Occurrence and Antimicrobial Sensitivity in Staphylococci Isolated from Goat, Sheep and Cow’s Milk

VYLETĚLOVÁ, M., HANUŠ, O., KARPÍŠKOVÁ, R., ŠŤÁSTKOVÁ, Z.: Occurrence and antimicrobial sensitivity in staphylococci isolated from goat, sheep and cow’s milk. Acta univ. agric. et silvic. Mendel. Brun., 2011, LIX, No. 3, pp. 209–214 The aim of this study was to compare the sensitivity to selected antibiotics in staphylococci isolated from goat (n = 60), sheep (n = 60) and cow’s milk (n = 120). The individual milk samples were inoculated onto Blood agar cultivated at 36 °C/24 h.The isolated species of staphylococci were identifi ed using biochemical tests, namely STAPHYtest and identifi cation program TNW pro 6.5. We examined the sensitivity of strains to the spectrum of antibiotics, as follows: vancomycin (VA), amoxicillin/ clavulanic acid (AMC), penicillin (P), rifampicin (RD), oxacillin (OX), tetracycline (TE), erythromycin (E), chloramphenicol (C), clindamycin (DA), gentamicin (CN), ciprofl oxacin (CIP), teicoplanin (TEC), cefoxitin (FOX) and novobiocin (NOV). Altogether, 97 staphylococcal isolates were obtained; 70 from cow’s milk, 11 from goat’s milk and 16 from sheep’ milk. Staphylococcus aureus was the most frequent species in milk of all animal origin tested, was detected in 54 (45%) cow’s milk, 10 (17%) goat’s and 15 (25%) sheep’ milk samples. S. lentus was identifi ed only in goat’s and sheep’ milk whereas in cow’s milk there were representation of staphylococcal species as follows: S. haemolyticus (n = 7), S. chromogenes (n = 2), S. warneri (n = 2), S. xylosus (n = 2), S. epidermidis (n = 2) and unclassifi ed staphylococci (n = 1). The results of S. aureus sensitivity are similar for all tested antibiotics and for all monitored milk: No resistance to vancomycin, rifampicin, chloramphenicol and teicoplanin was recorded in obtained S. aureus isolates whereas the resistance to ciprofl oxacin was found out most o en. milk, Staphylococcus spp., antibiotics Quality of raw milk is closely related to production disorders in animals such as mastitis. Milk obtained from the infected udder is signifi cantly of lower quality than that of the uninfected cows. Mastitis causes the changes in somatic cell count and N-acetyl-ß-D-glucosaminidase activity which are signifi cantly higher in the infected udder. Lactose concentration is lower, whey protein and albumin concentrations are signifi cantly higher (Leitner et al., 2004). Staphylococcus aureus forms the constituent part of microorganisms which participate in mastitis pathogenesis and plays the most important role (in animal and human medicine) among all the species of Staphylococcus genus. In study by Vyletělová et al. (2010), the occurrence of S. aureus was observed within 10 years and it was found out that S. aureus (together with Streptococcus uberis) persists as the main pathogen causing mastitis. Wilson and Richards (1980) indicated 77% of positive fi ndings of S. aureus in subclinical mastitis in cows in Great Britain. The same results (77% positive fi ndings of S. aureus) were reported in Neetherlands (Grootenhuis, 1976). Roberson et al. (1994) performed the ecological study of S. aureus at a cow breeding farm. In this study S. aureus was occurred in the whole dairy chain within the farm (dairy cows, bedding, air, insect, staff , milking system etc.), the highest risk for transfer of this bacteria are posed by the mammary gland and the animal body skin. In addition to the 210 M. Vyletělová, O. Hanuš, R. Karpíšková, Z. Šťástková fi ndings of S. aureus other isolates of coagulasenegative staphylococci (CNS) were recorded, especially S. haemolyticus. In study carried out by Pitkälä et al. (2004) 4 237 positive quarter samples (from total number 12 661 samples – 3 282 cows) were confi rmed and CNS were identifi ed in 2 103 samples (49.6%). The aim of this study was to compare the occurrence of staphylococci in raw milk of caw, goat and sheep origin and its resistance to selected antimicrobial agents. MATERIALS AND METHOD Herd characterization and milk sample collection Individual milk samples were collected from one goat herd (W; White short-haired breed; n = 60), one sheep herd (C; Tsigai breed; n = 60) and one farm keeping two herds of Holstein cows (n = 50 and 70) were used in this study. Goats and sheep were bred in free stables with separate milking parlour and were kept in one farm which was located in the less favorable area in the Czech Republic. The milk samples from these small ruminants were collected from all animals in lactation in herds. Holstein cows were bred also in the free stables with separate milking parlour and samples were collected from animals with clinical (n = 2) and mostly subclinical (n = 117) mastitis. Staphylococci detection and identifi cation Milk samples (0.05 ml) were inoculated onto Blood agar (Oxoid, UK) and cultivated at 36 °C for 24 h. Suspect colonies were isolated again on the Blood agar, cultivated at 36 °C for 24 h and identifi ed biochemically using the STAPHYtest and identifi cation program TNW Pro 7.5 (PlivaLachema). In the case of doubtful results strains were sent to the Czech Strain Collection of Microorganisms in Brno for more accurate identifi cation (using specifi c biochemical tests or molecular methods). Antibiotic susceptibility of S. aureus isolates For antimicrobial susceptibility disc diff usion method has been used with antibiotic discs (Oxoid) as follows: vancomycin, amoxicillin/clavulanic acid, penicillin, rifampicin, oxacillin, tetracycline, erythromycin, chloramphenicol, clindamycin, gentamicin, ciprofl oxacin, teicoplanin, cefoxitin and novobiocin (Table I). Mueller Hinton Agar (HiMedia, India) was used for performing the assay. Restults were interpreted according to CLSI standard (Table I). S. aureus isolates were confi rmed by the multiplex PCR method for detection of the species specifi c fragment SA442 (Martineau et al., 1998) and mecA gene which encodes resistance to methicillin (Bosgelmez-Tinaz et al., 2006). RESULTS AND DISCUSSION List of Staphylococcus species detected in milk samples is shown in Table II. In total, 97 isolates of staphylococci were identifi ed. Staphylococcus aureus was the most frequent species in milk of all origin. S. aureus was found in 45% of examined cow’s milk samples. Goat’s milk and sheep’milk samples were positive for S. aureus in 17% and 25% respectively. S. aureus is the main pathogen causing mastitis in cows in the Czech Republic (Vyletělová et al., 2010). Staphylococcus aureus is also the most frequently isolated from the clinical cases of mastitis in small ruminants (Bergonier et al., 2003; Vyletělová, 2009). Da Silva et al. (2004) detected that S. aureus represented 37% of the isolates from subclinical mastitis in goats and coagulase-negative I: List of antimicrobial agents tested and interpretation according to CLSI standard Antimicrobial agents Symbol Volume Resistant Intermediate Sensitive