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2 (Otto, 1994), and vegetables (Hopkins, 1993) prompted researchers to look at environmental sources as vectors for human infection. Previous efforts suggest the presence of H. pylori in ground water, surface water, and other drinking water (Mazari-Hiriart, 2001; Yingzhi, 2002; Hegarty, 1999; Hulten, 1998; Hulten, 1995; Shahamat, 1993), therefore implying a waterborne route of transmission to humans. The complex methods used in these studies (polymerase chain reaction; immunomagnetic separation; autoradiography; enzyme immunoassay) are expensive to run, subject to numerous interferences and do not differentiate between viable and non-viable organisms. A low cost and effective test to isolate viable H. pylori, similar to selective media used for Salmonella or E. coli, from ground and surface water would enable the drinking water industry to routinely screen samples. This study focused on efforts towards development of a plating media that selects viable H. pylori from real world water samples containing mixed microbial populations. MATERIALS & METHODS Media development Bacterial strain management Five clinical infection (no environmental isolates are available in U.S. or European type culture collections) Helicobacter pylori strains were obtained from the American Type Culture Collection (ATCC; Manassas, VA) or the Wisconsin State Laboratory of Hygiene culture collection (WSLH; Madison, WI; Table 1) and cultured on Brain Heart Infusion agar (BHI; Becton Dickinson, Sparks, MD) supplemented with 7% calf serum. The plates were incubated under a microaerophillic gas mixture (5% C02; 10% H2; 85% N2; Praxair, Inc., Danbury, CT) at 37oC. Frozen stock cultures were prepared by picking isolated colonies from agar plates and homogenizing in sodium phosphate buffer to about 107 colony forming units (cfu)/ml using a McFarland nephelometer. Each of the five isolates was then frozen in BHI broth containing 10% glycerol. Sufficient quantities were prepared to complete the entire study in order to avoid multiple passing of strains, which can sometimes lead to phenotypic variability. The non- Helicobacter bacteria used to create the complex spiked water samples (Table 1) were obtained from the WSLH culture collection, grown on BHI agar slants at 35oC and then stored at 4oC for up to 3 weeks before re-culturing. The bacteria are listed in table 1. TABLE 1. Bacterial strains and sources used in preparing water samples containing known levels of contaminants. Helicobacter pylori aATCC 43504 Helicobacter pylori ATCC 700392 Helicobacter pylori ATCC 49503 Helicobacter pylori bWSLH 95-10882 Helicobacter pylori WSLH 409013 Acinetobacter WSLH Aeromonas WSLH E. coli WSLH Pseudomonas aeruginosa WSLH Enterobacter cloacea WSLH Enterococcus faecalis WSLH Bacillus WSLH a American Type Culture Collection. b Wisconsin State Laboratory of Hygiene culture collection.
3 Preparation of conventional media Conventional dehydrated media preparations were chosen for selection of H. pylori from mixed microbial populations based on the published clinical literature. The five types of media chosen for evaluation were BHI + 7% calf serum (Osaki, 1998), Brucella Agar (Poms, 2001), Campylobacter Agar Kit Skirrow (Corry, 1995), Columbia Blood Agar Base (Baronn, 1994; Becton Dickinson, Sparks, MD), and HPSPA (Jiang, 2000; Stevenson, 2000). Table 2. compares ingredients of these media to demonstrate the common and unique features of each. The inclusion of either Helicobacter pylori selective supplement (Oxoid Limited, Hampshire, England) or Campylobacter selective supplement S (Becton Dickinson) provided antibiotics to prevent background while permitting H. pylori growth. Positive control consisted of the five conventional media without the selective supplements while negative controls consisted of uninoculated plates. All media were prepared according to manufacturer's or authors' instructions. TABLE 2. Formulations of conventional media used to culture and select Helicobacter pylori. Brain Heart Infusion Brucella Agar Columbia Agar aHPSPA Campylobacter Agar 10 g proteose peptone 10 g peptamin 10 g pantone 15 g spec. peptone 15 g proteose peptone NA beef heart inf. 10 g tryptone 10 g bitone 2 g porcine mucin 2.5. ml liver digest NA calf brains inf. 2 g yeast extract 3 g beef heart dig. 5 g yeast extract 5 g yeast extract 2 g dextrose 1 g dextrose 1 g dextrose 5 g beef extract 5 g NaCl 5 g NaCl 5 g NaCl 5g NaCl 5g NaCl 15 g agar 15 g agar 15 g agar 15 g agar 12 g agar 2.5 g disodium phos. 0.1 g Na bisulfate 0.5 g ferrous sulfate 70 ml calf serum w/fe 70 ml calf serum w/fe 0.6 g urea 0.5 g Na pyruvate bselective supplement selective supplement selective supplement selective supplement cselective supplement a Formula published by Jiang and Doyle. J. Clin. Microbiol. 2000. b Vancomycin (10 mg/L), Cefsulodin (5 mg/L), Trimethoprim (5 mg/L), and Amphotericin B (5 mg/L). c Vancomycin (10 mg/L), Trimethoprim (5 mg/L), and Polymyxin B (2500 IU/L) Initial conventional test media screen Evaluation of the efficacy of the conventional media formulations used in clinical microbiology laboratories was carried out as follows. Each strain listed in Table 1 was separately cultured on each of the five media listed in Table 2 in order to evaluate the growth and inhibition spectra of individual formulations. First, pure colonies of each strain were picked from solid growth media (BHI agar plates) and homogenized in sodium phosphate buffer (4%). Serial dilutions of each pure homogenate were immediately spread (0.1ml/plate) onto each type of solid media with and without selective supplement added. All plates were incubated under microaerophillic conditions at 37oC for up to seven days and examined to determine the presence or absence of colonies on the media compared to the positive control of a non-selective media. Selectivity of media formulations for H. pylori from samples spiked with a cocktail of non H. pylori bacteria. Well water samples containing native flora (Table 5; identified using API 20 E identification system; Biomerieux Vitek, Inc., Hazelwood, MO) were further spiked with the seven strains of background bacteria described above, and the H. pylori, to represent a highly contaminated water sample (10,000 cells per strain per 100 mls). The spiked water sample was then serially diluted and 0.1 ml of each dilution was spread onto duplicate plates of each of the