scholarly journals Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number–qPCR Assay

2016 ◽  
Vol 82 (15) ◽  
pp. 4743-4756 ◽  
Author(s):  
Graham S. Banting ◽  
Shannon Braithwaite ◽  
Candis Scott ◽  
Jinyong Kim ◽  
Byeonghwa Jeon ◽  
...  
Keyword(s):  
Irrigation Water ◽  
Water Samples ◽  
Qpcr Assay ◽  
Most Probable Number ◽  
Quantitative Microbial Risk Assessment ◽  
Detection Rates ◽  
Probable Number ◽  
Content Type ◽  
Microbial Risk Assessment ◽  
Microbial Risk

ABSTRACTCampylobacterspp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)–quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly usedCampylobactermolecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and JensenglyAqPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, withArcobacterspp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053–1066, 2010,http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific forCampylobacterdetection in water.Campylobacteroccurrence in irrigation water was found to be very low (<2 MPN/300 ml) when thisCampylobacter-specific qPCR was used, with the most commonly detected species beingC. jejuni,C. coli, andC. lari. Campylobacters in raw sewage were present at ∼102/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, whenCampylobacterprevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification ofCampylobacterspp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures.IMPORTANCEThe results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring forCampylobacterwhen using molecular biology-based assays. Previous studies describingCampylobacterprevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification ofArcobacterspp. As such,Campylobacterprevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination ofCampylobacterconcentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment.

scholarly journals Quantification of the health risk associated with wastewater reuse in Accra, Ghana: a contribution toward local guidelines

2008 ◽  
Vol 6 (4) ◽  
pp. 461-471 ◽  
Author(s):  
Razak Seidu ◽  
Arve Heistad ◽  
Philip Amoah ◽  
Pay Drechsel ◽  
Petter D. Jenssen ◽  
...  
Keyword(s):  
Irrigation Water ◽  
Health Hazard ◽  
Wastewater Reuse ◽  
Quantitative Microbial Risk Assessment ◽  
Microbial Risk Assessment ◽  
Microbial Risk ◽  
Post Harvest ◽  
Pipe Water ◽  
Tolerable Risk ◽  
Rotavirus Infections

Quantitative Microbial Risk Assessment (QMRA) models with 10,000 Monte Carlo simulations were applied to ascertain the risks of rotavirus and Ascaris infections for farmers using different irrigation water qualities and consumers of lettuce irrigated with the different water qualities after allowing post-harvest handling. A tolerable risk (TR) of infection of 7.7 × 10−4 and 1 × 10−2 per person per year were used for rotavirus and Ascaris respectively. The risk of Ascaris infection was within a magnitude of 10−2 for farmers accidentally ingesting drain or stream irrigation water; ∼100 for farmers accidentally ingesting farm soil and 100 for farmers ingesting any of the irrigation waters and contaminated soil. There was a very low risk (10−5) of Ascaris infection for farmers using pipe−water. For consumers, the annual risks of Ascaris and rotavirus infections were 100 and 10−3 for drain and stream irrigated lettuce respectively with slight increases for rotavirus infections along the post-harvest handling chain. Pipe irrigated lettuce recorded a rotavirus infection of 10−4 with no changes due to post harvest handling. The assessment identified on-farm soil contamination as the most significant health hazard.

scholarly journals Sapovirus in Wastewater Treatment Plants in Tunisia: Prevalence, Removal, and Genetic Characterization

2018 ◽  
Vol 84 (6) ◽  
pp. e02093-17 ◽  
Author(s):  
Miguel F. Varela ◽  
Imen Ouardani ◽  
Tsuyoshi Kato ◽  
Syunsuke Kadoya ◽  
Mahjoub Aouni ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Wastewater Treatment ◽  
Bayesian Estimation ◽  
Quantitative Pcr ◽  
Censored Data ◽  
Wastewater Treatment Plants ◽  
Quantitative Microbial Risk Assessment ◽  
Content Type ◽  
Microbial Risk Assessment ◽  
Microbial Risk

