is limited. Bacterial communities in the bacteriomes and organs of reproductive, digestive and excretory systems of two cicada species (Platypleura kaempferi and Meimuna mongolica) were investigated using different methods, and the bacterial diversity and distribution patterns of dominant bacteria in different tissues were compared. Within each species, the bacterial communities of testes are significantly different from those of bacteriomes and ovaries. The dominant endosymbiont Candidatus Sulcia muelleri is found not only in the bacteriomes and reproductive organs, but also in the "filter chamber + conical segment" of both species. The transmission mode of this endosymbiont in the alimentary canal and its effect on physiological processes merits further study. A novel bacterium of Rhizobiales, showing ~80% similarity to Candidatus Hodgkinia cicadicola, is dominant in the bacteriomes and ovaries of P. kaempferi. Given that the genome of H. cicadicola exhibits rapid sequence evolution, it is possible that this novel bacterium is a related endosymbiont with beneficial trophic functions similar to that of H. cicadicola in some other cicadas. Failure to detect H. cicadicola in M. mongolica suggests that it has been subsequently replaced by another bacterium, a yeast or gut microbiota which compensates for the loss of H. cicadicola. The distribution of this novel Rhizobiales species in other cicadas and its identification require further investigation to help establish the definition of the bacterial genus Candidatus Hodgkinia and to provide more information on sequence divergence of related endosymbionts of cicadas. Our results highlight the complex bacterial communities of cicadas, and are informative for

Quantification, Distribution, and Possible Source of Bacterial Biofilm in Mouse Automated Watering Systems

PubMed Central

Meier, Thomas R; Maute, Carrie J; Cadillac, Joan M; Lee, Ji Young; Righter, Daniel J; Hugunin, Kelly MS; Deininger, Rolf A; Dysko, Robert C

2008-01-01

The use of automated watering systems for providing drinking water to rodents has become commonplace in the research setting. Little is known regarding bacterial biofilm growth within the water piping attached to the racks (manifolds). The purposes of this project were to determine whether the mouse oral flora contributed to the aerobic bacterial component of the rack biofilm, quantify bacterial growth in rack manifolds over 6 mo, assess our rack sanitation practices, and quantify bacterial biofilm development within sections of the manifold. By using standard methods of bacterial identification, the aerobic oral flora of 8 strains and stocks of mice were determined on their arrival at our animal facility. Ten rack manifolds were sampled before, during, and after sanitation and monthly for 6 mo. Manifolds were evaluated for aerobic bacterial growth by culture on R2A and trypticase soy agar, in addition to bacterial ATP quantification by bioluminescence. In addition, 6 racks were sampled at 32 accessible sites for evaluation of biofilm distribution within the watering manifold. The identified aerobic bacteria in the oral flora were inconsistent with the bacteria from the manifold, suggesting that the mice do not contribute to the biofilm bacteria. Bacterial growth in manifolds increased while they were in service, with exponential growth of the biofilm from months 3 to 6 and a significant decrease after sanitization. Bacterial biofilm distribution was not significantly different across location quartiles of the rack manifold, but bacterial levels differed between the shelf pipe and connecting elbow pipes. PMID:18351724

Quantification, distribution, and possible source of bacterial biofilm in mouse automated watering systems.

PubMed

Meier, Thomas R; Maute, Carrie J; Cadillac, Joan M; Lee, Ji Young; Righter, Daniel J; Hugunin, Kelly M S; Deininger, Rolf A; Dysko, Robert C

2008-03-01

The use of automated watering systems for providing drinking water to rodents has become commonplace in the research setting. Little is known regarding bacterial biofilm growth within the water piping attached to the racks (manifolds). The purposes of this project were to determine whether the mouse oral flora contributed to the aerobic bacterial component of the rack biofilm, quantify bacterial growth in rack manifolds over 6 mo, assess our rack sanitation practices, and quantify bacterial biofilm development within sections of the manifold. By using standard methods of bacterial identification, the aerobic oral flora of 8 strains and stocks of mice were determined on their arrival at our animal facility. Ten rack manifolds were sampled before, during, and after sanitation and monthly for 6 mo. Manifolds were evaluated for aerobic bacterial growth by culture on R2A and trypticase soy agar, in addition to bacterial ATP quantification by bioluminescence. In addition, 6 racks were sampled at 32 accessible sites for evaluation of biofilm distribution within the watering manifold. The identified aerobic bacteria in the oral flora were inconsistent with the bacteria from the manifold, suggesting that the mice do not contribute to the biofilm bacteria. Bacterial growth in manifolds increased while they were in service, with exponential growth of the biofilm from months 3 to 6 and a significant decrease after sanitization. Bacterial biofilm distribution was not significantly different across location quartiles of the rack manifold, but bacterial levels differed between the shelf pipe and connecting elbow pipes.

Quantification and risks associated with bacterial aerosols near domestic greywater-treatment systems.

PubMed

Benami, Maya; Busgang, Allison; Gillor, Osnat; Gross, Amit

2016-08-15

Greywater (GW) reuse can alleviate water stress by lowering freshwater consumption. However, GW contains pathogens that may compromise public health. During the GW-treatment process, bioaerosols can be produced and may be hazardous to human health if inhaled, ingested, or come in contact with skin. Using air-particle monitoring, BioSampler®, and settle plates we sampled bioaerosols emitted from recirculating vertical flow constructed wetlands (RVFCW) - a domestic GW-treatment system. An array of pathogens and indicators were monitored using settle plates and by culturing the BioSampler® liquid. Further enumeration of viable pathogens in the BioSampler® liquid utilized a newer method combining the benefits of enrichment with molecular detection (MPN-qPCR). Additionally, quantitative microbial risk assessment (QMRA) was applied to assess risks of infection from a representative skin pathogen, Staphylococcus aureus. According to the settle-plate technique, low amounts (0-9.7×10(4)CFUm(-2)h(-1)) of heterotrophic bacteria, Staphylococcus spp., Pseudomonas spp., Klebsiella pneumoniae, Enterococcus spp., and Escherichia coli were found to aerosolize up to 1m away from the GW systems. At the 5m distance amounts of these bacteria were not statistically different (p>0.05) from background concentrations tested over 50m away from the systems. Using the BioSampler®, no bacteria were detected before enrichment of the GW-aerosols. However, after enrichment, using an MPN-qPCR technique, viable indicators and pathogens were occasionally detected. Consequently, the QMRA results were below the critical disability-adjusted life year (DALY) safety limits, a measure of overall disease burden, for S. aureus under the tested exposure scenarios. Our study suggests that health risks from aerosolizing pathogens near RVFCW GW-treatment systems are likely low. This study also emphasizes the growing need for standardization of bioaerosol-evaluation techniques to provide more accurate

Expansion of space station diagnostic capability to include serological identification of viral and bacterial infections

NASA Technical Reports Server (NTRS)

Hejtmancik, Kelly E.

1987-01-01

It is necessary that an adequate microbiology capability be provided as part of the Health Maintenance Facility (HMF) to support expected microbial disease events during long periods of space flight. The applications of morphological and biochemical studies to confirm the presence of certain bacterial and fungal disease agents are currently available and under consideration. This confirmation would be greatly facilitated through employment of serological methods to aid in the identification for not only bacterial and fungal agents, but viruses as well. A number of serological approached were considered, particularly the use of Enzyme Linked Immunosorbent Assays (ELISAs), which could be utilized during space flight conditions. A solid phase, membrane supported ELISA for the detection of Bordetella pertussis was developed to show a potential model system that would meet the HMF requirements and specifications for the future space station. A second model system for the detection of Legionella pneumophilia, an expected bacterial disease agent, is currently under investigation.

An efficient tag derived from the common epitope of tospoviral NSs proteins for monitoring recombinant proteins expressed in both bacterial and plant systems.

PubMed

Cheng, Hao-Wen; Chen, Kuan-Chun; Raja, Joseph A J; Li, Jian-Xian; Yeh, Shyi-Dong

2013-04-15

NSscon (23 aa), a common epitope in the gene silencing suppressor NSs proteins of the members of the Watermelon silver mottle virus (WSMoV) serogroup, was previously identified. In this investigation, we expressed different green fluorescent protein (GFP)-fused deletions of NSscon in bacteria and reacted with NSscon monoclonal antibody (MAb). Our results indicated that the core 9 amino acids, "(109)KFTMHNQIF(117)", denoted as "nss", retain the reactivity of NSscon. In bacterial pET system, four different recombinant proteins labeled with nss, either at N- or C-extremes, were readily detectable without position effects, with sensitivity superior to that for the polyhistidine-tag. When the nss-tagged Zucchini yellow mosaic virus (ZYMV) helper component-protease (HC-Pro) and WSMoV nucleocapsid protein were transiently expressed by agroinfiltration in tobacco, they were readily detectable and the tag's possible efficacy for gene silencing suppression was not noticed. Co-immunoprecipitation of nss-tagged and non-tagged proteins expressed from bacteria confirmed the interaction of potyviral HC-Pro and coat protein. Thus, we conclude that this novel nss sequence is highly valuable for tagging recombinant proteins in both bacterial and plant expression systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Systemic acquired tolerance to virulent bacterial pathogens in tomato.

PubMed

Block, Anna; Schmelz, Eric; O'Donnell, Phillip J; Jones, Jeffrey B; Klee, Harry J

2005-07-01

Recent studies on the interactions between plants and pathogenic microorganisms indicate that the processes of disease symptom development and pathogen growth can be uncoupled. Thus, in many instances, the symptoms associated with disease represent an active host response to the presence of a pathogen. These host responses are frequently mediated by phytohormones. For example, ethylene and salicylic acid (SA) mediate symptom development but do not influence bacterial growth in the interaction between tomato (Lycopersicon esculentum) and virulent Xanthomonas campestris pv vesicatoria (Xcv). It is not apparent why extensive tissue death is integral to a defense response if it does not have the effect of limiting pathogen proliferation. One possible function for this hormone-mediated response is to induce a systemic defense response. We therefore assessed the systemic responses of tomato to Xcv. SA- and ethylene-deficient transgenic lines were used to investigate the roles of these phytohormones in systemic signaling. Virulent and avirulent Xcv did induce a systemic response as evidenced by expression of defense-associated pathogenesis-related genes in an ethylene- and SA-dependent manner. This systemic response reduced cell death but not bacterial growth during subsequent challenge with virulent Xcv. This systemic acquired tolerance (SAT) consists of reduced tissue damage in response to secondary challenge with a virulent pathogen with no effect upon pathogen growth. SAT was associated with a rapid ethylene and pathogenesis-related gene induction upon challenge. SAT was also induced by infection with Pseudomonas syringae pv tomato. These data show that SAT resembles systemic acquired resistance without inhibition of pathogen growth.

Detecting macroecological patterns in bacterial communities across independent studies of global soils.

PubMed

Ramirez, Kelly S; Knight, Christopher G; de Hollander, Mattias; Brearley, Francis Q; Constantinides, Bede; Cotton, Anne; Creer, Si; Crowther, Thomas W; Davison, John; Delgado-Baquerizo, Manuel; Dorrepaal, Ellen; Elliott, David R; Fox, Graeme; Griffiths, Robert I; Hale, Chris; Hartman, Kyle; Houlden, Ashley; Jones, David L; Krab, Eveline J; Maestre, Fernando T; McGuire, Krista L; Monteux, Sylvain; Orr, Caroline H; van der Putten, Wim H; Roberts, Ian S; Robinson, David A; Rocca, Jennifer D; Rowntree, Jennifer; Schlaeppi, Klaus; Shepherd, Matthew; Singh, Brajesh K; Straathof, Angela L; Bhatnagar, Jennifer M; Thion, Cécile; van der Heijden, Marcel G A; de Vries, Franciska T

2018-02-01

The emergence of high-throughput DNA sequencing methods provides unprecedented opportunities to further unravel bacterial biodiversity and its worldwide role from human health to ecosystem functioning. However, despite the abundance of sequencing studies, combining data from multiple individual studies to address macroecological questions of bacterial diversity remains methodically challenging and plagued with biases. Here, using a machine-learning approach that accounts for differences among studies and complex interactions among taxa, we merge 30 independent bacterial data sets comprising 1,998 soil samples from 21 countries. Whereas previous meta-analysis efforts have focused on bacterial diversity measures or abundances of major taxa, we show that disparate amplicon sequence data can be combined at the taxonomy-based level to assess bacterial community structure. We find that rarer taxa are more important for structuring soil communities than abundant taxa, and that these rarer taxa are better predictors of community structure than environmental factors, which are often confounded across studies. We conclude that combining data from independent studies can be used to explore bacterial community dynamics, identify potential 'indicator' taxa with an important role in structuring communities, and propose hypotheses on the factors that shape bacterial biogeography that have been overlooked in the past.

Automatic counting and classification of bacterial colonies using hyperspectral imaging

USDA-ARS?s Scientific Manuscript database

Detection and counting of bacterial colonies on agar plates is a routine microbiology practice to get a rough estimate of the number of viable cells in a sample. There have been a variety of different automatic colony counting systems and software algorithms mainly based on color or gray-scale pictu...

Evidence against a bacterial endotoxin masking effect in biologic drug products by limulus amebocyte lysate detection.

PubMed

Bolden, Jay S; Claerbout, Mark E; Miner, Matthew K; Murphy, Marie A; Smith, Kelly R; Warburton, Rob E

2014-01-01

The inability to detect endotoxin using compendia methods is a potential safety concern for patients due to the lack of endotoxin removal capabilities at the fill-finish stage in typical aseptic biologic drug product manufacturing. We have successfully demonstrated endotoxin challenge study recovery methodology using mammalian cell-produced biologic drug products and drug substances in citrate, histidine, phosphate, and sodium acetate buffer formulations containing polysorbate, challenged with an endotoxin analyte, for up to 6 months of storage. Successful recovery was similarly demonstrated for a preserved, peptide-containing drug product formulation. To isolate a potential masking-or low-endotoxin recovery-source, additional studies were performed to evaluate factors including product manufacturing contact surfaces, drug product matrix with and without polysorbate, individual matrix components, protein concentration, reagent suppliers, an orthogonal test method, and storage conditions. In all cases, acceptable recoveries were observed. Bacterial endotoxin is known to be chemically stable at physiological conditions. Purified endotoxin in aqueous conditions is likely to self-aggregate or bind to surfaces. Neither the nature of, nor the storage conditions of, the studied formulation matrices were shown experimentally to render the challenge endotoxin biologically inactive. The results highlight the importance of appropriate study design in assessing the recovery of endotoxins. Bacterial endotoxin is a Gram-negative bacterial cell wall component that is harmful to humans at threshold concentrations, and it is not expected to be in aseptically-produced pharmaceutical medicines. It has been suggested that endotoxin cannot be detected over time in certain biopharmaceutical drug product formulations containing citrate, phosphate, and polysorbate components via an unknown masking mechanism. We have generated and present data here that indicate that endotoxin can be

Influence of hydraulic regimes on bacterial community structure and composition in an experimental drinking water distribution system.

PubMed

Douterelo, I; Sharpe, R L; Boxall, J B

2013-02-01

Microbial biofilms formed on the inner-pipe surfaces of drinking water distribution systems (DWDS) can alter drinking water quality, particularly if they are mechanically detached from the pipe wall to the bulk water, such as due to changes in hydraulic conditions. Results are presented here from applying 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene to investigate the influence of different hydrological regimes on bacterial community structure and to study the potential mobilisation of material from the pipe walls to the network using a full scale, temperature-controlled experimental pipeline facility accurately representative of live DWDS. Analysis of pyrosequencing and water physico-chemical data showed that habitat type (water vs. biofilm) and hydraulic conditions influenced bacterial community structure and composition in our experimental DWDS. Bacterial community composition clearly differed between biofilms and bulk water samples. Gammaproteobacteria and Betaproteobacteria were the most abundant phyla in biofilms while Alphaproteobacteria was predominant in bulk water samples. This suggests that bacteria inhabiting biofilms, predominantly species belonging to genera Pseudomonas, Zooglea and Janthinobacterium, have an enhanced ability to express extracellular polymeric substances to adhere to surfaces and to favour co-aggregation between cells than those found in the bulk water. Highest species richness and diversity were detected in 28 days old biofilms with this being accentuated at highly varied flow conditions. Flushing altered the pipe-wall bacterial community structure but did not completely remove bacteria from the pipe walls, particularly under highly varied flow conditions, suggesting that under these conditions more compact biofilms were generated. This research brings new knowledge regarding the influence of different hydraulic regimes on the composition and structure of bacterial communities within DWDS and the implication that this

Near-infrared surface-enhanced-Raman-scattering (SERS) mediated identification of single optically trapped, bacterial spores

NASA Astrophysics Data System (ADS)

Alexander, Troy A.; Gillespie, James B.; Pellegrino, Paul M.; Fell, Nicholas F., Jr.; Wood, Gary L.; Salamo, Gregory J.

2003-03-01

A novel methodology has been developed for the investigation of bacterial spores. Specifically, this method has been used to probe the spore coat composition of several Bacillus species. This technique may be useful in many applications; most notably, development of novel detection schemes toward potentially harmful biological agents. This method would also be useful as an ancillary environmental monitoring system where sterility is of importance (i.e., food preparation areas as well as invasive and minimally invasive medical applications). This unique detection scheme is based on the near-infrared (NIR) Surface-Enhanced-Raman-Scattering (SERS) from single, optically trapped, bacterial spores. The SERS spectra of several bacterial spores in aqueous media have been measured using SERS substrates based on 60-nm diameter gold colloids bound to 3-Aminopropyltriethoxysilane derivatized glass. The light from a 785-nm laser diode was used to capture/manipulate as well as simultaneously excite the SERS of an individual bacterial spore. The collected SERS spectra were examined for uniqueness and the applicability of this technique for the species identification of bacterial spores.

A DNA pooling based system to detect Escherichia coli virulence factors in fecal and wastewater samples

PubMed Central

Luz María Chacón, J; Lizeth Taylor, C; Carmen Valiente, A; Irene Alvarado, P; Ximena Cortés, B

2012-01-01

The availability of a useful tool for simple and timely detection of the most important virulent varieties of Escherichia coli is indispensable. To this end, bacterial DNA pools which had previously been categorized were obtained from isolated colonies as well as selected in terms of utilized phenotype; the pools were assessed by two PCR Multiplex for the detection of virulent E. coli eaeA, bfpA, stx1, stx2, ipaH, ST, LT, and aatA genes, with the 16S gene used as DNA control. The system was validated with 66 fecal samples and 44 wastewater samples. At least one positive isolate was detected by a virulent gene among the 20 that were screened. The analysis of fecal samples from children younger than 6 years of age detected frequencies of 25% LT positive strains, 8.3% eae, 8.3% bfpA, 16.7% ipaH, as well as 12.5 % aatA and ST. On the other hand, wastewater samples revealed frequencies of 25.7% eaeA positive, 30.3% stx1, 15.1% LT and 19.7% aatA. This study is an initial step toward carrying out epidemiological field research that will reveal the presence of these bacterial varieties. PMID:24031959

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

Chemical interference with iron transport systems to suppress bacterial growth of Streptococcus pneumoniae.

PubMed

Yang, Xiao-Yan; Sun, Bin; Zhang, Liang; Li, Nan; Han, Junlong; Zhang, Jing; Sun, Xuesong; He, Qing-Yu

2014-01-01

Iron is an essential nutrient for the growth of most bacteria. To obtain iron, bacteria have developed specific iron-transport systems located on the membrane surface to uptake iron and iron complexes such as ferrichrome. Interference with the iron-acquisition systems should be therefore an efficient strategy to suppress bacterial growth and infection. Based on the chemical similarity of iron and ruthenium, we used a Ru(II) complex R-825 to compete with ferrichrome for the ferrichrome-transport pathway in Streptococcus pneumoniae. R-825 inhibited the bacterial growth of S. pneumoniae and stimulated the expression of PiuA, the iron-binding protein in the ferrichrome-uptake system on the cell surface. R-825 treatment decreased the cellular content of iron, accompanying with the increase of Ru(II) level in the bacterium. When the piuA gene (SPD_0915) was deleted in the bacterium, the mutant strain became resistant to R-825 treatment, with decreased content of Ru(II). Addition of ferrichrome can rescue the bacterial growth that was suppressed by R-825. Fluorescence spectral quenching showed that R-825 can bind with PiuA in a similar pattern to the ferrichrome-PiuA interaction in vitro. These observations demonstrated that Ru(II) complex R-825 can compete with ferrichrome for the ferrichrome-transport system to enter S. pneumoniae, reduce the cellular iron supply, and thus suppress the bacterial growth. This finding suggests a novel antimicrobial approach by interfering with iron-uptake pathways, which is different from the mechanisms used by current antibiotics.

Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

PubMed Central

Close, Dan M.; Ripp, Steven; Sayler, Gary S.

2009-01-01

Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed. PMID:22291559

Encyclopedia of bacterial gene circuits whose presence or absence correlate with pathogenicity--a large-scale system analysis of decoded bacterial genomes.

PubMed

Shestov, Maksim; Ontañón, Santiago; Tozeren, Aydin

2015-10-13

Bacterial infections comprise a global health challenge as the incidences of antibiotic resistance increase. Pathogenic potential of bacteria has been shown to be context dependent, varying in response to environment and even within the strains of the same genus. We used the KEGG repository and extensive literature searches to identify among the 2527 bacterial genomes in the literature those implicated as pathogenic to the host, including those which show pathogenicity in a context dependent manner. Using data on the gene contents of these genomes, we identified sets of genes highly abundant in pathogenic but relatively absent in commensal strains and vice versa. In addition, we carried out genome comparison within a genus for the seventeen largest genera in our genome collection. We projected the resultant lists of ortholog genes onto KEGG bacterial pathways to identify clusters and circuits, which can be linked to either pathogenicity or synergy. Gene circuits relatively abundant in nonpathogenic bacteria often mediated biosynthesis of antibiotics. Other synergy-linked circuits reduced drug-induced toxicity. Pathogen-abundant gene circuits included modules in one-carbon folate, two-component system, type-3 secretion system, and peptidoglycan biosynthesis. Antibiotics-resistant bacterial strains possessed genes modulating phagocytosis, vesicle trafficking, cytoskeletal reorganization, and regulation of the inflammatory response. Our study also identified bacterial genera containing a circuit, elements of which were previously linked to Alzheimer's disease. Present study produces for the first time, a signature, in the form of a robust list of gene circuitry whose presence or absence could potentially define the pathogenicity of a microbiome. Extensive literature search substantiated a bulk majority of the commensal and pathogenic circuitry in our predicted list. Scanning microbiome libraries for these circuitry motifs will provide further insights into the complex

A broad range assay for rapid detection and etiologic characterization of bacterial meningitis: performance testing in samples from sub-Sahara.

PubMed

Won, Helen; Yang, Samuel; Gaydos, Charlotte; Hardick, Justin; Ramachandran, Padmini; Hsieh, Yu-Hsiang; Kecojevic, Alexander; Njanpop-Lafourcade, Berthe-Marie; Mueller, Judith E; Tameklo, Tsidi Agbeko; Badziklou, Kossi; Gessner, Bradford D; Rothman, Richard E

2012-09-01

This study aimed to conduct a pilot evaluation of broad-based multiprobe polymerase chain reaction (PCR) in clinical cerebrospinal fluid (CSF) samples compared to local conventional PCR/culture methods used for bacterial meningitis surveillance. A previously described PCR consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by Gram typing, and pathogen-specific probes was designed targeting variable regions of the 16S rRNA gene. The diagnostic performance of the 16S rRNA assay in "127 CSF samples was evaluated in samples from patients from Togo, Africa, by comparison to conventional PCR/culture methods. Our probes detected Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Uniprobe sensitivity and specificity versus conventional PCR were 100% and 54.6%, respectively. Sensitivity and specificity of uniprobe versus culture methods were 96.5% and 52.5%, respectively. Gram-typing probes correctly typed 98.8% (82/83) and pathogen-specific probes identified 96.4% (80/83) of the positives. This broad-based PCR algorithm successfully detected and provided species level information for multiple bacterial meningitis agents in clinical samples. Copyright © 2012 Elsevier Inc. All rights reserved.

Detection of Mixed Infection from Bacterial Whole Genome Sequence Data Allows Assessment of Its Role in Clostridium difficile Transmission

PubMed Central

Eyre, David W.; Cule, Madeleine L.; Griffiths, David; Crook, Derrick W.; Peto, Tim E. A.

