Complete Genome Sequences of Cluster F1 Mycobacteriophages Akhila and MilanaBonita.

Akhila and MilanaBonita are mycobacteriophages that were isolated from soil in New York using Mycobacterium smegmatis. Both phages have genomes that are 56,251 bp long and contain 99 genes; the genomes differ by only 1 nucleotide. Based on gene content similarity to phages in the Actinobacteriophage Database, both phages are assigned to cluster F1.

Complete Genome Sequences of Mycobacteriophages HarryOW and Peeb.

HarryOW and Peeb are Mycobacterium smegmatis mc2 155 Siphoviridae temperate phages with 52,935 and 41,876 base pairs in genome length, respectively. HarryOW belongs to the A1 subcluster and Peeb to the G1 subcluster. They were isolated and annotated by students from the SUNY Old Westbury Science and Technology Entry Program.

Complete Genome Sequence of Rahel, a C1 Cluster Mycobacteriophage.

Rahel is a lytic Myoviridae bacteriophage that infects Mycobacterium smegmatis mc2155. It has 1,555,955 bp and 64.7% G+C content. Rahel has a circularly permuted genome with 270 genes, 53 of them of known function, 33 tRNAs, and 1 transfer-messenger RNA (tmRNA). Only five genes are coded on the reverse strand.

Antibiotic Resistance of Helicobacter pylori Among Male United States Veterans.

BACKGROUND & AIMS: The most recent information published on resistance of Helicobacter pylori to antibiotics in a large population in the United States is more than 10 years old. We assessed the susceptibility of H pylori to antibiotics among patients in a large metropolitan hospital, as well as demographic, clinical, and lifestyle factors associated with antimicrobial resistance. METHODS: We performed a cross-sectional study of a random sample of 656 patients (90.2% men) from a cohort of 1559 undergoing esophagogastroduodenoscopy with collection of gastric biopsies from 2009 through 2013 at the Houston Veterans Affairs Medical Center. We performed culture analyses of gastric tissues to detect H pylori. The minimum inhibitory concentrations of amoxicillin, clarithromycin, metronidazole, levofloxacin, and tetracycline were determined by the Epsilometer test. Logistic regression analysis was performed to estimate the association between risk factors and antimicrobial resistance. RESULTS: Biopsies from 135 subjects (20.6%) tested positive for H pylori; 128 of these were from men (94.8%). Only 65 strains were susceptible to all 5 antibiotics. The prevalence of resistance to levofloxacin was 31.3% (95% confidence interval [CI], 23.1%-39.4%), to metronidazole it was 20.3% (95% CI, 13.2%-27.4%), to clarithromycin it was 16.4% (95% CI, 9.9%-22.9%), and to tetracycline it was 0.8% (95% CI, 0.0%-2.3%). No isolate was resistant to amoxicillin. Clarithromycin resistance increased from 9.1% in 2009-2010 to 24.2% in 2011-2013. In multivariate analysis, prior treatment of H pylori infection and use of fluoroquinolones were significantly associated with clarithromycin and levofloxacin resistance, respectively. CONCLUSIONS: H pylori resistance to clarithromycin increased between 2009 and 2013; resistance to metronidazole remains high in infected men in the United States. The high frequency of resistance to levofloxacin is a new and concerning finding.

Improved allele-specific PCR assays for detection of clarithromycin and fluoroquinolone resistant of Helicobacter pylori in gastric biopsies: identification of N87I mutation in GyrA.

Molecular testing can rapidly detect Helicobacter pylori susceptibility using gastric biopsies. Allele-specific polymerase chain reaction (ASP-PCR) was used to identify H. pylori 23S rRNA and gyrA mutation using gastric biopsies from Colombian patients and confirmed by PCR and sequencing of the 23S rRNA and gyrA genes. The sensitivity and specificity of ASP-PCR were compared with susceptibilities measured by agar dilution. Samples included gastric biopsies from 107 biopsies with H. pylori infections and 20 H. pylori negative. The sensitivity and specificity of ASP-PCR for the 23S rRNA gene were both 100%. The sensitivity and specificity of ASP-PCR for the gyrA gene, published in 2007 by Nishizawa et al., were 52% and 92.7%, respectively; the lower sensitivity was due to the presence of mutation N87I in our samples, which were not detected by the test. In this study, we designed new primers to detect the mutation N87I in GyrA. The ASP-PCR was performed with the original primers plus the new primers. The molecular test with the new primers improved the sensitivity to 100%. In conclusion, ASP-PCR provides a specific and rapid means of predicting resistance to clarithromycin and levofloxacin in gastric biopsies.

