Attempts to Isolate Helicobacter from Cattle and Survival of Helicobacter pylori in Beef Products

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1 174 Journal of Food Protection, Vol. 63, No. 2, 2000, Pages Copyright, International Association for Food Protection Attempts to Isolate Helicobacter from Cattle and Survival of Helicobacter pylori in Beef Products TIMOTHY H. STEVENSON, 1 NATE BAUER, 2 LISA M. LUCIA, 1 AND GARY R. ACUFF 1 * 1 Texas A&M University, Department of Animal Science, 310 Kleberg, College Station, Texas 77843; and 2 U.S. Department of Agriculture Food Safety and Inspection Service, Donald L. Houston Center for Meat and Poultry Inspection Sciences, 200 Discovery Drive, College Station, Texas 77845, USA MS : Received 7 July 1999/Accepted 28 September 1999 ABSTRACT This study focused on important factors related to the potential of cattle and beef products to transmit Helicobacter pylori to humans. Mucosal samples were collected from the rumen and abomasum of 105 cattle and were plated on a selective medium to isolate Helicobacter spp.; none of the samples examined contained these bacteria. Studies were also conducted to determine how long H. pylori survives in refrigerated or frozen ground beef; results indicated that the microorganism dies rapidly in ground beef, whether refrigerated or frozen. Packaging in vacuum or air had little effect on survival of the organism. The number of H. pylori decreased in refrigerated samples from 3.3 log 10 CFU/g on day 0 to 1.4 log 10 CFU/g on day 6. H. pylori died even more rapidly when frozen, decreasing from 3.3 log 10 CFU/g on day 0 to 0.5 log 10 CFU/g on day 6. Retail beef cuts (n 20) were also examined for the presence of H. pylori by direct plating on a selective medium and by incubation in an enriched broth followed by plating on a selective medium. None of the retail samples contained H. pylori. This research suggests that transmission of H. pylori from beef and beef products is not a primary factor in the high prevalence of this bacterium in humans. Helicobacter pylori was first isolated in 1982 (23) from the stomach of humans with gastric ulcers. Since then, it has been determined that H. pylori is the most prevalent pathogenic bacterial infection in humans, infecting approximately half of the world s population (12). Infection with H. pylori causes numerous medical conditions, including gastritis, peptic ulcers, gastric carcinomas, and mucosa-associated lymphoid-tissue tumors (3, 11, 13). The definitive reservoir for the bacterium is assumed to be humans (4), but the mode of transmission has yet to be defined. Proposed methods of infection include direct oral-oral transfer, indirect transfer via fecal-oral routes, zoonotic transmission from animals, and transmission via such vehicles as food and water (7, 9, 22). The bacterium has been shown to remain active but nonculturable in the coccoid form for more than 20 days in river water (16), suggesting that a waterborne route of infection may be possible when combined with unsanitary living conditions (10). Begue et al. (1) found a correlation between increased consumption of food from street vendors and H. pylori infection, suggesting that preparation of food in unhygienic conditions could serve as a mode of transmission. Not only have Helicobacter spp. been isolated from humans, but similar spiral bacteria have also been isolated from gastric specimens of naturally infected primates, ferrets, pigs, cats, dogs, cheetahs, mice, and other laboratory animals (5, 7, 8, 13, 26). Because many types of animals have been shown to carry Helicobacter spp. and because * Author for correspondence. Tel: ; Fax: ; gacuff@tamu.edu. numerous factors conducive to growth of the bacterium exist in the gastrointestinal tract of cattle, it seemed prudent to determine whether bovines could maintain and transmit Helicobacter spp. The objectives of this study were (i) to determine the likelihood that beef cattle are reservoirs of H. pylori by attempting to culture the organism from the rumen and abomasum of cattle, (ii) to establish the survival potential of the bacterium in ground beef, and (iii) to examine raw beef products from retail sources for the presence of H. pylori. MATERIALS AND METHODS Receipt and maintenance of cultures. Strains of