Tigecycline resistance among carbapenem-resistant Klebsiella Pneumoniae: Clinical characteristics and expression levels of efflux pump genes

RESEARCH ARTICLE Tigecycline resistance among carbapenemresistant Klebsiella Pneumoniae: Clinical characteristics and expression levels of efflux pump genes Sheng-Kang Chiu1,2, Ming-Chin Chan3, Li-Yueh Huang4, Yi-Tsung Lin5, Jung-Chung Lin1, Po-Liang Lu6, L. Kristopher Siu1,4,7, Feng-Yee Chang1, Kuo-Ming Yeh1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC, 2 Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC, 3 Infection Control Office, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC, 4 Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Miaoli, Taiwan, ROC, 5 Section of Infectious Diseases, Department of Medicine, Taipei Veterans General Hospital, National Yan-Ming University, Taipei, Taiwan, ROC, 6 Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC, 7 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC * OPEN ACCESS Citation: Chiu S-K, Chan M-C, Huang L-Y, Lin Y-T, Lin J-C, Lu P-L, et al. (2017) Tigecycline resistance among carbapenem-resistant Klebsiella Pneumoniae: Clinical characteristics and expression levels of efflux pump genes. PLoS ONE 12(4): e0175140. https://doi.org/10.1371/journal. pone.0175140 Editor: Yung-Fu Chang, Cornell University, UNITED STATES Received: December 13, 2016 Accepted: March 21, 2017 Published: April 7, 2017 Copyright: © 2017 Chiu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Objectives Tigecycline is a treatment option for infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). Emerging tigecycline resistance in CRKP represents a growing threat. Knowledge of the clinical, microbiological, and molecular characteristics of tigecycline- and carbapenem-resistant Klebsiella pneumoniae (TCRKP) is limited. Methods Patients infected with TCRKP were identified from a Taiwanese national surveillance study. Clinical data were collected from medical records. We performed susceptibility tests, carbapenemase gene detection, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Furthermore, we performed quantitative real-time polymerase chain reaction (qRT-PCR) analyses to assess the expression levels of the efflux pump genes acrB and oqxB. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Results Funding: The work was supported by research grants from the Centers for Disease Control, Taiwan (MOHW104-CDC-C-114-144406; http:// www.cdc.gov.tw/english/index.aspx; JCL) and TriService General Hospital (TSGH-C103-124; http:// www.tsgh.ndmctsgh.edu.tw/; SKC). The funders had no role in study design, data collection and We identified 16 patients infected with TCRKP, with urinary tract infection (UTI) being the most common type of infection (63%). The all-cause 30-day mortality rate was 44% in patients with TCRKP infection. Patients with a site of infection other than the urinary tract had a significantly higher mortality rate than patients with UTIs (83% vs. 20%, p = 0.035). PFGE and MLST revealed no dominant clone or sequence type. Using qRT-PCR, overexpression of both the acrB and oqxB genes was identified in seven isolates, and overexpression of the PLOS ONE | https://doi.org/10.1371/journal.pone.0175140 April 7, 2017 1 / 15 Tigecycline- and Carbapenem-Resistant Klebsiella Pneumoniae analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. oqxB gene was observed in another seven. There was poor correlation between acrB or oqxB expression and tigecycline MICs (r = -0.038 and -0.166, respectively). Conclusions The mortality rate in patients infected with TCRKP in this study was 44% and this is an important subset of patients. The absence of a linear relationship between efflux pump genes expression and MICs indicates that tigecycline resistance may be mediated by other factors. Continuous monitoring of tigecycline resistance among CRKP isolates and resistance mechanisms are necessary. Introduction Klebsiella pneumoniae is an important pathogen that causes various infections including bacteremia, pneumonia, liver abscesses, and urinary tract infections [1]. The prevalence of carbapenem-resistant K. pneumoniae (CRKP) has been increasing globally, making antimicrobial treatment difficult and causing higher disease-related mortality rates [2,3]. Tigecycline is one of the few available choices for treating carbapenem-resistant bacterial infections [4]. Once CRKP develops resistance to tigecycline, the treatment options are much more limited. To date, studies on the clinical characteristics and outcome of tigecycline resistance superimposed to CRKP are very limited [5–7]. The nonsusceptibility rate of tigecycline against CRKP was 9.1% in a surveillance study from Taiwan [8]. The resistance rate of tigecycline among carbapenemase-producing K. pneumoniae was reported to be 11.2% in China [9] and 14.5% in Korea [10]. In their study, van Duin et al. found that the rate of tigecycline resistance among patients with CRKP was 18% in a multicenter, prospective cohort of hospitalized patients in the USA [7]. The increasing prevalence of tigecycline resistance, after its launch in 2005, is a growing concern [11]. The mechanism of tigecycline resistance is complex and has not yet been fully elucidated [12]. Previous studies have reported that increased expression of efflux pumps such as AcrAB and OqxAB play an essential role in the tigecycline resistance mechanisms of K. pneumoniae [9,13]. The AcrAB efflux pump is a tripartite complex consisting of the large cytoplasmic membrane protein AcrB, the membrane fusion protein AcrA and a channel, TolC; this pump expels a variety of antibiotics including β-lactams, chloramphenicol, erythromycin fluoroquinolones, fusidic acid and tetracycline [14]. The OqxAB efflux pump is chromosomally located in K. pneumoniae [15] and requires a functional AcrAB efflux pump, although its natural function remains unknown [11]. In the present study, we collected clinical isolates of carbapenem non-susceptible K. pneumoniae (CnSKP) between January 2012 and December 2014 in a Taiwanese surveillance study and identified 16 isolates of tigecycline- and carbapenem-resistant K. pneumoniae (TCRKP). We investigated the clinical characteristics and outcome of patients with TCRKP infection, efflux pumps expression among the TCRKP isolates, and elucidated the possible role of Acr (...truncated)