2013-01-01

Bacterial whole genome sequencing offers the prospect of rapid and high precision investigation of infectious disease outbreaks. Close genetic relationships between microorganisms isolated from different infected cases suggest transmission is a strong possibility, whereas transmission between cases with genetically distinct bacterial isolates can be excluded. However, undetected mixed infections—infection with ≥2 unrelated strains of the same species where only one is sequenced—potentially impairs exclusion of transmission with certainty, and may therefore limit the utility of this technique. We investigated the problem by developing a computationally efficient method for detecting mixed infection without the need for resource-intensive independent sequencing of multiple bacterial colonies. Given the relatively low density of single nucleotide polymorphisms within bacterial sequence data, direct reconstruction of mixed infection haplotypes from current short-read sequence data is not consistently possible. We therefore use a two-step maximum likelihood-based approach, assuming each sample contains up to two infecting strains. We jointly estimate the proportion of the infection arising from the dominant and minor strains, and the sequence divergence between these strains. In cases where mixed infection is confirmed, the dominant and minor haplotypes are then matched to a database of previously sequenced local isolates. We demonstrate the performance of our algorithm with in silico and in vitro mixed infection experiments, and apply it to transmission of an important healthcare-associated pathogen, Clostridium difficile. Using hospital ward movement data in a previously described stochastic transmission model, 15 pairs of cases enriched for likely transmission events associated with mixed infection were selected. Our method identified four previously undetected mixed infections, and a previously undetected transmission event, but no direct transmission between

Reduction of Airborne Bacterial Burden in the OR by Installation of Unidirectional Displacement Airflow (UDF) Systems.

PubMed

Fischer, Sebastian; Thieves, Martin; Hirsch, Tobias; Fischer, Klaus-Dieter; Hubert, Helmine; Beppler, Steffen; Seipp, Hans-Martin

2015-08-13

Intraoperative bacterial contamination is a major risk factor for postoperative wound infections. This study investigated the influence of type of ventilation system on intraoperative airborne bacterial burden before and after installation of unidirectional displacement air flow systems. We microbiologically monitored 1286 surgeries performed by a single surgical team that moved from operating rooms (ORs) equipped with turbulent mixing ventilation (TMV, according to standard DIN-1946-4 [1999], ORs 1, 2, and 3) to ORs with unidirectional displacement airflow (UDF, according to standard DIN-1946-4, annex D [2008], ORs 7 and 8). The airborne bacteria were collected intraoperatively with sedimentation plates. After incubation for 48 h, we analyzed the average number of bacteria per h, peak values, and correlation to surgery duration. In addition, we compared the last 138 surgeries in ORs 1-3 with the first 138 surgeries in ORs 7 and 8. Intraoperative airborne bacterial burden was 5.4 CFU/h, 5.5 CFU/h, and 6.1 CFU/h in ORs 1, 2, and 3, respectively. Peak values of burden were 10.7 CFU/h, 11.1 CFU/h, and 11.0 CFU/h in ORs 1, 2, and 3, respectively). With the UDF system, the intraoperative airborne bacterial burden was reduced to 0.21 CFU/h (OR 7) and 0.35 CFU/h (OR 8) on average (p<0.01). Accordingly, peak values decreased to 0.9 CFU/h and 1.0 CFU/h in ORs 7 and 8, respectively (p<0.01). Airborne bacterial burden increased linearly with surgery duration in ORs 1-3, but the UDF system in ORs 7 and 8 kept bacterial levels constantly low (<3 CFU/h). A comparison of the last 138 surgeries before with the first 138 surgeries after changing ORs revealed a 94% reduction in average airborne bacterial burden (5 CFU/h vs. 0.29 CFU/h, p<0.01). The unidirectional displacement airflow, which fulfills the requirements of standard DIN-1946-4 annex D of 2008, is an effective ventilation system that reduces airborne bacterial burden under real clinical conditions by more than 90

Spore-forming organisms in platelet concentrates: a challenge in transfusion bacterial safety.

PubMed

Störmer, M; Vollmer, T; Kleesiek, K; Dreier, J

2008-12-01

Bacterial detection and pathogen reduction are widely used methods of minimizing the risk of transfusion-transmitted bacterial infection. But, bacterial spores are highly resistant to chemical and physical agents. In this study, we assessed the bacterial proliferation of spore-forming organisms seeded into platelet concentrates (PCs) to demonstrate that spores can enter the vegetative state in PCs during storage. In the in vitro study, PCs were inoculated with 1-10 spores mL(-1)of Bacillus cereus (n = 1), Bacillus subtilis (n = 2) and Clostridium sporogenes (n = 2). Sampling was performed during 6-day aerobic storage at 22 degrees C. The presence of bacteria was assessed by plating culture, automated culture and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Spores of the C. sporogenes do not enter the vegetative phase under PC storage conditions, whereas B. subtilis and B. cereus showed growth in the PC and could be detected using RT-PCR and automated culture. Depending on the species and inoculums, bacterial spores may enter the vegetative phase during PC storage and can be detected by bacterial detection methods.

Viral-bacterial associations in acute apical abscesses.

PubMed

Ferreira, Dennis C; Rôças, Isabela N; Paiva, Simone S M; Carmo, Flávia L; Cavalcante, Fernanda S; Rosado, Alexandre S; Santos, Kátia R N; Siqueira, José F

2011-08-01

Viral-bacterial and bacterial synergism have been suggested to contribute to the pathogenesis of several human diseases. This study sought to investigate the possible associations between 9 candidate endodontic bacterial pathogens and 9 human viruses in samples from acute apical abscesses. DNA extracts from purulent exudate aspirates of 33 cases of acute apical abscess were surveyed for the presence of 9 selected bacterial species using a 16S ribosomal RNA gene-based nested polymerase chain reaction (PCR) approach. Single or nested PCR assays were used for detection of the human papillomavirus (HPV) and herpesviruses types 1 to 8. Two-thirds of the abscess samples were positive for at least one of the target viruses. Specifically, the most frequently detected viruses were HHV-8 (54.5%); HPV (9%); and varicella zoster virus (VZV), Epstein-Barr virus (EBV), and HHV-6 (6%). Bacterial DNA was present in all cases and the most prevalent bacterial species were Treponema denticola (70%), Tannerella forsythia (67%), Porphyromonas endodontalis (67%), Dialister invisus (61%), and Dialister pneumosintes (57.5%). HHV-8 was positively associated with 7 of the target bacterial species and HPV with 4, but all these associations were weak. Several bacterial pairs showed a moderate positive association. Viral coinfection was found in 6 abscess cases, but no significant viral association could be determined. Findings demonstrated that bacterial and viral DNA occurred concomitantly in two-thirds of the samples from endodontic abscesses. Although this may suggest a role for viruses in the etiology of apical abscesses, the possibility also exists that the presence of viruses in abscess samples is merely a consequence of the bacterially induced disease process. Further studies are necessary to clarify the role of these viral-bacterial interactions, if any, in the pathogenesis of acute apical abscesses. Copyright © 2011 Mosby, Inc. All rights reserved.

Phytoplankton production systems in a shellfish hatchery: variations of the bacterial load and diversity of vibrios.

PubMed

Dubert, J; Fernández-Pardo, A; Nóvoa, S; Barja, J L; Prado, S

2015-06-01

Outbreaks of disease caused by some Vibrio species represent the main production bottleneck in shellfish hatcheries. Although the phytoplankton used as food is one of the main sources of bacteria, studies of the associated bacterial populations, specifically vibrios, are scarce. The aim of the study was the microbiological monitoring of the microalgae as the first step in assessing the risk disease for bivalve cultures. Two phytoplankton production systems were sampled weekly throughout 1-year period in a bivalve hatchery. Quantitative analysis revealed high levels of marine heterotrophic bacteria in both systems throughout the study. Presumptive vibrios were detected occasionally and at low concentrations. In most of the cases, they belonged to the Splendidus and Harveyi clades. The early detection of vibrios in the microalgae may be the key for a successful bivalve culture. Their abundance and diversity were affected by factors related to the hatchery environment. This work represents the first long study where the presence of vibrios was evaluated rigorously in phytoplankton production systems and provides a suitable microbiological protocol to control and guarantee the quality of the algal cultures to avoid the risk of transferring potential pathogens to shellfish larvae and/or broodstock. © 2015 The Society for Applied Microbiology.

Visual Estimation of Bacterial Growth Level in Microfluidic Culture Systems.

PubMed

Kim, Kyukwang; Kim, Seunggyu; Jeon, Jessie S

2018-02-03

Microfluidic devices are an emerging platform for a variety of experiments involving bacterial cell culture, and has advantages including cost and convenience. One inevitable step during bacterial cell culture is the measurement of cell concentration in the channel. The optical density measurement technique is generally used for bacterial growth estimation, but it is not applicable to microfluidic devices due to the small sample volumes in microfluidics. Alternately, cell counting or colony-forming unit methods may be applied, but these do not work in situ; nor do these methods show measurement results immediately. To this end, we present a new vision-based method to estimate the growth level of the bacteria in microfluidic channels. We use Fast Fourier transform (FFT) to detect the frequency level change of the microscopic image, focusing on the fact that the microscopic image becomes rough as the number of cells in the field of view increases, adding high frequencies to the spectrum of the image. Two types of microfluidic devices are used to culture bacteria in liquid and agar gel medium, and time-lapsed images are captured. The images obtained are analyzed using FFT, resulting in an increase in high-frequency noise proportional to the time passed. Furthermore, we apply the developed method in the microfluidic antibiotics susceptibility test by recognizing the regional concentration change of the bacteria that are cultured in the antibiotics gradient. Finally, a deep learning-based data regression is performed on the data obtained by the proposed vision-based method for robust reporting of data.

Nest Material Shapes Eggs Bacterial Environment.

PubMed

Ruiz-Castellano, Cristina; Tomás, Gustavo; Ruiz-Rodríguez, Magdalena; Martín-Gálvez, David; Soler, Juan José

2016-01-01

Selective pressures imposed by pathogenic microorganisms to embryos have selected in hosts for a battery of antimicrobial lines of defenses that includes physical and chemical barriers. Due to the antimicrobial properties of volatile compounds of green plants and of chemicals of feather degrading bacteria, the use of aromatic plants and feathers for nest building has been suggested as one of these barriers. However, experimental evidence suggesting such effects is scarce in the literature. During two consecutive years, we explored experimentally the effects of these nest materials on loads of different groups of bacteria (mesophilic bacteria, Enterobacteriaceae, Staphylococcus and Enterococcus) of eggshells in nests of spotless starlings (Sturnus unicolor) at the beginning and at the end of the incubation period. This was also explored in artificial nests without incubation activity. We also experimentally increased bacterial density of eggs in natural and artificial nests and explored the effects of nest lining treatments on eggshell bacterial load. Support for the hypothetical antimicrobial function of nest materials was mainly detected for the year and location with larger average values of eggshell bacterial density. The beneficial effects of feathers and plants were more easily detected in artificial nests with no incubation activity, suggesting an active role of incubation against bacterial colonization of eggshells. Pigmented and unpigmented feathers reduced eggshell bacterial load in starling nests and artificial nest boxes. Results from artificial nests allowed us to discuss and discard alternative scenarios explaining the detected association, particularly those related to the possible sexual role of feathers and aromatic plants in starling nests. All these results considered together confirm the antimicrobial functionality mainly of feathers but also of plants used as nest materials, and highlight the importance of temporally and geographically

Nest Material Shapes Eggs Bacterial Environment

PubMed Central

Ruiz-Castellano, Cristina; Tomás, Gustavo; Ruiz-Rodríguez, Magdalena; Martín-Gálvez, David; Soler, Juan José

2016-01-01

Selective pressures imposed by pathogenic microorganisms to embryos have selected in hosts for a battery of antimicrobial lines of defenses that includes physical and chemical barriers. Due to the antimicrobial properties of volatile compounds of green plants and of chemicals of feather degrading bacteria, the use of aromatic plants and feathers for nest building has been suggested as one of these barriers. However, experimental evidence suggesting such effects is scarce in the literature. During two consecutive years, we explored experimentally the effects of these nest materials on loads of different groups of bacteria (mesophilic bacteria, Enterobacteriaceae, Staphylococcus and Enterococcus) of eggshells in nests of spotless starlings (Sturnus unicolor) at the beginning and at the end of the incubation period. This was also explored in artificial nests without incubation activity. We also experimentally increased bacterial density of eggs in natural and artificial nests and explored the effects of nest lining treatments on eggshell bacterial load. Support for the hypothetical antimicrobial function of nest materials was mainly detected for the year and location with larger average values of eggshell bacterial density. The beneficial effects of feathers and plants were more easily detected in artificial nests with no incubation activity, suggesting an active role of incubation against bacterial colonization of eggshells. Pigmented and unpigmented feathers reduced eggshell bacterial load in starling nests and artificial nest boxes. Results from artificial nests allowed us to discuss and discard alternative scenarios explaining the detected association, particularly those related to the possible sexual role of feathers and aromatic plants in starling nests. All these results considered together confirm the antimicrobial functionality mainly of feathers but also of plants used as nest materials, and highlight the importance of temporally and geographically

Complexity of Bacterial Communities in a River-Floodplain System (Danube, Austria)

PubMed Central

Besemer, Katharina; Moeseneder, Markus M.; Arrieta, Jesus M.; Herndl, Gerhard J.; Peduzzi, Peter

2005-01-01

Natural floodplains play an essential role in the processing and decomposition of organic matter and in the self-purification ability of rivers, largely due to the activity of bacteria. Knowledge about the composition of bacterial communities and its impact on organic-matter cycling is crucial for the understanding of ecological processes in river-floodplain systems. Particle-associated and free-living bacterial assemblages from the Danube River and various floodplain pools with different hydrological characteristics were investigated using terminal restriction fragment length polymorphism analysis. The particle-associated bacterial community exhibited a higher number of operational taxonomic units (OTUs) and was more heterogeneous in time and space than the free-living community. The temporal dynamics of the community structure were generally higher in isolated floodplain pools. The community structures of the river and the various floodplain pools, as well as those of the particle-associated and free-living bacteria, differed significantly. The compositional dynamics of the planktonic bacterial communities were related to changes in the algal biomass, temperature, and concentrations of organic and inorganic nutrients. The OTU richness of the free-living community was correlated with the concentration and origin of organic matter and the concentration of inorganic nutrients, while no correlation with the OTU richness of the particle-associated assemblage was found. Our results demonstrate the importance of the river-floodplain interactions and the influence of damming and regulation on the bacterial-community composition. PMID:15691909

Bench-to-bedside review: Quorum sensing and the role of cell-to-cell communication during invasive bacterial infection

PubMed Central

Asad, Shadaba; Opal, Steven M

2008-01-01

Bacteria communicate extensively with each other and employ a communal approach to facilitate survival in hostile environments. A hierarchy of cell-to-cell signaling pathways regulates bacterial growth, metabolism, biofilm formation, virulence expression, and a myriad of other essential functions in bacterial populations. The notion that bacteria can signal each other and coordinate their assault patterns against susceptible hosts is now well established. These signaling networks represent a previously unrecognized survival strategy by which bacterial pathogens evade antimicrobial defenses and overwhelm the host. These quorum sensing communication signals can transgress species barriers and even kingdom barriers. Quorum sensing molecules can regulate human transcriptional programs to the advantage of the pathogen. Human stress hormones and cytokines can be detected by bacterial quorum sensing systems. By this mechanism, the pathogen can detect the physiologically stressed host, providing an opportunity to invade when the patient is most vulnerable. These rather sophisticated, microbial communication systems may prove to be a liability to pathogens as they make convenient targets for therapeutic intervention in our continuing struggle to control microbial pathogens. PMID:19040778

Bacterial Community Shift Drives Antibiotic Resistance Promotion during Drinking Water Chlorination.

PubMed

Jia, Shuyu; Shi, Peng; Hu, Qing; Li, Bing; Zhang, Tong; Zhang, Xu-Xiang

2015-10-20

For comprehensive insights into the effects of chlorination, a widely used disinfection technology, on bacterial community and antibiotic resistome in drinking water, this study applied high-throughput sequencing and metagenomic approaches to investigate the changing patterns of antibiotic resistance genes (ARGs) and bacterial community in a drinking water treatment and distribution system. At genus level, chlorination could effectively remove Methylophilus, Methylotenera, Limnobacter, and Polynucleobacter, while increase the relative abundance of Pseudomonas, Acidovorax, Sphingomonas, Pleomonas, and Undibacterium in the drinking water. A total of 151 ARGs within 15 types were detectable in the drinking water, and chlorination evidently increased their total relative abundance while reduced their diversity in the opportunistic bacteria (p < 0.05). Residual chlorine was identified as the key contributing factor driving the bacterial community shift and resistome alteration. As the dominant persistent ARGs in the treatment and distribution system, multidrug resistance genes (mainly encoding resistance-nodulation-cell division transportation system) and bacitracin resistance gene bacA were mainly carried by chlorine-resistant bacteria Pseudomonas and Acidovorax, which mainly contributed to the ARGs abundance increase. The strong correlation between bacterial community shift and antibiotic resistome alteration observed in this study may shed new light on the mechanism behind the chlorination effects on antibiotic resistance.

Bacterial biomarkers thermally released from dissolved organic matter

USGS Publications Warehouse

Greenwood, P.F.; Leenheer, J.A.; McIntyre, C.; Berwick, L.; Franzmann, P.D.

2006-01-01

Hopane biomarker products were detected using microscale sealed vessel (MSSV) pyrolysis gas chromatography-mass spectrometry (GC-MS) analysis of dissolved organic matter from natural aquatic systems colonised by bacterial populations. MSSV pyrolysis can reduce the polyhydroxylated alkyl side chain of bacteriohopanepolyols, yielding saturated hopane products which are more amenable to GC-MS detection than their functionalised precursors. This example demonstrates how the thermal conditions of MSSV pyrolysis can reduce the biologically-inherited structural functionality of naturally occurring organic matter such that additional structural fragments can be detected using GC methods. This approach complements traditional analytical pyrolysis methods by providing additional speciation information useful for establishing the structures and source inputs of recent or extant organic material. ?? 2006.

Precise, High-throughput Analysis of Bacterial Growth.

PubMed

Kurokawa, Masaomi; Ying, Bei-Wen

2017-09-19

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Plant Pest Detection Using an Artificial Nose System: A Review.

PubMed

Cui, Shaoqing; Ling, Peter; Zhu, Heping; Keener, Harold M

2018-01-28

This paper reviews artificial intelligent noses (or electronic noses) as a fast and noninvasive approach for the diagnosis of insects and diseases that attack vegetables and fruit trees. The particular focus is on bacterial, fungal, and viral infections, and insect damage. Volatile organic compounds (VOCs) emitted from plants, which provide functional information about the plant's growth, defense, and health status, allow for the possibility of using noninvasive detection to monitor plants status. Electronic noses are comprised of a sensor array, signal conditioning circuit, and pattern recognition algorithms. Compared with traditional gas chromatography-mass spectrometry (GC-MS) techniques, electronic noses are noninvasive and can be a rapid, cost-effective option for several applications. However, using electronic noses for plant pest diagnosis is still in its early stages, and there are challenges regarding sensor performance, sampling and detection in open areas, and scaling up measurements. This review paper introduces each element of electronic nose systems, especially commonly used sensors and pattern recognition methods, along with their advantages and limitations. It includes a comprehensive comparison and summary of applications, possible challenges, and potential improvements of electronic nose systems for different plant pest diagnoses.

Engineering Rugged Field Assays to Detect Hazardous Chemicals Using Spore-Based Bacterial Biosensors.

PubMed

Wynn, Daniel; Deo, Sapna; Daunert, Sylvia

2017-01-01

Bacterial whole cell-based biosensors have been genetically engineered to achieve selective and reliable detection of a wide range of hazardous chemicals. Although whole-cell biosensors demonstrate many advantages for field-based detection of target analytes, there are still some challenges that need to be addressed. Most notably, their often modest shelf life and need for special handling and storage make them challenging to use in situations where access to reagents, instrumentation, and expertise are limited. These problems can be circumvented by developing biosensors in Bacillus spores, which can be engineered to address all of these concerns. In its sporulated state, a whole cell-based biosensor has a remarkably long life span and is exceptionally resistant to environmental insult. When these spores are germinated for use in analytical techniques, they show no loss in performance, even after long periods of storage under harsh conditions. In this chapter, we will discuss the development and use of whole cell-based sensors, their adaptation to spore-based biosensors, their current applications, and future directions in the field. © 2017 Elsevier Inc. All rights reserved.

Bacterial nitrification in chloraminated water supplies.

PubMed Central

Cunliffe, D A

1991-01-01

Nitrifying bacteria were detected in 64% of samples collected from five chloraminated water supplies in South Australia and in 20.7% of samples that contained more than 5.0 mg of monochloramine per liter. Laboratory experiments confirmed that nitrifying bacteria are relatively resistant to the disinfectant. Increased numbers of the bacteria were associated with accelerated decays of monochloramine within distribution systems. The combination of increased concentrations of oxidized nitrogen with decreased total chlorine residuals can be used as a rapid indicator of bacterial nitrification. PMID:1781698

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks.

PubMed

Jers, Carsten; Soufi, Boumediene; Grangeasse, Christophe; Deutscher, Josef; Mijakovic, Ivan

2008-08-01

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.

Measurement of Bacterial Bioluminescence Intensity and Spectrum: Current Physical Techniques and Principles.

PubMed

Jia, Kun; Ionescu, Rodica Elena

2016-01-01

: Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

Defense islands in bacterial and archaeal genomes and prediction of novel defense systems.

PubMed

Makarova, Kira S; Wolf, Yuri I; Snir, Sagi; Koonin, Eugene V

2011-11-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic "sinks" that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.

Bacterial reduction of Cr(VI) at technical scale--the Malaysian experience.

PubMed

Zakaria, Zainul Akmar; Ahmad, Wan Azlina; Zakaria, Zainoha; Razali, Firdausi; Karim, Norsuhada Abdul; Sum, Mohamad Md; Sidek, Mohd Saufi Mohd

2012-07-01

The bacterial reduction of Cr(VI) from industrial wastewater was evaluated using a 2.0-m(3) bioreactor. Liquid pineapple waste was used as a nutrient for the biofilm community formed inside the bioreactor. The use of rubber wood sawdust as packing material was able to immobilize more than 10(6) CFU mL(-1) of Acinetobacter haemolyticus cells after 3 days of contact time. Complete reduction of 15-240 mg L(-1) of Cr(VI) was achieved even after 3 months of bioreactor operation. Cr(VI) was not detected in the final effluent fraction indicating complete removal of Cr from solution from the flocculation/coagulation step and the unlikely re-oxidation of Cr(III) into Cr(VI). Impatiens balsamina L. and Gomphrena globosa L. showed better growth in the presence of soil-sludge mixture compared to Coleus scutellarioides (L.) Benth. Significant amounts of Cr accumulated at different sections of the plants indicate its potential application in Cr phytoremediation effort. The bacterial-based system was also determined not to be detrimental to human health based on the low levels of Cr detected in the hair and nail samples of the plant operators. Thus, it can be said that bacterial-based Cr(VI) treatment system is a feasible alternative to the conventional system especially for lower Cr(VI) concentrations, where sludge generated can be used as growth supplement for ornamental plant as well as not detrimental to the health of the workers.

