Biofilm Production and In Vitro Susceptibility to Disinfectants of Staphylococcus Coagulase Positive Isolated from Bovine Milk

Aila Fabiane Peixoto, Elizabeth Sampaio de Medeiros, Rinaldo Aparecido Mota, José Wilton Pinheiro Júnior, Karla Danielle Almeida Soares, Daniel Dias da Silva, Thayná Milena Siqueira Silva Souza, Tamyres Izarelly Barbosa da Silva, Renato Mesquita Peixoto

Abstract


Background: Mastitis is one of the diseases that cause damage to dairy cattle, and coagulase positive Staphylococcus (CPS), are its main etiological agents. The production of biofilms gives these microorganisms a physiological advantage, being an important virulence factor, as it confers their resistance to the action of disinfectants used in hygiene procedures. Therefore, the objective of this study was to evaluate the capacity of CPS, isolated from milk samples from cases of bovine subclinical mastitis in the Alagoas dairy basin, biofilm production and its sensitivity to disinfectant agents used before and after dipping.

Materials, Methods & Results: In the Alagoas Milk Basin Region, 10 properties were selected from the municipalities of Batalha, Cacimbinhas, Major Isidoro and Minador do Negrão, which used a mechanical milking system and supplied milk to a dairy under federal inspection. The California Mastitis Test (CMT) was performed on 1155 cows. The animals diagnosed with subclinical mastitis were classified as: mild (+); moderate (++) and severe (+++). CMT positive samples were collected from 2+, totaling 891 samples. After cleaning the papillary ostium, 2 to 5 mL of milk was collected from each fourth breast before milking began. Subsequently, 0.5 µL of each sample was streak plated by exhaustion on 5% Sheep Blood Agar and incubated at 37°C, with readings at 24 and 48 h. The macroscopic and microscopic characteristics of the colonies, catalase and coagulase test were evaluated. Biofilm formation was determined by the microplate adhesion test. The efficacy of the sanitizers was evaluated with the active ingredients: lactic acid (2%), allantoin (0.05%), iodine (0.5%), chlorhexidine (2.0%), chlorine (2.5%), at times 15, 30 and 60 seconds. It was observed that 91.2% of the samples were able to form biofilm; Of these, 22.2%, 29.6% and 48.2% were strong, moderate and weak producers, respectively. The sensitivity profile of chlorhexidine isolates was 79.73%, 80.41% and 83.78%; iodine was 59.46%, 65.54% and 66.22% sensitivity over the evaluated time. For allantoin, the isolates presented sensitivity of 22.30%, 27.03% and 37.74%, for lactic acid, 18.92%, 20.95% and 22.30% of the samples were sensitive, and to chlorine, 12.6%, 15.58% and 17.57% of the isolates were sensitive. Moreover, it was observed that the longer the exposure to the disinfectant, the greater the effectiveness of the same.

Discussion: The results are in line with literature data. Staphylococcus spp. from milk samples of animals with subclinical mastitis are able to form biofilm, thus being an important virulence factor. The heterogeneity regarding the biofilm production potential of the isolates is due to the different species of Staphylococcus spp. that, through distinct mechanisms, triggers a greater or lesser formation of this virulent factor, as well as having their adhesion tested, end up becoming more strongly attached to the plate. Among the disinfectants tested, it was confirmed that chlorhexidine and iodine are more effective than SCP, which tend to have high levels of sensitivity to these disinfectants. However, although chlorhexidine was more sensitive to SCP, its use in the field should be done with caution, as its effectiveness may be reduced in the presence of chlorinated water or excess organic matter. It is concluded that SCP isolates from bovine milk from subclinical mastitis mammary quarters were able to produce biofilms. These isolates were also in vitro sensitive to chlorhexidine and iodine, but periodic evaluation of the efficacy of disinfectants used before and after milking is necessary.



Full Text:

PDF

References


Amaral L.A., Romano A.P.M., Nader filho A. & Rossi Junior O.D. 2004. Avaliação da eficiência da desinfecção de teteiras e dos tetos no processo de ordenha mecânica de vacas. Pesquisa Veterinária Brasileira. 24(4): 173-177.

Boari C.A., Alves M.P., Tebaldi V.M.R., Savian T.V. & Piccoli R.H. 2009. Formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo. Ciência e Tecnologia de Alimentos. 29(4): 886-895.

Bussab W.O. & Marettin P.A. 2004. Estatística Básica. 5ed. São Paulo: Saraiva, 120p.

Carter G.R. 1988. Fundamentos de Bacteriologia e Micologia Veterinária. São Paulo: Roca, 250p.

Costa E.O., Ribeiro A.R., Watanabe E.T., Garino Junior F., Silva J.A.B. & Thiers S F.O. 1998. Avaliação in vitro dos desinfetantes utilizados na pós-ordenha (teat dipping) para controle da mastite bovina. Revista Napgama. 1(1): 18-22.

Costerton J.W., Stewart P.S. & Greenberg E.P. 1999. Bacterial Biofilms: A Common Cause of Persistent Infections. Science. 284 (5418): 1318-1322.

Coutinho L.C.A., Medeiros E.S., Silveira N.S.S., Silva L.B.G. & Mota R.A. 2012. Eficácia in vitro de desinfetantes utilizados na anti-sepsia dos tetos frente a leveduras isoladas do leite de vaca com mastite. Pesquisa Veterinária Brasileira. 32 (1): 61-65.

Jain A. & Agarwal A. 2009. Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci. Journal of Microbiological Methods. 76: 88-92.

