Rita Reddy

Helicobacter pylori outer membrane proteins and gastroduodenal disease

Background and aims: A number of Helicobacter pylori outer membrane proteins (OMPs) undergo phase variations. This study examined the relation between OMP phase variations and clinical outcome. Methods: Expression of H pylori BabA, BabB, SabA, and OipA proteins was determined by immunoblot. Multiple regression analysis was performed to determine the relation among OMP expression, clinical outcome, and mucosal histology. Results:H pylori were cultured from 200 patients (80 with gastritis, 80 with duodenal ulcer (DU), and 40 with gastric cancer). The most reliable results were obtained using cultures from single colonies of low passage number. Stability of expression with passage varied with OipA > BabA > BabB > SabA. OipA positive status was significantly associated with the presence of DU and gastric cancer, high H pylori density, and severe neutrophil infiltration. SabA positive status was associated with gastric cancer, intestinal metaplasia, and corpus atrophy, and negatively associated with DU and neutrophil infiltration. The Sydney system underestimated the prevalence of intestinal metaplasia/atrophy compared with systems using proximal and distal corpus biopsies. SabA expression dramatically decreased following exposure of H pylori to pH 5.0 for two hours. Conclusions: SabA expression frequently switched on or off, suggesting that SabA expression can rapidly respond to changing conditions in the stomach or in different regions of the stomach. SabA positive status was inversely related to the ability of the stomach to secrete acid, suggesting that its expression may be regulated by changes in acid secretion and/or in antigens expressed by the atrophic mucosa.

Studies Regarding the Mechanism of False Negative Urea Breath Tests With Proton Pump Inhibitors

OBJECTIVE:The mechanism of false negative urea breath tests (UBTs) results among proton pump inhibitor (PPI) users is unknown. We studied the time course of PPI-associated negative UBT, the relation to Helicobacter pylori density, and whether gastric acidification would prevent false negative UBT results.METHOD:In the UBT experiment, H. pylori–infected volunteers received omeprazole 20 mg b.i.d. for 13.5 days. UBTs with citric acid were done before, after 6.5 days of PPI, and 1, 2, 4, 7, and 14 days after therapy. In the culture and histology experiment, after a wash-out of >5 months, nine of the original subjects were rechallenged with omeprazole for 6.5 days. Antral and corpus biopsies for histology and culture were done before and 1 day after PPI administration.RESULTS:Thirty subjects (mean age 42 yr) were enrolled. UBTs were significantly reduced on day 6.5 (p = 0.031); 10 subjects (33%) developed transient negative UBTs. The UBT recovered in all but one subject by the fourth day post-PPI and in all subjects by day 14. In the culture and histology experiment, upon PPI rechallenge, three of nine subjects (33%) had negative UBTs. H. pylori density, whether measured by culture or histology, decreased with PPI therapy; antral biopsies became histologically negative in five subjects and corpus biopsies in three subjects.CONCLUSION:PPI-induced negative UBT results were related to the anti-H. pylori effect of the PPI. Acidification of the stomach did not prevent false negative UBT results. Three days is likely the minimum delay from stopping PPI until one should perform a test for active infection. A delay of 14 days is preferred.

Analysis of rdxA and Involvement of Additional Genes Encoding NAD(P)H Flavin Oxidoreductase (FrxA) and Ferredoxin-Like Protein (FdxB) in Metronidazole Resistance of Helicobacter pylori

