Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

Viviane Figueira Marques Cássia Couto da Motta Bianca da Silva Soares Dayanne Araújo de Melo Shana de Mattos de Oliveira Coelho Irene da Silva Coelho Helene Santos Barbosa Miliane Moreira Soares de Souza About the authors

Abstract

Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

Keywords:
Biofilm; Agr types; Antimicrobial resistance; Mastitis

Introduction

Staphylococcus spp. play an important role in the etiology of intramammary infections of dairy cattle. Staphylococcus aureus stands out among the prevalent etiologic agents in this type of infection due to its ability to produce a wide array of virulence factors that contribute to the bacterial invasion.11 Saei HD. coa types and antimicrobial resistance profile of S. aureus isolates from cases of bovine mastitis. Comp Clin Pathol. 2012;21:301-307. Of them, the production of slime, an extracellular mucopolysaccharide, appears to play a crucial role in the adhesion and colonization of the microorganism on the mammary glandular epithelium; this not only favors biofilm formation and their extracellular persistence but also ensures success in their installation and maintenance in the host tissues.22 Coelho SMO, Pereira IA, Soares LC, Pribul BR, Souza MMS. Profile of virulence factors of S. aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil. J Dairy Sci. 2011;94(7):3305-3310. Slime is composed of a high-molecular-weight polysaccharide intercellular adhesin. Its production is mediated by the intercellular adhesion operon (ica) formed by the genes icaA, icaB, icaC, and icaD and a regulator gene, icaR, which encodes the ICAA, ICAB, ICAC, and ICAD proteins.33 Gad GFM, El-Feky MA, El-Rehewy MS, Hassan MA, Aboella H, El-Baky RM. Detection of icaA, icaD genes and biofilm production by Staphylococcus aureus and Staphylococcus epidermidis isolated from urinary tract catheterized patients. J Infect Dev Ctries. 2009;3(5):342-351.

Furthermore, ica-independent mechanisms possibly play an essential role in bacterial biofilm formation. For example, the function of bap, which encodes for the surface protein Bap, is to assist in intercellular adhesion and biofilm formation. This gene has been primarily studied in isolates from bovine mastitis.44 Cucarella C, Solano C, Valle J, Amorena B, Lasa INI, Penadés JR. Bap, a S. aureus surface protein involved in biofilm formation. J Bacteriol. 2001;183(9):2888-2896.

The repression of agr quorum-sensing system is necessary for biofilm formation. Its reactivation in established biofilms through auto inducing peptides (AIPs) addition or glucose depletion triggers biofilm detachment.55 Boles BR, Horswill AR. agr-Mediated dispersal of S. aureus biofilms. PLoS Pathog. 2008;4(4):e1000052. The agr system includes AgrD, the signaling octapeptide produced in high cell density; AgrB, a transmembrane protein responsible for secretion, export, and processing of active AgrD; and AgrC, a membrane receptor that triggers AgrA phosphorylation mechanism when bound to AgrD. The phosphorylated AgrA positively regulates the production of the effector molecule RNA III.66 Geisinger E, Chen J, Novick RP. Allele-dependent differences in quorum-sensing dynamics result in variant expression of virulence genes in S. aureus. J Bacteriol. 2012;194(11):2854-2864.S. aureus can be classified into four polymorphic Agr types (AgrI, AgrII, AgrIII, and AgrIV) based on the specificity of AIP to the signal receptor AgrC.77 Shopsin B, Mathema B, Alcabes P, et al. Prevalence of agr specificity groups among S. aureus strains colonizing children and their guardians. J Clin Microbiol. 2003;41(1):456-459.

Moreover, biofilm production in S. aureus from mastitis can be associated with antimicrobial resistance.88 Cucarella C, Tormo MA, Úbeda C, et al. Role of biofilm-associated protein Bap in the pathogenesis of bovine S. aureus. Infect Immun. 2004;72(4):2177-2185. The mechanisms responsible for this resistance include the physical and chemical diffusion barrier formed by the exopolysaccharide matrix, which hinders antimicrobial penetration, the existence of microenvironments that antagonize the antibiotic action, the activation of stress responses that cause changes in bacterial physiology, and the stable and slower growth of these microorganisms due to nutrient limitation and the absence of antimicrobial targets.22 Coelho SMO, Pereira IA, Soares LC, Pribul BR, Souza MMS. Profile of virulence factors of S. aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil. J Dairy Sci. 2011;94(7):3305-3310.

