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Cited by 17Antibiotic Resistance in BacteriaMicrobial infections and disease researchAntimicrobial Resistance in Staphylococcus

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Rifampin resistance and its fitness cost in Riemerella anatipestifer

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Sun, Jiakai, Zhu, Dekang, Xu, Jinge, Jia, Renyong, Chen, Shun, Liu, Mafeng, Zhao, Xinxin, Yang, Qiao, Wu, Ying, Zhang, Shaqiu, Liu, Yunya, Zhang, Ling, Yu, Yanling, You, Yu, Wang, Mingshu, Cheng, Anchun·2019·BMC Microbiology
DOI 10.1186/s12866-019-1478-7PMC PMC6533769
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Abstract

Riemerella anatipestifer ( R. anatipestifer ) is one of the most important poultry pathogens worldwide, with associated infections causing significant economic losses. Rifampin Resistance is an important mechanism of drug resistance. However, there is no information about rpoB mutations conferring rifampin resistance and its fitness cost in Riemerella anatipestifer . Comparative analysis of 18 R.anatipestifer rpoB sequences and the determination of rifampin minimum inhibitory concentrations showed that five point mutations, V382I, H491N, G502K, R494K and S539Y, were related to rifampin resistance. Five overexpression strains were constructed using site-directed mutagenesis to validate these sites. To investigate the origin and fitness costs of the rpoB mutations, 15 types of rpoB mutations were isolated from R. anatipestifer ATCC 11845 by using spontaneous mutation in which R494K was identical to the type of mutation detected in the isolates. The mutation frequency of the rpoB gene was calculated to be 10 − 8 . A total of 98.8% (247/250) of the obtained mutants were located in cluster I of the rifampin resistance-determining region of the rpoB gene. With the exception of D481Y, I537N and S539F, the rifampin minimum inhibitory concentrations of the remaining mutants were at least 64 μg/mL. The growth performance and competitive experiments of the mutant strains in vitro showed that H491D and 485::TAA exhibit growth delay and severely impaired fitness. Finally, the colonization abilities and sensitivities of the R494K and H491D mutants were investigated. The sensitivity of the two mutants to hydrogen peroxide (H 2 O 2) and sodium nitroprusside (SNP) increased compared to the parental strain. The number of live colonies colonized by the two mutants in the duckling brain and trachea were lower than that of the parental strain within 24 h. Mutations of rpoB gene in R. anatipestifer mediate rifampin resistance and result in fitness costs. And different single mutations confer different levels of fitness costs. Our study provides, to our knowledge, the first estimates of the fitness cost associated with the R. anatipestifer rifampin resistance in vitro and in vivo .