Lopes L., Lacerda M. & Ronda J. 2013. Eficiência de desinfetantes em manejo de ordenha em vacas leiteiras na prevenção de mastites. Revista Científica Eletrônica de Medicina Veterinária. 21(1): 1-9.

Medeiros E.S., Santos, M.V., Pinheiro Júnior, J.W., Faria E.B., Wanderley G.G., Teles J.A.A. & Mota R.A. 2009. Avaliação in vitro da eficácia de desinfetantes comerciais utilizados no pré e pós-dipping frente amostras de Staphylococcus spp. isoladas de mastite bovina. Pesquisa Veterinária Brasileira. 29(1): 71-75.

Melo P.C. 2008. Estudo fenotípico e genotípico da produção de biofilmes por estirpes de Staphylococcus aureus isoladas dos casos de mastite subclínica bovina. 2008. 122f. Jaboticabal, SP. Dissertação (Mestrado em Medicina Veterinária Preventiva) - Faculdade de Ciências Agrárias e Veterinárias – Universidade Estadual Paulista “Julio de Mesquita Filho”.

Melo P.C., Ferreira L.M., Nader-Filho A., Zafalon L.F. & 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. Bioscience Journal. 28(1): 94-99.

Melo P.C., Ferreira L.M., Nader Filho A., Zafalon L.F., Vicente H.I.G. & Souza V. 2013. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis. Brazilian Journal of Microbiology. 44(1): 119-124.

Merino N., Toledo-Arana A., Vergara-Irigaray M., Valle J., Solano C., Calvo E., Lopez J.A., Foster T.J., Penadés J.R. & Lasa I. 2009. Protein A-Mediated Multicellular Behavior in Staphylococcus aureus. Journal of Bacteriology. 191(3): 832-843.

National Mastitis Council (NMC). 2004. Recommended protocols for evaluating efficacy of postmilking teal germicides. The 46th Annual Meeting Proceedings (San Antonio, USA). pp.179-399.

Noel C.C., Motta F.S., Francisco N.L.F.S., Almeida N.R. & Soares L.C. 2016. Perfil de Suscetibilidade Antimicrobiana e Produção de “Slime” de Isolados de Staphylococcus spp. Provenientes de casos de Mastite Bovina na Região Sul-Fluminense. Revista de Saúde. 7(1): 22-26.

Quadros D.G., Andrade A.P., Silva G.A.V. & Kanematsu C.H. 2019. Maior nível tecnológico e escala de produção propiciam melhor qualidade do leite e menor ocorrência de

mastite bovina? Revista Acadêmica de Ciência Animal. 17: e17003.

Ramalho A.C., Soares K.D.A., Silva D.F., Barros M.R.C., José W., Pinheiro Júnior J.W., Oliveira J.M.B., Mota R.A. & Medeiros E.S. 2012. Eficácia in vitro de desinfetantes comerciais utilizados no pré e pós-dipping frente a Staphylococcus spp. isolados em rebanhos leiteiros. Pesquisa Veterinária Brasileira. 32(12): 1285-1288.

Saeki E.K., Peixoto E.C.T.M., Matsumoto L.S., Marcusso P.F. & Monteiro R.M. 2011. Mastite bovina por Staphylococcus aureus: sensibilidade às drogas antimicrobianas e ao extrato alcoólico de própolis. Acta Veterinaria Brasilica. 5(3): 284-290.

Santana E.H.W., Beloti V., Aragon-alegro L.C. & Mendonça M.B.O.C. 2010. Estafilococos em Alimentos. Arquivos do Instituto Biológico. 77(3): 545-554.

Santos S.S. 2009. Investigação da presença e da formação de Biofilmes por estafilococos em micro-usina de beneficiamento de leite. 2009. 76f. Jaboticabal, SP. Dissertação (Mestrado em Medicina Veterinária Preventiva) - Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista “Julio de Mesquita Filho”.

Santos L.L., Pedroso T.F.F. & Guirro E. 2010. Perfil etiológico da mastite bovina na bacia de Santa Isabel do Oeste, Paraná. Ciência Animal Brasileira. 11(4): 860-866.

Santos I.C., Silva D.R., Oliveira A.F., Oliveira V.R. & Martins L.A. 2018. Eficácia in vitro de desinfetantes utilizados no pré-dipping frente a amostras de Staphylococcus spp. Jornal Interdisciplinar de Biociências. 3(1): 1-4.

Stepanovic S., Vukovic D., Davic I., Savic B. & Vlahovic M.S. 2000. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. Journal of Microbiological Methods. 40(2):175-179.

Stepanovic S., Cirkovic I., Mijac V. & Svabic-Vlahovic M. 2003. Influence of the incubation temperature, atmosphere and dynamic conditions on biofilm formation by Salmonella spp. Food Microbiology. 20(3): 339-343.

Yamazi A.K., Moraes P.M., Viçosa G.N., Ortolani M.B.T. & Nero L.A. 2010. Práticas de produção aplicadas no controle de contaminação microbiana na produção de leite cru. Bioscience Journal. 26(4): 610- 618.




DOI: https://doi.org/10.22456/1679-9216.97253

Copyright (c) 2019 Aila Fabiane Peixoto, Elizabeth Sampaio de Medeiros, Rinaldo Aparecido Mota, José Wilton Pinheiro Júnior, Karla Danielle Almeida Soares, Daniel Dias da Silva, Thayná Milena Siqueira Silva Souza, Tamyres Izarelly Barbosa da Silva, Renato Mesquita Peixoto

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.