Mutations of the Helicobacter pylori genes rdxA and pbp1 cause resistance against metronidazole and amoxicillin

To investigate amoxicillin and metronidazole resistance of Helicobacter pylori, we compared putative resistance genes between resistant strains obtained in vitro and their sensitive parent strain. All metronidazole-resistant strains had rdxA mutations, and an amoxicillin-resistant strain had pbp1 and pbp2 mutations. By transforming PCR products of these mutated genes into antibiotic-sensitive strains, we showed that rdxA null mutations were sufficient for metronidazole resistance, while pbp1 mutations contributed to amoxicillin resistance of H. pylori.     

Alterations in penicillin-binding protein 1A confer resistance to beta-lactam antibiotics in Helicobacter pylori

Most Helicobacter pylori strains are susceptible to amoxicillin, an important component of combination therapies for H. pylori eradication. The isolation and initial characterization of the first reported stable amoxicillin-resistant clinical H. pylori isolate (the Hardenberg strain) have been published previously, but the underlying resistance mechanism was not described. Here we present evidence that the beta-lactam resistance of the Hardenberg strain results from a single amino acid substitution in HP0597, a penicillin-binding protein 1A (PBP1A) homolog of Escherichia coli. Replacement of the wild-type HP0597 (pbp1A) gene of the amoxicillin-sensitive (Amx(s)) H. pylori strain 1061 by the Hardenberg pbp1A gene resulted in a 100-fold increase in the MIC of amoxicillin. Sequence analysis of pbp1A of the Hardenberg strain, the Amx(s) H. pylori strain 1061, and four amoxicillin-resistant (Amx(r)) 1061 transformants revealed a few amino acid substitutions, of which only a single Ser(414)-->Arg substitution was involved in amoxicillin resistance. Although we cannot exclude that mutations in other genes are required for high-level amoxicillin resistance of the Hardenberg strain, this amino acid substitution in PBP1A resulted in an increased MIC of amoxicillin that was almost identical to that for the original Hardenberg strain.     

Primary resistance in <Emphasis Type="Italic">Helicobacter pylori</Emphasis> isolated in children from Iran

Helicobacter pylori-associated infection is extre-mely common in Iran, as in other developing countries, but few data exist on the susceptibility of H. pylori to antimicrobials commonly used in the eradication schedules in this country. This study was performed to determine the resistance rate to six antimicrobial agents used in the treatment of H. pylori infection in dyspeptic Iranian children and to recommend an updated anti-H. pylori treatment regimen to use in children. All H. pylori isolated from children who were undergoing gastroscopy were prospectively collected and subcultured to yield their susceptibility to six antimicrobial agents, by E test and disk diffusion methods. Demographic data and presenting symptoms were also collected. A prospective study was carried out from January 2003 to January 2005 with 100 strains of H. pylori isolated from children (40 girls and 60 boys; age range, 1.5 to 16 years [mean, 9.22 ± 3.25 years]); the strains had been successfully subcultured to yield antimicrobial sensitivity. Overall the H. pylori resistance rate was 95% to metronidazole, 59% to amoxicillin, 16% to clarithromycin, 9% to furazolidone, 7% to ciprofloxacin, and 5% to tetracycline. The most common presenting symptom was abdominal pain. There were no statistically significant differences in antimicrobial resistance rates related to age, sex, or clinical presentation. In the Iranian children, the prevalence of H. pylori resistance was very high to metronidazole and amoxicillin, moderate to clarithromycin, and low to ciprofloxacin and tetracycline.     