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Fig. 1 A schematic representation of the rpoB gene which encodes the β subunit of RNAP is shown (adapted from Jin DJ al. [3]). The shaded region on the β subunit is the RRDRs. The amplified portion is the region of the mutation involved in this study. The sequence consists of amino acid sequences of R. anatipestifer, F. psychrophilum, E. coli and M. tuberculosis, with the region RRDRs underlined. The asterisk at the bottom indicates that the corresponding amino acid at the top is conserved in the four strains. The mutation types associated with rifampin resistance in this study are listed above the sequence. The type identified in the isolates is underlined. The rest are spontaneous mutation types, and insert mutation types are framed. R494K was shared by both
Fig. 1 A schematic representation of the rpoB gene which encodes the β subunit of RNAP is shown (adapted from Jin DJ al. [3]). The shaded region on the β subunit is the RRDRs. The amplified portion is the region of the mutation involved in this study. The sequence consists of amino acid sequences of R. anatipestifer, F. psychrophilum, E. coli and M. tuberculosis, with the region RRDRs underlined. The asterisk at the bottom indicates that the corresponding amino acid at the top is conserved in the four strains. The mutation types associated with rifampin resistance in this study are listed above the sequence. The type identified in the isolates is underlined. The rest are spontaneous mutation types, and insert mutation types are framed. R494K was shared by bothCC BY · PubMed Central
Fig. 2 Growth curves of the spontaneous mutant strains and the parental strain. (A~H) Different mutation types at the same locus were plotted on the same graph. (I) Two insertion mutation types were plotted on the same graph. Error bars represent the standard deviation of three independent experiments
Fig. 2 Growth curves of the spontaneous mutant strains and the parental strain. (A~H) Different mutation types at the same locus were plotted on the same graph. (I) Two insertion mutation types were plotted on the same graph. Error bars represent the standard deviation of three independent experimentsCC BY · PubMed Central
Fig. 3 In vitro evaluation of the susceptibility of the wild-type ATCC 11845, mutants R494K and H491D to oxidative damage and nitrification stress and their ability to colonize ducklings. a and b Colonization capacity of wild-type ATCC 11845 and the mutants R494K and H491D in the duck brain and trachea. Values are the means of four independent experiments, and the results are shown as the relative number of CFU per gram of organs. c Effect of R494K and H491D substitutions on the sensitivity to oxidative damage. d Effect of R494K and H491D substitutions on the sensitivity to nitrificative stress. When the SNP concentration reached 0.06 mmol/L, none of the three strains could grow. Data are shown as the mean and SDs from four independent experiments, each with triplicate samples. The statistical significance of all the above tests was evaluated by Student’s t-test. The asterisk represents statistical significance (*, P < 0.05; **, P < 0.01)
Fig. 3 In vitro evaluation of the susceptibility of the wild-type ATCC 11845, mutants R494K and H491D to oxidative damage and nitrification stress and their ability to colonize ducklings. a and b Colonization capacity of wild-type ATCC 11845 and the mutants R494K and H491D in the duck brain and trachea. Values are the means of four independent experiments, and the results are shown as the relative number of CFU per gram of organs. c Effect of R494K and H491D substitutions on the sensitivity to oxidative damage. d Effect of R494K and H491D substitutions on the sensitivity to nitrificative stress. When the SNP concentration reached 0.06 mmol/L, none of the three strains could grow. Data are shown as the mean and SDs from four independent experiments, each with triplicate samples. The statistical significance of all the above tests was evaluated by Student’s t-test. The asterisk represents statistical significance (*, P < 0.05; **, P < 0.01)CC BY · PubMed Central

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Cited by

Metadata

Authors
Sun, J., Zhu, D., Xu, J., Jia, R., Chen, S., Liu, M., Zhao, X., Yang, Q., Wu, Y., Zhang, S., Liu, Y., Zhang, L., Yu, Y., You, Y., Wang, M., & Cheng, A.
Affiliations
Sichuan Agricultural University (CN)
Corresponding author
  • Wang, Mingshu
  • Cheng, Anchun
ORCID
Venue
BMC Microbiology
Publisher
Springer Science and Business Media LLC
Published
2019-05-23
Volume
19
Issue
1
Pages
107-107
ISSN
1471-2180 (ISSN-L)
DOI
10.1186/s12866-019-1478-7
PMC
PMC6533769
OpenAlex
W2947557968
Cited by
17
References
1
Citations by year
2025: 3 · 2024: 2 · 2023: 5 · 2022: 1 · 2021: 3 · 2020: 3
FWCI
0.61
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Microbiology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Key Research and Development Program of China (2017YFD050080)
  • Agriculture Research System of China (CARS-42-17)
  • International S&T Cooperation Program of Sichuan Province (2017HH0026)
  • Special Fund for Key Laboratory of Animal Disease and Human Health of Sichuan Province (2016JPT0004)
  • Science and Technology Innovation Program of Guizhou Academy of Agricultural Science ([2017]03)
Topics
  • Health Sciences › Medicine › Microbiology (medical) › Antibiotic Resistance in Bacteria
  • Life Sciences › Immunology and Microbiology › Microbiology › Microbial infections and disease research
  • Health Sciences › Medicine › Infectious Diseases › Antimicrobial Resistance in Staphylococcus
Keywords
resistance, Riemerella anatipestifer, rpoB mutations, fitness cost, drug resistance, minimum inhibitory concentration, spontaneous mutation, competitive fitness, colonization ability, hydrogen peroxide sensitivity, poultry pathogens
MeSH terms
Animals, Bacterial Proteins, Brain, Ducks, Hydrogen Peroxide, Microbial Sensitivity Tests, Nitroprusside, Rifampin, DNA-Directed RNA Polymerases, Trachea, Point Mutation, Drug Resistance, Bacterial, Riemerella

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