scholarly journals Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos

2015 ◽  
Vol 81 (11) ◽  
pp. 3722-3727 ◽  
Author(s):  
Michael Knappik ◽  
David A. B. Dance ◽  
Sayaphet Rattanavong ◽  
Alain Pierret ◽  
Olivier Ribolzi ◽  
...  
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
Environmental Samples ◽  
Burkholderia Pseudomallei ◽  
Molecular Detection ◽  
Culture Methods ◽  
Environmental Distribution ◽  
Content Type ◽  
Culture Techniques ◽  
Enrichment Step

ABSTRACTBurkholderia pseudomalleiis the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution ofB. pseudomalleiworldwide and within countries where it is endemic, such as the Lao People's Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology ofB. pseudomalleiand to facilitate public health interventions. Sensitive and specific methods to detectB. pseudomalleiin environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection ofB. pseudomalleiin soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100;P< 0.001). Similarly, qPCR following enrichment was the most sensitive method for filtered river water compared with the sensitivity of the individual methods and all individual methods combined. In conclusion, molecular detection following an enrichment step has proven to be a sensitive and reliable approach forB. pseudomalleidetection in Lao environmental samples and is recommended as the preferred method for future surveys.

scholarly journals Distribution and Differential Survival of Traditional and Alternative Indicators of Fecal Pollution at Freshwater Beaches

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Danielle D. Cloutier ◽  
Sandra L. McLellan
Keyword(s):  
Water Samples ◽  
Environmental Samples ◽  
High Sensitivity ◽  
Fecal Pollution ◽  
Source Material ◽  
Beach Sand ◽  
Differential Survival ◽  
Content Type ◽  
Volume Comparison ◽  
E Coli

ABSTRACT Alternative indicators have been developed that can be used to identify host sources of fecal pollution, yet little is known about how their distribution and fate compare to traditional indicators. Escherichia coli and enterococci were widely distributed at the six beaches studied and were detected in almost 95% of water samples (n = 422) and 100% of sand samples (n = 400). Berm sand contained the largest amount of E. coli (P < 0.01), whereas levels of enterococci were highest in the backshore (P < 0.01). E. coli and enterococci were the lowest in water, using a weight-to-volume comparison. The gull-associated Catellicoccus marimammalium (Gull2) marker was found in over 80% of water samples, regardless of E. coli levels, and in 25% of sand samples. Human-associated Bacteroides (HB) and Lachnospiraceae (Lachno2) were detected in only 2.4% of water samples collected under baseflow and post-rain conditions but produced a robust signal after a combined sewage overflow, despite low E. coli concentrations. Burdens of E. coli and enterococci in water and sand were disproportionately high in relation to alternative indicators when comparing environmental samples to source material. In microcosm studies, Gull2, HB, and Lachno2 quantitative PCR (qPCR) signals were reduced twice as quickly as those from E. coli and enterococci and approximately 20% faster than signals from culturable E. coli. High concentrations of alternative indicators in source material illustrated their high sensitivity for the identification of fecal sources; however, differential survival and the potential for long-term persistence of traditional fecal indicators complicate the use of alternative indicator data to account for the levels of E. coli and enterococci in environmental samples. IMPORTANCE E. coli and enterococci are general indicators of fecal pollution and may persist in beach sand, making their use problematic for many applications. This study demonstrates that gull fecal pollution is widespread at Great Lakes beaches, whereas human and ruminant contamination is evident only after major rain events. An exploration of sand as a reservoir for indicators found that E. coli was ubiquitous, while gull host markers were detected in only 25% of samples. In situ sand beach microcosms provided decay rate constants for E. coli and enterococci relative to alternative indicators, which establish comparative benchmarks that would be helpful to distinguish recent from past pollution. Overall, alternative indicators are useful for identifying sources and assessing potentially high health risk contamination events; however, beach managers should be cautious in attempting to directly link their detection to the levels of E. coli or enterococci.