Antimicrobials such as beta-lactams are preferred for the treatment of staphylococcal infections. However, production of beta-lactamase enzymes, coded by blaZ that hydrolyzes the beta-lactamic ring, and production of low-affinity penicillin binding protein (PBP2a), coded by mecA, may lead to antimicrobial resistance.99 Pehlivanoglu F, Yardimci H. Detection of methicillin and vancomycin resistance in Staphylococcus strains isolated from bovine milk samples with mastitis. Kafkas Univ Vet Fak. 2012;18(5):849-855.

This study aimed to detect the phenotypic expression of biofilm and the presence of structural and regulatory genes involved in the production of this virulence factor. In addition, the stages of biofilm synthesis along the growth curve were evaluated by scanning electron microscopy (SEM), and pheno-genotypic resistance to beta-lactamic and its possible relation to biofilm production were evaluated.

Materials and methods

Sampling and pheno-genotypic and proteomic identification

Three dairy cattle farms located in an important milk production region of Rio de Janeiro, Brazil, were selected owing to the high prevalence of subclinical mastitis on the farms, identified through the California mastitis test and somatic cell count. In total, 120 milk samples were collected in October and November 2012. Fifty nine Staphylococcus spp. were isolated, of which 41 were S. aureus strains.

After phenotypic identification, all 41 strains were submitted to polymerase chain reaction (PCR) for 16S rRNA to confirm the Staphylococcus spp.1010 Zhang K, Sparling J, Chow BL, et al. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of S. aureus from coagulase-negative staphylococci. J Clin Microbiol. 2004;42(11):4947-4955. PCR for coa,1111 Hookey JV, Richardson JF, Cookson BD. Molecular typing of S. aureus based on PCR Restriction Fragment Length Polymorphism and DNA sequence analysis of the coagulase gene. J Clin Microbiol. 1998;36(4):1083-1089.nuc,1212 Ciftci A, Findik A, Onuk EE, Savasan S. Detection of methicillin resistance and slime factor production of S. aureus in bovine mastitis. Braz J Microbiol. 2009;40:254-261. and 23S rDNA1313 Straub JA, Hertel C, Hammes WP. A 23S RNAr-targeted polymerase chain reaction-based system for detection of S. aureus in meat started cultures and dairy products. J Food Prot. 1999;62:1150-1156. genes were performed to characterize S. aureus. The ATCC 29213 S. aureus was used as quality control. Furthermore, all isolates were evaluated by the matrix-assisted laser desorption ionization-time of flight mass spectrometry, as described by Motta et al.,1414 Motta CC, Rojas ACM, Dubenczuk FC, et al. Verification of molecular characterization of coagulase positive Staphylococcus from bovine mastitis with matrix-assisted laser desorption ionization, time-offlight mass spectrometry (MALDI-TOF MS) mass spectrometry. Afr J Microbiol Res. 2014;8(48):3861-3866. considering the accepted values for matches ≥2.

The S. aureus isolates were subjected to disk diffusion tests using amoxicillin (10 µg), ampicillin (10 µg), azithromycin (15 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), cefepime (30 µg), enrofloxacin (5 mcg), erythromycin (15 µg), streptomycin (10 µg), moxifloxacin (5 µg), neomycin (30 mcg), novobiocin (5 mcg), cotrimoxazole (25 µg), and tetracycline (30 µg) disks. After overnight incubation at 35 °C, followed by inhibition zone measurement, the results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) standards.1515 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; Approved Standards; Twenty-fourth Informational Supplement, M100-S24; 2014.

