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The environmental contamination plays a significant role in the emergence of antimicrobial resistance. In this study, we report a genomic analysis of an extensively drug-resistant and bla NDM-1 -producing Klebsiella pneumoniae (EW807) strain recovered from a surface water sample. Strain EW807 belonged to sequence type (ST) 340 and serotype O4:KL15, a high-risk clone of the clonal group 258. This strain carried a broad resistome, including bla NDM-1 and bla CTX-M-15 . The core genome multilocus sequence typing phylogenetic analysis revealed that the EW807 strain was most related to strains from Brazil and the USA. An IncX3 plasmid was identified harboring the bla NDM-1 gene, while an IncFIB(K) plasmid was detected carrying the bla CTX-M-15 in addition to multidrug resistance and multimetal tolerance regions. IncX3 and IncFIB(K) plasmids shared high similarity with plasmids from a human in China and a dog in Brazil, respectively. The regions harboring the bla NDM-1 and bla CTX-M-15 genes contained sequences from the Tn3 family. These findings suggest that IncX3 plasmid could play a role in the spread of NDM-1 in a post-pandemic scenario. To the best of our knowledge, this is the first report of bla NDM-1 -producing K. pneumoniae ST340 O4:KL15 strain in the environment. Therefore, the presence of high-risk clones of K. pneumoniae carrying carbapenemases in the environment requires strict surveillance., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

BackgroundSince 2021, an emergence of New Delhi metallo-β-lactamase (NDM)-14-producing Klebsiella pneumoniae has been identified in France. This variant with increased carbapenemase activity was not previously detected in Enterobacterales.AimWe investigated the rapid dissemination of NDM-14 producers among patients in hospitals in France.MethodsAll NDM-14-producing non-duplicate clinical isolates identified in France until June 2022 (n = 37) were analysed by whole genome sequencing. The phylogeny of NDM-14-producers among all K. pneumoniae sequence type (ST) 147 reported in France since 2014 (n = 431) was performed. Antimicrobial susceptibility testing, conjugation experiments, clonal relationship and molecular clock analysis were performed.ResultsThe 37 NDM-14 producers recovered in France until 2022 belonged to K. pneumoniae ST147. The dissemination of NDM-14-producing K. pneumoniae was linked to a single clone, likely imported from Morocco and responsible for several outbreaks in France. The gene bla NDM-14 was harboured on a 54 kilobase non-conjugative IncFIB plasmid that shared high homology with a known bla NDM-1 -carrying plasmid. Using Bayesian analysis, we estimated that the NDM-14-producing K. pneumoniae ST147 clone appeared in 2020. The evolutionary rate of this clone was estimated to 5.61 single nucleotide polymorphisms per genome per year. The NDM-14 producers were highly resistant to all antimicrobials tested except to colistin, cefiderocol (minimum inhibitory concentration 2 mg/L) and the combination of aztreonam/avibactam.ConclusionHighly resistant NDM-14 producing K. pneumoniae can rapidly spread in healthcare settings. Surveillance and thorough investigations of hospital outbreaks are critical to evaluate and limit the dissemination of this clone.

The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: bla NDM-5 and two copies of bla OXA-181 in the chromosome, and a second copy of bla NDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured bla CTX-M . All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal bla NDM-5 . Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo-spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

Surgical site infections in instrumented posterior lumbar interbody fusion surgery are normally due to gram-positive bacteria, but gram-negative bacteria can cause infections in cases involving lower lumbar interventions as its closer to the perianal area. Here we report an uncommon fatal wound infection caused by a multidrug-resistant Klebsiella pneumoniae after an elective spine surgery. In silico analysis revealed that LWI_ST16 belonged to ST16, an emergent international clone notable for its increased virulence potential. We also observed that this strain carried a conjugative IncF plasmid encoding resistance genes to beta-lactams (bla KPC-2 and bla OXA-1 ), tetracycline (tetA), aminoglycosides and fluoroquinolones (aac(6')-Ib-cr). The carbapenemase encoding gene bla KPC-2 was located on a Tn4401e transposon previously characterized to increase bla KPC expression. LWI_ST16 is a strong biofilm producer on polystyrene and capable of forming tower-like structures on a titanium device like the one inserted in the patient's spine. Our findings strengthen the valuable contribution of continuous surveillance of multidrug-resistant and high-risk K. pneumoniae clones to avoid unfavourable clinical outcomes., (Copyright © 2021. Published by Elsevier B.V.)