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis

2011 ◽  
Vol 64 (12) ◽  
pp. 2453-2459 ◽  
Author(s):  
G. N. van Blerk ◽  
L. Leibach ◽  
A. Mabunda ◽  
A. Chapman ◽  
D. Louw
Keyword(s):  
Surface Water ◽  
High Resolution ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
Curve Analysis ◽  
Pcr Assay ◽  
High Resolution Melt ◽  
Gene Target ◽  
Enrichment Step

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16–18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

scholarly journals The prevalence of Francisella spp. in different natural surface water samples collected from northwest of Iran

2019 ◽  
Author(s):  
Mahdi Rohani ◽  
Abdolrazagh Hashemi Shahraki ◽  
Ahmad Ghasemi ◽  
Saber Esmaeili ◽  
Aynur Karadenizli ◽  
...  
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
Environmental Samples ◽  
Bacterial Species ◽  
Wide Distribution ◽  
Bacterial Dna ◽  
Taqman Pcr ◽  
Northwest Of Iran ◽  
Surface Water Samples ◽  
North Western

Background and Objectives: Francisella tularensis has a wide distribution in northern hemisphere of the world. Up to now, there was little information about the Francisella spp. situation in the environmental samples in Iran. In this study we aimed to determine the prevalence of Francisella spp. in the environmental samples in northwest of Iran. Materials and Methods: A total of 237 natural water samples from ponds, rivers, lakes, springs and other surface waters from north western provinces of Iran (Kurdistan and Western Azerbaijan) were collected from September to November 2015. All samples were cultured for Francisella and other bacterial species and Real Time TaqMan PCR was performed on the concentrated and DNA extracted samples. For detection of the presence of bacterial DNA in the samples, two different targets in the genome of Francisella, ISFtu2 and fopA were used. Results: Among the tested surface water samples, 40 (17.09%; 95% CI: 12.67-22.33%) and 12 (5.13%; 95%CI: 2.81-8.56%) samples were positive for ISFtu2 and fopA respectively. None of them was positive in culture. Conclusion: The prevalence of Francisella spp. in the environmental samples in the west of Iran is high and it is comparable with Turkey, Iran’s neighboring country. Use of higher copy number genes or IS like ISFtu2 could improve the detection of this organism in the environmental samples.

scholarly journals Spatiotemporal Analysis of Cryptosporidium Species/Genotypes and Relationships with Other Zoonotic Pathogens in Surface Water from Mixed-Use Watersheds

2012 ◽  
Vol 79 (2) ◽  
pp. 434-448 ◽  
Author(s):  
Graham Wilkes ◽  
Norma J. Ruecker ◽  
Norman F. Neumann ◽  
Victor P. J. Gannon ◽  
Cassandra Jokinen ◽  
...  
Keyword(s):  
Land Use ◽  
Surface Water ◽  
Water Samples ◽  
Regression Tree ◽  
Infection Risk ◽  
Human Infection ◽  
Classification And Regression Tree ◽  
Odds Ratios ◽  
Content Type ◽  
Zoonotic Pathogens

ABSTRACTNearly 690 raw surface water samples were collected during a 6-year period from multiple watersheds in the South Nation River basin, Ontario, Canada.Cryptosporidiumoocysts in water samples were enumerated, sequenced, and genotyped by detailed phylogenetic analysis. The resulting species and genotypes were assigned to broad, known host and human infection risk classes. Wildlife/unknown, livestock, avian, and human host classes occurred in 21, 13, 3, and <1% of sampled surface waters, respectively.Cryptosporidium andersoniwas the most commonly detected livestock species, while muskrat I and II genotypes were the most dominant wildlife genotypes. The presence ofGiardiaspp.,Salmonellaspp.,Campylobacterspp., andEscherichia coliO157:H7 was evaluated in all water samples. The greatest significant odds ratios (odds of pathogen presence when host class is present/odds of pathogen presence when host class is absent) forGiardiaspp.,Campylobacterspp., andSalmonellaspp. in water were associated, respectively, with livestock (odds ratio of 3.1), avian (4.3), and livestock (9.3) host classes. Classification and regression tree analyses (CART) were used to group generalized host and human infection risk classes on the basis of a broad range of environmental and land use variables while tracking cooccurrence of zoonotic pathogens in these groupings. The occurrence of livestock-associatedCryptosporidiumwas most strongly related to agricultural water pollution in the fall (conditions also associated with elevated odds ratios of other zoonotic pathogens occurring in water in relation to all sampling conditions), whereas wildlife/unknown sources ofCryptosporidiumwere geospatially associated with smaller watercourses where urban/rural development was relatively lower. Conditions that support wildlife may not necessarily increase overall human infection risks associated withCryptosporidiumsince mostCryptosporidiumgenotypes classed as wildlife in this study (e.g., muskrat I and II genotype) do not pose significant infection risks to humans. Consequently, from a human health perspective, land use practices in agricultural watersheds that create opportunities for wildlife to flourish should not be rejected solely on the basis of their potential to increase relative proportions of wildlife fecal contamination in surface water. The present study suggests that mitigating livestock fecal pollution in surface water in this region would likely reduce human infection risks associated withCryptosporidiumand other zoonotic pathogens.