16 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk and Diluition Susceptibility Tests for Bacteria Isolated From Animals; Approved Standards – 4 Ed, VET01-A4; 2013.
-1717 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals; Approved Standards; Second Informational Supplement, VET01-S2; 2013. These isolates were subjected to DNA extraction and amplification of hlA and hlB,1818 Nilsson IM, Hartford O, Foster T, Tarkowski A. Alpha-toxin and gamma-toxin jointly promote Staphylococcus aureus virulence in murine septic arthritis. Infect Immun. 1999;67:1045-1049.fbnA and fbnB,1919 El-Sayed A, Alber J, Lammer C, Jager S, Wolter W, Vázquez HC. Comparative study on genotypic properties of Staphylococcus aureus isolated from clinical and subclinical mastitis in Mexico. Vet Méx. 2006;37(2):165-179. and cap5 and cap8,1919 El-Sayed A, Alber J, Lammer C, Jager S, Wolter W, Vázquez HC. Comparative study on genotypic properties of Staphylococcus aureus isolated from clinical and subclinical mastitis in Mexico. Vet Méx. 2006;37(2):165-179. according to the protocol described by Marques et al.2020 Marques VF, Souza MMS, Mendonça ECL, et al. Análise fenotípica e genotípica da virulência em Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina em regiões do Estado do Rio de Janeiro. Pesq Vet Bras. 2013;33(2):161-170. and Tito et al.2121 Tito TM, Rodrigues NMB, Coelho SMO, Souza MMS, Zonta E, Coelho IS. Choice of DNA extraction protocols from Gram negative and positive bacteria and directly from the soil. Afr J Microbiol Res. 2015;9(12):863-871. To study the biofilm production, 20 S. aureus strains were selected considering their antibiotic resistance profiles and the presence of virulence genes.

Qualitative and quantitative biofilm assay

Biofilm production was measured using qualitative and quantitative assays, described by Marques et al.2020 Marques VF, Souza MMS, Mendonça ECL, et al. Análise fenotípica e genotípica da virulência em Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina em regiões do Estado do Rio de Janeiro. Pesq Vet Bras. 2013;33(2):161-170. All the 20 S. aureus isolates were transferred to sheep blood agar for 24 h at 35 °C. The grown colonies were inoculated into tryptic soy broth (TSB) containing 0.24% glucose to stimulate slime production for 24 h at 35 °C. The bacterial cultures were adjusted to a 0.5 McFarland scale and diluted 1:10 in TSB with the addition of 0.24% glucose. Aliquots of this suspension (200 µL) were inoculated into sterile polystyrene 96-wellmicroplates for 24 h at 35 °C without agitation. After discarding this material, the wells were washed twice with 200 µL sterile saline, oven dried at 65 °C for 1 h, and stained with 200 µL safranin 1% for 15 min. Subsequently, the wells were washed three times with distilled water and dried at room temperature. The absorbance was determined at 490 nm in an ELISA reader (BIO RAD MODEL 680). Uninoculated wells containing TSB broth with 0.24% glucose were used as controls. The tests were performed in triplicate. The strains were classified according to the following OD values: strong ≥0.3, moderate ≥0.2 and <0.3, weak ≥0.1 and <0.2, and negative <0.1.2020 Marques VF, Souza MMS, Mendonça ECL, et al. Análise fenotípica e genotípica da virulência em Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina em regiões do Estado do Rio de Janeiro. Pesq Vet Bras. 2013;33(2):161-170.

Biofilm gene and Agr types

All isolates were subjected to DNA extraction and amplification of icaA and icaD,2222 Vasudevan P, Nair MKM, Annamalai T, Venkitanarayana KS. Phenotypic and Genotipic characterization of bovine mastitis isolates S. aureus for biofilm formation. Vet Microbiol. 2003;92:179-185.bap,88 Cucarella C, Tormo MA, Úbeda C, et al. Role of biofilm-associated protein Bap in the pathogenesis of bovine S. aureus. Infect Immun. 2004;72(4):2177-2185. and agr RNAIII,2323 Reinoso EB (Tese de Doutorado) Análisis epidemiológico y molecular de cepas de S. aureus de distintosorígenes. Rio Cuarto, Argentina: Instituto de Microbiologia, UNRC; 2004, 199 pp. according to the protocol described by Marques et al.2020 Marques VF, Souza MMS, Mendonça ECL, et al. Análise fenotípica e genotípica da virulência em Staphylococcus spp. e de sua dispersão clonal como contribuição ao estudo da mastite bovina em regiões do Estado do Rio de Janeiro. Pesq Vet Bras. 2013;33(2):161-170. and Tito et al.2121 Tito TM, Rodrigues NMB, Coelho SMO, Souza MMS, Zonta E, Coelho IS. Choice of DNA extraction protocols from Gram negative and positive bacteria and directly from the soil. Afr J Microbiol Res. 2015;9(12):863-871. (Table 1).