Enterobacteria producing NDM carbapenemases represent a severe diagnostic and therapeutic challenge in healthcare settings. Infections caused by NDM-positive strains are usually associated with high mortality rates and very limited treatment options. A total number of 33 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates were included in this study, comprising 30 recovered from clinical diagnostic samples and 3 cultured from screening rectal swabs taken at patient admission. Bacterial identification was performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) and antibiotic susceptibility testing was performed by reference broth microdilution and a commercial automated method. Isolates were investigated for carbapenemase production using the β-CARBA test, the modified carbapenem inactivation method (mCIM) and, for the 30 clinical isolates, by MALDI-TOF/MS, using the MBT STAR Ⓡ -Carba IVD Kit. Carbapenem resistance genes were characterised by PCR and sequencing. Seven different bla NDM gene variants were identified in 94% of the isolates, whilst three variants of bla OXA-48-like were detected in 27% of the isolates. Most CRKP corresponded to high-risk clones (ST147, ST11 and ST15). Novel ST4497 is reported for the first time in this study as well as the first emergence of K. pneumoniae ST231 producing OXA-232 in Egypt. These results indicate an ongoing evolution of the bla NDM genes in our area and confirm the need for a maintained surveillance system in order to monitor the spread of these mobile bla NDM genes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

An emerging multidrug-resistant Klebsiella pneumoniae high-risk clone of sequence type 307 (ST307) has been increasingly reported worldwide. Here, we described the genomic characteristics of an IMP-38-producing ST307 K. pneumoniae strain and investigated the prevalence of bla IMP-38 among carbapenem-resistant Klebsiella pneumoniae isolates from a tertiary care hospital in central China. A total of 14 IMP-38-producing ST307 K. pneumoniae strains were identified from 2013 to 2016, with 13 strains isolated from patients with neonatal sepsis in the neonatal ward. PacBio and Illumina whole-genome sequencing analysis performed on a representative IMP-38-producing K. pneumoniae strain, WCGKP294, showed that it contained a circular chromosome and two plasmids. Carbapenemase gene bla IMP-38 is colocated with bla CTX-M-3 in transposon Tn 6382 on an IncHI5 plasmid (pWCGKP294-2). WCGKP294 harbors another IncFIB plasmid, pWCGKP294-1, carrying three copies of tandem-repeated IS 26 - bla SHV-2A - deoR - ygbJ - ygbK - fucA -IS 26 composite transposon elements. Phylogenetic analysis placed WCGKP294 in the global ST307 cluster, distant from the U.S. (Texas) and South Africa clusters. Nevertheless, WCGKP294 does not contain the chromosomal fluoroquinolone resistance-associated mutations and IncFIIK/IncFIBK plasmid-associated bla CTX-M-15 gene that are frequently found in other global ST307 strains. IMPORTANCE We described the genome and resistome characterization of a carbapenem-resistant Klebsiella pneumoniae ST307 strain carrying bla IMP-38 in China. This report highlights that the high-risk ST307 clone continues to acquire different antimicrobial resistance genes, posing significant challenges to clinical practice, and should be closely monitored., (Copyright © 2020 Wang et al.)

Objectives: Klebsiella pneumoniae sequence type 307 (ST307) has emerged as a new high-risk clone worldwide. The aim of this study was to report the draft genome sequence of a multidrug-resistant CTX-M-15-positive K. pneumoniae strain causing urinary tract infection in a companion animal in Brazil., Methods: Whole-genome sequencing was performed on an Illumina NextSeq platform and the genome was de novo assembled using SPAdes. Data analysis was performed using online tools from the Center for Genomic Epidemiology., Results: The genome size of K. pneumoniae strain PRETA was calculated at 5 450 575 bp, containing a total of 5355 protein-coding sequences. The strain was assigned to the high-risk ST307 and carried the clinically relevant bla CTX-M-15 gene, besides other genes conferring resistance to aminoglycosides, tetracyclines, quinolones, trimethoprim and fosfomycin., Conclusion: These data offer novel information for comparative genomic analyses in order to track the transmission dynamics and epidemiology of the newly emerging high-risk clone ST307., (Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.)

