Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of blaNDM-1 in India, Sweden, and the United Kingdom.
ABSTRACT: Clinical isolates of Klebsiella pneumoniae producing NDM-1 carbapenemase from India (n = 22), the United Kingdom (n = 13), and Sweden (n = 4) were subjected to multilocus sequence typing (MLST), automated repetitive sequence-based PCR (rep-PCR), serotyping, virulence gene screening, and plasmid replicon typing. The most frequently detected MLST sequence types (STs) were ST14 (n = 13; all serotype K2), ST11, ST149, ST231, and ST147. The correlation between MLST and automated rep-PCR was excellent. IncA/C was the most frequently detected plasmid replicon type (n = 14). ST14, ST11, and other successful clones may be important for the dissemination of bla(NDM-1).
Project description:For addressing the issue of antimicrobial drug resistance in developing countries, it is important to investigate the characteristics of carbapenemase-producing organisms. We aimed to genetically characterize a carbapenemase-producing <i>Klebsiella pneumoniae</i> (CPKP) isolated in the intensive care unit of a tertiary hospital in Bangladesh. The number of CPKP isolates were 43/145 (30%), of which pandrug-resistant (PDR) strains were 14%. These carbapenemases were New Delhi metallo-beta-lactamase (NDM)-1 (53%), NDM-5 (14%), oxacillinase (OXA)-181 (12%), OXA-232 (10%), NDM-5 + OXA-181 (5%), and NDM-5 + OXA-232 (2%). Many CPKP isolates harbored a variety of resistance genes, and the prevalence of 16S rRNA methyltransferase was particularly high (91%). The 43 CPKP isolates were classified into 14 different sequence types (STs), and the common STs were ST34 (26%), ST147 (16%), ST11 (9%), ST14 (9%), ST25 (7%), and ST231 (7%). In this study, PDR strains were of three types, ST147, ST231, and ST14, and their PDR rates were 57, 33, and 25%, respectively. The spread of the antimicrobial drug resistance of CPKP in Bangladesh was identified. In particular, the emergence of PDR is problem, and there may be its spread as a superbug of antimicrobial treatment.
Project description:Background & objectives:Klebsiella pneumoniae (KP), a common cause of invasive infections, is often extensively drug resistant in India. At present, studies on resistance mechanism and clonal relationship of KP from India are limited. The present study was undertaken to determine the resistance mechanism and clonal relationship of colistin-resistant isolates obtained from various specimens. Carbapenemases were also determined since the isolates were carbapenem resistant. Methods:Sixty five isolates from blood, exudates and respiratory specimens collected between 2016 and 2017 were studied. Colistin minimum inhibitory concentration (MIC) was performed by broth-micro dilution method. Multiplex PCR was carried out to determine carbapenemases. Targeted sequencing was performed to determine mutations in mgrB, phoP, phoQ and multilocus sequence typing was performed to determine the prevalent clones. Results:Colistin MIC ranged from 4 to 256 ?g/ml. SHV, TEM and CTX-M were co-produced in 60 per cent and OXA48-like in 71 per cent. Thirteen isolates had mutations in mgrB. Mutations included a premature stop codon at 21st amino acid, the presence of insertion sequences such as IS903, IS Kpn 14 and ISK pn 26; and elongation of mgrB. Novel mutations were also observed among phoP and phoQ genes. Colistin resistance due to mcr genes was absent. Fifteen clonal types were seen with ST231, ST14 and ST2096 being predominant. Interpretation & conclusions:This study revealed the changing trend of carbapenem resistance mechanism predominantly to OXA48-like from NDM. Known mgrB mutations and novel mutations in phoP and phoQ were detected. There was no plasmid-mediated colistin resistance. ST14 and ST231 were international clones associated with carbapenem resistance. Colistin-resistant KP was of diverse clones with predominantly ST231, ST14 and ST2096.
