Dataset Information

Analysis of drug resistance determinants in Klebsiella pneumoniae isolates from a tertiary-care hospital in Beijing, China.

ABSTRACT:

Background

The rates of multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) isolates among Enterobacteriaceae isolates, particularly Klebsiella pneumoniae, have risen substantially worldwide.

Methodology/principal findings

To better understand the molecular mechanisms of drug resistance in K. pneumoniae, we analyzed the drug resistance determinants for K. pneumoniae isolates collected from the 306 Hospital, a tertiary-care hospital in Beijing, China, for the period of September 1, 2010-October 31, 2011. Drug susceptibility testing, PCR amplification and sequencing of the drug resistance determinants were performed. Conjugation experiments were conducted to examine the natural ability of drug resistance to disseminate among Enterobacteriaceae strains using a sodium azide-resistant Escherichia coli J53 strain as a recipient. Among the 223 consecutive non-repetitive K. pneumoniae isolates included in this study, 101 (45.3%) were extended-spectrum beta-lactamases (ESBLs) positive. The rates of MDR, XDR, and PDR isolates were 61.4% (n = 137), 22.0% (n = 49), and 1.8% (n = 4), respectively. Among the tested drug resistance-associated genes, the following ones were detected at relatively high rates bla(CTX-M-10) (80, 35.9%), aacC2 (73, 32.7%), dhfr (62, 27.8%), qnrS (58, 26.0%), aacA4 (57, 25.6%), aadA1 (56, 25.1%). Results from conjugation experiments indicate that many of the drug resistance genes were transmissible.

Conclusions/significance

Our data give a "snapshot" of the complex genetic background responsible for drug resistance in K. pneumoniae in China and demonstrate that a high degree of awareness and monitoring of those drug resistance determinants are urgently needed in order to better control the emergence and transmission of drug-resistant K. pneumoniae isolates in hospital settings.

SUBMITTER: Li B

PROVIDER: S-EPMC3409176 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Analysis of drug resistance determinants in Klebsiella pneumoniae isolates from a tertiary-care hospital in Beijing, China.

Li Bin B Yi Yong Y Wang Qi Q Woo Patrick C Y PC Tan Lin L Jing Hua H Gao George F GF Liu Cui Hua CH

PloS one 20120731 7

<h4>Background</h4>The rates of multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) isolates among Enterobacteriaceae isolates, particularly Klebsiella pneumoniae, have risen substantially worldwide.<h4>Methodology/principal findings</h4>To better understand the molecular mechanisms of drug resistance in K. pneumoniae, we analyzed the drug resistance determinants for K. pneumoniae isolates collected from the 306 Hospital, a tertiary-care hospital in Beijing, C ...[more]