Silver is used extensively in both hospitals and outpatient clinics as a disinfectant coating agent on various devices. Resistance to silver was recently reported as an emerging problem in Enterobacteriaceae. Multidrug-resistant high-risk clones of Klebsiella pneumoniae are common causes of serious healthcare-associated infections worldwide posing a serious threat to patients. In this study, we investigated the capacity of both high-risk (CG14/15 and CG258) and minor clone strains of K. pneumoniae to develop resistance to silver. Resistance was induced in vitro in silver-susceptible but otherwise multidrug-resistant clinical isolates. Genetic alterations in the silver-resistant derivative strains with regard to the silver-susceptible isolates were investigated by whole-genome sequencing. The transferability of high-level resistance to silver was also tested. We demonstrated that the high-level resistance to silver can quickly evolve as a consequence of a single-point mutation either in the cusS gene of the chromosomally encoded CusCFBARS efflux system and/or in the silS gene of the plasmid-encoded Copper Homeostasis and Silver Resistance Island (CHASRI) coding also for a metallic efflux. The minimal inhibitory concentrations (MICs) of the strains increased from 4 mg/L (23.5 μM) AgNO 3 to >8,500 mg/L (>50,000 μM) AgNO 3 during induction. Harboring the CHASRI proved an important selective asset for K. pneumoniae when exposed to silver. Successful conjugation experiments using Escherichia coli K12 J5-3 Rif as recipient showed that high-level silver resistance can transmit between strains of high-risk clones of K. pneumoniae (ST15 and ST11) and isolates from additional species of Enterobacteriaceae. The lack of fitness cost associated with the carriage of the CHASRI in a silver-free environment and the presence of the RelEB toxin-antitoxin system on the conjugative plasmids could advance the dissemination of silver resistance. Our results show that multidrug-resistant high-risk clones of K. pneumoniae are capable of evolving and transmitting high-level resistance to silver. This observation should warrant a more judicious use of silver coated-devices to prevent the extensive dissemination of silver resistance.

