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Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline.

ABSTRACT: Based on its important role in last-line therapeutics against multidrug-resistant bacteria, tigecycline has been increasingly important in treating infections. However, mounting reports on tigecycline-resistant bacterial strains isolated from different sources are of concern, and molecular mechanisms regarding tigecycline resistance are poorly understood. Riemerella anatipestifer is a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, which causes fibrinous pericarditis, perihepatitis, and meningitis in infected ducks. We previously constructed a random transposon mutant library using Riemerella anatipestifer strain CH3, in present study, we described that Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline. Using the minimum inhibitory concentration assay, a mutant strain showed significantly increased (about six-fold) tigecycline susceptibility. Subsequently, the knocked-down gene was identified as M949_0459, a putative flavin adenine dinucleotide-dependent oxidoreductase. To confirm the resistance function, M949_0459 gene was overexpressed in Escherichia coli strain BL21, and the minimum inhibitory concentration analysis showed that the gene product conferred resistance to tigecycline. Additionally, expression of the M949_0459 gene under treatment with tigecycline was measured with quantitative real-time PCR. Results showed that the mRNA expression of M949_0459 gene was elevated under tigecycline treatment with dose range of 1-10 mg/L, and peaked at 4 mg/L. Moreover, two kinds of efflux pump inhibitors, carbonyl cyanide m-chlorophenyl hydrazone and phenylalanine arginyl ?-naphthylamide were tested, which showed no function on tigecycline resistance in the strain CH3. Our results may provide insights into molecular mechanisms for chemotherapy in combating Riemerella anatipestifer infections.

SUBMITTER: Li T

PROVIDER: S-EPMC5722508 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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<i>Riemerella anatipestifer M949_0459</i> gene is responsible for the bacterial resistance to tigecycline.

Li Tao T Shan Min M He Jing J Wang Xiaolan X Wang Shaohui S Tian Mingxing M Qi Jingjing J Luo Tingrong T Shi Yonghong Y Ding Chan C Yu Shengqing S

Oncotarget 20170727 57

Based on its important role in last-line therapeutics against multidrug-resistant bacteria, tigecycline has been increasingly important in treating infections. However, mounting reports on tigecycline-resistant bacterial strains isolated from different sources are of concern, and molecular mechanisms regarding tigecycline resistance are poorly understood. <i>Riemerella anatipestifer</i> is a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, which causes fibrinous pericarditis, ...[more]

PMID: 29228556

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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.

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.

Dataset Information

Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline.

Publications

<i>Riemerella anatipestifer M949_0459</i> gene is responsible for the bacterial resistance to tigecycline.

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