Faculty: Pharmaceutical Sciences
Department: Pharm. Microbio & Biotech


Ejikeugwu, E. P. C.
Esimone, C.O.
Iroha, I.R.


Gram negative bacteria represent the most relevant reservoir of resistance to antibiotics in the environment. These non-hospital sources of pathogens are of public health importance due to the possibility of transmission of resistant bacteria from these animals to humans through consumption of meat and other animal products. However, the excessive use of antibiotics in the rearing of animals allows bacteria to develop antibiotic resistance through natural selection, and this undermines the efficacy of some available antibiotics due to failure in treatment. This study was aimed at detecting the occurrence of metallo β-lactamase (MBL) and AmpC β-lactamase genes from environmental isolates of Escherichia coli, Klebsiella species and Pseudomonas aeruginosa.
The isolation of E. coli, Klebsiella species and P. aeruginosa was carried out using MacConkey agar, Eosin methylene blue (EMB) agar and cetrimide selective agar. Indole test, urease test, methyl red test, Voges-Proskauer test, oxidase test and citrate test were used for biochemical identification of the isolated organisms. β-lactamase characterization was carried out using nitrocefin sticks. Antimicrobial susceptibility studies were carried out using the Kirby-Bauer disk diffusion technique. Metallo-β-lactamase and AmpC β-lactamase production was phenotypically carried out using ceftazidime-imipenem antagonism test (CIAT), cefoxitin-cloxacillin double disk synergy test (CC-DDST), modified Hodges test, and inhibition based assay. Plasmid DNA was isolated from the bacterial isolates using Zymo® plasmid DNA miniprep kit. Multiplex PCR technique was used to determine the presence of MBL and AmpC genes in the test bacterial isolates. Plasmid curing analysis was carried out to determine the location of the MBL and AmpC genes.
The results of this study showed that from the 370 environmental samples bacteriologically analyzed, a total of 168 isolates of E. coli were isolated while 147 P. aeruginosa isolates and 141 isolates of Klebsiella species were recovered from the environmental samples. The test isolates showed varying levels of susceptibility to the test antibiotics. Metallo-β-lactamase was phenotypically detected in 22 E. coli isolates, 22 P. aeruginosa isolates and 18 isolates of Klebsiella species. However, only 11 E. coli isolates, 24 P. aeruginosa isolates and 18 Klebsiella species were phenotypically confirmed to be AmpC producers. Metallo-β-lactamase encoding genes (particularly the blaIMP-1 genes and blaIMP-2 genes) was detected by PCR in 12 (54.6 %) isolates of E. coli, 15 (83.3 %) isolates of Klebsiella species and 16 (72.7 %) isolates of P. aeruginosa. AmpC genes (particularly the CMY-1 genes and FOX-1 genes) were found in a total of 5 (29.4 %) E. coli isolates, 5 (27.8 %) isolates of Klebsiella species and 10 (41.7 %) isolates of P. aeruginosa. The molecular types of AmpC genes detected were predominantly the CMY-1 genes and the FOX-1 genes which were found specifically in 3 isolates of E. coli, 4 isolates of Klebsiella species, 7 isolates of P. aeruginosa for the CMY-1 genes, and 2 isolates of E. coli, one isolate of Klebsiella species and 3 isolates of P. aeruginosa for the FOX-1 genes.
Conclusively, this study detected the presence of blaIMP-1, blaIMP-2, FOX-1 and CMY-1 genes, which are responsible for MBL and AmpC enzyme production in Gram-negative bacteria. The molecular identification of these genes in Gram-negative bacteria is vital for a reliable epidemiological investigation and forestalling of the emergence and spread of these organisms through the food chain especially through abattoirs and poultry sources