Objectives: Carbapenem-resistant Enterobacteriaceae (CRE) are identified as a major global health concern. The success of CRE is facilitated by the emergence, acquisition and spread of successful clones carrying plasmid-encoded resistance genes. In this study, an outbreak of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in patients hospitalised in Brunei Darussalam was investigated., Methods: Over a 3-month period (May-July 2015), five multidrug-resistant K. pneumoniae were recovered from individual patients admitted to intensive care units at two hospitals (RIPAS and PMMPMHAB) in Brunei. Antimicrobial susceptibility was determined by broth microtitre dilution using a Micronaut-S β-lactamase VII kit or by Etest. Carbapenemase production was confirmed using the RAPID CARB Blue screen, and classes A-D β-lactamases were detected by multiplex PCR. Molecular typing was performed by random amplified polymorphic DNA (RAPD) PCR and multilocus sequence typing (MLST), with associated virulence and capsular types identified by PCR and sequencing. Plasmids were extracted, sized and characterised by PCR-based replicon typing., Results: All isolates were resistant to cephalosporins, carbapenems, aminoglycosides, quinolones and sulfonamides but remained susceptible to polymyxins. Isolates were indistinguishable by RAPD-PCR and all belonged to sequence type (ST231). Resistance was due to the production of OXA-232 and CTX-M-15 β-lactamases, with the bla OXA-232 carbapenemase gene located on a ColE-like plasmid., Conclusions: This is the first report of plasmid-encoded OXA-232-producing CRKP in Brunei hospitals. All isolates were members of ST231, which may be representatives of a high-risk CRKP clone disseminating across Southeast Asia., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Background: The high prevalence and rapid emergence of antibiotic resistance in high-risk Klebsiella pneumoniae (KP) ST147 clones is a global health concern and warrants molecular surveillance. Methods: A pangenome analysis was performed using publicly available ST147 complete genomes. The characteristics and evolutionary relationships among ST147 members were investigated through a Bayesian phylogenetic analysis. Results: The large number of accessory genes in the pangenome indicates genome plasticity and openness. Seventy-two antibiotic resistance genes were found to be linked with antibiotic inactivation, efflux, and target alteration. The exclusive detection of the blaOXA-232 gene within the ColKp3 plasmid of KP_SDL79 suggests its acquisition through horizontal gene transfer. The association of seventy-six virulence genes with the acrAB efflux pump, T6SS system and type I secretion system describes its pathogenicity. The presence of Tn6170, a putative Tn7-like transposon in KP_SDL79 with an insertion at the flanking region of the tnsB gene, establishes its transmission ability. The Bayesian phylogenetic analysis estimates ST147's initial divergence in 1951 and the most recent common ancestor for the entire KP population in 1621. Conclusions: Present study highlights the genetic diversity and evolutionary dynamics of high-risk clones of K. pneumoniae. Further inter-clonal diversity studies will help us understand its outbreak more precisely and pave the way for therapeutic interventions. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the frequency of carbapenem resistant Klebsiella pneumoniae (CRKP) sequence types (STs) in Iran. Samples were collected from three university hospitals in Sanandaj, Iran, from December 2016 to March 2018. Antibiotic susceptibility testing, phenotypic and genotypic detection of carbapenemases were performed. Common K. pneumoniae capsular types were sought for all isolates. The genetic relatedness of isolates was investigated by multilocus sequence typing (MLST). Plasmids were detected by PCR-based Replicon Typing (PBRT). During the study, 67 K. pneumoniae isolates were identified. Of which, 18 (26.9%) isolates were detected as carbapenem-resistant. The most effective antibacterial agent was tigecycline (97%, 65 isolates) followed by imipenem and ertapenem (73.13%, 49 isolates). PCR showed that 13 isolates (19.4%) had bla NDM-1 gene and 5 (7.5%) harbored bla OXA-48. Examination of common capsular types showed that 2 isolates had K2 and 2 others had K54. REP-PCR revealed 10 clones and 11 singleton strains. MLST analysis of CRKP found ST15 as the most common type (13 isolates, 72.2%), but other STs were also detected namely, ST19, ST117, ST1390, and ST1594. ColE1 and IncL/M plasmids were the carriers of bla NDM-1 and bla OXA-48, respectively. The results showed that CRKP spread in our health centers. Our results, therefore, indicate a worrying trend of resistance to carbapenems in K. pneumoniae. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is considered an opportunistic pathogen frequently involved with healthcare-associated infections. The genome of K. pneumoniae is versatile, harbors diverse virulence factors and easily acquires and exchanges resistance plasmids, facilitating the emergence of new threatening clones. In the last years, ST16 has been described as an emergent, clinically relevant strain, increasingly associated with outbreaks, and carrying virulence factors (such as ICEKp, iuc, rmpADC/2) and a diversity of resistance genes. However, a far-reaching phylogenetic study of ST16, including geographically, clinically and temporally distributed isolates is not available. In this work, we analyzed all publicly available ST16 K. pneumoniae genomes in terms of virulence factors, including capsular lipopolysaccharide and polysaccharide diversity, plasmids and antimicrobial resistance genes. A core genome SNP analysis shows that less than 1% of studied sites were variant sites, with a median pairwise single nucleotide polymorphism difference of 87 SNPs. The number and diversity of antimicrobial resistance genes, but not of virulence-related genes, increased consistently in ST16 strains during the studied period. A genomic comparison between ST16 and the high-risk clone ST11 K. pneumoniae, showed great similarities in their capacity to acquire resistance and virulence markers, differing mostly in the great diversity of capsular lipopolysaccharide and polysaccharide types in ST11, in comparison with ST16. While virulence and antimicrobial resistance scores indicated that ST11 might still constitute a more difficult-to-manage strain, results presented here demonstrate the great potential of the ST16 clone becoming critical in public health. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to characterize the clonal spread of resistant bacteria and dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at a tertiary hospital in Catalonia, Spain. Methods: Isolates were recovered from surveillance rectal swabs and diagnostic samples. Species identification was by matrix-assisted laser desorption ionization-time time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of bla genes was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and long-read sequencing were used to characterize resistance plasmids. Results: From July 2018 to February 2019, 125 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies identified a major clone of Klebsiella pneumoniae belonging to sequence type ST15 and additional isolates of K. pneumoniae , Escherichia coli and Enterobacter sp. from different STs. All isolates but one carried the bla KPC–2 allelic variant. The bla KPC–2 gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn 4401 element designated NTEKPC-pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTEKPC-pMC-2-1 module was highly conserved. Conclusion: We report a hospital outbreak caused by the clonal dissemination of KPC-producing ST15 K. pneumoniae but also the intra- and inter-species transmission of the bla KPC–2 gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant high-risk clones. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is considered an opportunistic pathogen frequently involved with healthcare-associated infections. The genome of K. pneumoniae is versatile, harbors diverse virulence factors and easily acquires and exchanges resistance plasmids, facilitating the emergence of new threatening clones. In the last years, ST16 has been described as an emergent, clinically relevant strain, increasingly associated with outbreaks, and carrying virulence factors (such as ICEKp, iuc, rmpADC/2) and a diversity of resistance genes. However, a far-reaching phylogenetic study of ST16, including geographically, clinically and temporally distributed isolates is not available. In this work, we analyzed all publicly available ST16 K. pneumoniae genomes in terms of virulence factors, including capsular lipopolysaccharide and polysaccharide diversity, plasmids and antimicrobial resistance genes. A core genome SNP analysis shows that less than 1% of studied sites were variant sites, with a median pairwise single nucleotide polymorphism difference of 87 SNPs. The number and diversity of antimicrobial resistance genes, but not of virulence-related genes, increased consistently in ST16 strains during the studied period. A genomic comparison between ST16 and the high-risk clone ST11 K. pneumoniae, showed great similarities in their capacity to acquire resistance and virulence markers, differing mostly in the great diversity of capsular lipopolysaccharide and polysaccharide types in ST11, in comparison with ST16. While virulence and antimicrobial resistance scores indicated that ST11 might still constitute a more difficult-to-manage strain, results presented here demonstrate the great potential of the ST16 clone becoming critical in public health.