PMID: 22860106

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Project description:BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is an important pathogen causing hospital-associated outbreaks worldwide. The spread of K. pneumoniae carbapenemase-2 (KPC-2)-producing CRKP is primarily associated with sequence type (ST) 11.MethodsA total of 152 KPC-2-producing K. pneumoniae ST11 isolates were collected from the respiratory department of a tertiary care hospital in Beijing, China between 2009 and 2018. The genome sequencing of these isolates was performed on the HiSeq X Ten sequencer. Multilocus sequence typing (MLST), capsular type, plasmid replicon types and resistance genes were identified. Fifteen isolates were selected for the subsequent single-molecule real-time (SMRT) sequencing on the PacBio RS II. Alignment of the complete sequences of the plasmids carrying bla KPC-2 and/or virulence genes was performed by using BRIG and Easyfig.ResultsFrom 2012 to 2018, the detection rate of the bla KPC-2-carrying CRKP rose rapidly from 3.3 to 28.1%. KPC-2-producing K. pneumoniae ST11 isolates were dominant in CRKP, which emerged in 2012 and caused several outbreaks. Most isolates exhibited multidrug-resistant to commonly used antibiotics, while all the isolates remained susceptible to tigecycline and polymyxin B. The single nucleotide polymorphism (SNP) analysis showed that all these 152 KPC-2-producing K. pneumoniae ST11 isolates could be divided into three genetically distinct clades (A, B, and C) and eleven subclades (A1-A9 and B1-B2). The majority belonged to clade A with KL47 serotype (n = 117, 77.0%), while KL64 and KL16 were identified in clades B and C, respectively. The bla KPC-2-carrying plasmids exhibited diverse types, namely, IncFII (pHN7A8)/IncR(6/15), IncFII (pHN7A8)/IncpA1763-KPC (5/15), IncFII (pHN7A8) (1/15), IncR (1/15), and IncpA1763-KPC (1/15). The genetic environment of bla KPC-2 showed nine IS26-based composite transposons, which had a basic core structure ISKpn27-bla KPC-2-ΔISKpn6. About 27.6% (42/152) isolates co-carried 2 to 4 virulence marker genes (namely, peg344, iucA, iroB, rmpA, and rmpA2) for hvKp strains. At least three isolates were identified to harbor virulence gene-carrying plasmids.ConclusionKPC-2-producing K. pneumoniae ST11 was highly heterogeneous in our hospital. Transmission of these strains was mainly mediated by twelve high-risk clones. The bla KPC-2-carrying plasmids and genetic environment of bla KPC-2 genes exhibited active evolution in K. pneumoniae ST11. More attention should be paid to the tendency of KPC-2-ST11 to acquire hypervirulent plasmids.

Project description:Multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) is a major bacterial pathogen responsible for hospital outbreaks worldwide, mainly via the spread of high-risk clones and epidemic resistance plasmids. In this study, we evaluated the molecular epidemiology and β-lactam resistance mechanisms of MDR-Kp strains isolated in a Brazilian academic care hospital. We used whole-genome sequencing to study drug resistance mechanisms and their relationships with a K. pneumoniae carbapenemase-producing (KPC) Kp outbreak. Forty-three Kp strains were collected between 2003 and 2012. Antimicrobial susceptibility testing was performed for 15 antimicrobial agents, and polymerase chain reaction (PCR) was used to detect 32 resistance genes. Mutations in ompk35, ompk36, and ompk37 were evaluated by PCR and DNA sequencing. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to differentiate the strains. Based on distinct epidemiological periods, six Kp strains were subjected to whole-genome sequencing. β-lactamase coding genes were widely distributed among isolates. Almost all isolates had mutations in porin genes, particularly ompk35. The presence of bla KPC promoted a very high increase in carbapenem minimum inhibitory concentration only when ompk35 and ompk36 were interrupted by insertion sequences. A major cluster was identified by PFGE analysis and all isolates from this cluster belonged to clonal group (CG) 258. We have also identified a large repertoire of resistance genes in the sequenced isolates. A bla KPC-2-bearing plasmid (pUFPRA2) was also identified, which was very similar to a plasmid previously described in the first Brazilian KPC-Kp (2005). We found high-risk clones (CG258) and an epidemic resistance plasmid throughout the duration of the study (2003 to 2012), emphasizing a persistent presence of MDR-Kp strains in the hospital setting. Finally, we found that horizontal transfer of resistance genes between clones may have played a key role in the evolution of the outbreak.

