Helicobacter pylori resistance rates for levofloxacin, tetracycline and rifabutin among Irish isolates at a reference centre.

INTRODUCTION: Helicobacter pylori eradication rates using conventional triple therapies are falling, making viable second-line and rescue regimens necessary. Levofloxacin, tetracycline and rifabutin are three efficacious antibiotics for rescue therapy. AIM: We aimed to assess the resistance rates for H. pylori against these antibiotics in an Irish cohort. METHODS: Gastric biopsies were collected from 85 patients infected with H. pylori (mean age 46 years) in the Adelaide and Meath Hospital, Dublin in 2008 and 2009. Susceptibility to antibiotics was tested using the Etest. Clinical information was obtained from endoscopy reports and chart review. RESULTS: 50.6 % of patients were females. Mean age was 47 years. Ten had prior attempts at eradication therapy with amoxicillin-clarithromycin-PPI, two had levofloxacin-based second-line therapy. 11.7 % [95 % CI (6.5-20.3 %)] (N = 10) had strains resistant to levofloxacin. There were no strains resistant to rifabutin or tetracycline. Levofloxacin resistance in the under 45 age group was 2.6 % (1/38) compared to 19.1 % (9/47) of above 45 age group (p = 0.02). DISCUSSION: The levofloxacin rates illustrated in this study are relatively low by European standards and in line with other studies from the United Kingdom and Germany, with younger patients having very low levels of resistance. Levofloxacin, tetracycline and rifabutin are all valid options for H. pylori eradication in Irish patients but the importance of compliance cannot be underestimated.

Analysis of drug resistance and virulence-factor genotype of Irish Helicobacter pylori strains: is there any relationship between resistance to metronidazole and cagA status?

BACKGROUND: Helicobacter pylori infection is eradicated with antimicrobial agents and drug-resistant strains make successful treatment difficult. Geographical variations in virulence-factor genotype also exist. AIM: To evaluate prevalence of drug resistance and virulence-factor genotype in Irish H. pylori strains and to investigate if there is any relationship between drug resistance and genotype. METHODS: Helicobacter pylori strains isolated from 103 patients were examined. Antimicrobial susceptibilities were tested by Etest. The virulence-factor genotypes were determined using PCR. Frequencies of spontaneous metronidazole-resistance were measured in vitro. RESULTS: Metronidazole resistance was present in 37.9% of strains examined. 16.5% of strains were clarithromycin-resistant and resistance to both agents observed was found in 12.6% of strains. 68% of strains were cagA(+). The dominant vacA type was s1/m2, followed by s1/m1 and s2/m2. The metronidazole resistance rate in cagA(-) group was significantly higher than in cagA(+) (P = 0.0089). Spontaneous resistance to metronidazole in cagA(-) occurred in higher frequency when compared with cagA(+). CONCLUSIONS: cagA(+) and vacAs1/m2 type was the dominant genotype in Irish H. pylori strains. Significant rates of metronidazole resistance were observed in cagA(-) group. cagA(-) strains tend to acquire metronidazole resistance in vitro. Absence of cagA might be a risk factor in development of metronidazole resistance.

Genetic heterogeneity of the dnaK gene locus including transcription terminator region (TTR) in Campylobacter lari.