ABSTRACT Metronidazole (Mtz) is a critical ingredient of modern multidrug therapies for Helicobacter pylori infection. Mtz resistance reduces the effectiveness of these combinations. Although null mutations in a rdxA gene that encodes oxygen-insensitive NAD(P)H nitroreductase was reported in Mtz-resistant H. pylori, an intact rdxA gene has also been reported in Mtz-resistant H. pylori, suggesting that additional Mtz resistance mechanisms exist in H. pylori. We explored the nature of Mtz resistance among 544 clinical H. pyloriisolates to clarify the role of rdxA inactivation in Mtz resistance and to identify another gene(s) responsible for Mtz resistance in H. pylori. Mtz resistance was present in 33% (181 of 544) of the clinical isolates. There was marked heterogeneity of resistance, with Mtz MICs ranging from 8 to ≥256 μg/ml.rdxA inactivation resulted in Mtz MICs of up to 32 μg/ml for 6 Mtz-sensitive H. pylori strains and 128 μg/ml for one Mtz-sensitive strain. Single or dual (with rdxA) inactivation of genes that encode ferredoxin-like protein (designatedfdxB) and NAD(P)H flavin oxidoreductase (frxA) also increased the MICs of Mtz for sensitive and resistant strains with low to moderate levels of Mtz resistance. fdxB inactivation resulted in a lower level of resistance than that from rdxAinactivation, whereas frxA inactivation resulted in MICs similar to those seen with rdxA inactivation. Further evidence for involvement of the frxA gene in Mtz resistance included the finding of a naturally inactivated frxA but an intact rdxA in an Mtz-resistant strain, complementation of Mtz sensitivity from an Mtz-sensitive strain to an Mtz-resistant strain or vice versa by use of naturally inactivated or functionalfrxA genes, respectively, and transformation of an Mtz-resistant Escherichia coli strain to an Mtz sensitive strain by a naturally functional frxA gene but not an inactivated frxA gene. These results are consistent with the hypothesis that null mutations in fdxB,frxA, or rdxA may be involved in Mtz resistance.

Regional Differences in Metronidazole Resistance and Increasing Clarithromycin Resistance among Helicobacter pylori Isolates from Japan

ABSTRACT The patterns of antibiotic resistance in Helicobacter pylori were assessed in two different regions in Japan. Overall, prevalences of resistance to metronidazole and clarithromycin were 12.4 and 12.9%, respectively. While there was no difference in clarithromycin resistance, the prevalence of metronidazole resistance was significantly higher in Kyoto (23.8%) than in Sapporo (8.1%). From 1996 to 1999, the prevalence of metronidazole resistance did not change but the prevalence of clarithromycin resistance doubled (from 9.1 to 18.7%).

Comparison of the Etest and the NCCLS-approved agar dilution method to detect metronidazole and clarithromycin resistant Helicobacter pylori.

Although the NCCLS has approved the agar dilution method as the test of choice for antimicrobial susceptibility testing of Helicobacter pylori, a critical evaluation of this method in clinical trials to detect antibiotic resistance has not been performed. This study compares the Etest and agar dilution methods for detection of metronidazole and clarithromycin resistance in clinical isolates of H. pylori. MIC data were gathered from US-based clinical trials. The Etest was performed on Mueller-Hinton sheep blood agar plates following incubation for 4 days under 12% CO(2). The agar dilution test was performed according to the recently approved NCCLS methodology using aged sheep blood in a Mueller-Hinton agar base. Metronidazole resistance as determined by Etest was significantly higher than that determined by agar dilution (39%; 690/1768 vs. 25. 1%; 367/1465)(P<0.01). Clarithromycin resistance as determined by Etest was higher than that determined by agar dilution, but was not significantly different (12.5%; 209/1671 vs. 10.6%; 150/1414)(P>0.5). Inter-patient metronidazole resistance showed that the MIC values for identical isolates tested by both methods were equivalent in 58% (109/188). Of the 42% with a >2log(2) difference in MIC values, 17. 6% had a change in susceptibility pattern. For clarithromycin, 71.4% (237/332) of the MIC values for identical isolates tested by both methods had equivalent MIC values. Of the MIC values with a >2log(2) difference in MIC values, only 3% showed a change in susceptibility pattern. Intra-patient variability, i.e. paired isolates from the same patient, was assessed only for metronidazole. Of the 1393 paired isolates tested by Etest, 38.8% were shown to be resistant. Almost 69% of the Etest MIC determinations were deemed equivalent and 16.7% had a change in susceptibility pattern. Of the 639 paired isolates tested by agar dilution, 23.9% were resistant to metronidazole. Almost 72% of the agar dilution MIC values were equivalent and 11.3% of the determinations had a change in susceptibility pattern. Clarithromycin resistance rates are similar, when determined by either test method. The Etest yields a significantly higher prevalence of metronidazole resistance among H. pylori compared with the agar dilution method and both methods yield discordant results, when isolates from different parts of the same stomach are compared. Neither method is reliable in determining metronidazole resistance in H. pylori.

