Genotypic Characterization of Coagulase-negative Staphylococci Isolated from Sheep Milk in Slovakia

Hitherto very few reports are available presenting identification and molecular characterization of the coagulase negative staphylococci (CNS) from sheep milk in the subclinical stage of mastitis. Furthermore, very scanty data are available on the epidemiological status of CNS in different Slovak provinces. Milk samples from 54 sheep farms located in eastern Slovak region were screened. A total 240 CNS were identified with series of biochemical testes (STAPH-API) and subjected further for genotyping with the help of pulse field gel electrophoresis (PFGE). The most frequently occurring CNS species according the biochemical characterization were: S. epidermidis (36.3 %), S. caprae (21.3 %), S. hominis (6.6 %), S. chromogenes (6.3 %), S. xylosus (5.8 %), S. warneri (5.0 %) and S. capitis (4.6 %). Further PFGE-based characterization of these isolates revealed six pulsotypes of the S. epidermidis, two of S. caprae, three of S. chromogenes, nine of S. hominis, five of S. capitis and seven of S. xylosus. These results contribute to knowledge of the epidemiological situation of the CNS from the subclinical form of mastitis in Slovakia. CNS, PFGE, pulsotype, mastitis, sheep Clinical and subclinical mastitis presents potential health hazard due to the increase in total bacterial count in milk (Fthenakis and Jones 1990; Gonzalo et al. 2006). In case of subclinical mastitis, coagulase negative staphylococci (CNS) are the most frequently isolated bacteria from milk (Ariznabarreta et al. 2002; Bergonier et al. 2003; Burriel 1997, 1998; Deinhofer and Pernthaner 1993; Gonzalo et al. 2002; Lafi et al. 1998; Pengov 2001; Watkins et al. 1991). Traditionally, CNS have been considered to show of these bacteria as the main aetiological agent of mastitis in sheep has been documented (Fthenakis and Jones 1990; Poutrel 1984). The primary impact of CNS-mediated subclinical mastitis is an increased number of somatic cells (Gougoulis et al. 2008; Leitner et al. 2000; Santos et al. 2008). Furthermore, CNS may sensitize and increase susceptibility of mammary cells to other infections (Jarp 1991; Oliver and Jayarao 1997; Seegers et al. 2003; Waage et al. 1999). CNS produce few virulence factors; however, they can cause infections in healthy host tissue. They are opportunists and adhere to metal devices to produce a protective biofilm. Production of biofilm reduces the organism’s susceptibility to antimicrobials (John and Harvin 2007; Vuong et al. 2003). Widely used antibiotics including penicillins, particularly semi-synthetic penicillins, cephalosporins, macrolides, aminoglycosides and tetracyclines are ineffective to control CNS (Cerca et al. 2005; de Allori et al. 2006). The ability to resist antimicrobials and produce biofilm enables CNS to persist on metal devices, milking equipments as well as on the milker’s hands, which serve a major source of staphylococcal spread. Unlike coagulase-positive staphylococci such as S. aureus, epizootiological studies focused on CNS are sporadic. Among CNS, S. epidermidis is the most commonly isolated Address for correspondence: Dr. Mangesh Bhide, Ph.D. Laboratory of Biomedical Microbiology and Immunology Department of Microbiology and immunology, University of Veterinary Medicine Komenského 73, 04181, Košice, Slovakia Phone: +421 904 461 705 Fax: +421 556 323 173 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm ACTA VET. BRNO 2010, 79: 269-275; doi:10.2754/avb201079020269 low pathogenicity for the mammary gland of domestic ruminants. However, the importance staphylococcal species in the subclinical form of mastitis (Aarestrup et al. 1997; Birgersson et al. 1992). Some authors have reported the prevalence of S. epidermidis even higher than 50% of the total CNS isolated from the milk (Ariznabarreta et al. 2002; Moroni et al. 2005a; Moroni et al. 2005b). However, some authors (Chaffer et al. 1999; Taponen et al. 2007) have reported abundance