scholarly journals Rapid Filter-Based Detection and Culture of Burkholderia pseudomallei from Small Volumes of Urine

2017 ◽  
Vol 55 (9) ◽  
pp. 2698-2707 ◽  
Author(s):  
Pierre A. Michel ◽  
Christine Lascols ◽  
Jay E. Gee ◽  
Linda M. Weigel ◽  
David Sue
Keyword(s):  
Laboratory Testing ◽  
Burkholderia Pseudomallei ◽  
Dna Isolation ◽  
Urine Culture ◽  
Colony Growth ◽  
Detection Sensitivity ◽  
Culture Methods ◽  
Content Type ◽  
New Methods ◽  
Low Concentrations

ABSTRACTClinical outcomes of melioidosis patients improve when the infecting agent,Burkholderia pseudomallei, is rapidly detected and identified by laboratory testing. Detection ofB. pseudomalleiDNA or recovery of the pathogen by culture from urine can support a diagnosis of melioidosis and guide patient care. Two new methods, designated filter-capture DNA isolation (FCDI) and filter cellular recovery (FCR), were developed to increase the sensitivity of detection and recovery of viableB. pseudomalleicells from small volumes (0.45 ml) of urine. DNA from eight strains ofB. pseudomalleithat were spiked into synthetic urine at low concentrations (1 × 102CFU/ml) was detected in FCDI cell lysates using real-time PCR with greater consistency than with preparations from a QIAamp DNA Blood minikit. The FCR method showed greaterB. pseudomalleidetection sensitivity than conventional urine culture methods and resulted in typical colony growth at 24 h from as few as 1 × 102CFU/ml. In addition, the FCR method does not rely on precipitation of a urine pellet by centrifugation and requires a smaller volume of urine. The FCDI and FCR methods described here could improve time-to-results and decrease the number of negativeB. pseudomalleireports that are currently observed from urine culture as a consequence of samples containing low or variable bacterial cell concentrations.

scholarly journals Multi-drug resistant pathogenic Listeria monocytogenes in surface water and soil samples of Dhaka city

2018 ◽  
Vol 33 (1-2) ◽  
pp. 39-42 ◽  
Author(s):  
Md Shifat E Manjur ◽  
Shahariar Siddique ◽  
Sangita Ahmed
Keyword(s):  
Listeria Monocytogenes ◽  
Surface Water ◽  
Water Samples ◽  
Soil Samples ◽  
Drug Resistant ◽  
Biochemical Tests ◽  
Environmental Distribution ◽  
Antibiotic Resistance Profile ◽  
Water And Soil Samples ◽  
Surface Water Samples

Listeria monocytogenes, the causative agent of human listeriosis is widely distributed in nature and a major threat to human health. Aiming to investigate the environmental distribution of this pathogen in Bangladesh, the current study conducted an initial investigation on 30 soil samples and 20 surface water samples for the presence of the pathogenic Listeria monocytogenes. Based on cultural, morphological, biochemical tests and presence of the virulent gene hly, 7 (14%) Listeria monocytogenes were obtained of which, surface water samples contained four Listeria monocytogenes (15%) while, three Listeria spp. (13.33%) were isolated from soil samples. Antibiotic resistance profile of the Listeria isolates showed that 100% isolates were resistant to erythromycin and resistance to oxacillin, ampicillin, sulphamethoxazole-trimethoprim and penicillin was 71%, 57%, 43% and 43% respectively. 71% Listeira monocytogenes isolates were sensitive to vancomycin and 100% sensitivity was observed to imipenem. This study shows that multi-drug resistant pathogenic Listeria monocytogenes is widely spread in soil and water samples in Dhaka and imposes great risk to public health. Bangladesh J Microbiol, Volume 33, Number 1-2, June-Dec 2016, pp 39-42

scholarly journals Evaluation of the Biotoxis qPCR Detection Kit for Francisella tularensis Detection in Clinical and Environmental Samples

2020 ◽  
Vol 59 (1) ◽  
pp. e01434-20
Author(s):  
Aurélie Hennebique ◽  
Fabienne Gas ◽  
Hélène Batina ◽  
Cécilia De Araujo ◽  
Karine Bizet ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Water Samples ◽  
Environmental Samples ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Environmental Water ◽  
Type B ◽  
Content Type ◽  
Clinical Specimens ◽  
Real Time Quantitative Pcr

