All biological material used to perform the present study was collected for diagnostic purposes. The study was performed in accordance with the guidelines for the care and use of animals of the Department of Veterinary Science of the University of Turin. Previous informed consent was obtained by the owners.
Animals and sampling
The study was carried out from July 2012 to July 2013 and included samples collected from 27 postpartum bitches of different breeds, either housed in two breeding kennels (Kennel 1: N = 12; Kennel 2: N = 8) or privately owned (N = 7). Twenty bitches whelped spontaneously, while seven underwent Caesarean section and were prophylactically treated with cephazolin during the following 5 days. On postpartum day 1, 7 and 15, samples of colostrum (D1) and milk (D7 and D15) were collected from the inguinal mammary glands for cytological and bacteriological exams, in order to assess health status and to diagnose overt or subclinical mastitis. For bacteriological samples, after cleaning the area with saline solution and drying it with a clean towel, each gland was milked; the first drops of secretion were discarded, and the following 3–4 drops were intercepted on a bacteriological swab and placed in Amies medium (Copan Innovation, Brescia, Italy).
The health conditions of the mammary glands and pups were checked daily, and neonatal mortality was recorded at D15.
Isolation and identification of coagulase-positive staphylococci
Swabs were seeded on Blood Agar Base N°2 (Biolife, Milano, Italy) with 5 % defibrinated sheep blood (Allevamento Blood, Teramo, Italy) and inoculated in broth containing 6.5 % sodium chloride (Mueller-Hinton broth, Biokar Diagnostics, Alonne, FR). After an incubation period at 37 °C ± 1 °C for 18–24 h, up to five colonies were recovered from blood agar for preliminary identification as coagulase positive staphylococci. Preliminary identification was based on colony morphology, gram stain appearance, catalase test, hemolysis, pigment production and coagulase enzymatic activity, evaluated by a tube coagulase test with rabbit plasma (Istituto Zooprofilattico delle Venezie, Legnaro, Italy).
The pre-enrichment broth was seeded in a methicillin-resistant staphylococci selective medium, CHROMagar® MRSA II (BD BBL™, New Jersey, USA). Selective plates were incubated at 35 °C ± 1 °C for 24–48 h in aerobic conditions. Growth on CHROMagar® MRSA II as pale pink colonies represented the first sign of methicillin resistance. Coagulase positivity was also tested.
Coagulase positive staphylococci species identification was performed by a MALDI-TOF MS: Microflex LT instrument (MALDI Biotyper, Bruker Daltonics) equipped with FlexControl software (version 3.3, Bruker Daltonics).
Antimicrobial susceptibility testing
Colonies grown on blood agar and on CHROMagar® MRSA II were tested for susceptibility to sixteen antimicrobial agents. The test was performed by the disk diffusion method on Mueller-Hinton agar (KIMA S.A.S, Padova, Italy) according to the guidelines of the Clinical Laboratory Standards Institute (CLSI) [17, 18]. Oxacillin was used as a marker to detect mecA-mediated methicillin resistance [17, 19].
Discs of penicillin G (10 IU), ampicillin (10 μg), amoxicillin-clavulanic acid (20 + 10 μg), oxacillin (1 μg), cefalotin (30 μg), spiramycin (100 μg), erythromycin (15 μg), tetracycline (30 μg), tilmicosin (15 μg), tylosin (30 μg), enrofloxacin (5 μg), licosamides (clindamycin CC 2 μg), tiamulin (30 μg), trimethoprim-sulfamethoxazole (1.25 + 23.75 μg) (BD BBL, New Jersey, USA), and cefquinone (30 μg) (Oxoid Ltd, Basingstoke, UK) were used for the antimicrobial sensitivity test. Interpretive criteria for the inhibition zone diameters provided by CLSI [17, 18], or alternatively by the manufacturers (i.e., tylosin), were followed.
Isolates resistant to three or more classes of antimicrobial agents were considered multidrug-resistant .
Identification of MRSP
Oxacillin-resistant S. pseudintermedius strains were confirmed as MRSP after the detection of the mecA gene by PCR . Staphylococcus aureus DSMZ 11729 was used as a control.
Genetic typing of methicillin-resistant S. pseudintermedius (MRSP) strains [Pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing and Multilocus Sequence Typing (MLST)]
spa- typing was performed according to the scheme developed by Moodley et al.  using sequence signatures 5’-AATAATTCA and 3’-GACAAGCG . Multilocus Sequence Typing (MLST) was performed according to Solyman et al. .
SCCmec types I–V, SCCmec III lacking SCC-Hg, II-III, and VII-241 were determined as previously described .
Preparation of chromosomal DNA and plugs (Agarose Prep; Amersham Biosciences, Uppsala, Sweden) for pulsed-field gel electrophoresis (PFGE) were performed according to the Harmony protocol , and S. aureus NCTC8325 was used as a control. DNA was fragmented using 20U SmaI (Fermentas, Vilnius, Lithuania), and the fragments were separated in a CHEF-DR-II system (BIO-Rad Lab, Hercules, CA) with a 1.2 % agarose gel (Agarose NA; GE Healthcare, Uppsala, Sweden). The gel was run for 24 h at 5.6 V cm-1 with pulsed-time ramping 2–5 s at 14 ° C. The analysis of the fragment pattern was performed in BioNumerics® version 7.1 (Applied Maths, Gent, Belgium) on fragments between 9 and 117 kb using the Dice coefficient and Unweighted Pair Group Method with Arithmetic Mean cluster analysis, with position optimization set at 0.5 % and tolerance at 1.2 %.
Analysis of data
The frequency of isolation of coagulase-positive staphylococci and of methicillin-resistant strains was compared between bitches that had been treated or untreated with antimicrobials (Fisher’s exact test) and among successive samples (Chi-squared test). The same test was used to assess the association between antimicrobial treatment and isolation of methicillin-resistant strains. The analysis of data was performed with GraphPad Prism software (GraphPad Software 4.00, California, USA.). P < 0.05 was considered statistically significant.