Significance and Impact of the Study: This study demonstrates that poultry environment of Karachi city harbours a diverse reservoir of Enterococcus spp. with multiple antibiotic resistance and virulence determinants. It is expected that the results will help in assessing the impact of multiple antibiotic resistance and virulent enterococci on public health, improvement of farm management practices and preventing their dissemination. Our findings strongly suggest the need for reducing antibiotic usage during poultry production, particularly those that are being used to treat human infections. AbstractEnterococci are well-established causative agents of a number of diseases in humans and animals alike. A total of 1047 isolates (838 presumptive enterococci and 209 non-enterococci), related to poultry environment (faeces/ manure, feed, water and air), were evaluated for species diversity, virulence potential, antibiotic resistance and associated threats. Multiplex PCR of 204 presumptive enterococci using genus (16s rDNA)-and species-specific (superoxide dismutase) primer set leads to identification of six Enterococcus spp., i.e. Ent. faecium, Ent. faecalis, Ent. mundtti, Ent. hirae, Ent. gallinarum and Ent. casseliflavus. BOX-PCR analysis of the representative strains confirmed inter-strain variation among species. Although enterococci generally appear to be catalase negative, Ent. faecalis from some faecal, water and air samples showed catalase production. In vitro assays identified poultry environment as a reservoir of MVD and MAR enterococci and non-enterococci. In addition to vancomycin, high-level-ampicillin-, clarithromycin-, erythromycin-, kanamycinand gentamicin-resistant enterococci and non-enterococci are also indicated. Our results show that virulence potential and resistance vary with the source of isolation. Our studies on MAR and MVD enterococci in environment, especially in air and water, clearly indicate the need for a serious commitment to deal with pathogenic enterococci associated with poultry for preventing transmission of infectious agent to humans.

This study aimed to investigate the zoonotic potential by virtue of phylogenetic analysis, virulence and resistance gene profiles of Enterococcus faecalis originating from poultry environment. The ERIC, BOX and RAPD PCR analysis showed the clustering of E. faecalis strains (n = 74) into five groups (G1-G5) and fifteen sub-clusters (B1-B15), which share 50%-80% similarities with ATCC E. faecalis and clinical strains of human infection. E. faecalis strains harboured seven enterocins genes including ent1097 (85%), entB (84%), enterolysinA (51%), entSEK4 (51%), entL50 (31%), entA (25.7%) and ent1071 (14.9%). The highest prevalence of gelE-sprE (90%), lip-fl (90%) followed by cylL (62%), hyl (60%), katA (16%) and cylA (5.4%) was observed in poultry isolates. The fsr operon and gelE-sprE was co-associated in 66.2% strains. E. faecalis also harboured biofilm and endocarditis-associated genes, including efaAfs (97%), ebp-pilli (ebpABC and srtC 69.9%-80%), asa1 (71%), agg (55%), ace (54%) and esp-Tim (3%). Despite all found sensitive to vancomycin, 98.6% strains were multi-drug resistant to five to twelve tested antimicrobials. An increased-level of resistance (≥32 μg/ml) was observed to ampicillin (8.1%), meropenem (21.6%), chloramphenicol (73.4%), erythromycin (90.5%), tetracycline (100%) and high-level resistance to kanamycin (79.7%) and gentamicin (52.7%). The multi-drug resistant E. faecalis (MDRe.f) were carried pbp4 (90%), tetL (90%), tetM (70%), ermB (81%), cat (52.7%), acc6-aph2 (58.1%), aaph(3)-III (49.9%), gyrA (97%) and parC (98%) genes. Moreover, these MDRe.f were also harboured, hospital-associated marker IS16 (58%) and pheromone responsive genes, that is ccf (88%), cpd (74%), cob (62%) and eep (66%). Thus, regardless of the distinct phylogenetic background of E. faecalis of poultry origin, ATCC E. faecalis and clinical strains of human origin, we found major similarities in virulence, resistance gene profiles and mobile genetic elements (IS16 and pheromone responsive plasmids), supporting the zoonotic/reverse zoonotic risk associated with this organism.

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