MOLECULAR CHARACTERISATION OF MULTIPLE ANTIBIOTICS RESISTANT ENTEROBACTER SPECIES ISOLATED FROM POULTRY DROPPINGS OF SELECTED FARMS IN SOUTHWEST NIGERIA

Abstract

Indiscriminate use of antibiotics in poultry production is among the factors responsible for antibiotic resistance by microorganisms. Large amounts of poultry droppings are generated annually which are used in fish feeding and as manure in agricultural farms. However, there is a dearth of information on the antibiotic resistance profile of Enterobacter species, a member of pathogens on the priority list of the World Health Organisation for developing new antibiotics: Enterococcus faecium- Staphylococcus aureus- Klebsiella pneumoniae- Acinetobacter baumannii- Pseudomonas aeruginosa- Enterobacter species, from poultry origin. Therefore, the aim of this study was to determine the antibiotic resistance pattern of Enterobacter species isolated from poultry droppings of selected farms in southwest Nigeria. Poultry dropping samples from layer chickens (24), broiler chickens (16), cockerels (8) and Noilers (4) were aseptically collected from 27 farms across the six states of southwest, Nigeria. Total Heterotrophic Bacterial Count (THBC) was done using pour plate method, while the isolation of Enterobacter species was carried out using standard method. The isolates were identified using the conventional method and Matrix-Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry (MALDI-TOF-MS). Antibiotic susceptibility of the isolates on 20 antibiotics was determined using Kirby-Bauer’s disc diffusion method. Extended Spectrum Beta-Lactamase (ESBL) production of the isolates was determined using phenotypic methods. The ESBL and Antibiotic Resistance (AR) genes were detected with specific primers using polymerase chain reaction. Selected multiple antibiotic resistant isolates from chicken droppings were genome sequenced using Illumnia technology (Mi-Seq). Pathosystems Resource Integration and Centre for Genomic Epidemiology Database were used for genomic analysis. Data were analysed using descriptive statistics. The THBC ranged 8.8×106 ±0.3 (Noilers) to 9.6×106±2.1 CFU/g (layer chickens), while the 72 Enterobacter spp. isolated comprised E. cloacae (52), E. asburiae (12), E. kobei (7) and E. ludwigii (1). The resistance patterns of the Enterobacter spp. showed that all the isolates were resistant to cefpodoxime, cefixime and amoxicillin across the states, while the least resistance was to ciprofloxacin (8.3%). Forty-two of the Enterobacter spp. were ESBL producers out of which 71.4% haboured at least one of the ESBL genes (blaCTX-M, blaTEM and blaSHV). The ampC, qnrB, dfrA1 and ermB, were detected in 52.8% of the Enterobacter species, which are of public health importance. Enterobacter cloacae (ILB8) genome revealed a close relationship with the pathogenic E. hormaechei and E. mori from humans and plants, respectively and contained virulence genes of clinical importance. Forty AR genes were detected in the E. cloacae (ILB8). A class C betalactamase gene (blaACT-16_AB737978) had been identified in another E cloacae strain from a septicaemic neonate, while fosfomycin gene (fosA) had also been identified in E. mori from a diseased Morus alba plant. There is the possibility of the spread of AR genes from bacteria present in poultry droppings to humans and plants through contact with the environment. Enterobacter species from poultry droppings in the southwest Nigeria were multiple antibiotic resistant. Extended Spectrum Beta-Lactamase-producing Enterobacter species had antibiotic resistance genes.