β-Lactamase inhibitors have antibacterial activities against Helicobacter pylori

Recently, it was reported that amoxicillin-clavulanate has slightly higher activity than amoxicillin against Helicobacter pylori. In this study, we evaluated the in-vitro antibacterial activity of β-lactamase inhibitors against H. pylori. We investigated the susceptibility of 30 H. pylori strains to β-lactamase inhibitors, including clavulanate, sulbactam, and tazobactam. In short-term bactericidal studies, a clinical isolate of H. pylori NU27 was exposed to 1 × minimum inhibitory concentration (MIC) of the β-lactamase inhibitors, amoxicillin, clarithromycin, and amoxicillin-clavulanate for 3 and 6 h. The MICs₉₀ for these β-lactamase inhibitors were 2, 4, and 2 mg/l, respectively. The short-term bactericidal studies showed that these β-lactamase inhibitors decreased viable counts of H. pylori during 6-h exposure at 1 × MIC. Our results suggest that β-lactamase inhibitors have in-vitro antibacterial activity against H. pylori. Amoxicillin and clavulanate used in combination resulted in increased antibacterial activity. Received: July 1, 1999 / Accepted: September 13, 1999     

New definitions of extended‐spectrum β‐lactamase conferring worldwide emerging antibiotic resistance

Although there is no consensus of the precise definition of ESBL, three kinds of ESBL definitions have been proposed. First, the classical definition includes variants derived from TEM‐1, TEM‐2, or SHV‐1; K1 (KOXY) of Klebsiella oxytoca. Second, the broadened definition has stretched the classical definition of ESBL to include: (1) β‐lactamases (CTX‐M‐ESBLs, GES‐ESBLs, and VEB‐ESBLs), with spectra similar to those of TEM and SHV variants (designated as TEM‐ and SHV‐ESBLs, respectively) but derived from other sources; (2) TEM and SHV variants with borderline ESBL activity; e.g., TEM‐12; and (3) various β‐lactamases conferring wider resistance than their parent types but not meeting the definition for group 2be; e.g., OXA‐types (OXA‐ESBLs) and mutant AmpC‐types (AmpC‐ESBLs), with increased activity against oxyimino‐cephalosporins and with resistance to clavulanic acid. Third, the all‐inclusive definition includes: (1) ESBL_A (named for class A ESBLs); (2) ESBL_M (miscellaneous ESBLs), which has been subdivided into ESBL_M‐C (class C; plasmid‐mediated AmpC) and ESBL_M‐D (class D); and (3) ESBL_CARBA (ESBLs with hydrolytic activity against carbapenems), which has been subdivided into ESBL_CARBA‐A (class A carbapenemases), ESBL_CARBA‐B (class B carbapenemases), and ESBL_CARBA‐D (class D carbapenemases). The consensus view about the ESBL definition is that the classical ESBL definition must be expanded to class A non‐TEM‐ and non‐SHV‐ESBLs (CTX‐M‐, GES‐, VEB‐ESBLs, etc.). However, these three definitions evoke rational debate on the question “Which would be included in the category of ESBLs among AmpC‐ESBLs, OXA‐ESBLs, and/or carbapenemases?” Therefore, there is a great need for consensus in the precise definition of ESBL. © 2010 Wiley Periodicals, Inc. Med Res Rev 32:216‐232, 2012     

Pharmacogenetics of esomeprazole or rabeprazole‐based triple therapy in Helicobacter pylori eradication in Hong Kong non‐ulcer dyspepsia Chinese subjects

Objective: Our study aimed to assess the effectiveness of esomeprazole or rabeprazole in combination with amoxicillin and clarithromycin for the eradication of Helicobacter pylori in Hong Kong non‐ulcer dyspepsia (NUD) patients. Methods: A prospective clinical trial was conducted at the Alice Ho Miu ling Nethersole Hospital outpatient endoscopy center from June 2004 to December 2005. Participants received amoxicillin 1 g, clarithromycin 500 mg, and, esomeprazole 20 mg (EAC) or rabeprazole 20 mg (RAC), all given twice daily for 1 week. The H. pylori status was determined by the [¹³C] urea breath test at least 4 weeks after completion of the treatment. Mutation status of CYP2C19 in exon 4 and exon 5 associated with the poor metabolizer phenotype was determined. Results: The intention‐to‐treat eradication rates in patients treated with RAC and EAC were 77% and 84·6% respectively, and per protocol‐based eradication rates were 83·7% and 88·9% respectively. The eradication rates did not vary with CYP2C19 phenotype found. For clarithromycin‐sensitive strains, the cure rates were statistically significant regardless of CYP2C19 polymorphism (P < 0·0001). Conclusion: Triple therapy with either EAC or RAC is effective for Hong Kong Chinese NUD patients with H. pylori infection. Success eradication was related to clarithromycin resistance and not CYP2C19 genotype.     

