Molecular epidemiology of Multi-Drug resistant bacteria isolated from the fecal matter of wild birds in the Islamabad region

Abstract

Introduction: Antibiotic resistance is a significant public health concern and contributes to reduced efficacy of the available antibiotic treatment regimens. This problem is considerably understood and has been reported primarily from the clinical settings, though now being recognized in non-clinical settings as well. Efflux of the antibiotic resistant bacteria and their associated resistance genes from clinic into various environmental settings has been reported around the globe but the phenomenon remains unexplored in our local environment. Particularly, the role of wildlife in disseminating the resistant bacteria and their resistance genes has been recognized and reported in various research studies but it has not been explored in Pakistani environment. To evaluate this, we studied the two main groups of bacteria, Staphylococci and Enterobacteriaceae in fecal matter of wild birds of Islamabad region. Aim of the study: We investigated the phenotypic and genotypic antibiotic resistance profiles, biofilm formation ability, typing of Staphylococci, Phylogrouping and pathotyping of Escherichia coli, and investigated the genomes of a small subset of isolates. Methodology: In this cross-sectional study, we collected 720 fecal droppings of wild birds from eight different environmental sites that included hospital sites, recreational parks and a university site during October 2016 to August 2018. Fecal samples were cultured on mannitol salt agar medium for the isolation of Gram-positive bacteria and on MacConkey agar for the isolation of Gram-negative bacteria. Based on the colony morphology, Gram positive isolates and lactose fermenting Gram negative isolates were subjected to a series of biochemical tests. A total of 394 Staphylococci and 556 Enterobacteriaceae were identified and processed for further analysis. Antibiotic susceptibility profile of Enterobacteriaceae was studied against 16 classes of antibiotics while that of Staphylococci was tested against eight classes of antibiotics of clinical and veterinary importance using Kirby Bauer disc diffusion method. Genotypic analysis of resistance genes in the resistant isolates in both bacterial groups was performed through PCR assays and biofilm formation abilities were assessed through microtitre plate assays. Sequence type and plasmid analysis was performed through whole genome sequencing of the diverse resistant isolates of Enterobacteriaceae. Results: Staphylococci: On antibiotic susceptibility testing, 55% of the Staphylococcal isolates exhibited resistance to one or more than one classes of antibiotics. Highest resistance was observed against erythromycin (40%) and tetracycline (21%), followed by cefoxitin (18%), xxii chloramphenicol (13%) and gentamicin (11%). Overall, 26% of the study isolates exhibited multidrug resistance pattern out of which 70 (18%) exhibited phenotypic methicillin resistance based on their resistance to cefoxitin. mecA gene was detected in 11% (45/394) MDR-Staphylococci where 22/45 (51%) were methicillin resistant Staphylococcus aureus (MRSA) and 23/45 (49%) were methicillin resistant coagulase negative Staphylococci (MRCoNS). SCCmec typing analysis of methicillin resistant Staphylococci (MRS) isolates revealed high prevalence of SCCmec type VI (42%) followed by type V (38%), type IV (7%), type II (7%). Based on SCCmec typing, 13% of the MRS isolates were categorized as Hospital acquired methicillin resistant Staphylococci (HAMRS), while 87% as community acquired methicillin resistant Staphylococci (CA-MRS). Fifty percent of the phenotypic methicillin resistant isolates harbored macrolides (erythromycin) coresistance (35/70); MRSA (54%) and MRCoNSwere46%. Prevalence of mefA resistance gene was 69% followed by ermC (50%) and msrA (31%). On biofilm formation assessment, 82% of MDRStaphylococci sensitive to methicillin (MDR-MSS) produced strong biofilm while in MDRStaphylococci resistant to methicillin resistant (MDR-MRS), 90% produced strong biofilm. Enterobacteriaceae: Out of 556 Enterobacteriaceae, 444 (80%) harbored resistance to either one or more than one class of antibiotics. Resistance was high against ampicillin (56%), nalidixic acid (34%), cefotaxime (33%), ceftraxione (32%), and tetracycline (32%). Among the resistant isolates, 256 (46%) had multi drug resistant pattern where 70% were E. coli, 21% Klebsiella pneumoniae. Hospital site 1 harbored the highest number of MDR Enterobacteriaceae (23%), followed by hospital site 2 (15%), Marghazar park 14%, Lake view park 12%. Among, cephalosporin resistant Enterobacteriaceae, 90% were confirmed as extended spectrum β-lactamases (ESBLs) producers through double disc synergy test. On PCR assay, 72% of the ESBLs- Enterobacteriaceae were found to carry blaCTX followed by blaTEM in 56% while blaSHV in 32% of the isolates. Among blaCTX positive ESBLs isolates, 52% belonged to CTX-M-1. Among plasmid mediated quinolone resistance qnrS was prevalent gene in 59% of ESBLs- Enterobacteriaceae followed by aac (6) lb-cr in 39% isolates while aac (6)-lb was in 68% of ESBLs- Enterobacteriaceae resistant to aminoglycosides. Metalloβ- lactamase production was confirmed in 71% carbapenem resistant Enterobacteriaceae. In MBLs, blaNDM was the prevalent gene detected in 55% isolates. On biofilm assessment, 32% of phenotypic ESBLs- Enterobacteriaceae produced strong biofilm among which E. coli were76% and K. pneumoniae (10%). Among MBLs producing isolates, 36% were strong biofilm formers among which 78% were E. coli while 14% were K. pneumoniae. xxiii In Phylogrouping, 23% of the ESBLs- Enterobacteriaceae belonged to B1 and 17% to A, hence belonged to commensals while 7% were in virulent group B2. On pathotyping, 25% of ESBL isolates were identified as atypical Enterohaemorrhagic E. coli (aEHEC) while 22% as extra intestinal E. coli (ExPEC). A subset of six isolates (4 E. coli, 2 K. pneumoniae) were analyzed by whole genome sequencing. Sequence types ST10, ST2178 and ST405 were found in E. coli isolates while ST995 and ST2113 were found in K. pneumoniae isolates. Six plasmid replicons were identified which belonged to incompatibility groups IncY, IncF and IncH. As per our knowledge, E. coli ST2178 is bring reported for the first time from Pakistani environment in the current study. Conclusion: A high prevalence of antibiotic resistant bacteria, carrying multiple genes of clinical importance by wild birds calls for inclusion of these agents in routine surveillance, and further investigations of antimicrobial resistance (AMR) action plans.