Metronidazole and clarithromycin resistance amongst Helicobacter pylori isolates from a large metropolitan hospital in the United States.

BACKGROUND Metronidazole and clarithromycin-based therapies are among the most efficacious treatment regimens for H. pylori infection. Resistance to metronidazole or clarithromycin is associated with impaired therapy with these agents. We conducted a retrospective review of susceptibility data to determine the frequency of primary metronidazole and clarithromycin resistance among H. pylori isolates from a single metropolitan hospital in the United States. The database comprised 933 patients who presented at the Digestive Diseases Clinic at the Veterans Affairs Medical Center in Houston between September 1988 and January 1997 with complaints of dyspepsia, abdominal pain and peptic ulcer disease. One hundred and seventy-nine of these patients had both pharmaceutical records available for evaluation and culture and antimicrobial susceptibility data for analysis. The MICs were determined by both E-test and broth microdilution tests. The frequency of primary metronidazole resistance was 37.4% (67/179). The level of primary clarithromycin resistance was 6.1%. Dual metronidazole and clarithromycin resistance was present in approx. 3%. The high level of primary metronidazole and clarithromycin resistance in H. pylori isolates from this metropolitan hospital is such that antimicrobial susceptibility data should be available so that informed choice can be made for specific eradication therapies, especially in patients who fail treatment.

False Negative Urea Breath Tests with H2‐Receptor Antagonists: Interactions Between Helicobacter pylori Density and pH

Background.  We studied the effects of famotidine, sodium bicarbonate, and citric acid on the 13C‐urea breath test (UBT).

Tetracycline-Resistant Clinical Helicobacter pylori Isolates with and without Mutations in 16S rRNA-Encoding Genes

ABSTRACT Tetracycline-resistant Helicobacter pylori strains have been increasingly reported worldwide. However, only a small number of tetracycline-resistant strains have been studied with regard to possible mechanisms of resistance and those studies have focused on mutations in the tetracycline binding sites of 16S rRNA-encoding genes. We here report studies of 41 tetracycline-resistant H. pylori strains (tetracycline MICs, 4 to 32 μg/ml) from North America (n = 12) and from East Asia (n = 29). DNA sequence analyses of 16S rRNA-encoding genes revealed that 22 (54%) of the resistant isolates carried one of five different single-nucleotide substitutions (CGA, GGA, TGA, AGC, or AGT) at the putative tetracycline binding site (AGA965-967). Single-nucleotide substitutions were associated with reduced ribosomal binding and with slightly increased tetracycline MICs (1 to 2 μg/ml). The 19 tetracycline-resistant isolates with no detectable mutations in the tetracycline binding site had normal tetracycline-ribosome binding. All tetracycline-resistant isolates, including those with and those without mutations in the tetracycline binding site, showed decreased accumulation of tetracycline. These results suggest that tetracycline resistance is multifactorial, involving alterations both in ribosomal binding and in membrane permeability.

Greater Than 95% Success with 14-day Bismuth Quadruple Anti- Helicobacter pylori Therapy: A Pilot Study in US Hispanics

A combination capsule of bismuth, metronidazole, and tetracycline plus omeprazole given as 10‐day therapy has an overall effectiveness of 92–93% in per‐protocol analysis (Grade B) with eradication of 86–91% of metronidazole‐resistant Helicobacter pylori. This study aimed to explore whether extending the duration to 14 days would improve overall effectiveness per protocol to ≥95% (Grade A) in a population in which metronidazole resistance was anticipated to exist.

Sequential therapy using high-dose esomeprazole-amoxicillin followed by gatifloxacin for Helicobacter pylori infection

The success rate of current anti‐Helicobacter pylori triple therapies in now generally 80% or less. Sequential therapy has proved superior.