H. pylori (ATCC 43504, 43629, and 43579) were obtained from the American Type Culture Collection (Rockville, Md.). These strains were maintained on H. pylori Special Peptone agar (HPSPA) plates and streaked onto fresh plates every 3 to 4 days (17). Plates were incubated at 37 C in 5.5-liter boxes (AnaeroPack, Mitsubishi Gas Chemical Co., New York, N.Y.) flushed with a microaerophilic gas mixture (6% O 2, 10% CO 2, and 84% N 2 ). The characteristics used to identify H. pylori (6) were typical colony morphology (clear, circular, entire, convex, and 0.5 to 1.5 mm in diameter), positive urease reaction on Christensen s urea agar (21), positive result on a catalase test, and typical curved-rod morphology after primary staining with crystal violet stain (Difco Laboratories, Detroit, Mich.). Standard and selective plating media. HPSPA was prepared by adding Special Peptone (10 g/liter, Oxoid Ltd., Basingstoke, UK), granulated agar (15 g/liter, Difco), sodium chloride (5 g/liter, EM Science, Gibbstown, N.J.), yeast extract (5 g/liter, Difco), beef extract (5 g/liter, Becton Dickinson and Co., Cockeysville, Md.), and pyruvic acid, sodium salt (0.5 g/liter, Sigma

2 J. Food Prot., Vol. 63, No. 2 HELICOBACTER PYLORI IN CATTLE AND BEEF PRODUCTS 175 Chemical Co., St. Louis, Mo.) (17). The selective agents added to the standard medium were vancomycin (10 mg/liter), amphotericin B (5 mg/liter), cefsulodin (10 mg/liter), polymyxin B sulfate (31,000 IU/liter), trimethoprim (40 mg/liter), and sulfamethoxazole (20 mg/liter) (18). Growth in selective enrichment broths. H. pylori Special Peptone broth (HPSPB), which contained the same ingredients as HPSPA except the agar, was supplemented with various concentrations and combinations of antibiotics to create a selective enrichment broth for isolating H. pylori. In each trial, six Erlenmeyer flasks containing 96 ml of HPSPB without antibiotics and six flasks containing HPSPB with various levels of antibiotics were inoculated with H. pylori (ATCC strains 43504, 43629, or 43579). For the first trial, the antibiotic combination tested was vancomycin (10 mg/liter), amphotericin B (10 mg/liter), cefsulodin (10 mg/liter), and trimethoprim (10 mg/liter). Samples were collected from the broths after 24 and 48 h of incubation, and 0.1 ml of appropriate decimal dilutions was surface-plated on HPSPA, providing a total of 12 replications for each HPSPB antibiotic formulation. After inoculation, all plates were incubated in a microaerophilic atmosphere for 4 days at 37 C, and the colonies were counted. Colonies from each replicate were confirmed as H. pylori as described above. The same methodology was used for three subsequent trials, in which additional antibiotic combinations were compared. Cattle sampling. A total of 105 cattle from abattoirs in different regions of Texas were sampled throughout the year. Initial samples were collected at the university abattoir from cattle reared and fed in south or central Texas. Subsequent samples were collected from cattle at an abattoir in central Texas (approximately 18 months old, steers and heifers) that had been fed a standard ration (steam-plate corn, whole-corn silage, supplement pellets, and liquid feed conditioner) at three feedlots in the Texas panhandle. After evisceration of the animals, 9-cm 2 sections of mucosa were obtained, using sanitized knives and forceps, from the rumen, the initial portion of the abomasum (omasoabomasal ostium), which corresponds to the cardia of the stomach of monogastric animals, and the pyloric antrum of the abomasum. Each sample was separately ground for 30 s with 5 ml of 0.1% peptone water (Difco) using a sterilized mortar and pestle, and 0.25 ml of the fluid was then surface-plated in duplicate on HPSPA containing the selective antibiotics listed above. Positive control plates were also included by collecting three colonies of H. pylori from an HPSPA plate with a sterile cotton swab and swirling the swab for 30 s in the fluid remaining in the mortar after the mucosa sample had been ground with the pestle. Inoculated sample fluid (0.25 ml) was then surface-plated on selective HPSPA. The plates were incubated as previously described and examined after 4 and 7 days of incubation for colonies with typical H. pylori morphology. If there were