Bacterial community composition of chronic periodontitis and novel oral sampling sites for detecting disease indicators

PubMed Central

2014-01-01

Background Periodontitis is an infectious and inflammatory disease of polymicrobial etiology that can lead to the destruction of bones and tissues that support the teeth. The management of chronic periodontitis (CP) relies heavily on elimination or at least control of known pathogenic consortia associated with the disease. Until now, microbial plaque obtained from the subgingival (SubG) sites has been the primary focus for bacterial community analysis using deep sequencing. In addition to the use of SubG plaque, here, we investigated whether plaque obtained from supragingival (SupG) and tongue dorsum sites can serve as alternatives for monitoring CP-associated bacterial biomarkers. Results Using SubG, SupG, and tongue plaque DNA from 11 healthy and 13 diseased subjects, we sequenced V3 regions (approximately 200 bases) of the 16S rRNA gene using Illumina sequencing. After quality filtering, approximately 4.1 million sequences were collapsed into operational taxonomic units (OTUs; sequence identity cutoff of >97%) that were classified to a total of 19 phyla spanning 114 genera. Bacterial community diversity and overall composition was not affected by health or disease, and multiresponse permutation procedure (MRPP) on Bray-Curtis distance measures only supported weakly distinct bacterial communities in SubG and tongue plaque depending on health or disease status (P < 0.05). Nonetheless, in SubG and tongue sites, the relative abundance of Firmicutes was increased significantly from health to disease and members of Synergistetes were found in higher abundance across all sites in disease. Taxa indicative of CP were identified in all three locations (for example, Treponema denticola, Porphyromonas gingivalis, Synergistes oral taxa 362 and 363). Conclusions For the first time, this study demonstrates that SupG and tongue dorsum plaque can serve as alternative sources for detecting and enumerating known and novel bacterial biomarkers of CP. This finding is clinically

New detection targets for amyloid-reactive probes: spectroscopic recognition of bacterial spores

NASA Astrophysics Data System (ADS)

Jones, Guilford, II; Landsman, Pavel

2005-05-01

We report characteristic changes in fluorescence of amyloid-binding dyes Thioflavin T (TfT), pinacyanol (PIN) and related dyes, caused by their interaction with suspended Bacillus spore cultures (B. subtilis, B thuringiensis). The gain in TfT emission in the presence of spores allowed their immediate detection in aqueous suspensions, with a sensitivity limit of < 105 spores per ml. The spectroscopic signatures are consistent with a large number of binding sites for the two dyes on spore coats. The possible structural relationship of these dye binding loci with characteristic motifs (β-stacks) of amyloid deposits and other misfolded protein formations suggests new designs for probing biocontamination and also for clinical studies of non-microbial human pathogens (e.g., amyloid-related protein aggregates in prion-related transmissible encephalopathies or in Alzheimer's disease). Also reported is a special screening technique that was designed and used herein for calibration of new detection probes and assays for spore detection. It employed spectroscopic interactions between the candidate amyloid stains and poly(vinylpyrrolidone)-coated colloid silica (Percoll) nanoparticles that also display remarkable parallelism with the corresponding dye-amyloid and dye-spore reactivities. Percoll may thus find new applications as a convenient non-biological structural model mimicking the putative probe-targeted motifs in both classes of bioanalytes. These findings are important in the design of new probes and assays for important human pathogens (i.e. bacterial spores and amyloidogenic protein aggregates).

The Use of Different Irrigation Techniques to Decrease Bacterial Loads in Healthy and Diabetic Patients with Asymptomatic Apical Periodontitis.

PubMed

Ghoneim, Mai; Saber, Shehab ElDin; El-Badry, Tarek; Obeid, Maram; Hassib, Nehal

2016-12-15

Diabetes mellitus is a multisystem disease which weakens the human's immunity. Subsequently, it worsens the sequelae of apical periodontitis by raising a fierce bacterial trait due to the impaired host response. This study aimed to estimate bacterial reduction after using different irrigation techniques in systemically healthy and diabetic patients with asymptomatic apical periodontitis. Enterococcus faecalis , Peptostreptococcus micros , and Fusobacterium necleatum bacteria were chosen, as they are the most common and prevailing strains found in periodontitis. Bacterial samples were retrieved from necrotic root canals of systemically healthy and diabetic patients, before and after endodontic cleaning and shaping by using two different irrigation techniques; the conventional one and the EndoVac system. Quantitive polymerase chain reaction (qPCR) was utilised to detect the reduction in the bacterial count. The EndoVac irrigation system was effective in reducing bacteria, especially Peptostreptococcus micros in the diabetic group when compared to conventional irrigation technique with a statistically significant difference. The EndoVac can be considered as a promising tool in combination with irrigant solution to defeat the bacterial colonies living in the root canal system. Additional studies ought to be done to improve the means of bacterial clearance mainly in immune-compromised individuals.

Phylogenetic characterization and in situ detection of bacterial communities associated with seahorses (Hippocampus guttulatus) in captivity.

PubMed

Balcázar, José L; Lee, Natuschka M; Pintado, José; Planas, Miquel

2010-03-01

Although there are several studies describing bacteria associated with marine fish, the bacterial composition associated with seahorses has not been extensively investigated since these studies have been restricted to the identification of bacterial pathogens. In this study, the phylogenetic affiliation of seahorse-associated bacteria was assessed by 16S rRNA gene sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rRNA analysis. Both methods revealed that Vibrionaceae was the dominant population in Artemia sp. (live prey) and intestinal content of the seahorses, while Rhodobacteraceae was dominant in water samples from the aquaculture system and cutaneous mucus of the seahorses. To our knowledge, this is the first time that bacterial communities associated with healthy seahorses in captivity have been described. Crown Copyright 2010. Published by Elsevier GmbH. All rights reserved.

Influence of sugarbeet tillage systems on rhizoctonia-bacterial root rot complex

USDA-ARS?s Scientific Manuscript database

The Rhizoctonia-bacterial root rot complex on sugarbeet caused by Rhizoctonia solani and Leuconostoc mesenteroides can cause significant yield losses. To investigate the impact of different tillage systems on this complex, field studies were conducted from 2009 to 2011. Split blocks with conventio...

Seasonal variation in detection of bacterial DNA in arthritic stifle joints of dogs with cranial cruciate ligament rupture using PCR amplification of the 16S rRNA gene.

PubMed

Muir, Peter; Fox, Robin; Wu, Qiang; Baker, Theresa A; Zitzer, Nina C; Hudson, Alan P; Manley, Paul A; Schaefer, Susan L; Hao, Zhengling

2010-02-24

An underappreciated cause and effect relationship between environmental bacteria and arthritis may exist. Previously, we found that stifle arthritis in dogs was associated with the presence of environmental bacteria within synovium. Cranial cruciate ligament rupture (CCLR) is often associated with stifle arthritis in dogs. We now wished to determine whether seasonal variation in detection of bacterial material may exist in affected dogs, and to also conduct analyses of both synovium and synovial fluid. We also wished to analyze a larger clone library of the 16S rRNA gene to further understanding of the microbial population in the canine stifle. Synovial biopsies were obtained from 117 affected dogs from January to December 2006. Using PCR, synovium and synovial fluid were tested for Borrelia burgdorferi and Stenotrophomonas maltophilia DNA. Broad-ranging 16S rRNA primers were also used and PCR products were cloned and sequenced for bacterial identification. Overall, 41% of arthritic canine stifle joints contained bacterial DNA. Detection of bacterial DNA in synovial fluid samples was increased, when compared with synovium (p<0.01). Detection rates were highest in the winter and spring and lowest in the summer period, suggesting environmental factors influence the risk of translocation to the stifle. Organisms detected were predominately Gram's negative Proteobacteria, particularly the orders Rhizobiales (32.8% of clones) and Burkholderiales (20.0% of clones), usually as part of a polymicrobial population. PCR-positivity was inversely correlated with severity of arthritis assessed radiographically and with dog age. Bacterial translocation to the canine stifle may be associated with changes to the indoor environment. Copyright 2009 Elsevier B.V. All rights reserved.

Probing the substrate specificity of the bacterial Pnkp/Hen1 RNA repair system using synthetic RNAs

PubMed Central

Zhang, Can; Chan, Chio Mui; Wang, Pei; Huang, Raven H.

2012-01-01

Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed. PMID:22190744

CHANGES IN BACTERIAL COMPOSITION OF BIOFILM IN A METROPOLITAN DRINKING WATER DISTRIBUTION SYSTEM

EPA Science Inventory

This study examined the development of bacterial biofilms within a metropolitan distribution system. The distribution system is fed with different source water (i.e., groundwater, GW and surface water, SW) and undergoes different treatment processes in separate facilities. The b...

Bacterial Community Structure and Physiological State within an Industrial Phenol Bioremediation System

PubMed Central

Whiteley, Andrew S.; Bailey, Mark J.

2000-01-01

The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the α, β and γ subclasses of the class Proteobacteria [α-, β-, and γ-Proteobacteria, respectively], the Cytophaga-Flavobacterium group, and the Pseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the Cytophaga-Flavobacterium group and to the γ-Proteobacteria. The α subclass Proteobacteria were of low relative abundance throughout the treatment system whilst the β subclass of the Proteobacteria exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The γ subclass of the Proteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of the Cytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

PubMed

Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

2012-01-01

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

Nucleic acid detection system and method for detecting influenza

DOEpatents

Cai, Hong; Song, Jian

2015-03-17

The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture.

PubMed

Kumar, Jyoti; Dixit, Shivendra Kumar; Kumar, Rajiv

2015-09-01

This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture. M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA), Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture. A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR) based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons. All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S rRNA gene sequence also revealed the identity of 98% with published

Bacterial Reduction after Gutta-Percha Removal with Single vs. Multiple Instrument Systems.

PubMed

Xavier, Felipe; Nevares, Giselle; Gominho, Luciana; Rodrigues, Renata; Cassimiro, Marcely; Romeiro, Kaline; Albuquerque, Diana

2018-01-01

The aim of this study was to evaluate the effectiveness of a reciprocating single-instrument system (Reciproc-REC) compared with combined continuously rotating multiple-instrument systems [D-Race (DR) and BioRace (BR)] in reducing Enterococcus faecalis (E.f.) after gutta-percha removal. Forty-six extracted human maxillary incisors were prepared and contaminated with E.f. strain (ATCC 29212) for 30 days. The samples were obturated and randomly divided into two experimental groups for gutta-percha removal ( n =23): a REC group (R50) and a DR/BR group (DR1, DR2 and BR6). A standardized irrigation with 0.9% saline solution was performed. Root canal samples were taken with paper points before (S1) and after (S2) the removal of gutta-percha to establish bacterial quantification by culture. The time required for gutta-percha removal was also recorded. Positive and negative control groups ( n =6) were used to test bacterial viability and control asepsis, respectively. Data were analysed using t -Student and one-way ANOVA tests (5% margin of error). The mean percentage of bacterial reduction was significantly higher in DR/BR group (84.2%) than in REC group (72.3%) ( P <0.05). The mean time for obturation removal was 74.00 sec in REC group and 107.53 sec in DR/BR group ( P <0.05). The combined continuously rotating multiple-instrument system was more effective in reducing bacteria after the removal of gutta-percha than the single-instrument system. None of the tested systems was able to completely eliminate root canal infection after gutta-percha removal. Thus, additional techniques should be considered.

Interplay of heritage and habitat in the distribution of bacterial signal transduction systems.

PubMed

Galperin, Michael Y; Higdon, Roger; Kolker, Eugene

2010-04-01

Comparative analysis of the complete genome sequences from a variety of poorly studied organisms aims at predicting ecological and behavioral properties of these organisms and helping in characterizing their habitats. This task requires finding appropriate descriptors that could be correlated with the core traits of each system and would allow meaningful comparisons. Using the relatively simple bacterial models, first attempts have been made to introduce suitable metrics to describe the complexity of organism's signaling machinery, which included introducing the "bacterial IQ" score. Here, we use an updated census of prokaryotic signal transduction systems to improve this parameter and evaluate its consistency within selected bacterial phyla. We also introduce a more elaborate descriptor, a set of profiles of relative abundance of members of each family of signal transduction proteins encoded in each genome. We show that these family profiles are well conserved within each genus and are often consistent within families of bacteria. Thus, they reflect evolutionary relationships between organisms as well as individual adaptations of each organism to its specific ecological niche.

Portable modular detection system

DOEpatents

Brennan, James S [Rodeo, CA; Singh, Anup [Danville, CA; Throckmorton, Daniel J [Tracy, CA; Stamps, James F [Livermore, CA

2009-10-13

Disclosed herein are portable and modular detection devices and systems for detecting electromagnetic radiation, such as fluorescence, from an analyte which comprises at least one optical element removably attached to at least one alignment rail. Also disclosed are modular detection devices and systems having an integrated lock-in amplifier and spatial filter and assay methods using the portable and modular detection devices.

Enteric bacterial pathogen detection in southern sea otters (Enhydra lutris nereis) is associated with coastal urbanization and freshwater runoff

PubMed Central

Miller, Melissa A.; Byrne, Barbara A.; Jang, Spencer S.; Dodd, Erin M.; Dorfmeier, Elene; Harris, Michael D.; Ames, Jack; Paradies, David; Worcester, Karen; Jessup, David A.; Miller, Woutrina A.

2009-01-01

Although protected for nearly a century, California’s sea otters have been slow to recover, in part due to exposure to fecally-associated protozoal pathogens like Toxoplasma gondii and Sarcocystis neurona. However, potential impacts from exposure to fecal bacteria have not been systematically explored. Using selective media, we examined feces from live and dead sea otters from California for specific enteric bacterial pathogens (Campylobacter, Salmonella, Clostridium perfringens, C. difficile and Escherichia coli O157:H7), and pathogens endemic to the marine environment (Vibrio cholerae, V. parahaemolyticus and Plesiomonas shigelloides). We evaluated statistical associations between detection of these pathogens in otter feces and demographic or environmental risk factors for otter exposure, and found that dead otters were more likely to test positive for C. perfringens, Campylobacter and V. parahaemolyticus than were live otters. Otters from more urbanized coastlines and areas with high freshwater runoff (near outflows of rivers or streams) were more likely to test positive for one or more of these bacterial pathogens. Other risk factors for bacterial detection in otters included male gender and fecal samples collected during the rainy season when surface runoff is maximal. Similar risk factors were reported in prior studies of pathogen exposure for California otters and their invertebrate prey, suggesting that land-sea transfer and/or facilitation of pathogen survival in degraded coastal marine habitat may be impacting sea otter recovery. Because otters and humans share many of the same foods, our findings may also have implications for human health. PMID:19720009

Quaternized magnetic nanoparticles-fluorescent polymer system for detection and identification of bacteria.

PubMed

Wan, Yi; Sun, Yan; Qi, Peng; Wang, Peng; Zhang, Dun

2014-05-15

Nanomaterial-based 'chemical nose' sensor with sufficient sensing specificity is a useful analytical tool for the detection of toxicologically important substances in complicated biological systems. A sensor array containing three quaternized magnetic nanoparticles (q-MNPs)-fluorescent polymer systems has been designed to identify and quantify bacteria. The bacterial cell membranes disrupt the q-MNP-fluorescent polymer, generating unique fluorescence response array. The response intensity of the array is dependent on the level of displacement determined by the relative q-MNP-fluorescent polymer binding strength and bacteria cells-MNP interaction. These characteristic responses show a highly repeatable bacteria cells and can be differentiated by linear discriminant analysis (LDA). Based on the array response matrix from LDA, our approach has been used to measure bacteria with an accuracy of 87.5% for 10(7) cfu mL(-1) within 20 min. Combined with UV-vis measurement, the method can be successfully performed to identify and detect eight different pathogen samples with an accuracy of 96.8%. The measurement system has a potential for further applications and provides a facile and simple method for the rapid analysis of protein, DNA, and pathogens. Copyright © 2013 Elsevier B.V. All rights reserved.

Bacterial and viral pathogens detected in sea turtles stranded along the coast of Tuscany, Italy.

PubMed

Fichi, G; Cardeti, G; Cersini, A; Mancusi, C; Guarducci, M; Di Guardo, G; Terracciano, G

2016-03-15

During 2014, six loggerhead turtles, Caretta caretta and one green turtle, Chelonia mydas, found stranded on the Tuscany coast of Italy, were examined for the presence of specific bacterial and viral agents, along with their role as carriers of fish and human pathogens. Thirteen different species of bacteria, 10 Gram negative and 3 Gram positive, were identified. Among them, two strains of Vibrio parahaemolyticus and one strain of Lactococcus garviae were recovered and confirmed by specific PCR protocols. No trh and tdh genes were detected in V. parahaemolyticus. The first isolation of L. garviae and the first detection of Betanodavirus in sea turtles indicate the possibility for sea turtles to act as carriers of fish pathogens. Furthermore, the isolation of two strains of V. parahaemolyticus highlights the possible role of these animals in human pathogens' diffusion. Copyright © 2016 Elsevier B.V. All rights reserved.

A Transcriptional Regulatory Mechanism Finely Tunes the Firing of Type VI Secretion System in Response to Bacterial Enemies.

PubMed

Lazzaro, Martina; Feldman, Mario F; García Véscovi, Eleonora

2017-08-22

The ability to detect and measure danger from an environmental signal is paramount for bacteria to respond accordingly, deploying strategies that halt or counteract potential cellular injury and maximize survival chances. Type VI secretion systems (T6SSs) are complex bacterial contractile nanomachines able to target toxic effectors into neighboring bacteria competing for the same colonization niche. Previous studies support the concept that either T6SSs are constitutively active or they fire effectors in response to various stimuli, such as high bacterial density, cell-cell contact, nutrient depletion, or components from dead sibling cells. For Serratia marcescens , it has been proposed that its T6SS is stochastically expressed, with no distinction between harmless or aggressive competitors. In contrast, we demonstrate that the Rcs regulatory system is responsible for finely tuning Serratia T6SS expression levels, behaving as a transcriptional rheostat. When confronted with harmless bacteria, basal T6SS expression levels suffice for Serratia to eliminate the competitor. A moderate T6SS upregulation is triggered when, according to the aggressor-prey ratio, an unbalanced interplay between homologous and heterologous effectors and immunity proteins takes place. Higher T6SS expression levels are achieved when Serratia is challenged by a contender like Acinetobacter , which indiscriminately fires heterologous effectors able to exert lethal cellular harm, threatening the survival of the Serratia population. We also demonstrate that Serratia 's RcsB-dependent T6SS regulatory mechanism responds not to general stress signals but to the action of specific effectors from competitors, displaying an exquisite strategy to weigh risks and keep the balance between energy expenditure and fitness costs. IMPORTANCE Serratia marcescens is among the health-threatening pathogens categorized by the WHO as research priorities to develop alternative antimicrobial strategies, and it was

[Laboratory diagnosis of bacterial meningitis: usefulness of various tests for the determination of the etiological agent].

PubMed

Carbonnelle, E

2009-01-01

Despite breakthroughs in the diagnosis and treatment of infectious diseases, meningitis still remains an important cause of mortality and morbidity. An accurate and rapid diagnosis of acute bacterial meningitis is essential for a good outcome. The gold-standard test for diagnosis is CSF analysis. Gram staining of CSF reveals bacteria in about 50 to 80 % of cases and cultures are positive in at best 80 % of cases. However, the sensitivity of both tests is less than 50 % in patients who are already on antibiotic treatment. CSF leukocyte count and concentration of protein and glucose lack specificity and sensitivity for the diagnosis of meningitis. Other biological tests are available for the diagnosis. Latex agglutination test were adapted for rapid and direct detection of soluble bacterial antigens in CSF of patients suspected with bacterial meningitis. This test is efficient in detecting antigens of most common central nervous system bateria but lacks sensibility. Furthermore, in the early phases of acute bacterial and viral meningitis, signs and symptoms are often non specific and it is not always possible to make a differential diagnosis. Markers like CRP, procalcitonin, or sTREM-1 may be very useful for the diagnosis and to differentiate between viral and bacterial meningitis. Bacterial meningitis diagnosis and management require various biological tests and a multidisciplinary approach.

Preliminary study on an innovative, simple mast cell-based electrochemical method for detecting foodborne pathogenic bacterial quorum signaling molecules (N-acyl-homoserine-lactones).

PubMed

Jiang, Donglei; Feng, Dongdong; Jiang, Hui; Yuan, Limin; Yongqi, Yin; Xu, Xin; Fang, Weiming

2017-04-15

This paper reports the a novel and simple mast cell-based electrochemical method for detecting of bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs), which can be utilized to preliminarily evaluate the toxicity of food-borne pathogenic bacteria. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide hydrogel were immobilized on a gold electrode, while mast cells as recognition elements were cultured in a 3D cell culture system. Electrochemical impedance spectroscopy (EIS) was utilized to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL). The results indicated that cellular activities such as cell viability, apoptosis, intracellular calcium, and degranulation were markedly influenced by the AHLs. Importantly, the exposure of 3OC 12 -HSL to mast cells induced a marked decrease in the electrochemical impedance signal in a dose-dependent manner. The detection limit for 3OC 12 -HSL was 0.034μM with a linear range of 0.1-1μM. These results were confirmed via conventional cell assay and transmission electron microscope (TEM) analysis. Altogether, the proposed method appears to be an innovative and effective approach to the quantitative measurement of Gram-negative bacterial quorum signaling molecules; to this effect, it also may serve as a primary evaluation of the cytotoxicity of food-borne pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Diagnostic Accuracy of Ascites Fluid Gross Appearance in Detection of Spontaneous Bacterial Peritonitis.

PubMed

Aminiahidashti, Hamed; Hosseininejad, Seyed Mohammad; Montazer, Hosein; Bozorgi, Farzad; Goli Khatir, Iraj; Jahanian, Fateme; Raee, Behnaz

2014-01-01

Spontaneous bacterial peritonitis (SBP) as a monomicrobial infection of ascites fluid is one of the most important causes of morbidity and mortality in cirrhotic patients. This study was aimed to determine the diagnostic accuracy of ascites fluid color in detection of SBP in cirrhotic cases referred to the emergency department. Cirrhotic patients referred to the ED for the paracentesis of ascites fluid were enrolled. For all studied patients, the results of laboratory analysis and gross appearance of ascites fluid registered and reviewed by two emergency medicine specialists. The sensitivity, specificity, positive and negative predictive value, and positive and negative likelihood ration of the ascites fluid gross appearance in detection of SBP were measured with 95% confidence interval. The present project was performed in 80 cirrhotic patients with ascites (52.5 female). The mean of the subjects' age was 56.25±12.21 years (35-81). Laboratory findings revealed SBP in 23 (29%) cases. Fifty nine (73%) cases had transparent ascites fluid appearance of whom 17 (29%) ones suffered from SBP. From 21 (26%) cases with opaque ascites appearance, 15 (71%) had SBP. The sensitivity and specificity of the ascites fluid appearance in detection of SBP were 46.88% (Cl: 30.87-63.55) and 87.50% (95% Cl: 75.3-94.14), respectively. It seems that the gross appearance of ascites fluid had poor diagnostic accuracy in detection of SBP and considering its low sensitivity, it could not be used as a good screening tool for this propose.

Diagnostic Accuracy of Ascites Fluid Gross Appearance in Detection of Spontaneous Bacterial Peritonitis

PubMed Central

Aminiahidashti, Hamed; Hosseininejad, Seyed Mohammad; Montazer, Hosein; Bozorgi, Farzad; Goli Khatir, Iraj; Jahanian, Fateme; Raee, Behnaz

2014-01-01

Introduction: Spontaneous bacterial peritonitis (SBP) as a monomicrobial infection of ascites fluid is one of the most important causes of morbidity and mortality in cirrhotic patients. This study was aimed to determine the diagnostic accuracy of ascites fluid color in detection of SBP in cirrhotic cases referred to the emergency department. Methods: Cirrhotic patients referred to the ED for the paracentesis of ascites fluid were enrolled. For all studied patients, the results of laboratory analysis and gross appearance of ascites fluid registered and reviewed by two emergency medicine specialists. The sensitivity, specificity, positive and negative predictive value, and positive and negative likelihood ration of the ascites fluid gross appearance in detection of SBP were measured with 95% confidence interval. Results: The present project was performed in 80 cirrhotic patients with ascites (52.5 female). The mean of the subjects’ age was 56.25±12.21 years (35-81). Laboratory findings revealed SBP in 23 (29%) cases. Fifty nine (73%) cases had transparent ascites fluid appearance of whom 17 (29%) ones suffered from SBP. From 21 (26%) cases with opaque ascites appearance, 15 (71%) had SBP. The sensitivity and specificity of the ascites fluid appearance in detection of SBP were 46.88% (Cl: 30.87-63.55) and 87.50% (95% Cl: 75.3-94.14), respectively. Conclusion: It seems that the gross appearance of ascites fluid had poor diagnostic accuracy in detection of SBP and considering its low sensitivity, it could not be used as a good screening tool for this propose. PMID:26495366

Microcontroller based driver alertness detection systems to detect drowsiness

NASA Astrophysics Data System (ADS)

Adenin, Hasibah; Zahari, Rahimi; Lim, Tiong Hoo

2018-04-01

The advancement of embedded system for detecting and preventing drowsiness in a vehicle is a major challenge for road traffic accident systems. To prevent drowsiness while driving, it is necessary to have an alert system that can detect a decline in driver concentration and send a signal to the driver. Studies have shown that traffc accidents usually occur when the driver is distracted while driving. In this paper, we have reviewed a number of detection systems to monitor the concentration of a car driver and propose a portable Driver Alertness Detection System (DADS) to determine the level of concentration of the driver based on pixelated coloration detection technique using facial recognition. A portable camera will be placed at the front visor to capture facial expression and the eye activities. We evaluate DADS using 26 participants and have achieved 100% detection rate with good lighting condition and a low detection rate at night.

PulseNet China, a model for future laboratory-based bacterial infectious disease surveillance in China.