We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1 -positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1 -positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring bla KPC– 2 and bla OXA– 48, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP. [ABSTRACT FROM AUTHOR]

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 β-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 β-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 blaNDM-1 23.9% (n=28), blaOXA-48 22.2% (n=26) and blaVIM 0.85% (n=1) while 12.7% (n=7) carried both blaNDM-1 and blaOXA-48 genes. The CRKP coharboring blaNDM-1 and blaOXA-48 genes (n=7) were positive for blaCTX-MblaSHV (n=3), blaSHV (n=1) and blaCTX-M (n=3). The novel CRKP with the coexistence of blaNDM-1, blaOXA-48, blaCTX-M and blaSHV 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 blaNDM-1, blaOXA-48, blaCTX-M and blaSHV 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. [ABSTRACT FROM AUTHOR]

Virulence plasmids are associated with hypervirulent types of Klebsiella pneumoniae, which generally do not carry antibiotic resistance genes. In contrast, nosocomial isolates are often associated with resistance, but rarely with virulence plasmids. Here, we describe virulence plasmids in nosocomial isolates of “high-risk” clones of sequence types (STs) 15, 48, 101, 147 and 383 carrying carbapenemase genes. The whole genome sequences were determined by long-read nanopore sequencing. The 12 isolates all contained hybrid plasmids containing both resistance and virulence genes. All carried rmpA/rmpA2 and the aerobactin cluster, with the virulence plasmids of two of three representatives of ST383 carrying blaNDM-5 and seventeen other resistance genes. Representatives of ST48 and ST15 had virulence plasmid-associated genes distributed between two plasmids, both containing antibiotic resistance genes. Representatives of ST101 were remarkable in all sharing virulence plasmids in which iucC and terAWXYZ were missing and iucB and iucD truncated. The combination of resistance and virulence in plasmids of high-risk clones is extremely worrying. Virulence plasmids were often notably consistent within a lineage, even in the absence of epidemiological links, suggesting they are not moving between types. However, there was a common segment containing multiple resistance genes in virulence plasmids of representatives of both STs 48 and 383.

