Dataset Information

Contribution of RaeB, a Putative RND-Type Transporter to Aminoglycoside and Detergent Resistance in Riemerella anatipestifer.

ABSTRACT: Riemerella anatipestifer is an important pathogenic bacterium that infects ducks. It exhibits resistance to multiple classes of antibiotics. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens and they are poorly understood in R. anatipestifer. In this study, a gene encoding the B739_0873 protein in R. anatipestifer CH-1, which belongs to the resistance-nodulation-cell division (RND) efflux pump family, was identified. With respect to the substrate specificity of B739_0873, the antibiotic susceptibility testing showed that the B739_0873 knockout strain was more sensitive to aminoglycosides and detergents than the wild-type strain. The transcription of B739_0873 was up-regulated when R. anatipestifer CH-1 was exposed to sub-inhibitory levels of these substrates. From the gentamicin accumulation assay, we concluded that B739_0873 was coupled to the proton motive force to pump out gentamicin. Furthermore, site-directed mutagenesis demonstrated that Asp 400, Asp 401, Lys 929, Arg 959, and Thr 966 were the crucial function sites of B739_0873 in terms of its ability to extrude aminoglycosides and detergents. Finally, we provided evidence that B739_0873 is co-transcribed with B739_0872, and that both B739_0872 and B739_0873 are required for aminoglycoside and detergent resistance. In view of these results, we designate B739_0873 as RaeB (Riemerella anatipestifer efflux).

SUBMITTER: Zhang X

PROVIDER: S-EPMC5727081 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Contribution of RaeB, a Putative RND-Type Transporter to Aminoglycoside and Detergent Resistance in Riemerella anatipestifer.

Zhang Xin X Wang Ming-Shu MS Liu Ma-Feng MF Zhu De-Kang DK Biville Francis F Jia Ren-Yong RY Chen Shun S Sun Kun-Feng KF Yang Qiao Q Wu Ying Y Zhao Xin-Xin XX Chen Xiao-Yue XY Cheng An-Chun AC

Frontiers in microbiology 20171208

Riemerella anatipestifer is an important pathogenic bacterium that infects ducks. It exhibits resistance to multiple classes of antibiotics. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens and they are poorly understood in R. anatipestifer. In this study, a gene encoding the B739_0873 protein in R. anatipestifer CH-1, which belongs to the resistance-nodulation-cell division (RND) efflux pump family, was identifie ...[more]

PMID: 29276505

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Project description:BackgroundRiemerella 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.ResultsComparative 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 (H2O2) 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.ConclusionsMutations 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.

Project description:Riemerella anatipestifer is an important pathogen in waterfowl, and is generally multidrug resistant. This study assessed the current status of Riemerella anatipestifer antibiotic resistance and antibiotic-resistance genes (ARGs), compared the results of different detection methods, and evaluated a new method of studying the association between antibiotic resistance and ARGs in Riemerella anatipestifer. In this study, 51 strains of Riemerella anatipestifer were isolated from ducks on several farms, their resistance to 28 antibiotics was assessed, and the isolates were subjected to whole-genome sequencing. The number of ARGs carried by Riemerella anatipestifer was predicted, compared, and analyzed, and the consistency between ARGs and antibiotic-resistance phenotypes was assessed. The potential for loss of resistance genes during the sequencing and assembly of genome-wide framework map was assessed, and a new ARG detection method was pilot tested. The 51 strains of Riemerella anatipestifer were multidrug resistant (MDR) and had high level of resistance to aminoglycosides, trimethoprim, lincosamides, polypeptides, and macrolides. Based on the genome-wide framework map of the 51 strains, 3 local databases of ABRicate software and 1 online database of CARD website were used to detect ARGs, and a mean of 4 to 5 ARGs were identified per isolate. Although the detection results differed according to the database used, the general performance was consistent. The online website detected more types of ARGs than the ABRicate software. The association between ARGs and antibiotic-resistance phenotypes was assessed, and the ermF gene was identified as a possible key ARGs regulating macrolide resistance of Riemerella anatipestifer. The method used to investigate and detect Riemerella anatipestifer ARGs was convenient and rapid, and had strong accuracy and pertinence. The ARGs detection method reported here combined the advantages of PCR and genome detection, and could greatly reduce workload and detect ARGs more precisely.

Project description:Resistance to trimethoprim is mainly mediated by the acquisition of mobile dfrA genes, and most of them were discovered in Enterobacteriales. A total of 139 Riemerella anatipestifer isolates were collected from different farms in China during 2014 to 2020. Whole genome sequencing (WGS) and genome analysis of R. anatipestifer isolates revealed a 504-bp open reading frame (ORF) encoding a putative dfrA gene. This DfrA variant shared 66.47% amino acid sequence identity with DfrA36 and shared ≤51.20% identity with any other previously identified DfrA proteins. The novel dfrA gene, designated dfrA49, conferred trimethoprim (TMP) resistance when cloned into Escherichia coli BL21(DE3). Thirty dfrA49-positive isolates were identified from Jiangsu and Guangdong province (5/38, 13.16%, and 25/101, 24.75%, respectively). Five of the 38 isolates had obtained the complete genome sequences. Genomic analysis showed that the dfrA49 gene was located on chromosomes or a plasmid (four of them were on chromosomes and one was located on a plasmid). The plasmid p20190305E2-2_2 carried dfrA49, catB, ermF, ereD, blaOXA (88.36% identity with blaOXA-209), Δarr, and tet(X18). Further research indicated that dfrA49 usually coexisted with catB in R. anatipestifer. In this study, a novel trimethoprim resistance gene, dfrA49, was identified and characterized in chromosome and plasmid sequences from R. anatipestifer using WGS and bioinformatic methods. It further expands knowledge about the pool of mobile dfrA genes that confer resistance to trimethoprim and provides information about antibiotic resistance genes in R. anatipestifer, where the resistance gene pool circulating is not well understood. IMPORTANCE Trimethoprim is a synthetic antimicrobial agent inhibiting dihydrofolate reductase (DHFR), which is encoded by the folA gene. Acquired genes that confer trimethoprim resistance due to mutations in the folA gene are designated dfr and divided into two main families including dfrA and dfrB. Resistance to trimethoprim is mainly mediated by the acquisition of mobile dfrA genes, and most of them were discovered in Enterobacteriales. R. anatipestifer belongs to the Flavobacteriaceae family, and the reservoir of dfrA resistance genes in R. anatipestifer has not been fully investigated. A novel trimethoprim resistance gene, dfrA49, which was identified and characterized in chromosome and plasmid sequences in this study, increased the MIC of TMP (>256-fold) in E. coli BL21(DE3). Our study expands knowledge about the pool of mobile dfrA genes that confer resistance to trimethoprim and broadens the understanding of the host spectrum of dfrA family genes.

Dataset Information

Contribution of RaeB, a Putative RND-Type Transporter to Aminoglycoside and Detergent Resistance in Riemerella anatipestifer.

Publications

Contribution of RaeB, a Putative RND-Type Transporter to Aminoglycoside and Detergent Resistance in <i>Riemerella anatipestifer</i>.

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