Dataset Information

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates.

ABSTRACT:

Background

The emergence and transmission of tigecycline- and carbapenem-resistant Klebsiella pneumoniae (TCRKP) have become a major concern to public health globally. Here, we investigated the molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant K pneumoniae (CRKP) isolates.

Methods

Forty-five non-duplicate CRKP isolates were collected from January 2017 to June 2019. We performed antimicrobial susceptibility tests, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). PCR and DNA sequencing were performed for the detection and mutation analysis of acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes, which are related to tigecycline resistance. The expression levels of efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed by quantitative real-time PCR.

Results

The resistance rate to tigecycline in CRKP isolates was 37.8% (17/45). K pneumoniae ST307 was a predominant clone type (70.6%, 12/17) among the TCRKP isolates. The expression levels of acrB (P < .001) and marA (P = .009) were significantly higher in the tigecycline-resistant group than in the tigecycline-intermediate and tigecycline-susceptible groups. Increased expression of acrB was associated with marA expression (r = 0.59, P = .013).

Conclusions

We found that the activated MarA-induced overexpression of AcrAB efflux pump plays an important role in the emergence of tigecycline resistance in CRKP isolates.

SUBMITTER: Park Y

PROVIDER: S-EPMC7755817 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Publications

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates.

Park Yumi Y Choi Qute Q Kwon Gye Cheol GC Koo Sun Hoe SH

Journal of clinical laboratory analysis 20200820 12

<h4>Background</h4>The emergence and transmission of tigecycline- and carbapenem-resistant Klebsiella pneumoniae (TCRKP) have become a major concern to public health globally. Here, we investigated the molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant K pneumoniae (CRKP) isolates.<h4>Methods</h4>Forty-five non-duplicate CRKP isolates were collected from January 2017 to June 2019. We performed antimicrobial susceptibility tests, multilocus sequence typing (ML ...[more]

PMID: 32815626

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Project description:Background:The increase in carbapenem-resistant Klebsiella pneumoniae (CRKP), especially the emergence of tigecycline-resistant K. pneumoniae (KP), is a serious public health concern. However, the underlying mechanism of tigecycline resistance is unclear. In this study, we evaluated the role of the CusS-CusR two-component system (TCS), which is associated with copper/silver resistance, in tigecycline resistance in CRKP. Methods:Following the in vitro evolution of tigecycline-resistant KP, the minimum inhibitory concentrations of tigecycline were determined using the micro-broth dilution method. RNA sequencing and data analysis were performed to identify differentially expressed genes. Quantitative PCR (qPCR) was performed to verify the genes of interest. Genes associated with tigecycline resistance, such as ramR, tex (T), and tet (A), were detected by PCR, and then mutants were confirmed by sequencing. Additionally, the efflux pump-associated genes soxS, oqxA, oqxB, acrE, and acrF were also analyzed by qPCR. CusR was deleted and complemented by the suicide vector pKO3-Km plasmid and pGEM-T-easy plasmid, respectively. Results:Nine strains of KP were evaluated in our study. Strains A2 and A3 were evolved from A1, B2, and B3 were evolved from B1, and C2 and C3 were evolved from C1. The tigecycline minimum inhibitory concentration for A1, B1, and C1 was 0.5 ?g/mL; that for A2, B2, and C3 was 16.0 ?g/mL; and that for A3, B3, and C3 was 32.0 ?g/mL. RNA-sequencing and qPCR confirmed that the differentially expressed genes cusE, cusS, cusR, cusC, cusF, cusB, and cusA showed higher expression in C2 and C3 than in C1. Genes related to the efflux pump AcrAB-TolC showed higher expression in B2 and B3 than in B1. No mutants of ramR, tex (T), or tet (A) were detected. SoxS, oqxA, oqxB, acrE, and acrF did not show increased expression in any group. After deletion and complementation of cusR among C3, the MIC of tigecycline decreased to 4 ?g/mL, and then recovered to 32 ?g/mL. The expression of cusFBCA, correspondingly decreased and increased significantly. Conclusion:In addition to its primary function in resistance to copper/silver, the CusS-CusR two-component system is associated with CRKP resistance to tigecycline.

Project description:Polymyxin has been the last resort to treat multidrug-resistant Klebsiella pneumonia. However, recent studies have revealed that polymyxin-resistant carbapenem-resistant Klebsiella pneumonia (PR-CRKP) emerged due to the mutations in chromosomal genes or the plasmid-harboring mcr gene, leading to lipopolysaccharide modification or efflux of polymyxin through pumps. Further surveillance was required. In the present study we collected PR-CRKP strains from 8 hospitals in 6 provinces/cities across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features by whole-genome sequencing (WGS). The broth microdilution method (BMD) was performed to determine the MIC of polymyxin. Of 662 nonduplicate CRKP strains, 15.26% (101/662) were defined as PR-CRKP; 10 (9.90%) were confirmed as Klebsiella quasipneumoniae by WGS. The strains were further classified into 21 individual sequence types (STs) by using multilocus sequence typing (MLST), with ST11 being prevalent (68/101, 67.33%). Five carbapenemase types were identified among 92 CR-PRKP, blaKPC-2 (66.67%), blaNDM-1 (16.83%), blaNDM-5 (0.99%), blaIMP-4 (4.95%), and blaIMP-38 (0.99%). Notably, 2 PR-CRKP strains harbored both blaKPC-2 and blaNDM-1. The inactivation of mgrB, associated significantly with high-level polymyxin resistance, was mainly caused by the insertion sequence (IS) insertion (62.96%, 17/27). Furthermore, acrR was inserted coincidently by ISkpn26 (67/101, 66.33%). The deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47 (capsule locus types), and diverse mutations of the ramR gene were identified. Only one strain carried the mcr gene. In summary, the high IS-inserted mgrB inactivation, the close relationship between ST11 and the deletion or splicing mutations of the crrCAB, and the specific features of PR-K. quasipneumoniae constituted notable features of our PR-CRKP strains in China. IMPORTANCE Polymyxin-resistant CRKP is a serious public health threat whose resistance mechanisms should be under continuous surveillance. Here, we collected 662 nonduplicate CRKP strains across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features. Polymyxin resistance mechanism in 101 PR-CRKP strains in China were also investigated, 9.8% of which (10/101) were K. quasipneumoniae, as determined via WGS, and inactivation of mgrB remained the most crucial polymyxin resistance mechanism, significantly related to high-level resistance. Deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47. Diverse mutations of the ramR gene were identified. The plasmid complementation experiment and mRNA expression analysis further confirmed that the mgrB promoter and ramR played a critical role in polymyxin resistance. This multicenter study contributed to the understanding of antibiotic resistance forms in China.

Dataset Information

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates.

Background

Methods

Results

Conclusions

Publications

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates.

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