Staphylococcus is the genus most commonly isolated from bovine mastitis in many countries. It may express several virulence factors including biofilm formation, which may protect the bacterial community from antimicrobials’ action, preventing these compounds from reaching its interior, where they reach subinhibitory concentrations (subMIC).
Most biofilm production assays are performed in static conditions, while studies regarding antimicrobial resistance usually do not resemble the udder environment because they are performed at high concentrations. In this study we evaluated the influence of dynamic conditions and media, including Mueller Hinton Broth (MHB) and UHT whole milk (WM), as well as the effect of subMIC concentrations of five different antimicrobial agents on biofilm formation by staphylococci isolated from subclinical mastitis. Results suggest that dynamic conditions and media may influence biofilm formation and revealed that milking simulation may significantly increase biofilm production. Sub-MIC concentrations decrease biofilm formation in MHB but increase in WM, suggesting a protective role of milk against antimicrobial compounds’ action. Therefore, in vitro conditions that simulate the udder environment and in vivo conditions should be included as one of the parameters in evaluation of biofilm producing strains, in order to provide more reliable results.
Amini B, Baghchesaraie H, Haji Ojagh Faghihi M (2009) Effect of different sub MIC concentrations of penicillin, vancomycin and ceftazidime on morphology and some biochemical properties of Staphylococcus aureus and Pseudomonas aeruginosa isolates. Iran J Microbiol 1: 43-47.
Amorena B, Gracia E, Monzón M, Leiva J, Oteiza C, Perez M, Alabart JL, Hernández-Yago J (1999) Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro. J Antimicrob Chemother 44: 43-55.
Barkema HW, Green MJ, Bradley AJ, Zadoks RN (2009) Invited review: The role of contagious disease in udder health. J Dairy Sci 92: 4717-4729.
Cerca N, Martins S, Pier GB, Oliveira R, Azeredo J (2005a) The relationship between inhibition of bacterial adhesion to a solid surface by sub-MICs of antibiotics and subsequent development of a biofilm. Res Microbiol 156: 650-655.
Cerca N, Martins S, Sillankorva S, Jefferson KK, Pier GB, Oliveira R, Azeredo J (2005b) Effects of growth in the presence of subinhibitory concentrations of dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus biofilms. Appl Environ Microbiol 71: 8677-8682.
Clinical and Laboratory Standards Institute (2007) Performance standards for antimicrobial susceptibility testing; 17th informational supplement; Document M100-S17. Wayne, PA.
Clutterbuck AL, Woods EJ, Knottenbelt DC, Clegg PD, Cochrane CA, Percival SL (2007) Biofilms and their relevance to veterinary medicine. Vet Microbiol 121: 1-17.
Kaplan JB, Izano EA, Gopal P, Karwacki MT, Kim S, Bose JL, Bayles KW, Horswill AR (2012) Low levels of β-lactam antibiotics induce extracellular DNA release and biofilm formation in Staphylococcus aureus. mBio 3: e00198-00112.
Kwasny SM, Opperman TJ (2010) Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery. Curr Protoc Pharmacol 50: 13A.18.11-13A.18.23.
Laureti L, Matic I, Gutierrez A (2013) Bacterial responses and genome instability induced by subinhibitory concentrations of antibiotics. Antibiotics 2: 100-114.
Melchior MB, Fink-Gremmels J, Gaastra W (2007) Extended antimicrobial susceptibility assay for Staphylococcus aureus isolates from bovine mastitis growing in biofilms. Vet Microbiol 125: 141-149.
Melchior MB, Vaarkamp H, Fink-Gremmels J (2006) Biofilms: a role in recurrent mastitis infections? Vet J 171: 398-407.
Melchior MB, van Osch MH, Graat RM, van Duijkeren E, Mevius DJ, Nielen M, Gaastra W, Fink-Gremmels J (2009) Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates: evidence for lack of penicillin-resistance in Agr-type II strains. Vet Microbiol 137: 83-89.
Oliveira M, Bexiga R, Nunes SF, Carneiro C, Cavaco LM, Bernardo F, Vilela CL (2006) Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates. Vet Microbiol 118: 133-140.
Oliveira M, Nunes SF, Carneiro C, Bexiga R, Bernardo F, Vilela CL (2007) Time course of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates. Vet Microbiol 124: 187-191.
Peeters E, Nelis HJ, Coenye T (2008) Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 72: 157-165.
Pérez-Giraldo C, Cruz-Villalón G, Sánchez-Silos R, Marttnez-Rubio R, Blanco MT, Gómez-Garcta AC (2003) In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation. J Appl Microbiol 95: 709-711.
Pettit RK, Weber CA, Kean MJ, Hoffmann H, Pettit GR, Tan R, Franks KS, Horton ML (2005) Microplate Alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing. Antimicrob Agents Chemother 49: 2612-2617.
Poutrel B, Gilbert FB, Lebrun M (1995) Effects of culture conditions on production of type 5 capsular polysaccharide by human and bovine Staphylococcus aureus strains. Clin Diagn Lab Immun 2: 166-171.
Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W (2000) Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis. Antimicrob Agents Chemother 44: 3357-3363.
Seixas R, Gabriel M, Machado J, Tavares L, Bernardo F, Oliveira M (2014a) Effect of simulated gastrointestinal conditions on biofilm formation by Salmonella 1,4,,12:i:-. Scientific World Journal 2014: 153956.
Seixas R, Santos JP, Bexiga R, Vilela CL, Oliveira M (2014b) Short communication: Antimicrobial resistance and virulence characterization of methicillin-resistant staphylococci isolates from bovine mastitis cases in Portugal. J Dairy Sci 97: 340-344.
Stepanović S, Cirković I, Ranin L, Svabić-Vlahović M (2004) Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Lett Appl Microbiol 38: 428-432.
Stepanović S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M (2000) A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 40: 175-179.
Stepanović S, Vukovic D, Jezek P, Pavlovic M, Svabic-Vlahovic M (2001) Influence of dynamic conditions on biofilm formation by staphylococci. Eur J Clin Microbiol Infect Dis 20: 502-504.
Taponen S, Pyorala S (2009) Coagulase-negative staphylococci as cause of bovine mastitis-not so different from Staphylococcus aureus? Vet Microbiol 134: 29-36.
Tremblay YD, Caron V, Blondeau A, Messier S, Jacques M (2014) Biofilm formation by coagulase-negative staphylococci: impact on the efficacy of antimicrobials and disinfectants commonly used on dairy farms. Vet Microbiol 172: 511-518.
Tremblay YD, Lamarche D, Chever P, Haine D, Messier S, Jacques M (2013) Characterization of the ability of coagulase-negative staphylococci isolated from the milk of Canadian farms to form biofilms. J Dairy Sci 96: 234-246.
Xue T, Chen X, Shang F (2014) Short communication: Effects of lactose and milk on the expression of biofilm-associated genes in Staphylococcus aureus strains isolated from a dairy cow with mastitis. J Dairy Sci 97: 6129-6134.