The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.Helicobacter pylori infection is chronic in nature, causes gastritis, and increases the risk of development of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer (for reviews, see references 13, 18, and 20). Increasing rates of resistance to the first-line antibiotic drugs (e.g., clarithromycin) are compromising the eradication of H. pylori and result in therapy failures (8). Thus, alternative treatments that include tetracyclines are recommended (1, 2, 14).Tetracyclines are bacteriostatic drugs which exert their antimicrobial effects by affecting the 30S subunit of the ribosome and block the binding of aminoacyl-tRNA, resulting in impaired protein biosynthesis (3,22). The resistance of H. pylori to tetracyclines is reported to be caused by mutations in the 16S rRNA. H. pylori isolates exhibiting AGA 926-928 3 TTC triple-base-pair mutations (4,5,22) show MICs higher than 4 mg/liter and probably represent the clinically most relevant strains, whereas single-or double-base-pair mutations were, rather, associated with MICs between 1 mg/liter and 4 mg/liter (3, 5).The susceptibility of H. pylori to tetracycline is routinely examined by agar diffusion (Etest) or agar dilution tests, which are accepted to be the "gold standards" (10). These methods are slow and time-consuming, and they sometimes fail due to a lack of growth of the infecting H. pylori strain or due to overgrowth with contaminating bacteria.It has already been shown that real-time PCR assays are elegant methods for prediction of resistance to clarithromycin (15, 16) or ciprofloxacin (7) in H. pylori. The major advantage of this technique over classical microbiological resistance testing is not in its enhanced speed and standardization but is mostly in the option to test for resistance in clinical specimens that no longer contain live bacteria.In this study, a real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline re...

The triple-base-pair 16S rDNA mutation AGA 926-928 3TTC mediates high-level tetracycline resistance in Helicobacter pylori. In contrast, single-and double-base-pair mutations mediated only low-level tetracycline resistance and decreased growth rates in the presence of tetracycline, explaining the preference for the TTC mutation in tetracycline-resistant H. pylori isolates.Tetracycline is a cheap and effective antibiotic for the treatment of Helicobacter pylori infections (7,8), but in the past few years the incidence of tetracycline resistance has significantly increased (1,5,6,9,13). The only known mechanism mediating tetracycline resistance in H. pylori involves mutations at positions 926 to 928 in both the 16S rRNA genes (2,4,12). H. pylori strains with high-level tetracycline resistance (Tet r ) carried the triple-base-pair mutation AGA 926-928 3TTC in both copies of the 16S rRNA genes (4, 12), whereas strains with low-level Tet r contained only single-and double-base-pair mutations in the exact same region (2). As the different mutations were present in unrelated strains, it is still unclear whether high-level tetracycline resistance requires the AGA 926-928 3 TTC mutation or whether single-or double-base-pair mutations at these positions may suffice for high-level tetracycline resistance. Therefore, we have created all possible combinations of single, double, and triple mutations at the 16S rRNA gene positions 926 to 928 in H. pylori strain 26695 and have determined the effects of the mutations on levels of tetracycline resistance, stability, and growth rate.Site-directed mutagenesis at positions 926 to 928 was carried out by using a three-step PCR approach (3, 10) with primers listed in Table 1, followed by natural transformation to H. pylori reference strain 26695 (4). Tet r H. pylori colonies were selected on plates containing tetracycline (1 g/ml). For each possible base pair mutation or combination thereof, eight Tet r transformants from at least two independent transformation experiments were selected. Both alleles of the 16S rRNA genes were amplified by PCR (4) to confirm the presence of the desired base pair mutations. With the exception of the AGC mutants, all mutants contained the desired mutations in both alleles of the 16S rRNA genes. All AGC mutants were heterozygous and contained the AGC mutation in the rrnA gene and a TTC mutation in the rrnB gene.The effects of the 16S rRNA mutations on both the stability and the level of tetracycline resistance were determined by subculturing two mutants of each type for 20 passages on Columbia agar plates supplemented with 7% lysed horse blood (BioTrading, Mijdrecht, The Netherlands) in either the presence or absence of tetracycline (1 g/ml). After each five rounds of subculturing, the MIC of tetracycline was determined by using E-test (AB Biodisk, Solna, Sweden) (4). In addition, for all mutants at time point zero (t 0 ) and after passage 20 (t 20 ), the 16S rRNA genes were sequenced. The stability of the various types of mutations and their effects on t...

