MOLECULAR STUDIES OF EXTENDED SPECTRUM BETA-LACTAMASE-PRODUCING BACTERIA ISOLATED FROM CLINICAL SAMPLES IN IMO STATE, SOUTH EASTERN NIGERIA.

Chinyere N. Ohalete* and Ifeanyi O. C. Obiajuru

ABSTRACT

Background: Antibiotic – resistant bacterial infections constitute a major health challenge in Nigeria. Many hospitals that encounter these infections lack facilities to detect beta lactamase producing bacteria in circulation. Lack of facilities for ESβL detection leads to incorrect identification of antibiotic resistance which may lead to inappropriate antibiotic prescription. This in turn may lead bacteria to produce new antibiotic - resistant genes by selective pressure. The objective of this study was to determine the existence of extended spectrum beta lactamase producing bacteria (ESβL) in clinical samples and carry out molecular studies on them to determine their nature. Methods: A total of 602 multi antibiotic resistant (MAR) gram negative bacterial isolates comprising, 294 Escherichia coli, 186 Klebsiella species and 122 Pseudomonas aeruginosa were collected from 3 tertiary health institutions in Imo State and examined for beta lactamase production. Antimicrobial resistance profile was determined by the Kirby-Bauer technique. Phenotypic expression of β-lactamase production was performed by the double disk diffusion method. Molecular studies were carried out on ESβL-producing strains of the bacterial isolates. Genomic DNA extraction was carried out by alkaline lysis method and hybridization effected with primers of the three β-lactamases genes, TEM, SHV and CTX-M. The isolated DNA and plasmids were analysed by agarose gel electrophoresis. Plasmid curing was effected with acridine-orange. Gel electrophoretic plasmid profiling was carried out using Thermo-Scientific extraction kit as described by the manufacturers. Polymerase chain reaction (PCR) amplification of extended spectrum beta lactamase gene was carried out using primers for SHV, TEM and CTX-M genes. Bacterial genomic DNA was used as substrate with specific primers for PCR reactions. The PCR amplicon size was calculated by comparison to molecular weight size marker. Results: The results showed that 391 (64. 9%) out of 602 multi antibiotic resistant bacterial isolates comprising of 67.6% Escherichia coli, 64.7% Klebsiella species and 57.7% Pseudomonas aeruginosa were positive for extended spectrum beta lactamase (ESβL) production. Thus the prevalence of (ESβL) bacteria in clinical bacterial isolates was highest (67.6%) amongst E. coli followed by Klebsiella species (64.7%) and P. aeruginosa (57.7%). Gel electrophoresis of the amplified (PCR) genomic products showed that 36.7% were positive for TEM, 66.7% for SHV, and 23.3% for CTX-M genes. Phenotypic screening of isolates for extended spectrum beta-lactamase production before curing showed high resistance to the β-lactam antimicrobials and the β-lactamases inhibitors of amoxicillin/clavulanic acid combination. However, analysis of post curing showed a great reduction in rates of ESβL positive isolates examined. E. coli 4(40%), P. aeruginosa 4(40%) and Klebsiella species 3 (30%) were found to habour ESβL genes as against, E. coli 8(80%), Klebsiella species 6 (60%) and P. aeruginosa 9(90%) before curing. Conclusion: Beta – lactamase – producing bacterial infections are common in Imo State, South eastern Nigeria. They are responsible for 64. 9% of multi antibiotic resistant bacterial infections encountered in hospitals in Imo State. The most common genes associated with ESβL production in Imo State are SHV, TEM and CTX – M.

Keywords: Escherichia coli, Pseudomonas aeruginosa.