Capacidade de adesão, invasão e produção de biofilme em superfície biótica e abiótica por Staphylococcus aureus isolados de leite de vacas com mastite subclínica
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Data
2016-07-28
Autores
Castilho, Ivana Giovannetti [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A mastite bovina é uma inflamação nas glândulas mamárias das vacas causada, principalmente, por bactérias e identificada a partir de amostras de leite, pelo aumento da contagem de células somáticas (CCS). Essa doença é a maior preocupação da indústria leiteira em todo o mundo por gerar grandes perdas econômicas. Vários micro-organismos podem causar mastite e Staphylococcus aureus é um dos principais patógenos envolvidos devido a vários fatores de virulência. Essa espécie é capaz de aderir e invadir as células do epitélio mamário bovino, constituindo os primeiros passos para o estabelecimento da mastite e deixando a bactéria fora do alcance dos mecanismos de defesa do hospedeiro e da ação de antibióticos. Os biofilmes são formados pela adesão das bactérias em superfícies, com multiplicação bacteriana e desenvolvimento de uma matriz polissacarídica, que envolve as multicamadas de bactérias, e vários genes estão envolvidos na sua formação, como o cluster ica e o gene bap. O objetivo do presente estudo foi testar 7 cepas de S. aureus, isolados de leite de vacas com mastite subclínica, com diferentes perfis dos genes bap, icaA e icaD quanto a capacidade de produzir biofilme in vitro (empregando-se caldo Tryptic Soy Broth – TSB-, TSB + 1% de glicose) e em superfícies bióticas (cultura celular de epitélio mamário bovino – BMEC – e HeLa) e abiótica (microplaca de poliestireno). O potencial de adesão e invasão e a susceptibilidade a antimicrobiano, na presença do biofilme, também foram testados. Além disso, investigou-se- a presença dos genes codificadores das adesinas MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules- Componentes da Superfície Microbiana Reconhecedores de Moléculas Adesivas da Matriz) e os isolados foram classificados molecularmente pelo o grupo agr. Todas as cepas foram capazes de invadir BMEC e HeLa, mas os índices de invasão em BMEC foram maiores que em HeLa. No teste de produção de biofilme em placa de poliestireno, houve diferença estatisticamente significativa entre o TSB +1% glicose e os demais meios, onde 5 das 7 cepas foram produtoras. A formação de biofilme em DMEM ocorreu somente na cepa que apresentou os genes bap, icaA e icaD simultaneamente, o que permitiu realizar o teste e produção de biofilme em superfície biótica somente com esse isolado. Em relação à produção de biofilme sobre as culturas celulares, foi possível observar sua presença apenas sobre as células HeLa, pois a cepa apresentou alta taxa de invasão em BMEC, destruindo o tapete celular e impossibilitando a visualização da matriz. Entre os genes codificadores das adesinas, todas as cepas produtoras de biofilme apresentaram em comum os genes fnbA, clfA, clfB, eno, fib e ebpS. Em todas as cepas produtoras, o biofilme protegeu as bactérias da ação do antimicrobiano sulfato de cefquinoma. Dentre todas as cepas testadas, não houve predominância de nenhum grupo agr, porém as cepas produtoras de biofilme pertenciam aos grupos agr II ou III. A presença de bap, icaA e icaD associada aos genes codificadores das MSCRAMMs confere às cepas de S. aureus, provenientes de mastite bovina subclínica, alta capacidade de adesão e produção de biofilme in vitro, o que contribui para a colonização e persistência desse patógeno.
