Antibiotic susceptibility patterns in Helicobacter pylori strains from patients with upper gastrointestinal pathology in western Nigeria

British Journal of Biomedical Science, 2009 by Oyedeji, K S, Smith, S I, Coker, A O, Arigbabu, A O

ABSTRACT

A total of 186 Helicobacter pylori isolates and 532 gastric biopsies recovered from 532 patients with varying degrees of gastroduodenal pathology are subjected to in vitro antibiotic susceptibility testing using the disc-diffusion method, Etest (MIC breakpoints) and molecular testing using the polymerase chain reaction (PCR). In the isolates studied, antibiotic resistance was as follows: piperacillin (72%), amoxicillin (66%), erythromycin (78%), tetracycline (100%) and metronidazole (95%). All isolates were sensitive to ofloxacin, ciprofloxacin and norfloxacin. None of the 245 amplicons (positive for H. pylori) from the biopsies were digested with the Bbs1 and Bsa1 restriction enzyme used in the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, showing sensitivity to clarithromycin. However, a 238 bp fragment from H. pylori chromosomal DNA (corresponding to the quinolone resistance determining region [QRDR]) of the gyrA gene was amplified successfully. Twelve (4.9%) of the 245 strains studied had the described mutation at position 91, from asparagine (Asn) to glycine (Gly). The study showed that all the H. pylori strains were sensitive to clarithromycin and ciprofloxacin. It also highlighted PCR as a potential tool for faster diagnosis and determination of antibiotic susceptibility (within 24 h) of H. pylori from biopsies and/or isolates recovered from peptic ulcer and gastritis patients.

KEY WORDS: Antibiotics.

Helicobacter pylori.

Polymerase chain reaction.

Polymorphism, restriction fragment length.

Quinolones.

Introduction

Eradication of Helicobacter pylori is the most effective means to cure peptic ulcer disease and prevent possible recurrent episodes. Common treatment regimens include a protonpump inhibitor and two antimicrobial agents (amoxicillin, clarithromycin, metronidazole or tetracycline).

H. pylori generally develops resistance to many antibiotics.1,2 Remarkably, the known mechanisms of antibiotic resistance in H. pylori are not plasmid-mediated but are due to mutations in chromosomal genes, even though plasmids can be detected in half of the isolates.3 In H. pylori, the genetic determinants for resistance to antibiotics such as clarithromycin, metronidazole, ciprofloxacin and rifampin have been determined.4

Clarithromycin resistance is associated with mutations in the 23S ribosomal RNA (rRNA) gene,5* which inhibits the binding of clarithromycin to the ribosome, while rifampin resistance results from mutations in the rpoB gene, encoding the B-subunit of RNA polymerase.7 In metronidazole resistance, although several different mechanisms may exist, the predominant determinant is mutational inactivation of the rdxA gene that encodes an oxygen-sensitive NADPH nitroreductase.8

Quinolones exert antimicrobial activity on H. pylori by inhibiting the enzyme DNA gyrase.9 The region of the mutation for resistance in quinolones has been termed the quinolone resistance determining region (QRDR).10 Resistance to quinolones by H. pylori has been attributed to specific mutations in the gyrA gene," which has been cloned and sequenced. The sequencing of the amplification product from ciprofloxacin-resistant mutants of H. pylori reveals four classes of mutations with substitutions at amino acid 87 (Asn[arrow right]Lys), 88 (Ala[arrow right]Val) and 91 (Asn[arrow right]Gly, Asp[arrow right]Tyr) and a double substitution at amino acids 91 and 97 (Ala[arrow right]Val).1

However, indications for treatment of H. pylori have been extended12 and the probability of emergence of resistant strains increases.13 The successful treatment of any infection lies with the results of in vitro susceptibility testing. In H. pylori infection, the progression of antibiotic resistance requires a faster approach in performing the susceptibility testing using modern molecular techniques that combine the direct detection of H. pylori infection on the biopsy as well as detection of antibiotic resistance.3

This study compares the antimicrobial susceptibility patterns of H. pylori strains isolated from dyspeptic patients in western Nigeria using conventional and molecular techniques.

Materials and methods

Study population

Biopsies collected from 532 patients presenting with varying degrees of gastroduodenal pathology from three hospitals in western Nigeria were studied. All patients provided informed consent and ethical approval was given by the ethical review committee of Lagos University Teaching Hospital, Lagos, and Obafemi Awolowo Teaching Hospital Complex, Ile-Ife, Nigeria.

Sample processing

Primary isolation of the organism was performed on Dent's medium14 consisting of Columbia agar (7%), laked horse blood and Dent's supplement (10 mg vancomycin, 5 mg trimethoprim, 5 mg cefsulodin and 5 mg amphotericin B per litre) (Oxoid). From the 532 biopsies cultured, 186 isolates were obtained for the disc-diffusion and Etest methods of antibiotic susceptibility testing.

Disc-diffusion method

The method of Baeur et a/.15 was employed. The antibiotic discs used were amoxicillin (10 µg), erythromycin (15 µg), piperacillin (100 µg), imipenem (10 µg), gentamicin (10 µg), tetracycline (10 µg), norfloxacin (10 µg), ofloxacin (5 µg), ciprofloxacin (5 µg), kanamycin (30 µg), metronidazole (50 µg) and cefuroxime (10 µg).