PubMed

Li, Wei; Lu, Shan; Cui, Zhigang; Cui, Jinghua; Zhou, Haijian; Wang, Yiqing; Shao, Zhujun; Ye, Changyun; Kan, Biao; Xu, Jianguo

2012-12-01

Surveillance is critical for the prevention and control of infectious disease. China's real-time web-based infectious disease reporting system is a distinguished achievement. However, many aspects of the current China Infectious Disease Surveillance System do not yet meet the demand for timely outbreak detection and identification of emerging infectious disease. PulseNet, the national molecular typing network for foodborne disease surveillance was first established by the Centers for Disease Control and Prevention of the United States in 1995 and has proven valuable in the early detection of outbreaks and tracing the pathogen source. Since 2001, the China CDC laboratory for bacterial pathogen analysis has been a member of the PulseNet International family; and has been adapting the idea and methodology of PulseNet to develop a model for a future national laboratory-based surveillance system for all bacterial infectious disease.We summarized the development progress for the PulseNet China system and discussed it as a model for the future of China's national laboratory-based surveillance system.

Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems.

PubMed

Gomaa, Ahmed A; Klumpe, Heidi E; Luo, Michelle L; Selle, Kurt; Barrangou, Rodolphe; Beisel, Chase L

2014-01-28

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in Escherichia coli as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of "smart" antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms. Controlling the composition of microbial populations is a critical aspect in medicine, biotechnology, and environmental cycles. While different antimicrobial strategies, such as antibiotics, antimicrobial peptides, and lytic bacteriophages, offer partial solutions

Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

PubMed Central

Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

2016-01-01

Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

Associations of periparturient plasma biochemical parameters, endometrial leukocyte esterase and myeloperoxidase, and bacterial detection with clinical and subclinical endometritis in postpartum dairy cows

PubMed Central

NAZHAT, Sayed Ahmad; KITAHARA, Go; KOZUKA, Nobutoshi; MIDO, Shogo; SADAWY, Mohammed; ALI, Hossam El-Sheikh; OSAWA, Takeshi

2018-01-01

This study was aimed at demonstrating associations between peripheral biochemical parameters, endometrial leukocyte esterase (LE) and myeloperoxidase (MPO), and bacterial detection with the degree of endometrial inflammation, and determining the best time postpartum for diagnosing endometritis to predict subsequent fertility in dairy cows. Plasma albumin, blood urea nitrogen (BUN), total cholesterol (T-cho), NEFA, and BHBA concentrations were analyzed in 43 Holstein cows at 3, 5 and 7 weeks postpartum (W3, W5 and W7). Endometrial samples were collected at W3, W5 and W7 to examine LE and MPO activities, bacterial detection rates, and PMN% profiles. The 43 cows were divided into healthy (HE), subclinical endometritis (SE), and clinical endometritis (CE) groups, classified differently at W3, W5 and W7 based on the definitions of SE and CE for each of the three weeks pp. LE level had an association with PMN% in all weeks pp (P<0.05). Albumin and BUN levels had weak negative associations with endometrial PMN% at W3. Pathogenic bacterial detection rates were higher in the cows with endometritis at W3 and W5. Conception rate at first artificial insemination tended to be lower (P=0.057) in the cows diagnosed with endometritis at W3 than in the healthy cows. In conclusion, associations were found between endometrial LE and endometritis, but not for MPO and endometritis. Diagnosing endometritis in W3 may be the best moment to predict subsequent fertility. PMID:29332864

Microelectrode-based technology for the detection of low levels of bacteria

NASA Technical Reports Server (NTRS)

Rogers, Tom D.; Hitchens, G. D.; Mishra, S. K.; Pierson, D. L.

1992-01-01

A microelectrode-based electrochemical detection method was used for quantitation of bacteria in water samples. The redox mediator, benzoquinone, was used to accept electrons from the bacterial metabolic pathway to create a flow of electrons by reducing the mediator. Electrochemical monitoring electrodes detected the reduced mediator as it diffused out of the cells and produced a small electrical current. By using a combination of microelectrodes and monitoring instrumentation, the cumulative current generated by a particular bacterial population could be monitored. Using commercially available components, an electrochemical detection system was assembled and tested to evaluate its potential as an emerging technology for rapid detection and quantitation of bacteria in water samples.

Failure of bacterial screening to detect Staphylococcus aureus: the English experience of donor follow-up.

PubMed

Brailsford, S R; Tossell, J; Morrison, R; McDonald, C P; Pitt, T L

2018-05-24

Between February 2011 and December 2016, over 1·6 million platelet units, 36% pooled platelets, underwent bacterial screening prior to issue. Contamination rates for apheresis and pooled platelets were 0·02% and 0·07%, respectively. Staphylococcus aureus accounted for 21 contaminations, including four pooled platelets, one confirmed transfusion-transmitted infection (TTI) and three 'near-miss' incidents detected on visual inspection which were negative on screening. We describe follow-up investigations of 16 donors for skin carriage of S. aureus and molecular characterisation of donor and pack isolates. Units were screened by the BacT/ALERT 3D detection system. Contributing donors were interviewed and consent requested for skin and nasal swabbing. S. aureus isolates were referred for spa gene type and DNA macrorestriction profile to determine identity between carriage strains and packs. Donors of 10 apheresis and two pooled packs screen positive for S. aureus were confirmed as the source of contamination; seven had a history of skin conditions, predominantly eczema; 11 were nasal carriers. The 'near-miss' incidents were associated with apheresis donors, two donors harboured strains indistinguishable from the pack strain. The TTI was due to a screen-negative pooled unit, and a nasal isolate of one donor was indistinguishable from that in the unit. Staphylococcus aureus contamination is rare but potentially harmful in platelet units. Donor isolates showed almost universal correspondence in molecular type with pack isolates, thus confirming the source of contamination. The importance of visual inspection of packs prior to transfusion is underlined by the 'near-miss' incidents. © 2018 International Society of Blood Transfusion.

The Use of Different Irrigation Techniques to Decrease Bacterial Loads in Healthy and Diabetic Patients with Asymptomatic Apical Periodontitis

PubMed Central

Ghoneim, Mai; Saber, Shehab ElDin; El-Badry, Tarek; Obeid, Maram; Hassib, Nehal

2016-01-01

BACKGROUND: Diabetes mellitus is a multisystem disease which weakens the human’s immunity. Subsequently, it worsens the sequelae of apical periodontitis by raising a fierce bacterial trait due to the impaired host response. AIM: This study aimed to estimate bacterial reduction after using different irrigation techniques in systemically healthy and diabetic patients with asymptomatic apical periodontitis. MATERIAL AND METHODS: Enterococcus faecalis, Peptostreptococcus micros, and Fusobacterium necleatum bacteria were chosen, as they are the most common and prevailing strains found in periodontitis. Bacterial samples were retrieved from necrotic root canals of systemically healthy and diabetic patients, before and after endodontic cleaning and shaping by using two different irrigation techniques; the conventional one and the EndoVac system. Quantitive polymerase chain reaction (qPCR) was utilised to detect the reduction in the bacterial count. RESULTS: The EndoVac irrigation system was effective in reducing bacteria, especially Peptostreptococcus micros in the diabetic group when compared to conventional irrigation technique with a statistically significant difference. CONCLUSION: The EndoVac can be considered as a promising tool in combination with irrigant solution to defeat the bacterial colonies living in the root canal system. Additional studies ought to be done to improve the means of bacterial clearance mainly in immune-compromised individuals. PMID:28028421

Evaluating the effects of variable water chemistry on bacterial transport during infiltration.

PubMed

Zhang, Haibo; Nordin, Nahjan Amer; Olson, Mira S

2013-07-01

Bacterial infiltration through the subsurface has been studied experimentally under different conditions of interest and is dependent on a variety of physical, chemical and biological factors. However, most bacterial transport studies fail to adequately represent the complex processes occurring in natural systems. Bacteria are frequently detected in stormwater runoff, and may present risk of microbial contamination during stormwater recharge into groundwater. Mixing of stormwater runoff with groundwater during infiltration results in changes in local solution chemistry, which may lead to changes in both bacterial and collector surface properties and subsequent bacterial attachment rates. This study focuses on quantifying changes in bacterial transport behavior under variable solution chemistry, and on comparing the influences of chemical variability and physical variability on bacterial attachment rates. Bacterial attachment rate at the soil-water interface was predicted analytically using a combined rate equation, which varies temporally and spatially with respect to changes in solution chemistry. Two-phase Monte Carlo analysis was conducted and an overall input-output correlation coefficient was calculated to quantitatively describe the importance of physiochemical variation on the estimates of attachment rate. Among physical variables, soil particle size has the highest correlation coefficient, followed by porosity of the soil media, bacterial size and flow velocity. Among chemical variables, ionic strength has the highest correlation coefficient. A semi-reactive microbial transport model was developed within HP1 (HYDRUS1D-PHREEQC) and applied to column transport experiments with constant and variable solution chemistries. Bacterial attachment rates varied from 9.10×10(-3)min(-1) to 3.71×10(-3)min(-1) due to mixing of synthetic stormwater (SSW) with artificial groundwater (AGW), while bacterial attachment remained constant at 9.10×10(-3)min(-1) in a constant

Seasonal dynamics of bacterial meningitis: a time-series analysis.

PubMed

Paireau, Juliette; Chen, Angelica; Broutin, Helene; Grenfell, Bryan; Basta, Nicole E

2016-06-01

Bacterial meningitis, which is caused mainly by Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae, inflicts a substantial burden of disease worldwide. Yet, the temporal dynamics of this disease are poorly characterised and many questions remain about the ecology of the disease. We aimed to comprehensively assess seasonal trends in bacterial meningitis on a global scale. We developed the first bacterial meningitis global database by compiling monthly incidence data as reported by country-level surveillance systems. Using country-level wavelet analysis, we identified whether a 12 month periodic component (annual seasonality) was detected in time-series that had at least 5 years of data with at least 40 cases reported per year. We estimated the mean timing of disease activity by computing the centre of gravity of the distribution of cases and investigated whether synchrony exists between the three pathogens responsible for most cases of bacterial meningitis. We used country-level data from 66 countries, including from 47 countries outside the meningitis belt in sub-Saharan Africa. A persistent seasonality was detected in 49 (96%) of the 51 time-series from 38 countries eligible for inclusion in the wavelet analyses. The mean timing of disease activity had a latitudinal trend, with bacterial meningitis seasons peaking during the winter months in countries in both the northern and southern hemispheres. The three pathogens shared similar seasonality, but time-shifts differed slightly by country. Our findings provide key insight into the seasonal dynamics of bacterial meningitis and add to knowledge about the global epidemiology of meningitis and the host, environment, and pathogen characteristics driving these patterns. Comprehensive understanding of global seasonal trends in meningitis could be used to design more effective prevention and control strategies. Princeton University Health Grand Challenge, US National Institutes of Health (NIH

Seasonal dynamics of bacterial meningitis: a time-series analysis

PubMed Central

Paireau, Juliette; Chen, Angelica; Broutin, Helene; Grenfell, Bryan; Basta, Nicole E

2017-01-01

Summary Background Bacterial meningitis, which is caused mainly by Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae, inflicts a substantial burden of disease worldwide. Yet, the temporal dynamics of this disease are poorly characterised and many questions remain about the ecology of the disease. We aimed to comprehensively assess seasonal trends in bacterial meningitis on a global scale. Methods We developed the first bacterial meningitis global database by compiling monthly incidence data as reported by country-level surveillance systems. Using country-level wavelet analysis, we identified whether a 12 month periodic component (annual seasonality) was detected in time-series that had at least 5 years of data with at least 40 cases reported per year. We estimated the mean timing of disease activity by computing the centre of gravity of the distribution of cases and investigated whether synchrony exists between the three pathogens responsible for most cases of bacterial meningitis. Findings We used country-level data from 66 countries, including from 47 countries outside the meningitis belt in sub-Saharan Africa. A persistent seasonality was detected in 49 (96%) of the 51 time-series from 38 countries eligible for inclusion in the wavelet analyses. The mean timing of disease activity had a latitudinal trend, with bacterial meningitis seasons peaking during the winter months in countries in both the northern and southern hemispheres. The three pathogens shared similar seasonality, but time-shifts differed slightly by country. Interpretation Our findings provide key insight into the seasonal dynamics of bacterial meningitis and add to knowledge about the global epidemiology of meningitis and the host, environment, and pathogen characteristics driving these patterns. Comprehensive understanding of global seasonal trends in meningitis could be used to design more effective prevention and control strategies. Funding Princeton University Health

Generalized Detectability for Discrete Event Systems

PubMed Central

Shu, Shaolong; Lin, Feng

2011-01-01

In our previous work, we investigated detectability of discrete event systems, which is defined as the ability to determine the current and subsequent states of a system based on observation. For different applications, we defined four types of detectabilities: (weak) detectability, strong detectability, (weak) periodic detectability, and strong periodic detectability. In this paper, we extend our results in three aspects. (1) We extend detectability from deterministic systems to nondeterministic systems. Such a generalization is necessary because there are many systems that need to be modeled as nondeterministic discrete event systems. (2) We develop polynomial algorithms to check strong detectability. The previous algorithms are based on observer whose construction is of exponential complexity, while the new algorithms are based on a new automaton called detector. (3) We extend detectability to D-detectability. While detectability requires determining the exact state of a system, D-detectability relaxes this requirement by asking only to distinguish certain pairs of states. With these extensions, the theory on detectability of discrete event systems becomes more applicable in solving many practical problems. PMID:21691432

A broad range assay for rapid detection and etiologic characterization of bacterial meningitis: performance testing in samples from sub-Sahara☆, ☆☆,★

PubMed Central

Won, Helen; Yang, Samuel; Gaydos, Charlotte; Hardick, Justin; Ramachandran, Padmini; Hsieh, Yu-Hsiang; Kecojevic, Alexander; Njanpop-Lafourcade, Berthe-Marie; Mueller, Judith E.; Tameklo, Tsidi Agbeko; Badziklou, Kossi; Gessner, Bradford D.; Rothman, Richard E.

2012-01-01

This study aimed to conduct a pilot evaluation of broad-based multiprobe polymerase chain reaction (PCR) in clinical cerebrospinal fluid (CSF) samples compared to local conventional PCR/culture methods used for bacterial meningitis surveillance. A previously described PCR consisting of initial broad-based detection of Eubacteriales by a universal probe, followed by Gram typing, and pathogen-specific probes was designed targeting variable regions of the 16S rRNA gene. The diagnostic performance of the 16S rRNA assay in “”127 CSF samples was evaluated in samples from patients from Togo, Africa, by comparison to conventional PCR/culture methods. Our probes detected Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Uniprobe sensitivity and specificity versus conventional PCR were 100% and 54.6%, respectively. Sensitivity and specificity of uniprobe versus culture methods were 96.5% and 52.5%, respectively. Gram-typing probes correctly typed 98.8% (82/83) and pathogen-specific probes identified 96.4% (80/83) of the positives. This broad-based PCR algorithm successfully detected and provided species level information for multiple bacterial meningitis agents in clinical samples. PMID:22809694

Spatial-Temporal Survey and Occupancy-Abundance Modeling To Predict Bacterial Community Dynamics in the Drinking Water Microbiome

PubMed Central

Pinto, Ameet J.; Schroeder, Joanna; Lunn, Mary; Sloan, William

2014-01-01

ABSTRACT Bacterial communities migrate continuously from the drinking water treatment plant through the drinking water distribution system and into our built environment. Understanding bacterial dynamics in the distribution system is critical to ensuring that safe drinking water is being supplied to customers. We present a 15-month survey of bacterial community dynamics in the drinking water system of Ann Arbor, MI. By sampling the water leaving the treatment plant and at nine points in the distribution system, we show that the bacterial community spatial dynamics of distance decay and dispersivity conform to the layout of the drinking water distribution system. However, the patterns in spatial dynamics were weaker than those for the temporal trends, which exhibited seasonal cycling correlating with temperature and source water use patterns and also demonstrated reproducibility on an annual time scale. The temporal trends were driven by two seasonal bacterial clusters consisting of multiple taxa with different networks of association within the larger drinking water bacterial community. Finally, we show that the Ann Arbor data set robustly conforms to previously described interspecific occupancy abundance models that link the relative abundance of a taxon to the frequency of its detection. Relying on these insights, we propose a predictive framework for microbial management in drinking water systems. Further, we recommend that long-term microbial observatories that collect high-resolution, spatially distributed, multiyear time series of community composition and environmental variables be established to enable the development and testing of the predictive framework. PMID:24865557

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.

Systemic cytokine signaling via IL-17 in smokers with obstructive pulmonary disease: a link to bacterial colonization?

PubMed Central

Andelid, Kristina; Tengvall, Sara; Andersson, Anders; Levänen, Bettina; Christenson, Karin; Jirholt, Pernilla; Åhrén, Christina; Qvarfordt, Ingemar; Ekberg-Jansson, Ann; Lindén, Anders

2015-01-01

We examined whether systemic cytokine signaling via interleukin (IL)-17 and growth-related oncogene-α (GRO-α) is impaired in smokers with obstructive pulmonary disease including chronic bronchitis (OPD-CB). We also examined how this systemic cytokine signaling relates to bacterial colonization in the airways of the smokers with OPD-CB. Currently smoking OPD-CB patients (n=60, corresponding to Global initiative for chronic Obstructive Lung Disease [GOLD] stage I–IV) underwent recurrent blood and sputum sampling over 60 weeks, during stable conditions and at exacerbations. We characterized cytokine protein concentrations in blood and bacterial growth in sputum. Asymptomatic smokers (n=10) and never-smokers (n=10) were included as control groups. During stable clinical conditions, the protein concentrations of IL-17 and GRO-α were markedly lower among OPD-CB patients compared with never-smoker controls, whereas the asymptomatic smoker controls displayed intermediate concentrations. Notably, among OPD-CB patients, colonization by opportunistic pathogens was associated with markedly lower IL-17 and GRO-α, compared with colonization by common respiratory pathogens or oropharyngeal flora. During exacerbations in the OPD-CB patients, GRO-α and neutrophil concentrations were increased, whereas protein concentrations and messenger RNA for IL-17 were not detectable in a reproducible manner. In smokers with OPD-CB, systemic cytokine signaling via IL-17 and GRO-α is impaired and this alteration may be linked to colonization by opportunistic pathogens in the airways. Given the potential pathogenic and therapeutic implications, these findings deserve to be validated in new and larger patient cohorts. PMID:25848245

PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

NASA Technical Reports Server (NTRS)

LaDuc, Myron T.; Venkateswaran, Kasthuri; Mohapatra, Bidyut

2012-01-01

The most common approach for assessing the abundance of viable bacterial endospores is the culture-based plating method. However, culture-based approaches are heavily biased and oftentimes incompatible with upstream sample processing strategies, which make viable cells/spores uncultivable. This shortcoming highlights the need for rapid molecular diagnostic tools to assess more accurately the abundance of viable spacecraft-associated microbiota, perhaps most importantly bacterial endospores. Propidium monoazide (PMA) has received a great deal of attention due to its ability to differentiate live, viable bacterial cells from dead ones. PMA gains access to the DNA of dead cells through compromised membranes. Once inside the cell, it intercalates and eventually covalently bonds with the double-helix structures upon photoactivation with visible light. The covalently bound DNA is significantly altered, and unavailable to downstream molecular-based manipulations and analyses. Microbiological samples can be treated with appropriate concentrations of PMA and exposed to visible light prior to undergoing total genomic DNA extraction, resulting in an extract comprised solely of DNA arising from viable cells. This ability to extract DNA selectively from living cells is extremely powerful, and bears great relevance to many microbiological arenas.

The DinJ/RelE Toxin-Antitoxin System Suppresses Bacterial Proliferation and Virulence of Xylella fastidiosa in Grapevine.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2017-04-01

Xylella fastidiosa, the causal agent of Pierce's disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf-scorching symptoms, senescence, and vine decline. It appears to be advantageous to this pathogen to avoid excessive blockage of xylem vessels, because living bacterial cells are generally found in plant tissue with low bacterial cell density and minimal scorching symptoms. The DinJ/RelE toxin-antitoxin system is characterized here for a role in controlling bacterial proliferation and population size during plant colonization. The DinJ/RelE locus is transcribed from two separate promoters, allowing for coexpression of antitoxin DinJ with endoribonuclease toxin RelE, in addition to independent expression of RelE. The ratio of antitoxin/toxin expressed is dependent on bacterial growth conditions, with lower amounts of antitoxin present under conditions designed to mimic grapevine xylem sap. A knockout mutant of DinJ/RelE exhibits a hypervirulent phenotype, with higher bacterial populations and increased symptom development and plant decline. It is likely that DinJ/RelE acts to prevent excessive population growth, contributing to the ability of the pathogen to spread systemically without completely blocking the xylem vessels and increasing probability of acquisition by the insect vector.

Bacteriophage-Based Pathogen Detection

NASA Astrophysics Data System (ADS)

Ripp, Steven

Considered the most abundant organism on Earth, at a population approaching 1031, bacteriophage, or phage for short, mediate interactions with myriad bacterial hosts that has for decades been exploited in phage typing schemes for signature identification of clinical, food-borne, and water-borne pathogens. With over 5,000 phage being morphologically characterized and grouped as to susceptible host, there exists an enormous cache of bacterial-specific sensors that has more recently been incorporated into novel bio-recognition assays with heightened sensitivity, specificity, and speed. These assays take many forms, ranging from straightforward visualization of labeled phage as they attach to their specific bacterial hosts to reporter phage that genetically deposit trackable signals within their bacterial hosts to the detection of progeny phage or other uniquely identifiable elements released from infected host cells. A comprehensive review of these and other phage-based detection assays, as directed towards the detection and monitoring of bacterial pathogens, will be provided in this chapter.

Auto-production of biosurfactants reverses the coffee ring effect in a bacterial system

NASA Astrophysics Data System (ADS)

Sempels, Wouter; de Dier, Raf; Mizuno, Hideaki; Hofkens, Johan; Vermant, Jan

2013-04-01

The deposition of material at the edge of evaporating droplets, known as the ‘coffee ring effect’, is caused by a radially outward capillary flow. This phenomenon is common to a wide array of systems including colloidal and bacterial systems. The role of surfactants in counteracting these coffee ring depositions is related to the occurrence of local vortices known as Marangoni eddies. Here we show that these swirling flows are universal, and not only lead to a uniform deposition of colloids but also occur in living bacterial systems. Experiments on Pseudomonas aeruginosa suggest that the auto-production of biosurfactants has an essential role in creating a homogeneous deposition of the bacteria upon drying. Moreover, at biologically relevant conditions, intricate time-dependent flows are observed in addition to the vortex regime, which are also effective in reversing the coffee ring effect at even lower surfactant concentrations.

Bacterial Community Structure Shifted by Geosmin in Granular Activated Carbon System of Water Treatment Plants.

PubMed

Pham, Ngoc Dung; Lee, Eun-Hee; Chae, Seon-Ha; Cho, Yongdeok; Shin, Hyejin; Son, Ahjeong

2016-01-01

We investigated the relation between the presence of geosmin in water and the bacterial community structure within the granular activated carbon (GAC) system of water treatment plants in South Korea. GAC samples were collected in May and August of 2014 at three water treatment plants (Sungnam, Koyang, and Yeoncho in Korea). Dissolved organic carbon and geosmin were analyzed before and after GAC treatment. Geosmin was found in raw water from Sungnam and Koyang water treatment plants but not in that from Yeoncho water treatment plant. Interestingly, but not surprisingly, the 16S rRNA clone library indicated that the bacterial communities from the Sungnam and Koyang GAC systems were closely related to geosmin-degrading bacteria. Based on the phylogenetic tree and multidimensional scaling plot, bacterial clones from GAC under the influence of geosmin were clustered with Variovorax paradoxus strain DB 9b and Comamonas sp. DB mg. In other words, the presence of geosmin in water might have inevitably contributed to the growth of geosmin degraders within the respective GAC system.

Antibody-based bacterial toxin detection

NASA Astrophysics Data System (ADS)

Menking, Darrell E.; Heitz, Jonathon M.; Anis, Nabil A.; Thompson, Roy G.

1994-03-01

Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a one step assay, antibodies against Cholera toxin or Staphylococcus Enterotoxin B were noncovalently immobilized on quartz fibers and probed with fluorescein-isothiocyanate (FITC)-labeled toxins. In the two-step assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified antitoxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or antitoxin IgG in a dose-dependent manner and the detection of the toxins was in the nanomolar range.

