Search

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains present a significant global public health threat due to their high mortality rates. This study investigated the genomic characteristics of seven ST11-K1 CR-hvKP isolates harboring highly homologous KPC-2-encoding multidrug-resistance plasmids. The strains were isolated from a Chinese tertiary hospital between 2017 and 2020. Whole-genome sequencing and bioinformatic analysis revealed various antibiotic resistance genes (ARGs) and virulence determinants. The blaKPC-2-bearing plasmids that contain multiple antibiotic-resistance genes were also identified in these strains. ISfinder and Orifinder were applied to identify insertion sequences (IS) and conjugation-related factors among these blaKPC-2-bearing plasmids. The blaKPC-2 was highly consistent in seven blaKPC-2-bearing plasmids (ISKpn6-blaKPC-2-ISKpn27-ISYps3-IS26). In addition, we found a region composed of ISIR, Tn5393, and IS26. It was located upstream of the blaCTX-M-15 gene and presented in six blaKPC-2-bearing plasmids, with pCR-hvKP221-KPC-P3 as an exception. Conjugation experiments demonstrated the horizontal transfer of resistance plasmids pCR-hvKP128-KPC-P1 and pCR-hvKP132-KPC-P1 across species. Notably, pLVPK-like virulence plasmids carrying virulence gene clusters pCR-hvKP173-Vir-P1, and pCR-hvKP221-Vir-P1 were also detected. A fusional plasmid pCR-hvKP221-Vir-P2, which carries virulence gene clusters and ARGs, was also identified. Five CR-hvKP strains displayed enhanced biofilm formation and high virulence in vivo infection models. Phylogenetic and single nucleotide polymorphism (SNP) analyses indicated a close genetic relationship among the isolates, suggesting a subclade. These findings highlight the complex genetic profiles and potential transmission mechanisms of CR-hvKP strains.IMPORTANCEWe reported seven CR-hvKP strains all carried a highly homologous blaKPC-2 integrated IncFⅡ-resistant plasmid, and two strains harbored virulence plasmids. Conjugation experiments confirmed the transferability of these plasmids, indicating a potential for resistance spread. Phylogenetic analysis clarified the relationship among the CR-hvKP isolates. This study provides insights into the phenotypic and genomic characteristics of seven ST11-K1 CR-hvKP strains. The high prevalence and potential for local outbreaks emphasize the need for effective control measures.

Ceftazidime-avibactam resistance attributable to the blaKPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harboured the blaKPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The blaKPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the blaKPC-2-positive cloned strain, only the MIC of ceftazidime increased against blaKPC-135-positive K. pneumoniae and wasn’t inhibited by avibactam (MIC 32 μg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harboured resistance and virulence genes, posing an elevated challenge in clinical treatment.

ABSTRACT Colistin is used as a last-line therapy against carbapenem-resistant Klebsiella pneumoniae (CRKP). However, colistin resistance in Klebsiella pneumoniae is increasingly reported worldwide. This study aims to investigate the instrumental role of insertion sequence (IS) elements in colistin resistance through mgrB disruption in K. pneumoniae during treatment. Five clinical isolates of CRKP, designated KPN1~KPN5 were collected from the lower respiratory tract of a patient with chest infection before and after treatment with colistin. Antimicrobial susceptibility testing was performed using the broth microdilution method. Whole genome sequencing and bioinformatics were used to analyze the sequence types (STs), resistance genes, and genetic characteristics of the five isolates of K. pneumoniae. Antimicrobial susceptibility testing indicated that all five K. pneumoniae isolates were resistant to cephalosporins (ceftriaxone, ceftazidime, and cefepime), several carbapenems (imipenem, meropenem), cefoperazone–sulbactam, piperacillin–tazobactam, ciprofloxacin, and fosfomycin, whereas they were sensitive to amikacin and tigecycline. In addition, three of these isolates were resistant to colistin, with minimum inhibitory concentration values of >8 mg/L. Whole genome sequencing revealed that all five K. pneumoniae isolates belonged to sequence type 1 (ST1), which shared an identical blaKPC-2. Notably, disruption of mgrB by the ISKpn26 insertion sequence was shown to be the primary colistin resistance mechanism during the treatment. To our knowledge, this is the first report of ISKpn26 element mediating mgrB disruption in the ST1 colistin and CRKP obtained from a patient with chest infection in mainland China. This study provides new research ideas to explore the clinical drug resistance mechanism of CRKP and the critical need to monitor and understand resistance mechanisms to preserve the efficacy of last-line antibiotics such as colistin.IMPORTANCEOf note, this chapter gives an update on colistin resistance in sequence type 1 Klebsiella pneumoniae, by focusing on the mgrB disrupted by ISKpn26 element.

