Molecular assessment of virulence determinants, hospital associated marker (IS16gene) and prevalence of antibiotic resistance in soil borne Enterococcus species Syed Abid Ali *, 1 , Hassan Bin-Asif 1 , Khwaja Ali Hasan, Marium Rehman, Atiya Abbasi H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan article info Article history: Received 3 October 2016 Received in revised form 25 February 2017 Accepted 27 February 2017 Available online 28 February 2017 Keywords: Enterococcus Soil Environmental health RAPD PCR Virulence factors abstract Enterococci, no more regarded as GRAS (Generally Recognized As Safe) organism, are emerging as an important source of nosocomial infections worldwide. The main contributors in pathogenesis of enterococci are the presence of various virulent factors and antibiotic resistance genes. We aimed to examine the prevalence, dissemination, antibiotic resistance and virulent factors associated with enterococci from bulk soil (BS). A total of 372 enterococci were isolated from 500 soil samples. PCR was used to identify the isolates up to species level and for carriage of 16 virulence genes including hospital associated marker (i.e. IS16). E. faecium (77%), E. faecalis (10%), E. hirae (4%) and E. casseliflavus (1%) were the major species isolated. The efaAfs was the most dominant gene (100%), followed by gelE (78.9%), sprE (76.3%) and esp (13%) in E. faecalis isolates. The E. faecium carried largely efaAfm (86.8%) and acm (50.3%) genes. Presence of entP (10%), entA (8.3%) and entB (6.9%) genes was detected mostly in E. faecium, while enlA (18%) and ef1097 (2.6%) was only detected in E. faecalis isolates. 50% E. faecalis and 2% E. faecium isolates harbored IS16, while five E. faecalis harbored both IS16 and espTIM genes providing strong evi- dence about the presence of espTIM gene on 64 Kb pathogenicity island. BOX and RAPD PCR analysis revealed high degree of genetic variation within the species. Degree of resistance against 12 major an- tibiotics showed chloramphenicol as the most effective and meropenom as the least effective antibiotic. Presence of multiple antibiotic resistant, virulent and hospital associated enterococci in bulk soil rep- resents a potential source for further dissemination to humans and animals and poses potential impact on public health. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction The essence of our ecosystem is the nature of soil: a very com- plex mixture of air, water, dead organic matter, and various types of living organisms responsible for its broad metabolic capacity [1]. Soils have a natural input of pathogenic microorganisms from different sources such as sewage, recipient water and fertilizers of animal origin which may possess a threat for the community [2]. The ecology of soil entertains numerous communities of patho- genic microorganisms such as Acinetobacter, Agraobacterium, Alcaligens, Arthrobacter, Bacillus, Brevibacterium, Caulobacter, Cellu- lomonas, Clostridium, Corynebacterium, Enterococcus, Fla- vobacterium, Micrococcus, Mycobacterium, Pseudomonas, Staphylococcus, Streptococcus, Xanthomonas and Actinomycetes [3]. It is reported that one gram of soil may contain 1  10 3 to 1  10 6 unique ‘‘species’’ of bacteria in general and 10 1 to 10 3 MPN (i.e. most probable number) of enterococci in particular [4]. Abundance of fecal indicator bacteria (FIB) especially enterococci in agricultural soil, beach sand, temporal or sedimentary soil have already been established whereas some studies also confirms the ubiquity of FIB (especially enterococci) in less impacted soils [5]. Enterococci, the gram-positive, catalase negative, non-spore forming and aero tolerant fermentative organisms form the sec- ond largest group of bacteria studied with reference to microbial source tracking [6]. In view of their ability to survive adverse environmental conditions and adaptable nature to revolutionize from low number commensals to a predominant population of host * Corresponding author. E-mail addresses: abid.ali@iccs.edu, dr.syedabidali@gmail.com (S.A. Ali), hassaanbinasif85@gmail.com (H. Bin-Asif), ali.biochemist@gmail.com (K.A. Hasan), maryam.abdulrehman@yahoo.com (M. Rehman), atiya786@super. net.pk (A. Abbasi). 1 Shared first authors. Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath http://dx.doi.org/10.1016/j.micpath.2017.02.041 0882-4010/© 2017 Elsevier Ltd. All rights reserved. Microbial Pathogenesis 105 (2017) 298e306