Proceeding of Veterinary and Animal Science Days 2018, 6th- 8th June, Milan, Italy HAF © 2013 Vol. V, No. 1s ISSN: 2283-3927 l Staphylococcus pseudintermedius (SP) has been associated with high antibiotic-resistance rates (e.g. methicillin) in European countries (Flemming and Wingender., 2010; Osland et al., 2010; Bardiau et al., 2013). This condition could be also related to the ability to produce biofilm (Flemming and Wingender., 2010). The aim of this study was to investigate the presence of methicillin-resistant SP strains and determine their ability to produce biofilm and some crucial virulence factors. Forty-two SP strains, previously determined as multi drug resistant (MDR) by the disk diffusion method using a panel of 17 antimicrobial agents, were selected from our collection and tested phenotypically for the minimum inhibitory concentration (MIC) of methicillin and genotypically for the presence of mecA and blaZ genes. The ability to produce biofilm was assessed phenotypically by two different assays: the Congo Red Agar plates (CRA) and the Microtiter Plate test (MtP) and genetically by the amplification of icaA and icaD genes. Three virulence factors genes coding for bicomponent leukocidin and enterotoxins (luk-I, seC, se-int) were searched. Thirty-six strains revealed a value of MIC major than 16 µg/mL (threshold for resistance); the remaining six isolates were sensible to this antibiotic. The 73% (31/42) were mecA-positive and 86% (36/42) resulted blaZ-positive; all the strains positive for mecA were also positive for blaZ. All SP strains resulted biofilm-producers by MtP assay (Figure 1) and classified as weakly producers (4.7%), moderate producers (47.6 %) and strongly producers (47.6 %). In contrast, only 35.7% of all strains were considered biofilm-producers by CRA method. The amplification of icaA and icaD gene occurred respectively in 66.6% and 97.6%; only one strain was negative for both genes. Almost all strains were positive for luk-I (95%), seC (74%) and se-int (84%). Our data reveal the pathogenicity potential of SP strains from dogs, suggesting that they could be considered zoonotic potential agents and confirming other previous studies (Osland et al., 2010; Singh et al., 2013; Stefanetti et al., 2017). In particular in this study we focused only on the dissemination of mecA and blaZ genes both coding for methicillin-resistance (mecA is present in Keywords Biofilm, methicillin-resistant, Staphylococcus pseudintermedius, icaA, icaD. CORRESPONDING AUTHOR Gabriele Meroni gabriele.meroni@unimi.it JOURNAL HOME PAGE riviste.unimi.it/index.php/haf Biofilm-forming ability and virulence factors of methicillin-resistant Staphylococcus pseudintermedius from canine pyoderma. G. Meroni1,*, P.A. Martino1 1 Department of Veterinary Medicine, Università degli Studi di Milano, Via dell’Università 6, 26900 Lodi, Italy. http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en Proceeding of Veterinary and Animal Science Days 2018, 6th- 8th June, Milan, Italy 56 HAF © 2013 Vol. V, No. 1s ISSN: 2283-3927 chromosome while blaZ in plasmid), but nowadays other authors reported the presence of methicillin-resistant SP strains mediated by mecC gene instead of mecA. Moreover could be observed a clear linkage between antibiotic-resistance and ability to produce biofilm. References Bardiau, M., Yamazaki, K., Ote, I., Misawa, N., Mainil, J.G., 2013.Characterization of methicillin-resistant isolated from dogs and cats. Microbiology and Immunology. 57, 496-501. Flemming, H.C., Wingender, J., 2010. The biofilm matrix. Nature Reviews Microbiology. 8, 623-633. Osland, A.M., Vestby, L.K., Fanuelsen, H.,Schau Slettemeas, J., Sunde M., 2010. Clonal diversity and biofilm-forming ability of methicillin-resistant Staphylococcus pseudintermedius. Journal of Antimicrobial Chemoterapy. 67, 841- 848. Singh, A., Waljer, M., Rousseau, J., Scott Weese J., 2013. Characterization of the biofilm forming ability of Staphylococcus pseudintermedius from dogs. BMC Veterinary Research. 9, 93-98. Stefanetti, V., Bietta, A., Pascucci, L., Marenzoni, M.L., Coletti, M., Franciosini, M.P., Passamonti, F., Casagrande Proietti, P., 2017. Investigation of the antibiotic resistance and biofilm formation of Staphylococcus pseudintermedius strains isolated from canine pyoderma. Veterinaria Italiana. 53, 289-296. Figure 1: A) Example of colourimetric appearance of colonies onto CRA: very red (I), bordeaux (II), almost black (III), black (IV); (B) MtP assay for detection of biofilm production in some strains (column 11 and 12: negative controls). http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en