Table 1
Primers and amplification conditions for the detection of resistance, biofilm genes, and agr genes.

The agr system groups were classified based on the hypervariable domain of the agr locus, according to Shopsin et al.77 Shopsin B, Mathema B, Alcabes P, et al. Prevalence of agr specificity groups among S. aureus strains colonizing children and their guardians. J Clin Microbiol. 2003;41(1):456-459. A forward primer, pan-agr, corresponding to the conserved sequences of agrB, was used in all the reactions. Furthermore, four reverse primers were used, each specific for the amplification of a single group of agr, based on the agr locus polymorphism. Duplex PCR was performed to classify the groups based on the following products: Agr I (440 bp) and Agr II (572 bp) and Agr III (406 bp) and Agr IV (588 bp) (Table 1). PCR products were separated by electrophoresis on 1% agarose gels using SYBR Green (Invitrogen®) diluted dye (1:100). The amplicons were visualized and documented using the image capturing system L-PIX EX (Loccus Biotecnologia®).

Growth curve estimation

A 1-mL aliquot of bacterial culture [106 CFU (colony forming unit)/mL] was diluted ten-fold in a simple broth (0.4% meat extract; 1% peptone, and 0.5% NaCl). Bacterial growth was evaluated considering the following intervals: 0, 2, 4, 6, 8, 10, 12, 24, 30, 36, and 48 h. After incubation at 35 °C for 18 h, the viable cells were counted in plate count agar and expressed as CFU per milliliter. The experiment was performed in triplicate. Further, the biofilm production of N-341 was evaluated considering the intervals determined in the bacterial growth curve.

Preparation of biofilm samples for SEM

Bacterial growth was observed on Petri dishes containing glass cover slips for biofilm adhesion. The isolates N-354, N-365, and N-341 were cultivated in TSA with 0.24% glucose overnight, adjusted to the 0.5 McFarland scale, and diluted 1:10 in TSA with 0.24% of glucose. The aliquots (2 mL each) were placed in each Petri dish containing three glass coverslips and were statically incubated at 35 °C for 4, 8, 12, and 24 h. After incubation, the Petri dish was washed three times with saline (0.85% NaCl) to remove all planktonic cells. The adherent cells were fixed with 5% glutaraldehyde for 5 h. After fixation, the plate was washed three times with 0.1 M sodium cacodylate buffer. For SEM, S. aureus cells were fixed for 30 min at room temperature with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) and post-fixed for 30 min at room temperature with 1% OsO4 solution containing 2.5 mM CaCl2 in the same buffer. The cells were dehydrated in an ascending acetone series and dried using the critical point method with CO2 (CPD 030, Balzers, Switzerland). Subsequently, the samples were mounted on aluminum stubs, coated with a 20-nm gold layer, and examined under a scanning electron microscope (Jeol JSM6390LV) at the Rudolf Barth Electron Microscopy Platform of Institute Oswaldo Cruz.

Evaluation of pheno-genotypic resistance to beta-lactamic

All the 20 S. aureus isolates were subjected to disk diffusion tests using cefoxitin (30 µg), oxacillin (10 µg), penicillin (10 UI), and amoxicillin + clavulanic acid (30 µg) disks. In addition, the “edge zone” test was used to evaluate the production of beta-lactamases. After overnight incubation at 35 °C, followed by inhibition zone measurement,1616 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk and Diluition Susceptibility Tests for Bacteria Isolated From Animals; Approved Standards – 4 Ed, VET01-A4; 2013.,1717 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals; Approved Standards; Second Informational Supplement, VET01-S2; 2013. the results were evaluated as per the interpretation criteria following the CLSI standards.1515 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; Approved Standards; Twenty-fourth Informational Supplement, M100-S24; 2014. PCR was performed for mecA using primers designed by Murakami et al.2424 Murakami KW, Minamide K, Wada W, Nakamura E, Teraoka H, Watanbe S. Identification of methicillin resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol. 1991;29:2240-2244. and Melo et al.2525 Melo DA, Coelho IS, Motta CC, et al. Impairments of mecA gene detection in bovine Staphylococcus spp.. Braz J Microbiol. 2014;45(3):1075-1082. and for blaZ according to Rosato et al.2626 Rosato AE, Kreiswirth BN, Graig WA, Eisner W, Climo MW, Aecher GL. mecA-blaZ corepressors in clinical S. aureus isolates. Antimicrob Agents Chemother. 2003;47:1463-1466. (Table 1).

