Pathogens In Water Samples Research Articles

분자생물학적 방법인 PCR-REBA를 이용한 대중목욕탕 수질 중 수인성병원성미생물 검출

수인성 병원성 미생물에 의한 공중목욕탕의 오염은 질병발생의 원인이 된다. 본 연구에서는 공중목욕탕내에 존재하는 수인성 병원성미생물들을 확인하고자 하였다. 서울지역내의 30 곳 공중목욕탕에서 욕조수 시료를 채수하여 진행하였다. 수인성 병원성미생물의 검출은 0.45 <TEX>${\mu}m$</TEX>의 여과막을 이용하여 전통적인 배양방법으로 분리 및 동정하였다. 분자생물학적 기법을 사용하기 위해 미생물학적인 배양을 하지 않고 핵산을 추출하여 16S rRNA유전자를 표적으로 polymerase chain reaction-reverse blot hybridization (PCR-REBA)을 실시하였다. 미생물학적 배양방법에서는 지표세균인 Escherichia coli와 Shigella spp.가 검출되었으며, 분자생물학적 기법인 PCR-REBA을 수행한 결과 E. coli, Shigella spp., Salmonella spp., Pseudomonas spp., Mycobacterium spp. 등의 수인성 병원성미생물이 7곳에서 검출되었다. 본 연구결과를 토대로 공중목욕탕의 욕조수내에 수인성병원성미생물에 의한 감염을 줄이기 위해 적절한 위생관리과 E. coli를 포함한 유해미생물을 선정하여 지속적인 모니터링이 필요할 것으로 사료된다. Contamination of public bath water by waterborne pathogens can cause disease outbreaks and contribute to background rates of disease. The aim of this study is to determine the prevalence of waterborne pathogens in public baths. A total of 30 water samples were collected from 30 different public baths in seoul, Korea. Pathogens in water samples were concentrated by 0.45 <TEX>${\mu}m$</TEX> nitrocellulose membrane filter, analyzed by both cultivation and polymerase chain reaction-reverse blot hybridization (PCR-REBA) of partial 16S rRNA gene. Various microorganisms including Escherichia coli and Shigella spp. were identified by microbiological cultivation. E. coli, Shigella spp., Salmonella spp., Pseudomonas spp. and Mycobacterium spp. were identified by PCR-REBA. Our results suggest that appropriate hygiene practice and continuous monitoring is needed for reducing health risk associated with public bath houses.

Quantification of Pathogens and Sources of Microbial Indicators for QMRA in Recreational Waters

This study has increased knowledge about relationships between pathogen indicators, source identifiers and pathogens to support Quantitative Microbial Risk Assessment (QMRA) efforts and the implementation of revised recreational water quality criteria. Data gaps pertaining to waterborne pathogens and indicators in fecally-impacted discharges to recreational waters were identified and filled by targeted monitoring campaigns in three geographic regions. Study design involved simultaneous detection of pathogens in water samples using a combination of genetic, culture- and microscopy-based methodologies for Salmonella, Campylobacter jejuni, Vibrio cholerae, Cryptosporidium, Giardia, Toxoplasma gondii, adenoviruses, enteroviruses, noroviruses, rotaviruses, Bacteroidales, Enterococcus, and Escherichia coli. The project team worked closely with experts and stakeholder groups in order to advance the understanding and applicability of QMRA for risk management purposes. QMRA analyses revealed Norovirus as the most dominant health risk followed by rotavirus. Norovirus and Enterococcus both had significant correlations with a number of pathogens in discharges. Using qPCR data on the fecal source identifier Bacteroidales a new model can predict the true amount of human fecal contamination in a water sample by relating a human-associated genetic marker to a universal assay for fecal sources. The model output can then be used to implement and evaluate management options intended to restore microbial water quality. This title belongs to WERF Research Report Series . ISBN: 9781843395430 (eBook)

Occurrence of Protozoan and Bacterial Pathogens in Water Samples from Small Water Systems in Mountainous Areas of Taiwan

Giardia, Cryptosporidium, Salmonella, and Shigella have emerged as waterborne pathogens of concern. Thirty water samples were collected from 10 small water systems in mountainous areas of Taiwan and investigated for the occurrence of these pathogens. In addition, the basic characteristics of each small water system were recorded, and each water sample was tested for several water quality parameters. The occurrence frequency was 10.0% for Giardia spp., 3.3% for Cryptosporidium spp. and Shigella flexneri, and 16.7% for Salmonella enterica. The highest correlation was found between the presence of Salmonella enterica and low water temperature, followed by the correlation between water turbidity and fecal coliform concentration. A low correlation was found between the presence of Giardia and the concentration of heterotrophic bacteria. The water samples from small water systems with filtration devices had a lower occurrence of pathogen microorganisms than those without filtration devices. The proportion of wa...

In Situ-Synthesized Virulence and Marker Gene Biochip for Detection of Bacterial Pathogens in Water

Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately -19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.

Rapid and sensitive enumeration of viable diluted cells of members of the family enterobacteriaceae in freshwater and drinking water.

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 10(8) nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.

PCR detection of specific pathogens in water: a risk-based analysis.

The relative concentration of pathogens in water samples collected from storm drains and adjacent surfaces was evaluated using established PCR-based protocols. Out of the 58 samples collected from 21 different storm drains, 22% were PCR positive for Escherichia coli ETEC, Salmonella, or adenovirus. The risk of swimming related illnesses associated with detection of E. coli ETEC and Salmonella ranged from 0.39 to 30:100 000 and 0.3-25:1000, respectively. The detection limit corresponding to a negative-PCR result was evaluated in reference to water quality standards developed using a risk-based approach that integrates human dose-response data with acceptable levels of risk promulgated by the U.S. EPA for recreational contact. The percent of samples with an acceptable detection limit ranged from 0% for Giardia lamblia and Shigella to 100% for E. coli ETEC. The principal factor influencing the detection limit of G. lamblia and Shigella was sample volume. The principal factor influencing the detection limit of the remaining bacteria and protozoa, including E. coli O157:H7, Salmonella, and Cryptosporidium parvum, was the presence of inhibitory compounds in the purified nucleic acid extracts. Both recovery and inhibition adversely impacted the detection limit of viruses. Ambient water quality standards based on the occurrence of specific pathogens enumerated with PCR-based assays could serve as a method of evaluating the biological quality of water but only after significant improvements in filtration and purification protocols. The risk-based methodology developed in this study can be used to evaluate future improvements in filtration and purification protocols.