Antimicrobial resistance in Helicobacter pylori isolates from children.

OBJECTIVE: To determine the resistance rate to four antimicrobial agents commonly used in the treatment of Helicobacter pylori infection in children. METHODS: Between July 1997 and January 2000, all H. pylori isolates from children undergoing gastroscopy were prospectively collected and subcultured to yield the susceptibility to four antimicrobial agents by E-test. In all, 23 isolates were tested. Demographic data, presenting symptoms, treatment regimen and clinical improvement after treatment were collected retrospectively. RESULTS: The resistance rate of H. pylori to metronidazole and clarithromycin were 43.5% and 8.7%, respectively. No H. pylori strains were resistant to amoxycillin or tetracycline. There were no statistically significant differences in age, sex, ethnicity, presenting symptoms or clinical improvement after treatment between antimicrobial-susceptible and antimicrobial-resistant groups. CONCLUSIONS: The frequent resistance of H. pylori to metronidazole and moderate resistance to clarithromycin in children are comparable with local adult data. The incidence of resistance tended to be higher in patients of non-European ethnicity, but this was not statistically significant. Given that the primary goal of therapy is eradication, and that local resistance rates are high, recommendations for H. pylori management may need to be modified to include sensitivity testing and/or determination of eradication in all patients.

Detection of extended spectrum beta-lactamases in the routine clinical microbiology laboratory.

AIMS: To compare three methods of confirming the presence of an extended spectrum beta-lactamase (ESBL) enzyme with the initial detection (i.e., screening) by the Vitek AMS. METHODS: Gram-negative bacteria which flagged as ESBL-positive in the Vitek GNS card, or were suspected of harbouring an enzyme, were further tested by each of the following methods: (a) combination disc test using cefpodoxime, ceftazidime and cefotaxime with and without clavulanate; (b) cefotaxime ESBL Etest; and (c) Jarlier keyhole method with cefpodoxime (10 microg), cefotaxime (5 microg) and aztreonam (30 microg) placed 15mm away from an augmentin (30 microg) disc. RESULTS: A total of 52 isolates were investigated, representing an 18-month time period. Fifty of these were positive by Vitek. Twenty-eight (56%) were confirmed by other methods (true positives). Of the 44% Vitek-positive/confirmatory test-negative (false positives), eight were Escherichia coli which was 53% of all E. coli tested. The majority of other false-positive isolates were Klebsiella oxytoca (24% overall) which were all Vitek- and Etest-positive but negative by the combination disc test. CONCLUSIONS: All ESBL-positive strains by Vitek should be confirmed by the combination disc test using all three antibiotics. This will enable differentiation of 'true' ESBLs from false-positive organisms, including K1 hyperbetalactamase-producing Klebsiella oxytoca and AmpC-producing organisms. The cefpodoxime combination discs gave the best differentiation in this study with only one ESBL organism being missed.

Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study.

Helicobacter pylori strains from 299 patients were tested in six laboratories in different countries. Macrolide susceptibility of the strains was determined by agar dilution (17.4%) or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA (rDNA) that are associated with macrolide resistance were analyzed by PCR and reverse hybridization (PCR-line probe assay [LiPA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and A2143C mutations in the 23S rDNA. vacA s-region (s1a, s1b, s1c, and s2) and m-region (m1, m2a, and m2b) genotypes and cagA status were also determined using another PCR-LiPA system. Of the 299 strains investigated by MIC testing, 130 (43.5%) were resistant and 169 (56.5%) were susceptible to clarithromycin. Of the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wild-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutations. Only five resistant strains contained the A2142C mutation (three of these in combination with the A2142G mutation), and the A2115G, G2141A, A2142T, and A2143C mutations were not found. MICs of clarithromycin for the A2142G mutant strains were significantly higher than MICs for the A2143G strains. Although there was no significant association between 23S rDNA mutations and the vacA and cagA status, clarithromycin-susceptible strains more often contained mixed vacA genotypes, indicating the presence of multiple H. pylori strains. In conclusion, our data confirmed the very strong association between 23S rDNA mutations and macrolide resistance and showed that the PCR-LiPA permits accurate and reliable diagnosis of macrolide resistance in H. pylori.

Bacteremia due to Leptotrichia trevisanii sp. nov.

A thin, filamentous, non-motile, aerotolerant, anaerobic, gram-negative bacterium was isolated from the blood of a 46-year-old man who was diagnosed as having acute myeloid leukemia. The organism had a positive catalase reaction but was negative in indole and oxidase tests. A commercially available system failed to identify the bacterium, but 16S rRNA gene sequencing showed it to be most closely related (97% similarity) to a recently isolated Leptotrichia sp. The DNA base composition was 29.7% mol G+C, and the organism produced lactate as the sole end-product of glucose fermentation. These data indicate the isolate is a new species of Leptotrichia for which the name Leptotrichia trevisanii sp. nov. is proposed.

