Development of a Plating Medium for Selection of Helicobacter pylori from Water Samples

Abstract

ABSTRACT The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori . Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms ( Acinetobacter , Aeromonas , Bacillus , Escherichia coli , Enterobacter , Enterococcus , Helicobacter pylori , and Pseudomonas strains) were individually plated onto each of these media. Three organisms ( Acinetobacter , E. coli , and H. pylori ) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora ( Flavobacterium , Serratia , Citrobacter , Pasteurella , Ochrobactrum , Rahnella , and unidentified molds) and were further adulterated with the eight strains listed above (10 6 CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37°C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.