Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin

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Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin. / Kuehne, Sarah A; Dempster, Andrew W; Collery, Mark M; Joshi, Nimitray; Jowett, Jamie; Kelly, Michelle L; Cave, Rory; Longshaw, Chris M; Minton, Nigel P.

In: Journal of Antimicrobial Chemotherapy, Vol. 73, No. 4, 04.2018, p. 973-980.

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Kuehne, Sarah A ; Dempster, Andrew W ; Collery, Mark M ; Joshi, Nimitray ; Jowett, Jamie ; Kelly, Michelle L ; Cave, Rory ; Longshaw, Chris M ; Minton, Nigel P. / Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin. In: Journal of Antimicrobial Chemotherapy. 2018 ; Vol. 73, No. 4. pp. 973-980.

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@article{7bb1375a337649709e3554ca151e670f,
title = "Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin",
abstract = "ObjectivesTo establish the role of specific, non-synonymous SNPs in the RNA polymerase β subunit (rpoB) gene in reducing the susceptibility of Clostridium difficile to fidaxomicin and to explore the potential in vivo significance of rpoB mutant strains.MethodsAllelic exchange was used to introduce three different SNPs into the rpoB gene of an erythromycin-resistant derivative (CRG20291) of C. difficile R20291. The genome sequences of the created mutants were determined and each mutant analysed with respect to growth and sporulation rates, toxin A/B production and cytotoxicity against Vero cells, and in competition assays. Their comparative virulence and colonization ability was also assessed in a hamster infection model.ResultsThe MIC of fidaxomicin displayed by three mutants CRG20291-TA, CRG20291-TG and CRG20291-GT was substantially increased (>32, 8 and 2 mg/L, respectively) relative to that of the parent strain (0.25 mg/L). Genome sequencing established that the intended mutagenic substitutions in rpoB were the only changes present. Relative to CRG20291, all mutants had attenuated growth, were outcompeted by the parental strain, had lower sporulation and toxin A/B production capacities, and displayed diminished cytotoxicity. In a hamster model, virulence of all three mutants was significantly reduced compared with the progenitor strain, whereas the degree of caecum colonization was unaltered.ConclusionsOur study demonstrates that particular SNPs in rpoB lead to reduced fidaxomicin susceptibility. These mutations were associated with a fitness cost in vitro and reduced virulence in vivo.",
author = "Kuehne, {Sarah A} and Dempster, {Andrew W} and Collery, {Mark M} and Nimitray Joshi and Jamie Jowett and Kelly, {Michelle L} and Rory Cave and Longshaw, {Chris M} and Minton, {Nigel P}",
year = "2018",
month = apr,
doi = "10.1093/jac/dkx486",
language = "English",
volume = "73",
pages = "973--980",
journal = "Journal of Antimicrobial Chemotherapy",
issn = "0305-7453",
publisher = "Oxford University Press",
number = "4",

}

RIS

TY - JOUR

T1 - Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin

AU - Kuehne, Sarah A

AU - Dempster, Andrew W

AU - Collery, Mark M

AU - Joshi, Nimitray

AU - Jowett, Jamie

AU - Kelly, Michelle L

AU - Cave, Rory

AU - Longshaw, Chris M

AU - Minton, Nigel P

PY - 2018/4

Y1 - 2018/4

N2 - ObjectivesTo establish the role of specific, non-synonymous SNPs in the RNA polymerase β subunit (rpoB) gene in reducing the susceptibility of Clostridium difficile to fidaxomicin and to explore the potential in vivo significance of rpoB mutant strains.MethodsAllelic exchange was used to introduce three different SNPs into the rpoB gene of an erythromycin-resistant derivative (CRG20291) of C. difficile R20291. The genome sequences of the created mutants were determined and each mutant analysed with respect to growth and sporulation rates, toxin A/B production and cytotoxicity against Vero cells, and in competition assays. Their comparative virulence and colonization ability was also assessed in a hamster infection model.ResultsThe MIC of fidaxomicin displayed by three mutants CRG20291-TA, CRG20291-TG and CRG20291-GT was substantially increased (>32, 8 and 2 mg/L, respectively) relative to that of the parent strain (0.25 mg/L). Genome sequencing established that the intended mutagenic substitutions in rpoB were the only changes present. Relative to CRG20291, all mutants had attenuated growth, were outcompeted by the parental strain, had lower sporulation and toxin A/B production capacities, and displayed diminished cytotoxicity. In a hamster model, virulence of all three mutants was significantly reduced compared with the progenitor strain, whereas the degree of caecum colonization was unaltered.ConclusionsOur study demonstrates that particular SNPs in rpoB lead to reduced fidaxomicin susceptibility. These mutations were associated with a fitness cost in vitro and reduced virulence in vivo.

AB - ObjectivesTo establish the role of specific, non-synonymous SNPs in the RNA polymerase β subunit (rpoB) gene in reducing the susceptibility of Clostridium difficile to fidaxomicin and to explore the potential in vivo significance of rpoB mutant strains.MethodsAllelic exchange was used to introduce three different SNPs into the rpoB gene of an erythromycin-resistant derivative (CRG20291) of C. difficile R20291. The genome sequences of the created mutants were determined and each mutant analysed with respect to growth and sporulation rates, toxin A/B production and cytotoxicity against Vero cells, and in competition assays. Their comparative virulence and colonization ability was also assessed in a hamster infection model.ResultsThe MIC of fidaxomicin displayed by three mutants CRG20291-TA, CRG20291-TG and CRG20291-GT was substantially increased (>32, 8 and 2 mg/L, respectively) relative to that of the parent strain (0.25 mg/L). Genome sequencing established that the intended mutagenic substitutions in rpoB were the only changes present. Relative to CRG20291, all mutants had attenuated growth, were outcompeted by the parental strain, had lower sporulation and toxin A/B production capacities, and displayed diminished cytotoxicity. In a hamster model, virulence of all three mutants was significantly reduced compared with the progenitor strain, whereas the degree of caecum colonization was unaltered.ConclusionsOur study demonstrates that particular SNPs in rpoB lead to reduced fidaxomicin susceptibility. These mutations were associated with a fitness cost in vitro and reduced virulence in vivo.

U2 - 10.1093/jac/dkx486

DO - 10.1093/jac/dkx486

M3 - Article

VL - 73

SP - 973

EP - 980

JO - Journal of Antimicrobial Chemotherapy

JF - Journal of Antimicrobial Chemotherapy

SN - 0305-7453

IS - 4

ER -