Caracterização de Staphyloccocus aureus em relação à resistência e virulência de estirpes isoladas de leite bovino mastítico
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Data
2021-02-18
Autores
Valmorbida, Mylena Karoline
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A mastite é umas das mais importantes enfermidades que acometem a bovinocultura leiteira e pode ser causada por diferentes patógenos amplamente difundidos, sendo que o mais importante deles é a bactéria Staphylococcus aureus. O S. aureus possui diferentes mecanismos para conseguir sobreviver no organismo dos animais infectados, dentre eles, diversos fatores de virulência e de resistência, produzidos respectivamente, para tentar evoluir o processo infeccioso e resistir aos diferentes princípios ativos de antimicrobianos utilizados numa tentativa de tratamento. Mediante essas informações os principais objetivos deste trabalho foram pesquisar genes de virulência e resistência a antimicrobianos de 65 estirpes de S. aureus de amostras de leite mastítico provenientes da cidade de Concórdia - Santa Catarina, assim como caracterizar a similaridade genética entre as estirpes. Essas estirpes inicialmente foram testadas quanto à presença da coagulase livre e logo depois foram submetidas à PCR para confirmação do gênero bacteriano, pela presença do gene tstag, e confirmação da espécie, pela pesquisa do gene cydB. Para a confirmação final da espécie bacteriana foi realizada a técnica de MALDI-TOF. Pesquisou-se os genes de virulência relacionados ao choque tóxico (tsst-1), enzima coagulase (coa) e enterotoxinas (sea, seb, sec, sed, see, seg, seh, sej). Posteriormente, foram pesquisados genes de resistência aos antimicrobianos, como os genes de resistência a aminoglicosídeos (mecA), a betalactâmicos (blaZ), a tetraciclina (tet) e o gene da resistência à eritromicina (ery). A caracterização da resistência fenotípica, foi realizado o Teste de Susceptibilidade aos antimicrobianos (Antibiograma). A Eletroforese em Gel de Campo Pulsado (PFGE) foi realizada para investigação da similaridade genética das estirpes. Neste trabalho, 88% dos isolados amplificaram o gene cydB e o gene coa apresentou 93,84% de amplificação. Cinco das oito enterotoxinas pesquisadas foram amplificadas, sendo elas: sec 3,07%, seg 6,15%, see 7,69%, seh 23,07% e sei 53,84%. Esses achados reforçam o alarme sobre as intoxicações alimentares que possuem alta prevalência nos seres humanos. As estirpes apresentaram alto grau de resistência no teste de antibiograma, sendo a Penicilina e a Tetraciclina com os maiores índices de resistência apresentados. Porém, na pesquisa dos genes de resistência relacionados, foi observada baixa frequência desses genes. Os isolados também apresentaram alto grau de variabilidade genética quando analisado o teste de PFGE. Os resultados obtidos são fundamentais para mostrar a importância desse agente no sistema produtivo leiteiro que possui alta prevalência, causando graves prejuízos. Fato que alerta sobre a tomada das devidas medidas de controle e prevenção da doença.
Mastitis is one of the most important diseases that affect dairy cattle and can be caused by different pathogens that are widespread, the most important of which is the bacterium Staphylococcus aureus. S. aureus has different mechanisms to survive in the organism of infected animals, among them, several virulence and resistance factors, produced respectively, to try to evolve the infectious process and resist the different active principles of antimicrobials used in an attempted treatment. Based on this information, the main objectives of this work were to search for virulence and resistance genes of 65 strains of S. aureus from mastitic milk samples from the city of Concórdia - Santa Catarina, as well to characterize the genetic similarity between the strains. These strains were initially tested for the presence of free coagulase and afterwards they underwent PCR to confirm the bacterial genus, through the presence of the tstag gene, and to confirm the species, through the cydB gene search. For the final confirmation of the bacterial species, the MALDI-TOF technique was made. Then, virulence genes related to toxic shock (tsst-1), coagulase enzyme (coa) and enterotoxins (sea, seb, sec, sed, see, sec, sem, seh, sej) were investigated. After that, antimicrobial resistance genes such as the aminoglycoside resistance gene (mecA), beta-lactam (blaZ), tetracycline (tet) and the erythromycin resistance gene (ery) were investigated. And for the characterization of phenotypic resistance, the Antimicrobial Susceptibility Test (Antibiogram) was performed. Pulsed Field Gel Electrophoresis (PFGE) was performed to investigate the genetic similarity of these strains. In this study, 88% of the isolates amplified the cydB gene and the coa gene showed 93.84% amplification. Five of eight enterotoxins surveyed were amplified, namely: sec 3.07%, seg 6.15%, see 7.69%, seh 23.07% and sei 53.84%. These findings reinforce the alarm about food poisoning that has a high prevalence in humans. The strains showed a high degree of resistance in the antibiogram test, with Penicillin and Tetracycline having the highest levels of resistance. However, in the search for resistance genes, the frequency of these related genes was observed. Strains also showed a high degree of genetic variability when analyzing the PFGE test. The results obtained are fundamental to show the importance of this agent in the dairy production system, which has a high prevalence, causing serious losses. Fact that warns about taking appropriate measures to control and prevent the disease.
Mastitis is one of the most important diseases that affect dairy cattle and can be caused by different pathogens that are widespread, the most important of which is the bacterium Staphylococcus aureus. S. aureus has different mechanisms to survive in the organism of infected animals, among them, several virulence and resistance factors, produced respectively, to try to evolve the infectious process and resist the different active principles of antimicrobials used in an attempted treatment. Based on this information, the main objectives of this work were to search for virulence and resistance genes of 65 strains of S. aureus from mastitic milk samples from the city of Concórdia - Santa Catarina, as well to characterize the genetic similarity between the strains. These strains were initially tested for the presence of free coagulase and afterwards they underwent PCR to confirm the bacterial genus, through the presence of the tstag gene, and to confirm the species, through the cydB gene search. For the final confirmation of the bacterial species, the MALDI-TOF technique was made. Then, virulence genes related to toxic shock (tsst-1), coagulase enzyme (coa) and enterotoxins (sea, seb, sec, sed, see, sec, sem, seh, sej) were investigated. After that, antimicrobial resistance genes such as the aminoglycoside resistance gene (mecA), beta-lactam (blaZ), tetracycline (tet) and the erythromycin resistance gene (ery) were investigated. And for the characterization of phenotypic resistance, the Antimicrobial Susceptibility Test (Antibiogram) was performed. Pulsed Field Gel Electrophoresis (PFGE) was performed to investigate the genetic similarity of these strains. In this study, 88% of the isolates amplified the cydB gene and the coa gene showed 93.84% amplification. Five of eight enterotoxins surveyed were amplified, namely: sec 3.07%, seg 6.15%, see 7.69%, seh 23.07% and sei 53.84%. These findings reinforce the alarm about food poisoning that has a high prevalence in humans. The strains showed a high degree of resistance in the antibiogram test, with Penicillin and Tetracycline having the highest levels of resistance. However, in the search for resistance genes, the frequency of these related genes was observed. Strains also showed a high degree of genetic variability when analyzing the PFGE test. The results obtained are fundamental to show the importance of this agent in the dairy production system, which has a high prevalence, causing serious losses. Fact that warns about taking appropriate measures to control and prevent the disease.
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Palavras-chave
Mastite, PFGE, Produção leiteira, Resistência, Toxinas