Project description:Purpose:The emergence of multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) is associated with the acquisition of multiple carbapenemases. Their clonal spread is a worldwide concern due to their critical role in nosocomial infections. Therefore, the identification of high-risk clones with antibiotic resistance genes is very crucial for controlling its global spread. Materials and Methods:A total of 227 K. pneumoniae strains collected during April 2018 to November 2019 were confirmed by PCR. Carbapenemases and extended-spectrum ?-lactamases (ESBL) were detected phenotypically. Confirmation of carbapenemases was carried out by PCR and Sanger sequencing. The clonal lineages were assigned to selected isolates by multilocus sequence typing (MLST), and the plasmid analysis was done by PCR-based detection of the plasmid replicon typing. Results:Of the total K. pneumoniae, 117 (51.5%) were carbapenem resistant (CRKP) and 140 (61.7%) were identified as ESBL producers. Intermediate to high resistance was detected in the tested ?-lactam drugs while polymyxin-B and tigecycline were found to be susceptible. Among CRKP, 91 (77.8%) isolates were detected as carbapenemase producing, while 55 (47%) were positive for bla NDM-1 23.9% (n=28), bla OXA-48 22.2% (n=26) and bla VIM 0.85% (n=1) while 12.7% (n=7) carried both bla NDM-1 and bla OXA-48 genes. The CRKP coharboring bla NDM-1 and bla OXA-48 genes (n=7) were positive for bla CTX-M bla SHV (n=3), bla SHV (n=1) and bla CTX-M (n=3). The novel CRKP with the coexistence of bla NDM-1, bla OXA-48, bla CTX-M and bla SHV genes were associated with the high-risk clone ST147 (n=5) and ST11 (n=2). The assigned replicon types were IncL/M, IncFII, IncA/C and IncH1. Conclusion:This is the first report of the coexistence of bla NDM-1, bla OXA-48, bla CTX-M and bla SHV genes on a high-risk lineage ST147 from Pakistan. This study highlights the successful dissemination of carbapenemase resistance genes in the high-risk clones that emphasizes the importance of monitoring and controlling the spread of these diverse clones globally.
Project description:Purpose:Plasmids of the incompatibility group X type 3 (IncX3) were described carrying various carbapenemase genes in carbapenemase-producing Enterobacteriaceae (CPE) worldwide and in the United Arab Emirates (UAE), as well. To understand the driving force behind the emergence of such plasmids in the UAE, the relationship between IncX3 plasmids encountered locally and globally was investigated. Methods:CPE strains isolated in the UAE during 2009-2014 were screened by X3 PCR-based replicon typing. The clonal relationship of CPE carrying IncX3 plasmids was determined by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Complete sequence of selected IncX3 plasmids was determined. Phylogenetic relationship between the carbapenemase carrying IncX3 plasmids from the UAE and of those reported worldwide was established by comparing the plasmid backbones. Results:10.2% of the 295 CPE tested were identified to carry IncX3 plasmids: 13 Escherichia coli, 13 Klebsiella pneumoniae, two Enterobacter cloacae, one Citrobacter freundii and one Morganella morganii isolate, respectively. Most of them were non-clonal; with small clusters of triplets and pairs of E. coli and K. pneumoniae, and a cluster of five K. pneumoniae ST11 exhibiting >90% similar PFGE patterns, respectively. The 30 isolates harbored either bla NDM-1, bla NDM-4, bla NDM-5, bla NDM-7, bla OXA-181 or bla KPC-2 carbapenemase genes on IncX3 plasmids. Phylogenetic analysis of the backbone region of IncX3 plasmids carrying various beta-lactamase genes from the UAE (n=23) and that of North-America, Europe, Asia and Australia (n=35) revealed three clusters based on the carbapenemase genes carried: plasmids harboring bla OXA-181 and bla NDM-5 formed two distinct groups, whereas backbones of plasmids with bla NDM-1, bla NDM-4 and bla NDM-7 clustered together. Each cluster contained plasmids of diverse geographical origin. Conclusion:The findings suggest that different carbapenemase gene carrying IncX3 plasmids encountered in the UAE do not evolve locally, rather are subtypes of this epidemic plasmid emerging in this country due to international transfer.
Project description:New Delhi metallo-?-lactamase (NDM) is a metallo-?-lactamase able to hydrolyze almost all ?-lactams. Twenty-four NDM variants have been identified in >60 species of 11 bacterial families, and several variants have enhanced carbapenemase activity. Klebsiella pneumoniae and Escherichia coli are the predominant carriers of bla NDM, with certain sequence types (STs) (for K. pneumoniae, ST11, ST14, ST15, or ST147; for E. coli, ST167, ST410, or ST617) being the most prevalent. NDM-positive strains have been identified worldwide, with the highest prevalence in the Indian subcontinent, the Middle East, and the Balkans. Most bla NDM-carrying plasmids belong to limited replicon types (IncX3, IncFII, or IncC). Commonly used phenotypic tests cannot specifically identify NDM. Lateral flow immunoassays specifically detect NDM, and molecular approaches remain the reference methods for detecting bla NDM Polymyxins combined with other agents remain the mainstream options of antimicrobial treatment. Compounds able to inhibit NDM have been found, but none have been approved for clinical use. Outbreaks caused by NDM-positive strains have been reported worldwide, attributable to sources such as contaminated devices. Evidence-based guidelines on prevention and control of carbapenem-resistant Gram-negative bacteria are available, although none are specific for NDM-positive strains. NDM will remain a severe challenge in health care settings, and more studies on appropriate countermeasures are required.
