Extended Spectrum Beta-lactamaseproducing Bacteria: Molecular Studies And Effects Of Medicinal Plants

ABSTRACT 

 Molecular studies of ESβL-producing bacteria isolated from clinical samples was determined as well as the effects of medicinal plants on the isolates. The study was conducted from September 2013 to September 2015. A total of 480 bacterial isolates (250 from urine, 110 from wound swab & 120 from HVS & US)  were used. The knowledge, attitude and practices (KAP) of health workers in Imo State towards multiple antibiotic resistant bacterial isolates and extended spectrum beta-lactamase producing bacteria was initially ascertained using structured questionnaire. Antimicrobial resistance profile was determined by the Kirby-Bauer technique. Phenotypic expression of β-lactamases production was performed by the double disk diffusion method.. Genomic DNA extraction was 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 the agarose gel electrophoresis. Plasmid curing was effected with acridine-orange. Extraction of the active components from plant material was conducted. Effects of the plant extracts on ESβL producing bacteria and the minimum inhibitory concentration were also determined.  Analysis of Variance (ANOVA) was used for the analysis of the data via SPSS software package. The result showed that the respondents had good knowledge of multiple antibiotic resistant bacterial infections and their causes. The results of screening the isolates with 12 antimicrobials showed that the isolates expressed high resistance rates. Examination of clinical samples showed higher prevalence of multiple antibiotic resistant (MAR) Escherichia coli (50.3%) than P. aeruginosa (43.3%) and Klebsiella species (36.6%). The prevalence of ESβL-producing isolates was highest (67.6%) amongst E. coli than 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 βlactamases 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%), Klebsiella species 3 (30%) and P. aeruginosa 4(40%) was found to habour ESβL genes as against, E. coli 8(80%), Klebsiella species 6 (60%) and P. aeruginosa  9(90%)  before curing.  Post curing results showed that half of the gene markers were borne on the plasmids while the other half were borne on the chromosomes. The plasmids and the beta-lactamase genes probably played important roles in the resistances identified. Studies on antibacterial effects of the medicinal plants showed that Ocimum gratissimum and Xylopia aethiopica exhibited higher growth inhibitory effects on the bacterial isolates than Allium sativum, Vernonia amygdalina and Garcinia kola.  The highest growth inhibitory effect was exhibited on E. coli and Klebsiella species by, Ocimum gratissimum and on Escherichia coli by Xylopia aethiopica. The minimal inhibitory concentrations of the selected medicinal plant extracts on the test bacterial isolates were higher for ESβL-bacteria than for non resistant isolates. Despite the high knowledge of MAR ESβL discovered in this study by healthcare practitioners, it appears that much attention is not given to MAR and ESβL-bacteria infections in the State. Extracts of commonly used medicinal plants in Imo State, Nigeria, such as those used in the present study are capable of inhibiting growth of MAR and ESβL-producing bacteria. Therefore there is need for further investigations in terms of toxicological studies and purification of active components with a view to exploiting the plants in novel drug development.    

Keywords: Beta-lactamase, clinical isolate, ESβL-producing bacteria, medicinal plants, Molecular