Class 1 integron containing metallo β-lactamase gene bla IMP-1 in carbapenem-resistant Pseudomonas aeruginosa in Thailand

We investigated the genetic properties of two carbapenem-resistant Pseudomonas aeruginosa isolates collected from a regional hospital in the north of Thailand. Both isolates demonstrated high-level resistance to extended-spectrum cephalosporins and carbapenems. Detection of the MBL genes was positive for bla _IMP in both isolates. Pulsed field gel electrophoresis (PFGE) analysis showed that the two P. aeruginosa isolates were nonclonal. Molecular analysis of the bla _IMP in isolate 837 showed the presence of a bla _IMP-1 gene inserted in a class 1 integron. The bla _IMP-1 was plasmid-mediated according to the transformation assay. This is the first case of the bla _IMP-1 gene in carbapenem-resistant P. aeruginosa in Thailand, suggesting the further dissemination of this gene in Southeast Asia.     

Helicobacter pylori infection and primary Sjögren's syndrome: a possible association and new therapeutic approach

The possible interrelationship between Helicobacter pylori infection and primary Sjögren's syndrome (pSS) and a new therapeutic approach to pSS were investigated. Biopsy samples from four patients with pSS were cultured for the presence of H pylori. Three patients who tested positive for H pylori were given concurrently omeprazole 40 mg once daily for 1 month, and amoxicillin 1000 mg and tinidazole 500 mg twice daily for 2 weeks. H pylori eradication was verified 6 to 8 weeks after therapy, and clinical symptoms of pSS, xerophthalmia and xerostomia, improved dramatically, suggesting that H pylori infection is associated with pSS. Further studies are needed to confirm the association and to investigate the efficacy of treatment of pSS with amoxicillin and tinidazole.     

Antibiotic resistance of Helicobacter pylori to 16 antibiotics in clinical patients

Background

Resistance of Helicobacter pylori (H. pylori) to antibiotics is increasing worldwide. To determine the status of H. pylori resistance and its patterns in clinical patients, an investigation utilizing susceptibility testing for commonly used antibiotics was needed.

Methods

Total of 2283 H. pylori strains were collected from 2013 to 2016. The resistance and its patterns of these strains were tested by agar dilution method. The resistance rate and minimal inhibition concentration (MIC) in different gender groups were also analyzed.

Results

The overall resistance rates were as following: amoxicillin (1.58%), clarithromycin (22.73%), levofloxacin (24.75%), furazolidone (1.49%), doxycycline (9.20%), cefetamet (97.20%), ceftriaxone (49.60%), cefuroxime (25.20%), gentamicin (3.73%), azithromycin (85.60%), rifampicin (2.80%), metronidazole (92.53%), ornidazole (94.27%), tinidazole (87.20%), ciprofloxacin (43.20%), and moxifloxacin (38.53%). There were only 64.08% strains pan‐susceptible to amoxicillin, clarithromycin, levofloxacin, and furazolidone, followed by mono resistance (23.17%), double resistance (11.13%), triple resistance (1.36%), and quadruple resistance (0.26%). Significant differences in the resistance rate and MIC were also observed in different gender groups.

Conclusion

Antibiotic resistance trends of H. pylori is increasing in clinical patients. With the increasing resistance, it is imperative to individualized therapy based on the results of drug susceptibility testing.