ABSTRACT: Objectives: Herein, we detected one multidrug-resistant Aeromonas hydrophila strain K522 co-carrying two blaKPC-2 genes together with a novel chromosomal integrative and mobilizable element (IME) Tn7548 from China. To reveal the genetic characteristics of the novel reservoir of blaKPC-2 and IME in Aeromonas, a detailed genomic characterization of K522 was performed, and a phylogenetic analysis of Tn7412-related IMEs was carried out. Methods: Carbapenemases were detected by using the immunocolloidal gold technique and antimicrobial susceptibility was tested by using VITEK 2. The whole-genome sequences of K522 were analysed using phylogenetics, detailed dissection, and comparison. Results: Strain K522 carried a Tn7412-related chromosomal IME Tn7548 and three resistance plasmids pK522-A-KPC, pK522-B-KPC, and pK522-MOX. A phylogenetic tree of 82 Tn7412-related IMEs was constructed, and five families of IMEs were divided. These IMEs shared four key backbone genes: int, repC, and hipAB, and carried various profiles of antimicrobial resistance genes (ARGs). pK522-A-KPC and pK522-B-KPC carried blaKPC-2 and belonged to IncG and unclassified type plasmid, respectively. The blaKPC-2 regions of these two plasmids were the truncated version derived from Tn6296, resulting in the carbapenem resistance of K522. Conclusion: We first reported A. hydrophila harbouring a novel Tn7412-related IME Tn7548 together with two blaKPC-2 carrying plasmids and a MDR plasmid. Three of these four mobile genetic elements (MGEs) discovered in A. hydrophila K522 were novel. The emergence of novel MGEs carrying ARGs indicated the rapid evolution of the resistance gene vectors in A. hydrophila under selection pressure and would contribute to the further dissemination of various ARGs in Aeromonas.

ABSTRACTHospital sewage serves as a crucial reservoir for antibiotic resistance genes. As colistin and carbapenems are the last-resort antibiotics, the emergence of their resistance genes has become a significant concern in clinical settings. In this study, we found that two novel mcr alleles (mcr-3.43 and mcr-7.2) with two carbapenemase genes (blaNDM-1 and blaKPC-2) were encoded in a single Aeromonas caviae strain isolated from hospital sewage. Our phylogenetic analysis revealed that the mcr-3.43 gene clustered with mcr-3.17 (with 95.55% amino acid identity), while the mcr-7.2 gene clustered with mcr-7.1 (with 68.68% amino acid identity). BLAST search against GenBank showed that mcr-7.2 was exclusively detected in Aeromonas spp. Mobile genetic elements were not found in the genetic context of mcr-7.2, suggesting that the dissemination of mcr-7.2 in Aeromonas spp. may be dependent on vertical transfer or recombination. The blaNDM-1 was adjacent to a recombinase gene and flanked by two IS91 elements, indicating a potential mobilization mechanism mediated by recombination and/or ISs. The blaKPC-2 gene was located on an IncU plasmid and adjacent to an ISKpn6. In summary, our study provides evidence for Aeromonas spp. as one of the potential reservoirs of colistin and carbapenem resistance genes.IMPORTANCEThe study discovered two novel mcr genes (mcr-3.43 and mcr-7.2) and two carbapenemase genes (blaNDM-1 and blaKPC-2) in a single Aeromonas caviae strain retrieved from hospital sewage. Using phylogenetic analysis and comparative data evaluation, the study revealed the genetic relatedness and dissemination potential of the detected resistance genes. With the exclusive discovery that mcr-7.2 is only present in Aeromonas spp. and the lack of mobile genetic elements in its genetic context, there is a strong indication of limited dissemination. The identification of these four resistance genes in a single strain of Aeromonas provided valuable insights into their potential presence in this genus. This study revealed that hospital sewage functions as a significant reservoir for antibiotic resistance genes, including colistin and carbapenem resistance genes.

