Bacterial Pathogen Gene Abundance and Relation to Recreational Water Quality at Seven Great Lakes Beaches

Abstract

Quantitative assessment of bacterial pathogens, their geographic variability, and distribution in various matrices at Great Lakes beaches are limited. Quantitative PCR (qPCR) was used to test for genes from E. coli O157:H7 (eaeO157), shiga-toxin producing E. coli (stx2), Campylobacter jejuni (mapA), Shigella spp. (ipaH), and a Salmonella enterica-specific (SE) DNA sequence at seven Great Lakes beaches, in algae, water, and sediment. Overall, detection frequencies were mapA>stx2>ipaH>SE>eaeO157. Results were highly variable among beaches and matrices; some correlations with environmental conditions were observed for mapA, stx2, and ipaH detections. Beach seasonal mean mapA abundance in water was correlated with beach seasonal mean log10 E. coli concentration. At one beach, stx2 gene abundance was positively correlated with concurrent daily E. coli concentrations. Concentration distributions for stx2, ipaH, and mapA within algae, sediment, and water were statistically different (Non-Detect and Data Analysis in R). Assuming 10, 50, or 100% of gene copies represented viable and presumably infective cells, a quantitative microbial risk assessment tool developed by Michigan State University indicated a moderate probability of illness for Campylobacter jejuni at the study beaches, especially where recreational water quality criteria were exceeded. Pathogen gene quantification may be useful for beach water quality management.

Contamination with bacterial zoonotic pathogen genes in U.S. streams influenced by varying types of animal agriculture

“…Four genes were tested: mapA specific for C. jejuni (Best et al, 2003); eae O157:H7 (an E. coli O157:H7-specific sequence of the eaeA gene tested by PCR; Ibekwe et al, 2002); stx2 (a short sequence in the longer sequence tested by PCR; Beutin et al, 2008); and a nonvirulence-associated sequence for Salmonella enterica (Wang et al, 2007). Assay details are described in Oster et al (2014). Briefly, the stx2, eae O157:H7, and S. enterica assays used SYBR green chemistry and the mapA assay used TaqMan® chemistry.…”

“…Standard curves were developed for each pathogen gene using E. coli plasmids generated using a TOPO TA Cloning Kit (Life Technologies, Grand Island, NY) containing the appropriate DNA fragment insert as described in Oster et al (2014). Plasmid DNA concentration was measured with a NanoDrop ND-1000 UV spectrophotometer, and gene copy numbers were calculated as described in Oster et al (2014). All standard curves with R 2 N 0.95, and efficiencies of 85-115% were considered acceptable for quantification of samples.…”

“…All sample, standard, and control qPCR assays were run in triplicate in 25 μL reaction volumes. Methods used to establish the limit of detection and limit of quantification, and final assignment for the triplicate qPCR results are described in detail in Oster et al (2014). Briefly, limits of detection (LOD) and limits of quantification (LOQ) were calculated for each assay.…”

Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study

“…The case study presented here is a duplex TaqMan real-time PCR (RT-PCR) for identification of Campylobacter jejuni and Campylobacter coli (19). Developed in the pre-WGS era using single gene sequences, the assay and variations thereof have been used for routine isolate identification to the species level (19–21); studies of Campylobacter isolates from humans (22–25), animals (26–35), and the environment (36); and outbreak investigations (37). For C.…”

Detection and Occurrence of Indicator Organisms and Pathogens

“…The geographic fluctuation and distribution of bacterial pathogens in various matrices at Great Lakes beaches were investigated byOster et al (2014). qPCR was used to quantify genes of E. coli O157:H7 (eaeO157), shiga-toxin producing E. coli (stx2), Campylobacter jejuni (mapA), Shigella spp.…”