Nucleotide sequences of approximately 3.1 kbp consisting of the full-length open reading frame (ORF) for grpE, a non-coding (NC) region and a putative ORF for the full-length dnaK gene (1860 bp) were identified from a urease-positive thermophilic Campylobacter (UPTC) CF89-12 isolate. Then, following the construction of a new degenerate polymerase chain reaction (PCR) primer pair for amplification of the dnaK structural gene, including the transcription terminator region of C. lari isolates, the dnaK region was amplified successfully, TA-cloned and sequenced in nine C. lari isolates. The dnaK gene sequences commenced with an ATG and terminated with a TAA in all 10 isolates, including CF89-12. In addition, the putative ORFs for the dnaK gene locus from seven UPTC isolates consisted of 1860 bases, and the four urease-negative (UN) C. lari isolates included C. lari RM2100 reference strain 1866. Interestingly, different probable ribosome binding sites and hypothetically intrinsic p-independent terminator structures were identified between the seven UPTC and four UN C. lari isolates, respectively. Moreover, it is interesting to note that 20 out of a total of 28 polymorphic sites occurred among amino acid sequences of the dnaK ORF from 11 C. lari isolates, identified to be alternatively UPTC-specific or UN C. lari-specific. In the neighbour-joining tree based on the nucleotide sequence information of the dnaK gene, C. lari forms two major distinct clusters consisting of UPTC and UN C. lari isolates, respectively, with UN C. lari being more closely related to other thermophilic campylobacters than to UPTC.

Molecular characterisation of urease genes from urease-positive thermophilic campylobacters (UPTC).

This study aims to clarify the molecular characteristics of the urease gene operon from urease-positive thermophilic campylobacters (UPTC) obtained from different sources and in various countries. Sequence heterogeneity was observed for the promoter structures at the -35-like region among the 12 isolates examined. The most probable TTG start codon was suggested for the ureB and ureH genes, and for the ureA, E, F and G genes, ATG was suggested among all the isolates examined. Overlap was detected between ureA and ureB and between ureB and ureE among all the isolates examined. UPTC is the first example of an overlap between the two structural genes ureA and ureB. When the completely sequenced open reading frames (ORFs) for ureE, ureF, ureG and ureH were identified, non-coding regions between ureE and ureF, ureF and ureG, and ureG and ureH were also demonstrated. All six start codons of the six urease genes were demonstrated to be preceded by Shine-Dalgarno sequences among all the isolates examined. The Cys-His sequence corresponding to urease active sites were aligned perfectly and fully conserved among the three UPTC isolates examined. A putative and intrinsic p-independent transcriptional terminator was identified to be identical among all the isolates examined. A partial and putative ORF of about 200 bp in length showing high sequence similarity to GTP cyclohydrolase I was observed downstream of ureH.

Molecular characterization of intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella equigenitalis.

Using two primer pairs constructed in silico for the amplification of the intervening sequences (IVSs) of the 23S rRNA gene sequences of the genus Taylorella, none of the three representative T. equigenitalis strains NCTC11184(T), Kentucky 188 and EQ59 was shown to contain any IVSs in the first quarter region. In the central region, all three strains possessed one approximately 70 bp IVS (TeIVS2) different from any IVSs found in T. asinigenitalis. The predicted secondary structure model of the IVSs contained stem and loop structures. The central region of the IVS-stem structure contains an identical double-stranded consensus 15-bp sequence. The purified RNA fraction from the three strains contained 16S and 4-5S RNA species but no 23S rRNA species. Thus, the primary 23S rRNA transcripts from the three strains would be cleaved into approximately 1.2- and 1.6-kb rRNA fragments and approximately 70-bp IVS. In addition, 16 other T. equigenitalis isolates were found to carry a similar 70-bp IVS in the central region and to produce fragmented 23S rRNA.

Cloning and structural analysis of the full-length cytolethal distending toxin (cdt) gene operon from Campylobacter lari.

Polymerase chain reaction (PCR) amplicons (approximately 2.5 kbp) encoding a cdt gene operon and two partial and putative open reading frames (ORFs) were identified in six urease-negative (UN) Campylobacter lari isolates using a new PCR primer pair constructed in silico. Three closely spaced and putative ORFs for cdtA, cdtB and cdtC, two putative promoters and a hypothetically intrinsic p-independent transcription terminator were found in the operon. Each ORF commenced with an ATG start codon and terminated with a TGA stop codon for cdtA and cdtB and a TAA for cdtC. Interestingly, an overlap of four nucleotides was detected between cdtA and cdtB and the non-coding region of six base pairs occurring between cdtB and cdtC. The start codons for the three cdt genes were preceded by Shine-Dalgarno sequences. Although nucleotide sequence differences were identified at seven loci in the cdtA gene, six in cdtB and two in cdtC among the seven isolates (including C. lari RM2100), no polymorphic sites occurred in the putative promoters, hypothetically intrinsic transcription terminator and the three ribosome binding sites among the seven isolates. All nine amino acid residues specific for both Escherichia coli cdtB and mammalian DNase I were completely conserved in the cdtB gene locus in the 26 C. lari isolates, as well as in C. jejuni and C. coli. No PCR amplicons were generated with urease-positive thermophilic campylobacters (UPTC; n=10) using the primer pair.