Project description:The continuous emergence of carbapenem-resistant Escherichia coli (CRECO) presents a great challenge to public health. New Delhi metallo-lactamase (NDM) variants are widely disseminated in China, so the research on the prevalence and transmission of diverse bla NDM variants is urgently needed. In the present study, 54 CRECO isolates were collected from 1,185 Escherichia coli isolates in five hospitals in Northern Jiangsu Province, China from September 2015 to August 2016. Antimicrobial susceptibility tests, PCR detection of resistance determinants, multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to characterize these strains. Plasmid conjugation experiments were carried out to determine the transferability of resistant genes from selected isolates. PCR-based replicon typing (PBRT), S1 nuclease-PFGE, and Southern blotting were conducted for plasmid profiling. Carbapenemase genes were detectable in all CRECO isolates, among which thirty-one CRECO isolates were found to carry bla NDM-5 (54.7%), while, bla NDM-1, bla NDM-7, bla NDM-4, bla NDM-9, and bla KPC-2 were identified in 14, five, two, one, and one isolates, respectively. MLST results revealed 15 different STs and four new STs were first reported to be linked with NDM-producing isolates. PFGE typing showed that no more than two isolates with the same ST appeared to the same band pattern except three ST410 isolates. Twenty-six selected NDM-producing isolates were successfully transferred to E. coli J53 by conjugation experiments. Notably, 50.0% (13/26) of blaNDM variants were found to be carried by ~55 kb IncX3 plasmid. Our study reported a high prevalence of blaNDM variants, especially bla NDM-5, in Northern Jiangsu province, China. Diverse bla NDM variants were mainly carried by ~55 kb IncX3 plasmids, suggesting that the fast evolution and high transferability of this kind of plasmid promote the high prevalence of bla NDM variants. Therefore, large-scale surveillance and effective infection control measures are also urgently needed to prevent diverse bla NDM variants from becoming epidemic in the future.
Project description:The aim of this study was to investigate the characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) collected during an outbreak in a Chinese teaching hospital and to provide insights into the prevention and control of nosocomial infection. We collected unique CRKP clinical isolates from 2009 to 2013. Antibiotic-resistant genes were identified by polymerase chain reaction (PCR) and sequencing. The isolates were typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmids were classified using a PCR-based incompatibility/replicon typing method and a replicon sequence typing method. Conjugation experiments were performed to evaluate the transferability of carbapenem-resistant genes. Whole genome sequencing (WGS) was conducted to further investigate the genetic background of the isolates. Infection control practices were reviewed throughout the study period. Klebsiella pneumoniae sequence type (ST) 11 emerged in 2010 and acquired the bla KPC-2 gene by 2011. From 2011 to 2013, ST11 KPC-2-producing CRKP (G type) prevailed as the most common CRKP in our hospital, causing a prolonged outbreak. The majority of these CRKP strains possess an IncFII plasmid, with Tn1721-bla KPC-2-?Tn3-IS26 bearing the genetic structure for bla KPC-2. Infection prevention control measures available at the time contained the initial outbreak, but had no effect on the spread of CRKP later. This study demonstrated the seriousness concerning the spread of KPC-2-producing ST11 CRKP in a Chinese hospital, indicating that current prevention and control strategies for carbapenem-resistant Enterobacteriaceae (CRE) nosocomial infection need to be investigated and adjusted.