ABSTRACT: Objectives: The emergence and spread of colistin resistance in carbapenem-resistant Enterobacteriaceae pose a serious threat to human and animal health. This work aimed to characterise the genetic features of antimicrobial resistance of the carbapenem- and colistin-resistant Enterobacter kobei strain SCLZS19, isolated from hospital sewage, by using whole genome sequencing. Methods: Antimicrobial susceptibility tests were performed using the disk diffusion method. Whole genome sequencing of SCLZS19 was carried out on the HiSeq 2000 combined with PacBio RSII platforms. Sequence type, plasmid incompatibility types, resistance genes, and insertion elements were identified using multilocus sequence typing, PlasmidFinder, ResFinder, and ISfinder, respectively. Conjugation assays were performed using both broth- and filter-based methods with the azide-resistant Escherichia coli J53 as the recipient. The function of the mcr-9-like variant was determined by gene cloning. Results: E. kobei SCLZS19 had a 4 862 177-bp circular chromosome and nine circular plasmids ranging in size from 4120 bp to 282 472 bp. It carried 11 antibiotic resistance genes, and 10 of them were located on plasmids. The colistin resistance gene mcr-10 was located on a 118 766-bp non-transferable IncF (Y3:A-:B-) plasmid. The carbapenemase gene blaKPC-2 was carried by a self-transmissible IncP6 plasmid, which is epidemic in China. In addition, SCLZS19 also carried an mcr-9-like variant on a IncHI2 (ST1) plasmid. The cloning assay showed that the mcr-9-like variant did not mediate colistin resistance in E. coli DH5α. Conclusion: The findings highlight that carbapenem- and colistin-resistant Enterobacterales from water environments may serve as a reservoir for clinically significant antibiotic resistance genes, and continuous surveillance is required.

ABSTRACT: Objectives: We isolated a strain of Enterobacter hormaechei, ECC2783, co-harbouring blaNDM-1, blaKPC-2 and mcr-9 plasmids from a bloodstream infection and investigated its biological features. Methods: The presence of carbapenemase genes and mcr-9 was confirmed by polymerase chain reaction amplification. Whole genome sequencing and genomic analysis were performed on ECC2783. Experiments assessing the conjugation and stability of plasmids carrying the carbapenemase gene were performed. We also performed a colistin resistance induction experiment and studied the fitness cost of transconjugants. Results: ECC2783 has an extensive drug resistance phenotype. Multilocus sequence typing analysis results showed that ECC2783 belongs to sequence type 93. Bioinformatics analysis confirmed that ECC2783 has four plasmids, of which pECC2783_a, carrying mcr-9, is the IncHI2 type, and pECC2783_c, carrying blaNDM-1, is the IncX3 type. pECC2783_d, carrying blaKPC-2, is an unclassified type. We successfully obtained two transconjugants (J53/ECC2783_1, carrying blaNDM-1, and J53/ECC2783_2, carrying blaKPC-2 and blaNDM-1). There was no statistically significant difference in the relative growth rate between J53 and J53/ECC2783_2. Conclusion: For the first time, we isolated carbapenem-resistant E. hormaechei plasmids co-harbouring blaNDM-1, blaKPC-2, and mcr-9 from a patient with a blood stream infection. This isolate has a survival advantage in a hospital environment, and its clinical monitoring should be strengthened.

BackgroundThe escalating resistance of Klebsiella pneumoniae, a prevalent pathogen in healthcare settings, especially its carbapenem-resistant K. pneumoniae (CRKP), to a wide array of antibiotics, notably β-lactams, constitutes a formidable challenge for healthcare and global public health management.MethodsThis research compared the resistance phenotypes and genomic profiles of CRKP and Non-CRKP isolates in a Beijing hospital, focusing on high-risk blaKPC-2 gene-bearing CRKP clones and the structure of mobile genetic elements facilitating their spread across hospital departments. Forty K. pneumoniae isolates were collected from various departments of the hospital and subjected to antimicrobial susceptibility testing and whole-genome sequencing to analyze their resistance phenotypes and genomic features.ResultsThe study revealed that among the 31 CRKP isolates, ST11 is the most common sequence type, with K47 and OL101 being the dominant capsule types, primarily observed in the respiratory department. In terms of antimicrobial susceptibility: 87.5% of the isolates exhibited multidrug resistance (MDR), with a high resistance rate of 30% against tigecycline. All CRKP isolates demonstrated resistance to multiple drug classes (≥5 CLSI classes). Non-CRKP isolates also showed high resistance rates to minocycline and doxycycline (77.8%). the ST11-KL47-OL101 type emerged as the predominant clone among the CRKP isolates carrying the blaKPC-2 gene. This dominance appears to be mediated by the pKpnR03_2 plasmid, which harbors not only blaKPC-2 and rmtb but also gene clusters pertinent to iron transport and arsenic resistance. These isolates, clustering in the C3 clade of the phylogenetic tree, exhibited minor genetic variations and close evolutionary relationships, suggesting a plasmid-driven spread across various hospital departments.ConclusionIn summary, our study highlights the extensive spread of antibiotic-resistant K. pneumoniae across various departments in our hospital, with a particular emphasis on the dominant clonal proliferation of the ST11-KL47-OL101 CRKP strain. This finding underscores the significant role of plasmid-mediated gene transfer in the evolution and dissemination of resistant strains within hospital environments. The study emphasizes the necessity for ongoing surveillance of antibiotic resistance and genomic analysis in hospital settings to effectively monitor and manage these challenges.