ABSTRACTRapid and reliable detection and identification of Francisella tularensis (a tier 1 select agent) are of primary interest for both medical and biological threat surveillance purposes. The Biotoxis qPCR detection kit is a real-time quantitative PCR (qPCR) assay designed for the detection of Bacillus anthracis, Yersinia pestis, and F. tularensis in environmental or biological samples. Here, we evaluated its performance for detecting F. tularensis in comparison to previously validated qPCR assays. The Biotoxis qPCR was positive for 87/87 F. tularensis subsp. holarctica (type B) strains but also for F. tularensis subsp. novicida. It was negative for Francisella philomiragia and 24/24 strains belonging to other bacterial species. For 31 tularemia clinical specimens, the Biotoxis qPCR displayed a sensitivity between 90.32% and 96.55%, compared to qPCR tests targeting ISFtu2 or a type B-specific DNA sequence, respectively. All 30 nontularemia clinical specimens were Biotoxis qPCR negative. For water samples, the Biotoxis qPCR limit of detection was 1,000 CFU/liter of F. tularensis. For 57 environmental water samples collected in France, the Biotoxis qPCR was positive for 6/15 samples positive for ISFtu2 qPCR and 4/4 positive for type B qPCR. In conclusion, the Biotoxis qPCR detection kit demonstrated good performances for F. tularensis detection in various biological and environmental samples, although cross-amplification of F. tularensis subsp. novicida must be considered. This plate format assay could be useful to test a large number of clinical or environmental specimens, especially in the context of natural or intentional tularemia outbreaks.

scholarly journals Characterization of Extended-Spectrum β-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm

2012 ◽  
Vol 78 (10) ◽  
pp. 3668-3673 ◽  
Author(s):  
Junying Ma ◽  
Jian-Hua Liu ◽  
Luchao Lv ◽  
Zhiyong Zong ◽  
Yan Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Environmental Samples ◽  
Genetic Relatedness ◽  
Antibiotic Resistance Genes ◽  
Hypothetical Protein ◽  
M Gene ◽  
Content Type ◽  
E Coli ◽  
Extended Spectrum

ABSTRACTIn this study, we focused on evaluating the occurrence of extended-spectrum β-lactamase (ESBL)-producingEscherichia coliin fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified asE. coliwere screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 hadE. coliisolates with ceftiofur MICs higher than 8 μg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples).blaCTX-M,blaSHV-12,blaCMY-2, andblaDHA-1were identified in 108, 5, 9, and 1 isolates, respectively. The most commonblaCTX-Mgenes wereblaCTX-M-27(n= 34),blaCTX-M-55(n= 27),blaCTX-M-24e(n= 22), andblaCTX-M-105(n= 20), followed byblaCTX-M-14a,blaCTX-M-14b,blaCTX-M-24a, andblaCTX-M-24b. Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed.blaCTX-Mgenes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M geneblaCTX-M-105was flanked by two hypothetical protein sequences, partial ISEcp1upstream and truncated IS903D,iroN,orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer ofblaCTX-Mgenes mediated by mobile elements and the clonal spread of CTX-M-producingE. coliisolates contributed to the dissemination ofblaCTX-Min the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.

scholarly journals Multitarget Quantitative PCR Improves Detection and Predicts Cultivability of the Pathogen Burkholderia pseudomallei

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Andre Göhler ◽  
Trinh Thanh Trung ◽  
Verena Hopf ◽  
Christian Kohler ◽  
Jörg Hartleib ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Burkholderia Pseudomallei ◽  
Detection Rate ◽  
Soil Samples ◽  
Single Type ◽  
Comparative Genomic ◽  
Environmental Distribution ◽  
Content Type ◽  
The World ◽  
Culture Positive