more than three colonies resembling H. pylori on each plate, one colony was selected, Gramstained, and examined for the presence of spiral rods. If there were fewer than three colonies resembling H. pylori on the plate, the colonies were transferred onto nonselective HPSPA plates. Transferred cultures were incubated for 4 days as previously described and then Gram-stained to determine the morphology of the organism. Colonies from positive control plates were confirmed as H. pylori as described above. After direct plating of the ground rumen and abomasum samples, 0.25 ml of the homogenate plus the remaining mucosa were transferred to a 600-ml bottle containing 192 ml of HPSPB, 8 ml of iron-supplemented calf serum (Biologos, Inc., Napersville, Ill.), and the antibiotics vancomycin (10 mg/liter), amphotericin B (5 mg/liter), cefsulodin (10 mg/liter), trimethoprim (20 mg/liter), and sulfamethoxazole (10 mg/liter). The broths were incubated in a microaerophilic atmosphere for 12 hours, the ph was adjusted to 4.0 with 1 N HCl, and urea (Sigma) was added to provide broth concentrations of 5 mm. After 24 and 48 h of incubation, 1 ml (divided over four plates) and 0.1 ml of the enrichment broths were surface-plated on the selective HPSPA plates. The plates were incubated for 4 days and examined as described above for the presence of typical H. pylori colonies. Survival in ground beef. Fresh beef trimmings (794 g) were coarsely ground through a sterile grinder with a plate that had 13- mm holes (Hobart, Troy, Ohio). The inoculum had been prepared previously by harvesting cells from HPSPA plates with 4-day-old cultures of H. pylori (ATCC strains 43504, 43629, and 43579). Cells from each plate were then used to inoculate 250-ml Erlenmeyer flasks (four flasks for each strain) containing 96 ml of HPSPB and 4 ml of iron-supplemented calf serum (Biologos), and the flasks were incubated in a microaerophilic atmosphere at 37 C for 48 h (17). The broths, containing 5.4 to 5.8 log CFU/ml, were then added to the coarsely ground meat and mixed for 2 min using a commercial mixer with a U-shaped pastry knife (Univex, Salem, N.H.). After mixing, the meat was ground through a plate with 6- mm holes and packaged in 110-g increments on plastic foam trays with polyvinyl chloride overwrap (oxygen transmission rate 4,650 cm 3 /m 2 /24hat22 C, Reynolds Metals Co., Richmond, Va.) or vacuum bags (oxygen transmission rate 54.3 cm 3 /m 2 /24 h at 22 C, Koch Supplies Inc., Kansas City, Mo.) and stored at 4 or 18 C. Uninoculated controls were processed as described above except that the inoculation step was omitted. Sampling of the ground beef was accomplished by placing 10 g of beef in a sterile stomacher bag. Ninety milliliters of 0.1% peptone water (Difco) was added, and the sample was pummeled for 1 min in a Stomacher-400 (Tekmar Co., Cincinnati, Ohio). Total plate counts were determined by spreading 1.0 ml (divided over four plates) or 0.1 ml of the beef-peptone homogenate on prepoured HPSPA with antibiotics (vancomycin [10 mg/liter], amphotericin B [5 mg/liter], cefsulodin [10 mg/liter], polymyxin B [62,000 IU/liter], trimethoprim [40 mg/liter], and sulfamethoxazole [20 mg/liter]). Additionally, 0.1 ml of appropriate decimal dilutions were plated as described above. Plates were incubated for 4 days, and colonies with typical H. pylori morphology were confirmed and enumerated. Triplicate samples of the ground beef were plated on day 0 to establish initial inoculum levels. Additional ground beef samples were stored at 4 or 18 C and sampled in triplicate on days 1, 3, 6, 10, 12, and 15. Prevalence in retail beef. A collection of beef cuts (two briskets, one package of round steaks, and one package of flank meat) was purchased from each of five retail supermarkets and transported to the laboratory in ice chests. Three 10-cm 2 surface samples were collected from each cut of meat, placed in a sterile stomacher bag with 99 ml of 0.1% peptone water (Difco), and pummeled for 1 min in a Stomacher-400. Samples were surfaceplated by spreading 1.0 ml of the homogenate on four plates of prepoured HPSPA containing antibiotics. The plates were incubated for 4 days and examined for the presence of typical H. pylori colonies. In addition to direct plating, three 10-cm 2 surface samples from each cut of meat were used to inoculate selective enrichment broths as described above. Positive controls were included by inoculating enrichment broths with three colonies of H. pylori obtained from HPSPA plates. After 12 h of microaerophilic incubation at 37 C, the ph of the broths was lowered to 4.0 with HCl, and urea (Sigma) was added to provide a 5-mM concentration in

