This study investigates the biofilm formation, presence and distribution of virulence genes and the capacity to induce an inflammatory response in strains of Staphylococcus aureus isolated from milk samples in Bahia, Brazil. A total of 132 samples of raw milk were collected from four dairy farms (designated A to D) located in southwestern Bahia, in the municipality of Vitória da Conquista, from October/2009 to September/2010. After processing of the samples, 94 (71.2%) S. aureus isolates were obtained. These strains were subjected to the antibiogram method MIC (Minimum Inhibitory Concentration). As for the pathogenicity, tests were performedin vitrobiofilm formation induced by glucose. Moreover, we performed PCR for their virulence genes: sea (enterotoxin A), seb (B), sec (C), pvl (Panton-Valentine Leukocidin), clfA (Clumping Factor A) and spa (protein A) and analysis of cytokine induction in the inflammatory response of J774 macrophages by exocellular lipoteichoic acid. No isolates were resistant to oxacillin and vancomycin. In biofilm production, 5.31% (5/94) isolates did not produce biofilm, 5.31% (5/94) of the samples were poor producers, 15.96% (15/94) strains were moderate producers, 18.09% (17/94) were producers and 55.32% (55/94) of isolates were strong biofilm producers. One (1.06%) isolate expressed the seb gene, one (1.06%) sec , 18 (19.2%) cflA and 44 (46.8%) had spa . There was no expression of sea and pvl between isolates analyzed. The analysis of cytokine induction in the inflammatory response did not show statistical difference in the levels of IL-6, TNF-α and IL-10 induction. However, there was statistical difference in IL-1 induction between isolates from different farms. Thus, it appears that the results obtained in this study show significant effects for the region studied, since it is an important dairy region, hence the need for further studies, with the intent of attracting funding that contributes to improving prevention and control in both dairy farms and dairy industries, since milk contamination poses a serious potential health risk to consumers.
Smith, E.M., Green, L.E., Medley, G.F., Bird, H.E. and Dowson, C.G. (2005) Multilocus Sequence Typing of Staphylococcus aureus Isolated from High-Somatic-Cell-Count Cows and the Environment of an Organic Dairy Farm in the United Kingdom. Journal of Clinical Microbiology, 43, 4731-4736. http://dx.doi.org/10.1128/JCM.43.9.4731-4736.2005
Veisseyre, R. (1998) Lactologia Técnica: Composición Recogida, Tratamento y Transformacio de la Leche. Acribia, Zaragoza, 1-10.
Amaral, L.A., Romano, A.P.M., Nader Filho, A. and Rossi Júnior, O.D. (2004) Qualidade da água em Propriedades Leiteiras como Fator de Risco à Qualidade do Leite e à Saúde da Glandula Mamária. Arquivos do Instituto Biológico, 71, 417-421.
Zecconi, A. and Hahn, G. (2000) Staphylococcus aureus in Raw Milk and Human Health Risk. Bulletin of the International Dairy Federation, 345, 15-18.
Langoni, H., Domingues, P.F. and Lucheis, S.B. (2008) Mastite Caprina: Seus Agentes e Sensibilidade Frente à Antimicrobianos. Revista Brasileira de Ciência Veterinária, 13, 51-54.
Wu, S.W., Lencastre, H. and Tomasz, A. (2001) Recruitment of the mecA Gene Homologue of Staphylococcus sciuri into a Resistance Determinant and Expression of the Resistant Phenotype in Staphylococcus aureus. Journal of Bacteriology, 183, 2417-2424. http://dx.doi.org/10.1128/JB.183.8.2417-2424.2001
Aung, M.S., Urushibara, N., Kawaguchiya, M., Aung, T.S., Mya, S., San, T., New, K.M. and Kobayashi, N. (2011) Virulence Factors and Genetic Characteristics of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates in Myanmar. Microbial Drug Resistance, 17, 525-535. http://dx.doi.org/10.1089/mdr.2011.0061
Antunes, A.L.S., Perez, L.R.R., Reiter, K.C., Secchi, C. and Freitas, A.L.P. (2007) Detecção da Produção de Biofilmes em Staphylococcus ssp. por Agar Congo Red. Revista de Saúde da UCPel, 1, 1.
Lim, Y., Jana, M., Luong, T.T. and Lee, C.Y. (2004) Control of Glucose- and NaCl-Induced Biofilm Formation by rbf in Staphylococcus aureus. Journal of Bacteriology, 186, 722-729. http://dx.doi.org/10.1128/JB.186.3.722-729.2004
Clinical and Laboratory Standards Institute (2009) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. CLSI, Pensylvania, 105 p.