New generations of fluoroquinolones, like levofloxacin and moxifloxacin, exhibit a broad-spectrum activity against Gram-positive and Gram-negative bacteria, and have been successfully introduced into the treatment of Helicobacter pylori infection. Based on a large body of evidence, current guidelines recommend the use of levofloxacin-or moxifloxacincontaining proton-pump inhibitor (PPI) triple therapies in second-line or rescue treatment of H. pylori infection. The efficacy of standard PPI triple therapies has substantially declined during the last decade, mainly due to increasing resistance against the key antibiotics clarithromycin and metronidazole. Therefore, alternative strategies for first-line therapy of H. pylori infection have been evaluated in a considerable number of clinical trials including sequential regimens, nonbismuth quadruple regimens, and quinolone-containing PPI triple therapy regimens. The aim of this paper is to summarize the current body of evidence of levofloxacin-and moxifloxacin-containing regimens in first-line treatment of H. pylori infection, and to discuss the risks and benefits of these strategies in the light of increasing resistance of H. pylori to quinolones.

Tetracycline is one of four antibiotics commonly used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing as the incidence of tetracycline resistance is increasing. In five Brazilian tetracycline-resistant (Tet(R)) H. pylori isolates, high-level tetracycline resistance is mediated by the triple-base-pair substitution AGA(926-928)-->TTC in both 16S rRNA genes, as was previously observed in two independent high-level Tet(R) H. pylori strains. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection of the AGA(926-928)-->TTC substitution, and confirmed the presence of the aforementioned triple-base-pair substitution in all five Brazilian Tet(R) isolates. This PCR-RFLP-based approach distinguishes the high-level Tet(R) isolates from low-level Tet(R) and Tet(S) H. pylori strains and thus allows the direct detection of Tet(R) H. pylori isolates.

No abstract

No abstract

Second-line/rescue H. pylori eradication therapy with esomeprazole, moxifloxacin, and amoxicillin is very effective and well tolerated. Fourteen days of treatment significantly increase the eradication rate but also the rate of adverse events.

Background:A postoperative pancreatic fistula (POPF) is the most common and potentially life-threatening surgical complication in pancreatic surgery. One possible pharmacological treatment could be the endoscopic injection of botulinum toxin (BTX) into the sphincter of Oddi to prevent POPF. Promising data reported a significantly reduced rate of clinically relevant POPF. We analyzed the effect of BTX injection in our patients undergoing distal pancreatectomy (DP).Methods:A retrospective analysis of patients undergoing DP was performed. Patients with preoperative endoscopic injection of BTX into the sphincter of Oddi were included. The end points were postoperative outcomes including POPF. BTX patients were compared with a historical cohort and matched in a 1:1 ratio using a propensity score analysis.Results:A total of 19 patients were treated with endoscopic injection of BTX before open (n=8) or laparoscopic (n=11) DP. The median age of the patients was 67 years and the mean body mass index was 25.9 kg/m2. In median, the intervention was performed 1 day (range, 0–14 days) before the operation. There were no intervention-related complications. The incidence of POPF was not statistically different between the two groups: a clinically relevant POPF grade (B/C) occurred in 32% (BTX) and 42% (control; p=0.737). Likewise, there were no significant differences in postoperative drain fluid amylase levels, morbidity, and mortality.Conclusion:The present study could not reproduce the published results of a significant lowering of grade B/C POPF. The explanations could be the timing of BTX injection before surgery and the endoscopic technique of BTX injection. However, the conflicting results after BTX injection in two high-volume centers prompt a randomized controlled multicenter trial with trained endoscopists.