Fatores de patogenicidade de Staphylococcus spp. em leite de vacas com tratamento não convencional da mastite
Data
2020-07-28
Autores
Ferreira, Elka Machado
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Objetivou-se caracterizar e comparar a formação de biofilme, suscetibilidade a antimicrobianos in vitro, presença e distribuição de genes de virulência e de resistência antimicrobiana, além de determinar a diversidade clonal de estirpes de Staphylococcus spp. isoladas em leite de vacas tratadas e não tratadas com homeopatia. Os isolados de Staphylococcus spp. foram identificados por reação em cadeia da polimerase-espaço transcrito interno (ITS-PCR) e foram investigados quanto a presença dos genes sea, seb, sec, sed, see, tsst-1, pvl e eta codificadores de toxinas, dos genes icaABCD, bap, aap, atlE e bhp relacionados com a produção de biofilme e dos genes blaZ e mecA associados com a resistência antimicrobiana. Amostras mecA positivas foram tipadas para o tipo SCCmec por Multiplex Polymerase Chain Reaction (PCR Multiplex), enquanto a pesquisa de produção in vitro de bioflme ocorreu pelo método de aderência em placas de poliestireno. O perfil de resistência antimicrobiana foi determinado pelo método de disco-difusão em ágar e o perfil de restrição enzimática por Pulsed-Field Gel Electroforesis (PFGE). Clusters de S. aureus e S. epidermidis com três ou mais isolados tiveram uma linhagem selecionada para tipagem por Multilocus Sequence Typing (MLST), cujas sequências foram analisadas por banco de dados (http://www.mlst.net). Verificou-se prevalência de S. aureus (50,5%), que apresentou distribuição semelhante nos dois grupos, enquanto Staphylococcus spp. coagulase-negativos (CoNS) (70,8%) foram frequentes em vacas tratadas (p=0,036). O gene sec ocorreu apenas em S. chromogenes, com maior frequência em animais tratados (p=0,004) e o gene pvl foi encontrado em 66,7% de S. aureus. O operon icaADBC e os genes icaA e icaD associados prevaleceram em S. aureus, porém o gene icaD foi predominante no grupo de vacas tratadas (p=0,012). Os genes atlE e aap foram carreados apenas por S. epidermidis. Entre as estirpes com produção de biofilme in vitro, 76,8% continham pelo menos um gene relacionado com esta característica. Os genes mecA e Blaz apresentaram distribuição similar em vacas tratadas e não tratadas, porém mecA foi identificado apenas em S. epidermidis (12,1%), todos tipados como SCCmec tipo I. A susceptibilidade antimicrobiana revelou um perfil de multirresistência em 29,3% das estirpes. Houve predominância de um cluster majoritário em S. epidermidis e S. chromogenes, o que não ocorreu para S. aureus. A sequência do tipo (ST) 81 foi predominante em S. epidermidis, enquanto as STs 1, 5 e 126 em S. aureus. A presença de genes de toxinas, resistência antimicrobiana e biofilme, bem como a produção in vitro deste fator de patogenicidade, aliados à persistência de perfis clonais bacterianos, demonstram que sistemas em transição para manejos orgânicos podem representar risco à saúde pública pela circulação de patógenos zoonóticos em leite bovino. Medidas eficazes devem ser tomadas no controle dos produtos lácteos e manejo dos animais.
