1264 MOLECULAR IDENTIFICATION AND ANTIBIOGRAM OF Enterococcus spp. ISOLATED ON ENTEROCOCCUS SELECTIVE DIFFERENTIAL (ESD) MEDIA FROM MEAT, MEAT PRODUCTS AND SEAFOOD IN LIBYA Hesham T. Naas 1 , Zaid Almajdoubi 1 , Aboubaker M. Garbaj 1 , Salah M. Azwai 2 , Fatim T. Gammoudi 2 , Said K. Abolghait 3 , Ashraf A. Moawad 4 , Ilaria Barbieri 5 , Hanan L. Eshamah 1 and Ibrahim M. Eldaghayes* 2 Address(es): 1 Department of Food Hygiene and Control, Faculty of Veterinary Medicine, University of Tripoli, P.O. Box 13662, Tripoli, Libya. 2 Department of Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Tripoli, P.O. Box 13662, Tripoli, Libya. 3 Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Suez Canal University, 41522 Ismailia, Egypt. 4 Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt. 5 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Via Bianchi, 9 - 25124 Brescia, Italy. *Corresponding author: ibrahim.eldaghayes@vetmed.edu.ly ABSTRACT Keywords: 16S rDNA, antibiogram, enterococci, food, Libya INTRODUCTION Enterococcus spp. is a genus of lactic acid bacteria of the phylum Firmicutes that possess Lancefield group D antigen as some of streptococci. Enterococci are Gram-positive cocci, often occur in pairs (diplococci) or short chains bacteria of the gastrointestinal tract of healthy human intestinal flora (Aarestrup et al., 2001). Enterococci are able to survive in extremes of temperature (5 to 60 °C), pH (4.6 to 9.9) and high sodium chloride (6.5% w/v) (Murray, 1990). They are capable of growth in the presence of bile salts (40% w/v) (Fisher and Phillips, 2009) and they commonly occur in foods, especially those of animal origin such as meat and milk (Giraffa, 2003). Previously, all streptococci of fecal origin that produce group D antigen were considered as enterococci (Hartman et al., 2001). Molecular biology studies (including oligonucleotide cataloging of 16S rRNA, DNA-DNA and DNA-rRNA hybridization), combined with physiological studies showed more detailed classification (Schleifer and Kilpper-Bälz, 1987). Members of this genus are: E. avium, E. casseliflavus, E. durans, E. faecalis, E. faecium, E. gallinarum, E. hirae, E. malodoratus and E. mundtii (Hartman et al., 2001). Previous studies have shown that meat and meat products represent a continuous supply of commensal bacteria, including enterococci (Choi and Woo, 2013; Sharifi et al., 2013; Sparo et al., 2013). E. faecalis and E. faecium are common commensal organisms in the intestines of humans were shown to be the predominant isolates in raw meat (beef and pork carcasses) (Knudtson and Hartman, 1993). Meanwhile, E. faecalis was the most frequent isolate among the Gram-positive cocci found in chicken meat (Turtura and Lorenzelli, 1994). In processed meat, the presence of enterococci reflects the extent of initial fecal contamination (Holley et al., 1988). Enterococci are recognized as opportunistic human pathogens and lately have distinguished themselves as major nosocomial pathogens causing bacteremia, endocarditis, urinary tract, central nervous system, intra-abdominal and pelvic infections (Franz et al., 1999). In addition, enterococci can be also used as an enteric contamination indicator (Foulquie Moreno et al., 2006). Enterococci are also known for their capability to exchange genetic information by conjugation (Dunny, 2007) and may spread antibiotic resistance genes among non-pathogenic organisms (Cocconcelli et al., 2003; Fisher and Phillips, 2009). Thus, there is a concern about their presence in uncooked fermented meats because of the contribution they may have to the baseline level of antibiotic resistance in other genera and the potential for transfer of antibiotic resistant bacteria from the indigenous animal microflora to the human gastrointestinal tract (Mathur and Singh, 2005), also leading causes of highly antibiotic-resistant and hospital-acquired infection (Aarestrup et al., 2001). Enterococci are recognized as opportunistic human pathogens, and as indicator for fecal contamination. Due to lack of good hygienic practice in the Libyan slaughterhouses and meat retail markets, therefore, the objectives of this study were to evaluate the presence of enterococci in meat, meat products of different animal species and seafood from different Libyan localities and for their antibiotic resistance profiles. MATERIAL AND METHODS Collection and preparation of samples A total of 104 samples (Table 1) included: raw meat samples (51), meat products (30) and seafood (23), were randomly collected from different cities in Libya (Tripoli, Regdalin, Janzour and Tobruk). The samples were packed in sterile plastic bags, stored in an insulated icebox and transferred as quickly as possible to Food Hygiene and Control Laboratory Department, Faculty of Veterinary Medicine, University of Tripoli. All samples were subjected to Enterococcus spp. microbiological enumeration and isolation techniques. Decimal dilutions, culturing and enumeration techniques were performed according to the methods described by the American Public Health Association (APHA) (Downes et al., 2001). Briefly, 25 g from each sample was aseptically transferred into a sterile stomacher bag (Seward Medicals, UK) and homogenized (Stomacher 400, Seaward Medicals, UK) with 225 mL of sterile peptone water 0.1% (w/v) (Park Scientific, UK) at 230 rpm for 2 min. This study was conducted to investigate the presence of Enterococcus spp. in meat, meat products and seafood. A hundred and four samples were randomly collected from different geographic localities in Libya. The samples were subjected to microbiological analysis for enumeration and isolation of Enterococcus spp. by conventional cultural and molecular identification using PCR and partial sequencing of 16S rDNA techniques. Out of 104 samples, 73 (70.2%) isolates were found to be enterococci based on their cultural characteristics on ESD medium. However, out of 36 samples subjected to molecular identification, only six isolates were confirmed to be Enterococcus spp. using PCR and partial sequencing of 16S rDNA technique. All enterococci strains tested for their antibiotic sensitivity profiles showed high percentage of multi-resistance phenotype. These results can be used for further studies on enterococci as an emerging food borne pathogen and its role in human infection in Libya and would suggest that meat, meat products and seafood might play a role in the spreading of enterococci through the food chain with antimicrobial resistance characteristics. ARTICLE INFO Received 7. 10. 2016 Revised 30. 3. 2017 Accepted 14. 5. 2017 Published 1. 6. 2017 Regular article doi: 10.15414/jmbfs.2017.6.6.1264-1268