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests were performed according to CLSI1515 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; Approved Standards; Twenty-fourth Informational Supplement, M100-S24; 2014. for the isolates N-354, N-365, and N-341 using different concentrations ranging from 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16, 32, 64, 128, 256 to 512 µg/mL in MH broth. For MBC determination, cefoxitin cultures with concentrations above the MIC were inoculated in AMH.2727 Mendonça ECL, Marques VF, Melo AD, et al. Caracterização fenogenotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite bovina. Pesq Vet Bras. 2012;31(9):859-864.

Results and discussion

The S. aureus isolates, which were identified by phenol-genotypic and proteomic techniques, were tested for sensitivity to certain antimicrobial agents and the presence of virulence genes for the fibronectin, hemolysin, and capsule. These results were used to create profiles highlighting the more distinct characteristics of the strains and representatives were collected. Therefore, 20 S. aureus strains were selected considering their antibiotic resistance profiles and the presence of virulence genes (Table 2).

Table 2
Antibiotyping profile and virulence genes of the 20 S. aureus strains included in this study.

All the 20 S. aureus strains were biofilm producers, classified as strong (55% = 11/20), moderate (30% = 6/20), and weak (15% = 3/20) producers. It is important to detect Staphylococcus spp. Isolates that produce biofilms because this virulence factor guarantees the installation and maintenance of the bacteria in the glandular breast tissue.2828 Melchior MB, Vaarkamp H, Fink-Gremmels J. Biofilms: a role in recurrent mastitis infections?. Vet J. 2006;171:398-407. Moreover, the polysaccharide mucus of biofilms facilitates bacterial adhesion to biomaterials, which is not removable despite repeated washings; therefore, its production is associated with infections caused by milking machines.2929 Dego K, Van Dijk JE, Nederbragt H. Factors involved in the early pathogenesis of bovine Staphylococcus aureus mastitis with emphasis on bacterial adhesion and invasion - a review. Vet Microbiol. 2002;24:181-198.icaA and icaD were detected in 17 (85%) and 19 (95%) isolates, respectively. Sixteen isolates tested positive for both the genes and only one (5%) tested positive for bap (Table 3). The low incidence of bap indicates that the ica-dependent mechanism, slime producer, may be primarily responsible for the adhesion and biofilm formation in the strains, as reported by Vautor et al.3030 Vautor E, Magnone V, Rios G, et al. Genetic differences among S. aureus isolates from dairy ruminant species: a single-dye DNA microarray approach. Vet Microbiol. 2008;133:105-114. All the biofilm-producing strains presented either icaA or icaD or both. However, no relation was observed between the biofilm formation intensity and number of amplified icaA and icaD. Arciola et al.3131 Arciola CR, Collamati S, Donati E, Montanaro L. A rapid PCR method for the detection of slime producing strains of Staphylococcus epidermidis and S. aureus in perioprosthesis infections. Diagn Mol Pathol. 2001;10:130-137. did not observe any relationship between slime production and the presence of these genes, suggesting that it could be a consequence of the experimental conditions such as lower sugar concentration in the agar or shorter incubation period.

Table 3
Biofilm production, agr system classification and presence of the genes icaA, icaD, bap, agr RNA III, blaZ and mecA in S. aureus strains isolated from bovine mastitis.

