Short communication
Genetic characterisation of clinical Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline: Role of the global regulator RamA and its local repressor RamR

https://doi.org/10.1016/j.ijantimicag.2014.12.022Get rights and content

Highlights

  • There was an association between expression of ramA and the minimum inhibitory concentration (MIC) of tigecycline (and other agents).

  • Tigecycline MICs were negatively correlated with the expression of marA and soxS.

  • Inactivation of ramA in one hyperexpressing isolate (with an insert in ramR) resulted in a 16-fold decrease in the tigecycline MIC.

  • Tigecycline resistance could develop in isolates with wild-type ramR and in isolates without increased expression of ramA.

  • Mutations in the rpsJ gene were not evident, and mutations in acrR did not account for tigecycline resistance.

Abstract

Laboratory-derived Klebsiella pneumoniae mutants demonstrated that the ramA locus mediated low-level tigecycline resistance. The aim of this study was to elucidate the underlying mechanisms of tigecycline resistance in clinical K. pneumoniae isolates. In total, 106 isolates with tigecycline MICs ranging from 0.125 mg/L to 16 mg/L were collected to determine the correlations between expression of the global regulon ramA, marA, soxS, the acrB pump gene and tigecycline MICs. PCR was used to determine whether mutations in ramR, acrR or the rpsJ gene encoding 30S ribosomal protein S10 were responsible for tigecycline resistance. ramA or ramR inactivation and corresponding trans-complemented strains were used to characterise the contribution of RamA and RamR to tigecycline resistance. Tigecycline MICs were correlated with transcriptional levels of ramA and acrB, but were negatively correlated with marA and soxS. Disrupting ramA strikingly reduced the tigecycline MIC by 16-fold, accompanied by a 0.5-fold downregulation of acrB expression and 3.14- and 3.80-fold upregulation of marA and soxS, respectively. Complementation with plasmid-borne ramA restored the original parental phenotype of decreased tigecycline susceptibility. Of 34 tigecycline-non-susceptible isolates, 21 harbouring diverse mutations in RamR led to ramA overexpression. Disrupting the mutated ramR gene and complementing the mutated ramR gene with a wild-type gene downregulated expression of ramA but maintained the same tigecycline-resistant phenotype as the parental strain; the complemented strain exhibited 4.21- and 27.51-fold increased expression of acrB and marA, respectively. In conclusion, for the majority of tigecycline-resistant K. pneumoniae, ramA, depressed by ramR, was the major factor accounting for tigecycline resistance.

Introduction

Tigecycline, belonging to the glycylcycline class of antibiotics, is one of the few therapeutic options available for difficult-to-treat infections such as those caused by carbapenem-resistant Gram-negative bacteria and multidrug-resistant (MDR) pathogens [1]. The AcrAB–TolC multidrug efflux pump, belonging to the resistance–nodulation–cell division (RND) family, has been shown to contribute to tigecycline resistance in Enterobacteriaceae, including Enterobacter cloacae, Escherichia coli, Salmonella enterica and Klebsiella pneumoniae [2], [3], [4], [5]. A previous study demonstrated that AcrR directly controls expression of the AcrAB efflux pump and consequently contributes to fluoroquinolone resistance [6] in K. pneumoniae, but no similar phenomenon had been found for tigecycline resistance in K. pneumoniae. Three large studies identified that tigecycline minimum inhibitory concentrations (MICs) were correlated with the transcriptional level of the regulator RamA [6], [7], [8] or SoxS [8] in clinical isolates of K. pneumoniae, whereas laboratory-derived tigecycline-resistant spontaneous mutants were found to be related to overexpression of marA and acrB (but not ramA and soxS) [8]. Previously, mutations within the ramR gene were shown to contribute to low-level tigecycline resistance [MIC = 2 mg/L according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines] in clinical K. pneumoniae isolates [9], but Rosenblum et al. [10] found that decreased tigecycline susceptibility was not always associated with ramA expression and that RamA was not always associated with RamR-mediated derepression. In addition, V57L mutation in the rpsJ gene, which encodes the 30S ribosomal protein S10, might be involved in tigecycline resistance among K. pneumoniae isolates that do not harbour mutations within the ramR gene [11]. Whether clinical K. pneumoniae isolates originating from different geographic locations possess different tigecycline resistance mechanisms remains unknown, as do the precise roles of these factors affecting tigecycline resistance.