ABSTRACT: Objectives: The worldwide dissemination of carbapenemase-producing Escherichia coli lineages belonging to high-risk clones poses a challenging public health menace. The aim of this work was to investigate genomic features of a colonizing multidrug-resistant strain of Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli from our institution. Methods: Whole-genome sequencing was done by Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Resistome, mobilome, plasmids, virulome, and integrons were analysed using ResFinder, AMRFinder, ISFinder, PlasmidFinder, MOB-suite, VirulenceFinder, and IntegronFinder. Sequence types (STs) were identified with pubMLST and BIGSdb databases. Conjugation assays were also performed. Results: Escherichia coli HA25pEc was isolated from a rectal swab sample taken within the framework of the hospital epidemiological surveillance protocol for detection of carbapenemase-producing Enterobacterales. Escherichia coli HA25pEc corresponded to the first report of ST648 co-harbouring blaKPC-2 and blaCTX-M-15 in Latin America from a colonized patient. It had 19 antibiotic resistance genes (ARGs), including blaKPC-2, located on a Tn4401a isoform. Conjugation assays revealed that blaKPC-2 was not transferred by conjugation to E. coli J53 under our experimental conditions. Conclusion: Escherichia coli ST648 has been detected previously in companion and farm animals as well as in hospital- and community-acquired infections worldwide. Although scarcely reported as KPC-producers, our finding in a culture surveillance with several acquired ARGs, including blaCTX-M-15, alerts the potential of this clone for worldwide unnoticed spreading of extreme drug resistance to β-lactams. These data reinforce the importance of carrying out molecular surveillance to identify reservoirs and warn about the dissemination of new international clones in carbapenemase-bearing patients.

Objectives: This study aimed to elucidate resistance to carbapenems and fluoroquinolones, the transmission mechanism of blaKPC-2, and the virulence characteristics of a Pseudomonas aeruginosa strain (TL3773) isolated in East China. Methods: The virulence and resistance mechanisms of TL3773 were investigated by whole genome sequencing (WGS), comparative genomic analysis, conjugation experiments, and virulence assays. Results: This study isolated carbapenem-resistant P. aeruginosa from blood resistant to carbapenems. The patient's clinical data showed poor prognosis compounded by multiple sites of infection. WGS showed that TL3773 carried aph (3′)-IIb, blaPAO, blaOXA-486, fosA, catB7, and two crpP resistance genes on chromosome, and the carbapenem resistance gene blaKPC-2 on plasmid. We identified a novel crpP gene named TL3773-crpP2. Cloning experiments proved that TL3773-crpP2 was not the primary cause of fluoroquinolone resistance in TL3773. GyrA and ParC mutations may confer fluoroquinolone resistance. The blaKPC-2 genetic environment was IS26-TnpR-ISKpn27-blaKPC-2-ISKpn6-IS26-Tn3-IS26, potentially mediating the transmission of blaKPC-2 in P. aeruginosa. The overall virulence of TL3773 was lower than that of PAO1. However, the pyocyanin and biofilm formation of TL3773 was higher than that of PAO1. WGS further indicated that TL3773 was less virulent than PAO1. Phylogenetic analysis showed that TL3773 was most similar to the P. aeruginosa isolate ZYPA29 from Hangzhou, China. These observations further indicate that ST463 P. aeruginosa is rapidly spreading. Conclusions: The threat of ST463 P. aeruginosa harbouring blaKPC-2 is emergent and may pose a threat to human health. More extensive surveillance and effective actions are urgently needed to control its further spread.

