• antimicrobial susceptibility
• CNS
• ovine milk
• Staphylococcus

Coagulase Negative Staphylococci (CoNS) represent one of the main responsible agents of subclinical mastitis in sheep. They are considered as opportunistic pathogens since they are usually present in the milking environment, equipment, and teat surface. CoNS can cause persistent infections leading to an increased number of somatic cells, changes in milk composition, and reduction of production. The aim of this study was to isolate, identify and characterize CoNS isolates from bulk tank ovine milk. One hundred and twenty ovine milk samples were collected from different sheep farms located in Tuscany and Lazio (Grosseto: 79; Pisa: 16; Viterbo: 10; Firenze: 8; Lucca: 2; Livorno: 2; Pistoia: 1; Arezzo: 1; Prato: 1). For the CoNS isolation MSA agar was employed (48 hours at 37°C). Subsequently, the colonies were streaked onto TSA agar to obtain pure cultures. Catalase, coagulase tests and Gram-staining were then performed. A total of 107 presumptively CoNS isolates were subjected to molecular identification via PCR-RFLP method. Out of the 107 isolates, 87 were identified as CoNS (36 S. epidermidis, 11 S. caprae, 11 S. simulans, 8 S. chromogenes, 5 S. haemolyticus, 4 S. gallinarum, 4 S. equorum, 2 S. arlettae, 2 S. auricularis, 2 S. xylosus, 1 S. jettensis and 1 S. muscae), 2 as Macrococcus caseolyticus and 17 remained unidentified. Among CoNS isolates, 73 isolates were selected for the study of the antimicrobial susceptibility by disc diffusion method following the EUCAST guidelines. Susceptibility against ampicillin (AM), cefoxitin (FOX), cephalothin (KF), cefotaxime (CTX), chloramphenicol (C), clindamycin (DA), gentamicin (CN), tetracycline (TE), trimethoprim-sulfamethoxazole (SXT), enrofloxacin (EN), kanamycin (K), and rifampicin (RA) was assessed. Thirty-four out of 73 isolates (46.6%) were susceptible to all the antimicrobials tested, while the remaining 39 isolates (53.4%) showed resistance to at least one antibiotic. All the isolates were susceptible to FOX, KF, CTX, SXT and ENR. The highest rate was recorded against AM (29/73; 39.7%), suggesting the frequent production of penicillases by CoNS. Resistant phenotypes were also obtained against TE (8/73; 10.9%), DA (6/73; 8.2%), CN (3/73; 4.1%), K (2/73; 2.7%), RA (1/73; 1.3%) and C (2/73, 2.7%). Moreover, 9 isolates out of 73 (12.3%) were resistant to more than one antibiotic. Subsequently, the CoNS isolates were subjected to PCR for the detection of the following resistance genes: blaZ, mecA, mecC, tetK, tetM, tetO, tetL, ermA, ermB, ermC, ermF, ermT, ermY and aac(6')-Ie-aph(2'')-Ia. The results revealed that 44 isolates (60.3%) were positive for at least one of the targeted genes. As suggested by the antibiograms results, a high percentage of isolates was positive for blaZ gene (46.6%). On the contrary, the mecA and mecC genes were never detected, which is consistent with the absence of positives isolates for the cefoxitin phenotype profile. As concerns tetracycline, 23.3% of the isolates harbored one of the targeted tet genes (tetK, tetL, tetM and tetO), with a prevalence of tetK, which was detected in all isolates except for the isolate 53C, which was instead positive for tetM. In addition, none of isolates harboured erm and aac(6')-Ie-aph(2'')-Ia genes.