Culture-based indicators of fecal contamination and molecular microbial indicators rarely correlate with Campylobacter spp. in recreational waters.

Campylobacter spp. are the leading cause of gastroenteritis worldwide. Most human infections result from contaminated food; however, infections are also caused by recreational waterway contamination. Campylobacter culture is technically challenging and enumeration by culture-based methods is onerous. Thus, we employed qPCR to quantify Campylobacter spp. in fresh- and marine-water samples, raw sewage and animal feces. Multiplex PCR determined whether Campylobacter jejuni or C. coli, most commonly associated with human disease, were present in qPCR-positive samples. Campylobacters were detected in raw sewage, and in feces of all avian and mammalian species tested. Campylobacter-positive concentrations ranged from 68 to 2.3 × 106 cells per 500 mL. Although C. jejuni and C. coli were rare in waterways, they were prevalent in sewage and feces. Campylobacter-specific qPCR screening of environmental waters did not correlate with the regulatory EPA method 1600 (Enterococcus culture), nor with culture-independent, molecular-based microbial source tracking indicators, such as human polyomavirus, human Bacteroidales and Methanobrevibacter smithii. Our results suggest that neither the standard EPA method nor the newly proposed culture-independent methods are appropriate surrogates for Campylobacter contamination in water. Thus, assays for specific pathogens may be necessary to protect human health, especially in waters that are contaminated with sewage and animal feces.

Optimization of a fluorescence sandwich enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 in apple juice.

Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 10(4) to 10(6) CFU/ml, whereas that for spiked phosphate-buffered saline was 10(6) to 10(8) CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.

Effects of chlorine injury, starvation, and Colitag(TM) enrichment on sandwich enzyme-linked immunosorbent assay (sELISA) detection of Escherichia coli O157:H7.

The validity of ELISA for detection of E. coli O157:H7 under many conditions is not proven. In this work, sELISA was able to detect bacteria after sub-lethal chlorine exposure and after seven days of starvation with little to no change in limit of detection and fluorescence signal as long as chlorine was not present in the sample or was neutralized by sodium thiosulfate. After Colitag enrichment, sELISA detected approximately 3 colony forming units/ml of originally added E. coli O157:H7. Thus, the present sELISA is valid for detection of E. coli O157:H7 in water sources, although sample matrices may interfere with assay.

Antibody-based detection of acid-shocked, acid-adapted, and apple juice-incubated Escherichia coli O157:H7.

Sandwich enzyme-linked immunosorbent assays (sELISA) allow for rapid detection of Escherichia coli (E. coli) O157:H7. Acidic conditions similar to those in certain foods and juices may reduce the ability to detect E. coli O157:H7. Growth of E. coli O157:H7 at pH 4 compared to pH 5-7 reduced fluorescent signal at the lower bacterial concentrations without altering the range of detection. Both acid-adaptation and a subsequent pH 7 incubation reversed sensitivity. Incubation in apple juice was not deleterious to sELISA detection. Exposure to acidic conditions can cause a small reduction in sELISA sensitivity used to detect E. coli O157:H7.