ABSTRACT: Objectives: Pseudomonas aeruginosa is a widely distributed opportunistic pathogen that can cause a variety of infections. The emergence of multidrug-resistant P. aeruginosa has complicated clinical treatment. Here, we report the genome sequence of a P. aeruginosa strain co-carrying blaKPC-2, blaOXA-246, and blaOXA-486. Genetic and phylogenetic characteristics of this strain were investigated. Methods: The Illumina NovaSeq 6000 platform was used to sequence the strain's genome. MLST v.2.22.1 was used to analyse the multilocus sequence typing (MLST). Antimicrobial resistance genes (ARGs) and virulence genes were identified using ABRicate v.1.0.1. Phylogenetic relationship analysis was performed using snippy. Results: The genomic sequence of P. aeruginosa PA23 consists of 229 contigs with a combined length of 7 193 462 bp. It was determined that PA23 belonged to ST463 and serotype O4, which is associated with a high-risk clone. The strain was resistant to almost all antibiotics tested, except colistin. The genome contained 23 antimicrobial resistance genes, including blaKPC-2, blaOXA-246, and blaOXA-486. A total of five P. aeruginosa strains both carrying blaKPC-2, blaOXA-246, and blaOXA-486 can be retrieved from the NCBI database. All of these strains are ST463 and serotype O4. With the exception of one strain, the other strains were spread across two neighbouring Chinese provinces and were clonal related. Conclusions: In conclusion, we reported the genome sequence of a multidrug-resistant P. aeruginosa ST463 strain containing 23 ARGs in China. This clone has the potential to become a dominant endemic clone in eastern China. To prevent clonal dissemination, continuous surveillance is necessary in the future.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales are prevalent worldwide and pose an alarming threat to public health. The incidence and transmission of blaKPC-2 gene via horizontal gene transfer (e.g. transposition) have been well documented. However, the dynamics of transposon structure bearing blaKPC-2 and their exact effects on the evolution and dissemination of blaKPC-2 gene are not well characterized. Here, we collected all 161 carbapenem-resistant Enterobacterales (CRE) isolates during the early stage of CRE pandemic. We observed that the prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and sequence types (STs), and that blaKPC-2 gene was located on three diverse variants of Tn1721 in multi-drug resistance (MDR) region of plasmid. Notably, the outbreak of KPC-2-producing plasmid is correlated with the dynamics of transposon structure. Furthermore, we experimentally demonstrated that replicative transposition of Tn1721 and IS26 promotes horizontal transfer of blaKPC-2 and the evolution of KPC-2-producing plasmid. The Tn1721 variants appearing concurrently with the peak of an epidemic (A2- and B-type) showed higher transposition frequencies and a certain superior ability to propagation. Overall, our work suggests replicative transposition contributes to the evolution and transmission of KPC-2-producing plasmid and highlights its important role in the inter- and intra-species dissemination of blaKPC-2 gene in Enterobacterales.

ABSTRACT: Objectives: Klebsiella pneumoniae carbapenemase (KPC)–producing sequence type (ST) 463 Pseudomonas aeruginosa are increasingly prevalent in China. This study aims to investigate how blaKPC-2 is acquired in ST463 P. aeruginosa during antimicrobial therapy. Methods: Two extensively drug-resistant P. aeruginosa strains, B1122 and U1121, were respectively isolated from blood and urine of a patient during carbapenem therapy. Whole-genome sequences were obtained, and minimum inhibitory concentrations (MICs) were determined. Plasmid transferability and stability were examined. Bacterial growth kinetics, biofilm formation, and virulence level was assessed. Results: U1121 and B1122 were only susceptible to amikacin and intermediately susceptible to colistin. They were isogenic ST463 P. aeruginosa strains and shared the same chromosome-encoded resistance genes, including blaAFM-1. This is the first report of chromosomal integration of blaAFM-1 in P. aeruginosa mediated by ISCR29. pU1121 and pB1122, which shared almost identical backbone, were the sole plasmids in U1121 and B1122, respectively, differing by an insertion region containing two copies of blaKPC-2 genes observed on pU1121. Sequence alignment revealed that pU1121 might evolve in vivo from pB1122 via IS26-mediated continuous genetic rearrangement in response to selective challenge from carbapenem. pU1121 was not self-transmissible and could be stably maintained in the host in the absence of antibiotic. Both U1121 and B1122 were hypervirulent, and no differences on virulence were recorded between them. However, U1121 exhibited significant impaired growth in comparison with B1122. Conclusion: ST463 P. aeruginosa can capture blaKPC-2 through horizontal transfer of insertion sequence under antibiotic selection pressure, which does decrease the fitness but does not impair the virulence of the ancestor.