of other species such as S. simulans, S. chromogenes and S. haemolyticus. The popularity of and consumer preference for sheep milk, cheese and related milk products is increasing continuously. This has brought the need to monitor the subclinical form of mastitis. The study and evaluation of the epidemiological situation of CNS in sheep is thus pivotal to avoid the threat of possible milk-born pathogens as well as to control mastitis. Among different methods of CNS characterization pulse field gel electrophoresis, PCR based assays and biochemical characterizations are commonly used methods. In particular, PFGE has profound discrimination power with excellent reproducibility. To date no comprehensive study has been done on the isolation and characterization (genotyping and pulso-typing) of coagulase negative staphylococci from sheep in Slovakia. On this background, this study was aimed to determine the prevalence and genetic diversity of CNS in the milk of subclinically affected sheep from eastern regions of Slovakia. Materials and Methods Animals and samples Milk samples (10 animals/farm) were collected from 54 different farms located in 7 regions of eastern Slovakia (Table 1) in the years 2005-2007. The farms were located 15–200 km apart from each other. 59.3% sheep were of Tsigai breed, 37% were Valachian and 3.7% were Tsigai × Merino. Majority (94.5%) of the farms practiced manual milking. Before milk collection, the udder was palpated carefully and milk was tested with NK test to rule out sampling from a sheep with clinical mastitis. During sample collection, teats were cleaned and disinfected, first streams of milk were discarded and 10 ml of milk were collected in sterile tube. Samples were taken from both teats. Milk samples were transported on ice and processed within 24 h. Ten μl of the sample were inoculated on Columbia blood agar (Oxoid, UK) and incubated at 37 °C for 24 h. Colonies were screened morphologically (colony characteristics and Gram staining) as well as biochemically (catalase and glucose fermentation Hugh-Leifson test, Oxoid, UK). Based on Gram staining and biochemical properties, suspected colonies on the blood agar, representing genus Staphylococcus were picked and subcultured on Baird-parker agar enriched with egg yolk (Oxoid, UK), a selective medium for staphylococci. Colonies on the Baird parker agar were subcultured and subjected for further biochemical tests. Biochemical characterization of the CNS Staphylococcal colonies were further screened for coagulase activity according to the manufacturer’s instructions (Oxoid, UK). Coagulase negative colonies were further subjected to biochemical analysis with the help of API-STAPH 32 ID commercial test kits (Bio-Mérieux, France). Pulsed field gel electrophoresis PFGE was performed as described previously (Fugett et al. 2006). Shortly, bacterial cells were suspended in TE buffer (Tris HCl 1M, EDTA 0.5M, OD ~ 1.3, all chemicals: Sigma, USA) and cells were lysed with lysis buffer (Tris HCl 1 M, EDTA 0.5 M and N-Lauroyl-Sarcosine 10%) for 10 min at 37 oC. Agarose blocks containing cell lysate were prepared (1.2% low melting agarose, 10% SDS, 1% proteinase K) as described earlier (Escudero et al. 2000) and blocks were subjected for the enzymatic digestion. Enzyme digestion of DNA was done either by SmaI or BspI (Fermentas, Amherst, NY) at 37 oC overnight as per the supplier’s instructions. A contour clamped homogenous electric pulse-field apparatus (Bio-Rad, Richmond, USA) was used for DNA fragment separation. Digested DNA was separated by pulse time ramped from 3 to 40 s, for 20 h at 200 volts and 14 oC. Separated DNA was visualized by ethidium bromide and analyzed by using Bio-Numerics software (USA). Phylogenetic analysis and dendrogram was constructed on the basis of arithmetic average clustering (UPGMA). To assess whether a correlation exists between biotyping (API-STAPH test) and pulsotyping (PFGE), kappa (K) test was employed by using Win-Episcope software 2.0 (CLIVE, UK).