Molecular fingerprinting of pathogenic Enterococcus isolates using random amplified polymorphic DNA Analysis

Enterococcus sp. is considered to be one of the most prevalent species in the oral cavity, particularly in endodontic infections. The aim of the present study was to investigate the molecular fingerprinting of Enterococcus sp in patient’s urine diversity by randomly amplified polymorphic DNA (RAPD-PCR) analysis of Enterococcus. Specific results for fingerprinting were obtained by the five primers retained for RAPD analysis produced different fragment patterns with varied number of bands. The primers yielded a total of 71 distinct bands 67.6 % were considered as polymorphic bands and 32.4% were considered as monomorphic bands. The OPA-01 primer has showed the highest polymorphism 76.9 % and total of 15 bands ranged from 300 bp-1700 bp. While, the OPD-05 primer has showed the lowest polymorphism 57.2 % and total of 14 bands ranged from 190 bp-2200 bp. Interestingly, OPA-01 primer was a unique negative marker for isolate 7 and, on the other hand, gives a positive one unique band only with isolate 9. The Dendrogram based on RAPD results grouped the thirteen Enterococcus isolates into two different clusters with about 82.2% genetic similarity. Moreover, the genetic distance among Enterococcus isolates was relatively low. The smallest genetic distance (0.167) was estimated between E 10 and E 13. Results indicate the presence of sufficient nuclear DNA level variations among studied Enterococcus isolates using RAPD markers, and the RAPD data might be a good source of information about the diversity of native Enterococcus isolates. *Corresponding Author:  khyate_99@yahoo.com Journal of Biodiversity and Environmental Sciences (JBES) ISSN: 2220-6663 (Print) 2222-3045 (Online) Vol. 8, No. 2, p. 208-215, 2016 http://www.innspub. net J. Bio. Env. Sci. 2016 209 | Eissa et al. Introduction Enterococci are Gram-positive, coccus-shaped bacteria and are common colonizers of different animal hosts, plants, soil, food, and water (Devriese et al., 2006; Fisher and Phillips, 2009). Previously, these bacteria were considered as normal commensals of the gastrointestinal tract, oral cavity, and vagina in humans (Klein, 2003; Kayaoglu and Qrstavik, 2004). However, there was recently an increase in nosocomial infections caused by enterococcal species, including urinary tract, bloodstream, endocardium, abdomen, biliary tract, burn wound, and endodontic infections, which were largely attributed to the antimicrobial resistance profiles (Sedgley et al., 2008; Santagati et al., 2012; Lins et al., 2013). Therefore, Enterococci now rank among the top three nosocomial bacterial human pathogens, and several multidrug-resistant strains have emerged that pose great therapeutic challenges (Souto and Colombo, 2008; Zoletti et al., 2011). Among various enterococcal species, Molecular-based methods have shown some promises in genotyping Enterococci bacteria strains (Scheidegger et al., 2009) and randomly amplified polymorphic DNA polymerase chain reactions (RAPD-PCR) assay has become a rapid and reliable tool in Enterococci taxonomy and for intraand interspecies genomic differentiation of various bacteria (Krawczyk et al., 2006; Hassan et al., 2014). RAPD techniques specific for LAB have been established and successfully applied (Nieto-Arribas et al., 2011; Buhnik-Rosenblau et al., 2013). The objectives of this study were to establish a molecular clustering of Enterococci isolates that obtained from Taif patient’s urine by using RAPD fingerprinting. Materials and methods This work was carried out in the Microbial Genetics laboratory, Biotechnology and Genetic Engendering Unit, Scientific Research Center, Taif University, KSA. Isolation and purification of clinical bacterial isolates Thirteen Enterococcus strains isolated from urine swabs that collected from hospitalized patients in the Taif hospitals, KSA according to Franklin and Matthew (2012). Sterile dry swabs were used for streaking of clinical samples onto sterile Petri dishes containing Nutrient agar media (biolife, USA). Inoculated streaked dishes were incubated at 28°C for 48 h. Single colonies were picked up by sterile inoculation needles and then sloped into cultures of nutrient agar media. Genomic DNA extraction The cell pellets from all isolates were used to extract genomic DNA using (Jena Bioscience, Germany) extraction kit following the manufacturer's instructions. RAPD analysis For RAPD analysis, five 10-mer random primers were used (supplied by Amersham Pharmacia Biotech. NJ. USA.). Names and the sequences of the primers are illustrated in Table (1). Following the experiments for optimization of component concentrations, PCR amplification of random primers were carried out according to (Williams et al., 1990; Hassan and Ismail 2014) in 25 μl volume containing 1μl (20 ng) of genomic DNA, 12.5μl of Go Taq® Green Master Mix, Promega, USA. 1μl of primer (20 p.mol), deionized distilled water (up to a total volume of 25 μl). For DNA amplification, the C1000TM Thermo Cycler Bio-Rad, Germany, was programmed under the conditions involving denaturation at 94°C for 5 min; 40 cycles of denaturation at 94°C for 30 Sec, primer annealing at 35°C for 1.5 min and primer extension at 72°C for 2.5 min; final extension step at 72°C for 7 min. Amplified DNA products were analyzed by electrophoresis in 1.5% agarose gel run in TBE. The gels were stained with ethidium bromide (5 μg ml-1). 100 pb. DNA Ladder RTU, (Gene Direx®) was used as a standard. DNA was visualized by UV illumination and then photographed by a Bio-Rad Gel Doc 2000 device.