Randomized trial of omeprazole and metronidazole with amoxycillin or clarithromycin for Helicobacter pylori eradication, in a region of high primary metronidazole resistance: the HERO study.

BACKGROUND: The efficacy of omeprazole-based eradication therapies has been determined mostly in populations with low to moderate prevalence of metronidazole resistant Helicobacter pylori, yet resistance is high in many regions. AIM AND METHODS: The H. pylori eradication and duodenal ulcer healing rates after 1 week of either omeprazole 40 mg mane, amoxycillin 500 mg t.d.s. and metronidazole 400 mg t.d.s. (OAM) or omeprazole 20 mg b.d., metronidazole 400 mg b. d. and clarithromycin 250 mg b.d. (OMC) were compared in a randomized trial in Australia and New Zealand. Patients had a further 1 week of omeprazole 20 mg. Outcome was assessed at 6 weeks with stringent criteria (endoscopy, biopsies and 13C-urea breath test). RESULTS: Of 220 subjects randomized, the H. pylori eradication rates (all patients treated/per protocol) were 82%/85% for OMC and 58%/63% for OAM (P= 0.001). Pre-treatment metronidazole resistance was present in 56% and clarithromycin resistance in 6%. The eradication rate for primary metronidazole resistance isolates treated with OMC was 80% (CI: 65-90%) compared with 45% (CI: 29-62%) for OAM, whereas for sensitive organisms, the eradication rates were 94% (CI: 79-99%) and 79% (CI: 62-91%), respectively. Duodenal ulcer healing was 96% for OMC and 87% for OAM. Compliance was excellent and both treatments were well-tolerated. CONCLUSIONS: OMC is a well-tolerated, effective therapy for H. pylori eradication and duodenal ulcer healing in this region despite the high metronidazole resistance rate. OAM is less effective, largely due to the impact of metronidazole resistance.

Use of a dry latex agglutination test to measure eradication of Helicobacter pylori infection.

BACKGROUND AND METHODS: A total antibody latex serology test was compared with enzyme immunoassay serology after treatment for Helicobacter pylori infection in 22 patients. RESULTS: Nineteen patients were cured of infection, but only nine (47%) were negative by the latex test after 6 months. However a significant decline in immunoglobulin (Ig)G was seen in 90% of the cured patients. CONCLUSIONS: Although the latex test is suitable for initial diagnosis of H. pylori infection, it is not suitable for monitoring treatment success. A decline in IgG of more than 40% correlates well with successful eradication of H. pylori.

Accurate diagnosis of Helicobacter pylori. Urease tests.

Because of their ease of use, rapidity, and cost-effectiveness, rapid urease biopsy tests continue to be an important diagnostic tool for the endoscopist. Various forms of these tests are available in different parts of the world, characteristically leading to sensitivities of 93% to 97% and specificities of 98%. When performing rapid urease tests, the endoscopist should be aware of factors that might lead to false-negative and false-positive results and should ensure that the biopsy specimens are of adequate size and taken from the correct location.

Geographic distribution of vacA allelic types of Helicobacter pylori.

BACKGROUND & AIMS: Distinct allelic types of Helicobacter pylori vacA have been defined. The geographic distribution of vacA alleles and cagA was assessed in this study. METHODS: A total of 735 cultures from patients in 24 countries were analyzed by polymerase chain reaction and reverse hybridization on a line probe assay (LiPA). RESULTS: In 124 (16.9%) of the 735 cultures, multiple vacA genotypes were detected, permitting analysis of 611 strains. In Europe, a distribution gradient of s1 subtypes was observed. In northern and eastern Europe, 89% were subtype s1a. s1a and s1b were equally present in France and Italy, whereas in Spain and Portugal 89% of strains were subtype s1b. s1a and s1b were approximately equally prevalent in North America. In Central and South America, virtually all s1 strains were subtype s1b. Subtype s1c was observed in 77% of the s1 isolates from East Asia. m1 and m2a have equal presence, except on the Iberian peninsula and in Central and South America, where m1 (86.2%) is more prevalent than m2 (13.8%). Subtype m2b was found exclusively among East Asian s1c strains. In all parts of the world, vacA s1/cagA-positive genotypes were associated with peptic ulcer disease (P < 0.001). CONCLUSIONS: These data indicate a geographic distribution of H. pylori genotypes and aid in understanding the relationship of H. pylori with disease.