Cloning, sequencing and molecular characterisation of a cryptic plasmid from a urease-positive thermophilic Campylobacter (UPTC) isolate.

Cloning, sequencing and molecular characterisation of a cryptic plasmid, pUPTC237, from a urease-positive thermophilic Campylobacter (UPTC) isolate obtained from the natural environment in Northern Ireland is reported in this study. Based on the determined DNA sequence, the pUPTC237 DNA was identified as a circular molecule of 3828 bp with a G+C content of 29.5%. As with other plasmid DNAs from Gram-negative bacteria, pUPTC237 contained an A+T-rich region (A+T content: 95%), followed by multiple direct tandem repeat units of 22 bp, characteristic of a replication origin and iteron sequence. A possible open reading frame (ORF)-1 was located upstream of the A+T-rich region and the iteron sequence that encoded a 460 amino acid protein similar to the mobilisation (mob) protein and two putative promoter structure sequences at the -35 and -10 regions and a possible ribosome binding site occurred upstream of the start codon for the ORF-1. Moreover, three possible ORFs (a short ORF-2 encoding 26 amino acids, similar to repA; an ORF-3 encoding 341 amino acids, similar to repB; and an ORF-4 encoding 96 amino acids with unknown function) were also identified. There are also two putative promoter structures for these three ORFs at the -35 and -10 regions upstream of the possible ORF-2. A possible transcription termination region was identified downstream of ORF-4. Northern blot hybridisation analysis suggested that these four ORFs constitute an operon and generate a messenger RNA (mRNA) transcript.

DNA analysis of nosocomial infection by Enterobacter aerogenes in three cases of septicaemia in Japan.

Ceftazidime-resistant Enterobacter aerogenes was isolated from blood cultures of three patients with fever. DNA analysis using pulsed-field gel electrophoresis and ribosomal RNA gene restriction digest pattern analysis revealed that the strains were clonally similar to each other with a 79.3-96.0% homology. The same strain of E. aerogenes was isolated from a three-way stopcock connected to the indwelling catheter in one of the patients at a concentration of 45 cfu/mL. A similar strain was also isolated from the urine of one other patient on the same floor. The data suggest that E. aerogenes caused septicaemia via low bacterial contamination of a three-way stopcock in a peripheral drip intravenous infusion system in at least one patient, and that the outbreak of E. aerogenes infections was due to clonally-related strains.

Helicobacter pylori intrafamilial infections: change in source of infection of a child from father to mother after eradication therapy.

Biopsy specimens of the antrum and corpus were obtained from four Helicobacter pylori-infected members of a family and from the same boy (son 1) in whom the infection reappeared after simultaneous successful eradication treatment of three family members, excluding the mother. A total of 18 to 60 H. pylori isolates were obtained from each specimen and subjected to rRNA gene restriction pattern analysis. The father's isolates and the initial isolates from son 1 showed the same HindIII type, which was divided into three HaeIII subtypes. Isolates from the mother and a brother (son 2) and posttreatment isolates from son 1 showed a distinct HindIII type (with one minor subtype), which was divided into six HaeIII subtypes. All subtypes of the initial isolates from son 1 were present in the father's isolates, and all subtypes of the posttreatment isolates from son 1 were present in the mother's isolates but not in son 2's. Electron microscopic analysis of the biopsy specimens demonstrated extremely high levels of H. pylori colonization in the father's gastric mucosa. H. pylori adherence with a ruffle formation was also demonstrated. The findings suggest that son 1 was infected initially with the H. pylori strain of the father and son 2 was infected with the H. pylori strain of the mother and that after eradication therapy son 1 was reinfected with the H. pylori strain of the mother, who did not undergo eradication therapy.

