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