Helicobacter ISSN 1523-5378 © 2009 The Authors 72 Journal compilation © 2009 Blackwell Publishing Ltd, Helicobacter 14: 72–79 Blackwell Publishing Ltd Oxford, UK HEL Helicobacter 1083-4389 1523-5378 © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Helicobacter XX: xx–xx XXX Original Article Helicobacter hepaticus Amoxicillin Resistance Belzer et al. The Helicobacter hepaticus hefA Gene is Involved in Resistance to Amoxicillin Clara Belzer, *§ Jeroen Stoof, * Simone Breijer, * Johannes G. Kusters, *¶ Ernst J. Kuipers *† and Arnoud H. M. van Vliet *‡ * Department of Gastroenterology and Hepatology, Erasmus MC – University Medical Center, ‘s Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands, † Department of Internal Medicine, Erasmus MC – University Medical Center, ‘s Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands, ‡ Institute of Food Research, Colney Lane, Norwich NR4 7UA, UK Abstract Background: Gastrointestinal infections with pathogenic Helicobacter species are commonly treated with combination therapies, which often include amoxicillin. Although this treatment is effective for eradication of Helicobacter pylori, the few existing reports are less clear about antibiotic susceptibility of other Helicobacter species. In this study we have determined the susceptibility of gastric and enterohepatic Helicobacter species to amoxicillin, and have investigated the mechanism of amoxicillin resistance in Helicobacter hepaticus. Materials and methods: The minimal inhibitory concentration (MIC) of antimicrobial compounds was determined by E-test and agar/broth dilution assays. The hefA gene of H. hepaticus was inactivated by insertion of a chloramphenicol resistance gene. Transcription was measured by quantitative real-time polymerase chain reaction. Results: Three gastric Helicobacter species (H. pylori, H. mustelae, and H. acinonychis) were susceptible to amoxicillin (MIC < 0.25 mg/L). In contrast, three enterohepatic Helicobacter species (H. rappini, H. bilis, and H. hepaticus) were resistant to amoxicillin (MIC of 8, 16, and 6–64 mg/L, respectively). There was no detectable β-lactamase activity in H. hepaticus, and inhibition of β- lactamases did not change the MIC of amoxicillin of H. hepaticus. A H. hepaticus hefA (hh0224) mutant, encoding a TolC-component of a putative efflux system, resulted in loss of amoxicillin resistance (MIC 0.25 mg/L), and also resulted in increased sensitivity to bile acids. Finally, transcription of the hefA gene was not responsive to amoxicillin, but induced by bile acids. Conclusions: Rodents are frequently colonized by a variety of enterohepatic Helicobacter species, and this may affect their global health status and intestinal inflammatory responses. Animal facilities should have treatment strategies for Helicobacter infections, and hence resistance of enterohepatic Helicobacter species to amoxicillin should be considered when designing eradication programs. Keywords amoxicillin, enteric Helicobacter species, treatment, efflux, antibiotic resistance, bile acids. Reprints request to: Arnoud H. M. van Vliet, Institute of Food Research, Colney Lane, Norwich NR4 7UA, UK. E-mail: arnoud.vanvliet@bbsrc.ac.uk § Present address: Department of Pathology, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA, USA. ¶ Present address: Tergooiziekenhuizen, Central Laboratory for Bacteriology and Serology, Hilversum, the Netherlands Members of the genus Helicobacter chronically colonize the different organs of the digestive tract in mammals. Helicobacter species are subdivided in the gastric Helicobacter species, which colonize the gastric mucosa (e.g. H. pylori in humans and primates), and the enterohepatic Helicobacter species, which colonize the intestines and/or hepatobiliary tract [1,2]. Colonization with enterohepatic Helicobacter species usually results in chronic intestinal and hepatic inflammation, and is associated with diseases like typhlocolitis, inflammatory bowel disease, hepatitis, and development of hepatic carcinoma [2–5] or can contribute to the formation of cholesterol gallstones [6–8]. Helicobacter hepaticus is the best studied enterohepatic Helicobacter species, and colonizes the intestine and bile ducts of rodents [3–5]. Colonization of laboratory mice with H. hepaticus or other enterohepatic Helicobacter species is a serious problem for many animal facilities worldwide [9]. Not much is known about the antimicrobial resistance of H. hepaticus or other enterohepatic Helicobacter species, although therapies for treatment of H. hepaticus in laboratory mice include a combination of antibiotics [10–13].