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.

A mastite é uma inflamação da glândula mamária causada principalmente por bactérias, dentre as quais o gênero Staphylococcus ocupa um papel importante. Bactérias pertencentes a este gênero são caracterizadas por expressar fatores de virulência que permitem sua persistência e disseminação no hospedeiro. O presente trabalho teve por objetivo avaliar fenogenotipicamente os fatores de virulência de isolados de Staphylococcus spp. a partir de casos de mastite bovina. Foram analisadas 272 amostras de leite provenientes de oito propriedades da região Sul-Fluminense do Estado do Rio de Janeiro. Após identificação, obteve-se um total de 250 isolados de Staphylococcus spp. Estes foram submetidos às provas fenotípicas de detecção da produção de "slime" em microplaca e em ágar vermelho congo; produção de hemolisinas e sinergismo hemolítico; produção de caseinase e DNase. Posteriormente foram submetidos à técnica de PCR para detecção dos genes de produção de cápsula (cap5 e cap8), fibronectina (fnbA,e fnbB), "slime" (icaA e icaD) e hemolisinas (hla e hlb). Do total avaliado, 58% (145/250) foi identificado como Staphylococcus spp. coagulase-negativos e 42% (105/250) como Staphylococcus spp. coagulase-positivos, destes 36,2% (38/105) foram identificados como S. aureus, 11,4% (12/105) como S. intermedius e 3,8% (4/105) como pertencentes ao grupo SIG. Apenas 6,4% (16/250) dos isolados foram produtores de α-hemólise, 4,8% (12/250) de β-hemólise e, 1,6% (4/250) de α e β-hemólise. A produção de caseinase foi observada em 66,4% (166/250), e a produção de "slime" avaliada pela técnica da microplaca em 76,8% (192/250) dos isolados, respectivamente. A DNase foi detectada em ECNs (38/145) e S. aureus (14/38). Os marcadores genéticos avaliados para a produção de slime, icaA e icaD apresentaram nenhuma ou leve concordância com a produção fenotípica, respectivamente, utilizando o coeficiente Kappa. Tal dado parece indicar que outros marcadores genéticos podem estar envolvidos com a expressão desta característica. Os demais genes detectados com frequência de 4% (10/250) para cap5 e para cap8, 32,8% (82/250) para fnbA, 4,4% (11/250) para fnbB, 19,2% (48/250) para hla e 18% (45/250) para hlb. O perfil circulante nas propriedades foi o 1: isolado produtor de "slime" e caseinase. O gene spaA foi positivo em todos os S. aureus, apresentando amplicons de tamanhos variados, sendo o tamanho prevalente o de 300pb. A amplificação do gene coa apresentou nove tipos polimórficos distintos, sendo prevalente o amplicon de 600pb. O gene agr foi detectado em todos os S. aureus, com amplicon de 200pb. Foi observado que os genes de virulência estudados estavam distribuídos de modo aleatório entreos 6 distintos perfis eletroforéticos obtidos através da Eletroforese em Gel de Campo Pulsado (PFGE).

Pesq. Vet. Bras. 32(9)

This study aims to detect the main virulence and antimicrobial resistance genes in Stapylococcus aureus from bovine mastitic milk as well as classifying them according to agr typing. A total of 55 strains from six dairy unities in the state of Rio de Janeiro were selected, of these 27.3% presented fbnA and 78,2% for fbnB genes, respectively. None of the strains tested were positive for cap5 gene, 3.6% were positive for cap8 gene. Additionally, 94.5% of strains had hlA gene and 89.1% had hlB gene while 67.3% of the strains had icaA gene and 87.3% had icaD gene. From these results it was possible to establish 12 different virulence profiles. Prevalence of agrII type was detected in 81.8% of the isolates. Concerning antimicrobial resistance evaluation, the studied strains were susceptible to all antibiotics tested except penicillin, 83.6% being resistant strains. None of the strains had mecA gene, however, 40% of the strains had blaZ gene. Associating virulence and resistance data made it possible to obtain 23 different profiles. This great diversity of strains shows wide array of bacterial strategies and the challenge of mastitis prevention in cattle. Despite antimicrobial susceptibility, these strains presented certain genes that allow its persistence in the herd.

Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.

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