     

Discriminatory Detection of Inhibitor-Resistant β-Lactamases in Escherichia coli by Single-Strand Conformation Polymorphism-PCR

Plasmid-mediated mechanisms, comprising TEM hyperproduction, TEM derivative production, and OXA production, lead to amoxicillin-clavulanic acid resistance in enterobacteria. The ability of the single-strand conformation polymorphism (SSCP)-PCR method to differentiate the genes encoding inhibitor-resistant β-lactamases was evaluated with three bla_TEM primer pairs. The bla_TEM genes, which were known to be different on the basis of their nucleotide sequences (bla_TEM-1A, bla_TEM-1B, bla_TEM-2, bla_TEM-30, bla_TEM-32, and bla_TEM-35), were identified as different by their electrophoretic mobilities. The bla_TEM-33, bla_TEM-34, bla_TEM-36, bla_TEM-37, bla_TEM-38, and bla_TEM-39 genes, whose sequence differences have been established by oligotyping, displayed different SSCP profiles for different fragments, suggesting genetic differences in addition to those defined by oligotyping. Confirmed by sequencing, these additional genetic events concerned silent mutations at certain positions and, notably, a G→T transversion at position 1 of the −10 consensus sequence in bla_TEM-34, bla_TEM-36, bla_TEM-37, and bla_TEM-39. Applied to eight clinical isolates of Escherichia coli resistant to amoxicillin-clavulanic acid, the SSCP method detected TEM-1 in three strains and TEM-30, TEM-32, and TEM-35 in three other strains, respectively. A novel TEM derivative (TEM-58) was detected in another strain, and the deduced amino acid sequence showed two substitutions: Arg244Ser, which is known to confer amoxicillin-clavulanic acid resistance in TEM-30, and Val261Ile, which has not been described previously. The eighth strain produced an OXA β-lactamase. Given the discriminatory power and the applicability of SSCP-PCR, this method can be proposed as a means of following the evolution of the frequencies of the different inhibitor-resistant β-lactamases.     

β-Lactamases in amoxicillin-clavulanate–resistant Escherichia coli strains isolated from a Chinese tertiary hospital

A total of 52 strains were resistant to amoxicillin-clavulanate by disk diffusion method in a Chinese tertiary hospital from July 2011 to December 2011. Among these isolates, 2 isolates possessed a phenotype consistent with production of inhibitor-resistant temoniera (TEM) (IRT) β-lactamase, and the TEM-type gene was cloned into strains of Escherichia coli JM109 cells. Both had no bla_TEM mutations and were identified as TEM-1 β-lactamase producers. As a result, no IRT β-lactamase was detected. Multiplex PCR detected most of these strains produced TEM-1 enzymes, and plasmid-mediated AmpC β-lactamase and oxacillinase-1 β-lactamases are important mechanisms of resistance as well.     

Clinical Application of the DiversiLab Microbial Typing System Using Repetitive Sequence‐Based PCR for Characterization of Helicobacter pylori in Japan

We evaluated the DiversiLab (DL) system with universal primers, a semiautomated repetitive extragenic palindromic sequence‐based polymerase chain reaction (PCR) (rep‐PCR) system, for the characterization of Helicobacter pylori in Japan. All 135 isolates from Japanese patients with gastric cancer (GC, n = 55) or non‐GC (n = 80) were used and subjected to the drug susceptibility examinations (amoxicillin, AMPC; metronidazole, MNZ; and clarithromycin, CAM) by E‐test. There were 28 MNZ‐resistant (20.7%), 35 CAM‐resistant (25.9%), and 16 MNZ/CAM‐resistant (11.9%) isolates. DL rep‐PCR fingerprinting analysis at the level of 95% similarity revealed five major groups (A–E) and the other including 45 isolates. The occupation rates of GC‐derived isolates in groups B (54.2%) and E (58.8%) were higher than in the other groups: A (26.7%), C (28.6%), D (30.0%), and the other (40.0%). Relative higher occupation rates of drug resistants, such as MNZ‐, CAM‐ and double MNZ/CAM‐resistant isolates, were observed in groups B (45.8%), C (42.6%), and D (40%). Five of eight GC‐derived isolates with MNZ/CAM resistance were significantly assigned to group B (P = 0.0312, χ²‐test). These results suggest that the isolates classified in group B have a potential to contribute to the development of severe gastric disorders. The DL system, rapid and high sensitive technology, would be widely available in clinical laboratory for pathological and epidemiological analyses even in H. pylori.     

