Academic journal article Journal of Health Population and Nutrition

Phenotypic and Genetic Characterization of Antimicrobial Profiles of Helicobacter Pylori Strains in Cuba

Academic journal article Journal of Health Population and Nutrition

Phenotypic and Genetic Characterization of Antimicrobial Profiles of Helicobacter Pylori Strains in Cuba

Article excerpt

INTRODUCTION

Helicobacter pylori is responsible for chronic gastritis, peptic ulcer disease, and gastric mucosa-associated lymphoid tissue lymphoma and is a major risk factor for the development of gastric adenocarcinoma (1). Eradication of such bacteria by treatment with two antimicrobial agents--clarithromycin (CLA) and amoxicillin (AMX) or metronidazole (MTZ)--and a proton pump inhibitor is recommended by various consensus groups (2).

Investigation on the susceptibility of H. pylori to antibiotics is one of the main factors associated with successful eradication therapy (3).

MTZ resistance in H. pylori is caused by null mutations in the rdxA gene and less frequently by mutations in frxA and fdxB genes. Resistance to CLA is associated with point mutations in the 23S rRNA gene (2).

The prevalence of H. pylori-resistant strains is high in naive patients and even higher in patients suffering unsuccessful eradication therapy (4).

In Cuba, the prevalence of H. pylori-associated infection among children and adults is 42.4% and 82.7% respectively (5); however, no information is available on antimicrobial susceptibility to commonly-used drugs for the treatment of infection due to H. pylori. This study was conducted to evaluate (a) resistance of five antimicrobial agents and (b) genetic basis for MTZ and CLA resistance in H. pylori strains from a hospital in Havana city, Cuba. The study also investigated the demographic and clinical factors associated with antibiotic resistance.

MATERIALS AND METHODS

Patients, gastric biopsies, and culture

In total, 70 consecutive patients aged 19-68 years were enrolled, and of them, 42 were male and 28 were female. They attended the hospital of the Institute Pedro Kouri, Havana, Cuba, for upper gastrointestinal endoscopy during the last four months of 2005. Of the patients, 51 had non-ulcer dyspepsia (NUD), and 19 had peptic ulcer (PU).

A gastric biopsy specimen obtained from the antrum of each patient was cultured onto selective Columbia agar plates (Oxoid, UK), with 10% sheep-blood and Dent supplement (Oxoid). The plates were incubated at 37[degrees]C in a micro-aerobic atmosphere (Campybag, Oxoid) for 3-5 days. A culture was considered positive if typical H. pylori colonies were observed and the microorganisms grown were positive in catalase, oxidase and urease tests (5).

Susceptibility testing

The minimum inhibitory concentrations (MICs) were determined for MTZ, CLA, AMX, ciprofloxacin (CIP), and tetracycline (TET) by the E-test method (AB Biodisk, Solna, Sweden) as described by the Clinical and Laboratory Standards Institute (CLSI). Mueller Hinton agar (Oxoid), with 5% of aged sheep-blood, was used as culture medium for determining antibiotic susceptibility (6). All tests were performed thrice, and H. pylori ATCC 43504 was used as a control strain.

Susceptibility to CLA was interpreted according to the guidelines of the CLSI (6). Since the CLSI has not designated breakpoints for other antimicrobials in H. pylori, the following MIC values were used in defining resistance: MTZ [greater than or equal to]8 mg/L, AMX and CIP [greater than or equal to]1 mg/L, and TET >2 mg/L (7).

PCR and DNA sequence analysis

Bacterial genomic DNA was extracted using the DNeasy tissue kit (QIAGEN, Japan). Resistant genes--rdxA, frxA, and 23S rRNA--were amplified using PCR with specific primers as previously described (8,9). Amplified DNA was purified using Wizard-SV gel and PCR Clean-Up System (Promega, USA) and was then sequenced in both directions using the ABI PRISM 3100 automatic sequencer (Applied Biosystems, USA). For identification of ribosomal mutations, sequences were compared with the genomic sequences of H. pylori reference strains J99 and 26695 (8). Comparisons were done using the software available over the Internet at the National Center for Biotechnology Information website (http://www. …

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