The bovine mastitis is defined as an inflammation of the mammary glands of cows, mainly caused by bacteria, and it can be identified in the milk samples due to the increase in somatic cell count (SCC). This disease is a major concern of the dairy industry throughout the world, causing severe economic losses. Several microorganisms may cause mastitis, however, Staphylococcus aureus is among the main pathogens involved in this framework, due to its virulence factors. This species can adhere and invade bovine mammary epithelial cells, the first step towards the establishment of mastitis and leaving the bacterium free from host defense mechanisms and the action of antibiotics and with a nutrient source guaranteed. Biofilms are formed by adhesion of bacteria on solid surfaces with bacterial multiplication and development of a polysaccharide matrix involving the multilayered cells. Several genes are involved in its formation, as the cluster ica and bap gene. The aim of this study was to test S. aureus strains isolated from the milk of cows with subclinical mastitis with different genetic profiles in relation to the genes involved in the production of biofilm (cluster ica and bap) for biofilm production in vitro, (using Tryptic Spy Broth –TSB-, TSB+1% glucose and DMEM) and in biotic (bovine mammary epithelial cells – BMEC- and HeLa cells culture) and abiotic surfaces (polystyrene microplates). The adhesive and invasive potential of these strains and the antimicrobial susceptibility in the presence of biofilms were also tested. Furthermore, we investigated de genes coding MSCRAMMs and characterized molecularly by agr group. All strains were able to invade HeLa and BMEC, but the rates of BMEC invasion were larger than to cells HeLa. In biofilm production in vitro test, there was statistically significant difference between TSB + glucose 1% and the other mediums, where 5 from 7 strains were producers. Biofilm production using DMEM occurred only by bap+icaA+icaD+ strain, so we observed the biofilm by electronic microscopy only by this strain. In the production of biofilm on cell cultures we observed its presence only on HeLa cells, because the strain presented a high invasion in BMEC, destroying the cells and preventing the visualization of the array. All biofilm -producing strains had in common the fnbA, cIfA, clfB , eno, fib and ebpS genes. In all producing strains, biofilms protect bacteria from the cefquinome sulphate action. Among all tested strains, no predominance of any agr group, but producing strains of biofilm belonged to agr II or III groups. The presence of bap, icaA and icaD associated with genes encoding the MSCRAMMs gives strains of S. aureus from subclinical bovine mastitis, high capacity for adhesion and biofilm production in vitro, which helps the colonization and persistence of this pathogen.
The bovine mastitis is defined as an inflammation of the mammary glands of cows, mainly caused by bacteria, and it can be identified in the milk samples due to the increase in somatic cell count (SCC). This disease is a major concern of the dairy industry throughout the world, causing severe economic losses. Several microorganisms may cause mastitis, however, Staphylococcus aureus is among the main pathogens involved in this framework, due to its virulence factors. This species can adhere and invade bovine mammary epithelial cells, the first step towards the establishment of mastitis and leaving the bacterium free from host defense mechanisms and the action of antibiotics and with a nutrient source guaranteed. Biofilms are formed by adhesion of bacteria on solid surfaces with bacterial multiplication and development of a polysaccharide matrix involving the multilayered cells. Several genes are involved in its formation, as the cluster ica and bap gene. The aim of this study was to test S. aureus strains isolated from the milk of cows with subclinical mastitis with different genetic profiles in relation to the genes involved in the production of biofilm (cluster ica and bap) for biofilm production in vitro, (using Tryptic Spy Broth –TSB-, TSB+1% glucose and DMEM) and in biotic (bovine mammary epithelial cells – BMEC- and HeLa cells culture) and abiotic surfaces (polystyrene microplates). The adhesive and invasive potential of these strains and the antimicrobial susceptibility in the presence of biofilms were also tested. Furthermore, we investigated de genes coding MSCRAMMs and characterized molecularly by agr group. All strains were able to invade HeLa and BMEC, but the rates of BMEC invasion were larger than to cells HeLa. In biofilm production in vitro test, there was statistically significant difference between TSB + glucose 1% and the other mediums, where 5 from 7 strains were producers. Biofilm production using DMEM occurred only by bap+icaA+icaD+ strain, so we observed the biofilm by electronic microscopy only by this strain. In the production of biofilm on cell cultures we observed its presence only on HeLa cells, because the strain presented a high invasion in BMEC, destroying the cells and preventing the visualization of the array. All biofilm -producing strains had in common the fnbA, cIfA, clfB , eno, fib and ebpS genes. In all producing strains, biofilms protect bacteria from the cefquinome sulphate action. Among all tested strains, no predominance of any agr group, but producing strains of biofilm belonged to agr II or III groups. The presence of bap, icaA and icaD associated with genes encoding the MSCRAMMs gives strains of S. aureus from subclinical bovine mastitis, high capacity for adhesion and biofilm production in vitro, which helps the colonization and persistence of this pathogen.
Descrição
Palavras-chave
S. aureus, Mastite, Bap, Cluster ica, MSCRAMM, Mastitis