Project description:Purpose:To characterize the clinical, resistance, and virulence features of carbapenem-resistant Klebsiella pneumonaie (CRKP) and hypervirulent Klebsiella pneumoniae (hvKP) and also provide an effective selection of drug in CRKP and hvKP treatment. Materials and Methods:Twelve strains were collected and investigated these isolates for their antimicrobial susceptibility and molecular features. Resistance mechanisms, virulence-associated genes, multilocus sequence typing (MLST), and serotypes were detected by PCR and sequencing. Next general sequencing (NGS) was carried out to determine the features of carbapenem resistance and virulence. The synergistic activity of tigecycline-imipenem (TGC+IPM), tigecycline-meropenem (TGC+MEM), and tigecycline-aztreonam (TGC+ATM) combinations were performed by microdilution checkerboard method. Results:Eleven CRKP and one hvKP strains were collected. All strains showed highly sensitive rates to tigecycline (TGC) and amikacin (AMK). NDM (33.3%, 4/12) was the main resistance mechanism and MLST assigned 3 of them to ST11. CTX-M-producing (n = 1) and KPC-2-producing (n = 1) isolates belonged to ST147 and ST11, respectively. The MICs of ATM and quinolones in NDM-1 CRKP and NDM-5 CRKP strains were different. The serotype of the majority strains was KL22KL137 (58.3%, 7/12), hvKP stain belonged to K64. CRKP strains harbored plasmid-mediated quinolone resistance genes (oqxA, oqxB, qnrS, qnrB), ?-lactams (bla CTX-M-3), aminoglycosides, type I and type III fimbriae genes, siderophore genes, and transporter and pumps. SIM-producing ST1764 K64 showed typical features of hvKP, showing hypermucoviscosity phenotype. The virulence genes, including rmpA2, alls and aerobactin genes, linked to hvKP, were found in ST1764 hvKP. hvKP was sensitive to quinolone; also, oqxA gene was detected. All TGC combinations showed highly synergistic effects and TGC+IPM was more effective treatment. Conclusion:We first identified the NDM-5-producing ST690 CRKP and SIM-producing ST1764 hvKP strains in Shanxi province. Tigecycline-carbapenem combinations were available treatments for CRKP.
Project description:Objective:This retrospective study was conducted to determine the prevalence and molecular epidemiology characteristics of carbapenem-resistant Escherichia coli (CRE). Methods:A total of 593 Escherichia coli (E. coli) isolates were recovered from pigs and urban river from 2009 to 2014 in Heilongjiang Province of China. Forty CRE including 22 strains isolated from fecal samples of pigs and 18 strains isolated from water samples were selected. PCR detection of resistance determinants, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and phylogenetic groups were performed to characterize CRE isolates. Conjugation experiments, plasmid stability testing, PCR-based replicon typing (PBRT), and PCR mapping were conducted to analyze bla NDM-carrying plasmids. In vitro time-growth studies and competition experiments were carried out to assess the fitness impact of NDM carriage. Results:Five NDM-1-positive E. coli isolates were identified from water samples. Genetic environment analysis revealed that a cluster of genes (ISAba125-bla NDM-1-ble MBL-?trpF) was detected in all of the NDM-1-positive isolates. Conjugation assays showed that bla NDM-1 could be successfully transferred to E. coli J53 from 5 donor strains at frequencies of 4.6×10-5 to 2.6×10-2. The plasmids from all transconjugants belonged to different plasmid replicon types including IncA/C (n=2), IncFII (n=1) and IncX3 (n=2). In vitro time-growth studies revealed that bla NDM-1 did not have a significant impact on cell proliferation. Meanwhile, competition experiments showed that the acquisition of bla NDM-1 can place an energy burden on the bacterial host and incur fitness cost. However, plasmid stability testing showed that bla NDM-1-carrying plasmid remained stable in the hosts after seven passages without antimicrobial selection. Conclusion:The study revealed the early molecular epidemiology and dissemination characteristics of CRE. In addition, the overall antimicrobial resistance in E. coli recovered from water samples is higher than the strains isolated from fecal samples of pigs. Furthermore, we isolated and identified five NDM-1-producing E. coli strains from water samples.
Project description:IntroductionCarbapenem-resistant Enterobacteriaceae (CRE) pose a serious threat to clinical patient management and public health, as they are generally resistant to most antibiotics and cause infections with high mortality rates. Klebsiella pneumoniae ranks second among Enterobacteriaceae species that cause nosocomial infections. In this study, we investigated the epidemic characteristics of carbapenem-resistant K. pneumoniae (CRKP) in the pediatric intensive care unit (PICU) of Yanbian University Hospital.Materials and MethodsA total of 14 non-duplicate CRKP strains, collected from March 2015 to November 2019, were subjected to automated microbial identification and antimicrobial susceptibility tests using the Phoenix-100 ID/AST system. The strains were also subjected to genotypic resistance testing, polymerase chain reaction assays to detect genes encoding carbapenemases and other ?-lactamases, multi-locus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE)-based homology analysis.ResultsTwo carbapenemase genes, KPC-2 and NDM-1 (in eight and six strains, respectively), were detected. MLST enabled the division of the strains into two sequence types, ST11 and ST1224 (containing eight and six strains, respectively). PFGE results classified the 14 strains into clonotypes A–D, of which clonotypes A and B belonged to ST11, while clonotypes C and D belonged to ST1224.ConclusionOur study reveals that epidemics of the KPC-2-ST11 and NDM-1-ST1224 strains occurred in the PICU of Yanbian University Hospital. Surveillance and strict implementation of prevention and control measures are crucial to prevent the occurrence and rapid spread of nosocomial infections.