Identification of the genes that contribute to lactate utilization in Helicobacter pylori.

Helicobacter pylori are Gram-negative, spiral-shaped microaerophilic bacteria etiologically related to gastric cancer. Lactate utilization has been implicated although no corresponding genes have been identified in the H. pylori genome. Here, we report that gene products of hp0137-0139 (lldEFG), hp0140-0141 (lctP), and hp1222 (dld) contribute to D- and L-lactate utilization in H. pylori. The three-gene unit hp0137-0139 in H. pylori 26695 encodes L-lactate dehydrogenase (LDH) that catalyzes the conversion of lactate to pyruvate in an NAD-dependent manner. Isogenic mutants of these genes were unable to grow on L-lactate-dependent medium. The hp1222 gene product functions as an NAD-independent D-LDH and also contributes to the oxidation of L-lactate; the isogenic mutant of this gene failed to grow on D-lactate-dependent medium. The parallel genes hp0140-0141 encode two nearly identical lactate permeases (LctP) that promote uptake of both D- and L-lactate. Interestingly an alternate route must also exist for lactate transport as the knockout of genes did not completely prevent growth on D- or L-lactate. Gene expression levels of hp0137-0139 and hp1222 were not enhanced by lactate as the carbon source. Expression of hp0140-0141 was slightly suppressed in the presence of L-lactate but not D-lactate. This study identified the genes contributing to the lactate utilization and demonstrated the ability of H. pylori to utilize both D- and L-lactate.

Greater than 95% success with 14-day bismuth quadruple anti- Helicobacter pylori therapy: a pilot study in US Hispanics.

BACKGROUND: 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. METHODS: A one-arm, open-label pilot study of H. pylori-infected, asymptomatic/mildly dyspeptic adults, Hispanic residents of El Paso, Texas, received a 14-day course of omeprazole, plus the combination capsule. We cultured and Gram-stained specimens obtained using a minimally invasive orogastric brush. Helicobacter pylori status was determined by (13)C-urea breath test at 4 or more weeks post-therapy. RESULTS: Forty-seven subjects (7 men and 40 women, average age 42 years) were entered. The per-protocol effectiveness was 97.1% (33/34) (95% mid-P CI: 86.3, 99.9); 100% of metronidazole-resistant strains were eradicated. Side effects were mild and self-limited but contributed to nonadherence. Therapy taken for <10 days was more likely to result in eradication failure (p < .001). Office-based orogastric brushing was well tolerated; positive cultures were obtained in 95%. Gram staining showed H. pylori-like forms in all specimens. CONCLUSIONS: This pilot study supports the concept that 14-day OBMT therapy is likely to be more efficacious for H. pylori eradication (Grade A, PP basis) than a 10-day course where metronidazole resistance is suspected. If confirmed, 14 days should be recommended in populations where metronidazole resistance is common.

Detection of clarithromycin resistance in Helicobacter pylori following noncryogenic storage of rapid urease tests for 30 days.

OBJECTIVE: Traditional Helicobacter pylori (H. pylori) eradication therapy has been undermined by increasing antimicrobial, especially clarithromycin, resistance. Susceptibility testing in some areas is difficult to achieve or unavailable. We aimed to determine whether gastric biopsy specimens stored at room temperature for rapid urease test (RUT) were suitable for clarithromycin susceptibility testing of H. pylori. METHODS: After 30 days of storage at room temperature, DNA was extracted from gastric biopsies present in RUTs (Hpfast). H. pylori status and clarithromycin susceptibility were evaluated using H. pylori-specific polymerase chain reaction (PCR) for ureA, vacA, and allele-specific primer-PCR of the 23S rRNA genes. The PCR results were compared with histology, RUT, and culture results. H. pylori positive was defined as RUT and either culture or histology positive; H. pylori negative as RUT, culture and histology negative. RESULTS: Samples from 31 patients were evaluated; 11 were H. pylori positive including 9 by culture; seven of which had allele-specific primer-PCR results from the RUT specimen for the detection of mutations of the 23S rRNA gene. When both tests were available, culture and PCR results were concordant in 7 cases. In 15 of the 20 histology, RUT and culture negative patients, three PCR were negative. In one patient, all of the three tests were positive; and in three only the 23S rRNA was positive and in one only ureA was positive. CONCLUSION: Gastric biopsy specimens stored in the gel of RUT for 30 days can be used for molecular testing to confirm the diagnosis of H. pylori infection and test for clarithromycin susceptibility.

