Development of a Rapid and Sensitive Method Combining a Cellulose Ester Microfilter and a Real-Time Quantitative PCR Assay To Detect Campylobacter jejuni and Campylobacter coli in 20 Liters of Drinking Water or Low-Turbidity Waters

Abstract

Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (؎22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (؎10.3%) and 78.5% (؎15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter.

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

“…Concentration of water samples by filtration may be more effective and less damaging to V. cholerae cells. Recovery rates obtained via filtration ranged from 51% to more than 90% (23,24) compared to the wide range of rates obtained by centrifugation (this study), 1.6% to 76.4%. However, because of the large amount of suspended solids in some of the investigated environmental waters, the use of a filter was impossible, because the filter was blocked after the passage of a few milliliters of water.…”

“…Global recovery (GR) was defined as the efficiency of the cell concentration and DNA extraction procedures in recovering bacteria spiked into environmental water samples (23,24). To test the reproducibility of the GR rates, three independent samples from the Neusiedler See were spiked with 5.4 ϫ 10 5 cells into 200 ml of lake water.…”

Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

“…As the cultivation of Campylobacter spp. is time-consuming and unsuitable for detecting viable but nonculturable (VBNC) cells, PCR-based methods have been developed for the detection and quantification of thermophilic Campylobacter in food, feed, or fecal or water samples (4)(5)(6)(7)(8). However, the lack of differentiation between viable and nonviable cells (due to the persistence of DNA after cell death) is a crucial point that limits the implementation of these approaches for routine diagnostic applications, even if a rapid quantification method is urgently needed (9).…”

The pattern of Campylobacter contamination on broiler farms; external and internal sources

“…was used to test water and feed samples (Tissier et al . ). A 1‐l volume of water from each house was collected at three different times throughout the cycle.…”

“…An ISO enrichment method for the detection and semiquantitative enumeration of thermo-tolerant Campylobacter spp. was used to test water and feed samples (Tissier et al 2012). A 1-l volume of water from each house was collected at three different times throughout the cycle.…”