ABSTRACT: Objectives: The emergence of blaKPC-2 within nosocomial settings has become a major public health crisis worldwide. Our aim was to perform whole-genome sequencing (WGS) of three KPC-producing Gram-negative bacilli (KPC-GNB) strains isolated from a hospitalized patient to identify acquired antimicrobial resistance genes (ARGs). Methods: WGS was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Bioinformatics analysis was done using Resfinder, AMRFinder, ISFinder, plasmidSPAdes, PlasmidFinder, MOB-suite, PLSDB database, and IntegronFinder. Conjugation assays were performed to assess the ability of blaKPC-2 to transfer via a plasmid-related mobilization mechanism. Results: High-risk clone KPC-producing Klebsiella pneumoniae sequence type (ST) 258 (HA3) was colonizing an inpatient who later was infected by KPC-producing Escherichia coli ST730 (HA4) and subsequently by KPC-producing K. pneumoniae ST11 (HA15) during hospitalization. Although belonging to different species, both strains causing infections harbored the same gene configuration for dissemination of blaKPC-2 in related IncM1 plasmids recently found in other KPC-GNB isolated from Hospital Alemán at Ciudad Autónoma de Buenos Aires. Conjugation assays revealed that only pDCVEA4-KPC from E. coli HA4 was successfully transferred with a conjugation frequency of 3.66 × 101. Conclusions: Interchange of multidrug-resistant K. pneumoniae lineages ST258 replaced by ST11 in the framework of colonization and infection by KPC-GNB of an inpatient from our institution was found. In addition, the transfer of the gene configuration of blaKPC–2 between infecting strains may have occurred in the nosocomial environment, but we cannot rule out that the event took place in vivo, within the patient, during hospitalization.

IntroductionThe raise of multi-drug resistant bacteria involving carbapenem, colistin, or tigecycline resistance constitutes a threat to public health, which partly results from the transmission of corresponding mobile resistance genes, such as blaKPC and blaNDM for carbapenem, mcr for colistin, and tmexCD-toprJ gene cluster for tigecycline. Herein, we described the emergence of an Aeromonas veronii strain HD6454 co-harboring blaKPC–2, mcr-3.17, and tmexC3.2-tmexD3.3-toprJ1b gene cluster from hospital sewage.MethodsWhole genome sequencing (WGS) was used to determine the genome sequence of HD6454, and the detailed genomic analysis of genetic elements or regions carrying key antimicrobial resistance genes (ARGs) from HD6454 were performed. Cloning experiment was conducted to confirm the function of key ARGs in mediating antimicrobial resistance. Conjugation experiment was conducted to determine the mobility of the plasmid.ResultsThe results showed that this strain belonged to a novel sequence type (ST) variant ST1016, and carried 18 important ARGs. Among them, the blaKPC–2 was carried by non-self-transmissible IncP-6 plasmid, while tmexC3.2-tmexD3.3-toprJ1b gene cluster and mcr-3.17 were carried by integrative and mobilizable element (IME) or IME-related region in chromosome. The mcr-3.17, mcr-3.6, and mcr-3-like3 genes were further inferred to originate from IMEs of Aeromonas species. Additionally, for the first time, the mcr-3.17 was confirmed to confer low-level resistance to colistin under inducible expression, while tmexC3.2-tmexD3.3-toprJ1b gene cluster was confirmed to confer low-level resistance to tigecycline.DiscussionThis is the first report of a strain co-harboring blaKPC–2, mcr-3.17, and tmexC3.2-tmexD3.3-toprJ1b gene cluster. Although the resistance and/or mobility of these ARGs are limited in this strain, the emergence of this multiple important ARGs-carrying strain deserves further attention.