Influence of sugarbeet tillage Systems on the rhizoctonia-bacterial root rot complex

USDA-ARS?s Scientific Manuscript database

The Rhizoctonia-bacterial root rot complex in sugarbeet caused by Rhizoctonia solani and Leuconostoc mesenteroides can cause significant yield losses. To investigate the impact of different tillage systems on this complex, field studies were conducted from 2009 to 2011. Split blocks with conventio...

Direct Detection and Identification of Bacterial Pathogens from Urine with Optimized Specimen Processing and Enhanced Testing Algorithm

PubMed Central

Huang, Bin; Zhang, Lei; Zhang, Weizheng; Liao, Kang; Zhang, Shihong; Zhang, Zhiquan; Ma, Xingyan; Chen, Jialong; Zhang, Xiuhong; Qu, Pinghua; Wu, Shangwei

2017-01-01

ABSTRACT Rapid and accurate detection and identification of microbial pathogens causing urinary tract infections allow prompt and specific treatment. We optimized specimen processing to maximize the limit of detection (LOD) by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and evaluated the capacity of combination of MALDI-TOF MS and urine analysis (UA) for direct detection and identification of bacterial pathogens from urine samples. The optimal volumes of processed urine, formic acid/acetonitrile, and supernatant spotted onto the target plate were 15 ml, 3 μl, and 3 μl, respectively, yielding a LOD of 1.0 × 105 CFU/ml. Among a total of 1,167 urine specimens collected from three hospital centers, 612 (52.4%) and 351 (30.1%) were, respectively, positive by UA and urine culture. Compared with a reference method comprised of urine culture and 16S rRNA gene sequencing, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MALDI-TOF MS alone and MALDI-TOF MS coupled with UA were 86.6% versus 93.4% (χ2 = 8.93; P < 0.01), 91.5% versus 96.3% (χ2 = 7.06; P < 0.01), 81.5% versus 96.4% (χ2 = 37.32; P < 0.01), and 94.1% versus 93.1% (χ2 = 0.40; P > 0.05), respectively. No significant performance differences were revealed among the three sites, while specificity and NPV of MALDI-TOF MS for males were significantly higher than those for females (specificity, 94.3% versus 77.3%, χ2 = 44.90, P < 0.01; NPV, 95.5% versus 86.1%, χ2 = 18.85, P < 0.01). Our results indicated that the optimization of specimen processing significantly enhanced analytical sensitivity and that the combination of UA and MALDI-TOF MS provided an accurate and rapid detection and identification of bacterial pathogens directly from urine. PMID:28249997

Direct Detection and Identification of Bacterial Pathogens from Urine with Optimized Specimen Processing and Enhanced Testing Algorithm.

PubMed

Huang, Bin; Zhang, Lei; Zhang, Weizheng; Liao, Kang; Zhang, Shihong; Zhang, Zhiquan; Ma, Xingyan; Chen, Jialong; Zhang, Xiuhong; Qu, Pinghua; Wu, Shangwei; Chen, Cha; Tang, Yi-Wei

2017-05-01

Rapid and accurate detection and identification of microbial pathogens causing urinary tract infections allow prompt and specific treatment. We optimized specimen processing to maximize the limit of detection (LOD) by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and evaluated the capacity of combination of MALDI-TOF MS and urine analysis (UA) for direct detection and identification of bacterial pathogens from urine samples. The optimal volumes of processed urine, formic acid/acetonitrile, and supernatant spotted onto the target plate were 15 ml, 3 μl, and 3 μl, respectively, yielding a LOD of 1.0 × 10 5 CFU/ml. Among a total of 1,167 urine specimens collected from three hospital centers, 612 (52.4%) and 351 (30.1%) were, respectively, positive by UA and urine culture. Compared with a reference method comprised of urine culture and 16S rRNA gene sequencing, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MALDI-TOF MS alone and MALDI-TOF MS coupled with UA were 86.6% versus 93.4% (χ 2 = 8.93; P < 0.01), 91.5% versus 96.3% (χ 2 = 7.06; P < 0.01), 81.5% versus 96.4% (χ 2 = 37.32; P < 0.01), and 94.1% versus 93.1% (χ 2 = 0.40; P > 0.05), respectively. No significant performance differences were revealed among the three sites, while specificity and NPV of MALDI-TOF MS for males were significantly higher than those for females (specificity, 94.3% versus 77.3%, χ 2 = 44.90, P < 0.01; NPV, 95.5% versus 86.1%, χ 2 = 18.85, P < 0.01). Our results indicated that the optimization of specimen processing significantly enhanced analytical sensitivity and that the combination of UA and MALDI-TOF MS provided an accurate and rapid detection and identification of bacterial pathogens directly from urine. Copyright © 2017 American Society for Microbiology.

Kynetic resazurin assay (KRA) for bacterial quantification of foodborne pathogens

NASA Astrophysics Data System (ADS)

Arenas, Yaxal; Mandel, Arkady; Lilge, Lothar

2012-03-01

Fast detection of bacterial concentrations is important for the food industry and for healthcare. Early detection of infections and appropriate treatment is essential since, the delay of treatments for bacterial infections tends to be associated with higher mortality rates. In the food industry and in healthcare, standard procedures require the count of colony-forming units in order to quantify bacterial concentrations, however, this method is time consuming and reports require three days to be completed. An alternative is metabolic-colorimetric assays which provide time efficient in vitro bacterial concentrations. A colorimetric assay based on Resazurin was developed as a time kinetic assay (KRA) suitable for bacterial concentration measurements. An optimization was performed by finding excitation and emission wavelengths for fluorescent acquisition. A comparison of two non-related bacteria, foodborne pathogens Escherichia coli and Listeria monocytogenes, was performed in 96 well plates. A metabolic and clonogenic dependence was established for fluorescent kinetic signals.

Optoacoustic sensing of ocular bacterial antigen using targeted gold nanorods

NASA Astrophysics Data System (ADS)

Maswadi, Saher; Page, Leland; Woodward, Lee; Glickman, Randolph D.; Barsalou, Norman

2008-02-01

Bacterial contamination can be detected using a minimally invasive optical method, based on laser-induced optoacoustic spectroscopy, to probe for specific antigens associated with a specific infectious agent. As a model system, we have used a surface antigen (Ag), isolated from Chlamydia trachomatis, and a complementary antibody (Ab). A preparation of 0.2 mg/ml of monoclonal Ab specific to the C. trachomatis surface Ag was conjugated to gold nanorods using standard commercial reagents, in order to produce a targeted contrast agent with a strong optoacoustic signal. The C. trachomatis Ag was absorbed in standard plastic microwells, and the binding of the complementary Ab-nanorod conjugate was tested in an immunoaffinity assay. Optoacoustic signals were elicited from the bound nanorods, using an optical parametric oscillator (OPO) laser system as the optical pump. The wavelength tuneability of the OPO optimized the spectroscopic measurement by exciting the nanorods at their optical absorption maxima. Optoacoustic responses were measured in the microwells using a probe beam deflection technique. Immunoaffinity assays were performed on several dilutions of purified C. trachomatis antigen ranging from 50 μg/ml to 1 pg/ml, in order to determine the detection limit for the optoacoustic-based assay. Only when the antigen was present, and the complementary Ab-NR reagent was introduced into the microwell, was an enhanced optoacoustic signal obtained, which indicated specific binding of the Ab-NR complex. The limit of detection with the current system design is between 1 and 5 pg/ml of bacterial Ag.

Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens.

PubMed

Eldridge, Matthew J G; Shenoy, Avinash R

2015-02-01

Inflammasomes - molecular platforms for caspase-1 activation - have emerged as common hubs for a number of pathways that detect and respond to bacterial pathogens. Caspase-1 activation results in the secretion of bioactive IL-1β and IL-18 and pyroptosis, and thus launches a systemic immune and inflammatory response. In this review we discuss signal transduction leading to 'canonical' and 'non-canonical' activation of caspase-1 through the involvement of upstream caspases. Recent studies have identified a growing number of regulatory networks involving guanylate binding proteins, protein kinases, ubiquitylation and necroptosis related pathways that modulate inflammasome responses and immunity to bacterial infection. By being able to respond to extracellular, vacuolar and cytosolic bacteria, their cytosolic toxins or ligands for cell surface receptors, inflammasomes have emerged as important sentinels of infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

ISYMOD: a knowledge warehouse for the identification, assembly and analysis of bacterial integrated systems.

PubMed

Chabalier, Julie; Capponi, Cécile; Quentin, Yves; Fichant, Gwennaele

2005-04-01

Complex biological functions emerge from interactions between proteins in stable supra-molecular assemblies and/or through transitory contacts. Most of the time protein partners of the assemblies are composed of one or several domains which exhibit different biochemical functions. Thus the study of cellular process requires the identification of different functional units and their integration in an interaction network; such complexes are referred to as integrated systems. In order to exploit with optimum efficiency the increased release of data, automated bioinformatics strategies are needed to identify, reconstruct and model such systems. For that purpose, we have developed a knowledge warehouse dedicated to the representation and acquisition of bacterial integrated systems involved in the exchange of the bacterial cell with its environment. ISYMOD is a knowledge warehouse that consistently integrates in the same environment the data and the methods used for their acquisition. This is achieved through the construction of (1) a domain knowledge base (DKB) devoted to the storage of the knowledge about the systems, their functional specificities, their partners and how they are related and (2) a methodological knowledge base (MKB) which depicts the task layout used to identify and reconstruct functional integrated systems. Instantiation of the DKB is obtained by solving the tasks of the MKB, whereas some tasks need instances of the DKB to be solved. AROM, an object-based knowledge representation system, has been used to design the DKB, and its task manager, AROMTasks, for developing the MKB. In this study two integrated systems, ABC transporters and two component systems, both involved in adaptation processes of a bacterial cell to its biotope, have been used to evaluate the feasibility of the approach.

Mechanical Homogenization Increases Bacterial Homogeneity in Sputum

PubMed Central

Stokell, Joshua R.; Khan, Ammad

2014-01-01

Sputum obtained from patients with cystic fibrosis (CF) is highly viscous and often heterogeneous in bacterial distribution. Adding dithiothreitol (DTT) is the standard method for liquefaction prior to processing sputum for molecular detection assays. To determine if DTT treatment homogenizes the bacterial distribution within sputum, we measured the difference in mean total bacterial abundance and abundance of Burkholderia multivorans between aliquots of DTT-treated sputum samples with and without a mechanical homogenization (MH) step using a high-speed dispersing element. Additionally, we measured the effect of MH on bacterial abundance. We found a significant difference between the mean bacterial abundances in aliquots that were subjected to only DTT treatment and those of the aliquots which included an MH step (all bacteria, P = 0.04; B. multivorans, P = 0.05). There was no significant effect of MH on bacterial abundance in sputum. Although our results are from a single CF patient, they indicate that mechanical homogenization increases the homogeneity of bacteria in sputum. PMID:24759710

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

Fungal Innate Immunity Induced by Bacterial Microbe-Associated Molecular Patterns (MAMPs)

PubMed Central

Ipcho, Simon; Sundelin, Thomas; Erbs, Gitte; Kistler, H. Corby; Newman, Mari-Anne; Olsson, Stefan

2016-01-01

Plants and animals detect bacterial presence through Microbe-Associated Molecular Patterns (MAMPs) which induce an innate immune response. The field of fungal–bacterial interaction at the molecular level is still in its infancy and little is known about MAMPs and their detection by fungi. Exposing Fusarium graminearum to bacterial MAMPs led to increased fungal membrane hyperpolarization, a putative defense response, and a range of transcriptional responses. The fungus reacted with a different transcript profile to each of the three tested MAMPs, although a core set of genes related to energy generation, transport, amino acid production, secondary metabolism, and especially iron uptake were detected for all three. Half of the genes related to iron uptake were predicted MirA type transporters that potentially take up bacterial siderophores. These quick responses can be viewed as a preparation for further interactions with beneficial or pathogenic bacteria, and constitute a fungal innate immune response with similarities to those of plants and animals. PMID:27172188

Biofilm bacterial communities in urban drinking water distribution systems transporting waters with different purification strategies.

PubMed

Wu, Huiting; Zhang, Jingxu; Mi, Zilong; Xie, Shuguang; Chen, Chao; Zhang, Xiaojian

2015-02-01

Biofilm formation in drinking water distribution systems (DWDS) has many adverse consequences. Knowledge of microbial community structure of DWDS biofilm can aid in the design of an effective control strategy. However, biofilm bacterial community in real DWDS and the impact of drinking water purification strategy remain unclear. The present study investigated the composition and diversity of biofilm bacterial community in real DWDSs transporting waters with different purification strategies (conventional treatment and integrated treatment). High-throughput Illumina MiSeq sequencing analysis illustrated a large shift in the diversity and structure of biofilm bacterial community in real DWDS. Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Nitrospirae, and Cyanobacteria were the major components of biofilm bacterial community. Proteobacteria (mainly Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) predominated in each DWDS biofilm, but the compositions of the dominant proteobacterial classes and genera and their proportions varied among biofilm samples. Drinking water purification strategy could shape DWDS biofilm bacterial community. Moreover, Pearson's correlation analysis indicated that Actinobacteria was positively correlated with the levels of total alkalinity and dissolved organic carbon in tap water, while Firmicutes had a significant positive correlation with nitrite nitrogen.

Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

PubMed

Kalb, Suzanne R; Boyer, Anne E; Barr, John R

2015-08-31

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems

PubMed Central

Gomaa, Ahmed A.; Klumpe, Heidi E.; Luo, Michelle L.; Selle, Kurt; Barrangou, Rodolphe; Beisel, Chase L.

2014-01-01

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in Escherichia coli as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of “smart” antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms. PMID:24473129

Molecular Detection of Leptospira in Two Returned Travelers: Higher Bacterial Load in Cerebrospinal Fluid versus Serum or Plasma

PubMed Central

Waggoner, Jesse J.; Soda, Elizabeth A.; Seibert, Ryan; Grant, Philip; Pinsky, Benjamin A.

2015-01-01

Leptospirosis is a potentially severe illness in returned travelers. Patients often present with fever, headache, and neck pain, which may lead to a workup for meningitis including the acquisition of cerebrospinal fluid (CSF). Although Leptospira DNA has been detected in CSF by polymerase chain reaction (PCR), little data exist regarding the utility of testing CSF in addition to serum or plasma obtained on presentation. In this report, we present two cases of leptospirosis in returned travelers presenting with fever and headache. Our first patient had neutrophilic meningitis, and Leptospira was detectable only in CSF obtained on admission. The second patient had a normal CSF profile, but Leptospira was detected in CSF at a bacterial load 5- to 10-fold higher than that in plasma. CSF is an important specimen for the diagnosis of Leptospira by molecular methods and may yield an actionable diagnosis in the absence of leptospiremia. PMID:26033024

Profiling bacterial communities associated with sediment-based aquaculture bioremediation systems under contrasting redox regimes

NASA Astrophysics Data System (ADS)

Robinson, Georgina; Caldwell, Gary S.; Wade, Matthew J.; Free, Andrew; Jones, Clifford L. W.; Stead, Selina M.

2016-12-01

Deposit-feeding invertebrates are proposed bioremediators in microbial-driven sediment-based aquaculture effluent treatment systems. We elucidate the role of the sediment reduction-oxidation (redox) regime in structuring benthic bacterial communities, having direct implications for bioremediation potential and deposit-feeder nutrition. The sea cucumber Holothuria scabra was cultured on sediments under contrasting redox regimes; fully oxygenated (oxic) and redox stratified (oxic-anoxic). Taxonomically, metabolically and functionally distinct bacterial communities developed between the redox treatments with the oxic treatment supporting the greater diversity; redox regime and dissolved oxygen levels were the main environmental drivers. Oxic sediments were colonised by nitrifying bacteria with the potential to remediate nitrogenous wastes. Percolation of oxygenated water prevented the proliferation of anaerobic sulphate-reducing bacteria, which were prevalent in the oxic-anoxic sediments. At the predictive functional level, bacteria within the oxic treatment were enriched with genes associated with xenobiotics metabolism. Oxic sediments showed the greater bioremediation potential; however, the oxic-anoxic sediments supported a greater sea cucumber biomass. Overall, the results indicate that bacterial communities present in fully oxic sediments may enhance the metabolic capacity and bioremediation potential of deposit-feeder microbial systems. This study highlights the benefits of incorporating deposit-feeding invertebrates into effluent treatment systems, particularly when the sediment is oxygenated.

Profiling bacterial communities associated with sediment-based aquaculture bioremediation systems under contrasting redox regimes

PubMed Central

Robinson, Georgina; Caldwell, Gary S.; Wade, Matthew J.; Free, Andrew; Jones, Clifford L. W.; Stead, Selina M.

2016-01-01

Deposit-feeding invertebrates are proposed bioremediators in microbial-driven sediment-based aquaculture effluent treatment systems. We elucidate the role of the sediment reduction-oxidation (redox) regime in structuring benthic bacterial communities, having direct implications for bioremediation potential and deposit-feeder nutrition. The sea cucumber Holothuria scabra was cultured on sediments under contrasting redox regimes; fully oxygenated (oxic) and redox stratified (oxic-anoxic). Taxonomically, metabolically and functionally distinct bacterial communities developed between the redox treatments with the oxic treatment supporting the greater diversity; redox regime and dissolved oxygen levels were the main environmental drivers. Oxic sediments were colonised by nitrifying bacteria with the potential to remediate nitrogenous wastes. Percolation of oxygenated water prevented the proliferation of anaerobic sulphate-reducing bacteria, which were prevalent in the oxic-anoxic sediments. At the predictive functional level, bacteria within the oxic treatment were enriched with genes associated with xenobiotics metabolism. Oxic sediments showed the greater bioremediation potential; however, the oxic-anoxic sediments supported a greater sea cucumber biomass. Overall, the results indicate that bacterial communities present in fully oxic sediments may enhance the metabolic capacity and bioremediation potential of deposit-feeder microbial systems. This study highlights the benefits of incorporating deposit-feeding invertebrates into effluent treatment systems, particularly when the sediment is oxygenated. PMID:27941918

Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

NASA Astrophysics Data System (ADS)

Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

2012-06-01

Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

Bacterial 'immunity' against bacteriophages.

PubMed

Abedon, Stephen T

2012-01-01

Vertebrate animals possess multiple anti-pathogen defenses. Individual mechanisms usually are differentiated into those that are immunologically adaptive vs. more "primitive" anti-pathogen phenomena described as innate responses. Here I frame defenses used by bacteria against bacteriophages as analogous to these animal immune functions. Included are numerous anti-phage defenses in addition to the adaptive immunity associated with CRISPR/cas systems. As these other anti-pathogen mechanisms are non-adaptive they can be described as making up an innate bacterial immunity. This exercise was undertaken in light of the recent excitement over the discovery that CRISPR/cas systems can serve, as noted, as a form of bacterial adaptive immunity. The broader goal, however, is to gain novel insight into bacterial defenses against phages by fitting these mechanisms into considerations of how multicellular organisms also defend themselves against pathogens. This commentary can be viewed in addition as a bid toward integrating these numerous bacterial anti-phage defenses into a more unified immunology.

Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.

PubMed

Yang, Samuel; Rothman, Richard E; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A

2008-04-01

To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.

Development of a multispectral light-scatter sensor for bacterial colonies

USDA-ARS?s Scientific Manuscript database

We report a multispectral elastic-light-scatter instrument that can simultaneously detect three-wavelength scatter patterns and associated optical densities from individual bacterial colonies, overcoming the limits of the single-wavelength predecessor. Absorption measurements on liquid bacterial sam...

Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

NASA Astrophysics Data System (ADS)

Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

2001-05-01

In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

Detection of Bacterial Pathogens from Broncho-Alveolar Lavage by Next-Generation Sequencing.

PubMed

Leo, Stefano; Gaïa, Nadia; Ruppé, Etienne; Emonet, Stephane; Girard, Myriam; Lazarevic, Vladimir; Schrenzel, Jacques

2017-09-20

The applications of whole-metagenome shotgun sequencing (WMGS) in routine clinical analysis are still limited. A combination of a DNA extraction procedure, sequencing, and bioinformatics tools is essential for the removal of human DNA and for improving bacterial species identification in a timely manner. We tackled these issues with a broncho-alveolar lavage (BAL) sample from an immunocompromised patient who had developed severe chronic pneumonia. We extracted DNA from the BAL sample with protocols based either on sequential lysis of human and bacterial cells or on the mechanical disruption of all cells. Metagenomic libraries were sequenced on Illumina HiSeq platforms. Microbial community composition was determined by k-mer analysis or by mapping to taxonomic markers. Results were compared to those obtained by conventional clinical culture and molecular methods. Compared to mechanical cell disruption, a sequential lysis protocol resulted in a significantly increased proportion of bacterial DNA over human DNA and higher sequence coverage of Mycobacterium abscessus , Corynebacterium jeikeium and Rothia dentocariosa , the bacteria reported by clinical microbiology tests. In addition, we identified anaerobic bacteria not searched for by the clinical laboratory. Our results further support the implementation of WMGS in clinical routine diagnosis for bacterial identification.

Drosophila Embryos as Model Systems for Monitoring Bacterial Infection in Real Time

PubMed Central

Evans, Iwan R.; Waterfield, Nicholas; ffrench-Constant, Richard H.; Wood, Will

2009-01-01

Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid ‘freezing’ phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors. PMID:19609447

Application of Recombinant Factor C Reagent for the Detection of Bacterial Endotoxins in Pharmaceutical Products.

PubMed

Bolden, Jay; Smith, Kelly

2017-01-01

Recombinant Factor C (rFC) is non-animal-derived reagent used to detect bacterial endotoxins in pharmaceutical products. Despite the fact that the reagent was first commercially available nearly 15 years ago, the broad use of rFC in pharmaceutical industry has long been lagging, presumably due to historical single-source supplier concerns and the lack of inclusion in worldwide pharmacopeias. Commercial rFC reagents are now available from multiple manufacturers, thus single sourcing is no longer an issue. We report here the successful validation of several pharmaceutical products by an end-point florescence-based endotoxin method using the rFC reagent. The method is equivalent or superior to the compendia bacterial endotoxins test method. Based on the comparability data and extenuating circumstances, the incorporation of the end point fluorescence technique and rFC reagent in global compendia bacterial endotoxins test chapters is desired and warranted. LAY ABSTRACT: Public health has been protected for over 30 years with the use of a purified blood product of the horseshoe crab, limulus amebocyte lysate. More recently, this blood product can be produced in biotech manufacturing processes, which reduces potential impacts to the horseshoe crab and related species dependent upon the crab, for example, migrating shorebirds. The pharmaceutical industry has been slow to adopt the use of this reagent, Recombinant Factor C (rFC), for various reasons. We evaluated the use of rFC across many pharmaceutical products, and in other feasibility demonstration experiments, and found rFC to be a suitable alternative to the animal-derived limulus amebocyte lysate. Incorporation of rFC and its analytical method into national testing standards would provide an equivalent or better test while continuing to maintain patient safety for those who depend on medicines and while securing pharmaceutical supply chains. In addition, widespread use of this method would benefit existing animal

Structure, function, and evolution of bacterial ATP-binding cassette systems

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

Davidson, A.L.; Dassa, E.; Orelle, C.

2010-07-27

The ATP-binding cassette (ABC) systems constitute one of the largest superfamilies of paralogous sequences. All ABC systems share a highly conserved ATP-hydrolyzing domain or protein (the ABC; also referred to as a nucleotide-binding domain [NBD]) that is unequivocally characterized by three short sequence motifs (Fig. 1): these are the Walker A and Walker B motifs, indicative of the presence of a nucleotide-binding site, and the signature motif, unique to ABC proteins, located upstream of the Walker B motif (426). Other motifs diagnostic of ABC proteins are also indicated in Fig. 1. The biological significance of these motifs is discussed inmore » Structure, Function, and Dynamics of the ABC. ABC systems are widespread among living organisms and have been detected in all genera of the three kingdoms of life, with remarkable conservation in the primary sequence of the cassette and in the organization of the constitutive domains or subunits (203, 420). ABC systems couple the energy of ATP hydrolysis to an impressively large variety of essential biological phenomena, comprising not only transmembrane (TM) transport, for which they are best known, but also several non-transport-related processes, such as translation elongation (62) and DNA repair (174). Although ABC systems deserve much attention because they are involved in severe human inherited diseases (107), they were first discovered and characterized in detail in prokaryotes, as early as the 1970s (13, 148, 238, 468). The most extensively analyzed systems were the high-affinity histidine and maltose uptake systems of Salmonella enterica serovar Typhimurium and Escherichia coli. Over 2 decades ago, after the completion of the nucleotide sequences encoding these transporters in the respective laboratories of Giovanna Ames and Maurice Hofnung, Hiroshi Nikaido and colleagues noticed that the two systems displayed a global similarity in the nature of their components and, moreover, that the primary sequences of

Susceptibility of metallic magnesium implants to bacterial biofilm infections.