Fan, H.H., Kleven, S.H. and Jackwood, M.W. (1995) Application of Polymerase Chain Reaction with Arbitrary Primers to Strain Identification of Mycoplasma gallisepticum. Avian Diseases, 39, 729-735. http://dx.doi.org/10.2307/1592409
Proietti, P.C., Coppola, G., Bietta, A., Marenzoni, M.L., Hyatt, D.R., Coletti, M. and Passamonti, F. (2010) Characterization of Genes Encoding Virulence Determinants and Toxins in Staphylococcus aureus from Bovine Milk in Central Italy. Journal of Veterinary Medical Science, 72, 1443-1448. http://dx.doi.org/10.1292/jvms.10-0158
Cassat, J.E., Lee, C.Y. and Smeltzer, M.S. (2007) Investigation of Biofilm Formation in Clinical Isolates of Staphylococcus aureus. Methods in Molecular Biology, 391, 127-144. http://dx.doi.org/10.1007/978-1-59745-468-1_10
Zecconi, A., Cesaris, L., Liandris, E., Daprà, V. and Piccinini, R. (2006) Role of Several Staphylococcus aureus Virulence Factors on the Inflammatory Response in Bovine Mammary Gland. Microbial Pathogenesis, 40, 177-183. http://dx.doi.org/10.1016/j.micpath.2006.01.001
Zafalon, L.F., Arcaro, J.R.P., Filho, A.N., Ferreira, L.M. and Veschi, J.L.A. (2009) Staphylococcus aureus Portadores de Genes de Toxinas Isolados em Amostras de Diferentes Fontes de Transmissão Durante a Ordenha. Revista do Instituto Adolfo Lutz, 68, 269-277.
Borges, M.F., Nassu, R.T., Pereira, J.L., de Andrade, A.P.C. and Kuaye, A.Y. (2008) Perfil de contaminação por Staphylococcus e suas enterotoxinas e monitorização das condições de higiene em uma linha de produção de queijo de coalho. Ciência Rural, 38, 1431-1438. http://dx.doi.org/10.1590/S0103-84782008000500037
Sumathi, B.R., Veeregowda, B.M. and Amitha, R.G. (2008) Prevalence and Antibiogram Profile of Bacterial Isolates from Clinical Bovine Mastitis. Vet World, 8, 237-238.
Unakal, C.G. and Kaliwal, B.B. (2010) Prevalence and Antibiotic Susceptibility of Staphylococcus aureus from Bovine Mastitis. Vet World, 3, 65-67.
Harmon, R.J. (1994) Physiology of Mastitis and Factors Affecting Somatic Cell Counts. Journal of Dairy Science, 77, 2103-2112. http://dx.doi.org/10.3168/jds.S0022-0302(94)77153-8
Fonseca, L.F. and Santos, M.V. (2000) Qualidade do Leite e Controle de Mastite. Lemos Editorial, São Paulo,175 p.
Reis, S.R., Silva, N. and Brescia, M.V. (2003) Antibioticoterapia para controle da mastite subclínica de vacas em lactação. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 55, 651-658. http://dx.doi.org/10.1590/S0102-09352003000600001
Eley, A.R. (1992) Intoxicaciones Alimentárias de Etiologia Microbiana. Acribia, S.A., Zaragoza, 208 p.
Amaral, L.A., Filho, A.N., Júnior, O.D.R. and Penha, L.H.C. (1995) Características microbiológicas da água utilizada no processo de obtenção do leite. Pesquisa Veterinária Brasileira, 15, 85-88.
Andrade, M.A. (1998) Mastite bovina subclínica: Prevalência, etiologia e testes de sensibilidade a drogas antimicrobianas. A Hora Veterinaria, 28, 29-42.
Annemuller, C., Lammler, C. and Zschock, M. (1999) Genotyping of Staphylococcus aureus Isolated from Bovine Mastitis. Veterinary Microbiology, 69, 217-224. http://dx.doi.org/10.1016/S0378-1135(99)00117-0
Fox, L.K., Zadoks, R.N. and Gaskins, C.T. (2005) Biofilm Production by Staphylococcus aureus Associated with Intramammary Infection. Veterinary Microbiology, 107, 295-299. http://dx.doi.org/10.1016/j.vetmic.2005.02.005
Vasudevan, P., Nair, M.K.M., Annamalai, T. and Venkitanarayanan, K.S. (2003) Phenotypic and Genotypic Characterization of Bovine Mastitis Isolates of Staphylococcus aureus for Biofilm Formation. Veterinary Microbiology, 92, 179-185. http://dx.doi.org/10.1016/S0378-1135(02)00360-7
Melo, P.C., Filho, A.N., Ferreira, L.M. and Vicente, H.I.G. (2012) Análise fenotípica e molecular da produção de biofilmes por estirpes de Staphylococcus aureus isoladas de casos de mastite subclínica bovina. Biosciences Journal, 28, 99-102.
Baselga, R., Albizu, I., De La Cruz, M., Del Caho, E., Barberan, M., Amorena, B., et al. (1993) Phase Variation of Slime Production in Staphylococcus aureus, Implications in Colonization and Virulence. Infection and Immunity, 61, 4857-4862.
