Molecular Characterization of Carbapenem Resistant Extended Spectrum Beta-Lactamase (ESBL) Producing Enterobacteriaceae

Abstract

Carbapenem resistance amongst the Extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae is increasing worldwide, leading to the Centre for Disease Control and Prevention declaring them an urgent public health threat. Animal species are believed to be a source of most disease causing pathogens in humans, this being the case, despite all the preventative measures in place. Carbapenem-resistant Enterobacteriaceae (CRE) are also believed to have originated from animals although, Carbapenems have not been approved for Veterinary use anywhere in the world. Carbapenem-resistant Enterobacteriaceae confer resistance to most clinically prescribed antibiotics, making it almost impossible to treat diseases they cause. This, together with the lack in newly developed antibiotics has led to use of previously phased out antibiotics associated with toxicity and efficacy in humans, such as fosfomycin, polymyxins and aminoglycosides. In this study Carbapenem-resistant extended spectrum β-lactamase producing Enterobacteriaceae from cattle faeces were isolated and characterized. Samples were collected from commercial and communal farms in the North West Province of South Africa. Three biochemical tests namely the oxidase test, Simmon's Citrate test and Triple Sugar Iron test were used as preliminary tests for screening isolates belonging to the Enterobacteriaceae family. The preliminary isolates were phenotypically tested for resistance to Carbapenem antibiotics using the disk diffusion test and also their growth on Brilliance TM Carbapenem Resistance Enterobacteriaceae agar. Modified Hodge Test was performed on all isolates that were either resistant or intermediate to Imipenem antibiotic determining their ability to produce Carbapenemase enzyme. Extended spectrum β-lactamase (ESBL) production amongst carbapenem resistant isolates was phenotypically tested based on chromogenic behaviour on Brilliance TM Extended Spectrum β-Lactamase (ESBL) agar. Multi drug resistance profiling was determined amongst the isolates using 13 antibiotics namely Piperacillin, Ticarcillin, Amoxicillin, Amoxicillin-clavulanate, Cephalothin, Cefoxitin, Cefuroxime, Cefotaxime, Ceftazidime, Ceftiofur, Cefepime, Aztreonam and Ciprofloxacin. The isolates were characterized as either Klebsiella pneumoniae, Escherichia coli, Salmonella species or Proteus mirabilis using species specific primers. Presence of both extended spectrum β-lactamase and Carbapenem resistance encoding genes was tested on all the resistant isolates. The 233 samples collected yielded 314 isolates of which a total of 280 presumptive isolates were obtained after the biochemical tests were performed. Some 67.5% (189) of the presumptive isolates were resistant to at least one Carbapenem antibiotic with Meropenem resistance at 68.57%, Imipenem 67.86%, Ertapenem 66.07% and Doripenem at 63.57%. The results on BrillianceTM Carbapenem Resistance Enterobacteriaceae agar however, showed a resistance of 69.29% (194), proving the unreliability and human error possibilities associated with phenotypic tests. Of these 53.10% were from the Klebsiella, Enterobacter, Serratia and Citrobacter (KESC) group, 35.03% as Escherichia coli, 8.25% as Non-Enterobacteriaceae and 3.61% belonging to Proteus, Morganella and Providencia group. Only 19.14% (31) of the isolates tested with Modified Hodge test were positive for Carbapenemase production. Of these isolates 151(77.84%) were positive for Extended Spectrum β-Lactamase (ESBL) production, when cultured on Brilliance TM Extended Spectrum β-Lactamase (ESBL) agar. Of these 54.97% belonged to Klebsiella, Enterobacter, Serratia and Citrobacter (KESC) group, 33.77% were Escherichia coli, 7.95% Salmonella, Acinetobacter and other and 3.31% were Proteus, Morganella and Providencia group. About 94.04% of the Carbapenem resistant Extended spectrum β-lactamase producing Enterobacteriaceae were multi-drug resistant where CTX was at 59.60%, ATM 54.30%, CXM 47.68%, PRL 43.71%, FOX 42.38%, KF 41.06%, CAZ 41.06%, EFT 39.74%, A 39.74%, TC 39.07%, CPM 19.87%, CIP 12.58%, and AMC 9.27% resistance. Some 66 (43.17%) of the isolates were identified as Klebsiella pneumoniae, 52 (33.77%) as Escherichia coli, 7 (4.6%) as Salmonella species and 2 (1.32%) as Proteus mirabilis using species specific Polymerase Chain Reaction (PCR). The most prevalent Carbapenem Resistant Enterobacteriaceae gene in the current study was blaKPC 35.76%, followed by blaNDM at 20.53%, blaGES at 17.88%, blaOXA-48 at 10.60%, blaVIM at 6.62% and blaOXA-23 with 3.31%. 8 (5.29%) of the isolates were found to harbour multiple genes with the combination of blaKPC+ blaOXA-23, blaKPC+ blaNDM and blaGES+ blaOXA-48 at 2.65%, 1.32% and 1.32% respectively. The most prevalent extended spectrum β-lactamase gene amongst the isolates was blaSHV at 33.11%, followed by blaTEM with 22.52%, then blaCTX-M with 20.53% and blaOXA at 11.26%. 19 (12.58%) of the isolates harboured multiple genes, where 7.28% had a blaOXA+ blaCTX-M combination and 5.30% had the blaSHV+ blaTEM combination. Similar to clinical cases Klebsiella pneumoniae has been found to be the most prevalent species in Carbapenem resistant extended spectrum β-lactamase producing Enterobacteriaceae isolated in this study. The isolation of multiple genes for both extended spectrum β-lactamase and Carbapenem resistant Enterobacteriaceae genes has been recorded in clinical isolates in South Africa and the current study proved their existence in animals also. Cattle from both commercial and communal farms were found to be carriers of multi-drug resistant Carbapenem resistant Extended spectrum β-lactamase producing Enterobacteriaceae in the current study. These multi drug resistant Carbapenem resistant Extended spectrum β-lactamase producing Enterobacteriaceae were successfully characterised and the genes they harbour identified from the commonly isolated species.