Helicobacter pylori virulence factor genotypes in children in the United States: clues about genotype and outcome relationships.

The most common Helicobacter pylori genotype among 37 U.S. children was cagA positive, vacA s1m1, and oipA "on" (n = 17, 45.9%), followed by cagA negative, vacA s2m2, and oipA "off" (n = 8, 21.6%), similar to the pattern in adults. cagA positivity was more common in blacks than in whites (i.e., 100% versus 56.5%, P = 0.032).

Practical rapid, minimally invasive, reliable nonendoscopic method to obtain Helicobacter pylori for culture.

BACKGROUND: Helicobacter pylori culture typically requires endoscopy. AIM: To develop a minimally invasive rapid and reliable method to obtain H. pylori cultures. METHODS: An extendable oro-gastric brush, contained within a plastic over-tube, was constructed (Baylor Brush, US Endoscopy). After topical oral anesthesia, the 5-mm diameter brush assembly was swallowed. The brush was extended in the stomach and the mucosa was brushed three or four times. The brush was then retracted into the protective sleeve and withdrawn from the patient. The brush was either cultured directly or placed in cysteine transport medium with 20% glycerol which was then sampled immediately or after freezing at -70 degrees C. RESULTS: Twenty-five adult H. pylori-infected subjects (13 male, 12 female) were studied. Helicobacter pylori recovery rate was 100% (11 of 11) when cultured immediately or after storage in transport medium at -70 degrees C for 1 or 2 weeks or after storage at 4 degrees C for 24 hours (four of four) or 72 hours (four of four) before being cultured. Freezing on dry ice and air shipment did not reduce recovery. CONCLUSION: Rapid, reliable, nonendoscopic culture of gastric mucus is a practical method to obtain culture of H. pylori for clinical or research purposes. The method is amenable to being performed in a doctor's office or in the field.

Tetracycline-resistant clinical Helicobacter pylori isolates with and without mutations in 16S rRNA-encoding genes.

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 microg/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 (AGA(965-967)). Single-nucleotide substitutions were associated with reduced ribosomal binding and with slightly increased tetracycline MICs (1 to 2 microg/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.

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). METHODS: Helicobacter pylori-infected volunteers received a UBT, 40 mg of famotidine at bedtime, and a second UBT (pudding test meal, 648 mg NaHCO3 tablet then 125 mg of urea in 200 ml of water containing 650 mg of NaHCO3). Experiment 2 consisted of four UBTs. Two were standard citric acid UBTs with 75 mg of urea and 2 g citric acid and two were sequential bicarbonate-citric acid UBTs. Sequential UBTs consisted of administration of a 648 mg bicarbonate tablet with 50 g of Polycose in 200 ml of water. Five minutes later, 125 mg of 13C-urea was given in 75 ml of water containing 650 mg of NaHCO3. Breath samples were collected after 15 minutes. Then, to acutely acidify the stomach, 4 g of citric acid was given in 200 ml of water. A second breath sample was collected 15 minutes after the citric acid. The standard UBTs were done before and after 6 days of famotidine (40 mg b.i.d.). Sequential UBTs were done after 1 and 6 days of famotidine therapy. Gastric biopsies for histology, culture, and mucosal cytokines were assessed before and after 6 days of famotidine. RESULTS: Eighteen subjects participated, 10 in each experiment; seven had endoscopy with biopsy. Famotidine/ bicarbonate resulted an approximately 50% fall in UBT values (p = .021) with 10% becoming negative. The gastric pH increased from 5.1 +/- 0.5 to 6.7 +/- 0.2 (p = .03) although no pH value predicted the occurrence of false negative results. Under famotidine acid suppression, NaHCO3 reduced the delta over baseline (DOB) by 63% (p = .021). This was reversed with citric acid. Histology showed a H2-receptor antagonist-associated increase in the depth of gastric corpus inflammation. CONCLUSIONS: H2-receptor antagonists differ from proton pump inhibitors as high intragastric pH may cause a reduction in urease activity, unrelated to a reduced bacterial load and reversed by citric acid.

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.