All the S. aureus isolates tested positive for agr RNAIII. Although RNAIII is not essential for S. aureus growth in vitro, it modulates the expression of genes involved in its pathogenesis.3232 Boisset M, Geissmann T, Huntzinger E, et al. S. aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism. Genes Dev. 2007;21:1353-1366. In fact, the roles of agr and quorum sensing in biofilm formation remain elusive. The Agr type II group was prevalent in 14 isolates, but the remaining six isolates could not be typed by the adopted technique. Moreover, Melchior et al.3333 Melchior MB, van Osch MHJ, Graat RM, et al. Biofilm formation and genotyping of S. aureus bovine mastitis isolates: evidence for lack of penicillin-resistance in Agr-type II strains. Vet Microbiol. 2009;137:83-89. found a high prevalence of Agr type II in 81% S. aureus isolates from bovine mastitis in the Netherlands, whereas 9% were Agr type I group. In addition, they suggested Agr type II strains are better adapted to the dairy environment than Agr type I strains. A study by Fabre-Klein et al.3434 Fabres-Klein MH, Santos MJC, Klein RC, Souza GN, Ribon AOB. An association between milk and slime increases biofilm production by bovine S. aureus. BMC Vet Res. 2015;11(3):. suggested that S. aureus strains increase biofilm production to adapt to the milk-filled environment of the udder. The absence of the agr system function facilitates the initial adhesion of staphylococci to surfaces, presumably due to the positive expression of adhesion molecules and negative expression of biofilm formation factors, which are released in the late stationary phase.3535 Vuong C, Saenz HL, Gotz F, Otto M. Impact of the agrquorum-sensing system on adherence to polystyrene in S. aureus. J Infect Dis. 2000;182:1688-1693.

The S. aureus isolate N-341was tested positive for icaA, icaD, and agr and presented a strong biofilm production in the assays; therefore, it was selected for the growth curve estimation. The biofilm production of N-341 in microplates was evaluated according to the time intervals of the growth curve assay considering the following phases: lag until 4 h, exponential reaching the plateau at 12 h, and death from 24 h. The biofilm production reached its peak at 12 h (OD = 0.596). At SEM, this isolate displayed a meshwork-like structure associated with the surface and with various gaps at 8 h of growth; this was not apparent at 4 h. At 12 h of growth, these gaps decreased in size and the cell layer became thicker, indicating the possible establishment of the biofilm. Within 24 h, the surface was filled with dense cell clusters (Fig. 1), probably indicating the next stage of biofilm formation. Using this model, it was possible to detect gradual changes in the biofilm complexity during the different stages of S. aureus growth. Therefore, our model used for biofilm formation and the procedure for cultivation of strains in TSA with 0.24% glucose in microplates and glass cover slips for 24 h at 35 °C without agitation can be satisfactorily used.

Fig. 1
Scanning electron micrographs of strain N-341 showing morphological changes associated with growth. Meshwork-like structures associated to the surface with various gaps were observed at 8 h of growth, these structures were not apparent at 4 h. At 12 h of growth, the gaps decreased in size and the cell layer became denser and at 24 h the surface was filled with dense cell clusters.

Recently, Savage et al.3636 Savage VJ, Chopra I, O’Neill AJ. S. aureus biofilms promote horizontal transfer of antibiotic resistance. J Antimicrob Antichem Agents. 2013;57(4):1968-1970. reported that biofilm mode of growth increases horizontal transfer of plasmid-borne antibiotic resistance determinants by conjugation in S. aureus. In addition, the biofilm can act as a barrier preventing the adsorption/penetration of antimicrobials, and the matrix promotes their dilution to subinhibitory concentrations. Moreover, the difference in bacterial physiology presented in the biofilm can influence the efficacy of antibiotics.3737 Raza A, Muhammad G, Sharif S, Atta A. Biofilm producing S. aureus and bovine mastitis: a review. Mol Microbiol Res. 2013;3:1-8. In this study, all isolates were sensitive to oxacillin and cefoxitin in the disk diffusion test, supporting the results of previous studies stating that the prevalence of oxacillin resistance in S. aureus isolates from bovine mastitis is low.3838 Krewer CC, Lacerda IP de, Amanso ES, et al. Etiology, antimicrobial susceptibility profile of Staphylococcus spp. and risk factors associated with bovine mastitis in the states of Bahia and Pernambuco. Pesq Vet Bras. 2013;33(5):601-606.,3939 Bardiau M, Yamazaki K, Duprez JN, Taminiau B, Mainil JG, Ote I. Genotypic and phenotypic characterization of methicillin resistant S. aureus (MRSA) isolated from milk of bovine mastitis. Lett Appl Microbiol. 2013;57:181-186. In the diffusion disk test, 15 isolates showed a sensitivity zone >29 mm, but five were resistant to penicillin. The CLSI indicates that staphylococci producing beta-lactamase may be phenotypically sensitive; therefore, before reporting their sensitivity, it is recommended that these isolates should be tested for beta-lactamase production. The recommended phenotypic test for the production of beta-lactamase in S. aureus, i.e., the edge zone test, interprets the growth on the border of the inhibition zone. This test is considered more sensitive for S. aureus than the nitrocefin test.1616 Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk and Diluition Susceptibility Tests for Bacteria Isolated From Animals; Approved Standards – 4 Ed, VET01-A4; 2013. In the edge zone test, 100% (15/15) of the sensitive isolates were positive and were reported as penicillin resistant. A previous study has reported high resistance to penicillin in coagulase-positive staphylococci isolated from bovine mastitis cases.3838 Krewer CC, Lacerda IP de, Amanso ES, et al. Etiology, antimicrobial susceptibility profile of Staphylococcus spp. and risk factors associated with bovine mastitis in the states of Bahia and Pernambuco. Pesq Vet Bras. 2013;33(5):601-606. However, this antibiotic is rarely considered a treatment option for mastitis, although some producers still insist on using it due to its low cost.