3 176 STEVENSON ET AL. J. Food Prot., Vol. 63, No. 2 TABLE 1. Effects of adding various antibiotic combinations to broths a inoculated with H. pylori measured by plate counts (log 10 CFU/ ml) b at 24 and 48 h Log 10 CFU/ml d Antibiotic concentrations (mg/liter) c 24 h 48 h No antibiotics 5.8 AB 6.1 A Vancomycin (10), amphotericin B (10), cefsulodin (10), and trimethoprim (10) 4.8 B 6.1 A Vancomycin (10), amphotericin B (10), cefsulodin (10), polymyxin B (62,000 IU/liter), and trimethoprim (20) 4.8 B 6.0 A Amphotericin B (10), cefsulodin (10), polymyxin B (62,000 IU/liter), trimethoprim (40), and sulfamethoxazole (20) 6.0 A 6.0 A Amphotericin B (2.5), cefsulodin (10), trimethoprim (20), and sulfamethoxazole (10) 4.8 B 5.6 AB Vancomycin (10), amphotericin B (2.5), cefsulodin (5), polymyxin B (31,000 IU/liter), trimethoprim (20), and sulfamethoxazole (10) 5.2 AB 4.7 B Vancomycin (10), amphotericin B (5), cefsulodin (10), polymyxin B (31,000 IU/liter), trimethoprim (20), and sulfamethoxazole (10) 5.5 AB 6.0 A a H. pylori Special Peptone broth was the standard medium, to which various antibiotic combinations were added. Broths were incubated at 37 C in a microaerophilic atmosphere. b From each 250-ml Erlenmeyer flask of broth, a 0.1-ml sample was surface-plated on HPSPA using appropriate decimal dilutions. The plates were incubated at 37 C for 4 days in a microaerophilic atmosphere, and the mean plate counts were calculated. c All antibiotic concentrations are expressed as milligrams per liter, except for polymyxin B, which is expressed as international units per liter. d Means in the same column followed by the same letter are not significantly different (P 0.05). the broth. The ph of the enrichment broths was also checked and adjusted to 4.0 after 24 and 48 h of incubation. Samples were collected from the enrichment broths after incubating for 48 and 72 h, and 1 ml (divided over four plates) was surface-plated on prepoured HPSPA with antibiotics. After inoculation, plates were incubated for 4 days and examined for the presence of typical H. pylori colonies. Statistical analysis. Plate counts were converted to log 10 colony-forming units per milliliter, and mean separation (P 0.05) was determined using Duncan s multiple range test of the general linear model procedure (analysis of variance) of SAS Institute, Inc. (15). RESULTS AND DISCUSSION Growth in selective enrichment broths. Comparison of the counts after only 24 h of incubation showed that three of the six antibiotic combinations inhibited growth in the broth by 1 log 10 CFU/ml when compared with growth in broth without antibiotics; the remaining three antibiotic combinations did not significantly inhibit growth (Table 1). However, after the broths were allowed to incubate for 48 h, five of the six antibiotic combinations showed no significant growth inhibition. These data indicate that growth of H. pylori may be delayed in the presence of the antibiotic combinations used in these trials but that the bacterium grows wells after it adjusts to these antibiotics. The antibiotic concentrations used in these trials (vancomycin, 10 mg/liter; amphotericin B, 5 mg/liter; cefsulodin, 10 mg/liter; polymyxin B, 62,000 IU/liter; trimethoprim, 40 mg/ liter; and sulfamethoxazole, 20 mg/liter) were very high in comparison to other selective media used to cultivate H. pylori (19, 24). Given the strict selectivity provided by these antibiotic combinations, this slight delay in growth seems acceptable for most situations in which high selectivity is required. In preliminary studies, the enrichment broth described above, which contained vancomycin (5 mg/liter), amphotericin B (2.5 mg/liter), cefsulodin (5 mg/liter), trimethoprim (20 mg/liter), and sulfamethoxazole (10 mg/liter), was used to successfully isolate H. pylori from inoculated abomasum and ground beef samples (data not shown), providing additional evidence that the