SOURCE:

Faculty: Biosences
Department: Applied Microbiology And Brewing

CONTRIBUTORS:

Ugbo, E.N;
Anyamene, C.O;

ABSTRACT:

Beta-lactam antibiotics are the largest and most commonly prescribed antimicrobial agents in Nigeria as well as in the whole world. Bacteria expressing extended-spectrum beta-lactamase (ESBL) break down beta-lactam rings, conferring resistance to extended-spectrum cephalosporins. The present study is aimed at phenotypic screening and molecular characterization of ESBL-producing organisms isolated from patients attending Federal Teaching Hospital Abakaliki in Ebonyi State, Nigeria. A total of 1000 clinical samples (wound, n=350; high vaginal, n=200 and urine, 450) were collected from male (n= 450) and female (n= 550) patients. The bacterial isolates were characterized and identified using standard microbiological identification techniques. Susceptibility patterns to different classes of beta-lactam antibiotics were studied using Kirby-Bauer disc diffusion method. Resistant isolates were screened for the presence of beta-lactamase and extended spectrum beta-lactamase production using nitrocefin stick, double disc synergy test and brilliant ESBL agar phenotypically according to Clinical Laboratory Standard Institute guidelines. Phenotypically positive ESBL isolates were molecularly characterized for the presence of blaTEM, blaSHV and blaCTX-M group genes by polymerase chain reaction method using specific primers. Plasmid analysis was done to check for the presence of ESBL genes in plasmid DNA of the isolates. Statistical analysis was performed using SPSS 16.0 version software package. The PCR products of 16S ribosomal ribonucleic acid (16S rRNA) region of the bacteria isolates were sequenced to identify the isolates to species level. Nucleotide sequences of the identified isolates were deposited into the GenBank and were assigned accession numbers. Phylogenetic tree was reconstructed to reveal the similarities between the isolates and the matched isolates in GenBank using Molecular Evolutionary Genetic Analysis. Four hundred and fifty four (454) clinical isolates (Staphylococcus aureus (n=178), Klebsiella pneumoniae (n=84) and Escherichia coli (n=192)) were obtained from males (n=146) and females (n=308) suspected of wound, high vaginal and urinary tract infection. Bacteria isolates in this study showed varying degrees of resistance to the tested antibiotics. All the bacterial isolates in this study showed high degrees of susceptibility to cefepime ranging from 60.9 % to 82.6 %. Different degrees of resistance were recorded for ceftazidime (23.8 % to 52.2 %), ceftriaxone (27.0 % to 59.6 %), cefpodoxime (43.2 % to 82.6 %), aztreoname (35.1 % to 69.6 %), cefotaxime (36.4 % to 73.9 %) and amoxycline/clavulanic acid (25.0 % to 59.5 %) antibiotics tested on the isolates. Statistical analysis showed significant difference in the antibiotic resistance frequencies of S. aureus, K. pneumoniae, and E. coli (p< 0.05). Out of 264 highly resistant isolates, S. aureus (92, 34.8 %), K. pneumoniae (53, 20.1 %) and E. coli (119, 45.1 %), 130 isolates were identified as beta-lactamase producers. Thus, 23 isolates, S. aureus (1, 4.4 %), K. pneumoniae (7, 30.4 %) and E. coli (15, 65.2 %) were phenotypically confirmed as ESBLs producers. ESBL genes were identified in (20, 86.9 %) of the isolates, (15, 75.0 %) of E. coli and (5, 25.0 %) of K. pneumoniae molecularly. The presences of blaTEM (n=11; 55.0 %), blaSHV (n=7; 35.0 %) and blaCTX-M (n=9; 45.0 %) (5 CTX-M-15, 2 CTX-M-14 and 1 CTX-M-25) genes were confirmed among the isolates. Notably, six (6) E. coli and one (1) K. pneumoniae had multiple ESBL genes. Plasmid analysis revealed presence of plasmid in all the isolates with molecular weights ranging from 1500bp to 21226bp. Molecular screening for the presence of ESBL genes in the plasmid DNA confirmed 13 (65.0 %) isolates to carry the ESBL genes in their plasmid DNA. This study revealed the presence of multiple ESBL genes in genomic DNA and plasmid DNA of clinical isolates and this may encourage high dissemination of ESBL genes. Prompt and introduction of routine clinical detection of ESBL-producing organisms, continuous monitoring of antibiotics resistance and regulating indiscriminate use of beta-lactam antibiotics is necessary in clinical settings for proper disease management and treatment.