British Journal of Surgery 1998, 85, 850–852 Oral colonization is unlikely to play an important role in Helicobacter pylori infection A. OSHOWO, M. TUNIO, D. GILLAM*, A. J. BOTHA, J. HOLTON†, P. BOULOS and M. HOBSLEY Departments of Surgery and †Microbiology, and *The Eastman Dental Institute, University College London Medical School, London, UK Correspondence to: Professor M. Hobsley, Department of Surgery, University College London Medical School, Charles Bell House, 67–73 Riding House Street, London W1P 7LD, UK Background This clinical and microbiological study investigated whether Helicobacter pylori colonizes the mouth en route to the pyloric antrum. It has been suggested that oral colonies are a source of reinfection after eradication of gastric infection. Methods Some 208 patients attending for routine diagnostic endoscopy for dyspepsia were recruited. Before endoscopy, samples were collected of saliva, supragingival and infragingival plaque, and swabs were taken from the tongue, mouth and pharynx. At endoscopy, gastric antral biopsies were taken for the rapid urease test, culture and histological examination. Gastric and duodenal juice samples were aspirated. Restriction nuclease digestion with HaeIII was employed on all specimens from patients in whom there was evidence of the organism in the mouth. Results H. pylori was observed in dental plaque in only 15 patients, all from the 116 who had evidence of the organism in the stomach. Restriction endonuclease digestion demonstrated that in 13 of the 15 patients the strains were identical in mouth and stomach. Conclusion Oral colonization is a rare event, but does occur. Its rarity suggests that it is not an important factor in reinfection. Helicobacter pylori is a Gram-negative, microaerophilic, curved, rod-shaped bacterium that colonizes human stomach. This colonization causes active chronic and acute gastritis 1,2 , is strongly associated with chronic duo- denal ulcer, and may be a risk factor for gastric cancer. Despite being one of the commonest infections in the world, the exact route of transmission is still unclear 3 . Some researchers have implicated the oral cavity 4–8 as a reservoir for systemic infection and perhaps a sanctuary where eradication therapy is ineffectual, thus leading to reinfection. Others have reported an absence of this organism from the mouth 9,10 . The aim of this study was to determine whether H. pylori colonizes the oral cavity and whether, if it does, the organism in the mouth is the same strain as that in the stomach. Patients and methods A total of 208 unselected dyspeptic patients referred for upper gastrointestinal endoscopy were recruited. Informed consent was obtained. The study was reviewed by the local ethics committee. Exclusion criteria included patients with a potential infection risk, a history of bleed- ing tendency, antibiotic therapy in the preceding 8 months and the requirement for antibiotic prophylaxis. Immedi- ately before endoscopy, the following specimens were collected: saliva, both supragingival and subgingival plaque using a sterile periodontal curette with a gentle upward scrape against the surface of the tooth, and a swab of the tongue, mouth and pharynx. During endos- copy, specimens were collected by aspiration via a trap collector of gastric juice and (separately, in 50 subjects only) duodenal juice down to the second part of the duo- denum. Three antral mucosal biopsy specimens were obtained with sterile forceps: one was used immediately for the rapid urease test, one was cultured, and the third was stored at - 70°C for subsequent analysis by poly- merase chain reaction (PCR) and possible restriction endonuclease digestion. Statistical analysis was by standard methods including the binomial sign test. Specimen preparation The dental plaque, saliva and oropharyngeal swab were vortexed in sterile solution, cultured and used for PCR. Gastric and duo- denal juice specimens were initially centrifuged at high speed and the sediments tested by PCR. One gastric antral biopsy was homogenized before culture and PCR. All specimens were cultured using Colombia agar with 5 per cent horse blood and amphotericin B 10 μg/ml. H. pylori was identified by growth condition, typical morphology in Gram-stained preparations, a positive oxidase and a positive urease reaction. Extraction of genomic DNA from all clinical isolates was carried out with Puregene DNA isolation kits (Gentra Systems, Minneapolis, Minnesota, USA) and applied according to the manufacturer’s instructions. Two 20-base oligonucleotide primers (Oswell DNA Service, Southampton, UK) designated HP1 (5'-CTG GAG AGA CTA AGC CCT CC-3') and HP2 (5'-ATT ACT GAC GCT GAT TGT GC-3') were used. The primers target areas of the 16S ribosomal RNA gene in which there is the least sequence homology, and amplify a 109-base pair product. PCR was performed in a 100-ml mixture containing 10 ml PCR buffer (Tris–hydrochloric acid 10 mmol/l, pH 8·3, potas- sium chloride 50 mmol/l), 16 μl 2'-deoxynucleotide 5'-triphos- phate 200 mmol/l, 1 μl AmpErase uracil N-glycosylase (Perlan Elmer, Branchburg, New Jersey, USA), 1 μl AmpliTaq DNA polymerase (Amersham International, Amersham, UK), 8 μl magnesium chloride 2·0 mmol/l, 10 μl HP1 and HP2 2 mmol/l, Presented to the Joint International Meeting of Surgical Research Societies, Nottingham, UK, July 1997 Paper accepted 18 November 1997 850 © 1998 Blackwell Science Ltd