Cucarella, C., Tormo, M.á., úbeda, C., Trotonda, M.P., Monzón, M., Peris, C., Amorena, B., Lasa, I. and Penades, J.R. (2004) Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus. Infection and Immunity, 72, 2177-2185. http://dx.doi.org/10.1128/IAI.72.4.2177-2185.2004
Melchior, M.B., Fink-Gremmels, J. and Gaastra, W. (2006) Comparative Assessment of the Antimicrobial Susceptibility of Staphylococcus aureus Isolates from Bovine Mastitis in Biofilm versus Planktonic Culture. Journal of Veterinary Medicine, Series B, 53, 326-332. http://dx.doi.org/10.1111/j.1439-0450.2006.00962.x
Zottola, E.A. (1994) Microbial Attachment and Biofilm Formation: A New Problem for the Food Industry? Food Technology, 48, 107-114.
Yang, F.L., Li, X.S., Liang, X.W., Zhang, X.F., Qin, G.S. and Yang, B.Z. (2012) Detection of Virulence-Associated Genes in Staphylococcus aureus Isolated from Bovine Clinical Mastitis Milk Samples in Guangxi. Tropical Animal Health and Production, 44, 1821-1826. http://dx.doi.org/10.1007/s11250-012-0143-z
Kalorey, D.R., Shanmugam, Y., Kurkure, N.V., Chousalkar, K.K. and Barbuddhe, S.B. (2007) PCR-Based Detection of Genes Encoding Virulence Determinants in Staphylococcus aureus from Bovine Subclinical Mastitis Cases. Journal of Veterinary Science, 8, 151-154. http://dx.doi.org/10.4142/jvs.2007.8.2.151
Spanu, V., Spanu, C., Virdis, S., Cossu, F., Scarano, C. and De Santis, E.P. (2012) Virulence Factors and Genetic Variability of Staphylococcus aureus Strains Isolated from Raw Sheep’s Milk Cheese. International Journal of Food Microbiology, 153, 53-57. http://dx.doi.org/10.1016/j.ijfoodmicro.2011.10.015
Ote, I., Taminiau, B., Duprez, J.N., Dizier, I. and Mainil, J.G. (2011) Genotypic Characterization by Polymerase Chain Reaction of Staphylococcus aureus Isolates Associated with Bovine Mastitis. Veterinary Microbiology, 153, 285-292. http://dx.doi.org/10.1016/j.vetmic.2011.05.042
Bhakdi, S., Klonisch, T., Nuber, P. and Fischer, W. (1991) Stimulation of Monokine Production by Lipoteichoic Acids. Infection and Immunity, 59, 4614-4620.
Jones, K.J., Perris, A.D., Vernallis, A.B., Worthington, T., Lambert, P.A. and Elliott, T.S. (2005) Induction of Inflammatory Cytokines and Nitric Oxide in J774.2 Cells and Murine Macrophages by Lipoteichoic Acid and Related Cell Wall Antigens from Staphylococcus epidermidis. Journal of Medical Microbiology, 54, 315-321. http://dx.doi.org/10.1099/jmm.0.45872-0
Lee, J.W., Bannerman, D.D., Paape, M.J., Huang, M.K. and Zhao, X. (2006) Characterization of Cytokine Expression in Milk Somatic Cells during Intramammary Infections with Escherichia coli or Staphylococcus aureus by Real-Time PCR. Veterinary Research, 37, 219-229. http://dx.doi.org/10.1051/vetres:2005051
Dauwalder, O., Thomas, D., Ferry, T., Debard, A.L., Badiou, C., Vandenesch, F., Etienne, J., Lina, G. and Monneret, G. (2006) Comparative Inflammatory Properties of Staphylococcal Superantigenic Enterotoxins SEA and SEG: Implications for Septic Shock. Journal of Leukocyte Biology, 80, 753-758. http://dx.doi.org/10.1189/jlb.0306232
Fijalkowski, K., Czernomysy-Furowicz, D., Irwin, J.A., Nawrotek, P. and Pobucewicz, A. (2011) Secretory Virulence Factors Produced by Staphylococcus aureus Isolates Obtained from Mastitic Bovine Milk—Effect on Bovine Polymorphonuclear Neutrophils. Research in Veterinary Science, 93, 82-87. http://dx.doi.org/10.1016/j.rvsc.2011.08.017
Fijalkowski, K., Czernomysy-Furowicz, D., Nawrotek, P. and Karakulska, J. (2011) Influence of S. aureus Exosecretions on Cytokine Profile in Bovine Leukocyte Cultures in Vitro. Research in Veterinary Science, 93, 1179-1184. http://dx.doi.org/10.1016/j.rvsc.2012.07.003
Oliveira, C.A.F., Fonseca, L.F.L. and Germano, P.M.L. (1999) Aspectos relacionados à produção que influenciam a qualidade do leite. Higiene Alimentar, 13, 10-16.