ABSTRACTSapovirus(SaV), from theCaliciviridaefamily, is a genus of enteric viruses that cause acute gastroenteritis. SaV is shed at high concentrations with feces into wastewater, which is usually discharged into aquatic environments or reused for irrigation without efficient treatments. This study analyzed the incidence of human SaV in four wastewater treatment plants from Tunisia during a period of 13 months (December 2009 to December 2010). Detection and quantification were carried out using reverse transcription-quantitative PCR (RT-qPCR) methods, obtaining a prevalence of 39.9% (87/218). Sixty-one positive samples were detected in untreated water and 26 positive samples in processed water. The Dekhila plant presented the highest contamination levels, with a 63.0% prevalence. A dominance of genotype I.2 was observed on 15 of the 24 positive samples that were genetically characterized. By a Bayesian estimation algorithm, the SaV density in wastewater was estimated using left-censored data sets. The mean value of log SaV concentration in untreated wastewater ranged between 2.7 and 4.5 logs. A virus removal efficiency of 0.2 log was calculated for the Dekhila plant as the log ratio posterior distributions between untreated and treated wastewater. Multiple quantitative values obtained in this study must be available in quantitative microbial risk assessment in Tunisia as parameter values reflecting local conditions.IMPORTANCEHuman sapovirus (SaV) is becoming more prevalent worldwide and organisms in this genus are recognized as emerging pathogens associated with human gastroenteritis. The present study describes novel findings on the prevalence, seasonality, and genotype distribution of SaV in Tunisia and Northern Africa. In addition, a statistical approximation using Bayesian estimation of the posterior predictive distribution (“left-censored” data) was employed to solve methodological problems related with the limit of quantification of the quantitative PCR (qPCR). This approach would be helpful for the future development of quantitative microbial risk assessment procedures for wastewater.

Application of Microbial Risk Assessment to the Development of Standards for Enteric Pathogens in Water Used To Irrigate Fresh Produce

2005 ◽  
Vol 68 (5) ◽  
pp. 913-918 ◽  
Author(s):  
SCOTT W. STINE ◽  
INHONG SONG ◽  
CHRISTOPHER Y. CHOI ◽  
CHARLES P. GERBA
Keyword(s):  
Risk Assessment ◽  
Irrigation Water ◽  
Fresh Produce ◽  
Enteric Bacteria ◽  
Irrigation Event ◽  
Risk Of Infection ◽  
Probable Number ◽  
Microbial Risk Assessment ◽  
Microbial Risk ◽  
Annual Risk

Microbial contamination of the surfaces of cantaloupe, iceberg lettuce, and bell peppers via contact with irrigation water was investigated to aid in the development of irrigation water quality standards for enteric bacteria and viruses. Furrow and subsurface drip irrigation methods were evaluated with the use of nonpathogenic surrogates, coliphage PRD1, and Escherichia coli ATCC 25922. The concentrations of hepatitis A virus (HAV) and Salmonella in irrigation water necessary to achieve a 1:10,000 annual risk of infection, the acceptable level of risk used for drinking water by the U.S. Environmental Protection Agency, were calculated with a quantitative microbial risk assessment approach. These calculations were based on the transfer of the selected nonpathogenic surrogates to fresh produce via irrigation water, as well as previously determined preharvest inactivation rates of pathogenic microorganisms on the surfaces of fresh produce. The risk of infection was found to be variable depending on type of crop, irrigation method, and days between last irrigation event and harvest. The worst-case scenario, in which produce is harvested and consumed the day after the last irrigation event and maximum exposure is assumed, indicated that concentrations of 2.5 CFU/100 ml of Salmonella and 2.5 × 10−5 most probable number per 100 ml of HAV in irrigation water would result in an annual risk of 1:10,000 when the crop was consumed. If 14 days elapsed before harvest, allowing for die-off of the pathogens, the concentrations were increased to 5.7 × 103 Salmonella per 100 ml and 9.9 × 10−3 HAV per 100 ml.

scholarly journals Quantitative Microbial Risk Assessment of Pathogenic Vibrios in Marine Recreational Waters of Southern California

2012 ◽  
Vol 79 (1) ◽  
pp. 294-302 ◽  
Author(s):  
Gregory Dickinson ◽  
Keah-ying Lim ◽  
Sunny C. Jiang
Keyword(s):  
Risk Assessment ◽  
Genetic Marker ◽  
Southern California ◽  
Toxin Gene ◽  
Quantitative Microbial Risk Assessment ◽  
Recreational Waters ◽  
Content Type ◽  
Microbial Risk Assessment ◽  
Microbial Risk ◽  
Illness Model

ABSTRACTThis study investigated the occurrence of three types of vibrios in Southern California recreational beach waters during the peak marine bathing season in 2007. Over 160 water samples were concentrated and enriched for the detection of vibrios. Four sets of PCR primers, specific forVibrio cholerae,V. parahaemolyticus, andV. vulnificusspecies and theV. parahaemolyticustoxin gene, respectively, were used for the amplification of bacterial genomic DNA. Of 66 samples from Doheny State Beach, CA, 40.1% were positive forV. choleraeand 27.3% were positive forV. parahaemolyticus, and 1 sample (1.5%) was positive for theV. parahaemolyticustoxin gene. Of the 96 samples from Avalon Harbor, CA, 18.7% were positive forV. cholerae, 69.8% were positive forV. parahaemolyticus, and 5.2% were positive for theV. parahaemolyticustoxin gene. The detection of theV. choleraegenetic marker was significantly more frequent at Doheny State Beach, while the detection of theV. parahaemolyticusgenetic marker was significantly more frequent at Avalon Harbor. A probability-of-illness model forV. parahaemolyticuswas applied to the data. The risk for bathers exposed to recreational waters at two beaches was evaluated through Monte Carlo simulation techniques. The results suggest that the microbial risk from vibrios during beach recreation was below the illness benchmark set by the U.S. EPA. However, the risk varied with location and the type of water recreation activities. Surfers and children were exposed to a higher risk of vibrio diseases. Microbial risk assessment can serve as a useful tool for the management of risk related to opportunistic marine pathogens.