Plasmid-mediated fluoroquinolone efflux pump gene, qepA, in Escherichia coli clinical isolates in Korea

The qepA gene was detected in 4 (0.6%) of 621 nonduplicate Escherichia coli clinical isolates collected from blood cultures in Korea. Three of the 4 qepA-positive isolates contained bla_CTX-M-14 and/or bla_TEM-1 genes, but not the E8700 isolate. The qepA gene was successfully transferred and conferred resistance to hydrophilic quinolones, such as norfloxacin and ciprofloxacin, in the recipient. The gene was located in part of IS26. Two isolates were linked to the truncated rmtB gene.     

Triple heterozygosity in the integrin αIIb subunit in a patient with Glanzmann's thrombasthenia

Summary. We report triple heterozygosity in the integrin α_IIb subunit in a 5‐year‐old Canadian girl with Glanzmann's thrombasthenia. The patient has a severe bleeding history possibly aggravated by low VWF suggestive of associated type 1 von Willebrand's disease. Platelet aggregation was absent or severely reduced for all physiologic agonists. Flow cytometry showed an ～ 4% residual surface expression of α_IIbβ₃. Western blotting confirmed a low platelet expression of both subunits. PCR‐SSCP and direct sequencing showed no abnormalities in the β₃ gene, but revealed a G→A transition at a splice site [IVS 19 (+1)] of exon 19 in the α_IIb gene. Of maternal inheritance, the splice site mutation was associated with intermediate levels of α_IIbβ₃ in carriers. Unexpectedly, two G→A transitions were detected in exon 29 of the α_IIb gene and led to V⁹⁵¹→M and A⁹⁵⁸→T amino acid substitutions. Family studies using restriction enzymes showed that both exon 29 mutations were paternal in origin and cosegregated across three generations. Transient expression in which mutated α_IIb was cotransfected with wild‐type β₃ in COS‐7 cells showed that V⁹⁵¹→M gave a much reduced surface expression of α_IIbβ₃ and a block in the maturation of pro‐α_IIb. In contrast, the A⁹⁵⁸ substitution appeared to be a novel polymorphism. Our studies highlight an unusual mixture of defects giving rise to severe bleeding in a child and describe the first pathological missense mutation affecting a C‐terminal residue of the calf‐2 domain of α_IIb.     

In vitro and in vivo anti- Helicobacter pylori activity of Y-904, a new fluoroquinolone