The present study aimed to characterize and compare biofilm formation, in vitro antimicrobial susceptibility, and the presence and distribution of virulence and antimicrobial resistance genes, as well as to determine the clonal diversity of Staphylococcus spp. strains isolated in milk from cows treated or not with homeopathy. The Staphylococcus spp. isolates were identified by polymerase chain reaction targeting the internal transcribed spacer (ITS-PCR) and were investigated regarding the presence of the sea, seb, sec, sed, see, tsst-1, pvl, and eta toxin-encoding genes, the icaABCD, bap, aap, atlE, and bhp genes, related to the production of biofilm, and the blaZ and mecA genes, which are associated with antimicrobial resistance. mecA-positive samples were typed for SCCmec by Multiplex Polymerase Chain Reaction (Multiplex PCR), while the assessment of in vitro biofilm production was carried out using the polystyrene plate adhesion method. The antimicrobial resistance profile was determined using the agar disc diffusion method, and the enzyme restriction profile, by Pulsed-Field Gel Electrophoresis (PFGE). Clusters of S. aureus and S. epidermidis with three or more isolates had one strain selected for typing by Multilocus Sequence Typing (MLST), and the sequences were analyzed on an online database (http://www.mlst.net). A 50.5% prevalence of S. aureus was observed, with similar distribution in both groups, while coagulase-negative Staphylococcus spp. (CoNS) were frequent (70.8%) in treated cows (p=0.036). The sec gene was found only in S. chromogenes and more frequently in treated animals (p=0.004), while the pvl gene was observed in 66.7% of the S. aureus. The icaADBC operon and the associated icaA and icaD genes prevailed in S. aureus, although the icaD gene was predominant in the treated cows group (p=0.012). The atlE and aap genes were carried only by S. epidermidis. Among the strains presenting in vitro biofilm production, 76.8% had at least one gene related to this trait. The mecA and Blaz genes exhibited similar distribution in treated and untreated cows, although mecA was identified only in S. epidermidis (12.1%), all of which were typed as SCCmec type I. The antimicrobial susceptibility assay revealed a multiresistance profile in 29.3% of the strains. There was a predominance of a major cluster in S. epidermidis and S. chromogenes, which did not occur for S. aureus. Sequence type (ST) 81 was predominant in S. epidermidis, while STs 1, 5, and 126 were prevalent in S. aureus. The presence of genes related to toxin production, antimicrobial resistance, and biofilm formation, as well as the in vitro production of this pathogenicity factor, combined with the persistence of bacterial clonal profiles, demonstrate that systems in transition to organic management can represent a risk to public health due to the circulation of zoonotic pathogens in bovine milk. Effective measures must be taken to control dairy products and animal management.
The present study aimed to characterize and compare biofilm formation, in vitro antimicrobial susceptibility, and the presence and distribution of virulence and antimicrobial resistance genes, as well as to determine the clonal diversity of Staphylococcus spp. strains isolated in milk from cows treated or not with homeopathy. The Staphylococcus spp. isolates were identified by polymerase chain reaction targeting the internal transcribed spacer (ITS-PCR) and were investigated regarding the presence of the sea, seb, sec, sed, see, tsst-1, pvl, and eta toxin-encoding genes, the icaABCD, bap, aap, atlE, and bhp genes, related to the production of biofilm, and the blaZ and mecA genes, which are associated with antimicrobial resistance. mecA-positive samples were typed for SCCmec by Multiplex Polymerase Chain Reaction (Multiplex PCR), while the assessment of in vitro biofilm production was carried out using the polystyrene plate adhesion method. The antimicrobial resistance profile was determined using the agar disc diffusion method, and the enzyme restriction profile, by Pulsed-Field Gel Electrophoresis (PFGE). Clusters of S. aureus and S. epidermidis with three or more isolates had one strain selected for typing by Multilocus Sequence Typing (MLST), and the sequences were analyzed on an online database (http://www.mlst.net). A 50.5% prevalence of S. aureus was observed, with similar distribution in both groups, while coagulase-negative Staphylococcus spp. (CoNS) were frequent (70.8%) in treated cows (p=0.036). The sec gene was found only in S. chromogenes and more frequently in treated animals (p=0.004), while the pvl gene was observed in 66.7% of the S. aureus. The icaADBC operon and the associated icaA and icaD genes prevailed in S. aureus, although the icaD gene was predominant in the treated cows group (p=0.012). The atlE and aap genes were carried only by S. epidermidis. Among the strains presenting in vitro biofilm production, 76.8% had at least one gene related to this trait. The mecA and Blaz genes exhibited similar distribution in treated and untreated cows, although mecA was identified only in S. epidermidis (12.1%), all of which were typed as SCCmec type I. The antimicrobial susceptibility assay revealed a multiresistance profile in 29.3% of the strains. There was a predominance of a major cluster in S. epidermidis and S. chromogenes, which did not occur for S. aureus. Sequence type (ST) 81 was predominant in S. epidermidis, while STs 1, 5, and 126 were prevalent in S. aureus. The presence of genes related to toxin production, antimicrobial resistance, and biofilm formation, as well as the in vitro production of this pathogenicity factor, combined with the persistence of bacterial clonal profiles, demonstrate that systems in transition to organic management can represent a risk to public health due to the circulation of zoonotic pathogens in bovine milk. Effective measures must be taken to control dairy products and animal management.
Descrição
Palavras-chave
Mastite, Homeopatia veterinária, Microbiologia dos laticínios, Eletroforese em campo pulsátil, Resistência à meticilina