scholarly journals Evaluation of a Disinfectant Wipe Intervention on Fomite-to-Finger Microbial Transfer

2014 ◽  
Vol 80 (10) ◽  
pp. 3113-3118 ◽  
Author(s):  
Gerardo U. Lopez ◽  
Masaaki Kitajima ◽  
Aaron Havas ◽  
Charles P. Gerba ◽  
Kelly A. Reynolds
Keyword(s):  
Escherichia Coli ◽  
Respiratory Pathogens ◽  
Quantitative Microbial Risk Assessment ◽  
Content Type ◽  
Microbial Risk Assessment ◽  
Surface Disinfection ◽  
Microbial Risk ◽  
Risk Assessment Models ◽  
The Mean ◽  
The Impact

ABSTRACTInanimate surfaces, or fomites, can serve as routes of transmission of enteric and respiratory pathogens. No previous studies have evaluated the impact of surface disinfection on the level of pathogen transfer from fomites to fingers. Thus, the present study investigated the change in microbial transfer from contaminated fomites to fingers following disinfecting wipe use.Escherichia coli(108to 109CFU/ml),Staphylococcus aureus(109CFU/ml),Bacillus thuringiensisspores (107to 108CFU/ml), and poliovirus 1 (108PFU/ml) were seeded on ceramic tile, laminate, and granite in 10-μl drops and allowed to dry for 30 min at a relative humidity of 15 to 32%. The seeded fomites were treated with a disinfectant wipe and allowed to dry for an additional 10 min. Fomite-to-finger transfer trials were conducted to measure concentrations of transferred microorganisms on the fingers after the disinfectant wipe intervention. The mean log10reduction of the test microorganisms on fomites by the disinfectant wipe treatment varied from 1.9 to 5.0, depending on the microorganism and the fomite. Microbial transfer from disinfectant-wipe-treated fomites was lower (up to <0.1% on average) than from nontreated surfaces (up to 36.3% on average, reported in our previous study) for all types of microorganisms and fomites. This is the first study quantifying microbial transfer from contaminated fomites to fingers after the use of disinfectant wipe intervention. The data generated in the present study can be used in quantitative microbial risk assessment models to predict the effect of disinfectant wipes in reducing microbial exposure.

A Most Probable Number Method for the Enumeration of Legionella Bacteria in Water

1991 ◽  
Vol 24 (2) ◽  
pp. 143-147 ◽  
Author(s):  
N. A. Grabow ◽  
R. Kfir ◽  
W. O. K. Grabow
Keyword(s):  
Water Samples ◽  
Membrane Filtration ◽  
Quantitative Method ◽  
Fluorescent Antibody ◽  
Most Probable Number ◽  
Probable Number ◽  
Comparative Tests ◽  
Antibody Staining ◽  
Direct Fluorescent Antibody ◽  
Yeast Extract Agar

A new quantitative method for the enumeration of Legionella bacteria in water is described. Appropriate tenfold serial dilutions of water samples concentrated by membrane filtration are plated in triplicate on buffered charcoal yeast extract agar. After incubation for 3 days representative smears from individual plates are tested for the presence of Legionella by direct fluorescent antibody staining. The number of positive plates in each dilution is used to calculate the Legionella count by means of conventional most probable number statistics. In comparative tests on a variety of water samples this method yielded significantly higher counts than previously used procedures.

Enteric virus presence in green vegetables and associated irrigation waters in a rural area from Argentina. A quantitative microbial risk assessment

LWT ◽  
2021 ◽  
Vol 144 ◽  
pp. 111201 ◽  
Author(s):  
Prez Verónica Emilse ◽  
Victoria Matías ◽  
Martínez Laura Cecilia ◽  
Giordano Miguel Oscar ◽  
Masachessi Gisela ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Rural Area ◽  
Enteric Virus ◽  
Quantitative Microbial Risk Assessment ◽  
Microbial Risk Assessment ◽  
Microbial Risk ◽  
Irrigation Waters
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