PubMed

Rahim, Muhammad Imran; Rohde, Manfred; Rais, Bushra; Seitz, Jan-Marten; Mueller, Peter P

2016-06-01

Magnesium alloys have promising mechanical and biological properties as biodegradable medical implant materials for temporary applications during bone healing or as vascular stents. Whereas conventional implants are prone to colonization by treatment resistant microbial biofilms in which bacteria are embedded in a protective matrix, magnesium alloys have been reported to act antibacterial in vitro. To permit a basic assessment of antibacterial properties of implant materials in vivo an economic but robust animal model was established. Subcutaneous magnesium implants were inoculated with bacteria in a mouse model. Contrary to the expectations, bacterial activity was enhanced and prolonged in the presence of magnesium implants. Systemic antibiotic treatments were remarkably ineffective, which is a typical property of bacterial biofilms. Biofilm formation was further supported by electron microscopic analyses that revealed highly dense bacterial populations and evidence for the presence of extracellular matrix material. Bacterial agglomerates could be detected not only on the implant surface but also at a limited distance in the peri-implant tissue. Therefore, precautions may be necessary to minimize risks of metallic magnesium-containing implants in prospective clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1489-1499, 2016. © 2016 Wiley Periodicals, Inc.

Apparatus and method for automated monitoring of airborne bacterial spores

NASA Technical Reports Server (NTRS)

Ponce, Adrian (Inventor)

2009-01-01

An apparatus and method for automated monitoring of airborne bacterial spores. The apparatus is provided with an air sampler, a surface for capturing airborne spores, a thermal lysis unit to release DPA from bacterial spores, a source of lanthanide ions, and a spectrometer for excitation and detection of the characteristic fluorescence of the aromatic molecules in bacterial spores complexed with lanthanide ions. In accordance with the method: computer-programmed steps allow for automation of the apparatus for the monitoring of airborne bacterial spores.

On-line detection of Escherichia coli intrusion in a pilot-scale drinking water distribution system.

PubMed

Ikonen, Jenni; Pitkänen, Tarja; Kosse, Pascal; Ciszek, Robert; Kolehmainen, Mikko; Miettinen, Ilkka T

2017-08-01

Improvements in microbial drinking water quality monitoring are needed for the better control of drinking water distribution systems and for public health protection. Conventional water quality monitoring programmes are not always able to detect a microbial contamination of drinking water. In the drinking water production chain, in addition to the vulnerability of source waters, the distribution networks are prone to contamination. In this study, a pilot-scale drinking-water distribution network with an on-line monitoring system was utilized for detecting bacterial intrusion. During the experimental Escherichia coli intrusions, the contaminant was measured by applying a set of on-line sensors for electric conductivity (EC), pH, temperature (T), turbidity, UV-absorbance at 254 nm (UVAS SC) and with a device for particle counting. Monitored parameters were compared with the measured E. coli counts using the integral calculations of the detected peaks. EC measurement gave the strongest signal compared with the measured baseline during the E. coli intrusion. Integral calculations showed that the peaks in the EC, pH, T, turbidity and UVAS SC data were detected corresponding to the time predicted. However, the pH and temperature peaks detected were barely above the measured baseline and could easily be mixed with the background noise. The results indicate that on-line monitoring can be utilized for the rapid detection of microbial contaminants in the drinking water distribution system although the peak interpretation has to be performed carefully to avoid being mixed up with normal variations in the measurement data. Copyright © 2017 Elsevier Ltd. All rights reserved.

40 CFR 799.9510 - TSCA bacterial reverse mutation test.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 32 2014-07-01 2014-07-01 false TSCA bacterial reverse mutation test... REQUIREMENTS Health Effects Test Guidelines § 799.9510 TSCA bacterial reverse mutation test. (a) Scope. This... mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect...

A New Experimental Design for Bacterial Microleakage Investigation at the Implant-Abutment Interface: An In Vitro Study.

PubMed

Zipprich, Holger; Miatke, Sven; Hmaidouch, Rim; Lauer, Hans-Christoph

2016-01-01

This study aimed to test bacterial microleakage at the implant-abutment interface (IAI) before and after dynamic loading using a new chewing simulation. Fourteen implant systems (n = 5 samples of each) were divided into two groups: (1) systems with conical implant-abutment connections (IACs), and (2) systems with flat IACs. For collecting samples without abutment disconnection, channels (Ø = 0.3 mm) were drilled into implants perpendicularly to their axes, and stainless-steel cannulas were adhesively glued inside these channels to allow a sterilized rinsing solution to enter the implant interior and to exit with potential contaminants for testing. Implants were embedded in epoxy resin matrices, which were supported by titanium cylinders with lateral openings for inward and outward cannulas. Abutments were tightened and then provided with vertically adjustable, threaded titanium balls, which were cemented using composite cement. Specimens were immersed in a bacterial liquid and after a contact time of 15 minutes, the implant interior was rinsed prior to chewing simulation (0 N ≘ static seal testing). Specimens were exposed to a Frankfurt chewing simulator. Two hundred twenty force cycles per power level (110 in ± X-axis) were applied to simulate a daily masticatory load of 660 chewing cycles (equivalent to 1,200,000 cycles/5 years). The applied load was gradually increased from 0 N to a maximum load of 200 N in 25-N increments. The implant interior was rinsed to obtain samples before each new power level. All samples were tested using fluorescence microscopy; invading microorganisms could be counted and evaluated. No bacterial contamination was detected under static loading conditions in both groups. After loading, bacterial contamination was detected in one sample from one specimen in group 1 and in two samples from two specimens in group 2. Controlled dynamic loading applied in this study simulated a clinical situation and enabled time-dependent analysis

Effects of bacterial pollution caused by a strong typhoon event and the restoration of a recreational beach: Transitions of fecal bacterial counts and bacterial flora in beach sand.

PubMed

Suzuki, Yoshihiro; Teranishi, Kotaro; Matsuwaki, Tomonori; Nukazawa, Kei; Ogura, Yoshitoshi

2018-05-28

To determine the effects of bacteria pollution associated with a strong typhoon event and to assess the restoration of the normal bacterial flora, we used conventional filtration methods and nextgeneration sequencing of 16S rRNA genes to analyze the transition of fecal and total bacterial counts in water and core sand samples collected from a recreational beach. Immediately after the typhoon event, Escherichia coli counts increased to 82 CFU/100 g in the surface beach sand. E. coli was detected through the surface to sand 85-cm deep at the land side point (10-m land side from the high-water line). However, E. coli disappeared within a month from the land side point. The composition of the bacterial flora in the beach sand at the land point was directly influenced by the typhoon event. Pseudomonas was the most prevalent genus throughout the sand layers (0-102-cm deep) during the typhoon event. After 3 months, the population of Pseudomonas significantly decreased, and the predominant genus in the surface layer was Kaistobacter, although Pseudomonas was the major genus in the 17- to 85-cm layer. When the beach conditions stabilized, the number of pollutant Pseudomonas among the 10 most abundant genera decreased to lower than the limit of detection. The bacterial population of the sand was subsequently restored to the most populous pre-event orders at the land point. A land-side beach, where users directly contact the sand, was significantly affected by bacterial pollution caused by a strong typhoon event. We show here that the normal bacterial flora of the surface sand was restored within 1 month. Copyright © 2018 Elsevier B.V. All rights reserved.

[Autochthonous acute viral and bacterial infections of the central nervous system (meningitis and encephalitis)].

PubMed

Pérez-Ruiz, Mercedes; Vicente, Diego; Navarro-Marí, José María

2008-07-01

Rapid diagnosis of acute viral and bacterial infections of the central nervous system (meningitis and encephalitis) is highly important for the clinical management of the patient and helps to establish early therapy that may solve life-threatening situations, to avoid unnecessary empirical treatments, to reduce hospital stay, and to facilitate appropriate interventions in the context of public health. Molecular techniques, especially real-time polymerase chain reaction, have become the fastest and most sensitive diagnostic procedures for autochthonous viral meningitis and encephalitis, and their role is becoming increasingly important for the diagnosis and control of most frequent acute bacterial meningitides. Automatic and closed systems may encourage the widespread and systematic use of molecular techniques for the diagnosis of these neurological syndromes in most laboratories.

Noninvasive forward-scattering system for rapid detection, characterization, and identification of Listeria colonies: image processing and data analysis

NASA Astrophysics Data System (ADS)

Rajwa, Bartek; Bayraktar, Bulent; Banada, Padmapriya P.; Huff, Karleigh; Bae, Euiwon; Hirleman, E. Daniel; Bhunia, Arun K.; Robinson, J. Paul

2006-10-01

Bacterial contamination by Listeria monocytogenes puts the public at risk and is also costly for the food-processing industry. Traditional methods for pathogen identification require complicated sample preparation for reliable results. Previously, we have reported development of a noninvasive optical forward-scattering system for rapid identification of Listeria colonies grown on solid surfaces. The presented system included application of computer-vision and patternrecognition techniques to classify scatter pattern formed by bacterial colonies irradiated with laser light. This report shows an extension of the proposed method. A new scatterometer equipped with a high-resolution CCD chip and application of two additional sets of image features for classification allow for higher accuracy and lower error rates. Features based on Zernike moments are supplemented by Tchebichef moments, and Haralick texture descriptors in the new version of the algorithm. Fisher's criterion has been used for feature selection to decrease the training time of machine learning systems. An algorithm based on support vector machines was used for classification of patterns. Low error rates determined by cross-validation, reproducibility of the measurements, and robustness of the system prove that the proposed technology can be implemented in automated devices for detection and classification of pathogenic bacteria.

Detection of a mixed infection in a culture-negative brain abscess by broad-spectrum bacterial 16S rRNA gene PCR.

PubMed

Keller, Peter M; Rampini, Silvana K; Bloemberg, Guido V

2010-06-01

We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis.

Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization▿ †

PubMed Central

Smolina, Irina; Lee, Charles; Frank-Kamenetskii, Maxim

2007-01-01

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes. PMID:17293504

Nanobiocomposite platform based on polyaniline-iron oxide-carbon nanotubes for bacterial detection.

PubMed

Singh, Renu; Verma, Rachna; Sumana, G; Srivastava, Avanish Kumar; Sood, Seema; Gupta, Rajinder K; Malhotra, B D

2012-08-01

The nanocomposite based on polyaniline (PANI)-iron oxide nanoparticles (nFe(3)O(4)) and multi walled carbon-nanotubes (CNT) has been fabricated onto indium tin oxide (ITO) coated glass plate via facile electrochemical synthesis of polyaniline in presence of nFe(3)O(4) (~20 nm) and CNT (20-80 nm in diameter). The results of transmission electron microscopic studies show evidence of coating of PANI and nFe(3)O(4) onto the CNT. The PANI-nFe(3)O(4)-CNT/ITO nanoelectrode has been characterized by Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy studies. The biotinylated nucleic acid probe sequence consisting of 20 bases has been immobilized onto PANI-nFe(3)O(4)-CNT/ITO nanoelectrode using biotin-avidin coupling. It is shown that the PANI-nFe(3)O(4)-CNT platform based biosensor can be used to specifically detect bacteria (N. gonorrhoeae) at minute concentration as low as (1×10(-19) M) indicating high sensitivity within 45 s of hybridization time at 298 K by differential pulse voltammetry using methylene blue as electroactive indicator. This bacterial sensor has also been tested with 4 positive and 4 negative PCR amplicons of gonorrhoea affected patient samples. The results of these studies have implications towards the fabrication of a handheld device for Neisseria gonorrhoeae detection that may perhaps result in a decrease in the human immunodeficiency virus infections. Copyright © 2012 Elsevier B.V. All rights reserved.

A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens.

PubMed

Rundell, Mark S; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H; Spitzer, Eric D; Golightly, Linnie; Barany, Francis

2014-06-01

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm.

PubMed

Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

2016-08-01

There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (D_{tot}) and the displacement distribution functions (P(r,t)) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ, which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ=0.65, while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ. We also investigate the distribution (P(θ,t)) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm

NASA Astrophysics Data System (ADS)

Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

2016-08-01

There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (Dtot) and the displacement distribution functions (P (r ,t ) ) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ , which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ =0.65 , while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ . We also investigate the distribution (P (θ ,t ) ) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

Distribution and diversity of bacterial secretion systems across metagenomic datasets.

PubMed

Barret, Matthieu; Egan, Frank; O'Gara, Fergal

2013-02-01

Bacteria can manipulate their surrounding environment through the secretion of proteins into other living organisms and into the extracellular milieu. In Gram stain negative bacteria this process is mediated by different types of secretion systems from type I through type VI secretion system (T1SS-T6SS). In this study the prevalence of these secretion systems in 312 publicly available microbiomes derived from a wide range of ecosystems was investigated by a gene-centric approach. Our analysis demonstrates that some secretion systems are over-represented in some specific samples. In addition, some T3SS and T6SS phylogenetic clusters were specifically enriched in particular ecological niches, which could indicate specific bacterial adaptation to these environments. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Homologs of the Acinetobacter baumannii AceI transporter represent a new family of bacterial multidrug efflux systems.

PubMed

Hassan, Karl A; Liu, Qi; Henderson, Peter J F; Paulsen, Ian T

2015-02-10

Multidrug efflux systems are a major cause of resistance to antimicrobials in bacteria, including those pathogenic to humans, animals, and plants. These proteins are ubiquitous in these pathogens, and five families of bacterial multidrug efflux systems have been identified to date. By using transcriptomic and biochemical analyses, we recently identified the novel AceI (Acinetobacter chlorhexidine efflux) protein from Acinetobacter baumannii that conferred resistance to the biocide chlorhexidine, via an active efflux mechanism. Proteins homologous to AceI are encoded in the genomes of many other bacterial species and are particularly prominent within proteobacterial lineages. In this study, we expressed 23 homologs of AceI and examined their resistance and/or transport profiles. MIC analyses demonstrated that, like AceI, many of the homologs conferred resistance to chlorhexidine. Many of the AceI homologs conferred resistance to additional biocides, including benzalkonium, dequalinium, proflavine, and acriflavine. We conducted fluorimetric transport assays using the AceI homolog from Vibrio parahaemolyticus and confirmed that resistance to both proflavine and acriflavine was mediated by an active efflux mechanism. These results show that this group of AceI homologs represent a new family of bacterial multidrug efflux pumps, which we have designated the proteobacterial antimicrobial compound efflux (PACE) family of transport proteins. Bacterial multidrug efflux pumps are an important class of resistance determinants that can be found in every bacterial genome sequenced to date. These transport proteins have important protective functions for the bacterial cell but are a significant problem in the clinical setting, since a single efflux system can mediate resistance to many structurally and mechanistically diverse antibiotics and biocides. In this study, we demonstrate that proteins related to the Acinetobacter baumannii AceI transporter are a new class of multidrug

Specific detection of common pathogens of acute bacterial meningitis using an internally controlled tetraplex-PCR assay.

PubMed

Farahani, Hamidreza; Ghaznavi-Rad, Ehsanollah; Mondanizadeh, Mahdieh; MirabSamiee, Siamak; Khansarinejad, Behzad

2016-08-01

Accurate and timely diagnosis of acute bacterial meningitis is critical for antimicrobial treatment of patients. Although PCR-based methods have been widely used for the diagnosis of acute meningitis caused by bacterial pathogens, the main disadvantage of these methods is their high cost. This disadvantage has hampered the widespread use of molecular assays in many developing countries. The application of multiplex assays and "in-house" protocols are two main approaches that can reduce the overall cost of a molecular test. In the present study, an internally controlled tetraplex-PCR was developed and validated for the specific detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in cerebrospinal fluid (CSF) samples. The analysis of a panel of other human pathogens showed no cross-reactivity in the assay. The analytical sensitivity of the in-house assay was 792.3 copies/ml, when all three bacteria were presentin the specimens. This value was calculated as 444.5, 283.7, 127.8 copies/ml when only S. pneumoniae, N. meningitidis and H. influenzae, respectively, were present. To demonstrate the diagnostic performance of the assay, a total of 150 archival CSF samples were tested and compared with a commercial multiplex real-time PCR kit. A diagnostic sensitivity of 92.8% and a specificity of 95.1% were determined for the present tetraplex-PCR assay. The results indicate that the established method is sensitive, specific and cost-effective, and can be used particularly in situations where the high cost of commercial kits prevents the use of molecular methods for the diagnosis of bacterial meningitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differentiation of bacterial colonies and temporal growth patterns using hyperspectral imaging

NASA Astrophysics Data System (ADS)

Mehrübeoglu, Mehrube; Buck, Gregory W.; Livingston, Daniel W.

2014-09-01

Detection and identification of bacteria are important for health and safety. Hyperspectral imaging offers the potential to capture unique spectral patterns and spatial information from bacteria which can then be used to detect and differentiate bacterial species. Here, hyperspectral imaging has been used to characterize different bacterial colonies and investigate their growth over time. Six bacterial species (Pseudomonas fluorescens, Escherichia coli, Serratia marcescens, Salmonella enterica, Staphylococcus aureus, Enterobacter aerogenes) were grown on tryptic soy agar plates. Hyperspectral data were acquired immediately after, 24 hours after, and 96 hours after incubation. Spectral signatures from bacterial colonies demonstrated repeatable measurements for five out of six species. Spatial variations as well as changes in spectral signatures were observed across temporal measurements within and among species at multiple wavelengths due to strengthening or weakening reflectance signals from growing bacterial colonies based on their pigmentation. Between-class differences and within-class similarities were the most prominent in hyperspectral data collected 96 hours after incubation.

Characterization of bacterial community structure in a drinking water distribution system during an occurrence of red water.

PubMed

Li, Dong; Li, Zheng; Yu, Jianwei; Cao, Nan; Liu, Ruyin; Yang, Min

2010-11-01

The role of bacteria in the occasional emergence of red water, which has been documented worldwide, has yet to be determined. To better understand the mechanisms that drive occurrences of red water, the bacterial community composition and the relative abundance of several functional bacterial groups in a water distribution system of Beijing during a large-scale red water event were determined using several molecular methods. Individual clone libraries of the 16S rRNA gene were constructed for three red water samples and one sample of normal water. Beta-, Alpha-, and Gammaproteobacteria comprised the major bacterial communities in both red water and normal water samples, in agreement with previous reports. A high percentage of red water clones (25.2 to 57.1%) were affiliated with or closely related to a diverse array of iron-oxidizing bacteria, including the neutrophilic microaerobic genera Gallionella and Sideroxydans, the acidophilic species Acidothiobacillus ferrooxidans, and the anaerobic denitrifying Thermomonas bacteria. The genus Gallionella comprised 18.7 to 28.6% of all clones in the three red water libraries. Quantitative real-time PCR analysis showed that the 16S rRNA gene copy concentration of Gallionella spp. was between (4.1 ± 0.9) × 10⁷ (mean ± standard deviation) and (1.6 ± 0.3) × 10⁸ per liter in red water, accounting for 13.1% ± 2.9% to 17.2% ± 3.6% of the total Bacteria spp. in these samples. By comparison, the percentages of Gallionella spp. in the normal water samples were 0.1% or lower (below the limit of detection), suggesting an important role of Gallionella spp. in the formation of red water.

System Detects Vibrational Instabilities

NASA Technical Reports Server (NTRS)

Bozeman, Richard J., Jr.

1990-01-01

Sustained vibrations at two critical frequencies trigger diagnostic response or shutdown. Vibration-analyzing electronic system detects instabilities of combustion in rocket engine. Controls pulse-mode firing of engine and identifies vibrations above threshold amplitude at 5.9 and/or 12kHz. Adapted to other detection and/or control schemes involving simultaneous real-time detection of signals above or below preset amplitudes at two or more specified frequencies. Potential applications include rotating machinery and encoders and decoders in security systems.

An automatically tuning intrusion detection system.

PubMed

Yu, Zhenwei; Tsai, Jeffrey J P; Weigert, Thomas

2007-04-01

An intrusion detection system (IDS) is a security layer used to detect ongoing intrusive activities in information systems. Traditionally, intrusion detection relies on extensive knowledge of security experts, in particular, on their familiarity with the computer system to be protected. To reduce this dependence, various data-mining and machine learning techniques have been deployed for intrusion detection. An IDS is usually working in a dynamically changing environment, which forces continuous tuning of the intrusion detection model, in order to maintain sufficient performance. The manual tuning process required by current systems depends on the system operators in working out the tuning solution and in integrating it into the detection model. In this paper, an automatically tuning IDS (ATIDS) is presented. The proposed system will automatically tune the detection model on-the-fly according to the feedback provided by the system operator when false predictions are encountered. The system is evaluated using the KDDCup'99 intrusion detection dataset. Experimental results show that the system achieves up to 35% improvement in terms of misclassification cost when compared with a system lacking the tuning feature. If only 10% false predictions are used to tune the model, the system still achieves about 30% improvement. Moreover, when tuning is not delayed too long, the system can achieve about 20% improvement, with only 1.3% of the false predictions used to tune the model. The results of the experiments show that a practical system can be built based on ATIDS: system operators can focus on verification of predictions with low confidence, as only those predictions determined to be false will be used to tune the detection model.

Role of quorum sensing in bacterial infections

PubMed Central

Castillo-Juárez, Israel; Maeda, Toshinari; Mandujano-Tinoco, Edna Ayerim; Tomás, María; Pérez-Eretza, Berenice; García-Contreras, Silvia Julieta; Wood, Thomas K; García-Contreras, Rodolfo

2015-01-01

Quorum sensing (QS) is cell communication that is widely used by bacterial pathogens to coordinate the expression of several collective traits, including the production of multiple virulence factors, biofilm formation, and swarming motility once a population threshold is reached. Several lines of evidence indicate that QS enhances virulence of bacterial pathogens in animal models as well as in human infections; however, its relative importance for bacterial pathogenesis is still incomplete. In this review, we discuss the present evidence from in vitro and in vivo experiments in animal models, as well as from clinical studies, that link QS systems with human infections. We focus on two major QS bacterial models, the opportunistic Gram negative bacteria Pseudomonas aeruginosa and the Gram positive Staphylococcus aureus, which are also two of the main agents responsible of nosocomial and wound infections. In addition, QS communication systems in other bacterial, eukaryotic pathogens, and even immune and cancer cells are also reviewed, and finally, the new approaches proposed to combat bacterial infections by the attenuation of their QS communication systems and virulence are also discussed. PMID:26244150

Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity

PubMed Central

Kalb, Suzanne R.; Boyer, Anne E.; Barr, John R.

2015-01-01

Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin. PMID:26404376

Influence of tillage systems on Rhizoctonia-bacterial root rot complex in sugar beet

USDA-ARS?s Scientific Manuscript database

The Rhizoctonia-bacterial root rot complex on sugarbeet caused by Rhizoctonia solani and Leuconostoc mesenteroides can cause significant yield losses. To investigate the impact of different tillage systems on this complex, field studies were conducted from 2009 to 2011. Split blocks with conventio...

Synthetic analogs of bacterial quorum sensors

DOEpatents

Iyer, Rashi [Los Alamos, NM; Ganguly, Kumkum [Los Alamos, NM; Silks, Louis A [Los Alamos, NM

2011-12-06

Bacterial quorum-sensing molecule analogs having the following structures: ##STR00001## and methods of reducing bacterial pathogenicity, comprising providing a biological system comprising pathogenic bacteria which produce natural quorum-sensing molecule; providing a synthetic bacterial quorum-sensing molecule having the above structures and introducing the synthetic quorum-sensing molecule into the biological system comprising pathogenic bacteria. Further is provided a method of targeted delivery of an antibiotic, comprising providing a synthetic quorum-sensing molecule; chemically linking the synthetic quorum-sensing molecule to an antibiotic to produce a quorum-sensing molecule-antibiotic conjugate; and introducing the conjugate into a biological system comprising pathogenic bacteria susceptible to the antibiotic.

Synthetic analogs of bacterial quorum sensors

DOEpatents

Iyer, Rashi S.; Ganguly, Kumkum; Silks, Louis A.