enrichment broth, followed by plating on selective agar, can be used to recover this fastidious organism from highly contaminated samples. Prevalence in cattle. No spiral-shaped rods were isolated on secondary cultures from any of the cattle. Numerous colonies with morphologic characteristics similar to those of H. pylori were transferred to nonselective media, but spiral-shaped rods typical of H. pylori were not isolated. The sampling plan was thorough, including cattle from a wide geographic area and sampling throughout the year. The number of cattle tested was sufficient to detect the organism with 99% certainty if the prevalence of H. pylori in cattle was greater than or equal to 5% (2). In humans, H. pylori is widespread and has an extremely high prevalence, affecting more than 90% of some populations (14). On the basis of epidemiologic evidence from other species, it is reasonable to expect that if Helicobacter spp. were present in cattle, the prevalence would likely be higher than 5%. Thus, there is no evidence at this time to support the hypothesis that cattle are significant reservoirs of H. pylori. Given that Helicobacter spp. have been isolated from many monogastric animals but were not isolated from ruminants in this study may indicate that the ecology of the ruminant gastrointestinal tract is not as favorable as a monogastric stomach for colonization of H. pylori. It is possible that the sections of the ruminant forestomach (reticulum, rumen, and omasum) that precede the abomasum may not provide a suitable environment for the passage of viable Helicobacter cells into the abomasum for subsequent infection with the bacterium.

4 J. Food Prot., Vol. 63, No. 2 HELICOBACTER PYLORI IN CATTLE AND BEEF PRODUCTS 177 TABLE 2. Mean plate counts a (log 10 CFU/g) of H. pylori in inoculated ground beef under various packaging and storage temperatures Packaging Polyvinyl chloride Vacuum Polyvinyl chloride Vacuum Storage temperature ( C) Plate counts (log 10 CFU/g) b Day 0 c Day 1 Day 3 Day 6 Day A 2.8 A 1.8 B 2.1 B 2.2 A 2.4 A 0.9 B 0.8 B 1.4 A a From each storage condition, triplicate samples were processed by mixing 90 ml of peptone water with 10 g of ground beef and pummeling for 1 min. A 0.1-ml sample of the homogenate was surface-plated on selective HPSPA using appropriate decimal dilutions. The plates were incubated at 37 C for 4 days in a microaerophilic atmosphere, and the mean plate counts from triplicate samples at each storage condition were calculated. b Means in the same column followed by the same letter are not significantly different (P 0.05). c Initial baseline levels after mixing, grinding, and packaging and before freezing or refrigerating. d No colonies were detected, so results indicate the midpoint between the detectable limit (1 log 10 CFU/g) and 0. Survival and prevalence in beef products. Preliminary experiments showed that the selective agar and enrichment broths developed for isolation of H. pylori from the bovine rumen and abomasum were also effective at isolating H. pylori from ground beef. After initial pilot studies (data not shown), a definitive experiment was completed to determine the survival of H. pylori in ground beef. The initial inoculum level of H. pylori in ground beef was 4.6 log 10 CFU/g. After mixing, grinding, and packaging, the counts decreased 1.3 log 10 CFU/g to 3.3 log 10 CFU/g. Counts decreased rapidly until day 9, when counts for all treatments were below the detection limit of 1 log 10 CFU/g. There was a statistically significant difference in the rate of survival between refrigerated and frozen samples, with greater survival in refrigerated samples (Table 2). In addition, by day 6, there was slightly greater survival in refrigerated samples packaged in polyvinyl chloride packaging than in refrigerated samples packaged in vacuum packaging. Previous experience in our laboratory indicates that even though H. pylori is microaerophilic, it tolerates the presence of atmospheric levels of oxygen better than it tolerates extremely low oxygen concentrations. This observation is supported by other research showing that some strains of H. pylori can be induced to grow aerobically (20, 25), which coincides with the data obtained in this experiment. 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