ABSTRACT Burkholderia pseudomallei is present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification of B. pseudomallei in the environment are a prerequisite for assessing the risk of infection. We recently reported the direct detection of B. pseudomallei in soil samples using a quantitative PCR (qPCR) targeting a single type three secretion system 1 (TTSS1) gene. Here, we extend the qPCR-based analysis of B. pseudomallei in soil by validating novel qPCR gene targets selected from a comparative genomic analysis. Two hundred soil samples from two rice paddies in northeast Thailand were evaluated, of which 47% (94/200) were B. pseudomallei culture positive. The TTSS1 qPCR and two novel qPCR assays that targeted open reading frames (ORFs) BPSS0087 and BPSS0745 exhibited detection rates of 76.5% (153/200), 34.5% (69/200), and 74.5% (150/200), respectively. The combination of TTSS1 and BPSS0745 qPCR increased the detection rate to 90% (180/200). Combining the results of the three qPCR assays and the BPSS1187 nested PCR previously published, all 200 samples were positive by at least one PCR assay. Samples positive by either TTSS1 (n = 153) or BPSS0745 (n = 150) qPCR were more likely to be direct-culture positive, with odds ratios of 4.0 (95% confidence interval [CI], 1.7 to 9.5; P < 0.001) and 9.0 (95% CI, 3.1 to 26.4; P < 0.001), respectively. High B. pseudomallei genome equivalents correlated with high CFU counts by culture. In conclusion, multitarget qPCR improved the B. pseudomallei detection rate in soil samples and predicted culture positivity. This approach has the potential for use as a sensitive environmental screening method for B. pseudomallei. IMPORTANCE The worldwide environmental distribution of the soil bacterium Burkholderia pseudomallei remains to be determined. So far, most environmental studies have relied on culture-based approaches to detect this pathogen. Since current culture methods are laborious, are time consuming, and have limited sensitivity, culture-independent and more sensitive methods are needed. In this study, we show that a B. pseudomallei-specific qPCR approach can detect significantly higher numbers of B. pseudomallei-positive soil samples from areas where it is endemic compared with that from culture. The use of multiple independent B. pseudomallei-specific qPCR targets further increased the detection rate of B. pseudomallei compared with that from single targets. Samples with a high molecular B. pseudomallei load were more likely to be culture positive. We conclude that our quantitative multitarget approach might be useful in defining areas where there is a risk of B. pseudomallei infections in different parts of the world.

scholarly journals Dead-End Ultrafiltration and DNA-Based Methods for Detection of Cyclospora cayetanensis in Agricultural Water

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Mauricio Durigan ◽  
Helen R. Murphy ◽  
Alexandre J. da Silva
Keyword(s):  
Surface Water ◽  
Water Samples ◽  
Protozoan Parasite ◽  
Environmental Water ◽  
Agricultural Water ◽  
Content Type ◽  
Cyclospora Cayetanensis ◽  
Dead End ◽  
Low Levels ◽  
Surface Water Samples

ABSTRACT Cyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne diarrheal illness outbreaks worldwide. Most of these outbreaks are associated with the consumption of fresh produce. Sensitive and specific methods to detect C. cayetanensis in agricultural water are needed to identify the parasite in agricultural water used to irrigate crops that have been implicated in outbreaks. In this study, a method to detect C. cayetanensis in water by combining dead-end ultrafiltration (DEUF) with sensitive and specific molecular detection was developed and evaluated. Triplicates of 10-liter agricultural water samples were seeded with 200, 100, 25, 12, and 6 C. cayetanensis oocysts. Surface water samples were also collected in the Mid-Atlantic region. All water samples were processed by DEUF and backflushed from the ultrafilters. DNA was extracted from concentrated samples and analyzed by quantitative PCR (qPCR) targeting the C. cayetanensis 18S rRNA gene. All water samples seeded with 12, 25, 100, and 200 oocysts were positive, and all unseeded samples were negative. Samples seeded with 6 oocysts had a detection rate of 66.6% (8/12). The method was also able to detect C. cayetanensis isolates in surface water samples from different locations of the Chesapeake and Ohio Canal (C&O Canal) in Maryland. This approach could consistently detect C. cayetanensis DNA in 10-liter agricultural water samples contaminated with low levels of oocysts, equivalent to the levels that may be found in naturally incurred environmental water sources. Our data demonstrate the robustness of the method as a useful tool to detect C. cayetanensis from environmental sources. IMPORTANCE Cyclospora cayetanensis is a protozoan parasite that causes foodborne and waterborne outbreaks of diarrheal illness worldwide. These foodborne outbreaks associated with the consumption of fresh produce and agricultural water could play a role in the contamination process. In this study, a method to detect C. cayetanensis in agricultural water by combining a robust filtration system with sensitive and specific molecular detection was developed and validated by the FDA. The results showed that this approach could consistently detect low levels of C. cayetanensis contamination in 10 liters of agricultural water, corresponding to the levels that may be found in naturally occurring environmental water sources. The method was also able to detect C. cayetanensis in surface water samples from a specific location in the Mid-Atlantic region. Our data demonstrate the robustness of the method to detect C. cayetanensis in agricultural water samples, which could be very useful to identify environmental sources of contamination.

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