Section snippets

Bacterial strains and plasmids

In total, 106 clinical isolates of K. pneumoniae, comprising 34 tigecycline-non-susceptible K. pneumoniae and 72 tigecycline-susceptible K. pneumoniae (including 16 randomly selected, multisusceptible K. pneumoniae isolates), were collected from two national surveillance programmes in China in 2011 and 2012. All of the strains were isolated from respiratory tract, blood or abdominal infections. The properties of the laboratory strains and vectors are listed in Table 1.

Antibiotic susceptibility testing

Susceptibility assays were

Antibiotic susceptibility testing and multilocus sequence typing

The susceptibility profiles of tigecycline-non-susceptible and tigecycline-susceptible K. pneumoniae isolates are shown in Supplementary Table S1, and the STs of tigecycline-non-susceptible K. pneumoniae isolates are shown in Supplementary Table S2. By eBURST algorithm, 20 of the 34 tigecycline-non-susceptible isolates, with diverse STs, clustered into CC37, whereas 56 of 72 tigecycline-susceptible K. pneumoniae isolates belonged to CC37, the dominant ST of which was ST11 (data not shown).

Correlations between tigecycline minimum inhibitory concentrations and the transcriptional levels of ramA, the acrB pump gene and other regulatory genes

As

Discussion

Tigecycline is one of the few remaining therapeutic options for treating infections caused by carbapenem-resistant or MDR Gram-negative bacilli. However, increasing rates of tigecycline-resistant Enterobacteriaceae are of growing concern, and the prevalence of these strains varies worldwide [15]. In this study, 7.17% (34/474) of K. pneumoniae isolates from nosocomial infections were resistant to tigecycline (data not shown). Although tigecycline retained a high susceptibility rate against

Funding

This work was supported by the Beijing Natural Science Foundation [grant no. 5122041], the Research Fund for the Doctoral Program of Higher Education of China (RFDP) [grant no. 20110001110043], the Beijing City Board of Education Science and Technology key project [grant no. KZ201210025025] and the Trans-Century Training Programme Foundation for the Talents by the State Education Commission [grant no. NCET-10-0205].

Competing interests

None declared.

Ethical approval

Not required.

Acknowledgments

The authors thank Dongshen Zhou of the State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology (Beijing, China), for kind guidance in the gene inactivation experiment and for donation of the pKO3-Km and pGEM®-T Easy-Km vectors, which were constructed by Jin-Town Wang's group at National Taiwan University Hospital (Taipei, Taiwan).

References (15)

  • Y. Sun et al.

    The emergence of clinical resistance to tigecycline

    Int J Antimicrob Agents

    (2013)
  • R. Rosenblum et al.

    Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae

    Int J Antimicrob Agents

    (2011)
  • D. Keeney et al.

    RamA, a transcriptional regulator, and AcrAB, a RND-type efflux pump, are associated with decreased susceptibility to tigecycline in Enterobacter cloacae

    Microb Drug Resist

    (2007)
  • D. Keeney et al.

    MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli

    J Antimicrob Chemother

    (2008)
  • T. Horiyama et al.

    Roles of Salmonella multidrug efflux pumps in tigecycline resistance

    J Antimicrob Chemother

    (2011)
  • Z.K. Sheng et al.

    Mechanisms of tigecycline resistance among Klebsiella pneumoniae clinical isolates

    Antimicrob Agents Chemother

    (2014)
  • T. Schneiders et al.

    Role of AcrR and RamA in fluoroquinolone resistance in clinical Klebsiella pneumoniae isolates from Singapore

    Antimicrob Agents Chemother

    (2003)
There are more references available in the full text version of this article.

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