 Y-904 is a new fluoroquinolone with a broad antimicrobial spectrum. In particular, it has anti-Helicobacter pylori activity superior to that of existing fluoroquinolones. In the present study it was examined for its in vitro antibacterial activity against 51 clinical isolates of H. pylori, including clarithromycin- and metronidazole-resistant strains. The minimum inhibitory concentration of Y-904 at which 90% of isolates were inhibited was close to that of amoxicillin and clarithromycin and lower than that of levofloxacin and metronidazole (0.1, 0.1, 0.2, 3.13, and 12.5 μg/ml, respectively). Y-904 showed equally strong activity at pH 5.5 as at pH 7.0. At 10 times the minimum inhibitory concentration, Y-904 decreased the viable count of H. pylori to below 10⁻⁵ within 2 h after exposure. No significant change in the minimum inhibitory concentration was observed when H. pylori, Staphylococcus aureus, and Escherichia coli were successively subcultured in medium containing subinhibitory concentrations of Y-904. Y-904 also strongly inhibited the supercoiling activity of DNA gyrase from H. pylori ATCC43504 (IC₅₀, 1.48 μg/ml). A study of Y-904 treatment in H. pylori-infected Mongolian gerbils using twice-daily oral administration for 7 days demonstrated that the complete clearance dose of Y-904 was 1 mg/kg and that its potency was around 10, 30, and 30 times that of amoxicillin, clarithromycin, and levofloxacin, respectively. These results indicate that Y-904 is a promising candidate for the eradication of H. pylori infection. Received: November 8, 2002 / Accepted: March 10, 2003 Acknowledgments We thank Dr. T. Sugiyama, Dr. M. Kato (Hokkaido University, Sapporo, Japan), and Dr. K. Sakurai (Showa University, Yokohama, Japan) for graciously providing the clinical isolates of H. pylori. We are also grateful to Mr. K. Honjo for assistance in the in vitro antibacterial studies and to Mr. S. Miyoshi for pharmacokinetic data.     

Probe ligation and real-time detection of KPC,OXA-48, VIM,IMP, and NDM carbapenemase genes

The Check-MDR Carba test (Check-Points, Wageningen, Netherlands), which is based on specific molecular recognition of bla_NDM, bla_KPC, bla_OXA-48, bla_VIM, and bla_IMP genes by DNA probe ligation and real-time PCR detection, was evaluated on 183 well-characterized Gram-negative rods. Representatives of the 5 gene families were accurately identified (specificities and sensitivities of 100%) within 4.5 hours. This test may be helpful to differentiate carbapenem resistance mediated by carbapenemases from those involving other mechanisms.     

Supplementing vitamins C and E to standard triple therapy for the eradication of Helicobacter pylori

What is known and Objective: Helicobacter pylori eradication rates of currently accepted triple therapy regimens vary between geographic locations and do not exceed 70–80%. Eradication rates are much lower in locations where uncontrolled antibiotic use is common such as Turkey. In the present study, we aimed to test whether supplementing vitamins C and E to standard triple therapy, including a proton pump inhibitor plus amoxicillin plus clarithromycin, increased the H. pylori eradication rate. Methods: Two hundred patients infected with H. pylori were randomized into two groups in an open‐label trial. In group A, patients (n = 160) were given standard triple therapy, including lansoprazole 30 mg BID plus amoxicillin 1000 mg BID plus clarithromycin 500 mg BID for 14 days, plus vitamin C 500 mg BID plus vitamin E 200 IU BID for 30 days. In group B, patients (n = 40) were given standard triple therapy for 14 days. The success of H. pylori eradication was defined as a negative ¹⁴C‐urea breath test result, 4–6 weeks after the completion of therapy. Comaprisons were by both intention‐to‐treat (ITT) and per‐protocol (PP) analysis. Results and Discussion: Two hundred patients (137 women, 63 men) were analysed using ITT analysis and 195 patients completed the study. In group A, H. pylori eradication was achieved in 132 of the 160 patients (82·5%) included in ITT analysis and 132 of the 157 patients (84%) included in PP analysis. In group B, H. pylori eradication was achieved in 18 of the 40 patients (45%) included in ITT analysis and 18 of the 38 patients (47·4%) included in PP analysis. Eradication rates were significantly higher in group A than in group B (P < 0·005). Eradication rates were not statistically significant between men and women in both groups. What is new and Conclusion: Adding vitamins C and E to standard triple therapy increases the eradication rate of H. pylori. Vitamins C and E may increase the eradication rate via increasing the effectiveness of the antibiotics by decreasing oxidative stress in the gastric mucosa and strengthening the immune system.     