Furthermore, the susceptibility to amoxicillin associated with the beta-lactamase inhibitor was 100%, suggesting that the mechanism of beta-lactamase production may lead to penicillin resistance. Among the isolates considered resistant to penicillin, 70% (14/20) possessed blaZ and were tested negative for mecA using two different primers. Of the six isolates tested negative for both blaZ and mecA, five displayed moderate or strong biofilm production.

The MIC and MBC for cefoxitin were analyzed for the isolates N-354, N-365, and N-341. The stronger biofilm producer N-341 presented the highest MIC and MBC (1and 64 µg/mL, respectively), followed by the moderate producer N-365 (<0.25and 4 µg/mL, respectively). The weaker biofilm producer N-354 presented the lowest MIC and MBC (<0.25 µg/mL for both). Since N-341 was negative for blaZ and mecA, the high cefoxitin MBC value may be associated with biofilm protection. Wells et al.4040 Wells CL, Henry-Stanley MJ, Barnes AMT, Dunny GM, Hess DJ. Relation between antibiotic susceptibility and ultrastructure of Staphylococcus aureus biofilms on surgical suture. Surg Infect. 2011;12(4):297-305. discussed that there is no universal acceptable methodology for evaluating antimicrobial resistance and biofilm production. Based on this, it appears that exopolysaccharides (EPS) secreted by the bacteria act as a barrier that may play a role in this resistance, preventing the adsorption and penetration of antimicrobials. Moreover, the EPS matrix could neutralize or bind these compounds, promoting their dilution to subinhibitory concentrations before they reach the cells. In addition, biofilms are composed of both dormant and active cell subpopulations. This difference in the bacterial physiology can influence the efficacy of antibiotics.3737 Raza A, Muhammad G, Sharif S, Atta A. Biofilm producing S. aureus and bovine mastitis: a review. Mol Microbiol Res. 2013;3:1-8.

In conclusion, all the 20 isolates were biofilm producers and mostly presented icaA and icaD; only the N-341 strain was positive for bap. agr RNAIII was detected in all the isolates, and Agr type II showed a significant prevalence. The N-341 strain showed gradual changes in the complexity of the biofilm along the phases of growth curve in SEM, reaching the peak at the stationary phase. Moreover, the detected penicillin resistance was related to the production of beta-lactamase due to the absence of mecA and sensitivity to amoxicillin + clavulanic acid in all the isolates. Finally, because N-341 was negative for the tested resistance genes, the cefoxitin MBC of 64 µg/mL may be associated with biofilm protection since this strain is a strong producer of this virulence factor.

Taken together, these data suggest that a greater understanding of biofilm formation may add to our knowledge on bacterial resistance in vivo. Thus, studies that uncover colonization factors in biofilm formation are important and will form the basis for the development of treatments for bacterial resistance in biofilms.

Acknowledgements

This study was supported by the National Council for Scientific and Technological Development (CNPq, Rio de Janeiro, Brazil - process 308528/2011-5), Foundation for Research Support in the State of Rio de Janeiro (FAPERJ; process E-26/112.658/2012) and Coordination for the Improvement of Higher Education Personal (CAPES).

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Publication Dates

  • Publication in this collection
    Jan-Mar 2017

History

  • Received
    5 June 2015
  • Accepted
    30 May 2016
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E-mail: bjm@sbmicrobiologia.org.br