2013-01-08

Bacterial quorum-sensing molecule analogs having the following structures: ##STR00001## and methods of reducing bacterial pathogenicity, comprising providing a biological system comprising pathogenic bacteria which produce natural quorum-sensing molecule; providing a synthetic bacterial quorum-sensing molecule having the above structures and introducing the synthetic quorum-sensing molecule into the biological system comprising pathogenic bacteria. Further is provided a method of targeted delivery of an antibiotic, comprising providing a synthetic quorum-sensing molecule; chemically linking the synthetic quorum-sensing molecule to an antibiotic to produce a quorum-sensing molecule-antibiotic conjugate; and introducing the conjugate into a biological system comprising pathogenic bacteria susceptible to the antibiotic.

Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology

PubMed Central

2017-01-01

ABSTRACT Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilis. IMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that

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.

Residual Agar Determination in Bacterial Spores by Electrospray Ionization Mass Spectrometry

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

Wahl, Karen L.; Colburn, Heather A.; Wunschel, David S.

2010-02-15

Presented here is an analytical method to detect residual agar from a bacterial spore sample as an indication of culturing on an agar plate. This method is based on the resolubilization of agar polysaccharide from a bacterial spore sample, enzymatic digestion, followed by electrospray ionization tandem mass spectrometry (ESI-MSn) analysis for detection of a specific agar fragment ion. A range of Bacillus species and strains were selected to demonstrate the effectiveness of this approach. The characteristic agar fragment ion was detected in the spores grown on agar that were washed from 1 to 5 times, irradiated or non-irradiated and notmore » in the spores grown in broth. A sample containing approximately 108 spores is currently needed for confident detection of residual agar from culture on agar plates in the presence of bacterial spores with a limit of detection of approximately 1 ppm agar spiked into a broth-grown spore sample. The results of a proficiency test with 42 blinded samples are presented demonstrating the utility of this method with no false positives and only 3 false negatives for samples that were below the detection level of the method as documented.« less

Near-infrared Surface-Enhanced-Raman-Scattering (SERS) mediated discrimination of single optically trapped bacterial spores

NASA Astrophysics Data System (ADS)

Alexander, Troy A.; Pellegrino, Paul M.; Gillespie, James B.

2004-03-01

A novel methodology has been developed for the investigation of bacterial spores. Specifically, this method has been used to probe the spore coat composition of two different Bacillus stearothermophilus variants. This technique may be useful in many applications; most notably, development of novel detection schemes toward potentially harmful bacteria. This method would also be useful as an ancillary environmental monitoring system where sterility is of importance (i.e., food preparation areas as well as invasive and minimally invasive medical applications). This unique detection scheme is based on the near-infrared (NIR) Surface-Enhanced-Raman- Scattering (SERS) from single, optically trapped, bacterial spores. The SERS spectra of bacterial spores in aqueous media have been measured using SERS substrates based on ~60-nm diameter gold colloids bound to 3-Aminopropyltriethoxysilane derivatized glass. The light from a 787-nm laser diode was used to trap/manipulate as well as simultaneously excite the SERS of an individual bacterial spore. The collected SERS spectra were examined for uniqueness and the applicability of this technique for the strain discrimination of Bacillus stearothermophilus spores. Comparison of normal Raman and SERS spectra reveal not only an enhancement of the normal Raman spectral features but also the appearance of spectral features absent in the normal Raman spectrum.

Intrusion Detection in Database Systems

NASA Astrophysics Data System (ADS)

Javidi, Mohammad M.; Sohrabi, Mina; Rafsanjani, Marjan Kuchaki

Data represent today a valuable asset for organizations and companies and must be protected. Ensuring the security and privacy of data assets is a crucial and very difficult problem in our modern networked world. Despite the necessity of protecting information stored in database systems (DBS), existing security models are insufficient to prevent misuse, especially insider abuse by legitimate users. One mechanism to safeguard the information in these databases is to use an intrusion detection system (IDS). The purpose of Intrusion detection in database systems is to detect transactions that access data without permission. In this paper several database Intrusion detection approaches are evaluated.

Regulation of the Expression of Bacterial Multidrug Exporters by Two-Component Signal Transduction Systems.

PubMed

Nishino, Kunihiko

2018-01-01

Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.

A model system for pathogen detection using a two-component bacteriophage/bioluminescent signal amplification assay

NASA Astrophysics Data System (ADS)

Bright, Nathan G.; Carroll, Richard J.; Applegate, Bruce M.

2004-03-01

Microbial contamination has become a mounting concern the last decade due to an increased emphasis of minimally processed food products specifically produce, and the recognition of foodborne pathogens such as Campylobacter jejuni, Escherichia coli O157:H7, and Listeria monocytogenes. This research investigates a detection approach utilizing bacteriophage pathogen specificity coupled with a bacterial bioluminescent bioreporter utilizing the quorum sensing molecule from Vibrio fischeri, N-(3-oxohexanoyl)-homoserine lactone (3-oxo-C6-HSL). The 3-oxo-C6-HSL molecules diffuse out of the target cell after infection and induce bioluminescence from a population of 3-oxo-C6-HSL bioreporters (ROLux). E. coli phage M13, a well-characterized bacteriophage, offers a model system testing the use of bacteriophage for pathogen detection through cell-to-cell communication via a LuxR/3-oxo-C6-HSL system. Simulated temperate phage assays tested functionality of the ROLux reporter and production of 3-oxo-C6-HSL by various test strains. These assays showed detection limits of 102cfu after 24 hours in a varietry of detection formats. Assays incorporating the bacteriophage M13-luxI with the ROLux reporter and a known population of target cells were subsequently developed and have shown consistent detection limits of 105cfu target organisms. Measurable light response from high concentrations of target cells was almost immediate, suggesting an enrichment step to further improve detection limits and reduce assay time.

Bacterial Pathogens Associated with Community-acquired Pneumonia in Children Aged Below Five Years.

PubMed

Das, Anusmita; Patgiri, Saurav J; Saikia, Lahari; Dowerah, Pritikar; Nath, Reema

2016-03-01

To determine the spectrum of bacterial pathogens causing community-acquired pneumonia in children below 5 years of age. Children aged below 5 years satisfying the WHO criteria for pneumonia, severe pneumonia or very severe pneumonia, and with the presence of lung infiltrates on chest X-ray were enrolled. Two respiratory samples, one for culture and the other for PCR analysis, and a blood sample for culture were collected from every child. Of the 180 samples processed, bacterial pathogens were detected in 64.4%. Streptococcus pneumoniae and Hemophilus influenzae were most frequently detected. The performance of PCR analysis and culture were identical for the typical bacterial pathogens; atypical pathogens were detected by PCR analysis only. S. pneumoniae and H. influenza were the most commonly detected organisms from respiratory secretions of children with community acquired pneumonia.

Flow Chamber System for the Statistical Evaluation of Bacterial Colonization on Materials

PubMed Central

Menzel, Friederike; Conradi, Bianca; Rodenacker, Karsten; Gorbushina, Anna A.; Schwibbert, Karin

2016-01-01

Biofilm formation on materials leads to high costs in industrial processes, as well as in medical applications. This fact has stimulated interest in the development of new materials with improved surfaces to reduce bacterial colonization. Standardized tests relying on statistical evidence are indispensable to evaluate the quality and safety of these new materials. We describe here a flow chamber system for biofilm cultivation under controlled conditions with a total capacity for testing up to 32 samples in parallel. In order to quantify the surface colonization, bacterial cells were DAPI (4`,6-diamidino-2-phenylindole)-stained and examined with epifluorescence microscopy. More than 100 images of each sample were automatically taken and the surface coverage was estimated using the free open source software g’mic, followed by a precise statistical evaluation. Overview images of all gathered pictures were generated to dissect the colonization characteristics of the selected model organism Escherichia coli W3310 on different materials (glass and implant steel). With our approach, differences in bacterial colonization on different materials can be quantified in a statistically validated manner. This reliable test procedure will support the design of improved materials for medical, industrial, and environmental (subaquatic or subaerial) applications. PMID:28773891

Particle detection systems and methods

DOEpatents

Morris, Christopher L.; Makela, Mark F.

2010-05-11

Techniques, apparatus and systems for detecting particles such as muons and neutrons. In one implementation, a particle detection system employs a plurality of drift cells, which can be for example sealed gas-filled drift tubes, arranged on sides of a volume to be scanned to track incoming and outgoing charged particles, such as cosmic ray-produced muons. The drift cells can include a neutron sensitive medium to enable concurrent counting of neutrons. The system can selectively detect devices or materials, such as iron, lead, gold, uranium, plutonium, and/or tungsten, occupying the volume from multiple scattering of the charged particles passing through the volume and can concurrently detect any unshielded neutron sources occupying the volume from neutrons emitted therefrom. If necessary, the drift cells can be used to also detect gamma rays. The system can be employed to inspect occupied vehicles at border crossings for nuclear threat objects.

Rapid detection of bacterial pathogens using flourescence spectroscopy and chemometrics

USDA-ARS?s Scientific Manuscript database

This work presents the development of a method for rapid bacterial identification based on the fluorescence spectroscopy combined with multivariate analysis. Fluorescence spectra of pure three different genera of bacteria (Escherichia coli, Salmonella, and Campylobacter) were collected from 200...

A uniform bacterial growth potential assay for different water types.

PubMed

Farhat, Nadia; Hammes, Frederik; Prest, Emmanuelle; Vrouwenvelder, Johannes

2018-06-06

The bacterial growth potential is important to understand and manage bacterial regrowth-related water quality concerns. Bacterial growth potential depends on growth promoting/limiting compounds, therefore, nutrient availability is the key factor governing bacterial growth potential. Selecting proper tools for bacterial growth measurement is essential for routine implementation of the growth potential measurement. This study proposes a growth potential assay that is universal and can be used for different water types and soil extract without restrictions of pure culture or cultivability of the bacterial strain. The proposed assay measures the sample bacterial growth potential by using the indigenous community as inocula. Flow cytometry (FCM) and adenosine tri-phosphate (ATP) were used to evaluate the growth potential of six different microbial communities indigenous to the sample being analyzed, with increasing carbon concentrations. Bottled mineral water, non-chlorinated tap water, seawater, river water, wastewater effluent and a soil organic carbon extract were analyzed. Results showed that indigenous bacterial communities followed normal batch growth kinetics when grown on naturally present organic carbon. Indigenous bacterial growth could detect spiked organic carbon concentrations as low as 10 μg/L. The indigenous community in all samples responded proportionally to the increase in acetate-carbon and proportional growth could be measured with both FCM and ATP. Bacterial growth was proportional to the carbon concentration but not the same proportion factor for the different water samples tested. The effect of inoculating the same water with different indigenous microbial communities on the growth potential was also examined. The FCM results showed that the highest increase in total bacterial cell concentration was obtained with bacteria indigenous to the water sample. The growth potential assay using indigenous bacterial community revealed consistent results

Utility of the serum C-reactive protein for detection of occult bacterial infection in children.

PubMed

Isaacman, Daniel J; Burke, Bonnie L

2002-09-01

To assess the utility of serum C-reactive protein (CRP) as a screen for occult bacterial infection in children. Febrile children ages 3 to 36 months who visited an urban children's hospital emergency department and received a complete blood cell count and blood culture as part of their evaluation were prospectively enrolled from February 2, 2000, through May 30, 2001. Informed consent was obtained for the withdrawal of an additional 1-mL aliquot of blood for use in CRP evaluation. Logistic regression and receiver operator characteristic (ROC) curves were modeled for each predictor to identify optimal test values, and were compared using likelihood ratio tests. Two hundred fifty-six patients were included in the analysis, with a median age of 15.3 months (range, 3.1-35.2 months) and median temperature at triage 40.0 degrees C (range, 39.0 degrees C-41.3 degrees C). Twenty-nine (11.3%) cases of occult bacterial infection (OBI) were identified, including 17 cases of pneumonia, 9 cases of urinary tract infection, and 3 cases of bacteremia. The median white blood cell count in this data set was 12.9 x 10(3)/ micro L [corrected] (range, 3.6-39.1 x10(3)/ micro L) [corrected], the median absolute neutrophil count (ANC) was 7.12 x 10(3)/L [corrected] (range, 0.56-28.16 x10(3)/L) [corrected], and the median CRP level was 1.7 mg/dL (range, 0.2-43.3 mg/dL). The optimal cut-off point for CRP in this data set (4.4 mg/dL) achieved a sensitivity of 63% and a specificity of 81% for detection of OBI in this population. Comparing models using cut-off values from individual laboratory predictors (ANC, white blood cell count, and CRP) that maximized sensitivity and specificity revealed that a model using an ANC of 10.6 x10(3)/L [corrected] (sensitivity, 69%; specificity, 79%) was the best predictive model. Adding CRP to the model insignificantly increased sensitivity to 79%, while significantly decreasing specificity to 50%. Active monitoring of emergency department blood cultures

Solar system fault detection

DOEpatents

Farrington, R.B.; Pruett, J.C. Jr.

1984-05-14

A fault detecting apparatus and method are provided for use with an active solar system. The apparatus provides an indication as to whether one or more predetermined faults have occurred in the solar system. The apparatus includes a plurality of sensors, each sensor being used in determining whether a predetermined condition is present. The outputs of the sensors are combined in a pre-established manner in accordance with the kind of predetermined faults to be detected. Indicators communicate with the outputs generated by combining the sensor outputs to give the user of the solar system and the apparatus an indication as to whether a predetermined fault has occurred. Upon detection and indication of any predetermined fault, the user can take appropriate corrective action so that the overall reliability and efficiency of the active solar system are increased.

Solar system fault detection

DOEpatents

Farrington, Robert B.; Pruett, Jr., James C.

1986-01-01

A fault detecting apparatus and method are provided for use with an active solar system. The apparatus provides an indication as to whether one or more predetermined faults have occurred in the solar system. The apparatus includes a plurality of sensors, each sensor being used in determining whether a predetermined condition is present. The outputs of the sensors are combined in a pre-established manner in accordance with the kind of predetermined faults to be detected. Indicators communicate with the outputs generated by combining the sensor outputs to give the user of the solar system and the apparatus an indication as to whether a predetermined fault has occurred. Upon detection and indication of any predetermined fault, the user can take appropriate corrective action so that the overall reliability and efficiency of the active solar system are increased.

Temperature variation, bacterial diversity and fungal infection dynamics in the amphibian skin.

PubMed

Longo, Ana V; Zamudio, Kelly R

2017-09-01

Host-associated bacterial communities on the skin act as the first line of defence against invading pathogens. Yet, for most natural systems, we lack a clear understanding of how temperature variability affects structure and composition of skin bacterial communities and, in turn, promotes or limits the colonization of opportunistic pathogens. Here, we examine how natural temperature fluctuations might be related to changes in skin bacterial diversity over time in three amphibian populations infected by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Our focal host species (Eleutherodactylus coqui) is a direct-developing frog that has suffered declines at some populations in the last 20 years, while others have not experienced any changes. We quantified skin bacterial alpha- and beta-diversity at four sampling time points, a period encompassing two seasons and ample variation in natural infections and environmental conditions. Despite the different patterns of infection across populations, we detected an overall increase in bacterial diversity through time, characterized by the replacement of bacterial operational taxonomic units (OTUs). Increased frog body temperatures possibly allowed the colonization of bacteria as well as the recruitment of a subset of indicator OTUs, which could have promoted the observed changes in diversity patterns. Our results suggest that natural environmental fluctuations might be involved in creating opportunities for bacterial replacement, potentially attenuating pathogen transmission and thus contributing to host persistence in E. coqui populations. © 2017 John Wiley & Sons Ltd.

Comprehensive Evaluation of the MBT STAR-BL Module for Simultaneous Bacterial Identification and β-Lactamase-Mediated Resistance Detection in Gram-Negative Rods from Cultured Isolates and Positive Blood Cultures.

PubMed

Lee, Annie W T; Lam, Johnson K S; Lam, Ricky K W; Ng, Wan H; Lee, Ella N L; Lee, Vicky T Y; Sze, Po P; Rajwani, Rahim; Fung, Kitty S C; To, Wing K; Lee, Rodney A; Tsang, Dominic N C; Siu, Gilman K H

2018-01-01

Objective: This study evaluated the capability of a MALDI Biotyper system equipped with the newly introduced MBT STAR-BL module to simultaneously perform species identification and β-lactamase-mediated resistance detection in bacteremia -causing bacteria isolated from cultured isolates and patient-derived blood cultures (BCs). Methods: Two hundred retrospective cultured isolates and 153 prospective BCs containing Gram-negative rods (GNR) were collected and subjected to direct bacterial identification, followed by the measurement of β-lactamase activities against ampicillin, piperacillin, cefotaxime, ceftazidime, and meropenem using the MBT STAR-BL module. The results and turnaround times were compared with those of routine microbiological processing. All strains were also characterized by beta-lactamase PCR and sequencing. Results: Using the saponin-based extraction method, MALDI-TOF MS correctly identified bacteria in 116/134 (86.6%) monomicrobial BCs. The detection sensitivities for β-lactamase activities against ampicillin, piperacillin, third-generation cephalosporin and meropenem were 91.3, 100, 97.9, and 100% for cultured isolates, and 80.4, 100, 68.8, and 40% for monomicrobial BCs ( n = 134) respectively. The overall specificities ranged from 91.5 to 100%. Furthermore, the MBT STAR-BL and conventional drug susceptibility test results were concordant in 14/19 (73.7%) polymicrobial cultures. Reducing the logRQ cut-off value from 0.4 to 0.2 increased the direct detection sensitivities for β-lactamase activities against ampicillin, cefotaxime and meropenem in BCs to 85.7, 87.5, and 100% respectively. The MBT STAR-BL test enabled the reporting of β-lactamase-producing GNR at 14.16 and 47.64 h before the interim and final reports of routine BCs processing, respectively, were available. Conclusion: The MALDI Biotyper system equipped with the MBT STAR-BL module enables the simultaneous rapid identification of bacterial species and �

Comprehensive Evaluation of the MBT STAR-BL Module for Simultaneous Bacterial Identification and β-Lactamase-Mediated Resistance Detection in Gram-Negative Rods from Cultured Isolates and Positive Blood Cultures

PubMed Central

Lee, Annie W. T.; Lam, Johnson K. S.; Lam, Ricky K. W.; Ng, Wan H.; Lee, Ella N. L.; Lee, Vicky T. Y.; Sze, Po P.; Rajwani, Rahim; Fung, Kitty S. C.; To, Wing K.; Lee, Rodney A.; Tsang, Dominic N. C.; Siu, Gilman K. H.

2018-01-01

Objective: This study evaluated the capability of a MALDI Biotyper system equipped with the newly introduced MBT STAR-BL module to simultaneously perform species identification and β-lactamase-mediated resistance detection in bacteremia -causing bacteria isolated from cultured isolates and patient-derived blood cultures (BCs). Methods: Two hundred retrospective cultured isolates and 153 prospective BCs containing Gram-negative rods (GNR) were collected and subjected to direct bacterial identification, followed by the measurement of β-lactamase activities against ampicillin, piperacillin, cefotaxime, ceftazidime, and meropenem using the MBT STAR-BL module. The results and turnaround times were compared with those of routine microbiological processing. All strains were also characterized by beta-lactamase PCR and sequencing. Results: Using the saponin-based extraction method, MALDI-TOF MS correctly identified bacteria in 116/134 (86.6%) monomicrobial BCs. The detection sensitivities for β-lactamase activities against ampicillin, piperacillin, third-generation cephalosporin and meropenem were 91.3, 100, 97.9, and 100% for cultured isolates, and 80.4, 100, 68.8, and 40% for monomicrobial BCs (n = 134) respectively. The overall specificities ranged from 91.5 to 100%. Furthermore, the MBT STAR-BL and conventional drug susceptibility test results were concordant in 14/19 (73.7%) polymicrobial cultures. Reducing the logRQ cut-off value from 0.4 to 0.2 increased the direct detection sensitivities for β-lactamase activities against ampicillin, cefotaxime and meropenem in BCs to 85.7, 87.5, and 100% respectively. The MBT STAR-BL test enabled the reporting of β-lactamase-producing GNR at 14.16 and 47.64 h before the interim and final reports of routine BCs processing, respectively, were available. Conclusion: The MALDI Biotyper system equipped with the MBT STAR-BL module enables the simultaneous rapid identification of bacterial species and �

Microfluidic system for the identification of bacterial pathogens causing urinary tract infections

NASA Astrophysics Data System (ADS)

Becker, Holger; Hlawatsch, Nadine; Haraldsson, Tommy; van der Wijngaart, Wouter; Lind, Anders; Malhotra-Kumar, Surbi; Turlej-Rogacka, Agata; Goossens, Herman

2015-03-01

Urinary tract infections (UTIs) are among the most common bacterial infections and pose a significant healthcare burden. The growing trend in antibiotic resistance makes it mandatory to develop diagnostic kits which allow not only the determination of a pathogen but also the antibiotic resistances. We have developed a microfluidic cartridge which takes a direct urine sample, extracts the DNA, performs an amplification using batch-PCR and flows the sample over a microarray which is printed into a microchannel for fluorescence detection. The cartridge is injection-molded out of COP and contains a set of two-component injection-molded rotary valves to switch between input and to isolate the PCR chamber during thermocycling. The hybridization probes were spotted directly onto a functionalized section of the outlet microchannel. We have been able to successfully perform PCR of E.coli in urine in this chip and perform a fluorescence detection of PCR products. An upgraded design of the cartridge contains the buffers and reagents in blisters stored on the chip.

Life detection systems.

NASA Technical Reports Server (NTRS)

Mitz, M. A.

1972-01-01

Some promising newer approaches for detecting microorganisms are discussed, giving particular attention to the integration of different methods into a single instrument. Life detection methods may be divided into biological, chemical, and cytological methods. Biological methods are based on the biological properties of assimilation, metabolism, and growth. Devices for the detection of organic materials are considered, taking into account an instrument which volatilizes, separates, and analyzes a sample sequentially. Other instrumental systems described make use of a microscope and the cytochemical staining principle.

Diagnosis of bacterial vaginosis by wet mount identification of bacterial morphotypes in vaginal fluid.

PubMed

Schmidt, H; Hansen, J G

2000-03-01

In order to develop a more practical way of diagnosing bacterial vaginosis (BV), we evaluated a scoring system, weighting small bacterial morphotypes versus lactobacillary morphotypes in wet mounts, assessed criteria for BV and normalcy from this scoring, and then evaluated their reproducibility and accuracy. We examined 754 women for pH, homogeneous vaginal discharge, amine odour, clue cells and the composite clinical diagnosis. We also examined wet mounts for small bacterial morphotypes and lactobacillary morphotypes, and weighted their quantitative presence as a bacterial morphotype score. The term 'small bacterial morphotypes' denotes a group of small bacillary forms comprising coccobacilli, tiny rods, and mobile curved rods. The different characteristics of BV were all gradually associated with increased bacterial morphotype scoring. We deemed a score of 0-1 as normal, 2-4 as intermediate phase, grade I, 5-6 as intermediate phase, grade II, and 7-8 indicative of BV. Reproducibility of the interpretation was high, both for the new grading system (weighted Kappa 0.90 in women perceiving and 0.81 in women not perceiving abnormal vaginal discharge) and for the new criterion for BV (non-weighted Kappa 0.91 and 0.84 in the 2 groups of women). The new criterion also proved highly concurrent with the composite clinical diagnosis (Kappa 0.91 and 0.81 in the 2 groups). In conclusion, the wet mount bacterial morphotype scoring is valid for grading of the disorder of the vaginal microbial ecosystem, and the new criterion for BV a more practical option than existing diagnostic methods.

Changes in diversity, abundance, and structure of soil bacterial communities in Brazilian Savanna under different land use systems.

PubMed

Rampelotto, Pabulo Henrique; de Siqueira Ferreira, Adão; Barboza, Anthony Diego Muller; Roesch, Luiz Fernando Wurdig

2013-10-01

The Brazilian Savanna, also known as "Cerrado", is the richest and most diverse savanna in the world and has been ranked as one of the main hotspots of biodiversity. The Cerrado is a representative biome in Central Brazil and the second largest biome in species diversity of South America. Nevertheless, large areas of native vegetation have been converted to agricultural land including grain production, livestock, and forestry. In this view, understanding how land use affects microbial communities is fundamental for the sustainable management of agricultural ecosystems. The aim of this work was to analyze and compare the soil bacterial communities from the Brazilian Cerrado associated with different land use systems using high throughput pyrosequencing of 16S rRNA genes. Relevant differences were observed in the abundance and structure of bacterial communities in soils under different land use systems. On the other hand, the diversity of bacterial communities was not relevantly changed among the sites studied. Land use systems had also an important impact on specific bacterial groups in soil, which might change the soil function and the ecological processes. Acidobacteria, Proteobacteria, and Actinobacteria were the most abundant groups in the Brazilian Cerrado. These findings suggest that more important than analyzing the general diversity is to analyze the composition of the communities. Since soil type was the same among the sites, we might assume that land use was the main factor defining the abundance and structure of bacterial communities.