• 8.6% (19/221) of pan-aminoglycoside-resistant Pseudomonas aeruginosa had 16S RMTase genes. • 16S RMTase gene variants rmtB4, rmtD3 and rmtF2 were detected. • 89.5% (17/19) of 16S RMTase gene-positive isolates harboured a carbapenemase gene. • P. aeruginosa 'high-risk' clones ST357, ST654 and ST773 harboured 16S RMTase genes. • Novel genetic environments for rmtB1, rmtC and rmtF2 were identified. 16S rRNA methyltransferase (16S RMTase) genes confer high-level aminoglycoside resistance, reducing treatment options for multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa isolates (n = 221) exhibiting high-level pan-aminoglycoside resistance (amikacin, gentamicin and tobramycin MICs ≥64, ≥32 and ≥32 mg/L, respectively) were screened for 16S RMTase genes to determine their occurrence among isolates submitted to a national reference laboratory from December 2003 to December 2015. 16S RMTase genes were identified using two multiplex PCRs, and whole-genome sequencing (WGS) was used to identify other antibiotic resistance genes, sequence types (STs) and the genetic environment of 16S RMTase genes. 16S RMTase genes were found in 8.6% (19/221) of isolates, with rmtB4 (47.4%; 9/19) being most common, followed by rmtD3 (21.1%; 4/19), rmtF2 (15.8%; 3/19) and single isolates harbouring rmtB1, rmtC and rmtD1. Carbapenemase genes were found in 89.5% (17/19) of 16S RMTase-positive isolates, with bla VIM (52.9%; 9/17) being most common. 16S RMTase genes were found in 'high-risk' clones known to harbour carbapenemase genes (ST233, ST277, ST357, ST654 and ST773). Analysis of the genetic environment of 16S RMTase genes identified that IS 6100 was genetically linked to rmtB1 ; IS 91 to rmtB4, rmtC or rmtD3; IS CR14 to rmtD1 ; and rmtF2 was linked to Tn 3 , IS 91 or Tn 1721. Although 16S RMTase genes explained only 8.6% of pan-aminoglycoside resistance in the P. aeruginosa isolates studied, the association of 16S RMTase genes with carbapenemase-producers and 'high-risk' clones highlights that continued surveillance is required to monitor spread as well as the importance of suppressing the emergence of dually-resistant clones in hospital settings. [ABSTRACT FROM AUTHOR]

• Returning travelers may be silent vectors of carbapenemase resistance genes • New carbapenemase gene in high-risk clone poses threat to antimicrobial resistance • E. coli ST167 harboring OXA-181 was introduced in a well-established IncX3 plasmid This is the first detection and genomic analysis of an OXA-181-carbapenemase-producing E. coli in Brazil, from a traveler returning from Sub-Saharan Africa. The ST167 isolate carries bla OXA-181 inserted in an IncX3 plasmid. This report illustrates the potential role of travelers as silent vectors for dissemination of high-risk resistant clones. [ABSTRACT FROM AUTHOR]

Virulence plasmids are associated with hypervirulent types of Klebsiella pneumoniae, which generally do not carry antibiotic resistance genes. In contrast, nosocomial isolates are often associated with resistance, but rarely with virulence plasmids. Here, we describe virulence plasmids in nosocomial isolates of "high-risk" clones of sequence types (STs) 15, 48, 101, 147 and 383 carrying carbapenemase genes. The whole genome sequences were determined by long-read nanopore sequencing. The 12 isolates all contained hybrid plasmids containing both resistance and virulence genes. All carried rmpA/rmpA2 and the aerobactin cluster, with the virulence plasmids of two of three representatives of ST383 carrying blaNDM-5 and seventeen other resistance genes. Representatives of ST48 and ST15 had virulence plasmid-associated genes distributed between two plasmids, both containing antibiotic resistance genes. Representatives of ST101 were remarkable in all sharing virulence plasmids in which iucC and terAWXYZ were missing and iucB and iucD truncated. The combination of resistance and virulence in plasmids of high-risk clones is extremely worrying. Virulence plasmids were often notably consistent within a lineage, even in the absence of epidemiological links, suggesting they are not moving between types. However, there was a common segment containing multiple resistance genes in virulence plasmids of representatives of both STs 48 and 383. [ABSTRACT FROM AUTHOR]