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Mechanisms of vancomycin resistance in Clostridioides difficile
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Clostridioides difficile é uma bactéria anaeróbica Gram-positiva e a principal causa de doenças gastrointestinais nosocomiais associadas à toma de antibióticos em adultos. A
infeção por C. difficile (ICD) está associada a elevadas taxas de recorrência, o que impõe um grande peso económico nos sistemas de saúde à escala mundial. Atualmente a vancomicina (VAN) é usada para o tratamento rotineiro de ICD. No entanto, estirpes com suscetibilidade diminuída à VAN têm sido descritas. A sua disseminação representaria um problema sério, pelo que é crucial entender através de que mecanismos a resistência à VAN pode surgir. Paradoxalmente, a maioria das estirpes de C. difficile é sensível à VAN apesar de possuir um cluster de genes van que noutros microrganismos é suficiente para conferir resistência ao antibiótico. O cluster é funcional quando introduzido noutro organismo, mas em C. difficile é críptico. Através de mutagénese química, obtivemos 27 isolados resistentes à VAN, 7 dos quais possuem mutações missense no gene vanS, que codifica para VanS, uma cinase sensora de histidina. Na presença de VAN, VanS fosforila o regulador de resposta VanR que por sua vez ativa a transcrição do cluster van. Todas as mutações resultaram em expressão constitutiva ou aumentada do cluster van e num aumento do MICVAN de 2 para 8 mg/L. Algumas das substituições ocorrem numa região denominada ATP-lid no domínio cinase de VanS e podem diminuir a sua capacidade de desfosforilar VanR~P, que permanecerá ativo mesmo na ausência de VAN. Enquanto que o cluster van contribui para a resistência, o aumento máximo relativamente modesto no valor de MIC, sugere a existência de bottlenecks adicionais. Outros isolados, sem mutações no gene vanS, apresentam fortes alterações da forma e divisão celular, sugerindo que a resistência à VAN em C. difficile pode surgir através de vários mecanismos e pode resultar num custo de fitness.
Clostridioides difficile is a Gram-positive anaerobic bacterium and the main cause of nosocomial acquired antibiotic-associated gastrointestinal diseases in adults. Clostridioides difficile infection (CDI) is linked to high levels of recurrence rates, which impose an economic burden in worldwide health care systems. Currently, vancomycin (VAN) is used for the routine treatment of CDI. Strains with decreased susceptibility to VAN, however, have been described. Their spreading would pose a serious problem and thus it is crucial to understand by what mechanisms VAN resistance may arise. Paradoxically, most strains of C. difficile are normally sensitive to VAN yet possess a van gene cluster that in other microorganisms is sufficient to confer resistance. The cluster is functional when introduced into another organism, but in C. difficile it is cryptic. Through chemical mutagenesis, we found 27 isolates with increased resistance to VAN, 7 of which carried missense mutations in vanS, coding for the VanS sensor histidine kinase. In the presence of VAN, VanS phosphorylates the VanR response regulator, which then activates transcription of the van cluster. All the mutations resulted in constitutive or increased expression of the van cluster and in an increase in the MIC value, from 2 to 8 mg/L. Some of them led to substitutions in the ATP lid region within the kinase domain of VanS, that may impair its ability to dephosphorylate VanR~P, that will remain active even in the presence of VAN. While suggesting that the van cluster contributes to resistance, the relatively modest maximum increase in the MIC value, suggests the existence of additional bottlenecks. Other isolates, with no mutations in vanS were strongly impaired in cell shape and cell division, indicating that VAN resistance in C. difficile may arise through various pathways with a potential fitness cost.
Clostridioides difficile is a Gram-positive anaerobic bacterium and the main cause of nosocomial acquired antibiotic-associated gastrointestinal diseases in adults. Clostridioides difficile infection (CDI) is linked to high levels of recurrence rates, which impose an economic burden in worldwide health care systems. Currently, vancomycin (VAN) is used for the routine treatment of CDI. Strains with decreased susceptibility to VAN, however, have been described. Their spreading would pose a serious problem and thus it is crucial to understand by what mechanisms VAN resistance may arise. Paradoxically, most strains of C. difficile are normally sensitive to VAN yet possess a van gene cluster that in other microorganisms is sufficient to confer resistance. The cluster is functional when introduced into another organism, but in C. difficile it is cryptic. Through chemical mutagenesis, we found 27 isolates with increased resistance to VAN, 7 of which carried missense mutations in vanS, coding for the VanS sensor histidine kinase. In the presence of VAN, VanS phosphorylates the VanR response regulator, which then activates transcription of the van cluster. All the mutations resulted in constitutive or increased expression of the van cluster and in an increase in the MIC value, from 2 to 8 mg/L. Some of them led to substitutions in the ATP lid region within the kinase domain of VanS, that may impair its ability to dephosphorylate VanR~P, that will remain active even in the presence of VAN. While suggesting that the van cluster contributes to resistance, the relatively modest maximum increase in the MIC value, suggests the existence of additional bottlenecks. Other isolates, with no mutations in vanS were strongly impaired in cell shape and cell division, indicating that VAN resistance in C. difficile may arise through various pathways with a potential fitness cost.
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Clostridioides difficile vancomycin antibiotic resistance vanS fitness cost