Although Helicobacter pylori infection is both a common and a serious bacterial infection, antimicrobial therapies have rarely been optimized, are prescribed empirically, and provide inferior results compared with antimicrobial therapies for other common infectious diseases. The effectiveness of many of the frequently recommended H. pylori infection treatment regimens has been increasingly compromised by antimicrobial resistance. Regional data on the susceptibility of strains of H. pylori to available antimicrobials are sorely needed. Noninvasive molecular methods are possible to assess clarithromycin susceptibility in isolates obtained from stool specimens. As a general rule, clinicians should prescribe therapeutic regimens that have a ≥90% or, preferably, ≥95% eradication rate locally. If no available regimen can achieve a ≥90% eradication rate, clinicians should use the most effective regimen(s) available locally. Eradication of infection should always be confirmed after treatment in order to provide feedback regarding local effectiveness and an early warning of increasing resistance. In most regions of the world, four-drug treatment regimens, including a PPI plus three antimicrobials (clarithromycin, metronidazole/tinidazole and amoxicillin), or a PPI plus a bismuth plus tetracycline and metronidazole provide the best results. Standard triple therapy (a PPI, amoxicillin and clarithromycin) should now be avoided owing to increasing resistance to this treatment.

The major cause of chemotherapy failure in patients with chronic gastritis and peptic ulcers caused by Helicobacter pylori is clarithromycin (CAM) resistance due to a mutation in the 23S rRNA gene. This study describes a non-invasive and accurate method for the detection of mixed CAM-resistant and -susceptible H. pylori by sequencing of the H. pylori 23S rRNA gene. Faeces were crushed with beads and the 23S rRNA gene was amplified using a nested PCR on the extracted DNA. Mutation analysis of this gene using this method showed that 20.4 % of patients carried mixed CAM-susceptible (wild type) and -resistant (A2142G or A2143G mutant) H. pylori. Furthermore, it was found that 66.6 % of patients who had been treated unsuccessfully carried one of these mutations in the 23S rRNA gene (including the mixed type), whilst standard culture detected CAM-resistant isolates in only 22.2 % of patients with unsuccessful treatment. These data suggest that, for successful therapy, the diagnosis method described here would more accurately detect CAM-resistant H. pylori, including mixed infections. INTRODUCTIONHelicobacter pylori is a Gram-negative, spiral bacterium found in the human stomach. This micro-organism causes chronic gastritis and peptic ulcers (Kuipers, 1997) and is linked to gastric cancer and other non-gastrointestinal diseases (Kusters et al., 2006;Leong & Sung, 2002). To treat H. pylori infections, a triple therapy is used comprising a combination of a proton pump inhibitor and two antimicrobial agents. This is reported to be the most successful method for eradication of infection (Malfertheiner et al., 2002). In Japan, a combination of lansoprazole, amoxicillin and clarithromycin (CAM) (the LAC regimen) is commonly used to eliminate H. pylori (Asaka et al., 2001). Although most patients are treated successfully with this triple therapy, some failure has necessitated the use of additional drugs such as levofloxacin (Asaka et al., 2001;Kato et al., 2000). The major cause of therapy failure in patients is resistance to CAM (Kato et al., 2000; Rimbara et al., 2005a). Therefore, a reliable diagnostic test to detect macrolide resistance would be useful in planning a therapeutic regimen. Standard culture is generally used to determine the susceptibility of H. pylori to antimicrobial agents; however, acquiring a test sample is invasive for the patient, as endoscopy is required to obtain gastric specimens. Faecal culture is not possible, as H. pylori coccoid forms are unculturable; however, there are some reliable non-invasive tests for the diagnosis of H. pylori infections (Graham et al., 1987;Leodolter et al., 2003;Shuber et al., 2002;Vaira et al., 1999).Resistance to CAM in H. pylori is due to a mutation in the 23S rRNA subunit in the 50S ribosome. The two most common mutations are an adenine-to-guanine transition at position 2142 or 2143, the latter leading to an adenineto-cytosine transversion at position 2142 (Alarcon et al., 2003;van Doorn et al., 2001). Resistance due to mutations at other positions has also been rep...

Objectives: To elucidate the relationship between the mutations of penicillin-binding protein (PBP)1, PBP2 and PBP3 and amoxicillin resistance in Helicobacter pylori. Methods:The mutations detected only in clinical amoxicillin-resistant strains were determined by comparison of the deduced amino acid sequences of PBP1(HP0597), PBP2(HP1556) and PBP3(HP1565) encoded by the pbp1, ftsI and pbp2 genes, respectively, in 13 clinical H. pylori strains and three ATCC strains. The contribution of the mutations in PBPs was analysed by the natural transformation of the amoxicillin-susceptible strain ATCC 700392 with various combinations of the pbp1, ftsI and pbp2 genes from the amoxicillin-resistant strain TH743 (MIC of amoxicillin: 8 mg/L). Results:We initially identified six, four and two mutations of PBP1, PBP2 and PBP3, respectively, which were detected only in amoxicillin-resistant strains. By the natural transformation of an amoxicillinsusceptible strain ATCC 700392, we found that mutations in PBP1 and PBP3 conferred higher resistance to amoxicillin than mutations in PBP1 and PBP2, or mutations only in PBP1. Furthermore, mutations in PBP1, PBP2 and PBP3 conferred a 256-fold higher amoxicillin resistance when compared with ATCC 700392.Conclusions: Multiple mutations in PBP2 and PBP3, in addition to mutations in PBP1, confer higher amoxicillin resistance in H. pylori.

Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.