Expanding allelic diversity of Helicobacter pylori vacA.

The diversity of the gene encoding the vacuolating cytotoxin (vacA) of Helicobacter pylori was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions of vacA, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from H. pylori isolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different vacA s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (cagA). This PCR-LiPA method permits rapid analysis of the vacA and cagA status of H. pylori strains for clinical and epidemiological studies and will facilitate identification of any further variations.

The actions of bismuth in the treatment of Helicobacter pylori infection.

Bismuth salts have been used in medicine for over three centuries, particularly in the treatment of dyspepsia. Commonly used agents include colloidal bismuth subcitrate (CBS), bismuth subsalicylate (BSS) and the newer ranitidine bismuth citrate (RBC). These are safe drugs which exert local effects on the gastroduodenal mucosa. Gastric mucosal levels of bismuth exceed the concentrations required to kill Helicobacter pylori in vitro. The mechanisms of actions of bismuth on gastrointestinal pathogens including H. pylori are complex and include inhibition of protein and cell wall synthesis, membrane function and ATP synthesis. Adherence of H. pylori to surface epithelial cells is also impaired. Bismuth monotherapy is effective in vivo to suppress H. pylori but cure rates are low. CBS, BSS and RBC have synergistic activity with one or two antibiotics and are effective in eradicating H. pylori. CBS and RBC also exert other effects on the mucosa including cytoprotective and ulcer healing properties. In addition, RBC is effective in inhibiting gastric acid secretion.

Bactericidal activity and synergy studies of proton pump inhibitors and antibiotics against Helicobacter pylori in vitro.

The bactericidal activity of the proton pump inhibitors omeprazole and lansoprazole alone and in combination with a beta-lactam or macrolide antibiotic were investigated in vitro. Time-kill curves against Helicobacter pylori NCTC 11637 and a recent clinical isolate revealed significant concentration-dependent killing with all drugs other than amoxycillin. Combinations of proton pump inhibitor and erythromycin showed synergic activity. In contrast, proton pump inhibitor plus amoxycillin showed additive activity against the clinical isolate only. Bactericidal investigations of anti-helicobacter drugs in vitro may suggest optimum treatment strategies for this common infectious disease.

Antimicrobial resistance testing of Helicobacter pylori: a comparison of Etest and disk diffusion methods.

Routine antimicrobial resistance testing of Helicobacter pylori is more commonly performed since the correlation between metronidazole resistance and failure to eradicate using this drug, has been made. While resistance testing of H. pylori by Etest is simple to perform, it is expensive compared to disk diffusion methods. In this study the Etest was compared with a modified Kirby-Bauer disk diffusion (NCCLS) method for routine resistance screening of H. pylori. Fifty one pre-treatment isolates were tested against amoxycillin, metronidazole, tetracycline and erythromycin by both Etest and disk diffusion using NCCLS guideline strength disks. Clarithromycin was tested by Etest only. Nitroimidazole and macrolide resistance were detected using the modified Kirby-Bauer disk diffusion method which correlated with Etest minimum inhibitory concentration (MIC). Resistance rates were 49% for metronidazole and 8% for clarithromycin. Cross resistance occurs with macrolides against H. pylori and allows testing of erythromycin to predict resistance to clarithromycin. The very low MICs obtained with H. pylori against amoxycillin and tetracycline require the use of Etest or lower strength disk methods to be used.

Novel bismuth compounds have in vitro activity against Helicobacter pylori.

The increasing antimicrobial resistance in Helicobacter pylori has led to the search for new therapeutic agents. In this in vitro study, novel compounds combining bismuth with sialic acid inhibitors were investigated for bactericidal activity using time-kill methodology. The activity of these compounds was compared to bismuth subcitrate against a type strain and a clinical isolate of H. pylori. The compounds tested showed cidal activity which was related to the bismuth component of each drug. These compounds may offer a potential advantage over current bismuth preparations with the sialic acid inhibitor moiety interfering with adhesion of H. pylori to gastric epithelium.

Oxygen concentration influences proton pump inhibitor activity against Helicobacter pylori in vitro.

Omeprazole and lansoprazole are proton pump inhibitors that have shown activity against Helicobacter pylori and other Helicobacter species when tested by agar dilution. Lansoprazole was more active against H. pylori than was omeprazole, and the activity was independent of urease production. Disk susceptibility tests and agar dilution MIC determinations were performed to investigate the effects of incubation under different sets of atmospheric conditions on H. pylori inhibition. Oxygen concentration was found to influence proton pump inhibitor activity in vitro, with higher concentrations leading to greater susceptibility. The method of testing is important in determining the anti-Helicobacter activity of proton pump inhibitors.