Development of a Selective Medium for Isolation of Helicobacter pylori from Cattle and Beef Samples

Abstract

Helicobacter pylori has been isolated from the human stomach with media containing only minimal selective agents. However, current research on the transmission and sources of infection requires more selective media due to the higher numbers of contaminants in environmental, oral, and fecal samples. The objective of this study was to develop and evaluate detection techniques that are sufficiently selective to isolate H. pylori from potential animal and food sources. Since H. pylori survives in the acidic environment of the stomach, low pH with added urea was studied as a potential selective combination. H. pylori grew fairly well on H. pylori Special Peptone plating medium supplemented with 10 mM urea at pH 4.5, but this pH did not sufficiently inhibit the growth of contaminants. Various antibiotic combinations were then compared, and a combination consisting of 10 mg of vancomycin per liter, 5 mg of amphotericin B per liter, 10 mg of cefsulodin per liter, 62,000 IU of polymyxin B sulfate per liter, 40 mg of trimethoprim per liter, and 20 mg of sulfamethoxazole per liter proved to be highly selective but still allowed robust colonies of H. pylori to grow. This medium was highly selective for recovering H. pylori from cattle and beef samples, and it is possible that it could be used to enhance the recovery of this bacterium from human and environmental samples, which may be contaminated with large numbers of competing microorganisms.In 1982 Warren and Marshall (17) isolated a new spiralshaped bacterium from the stomachs of humans with gastric ulcers, which was named Campylobacter pyloridis. Further research on this novel bacterium revealed significant morphological, biochemical, and genetic differences from other Campylobacter spp., and the microorganism was eventually renamed Helicobacter pylori (7). Since then, it has been determined that H. pylori is the most prevalent bacterial pathogen in the world and infects approximately one-half of the world's population (10). Infection with H. pylori causes numerous medical conditions, including gastritis, peptic ulcers, gastric carcinomas, and mucosa associated lymphoid tissue tumors (2, 9, 12).It has not been necessary for the media used to isolate H. pylori from the stomach to be highly selective because relatively few contaminants survive in the low-pH environment of the stomach (13). However, current research on the transmission and sources of infection requires more selective media. In several studies workers have tried to determine the prevalence of H. pylori in the oral cavities or feces of infected patients, but these efforts have been hindered by the large number of contaminants and the fastidious nature of the bacterium (3, 5, 16). Therefore, the need for a more selective medium has become increasingly important.The objective of this study was to develop and evaluate detection techniques that are selective enough so that H. pylori can be isolated from potential animal and food sources. Since H. pylori has the unique ability to survive in the highly acidic envi...

Shear Stress, Temperature, and Inoculation Concentration Influence the Adhesion of Water-Stressed Helicobacter pylori to Stainless Steel 304 and Polypropylene

“…The H. pylori concentrations in the flasks were determined by plating 100 l of the appropriate dilutions (1:10 dilutions in distilled water) onto three to five plates of HPSPA (27). Plates were incubated at 37°C for 6 days under the same microaerophilic conditions used for culture maintenance.…”

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

“…The antibiotic resistance spectrum of H. pylori is well defined, but the concentrations in media vary widely depending on the matrix in which the research is done. For example, trimethoprim and polymyxin B were used at concentrations of 5 mg/liter and 3,500 U/liter, respectively, to isolate H. pylori in a water matrix (18), but the concentrations were increased to 40 mg/liter and 62,000 U/liter for selection in cattle mucosa (22). In this study, we focused on antibiotic levels established by commercial vendors (Oxoid, Becton Dickinson) and adjusted these levels in order to achieve acceptable results.…”

“…Conventional dehydrated medium preparations were chosen for selection of H. pylori from mixed microbial populations based on previously published data. The five media chosen for evaluation were BHI containing 7% calf serum (16), brucella agar (19), campylobacter agar kit Skirrow (3), Columbia blood agar base (2) (Becton Dickinson), and HPSPA medium (9,22). Table 2 shows the ingredients of these media and the common and unique features of each medium.…”

Fate of Helicobacter pylori artificially inoculated in lettuce and carrot samples

“…pylori HP1 was recovered in modified CBA, and H. pylori ATCC 43629 in CBA and in HPSPA (Helicobacter pylori Special Peptone Agar) supplemented with vancomycin (10 mg/l), amphotericin (5 mg/l), cefsulodin (10 mg/l), polymyxin B sulfate (62,000 IU/l), trimethoprim (40 mg/l) and sulfamethoxazole (20 mg/l) (14,18). HPSPA and Columbia agar without supplements were also tested for the recovery of H. pylori ATCC.…”

“…Difficulties in elucidating the transmission routes of H. pylori are partially due to unsuitable detection methods available for the recovery of this pathogen from samples with a dense competing microflora (14,17,18). In this work we evaluated selective and non-selective media for the recovery of H. pylori from vegetable foods.…”