Inhibitory action of a novel proton pump inhibitor, rabeprazole, and its thioether derivative against the growth and motility of clarithromycin-resistant Helicobacter pylori.

BACKGROUND: Clarithromycin-resistant Helicobacter pylori (CRHP) has increasingly been isolated from patients in Japan. The aim of our study was to test whether proton pump inhibitors (PPIs) and their thioether derivatives, which are secreted into the gastric mucosa, could inhibit the growth and motility (a factor in colonization) of CRHP. MATERIALS AND METHODS: CRHP was isolated from patients who had experienced gastritis or peptic ulcers in Tokyo and Niigata. Drugs and related agents tested were omeprazole, lansoprazole, rabeprazole, the thioether derivative of rabeprazole (rabeprazole-TH), clarithromycin, amoxicillin and metronidazole. The MICs of the drugs and agents for H. pylori strains were determined by the agar dilution METHOD: Bacterial swimming in a liquid layer was examined under an inverted, phase-contrast microscope. RESULTS: The PPIs and rabeprazole-TH, but not the anti-H. pylori agents, inhibited the motility of CRHP at both pH 7.4 and 6.0. The concentrations (microg/ml) necessary to inhibit 50% of the motility at pH 7.4 were 0.25-0.5, 8-32, 8-16 and 128-256 for rabeprazole-TH, rabeprazole, lansoprazole and omeprazole, respectively. Rabeprazole-TH exhibited the strongest inhibitory effect against the growth of CRPH (MIC, 0.5 microg/ml). CONCLUSION: Rabeprazole-TH, which is secreted into the gastric mucosa, had the strongest inhibitory action against both the growth and motility of CRHP, suggesting that it is a potential novel agent for CRHP eradication.

A novel action of the proton pump inhibitor rabeprazole and its thioether derivative against the motility of Helicobacter pylori.

The motility of Helicobacter pylori was maximum at 37 degrees C and at pH 6. A newly developed proton pump inhibitor, rabeprazole (RPZ), and its thioether derivative (RPZ-TH) markedly inhibited the motility of H. pylori. The concentrations of the drug necessary to inhibit 50% of the motility were 0.25, 16, 16, and >64 microgram/ml for RPZ-TH, RPZ, lansoprazole, and omeprazole, respectively. No such inhibitory effects were observed with H(2) blockers or anti-H. pylori agents. The motilities of Campylobacter jejuni and C. coli-but not those of Vibrio cholerae O1 and O139, Vibrio parahaemolyticus, Salmonella enterica serovar Typhimurium, and Proteus mirabilis-were also inhibited. Prolonged incubation with RPZ or RPZ-TH inhibited bacterial growth of only H. pylori, except for a turbid colony mutant. The results indicate that RPZ and RPZ-TH have a characteristic inhibitory effect against the motility of H. pylori (spiral-shaped bacteria), which is distinguished from that against bacterial growth.

The sequences of enterohemorrhagic Escherichia coli and Yersinia pestis that are homologous to the enteroaggregative E. coli heat-stable enterotoxin gene: cross-species transfer in evolution.

The enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene is widely distributed among diarrheagenic E. coli. In this study, we examined the sequences of enterohemorrhagic E. coli (EHEC) strains by PCR and sequencing. All the EHEC strains possessed the EAST1 gene homologues but with two types of mutations. One of the mutation types was strongly associated with the large outbreak episodes in 1996 in Japan. Sequence comparison showed that the EHEC sequences are a branch of the EAST1 gene sequence family that showed the cross-species transfer in evolution among E. coli and Yersinia pestis.