Characterization of carbapenem-resistant Acinetobacter baumannii in Brazil (2008–2011): countrywide spread of OXA-23–producing clones (CC15 and CC79)

The study investigated the genetic relationship of carbapenem-resistant Acinetobacter baumannii clinical isolated from inpatients during 2008–2011 from 11 Brazilian states. Antimicrobial susceptibility profile was determined by disc diffusion method and Etest. Polymerase chain reaction was applied for carbapenemase genes, and ISAba1. Isolates were subjected to pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for molecular typing. Most of the isolates showed high resistance rates to antibiotics tested. The bla_OXA-51-like gene was found in all isolates, and 146 (94.2%) isolates were positive for bla_OXA-23-like. In the most OXA-23–producing isolates, the bla_OXA-23-like gene was accompanied by ISAba1. A total of 146 OXA-23–producing isolates were clustered into 28 genotypes by PFGE. Molecular analysis by MLST identified 13 sequence types (STs). The most prevalent PFGE profiles were designated as ST15 (CC15), ST1 (CC1), and ST79 (CC79). This study showed the widespread of clonal complexes of A. baumannii harboring the bla_OXA-23-like gene in different Brazilian states.     

Quadruple therapy with ecabet sodium,omeprazole, amoxicillin and metronidazole is effective for eradication of Helicobacter pylori after failure of first‐line therapy (KDOG0201 Study)

Background and object: An antiulcer agent, ecabet sodium, is active against Helicobacter pylori. The aim of the present study was to clinically examine whether eradication therapy, which includes ecabet sodium, is effective in eradication of H. pylori after failure of first‐line therapy. Methods: Patients with peptic ulcer who failed with first‐line triple eradication therapy containing clarithromycin received quadruple therapy with omeprazole (20 mg, twice daily), amoxicillin (750 mg, twice daily), metronidazole (500 mg, twice daily) and ecabet sodium (1000 mg, twice daily) for 14 days. Eradication of H. pylori was judged by ¹³C‐urea breath test 8 weeks later. Results: Fifty‐two patients (36 men and 16 women) were included. Their mean age was 51·4 years (range 28–73). One patient dropped out because of diarrhoea. The eradication rate was 98·0% (50/51) according to the per‐protocol analysis and 96·2% (50/52) according to the intention‐to‐treat analysis. Side effects occurred in seven patients, but none were serious. Conclusions: Quadruple therapy including ecabet sodium is useful as second‐line eradication treatment for H. pylori.     

A change in PBP1 is involved in amoxicillin resistance of clinical isolates of Helicobacter pylori

Reports on the isolation of amoxicillin-resistant Helicobacter pylori are increasing worldwide, which may cause serious problems in eradication therapy. To elucidate the mechanism of amoxicillin resistance of H. pylori, penicillin-binding proteins (PBPs) of amoxicillin-resistant strains isolated in Korea were analysed. Three PBPs (66, 63 and 60 kDa) were identified in both amoxicillin-resistant and -susceptible strains using biotinylated ampicillin, and the PBP profiles were very similar irrespective of the difference in amoxicillin susceptibility. We obtained clones with moderate resistance from an amoxicillin-susceptible strain, HPK5, by transformation with genomic DNA from an amoxicillin-resistant strain, HPA116. In a resistance-induced clone, HPO1, the affinity of PBP1 for amoxicillin was reduced. The pbp1 genes from HPA116, HPO1 and HPK5 were cloned and sequenced. The nucleotide sequences of pbp1 from HPA116 and HPO1 were almost identical, whereas that of HPK5 was quite different. Both the ORFs of HPA116 and HPO1 pbp1 have four substitutions and one insertion of amino acid residues compared with those of HPK5 and other sensitive strains. All the mutations, except one, are in the C-terminal half of the 659-amino-acid sequence containing the penicillin-binding modules. DNA fragments containing either full-length or a C-terminal half of pbp1 could transform HPK5 to have resistance, indicating that changes in the penicillin-binding core of PBP1 are involved in the amoxicillin resistance of H. pylori isolated in Korea.