Bacterial Associations with Diatoms Influence Host Health in a Xenic Model System

NASA Astrophysics Data System (ADS)

Baker, L.; Kemp, P. F.

2016-02-01

Diatoms are photosynthetic unicellular eukaryotes found ubiquitously in aquatic systems. Microorganisms such as bacteria are frequently found attached to diatoms and may influence the fitness of their host. The most commonly used model organisms in studies of diatom-bacterial associations are Alteromonas and Marinobacter. Some strains of Alteromonas are capable of parasitism, producing chitinases or having algicidal interactions; some strains of Marinobacter are capable of mutualism, providing its host with vital nutrients. In this study, multiple strains of Alteromonas and Marinobacter were isolated from the centric diatom Chaetoceros sp KBDT20. Isolates were added back in varying concentration to cultures of their original xenic diatom host, and to cultures of a smaller, xenic naïve host, Chaetoceros sp. KBDT32. The growth rate of the diatom host was monitored using flow cytometry to assess the impact of the added bacterial isolates on host health. Our results suggest that all strains of Alteromonas tested have an antagonistic relationship with both the original as well as the naïve host while all strains of Marinobacter tested have a synergistic relationship with both diatom cultures. The functional basis for these relationships is being explored by supplementing xenic diatom cultures with materials essential for diatom growth that may be contributed by bacteria, such as B-vitamins and bioavailable trace metals. The colonization rates and competitive interactions between bacteria are investigated through surface colonization studies. The goal of this study is to better inform our understanding of how bacterial associates of diatom populations may contribute to their health, success, or failure in aquatic systems.

Bacterial degradation of acetone in an outdoor model stream

USGS Publications Warehouse

Rathbun, R.E.; Stephens, D.W.; Tai, D.Y.

1993-01-01

Diurnal variations of the acetone concentration in an outdoor model stream were measured with and without a nitrate supplement to determine if the nitrate supplement would stimulate bacterial degradation of the acetone. Acetone loss coefficients were computed from the diurnal data using a fitting procedure based on a Lagrangian particle model. The coefficients indicated that bacterial degradation of the acetone was occurring in the downstream part of the stream during the nitrate addition. However, the acetone concentrations stabilized at values considerably above the limit of detection for acetone determination, in contrast to laboratory respirometer studies where the acetone concentration decreased rapidly to less than the detection limit, once bacterial acclimation to the acetone had occurred. One possible explanation for the difference in behavior was the limited 6-hour residence time of the acetone in the model stream.

Value of multiplex PCR to determine the bacterial and viral aetiology of pneumonia in school-age children.

PubMed

Aydemir, Yusuf; Aydemir, Özlem; Pekcan, Sevgi; Özdemir, Mehmet

2017-02-01

Conventional methods for the aetiological diagnosis of community-acquired pneumonia (CAP) are often insufficient owing to low sensitivity and the long wait for the results of culture and particularly serology, and it often these methods establish a diagnosis in only half of cases. To evaluate the most common bacterial and viral agents in CAP using a fast responsive PCR method and investigate the relationship between clinical/laboratory features and aetiology, thereby contributing to empirical antibiotic selection and reduction of treatment failure. In children aged 4-15 years consecutively admitted with a diagnosis of CAP, the 10 most commonly detected bacterial and 12 most commonly detected viral agents were investigated by induced sputum using bacterial culture and multiplex PCR methods. Clinical and laboratory features were compared between bacterial and viral pneumonia. In 78 patients, at least one virus was detected in 38 (48.7%) and at least one bacterium in 32 (41%). In addition, both bacteria and viruses were detected in 16 (20.5%) patients. Overall, the agent detection rate was 69.2%. The most common viruses were respiratory syncytial virus and influenza and the most frequently detected bacteria were S. pneumoniae and H. influenzae. PCR was superior to culture for bacterial isolation (41% vs 13%, respectively). Fever, wheezing and radiological features were not helpful in differentiating between bacterial and viral CAP. White blood cell count, CRP and ESR values were significantly higher in the bacterial/mixed aetiology group than in the viral aetiology group. In CAP, multiplex PCR is highly reliable, superior in detecting multiple pathogens and rapidly identifies aetiological agents. Clinical features are poor for differentiation between bacterial and viral infections. The use of PCR methods allow physicians to provide more appropriate antimicrobial therapy, resulting in a better response to treatment, and it may be possible for use as a routine service

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

Urban aerosols harbor diverse and dynamic bacterial populations

PubMed Central

Brodie, Eoin L.; DeSantis, Todd Z.; Parker, Jordan P. Moberg; Zubietta, Ingrid X.; Piceno, Yvette M.; Andersen, Gary L.

2007-01-01

Considering the importance of its potential implications for human health, agricultural productivity, and ecosystem stability, surprisingly little is known regarding the composition or dynamics of the atmosphere's microbial inhabitants. Using a custom high-density DNA microarray, we detected and monitored bacterial populations in two U.S. cities over 17 weeks. These urban aerosols contained at least 1,800 diverse bacterial types, a richness approaching that of some soil bacterial communities. We also reveal the consistent presence of bacterial families with pathogenic members including environmental relatives of select agents of bioterrorism significance. Finally, using multivariate regression techniques, we demonstrate that temporal and meteorological influences can be stronger factors than location in shaping the biological composition of the air we breathe. PMID:17182744

A Multiplex PCR/LDR Assay for Simultaneous Detection and Identification of the NIAID Category B Bacterial Food and Water-borne Pathogens

PubMed Central

Rundell, Mark S.; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H.; Spitzer, Eric D.; Golightly, Linnie; Barany, Francis

2014-01-01

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/LDR assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay was assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91 to 100% (median 100%) depending on the species. For the majority of organisms the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. PMID:24709368

Bacterial communities in full-scale wastewater treatment systems.

PubMed

Cydzik-Kwiatkowska, Agnieszka; Zielińska, Magdalena

2016-04-01

Bacterial metabolism determines the effectiveness of biological treatment of wastewater. Therefore, it is important to define the relations between the species structure and the performance of full-scale installations. Although there is much laboratory data on microbial consortia, our understanding of dependencies between the microbial structure and operational parameters of full-scale wastewater treatment plants (WWTP) is limited. This mini-review presents the types of microbial consortia in WWTP. Information is given on extracellular polymeric substances production as factor that is key for formation of spatial structures of microorganisms. Additionally, we discuss data on microbial groups including nitrifiers, denitrifiers, Anammox bacteria, and phosphate- and glycogen-accumulating bacteria in full-scale aerobic systems that was obtained with the use of molecular techniques, including high-throughput sequencing, to shed light on dependencies between the microbial ecology of biomass and the overall efficiency and functional stability of wastewater treatment systems. Sludge bulking in WWTPs is addressed, as well as the microbial composition of consortia involved in antibiotic and micropollutant removal.

Profile and Fate of Bacterial Pathogens in Sewage Treatment Plants Revealed by High-Throughput Metagenomic Approach.

PubMed

Li, Bing; Ju, Feng; Cai, Lin; Zhang, Tong

2015-09-01

The broad-spectrum profile of bacterial pathogens and their fate in sewage treatment plants (STPs) were investigated using high-throughput sequencing based metagenomic approach. This novel approach could provide a united platform to standardize bacterial pathogen detection and realize direct comparison among different samples. Totally, 113 bacterial pathogen species were detected in eight samples including influent, effluent, activated sludge (AS), biofilm, and anaerobic digestion sludge with the abundances ranging from 0.000095% to 4.89%. Among these 113 bacterial pathogens, 79 species were reported in STPs for the first time. Specially, compared to AS in bulk mixed liquor, more pathogen species and higher total abundance were detected in upper foaming layer of AS. This suggests that the foaming layer of AS might impose more threat to onsite workers and citizens in the surrounding areas of STPs because pathogens in foaming layer are easily transferred into air and cause possible infections. The high removal efficiency (98.0%) of total bacterial pathogens suggests that AS treatment process is effective to remove most bacterial pathogens. Remarkable similarities of bacterial pathogen compositions between influent and human gut indicated that bacterial pathogen profiles in influents could well reflect the average bacterial pathogen communities of urban resident guts within the STP catchment area.

QTLs for Resistance to Major Rice Diseases Exacerbated by Global Warming: Brown Spot, Bacterial Seedling Rot, and Bacterial Grain Rot.

PubMed

Mizobuchi, Ritsuko; Fukuoka, Shuichi; Tsushima, Seiya; Yano, Masahiro; Sato, Hiroyuki

2016-12-01

In rice (Oryza sativa L.), damage from diseases such as brown spot, caused by Bipolaris oryzae, and bacterial seedling rot and bacterial grain rot, caused by Burkholderia glumae, has increased under global warming because the optimal temperature ranges for growth of these pathogens are relatively high (around 30 °C). Therefore, the need for cultivars carrying genes for resistance to these diseases is increasing to ensure sustainable rice production. In contrast to the situation for other important rice diseases such as blast and bacterial blight, no genes for complete resistance to brown spot, bacterial seedling rot or bacterial grain rot have yet been discovered. Thus, rice breeders have to use partial resistance, which is largely influenced by environmental conditions. Recent progress in molecular genetics and improvement of evaluation methods for disease resistance have facilitated detection of quantitative trait loci (QTLs) associated with resistance. In this review, we summarize the results of worldwide screening for cultivars with resistance to brown spot, bacterial seedling rot and bacterial grain rot and we discuss the identification of QTLs conferring resistance to these diseases in order to provide useful information for rice breeding programs.

Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡

PubMed Central

Makarova, Kira S.; Wolf, Yuri I.; Snir, Sagi; Koonin, Eugene V.

2011-01-01

The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands. PMID:21908672

Stored Canine Whole Blood Units: What is the Real Risk of Bacterial Contamination?

PubMed

Miglio, A; Stefanetti, V; Antognoni, M T; Cappelli, K; Capomaccio, S; Coletti, M; Passamonti, F

2016-11-01

Bacterial contamination of whole blood (WB) units can result in transfusion-transmitted infection, but the extent of the risk has not been established and may be underestimated in veterinary medicine. To detect, quantify, and identify bacterial microorganisms in 49 canine WB units during their shelf life. Forty-nine healthy adult dogs. Forty-nine WB units were included in the study. Immediately after collection, 8 sterile samples from the tube segment line of each unit were aseptically collected and tested for bacterial contamination on days 0, 1, 7, 14, 21, 28, 35, and 42 of storage. A qPCR assay was performed on days 0, 21, and 35 to identify and quantify any bacterial DNA. On bacterial culture, 47/49 blood units were negative at all time points tested, 1 unit was positive for Enterococcus spp. on days 0 and 1, and 1 was positive for Escherichia coli on day 35. On qPCR assay, 26 of 49 blood units were positive on at least 1 time point and the bacterial loads of the sequences detected (Propionobacterium spp., Corynebacterium spp., Caulobacter spp., Pseudomonas spp., Enterococcus spp., Serratia spp., and Leucobacter spp.) were <80 genome equivalents (GE)/μL. Most of the organisms detected were common bacteria, not usually implicated in septic transfusion reactions. The very low number of GE detected constitutes an acceptable risk of bacterial contamination, indicating that WB units have a good sanitary shelf life during commercial storage. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

16S rRNA beacons for bacterial monitoring during human space missions.

PubMed

Larios-Sanz, Maia; Kourentzi, Katerina D; Warmflash, David; Jones, Jeffrey; Pierson, Duane L; Willson, Richard C; Fox, George E

2007-04-01

Microorganisms are unavoidable in space environments and their presence has, at times, been a source of problems. Concerns about disease during human space missions are particularly important considering the significant changes the immune system incurs during spaceflight and the history of microbial contamination aboard the Mir space station. Additionally, these contaminants may have adverse effects on instrumentation and life-support systems. A sensitive, highly specific system to detect, characterize, and monitor these microbial populations is essential. Herein we describe a monitoring approach that uses 16S rRNA targeted molecular beacons to successfully detect several specific bacterial groupings. This methodology will greatly simplify in-flight monitoring by minimizing sample handling and processing. We also address and provide solutions to target accessibility problems encountered in hybridizations that target 16S rRNA.

Postviral Complications: Bacterial Pneumonia.

PubMed

Prasso, Jason E; Deng, Jane C

2017-03-01

Secondary bacterial pneumonia after viral respiratory infection remains a significant source of morbidity and mortality. Susceptibility is mediated by a variety of viral and bacterial factors, and complex interactions with the host immune system. Prevention and treatment strategies are limited to influenza vaccination and antibiotics/antivirals respectively. Novel approaches to identifying the individuals with influenza who are at increased risk for secondary bacterial pneumonias are urgently needed. Given the threat of further pandemics and the heightened prevalence of these viruses, more research into the immunologic mechanisms of this disease is warranted with the hope of discovering new potential therapies. Published by Elsevier Inc.

Water system virus detection

NASA Technical Reports Server (NTRS)

Fraser, A. S.; Wells, A. F.; Tenoso, H. J.

1975-01-01

A monitoring system developed to test the capability of a water recovery system to reject the passage of viruses into the recovered water is described. A nonpathogenic marker virus, bacteriophage F2, is fed into the process stream before the recovery unit and the reclaimed water is assayed for its presence. Detection of the marker virus consists of two major components, concentration and isolation of the marker virus, and detection of the marker virus. The concentration system involves adsorption of virus to cellulose acetate filters in the presence of trivalent cations and low pH with subsequent desorption of the virus using volumes of high pH buffer. The detection of the virus is performed by a passive immune agglutination test utilizing specially prepared polystyrene particles. An engineering preliminary design was performed as a parallel effort to the laboratory development of the marker virus test system. Engineering schematics and drawings of a fully functional laboratory prototype capable of zero-G operation are presented. The instrument consists of reagent pump/metering system, reagent storage containers, a filter concentrator, an incubation/detector system, and an electronic readout and control system.

Volatiles in Inter-Specific Bacterial Interactions

PubMed Central

Tyc, Olaf; Zweers, Hans; de Boer, Wietse; Garbeva, Paolina

2015-01-01

The importance of volatile organic compounds for functioning of microbes is receiving increased research attention. However, to date very little is known on how inter-specific bacterial interactions effect volatiles production as most studies have been focused on volatiles produced by monocultures of well-described bacterial genera. In this study we aimed to understand how inter-specific bacterial interactions affect the composition, production and activity of volatiles. Four phylogenetically different bacterial species namely: Chryseobacterium, Dyella, Janthinobacterium, and Tsukamurella were selected. Earlier results had shown that pairwise combinations of these bacteria induced antimicrobial activity in agar media whereas this was not the case for monocultures. In the current study, we examined if these observations were also reflected by the production of antimicrobial volatiles. Thus, the identity and antimicrobial activity of volatiles produced by the bacteria were determined in monoculture as well in pairwise combinations. Antimicrobial activity of the volatiles was assessed against fungal, oomycetal, and bacterial model organisms. Our results revealed that inter-specific bacterial interactions affected volatiles blend composition. Fungi and oomycetes showed high sensitivity to bacterial volatiles whereas the effect of volatiles on bacteria varied between no effects, growth inhibition to growth promotion depending on the volatile blend composition. In total 35 volatile compounds were detected most of which were sulfur-containing compounds. Two commonly produced sulfur-containing volatile compounds (dimethyl disulfide and dimethyl trisulfide) were tested for their effect on three target bacteria. Here, we display the importance of inter-specific interactions on bacterial volatiles production and their antimicrobial activities. PMID:26733959

Multiplex PCR detection of problematic pathogens of clinically heterogeneous bacterial vaginosis in Bulgarian women

PubMed

Tosheva-Daskalova, Konstantsa; Strateva, Tanya Vasileva; Mitov, Ivan Gergov; Gergova, Raina Tzvetanova

2017-11-13

Background/aim: This study aimed to investigate the correlation between the prevalence of problematic pathogens and the clinical status of women with bacterial vaginosis (BV). Materials and methods: Gardnerella vaginalis, Atopobium vaginae, and Mobiluncus spp. were detected using a multiplex PCR assay, and their role in the infection of Bulgarian women with clinically heterogeneous BV was evaluated. Results: The predominant BV-associated pathogen identified was G. vaginalis with an incidence of 98.39%, followed by A. vaginae (68.05%) and Mobiluncus spp. at 17.01%. The coexistence of A. vaginae and G. vaginalis was more common in women with discharge (in 72.04%) and in patients with chronic recurrent BV than among asymptomatic or newly diagnosed BV cases (P < 0.05). Mobiluncus spp. was detected mostly in coinfections, in association with Trichomonas vaginalis. The coinfections were predominantly related to recurrent BV and with complications (P < 0.05). Conclusion: This is the first study about the correlation between problematic pathogens and clinically heterogeneous BV in Bulgarian women. High frequency of infection with key BV-related pathogens was observed in childbearing women. The incidence was shown to often correlate with coexistent T. vaginalis, with severity of infection, and with complicated and recurrent BV after unsuccessful treatments. Screening should be considered in reproductive health programs.

Core Principles of Bacterial Autoinducer Systems

PubMed Central

2015-01-01

SUMMARY Autoinduction (AI), the response to self-produced chemical signals, is widespread in the bacterial world. This process controls vastly different target functions, such as luminescence, nutrient acquisition, and biofilm formation, in different ways and integrates additional environmental and physiological cues. This diversity raises questions about unifying principles that underlie all AI systems. Here, we suggest that such core principles exist. We argue that the general purpose of AI systems is the homeostatic control of costly cooperative behaviors, including, but not limited to, secreted public goods. First, costly behaviors require preassessment of their efficiency by cheaper AI signals, which we encapsulate in a hybrid “push-pull” model. The “push” factors cell density, diffusion, and spatial clustering determine when a behavior becomes effective. The relative importance of each factor depends on each species' individual ecological context and life history. In turn, “pull” factors, often stress cues that reduce the activation threshold, determine the cellular demand for the target behavior. Second, control is homeostatic because AI systems, either themselves or through accessory mechanisms, not only initiate but also maintain the efficiency of target behaviors. Third, AI-controlled behaviors, even seemingly noncooperative ones, are generally cooperative in nature, when interpreted in the appropriate ecological context. The escape of individual cells from biofilms, for example, may be viewed as an altruistic behavior that increases the fitness of the resident population by reducing starvation stress. The framework proposed here helps appropriately categorize AI-controlled behaviors and allows for a deeper understanding of their ecological and evolutionary functions. PMID:25694124

Freshwater Recirculating Aquaculture System Operations Drive Biofilter Bacterial Community Shifts around a Stable Nitrifying Consortium of Ammonia-Oxidizing Archaea and Comammox Nitrospira

PubMed Central

Bartelme, Ryan P.; McLellan, Sandra L.; Newton, Ryan J.

2017-01-01

Recirculating aquaculture systems (RAS) are unique engineered ecosystems that minimize environmental perturbation by reducing nutrient pollution discharge. RAS typically employ a biofilter to control ammonia levels produced as a byproduct of fish protein catabolism. Nitrosomonas (ammonia-oxidizing), Nitrospira, and Nitrobacter (nitrite-oxidizing) species are thought to be the primary nitrifiers present in RAS biofilters. We explored this assertion by characterizing the biofilter bacterial and archaeal community of a commercial scale freshwater RAS that has been in operation for >15 years. We found the biofilter community harbored a diverse array of bacterial taxa (>1000 genus-level taxon assignments) dominated by Chitinophagaceae (~12%) and Acidobacteria (~9%). The bacterial community exhibited significant composition shifts with changes in biofilter depth and in conjunction with operational changes across a fish rearing cycle. Archaea also were abundant, and were comprised solely of a low diversity assemblage of Thaumarchaeota (>95%), thought to be ammonia-oxidizing archaea (AOA) from the presence of AOA ammonia monooxygenase genes. Nitrosomonas were present at all depths and time points. However, their abundance was >3 orders of magnitude less than AOA and exhibited significant depth-time variability not observed for AOA. Phylogenetic analysis of the nitrite oxidoreductase beta subunit (nxrB) gene indicated two distinct Nitrospira populations were present, while Nitrobacter were not detected. Subsequent identification of Nitrospira ammonia monooxygenase alpha subunit genes in conjunction with the phylogenetic placement and quantification of the nxrB genotypes suggests complete ammonia-oxidizing (comammox) and nitrite-oxidizing Nitrospira populations co-exist with relatively equivalent and stable abundances in this system. It appears RAS biofilters harbor complex microbial communities whose composition can be affected directly by typical system operations while

46 CFR 108.405 - Fire detection system.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false Fire detection system. 108.405 Section 108.405 Shipping... EQUIPMENT Fire Extinguishing Systems § 108.405 Fire detection system. (a) Each fire detection system and each smoke detection system on a unit must— (1) Be approved by the Commandant; and (2) Have a visual...

Evaluation of commercially prepared transport systems for nonlethal detection of Aeromonas salmonicida in salmonid fish

USGS Publications Warehouse

Cipriano, R.C.; Bullock, G.L.

2001-01-01

In vitro studies indicated that commercially prepared transport systems containing Amies, Stuart's, and Cary-Blair media worked equally well in sustaining the viability of the fish pathogen Aeromonas salmonicida, which causes furunculosis. The bacterium remained viable without significant increase or decrease in cell numbers for as long as 48 h of incubation at 18-20??C in Stuart's transport medium; consequently, obtaining mucus samples in such tubes were comparable to on-site detection of A. salmonicida by dilution plate counts on Coomassie Brilliant Blue agar. In three different assays of 100 samples of mucus from Atlantic salmon Salmo salar infected subclinically with A. salmonicida, dilution counts conducted on-site proved more reliable for detecting the pathogen than obtaining the samples in the transport system. In the on-site assays, dilution counts detected the pathogen in 34, 41, and 22 samples, whereas this was accomplished in only 15, 15, and 3 of the respective samples when the transport system was used. In an additional experiment, Arctic char Salvelinus alpinus sustaining a frank epizootic of furunculosis were sampled similarly. Here, too, dilution counts were more predictive of the prevalence of A. salmonicida and detected the pathogen in 46 mucus samples; in comparison, only 6 samples collected by using the transport system were positive. We also observed that the transport system supported the growth of the normal mucus bacterial flora. Particularly predominant among these were motile aeromonads and Pseudomonas fluorescens. In studies of mixed culture growth, two representatives of both of the latter genera of bacteria outgrew A. salmonicida - in some cases, to the total exclusion of the pathogen itself.

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird

PubMed Central

Rodríguez-Ruano, Sonia M.; Martín-Vivaldi, Manuel; Martín-Platero, Antonio M.; López-López, J. Pablo; Peralta-Sánchez, Juan M.; Ruiz-Rodríguez, Magdalena; Soler, Juan J.; Valdivia, Eva; Martínez-Bueno, Manuel

2015-01-01

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host. PMID:26445111

Engineering Approaches for the Detection and Control of Orthopaedic Biofilm Infections

PubMed Central

Ehrlich, Garth D.; Stoodley, Paul; Kathju, Sandeep; Zhao, Yongjun; McLeod, Bruce R.; Balaban, Naomi; Hu, Fen Ze; Sotereanos, Nicholas G.; Costerton, J. William; Stewart, Philip S.; Post, J. Christopher; Lin, Qiao

2005-01-01

Artificial joints are subject to chronic infections associated with bacterial biofilms, which only can be eradicated by the traumatic removal of the implant followed by sustained intravenous antibiotic therapy. We have adopted an engineering approach to develop electrical–current-based approaches to bacterial eradication and microelectromechanical systems that could be embedded within the implanted joint to detect the presence of bacteria and to provide in situ treatment of the infection before a biofilm can form. In the former case we will examine the combined bactericidal effects of direct and indirect electrical fields in combination with antibiotic therapy. In the latter case, bacterial detection will occur by developing a microelectromechanical–systems-based biosensor that can “eavesdrop” on bacterial quorum–sensing-based communication systems. Treatment will be effected by the release of a cocktail of pharmaceutical reagents contained within integral reservoirs associated with the implant, including a molecular jamming signal that competitively binds to the bacteria’s quorum sensing receptors (which will “blind” the bacteria, preventing the production of toxins) and multiple high dose antibiotics to eradicate the planktonic bacteria. This approach is designed to take advantage of the relatively high susceptibility to antibiotics that planktonic bacteria display compared with biofilm envirovars. Here we report the development of a generic microelectromechanical systems biosensor that measures changes in internal viscosity in a base fluid triggered by a change in the external environment. PMID:16056027

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