Project description:Hypervirulent and multidrug-resistant Klebsiella pneumoniae poses a significant threat to public health. We aimed to determine the common carbapenemase genotypes and the carriage patterns, main antibiotic resistance mechanisms, and in vitro susceptibility of clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) to ceftazidime/avibactam (CZA) for the reasonable selection of antimicrobial agents and determine whether hypermucoviscous (HMV) phenotype and virulence-associated genes are key factors for CRKP colonization and persistence. Antibiotics susceptibility of clinical CRKP isolates and carbapenemase types were detected. CRKP isolates were identified as hypermucoviscous K. pneumoniae (HMKP) using the string test, and detection of virulence gene was performed using capsular serotyping. The bla KPC-2, bla NDM, bla IMP, and/or bla OXA-48-like were detected in 96.4% (402/417) of the isolates, and the bla KPC-2 (64.7%, 260/402) was significantly higher (P<0.05) than those of bla NDM (25.1%), bla OXA-48-like (10.4%), and bla IMP (4.2%). Carriage of a single carbapenemase gene was observed in 96.3% of the isolates, making it the dominant antibiotic resistance genotype carriage pattern (P < 0.05). Approximately 3.7% of the isolates carried two or more carbapenemase genotypes, with bla KPC-2 + bla NDM and bla NDM + bla IMP being the dominant multiple antibiotic resistance genotype. In addition, 43 CRKP isolates were identified as HMKP, with a prevalence of 10.3% and 2.7% among CRKP and all K. pneumoniae isolates, respectively. Most clinical CRKP isolates were isolated from elderly patients, and carbapenemase production was the main mechanism of drug resistance. Tigecycline and polymyxin B exhibited exceptional antimicrobial activity against CRKP isolates in vitro. Furthermore, bla KPC-2, bla NDM, and bla OXA-48-like were the main carbapenemase genes carried by the CRKP isolates. CZA demonstrated excellent antimicrobial activity against isolates carrying the single bla KPC-2 or bla OXA-48-like genotype. Capsular serotype K2 was the main capsular serotype of the carbapenem-resistant HMKP isolates. Survival rates of Galleria mellonella injected with K. pneumoniae 1-7 were 20.0, 16.7, 6.7, 23.3, 16.7, 3.3, and 13.3, respectively. Therefore, worldwide surveillance of these novel CRKP isolates and carbapenem-resistant HMKP isolates as well as the implementation of stricter control measures are needed to prevent further dissemination in hospital settings.

Project description:The prevalence of multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKP) has gradually increased. It poses a severe threat to human health. However, polymyxin-resistant hvKP is rare. Here, we collected eight polymyxin B-resistant K. pneumoniae isolates from a Chinese teaching hospital as a suspected outbreak. The minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. HvKP was identified by detecting virulence-related genes and using a Galleria mellonella infection model. Their resistance to serum, growth, biofilm formation, and plasmid conjugation were analyzed in this study. Molecular characteristics were analyzed using whole-genome sequencing (WGS) and mutations of chromosome-mediated two-component systems pmrAB and phoPQ, and the negative phoPQ regulator mgrB to cause polymyxin B (PB) resistance were screened. All isolates were resistant to polymyxin B and sensitive to tigecycline; four were resistant to ceftazidime/avibactam. Except for KP16 (a newly discovered ST5254), all were of the K64 capsular serotype and belonged to ST11. Four strains co-harbored blaKPC-2, blaNDM-1, and the virulence-related genes prmpA, prmpA2, iucA, and peg344, and were confirmed to be hypervirulent by the G. mellonella infection model. According to WGS analysis, three hvKP strains showed evidence of clonal transmission (8-20 single nucleotide polymorphisms) and had a highly transferable pKOX_NDM1-like plasmid. KP25 had multiple plasmids carrying blaKPC-2, blaNDM-1, blaSHV-12, blaLAP-2, tet(A), fosA5, and a pLVPK-like virulence plasmid. Tn1722 and multiple additional insert sequence-mediated transpositions were observed. Mutations in chromosomal genes phoQ and pmrB, and insertion mutations in mgrB were major causes of PB resistance. Polymyxin-resistant hvKP has become an essential new superbug prevalent in China, posing a serious challenge to public health. Its epidemic transmission characteristics and mechanisms of resistance and virulence deserve attention.

Dataset Information

Analysis of drug resistance determinants in Klebsiella pneumoniae isolates from a tertiary-care hospital in Beijing, China.

Background

Methodology/principal findings

Conclusions/significance

Publications

Analysis of drug resistance determinants in Klebsiella pneumoniae isolates from a tertiary-care hospital in Beijing, China.

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