ABSTRACT: Objectives: Enterobacter cloacae complex (ECC) has awakened interest recently because of its increasing resistance to carbapenems codified by several genes all over the globe. Even though there are some sequence types (STs) which represent high-risk clones, there is substantial clonal diversity in the ECC. This work aimed to perform whole-genome sequencing (WGS), genomic analysis, and phylogenetic studies of a Klebsiella pneumoniae carbapenemase (KPC) -producing multidrug-resistant (MDR) ECC isolate from Argentina. Methods: We analysed the genome of an MDR KPC-producing ECC strain isolated from a urine sample from a patient in a hospital in Argentina. The WGS was done by Illumina MiSeq-I (Illumina, San Diego, CA). The genome was assembled with SPAdes 3.9.0, and annotated with PROKKA, RAST, and Blast. Plasmids were identified with PlasmidFinder. Antibiotic resistance genes were detected using RESfinder, CARD, and Blastn. STs were identified with pubMLST. Results: The strain was identified as Enterobacter hormaechei, an important emerging human pathogen. No ST could be assigned; six of seven alleles of multilocus sequence typing (MLST) were the same as for E. hormaechei ST66, which is a high-risk clone. We found multiple acquired antibiotic resistance genes, including blaKPC-2 in an IncM1 plasmid, and a secretion system VI, which can favour the prevalence of ECC strains while competing with other bacteria. Conclusion: Because of its MLST profile being so close to that of E. hormaechei ST66, the acquisition of multiple resistance genes, and the presence of the secretion systems, the potential of this strain for becoming a new high-risk clone cannot be discarded.

ABSTRACT: Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is increasingly reported worldwide and has posed a serious challenge for public health. Here we report the complete genome sequence of a multidrug-resistant (MDR) K. pneumoniae carrying one blaNDM-1 and two copies of blaKPC-2 genes isolated from a cerebrospinal fluid specimen in China. Methods: The minimal inhibitory concentrations (MICs) of 26 antimicrobial agents against K. pneumoniae strain KP46 were measured. The complete genome sequence of KP46 was determined using Illumina and Nanopore platforms. The derived short and long reads were assembled using Unicycler. Multilocus sequence typing (MLST), antimicrobial resistance genes, virulence genes, and plasmid replicons were predicted in silico using the BacWGSTdb server. The phylogenetic relationship between KP46 and 454 ST15 K. pneumoniae strains obtained from NCBI GenBank database was analysed based on a core genome MLST (cgMLST) strategy. Results: K. pneumoniae strain KP46 was resistant to all antimicrobial agents tested, except for tigecycline, colistin, cefiderocol, and fosfomycin. The genome sequence of KP46 belonged to sequence type 15 (ST15), which contained seven circularized contigs comprising 5 674 521 bp, including one chromosome and six plasmids. Serval antimicrobial resistance genes were identified, including a blaNDM-1 gene located in a 53 096 bp IncX3 plasmid, and two copies of blaKPC-2 gene located both in a 103 807 bp IncX6 and an 88 164 bp IncFII plasmid, respectively. The most closely related strain was another ST15 strain also isolated from China with five cgMLST loci differences. Conclusion: We reported the first complete genome sequence of a K. pneumoniae ST15 clinical isolate coharbouring blaNDM-1 and two copies of blaKPC-2 in China. This study will provide insight into the antimicrobial resistance mechanisms and phylogeny of carbapenem-resistant ST15 K. pneumoniae.

ABSTRACT: Objectives: This study investigated the underlying mechanism of the evolution of tigecycline resistance during treatment in a patient infected with Klebsiella pneumoniae harbouring blaKPC-2. Methods: A total of seven clonal K. pneumoniae strains were continuously isolated from a patient during hospitalisation. Antimicrobial resistance in the strains was determined by antimicrobial susceptibility testing. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to explore the homology of the isolates. Whole-genome shotgun (WGS) analysis and cloning experiments were used to investigate the underlying mechanism of the evolution of tigecycline resistance. Results: All of the isolates had a minimum inhibitory concentration (MIC) for tigecycline of 4 µg/mL, except strain FK6768 that had a MIC of 32 µg/mL. Carbapenem-resistant K. pneumoniae strains (FK6614, FK6768 and FK6809) were consecutively isolated from faeces at different times. Antimicrobial susceptibility testing indicated that tigecycline resistance increased in FK6768 and subsequently decreased in FK6809, which attracted our attention. WGS and further bioinformatics analysis showed a homology for the three faecal isolates of >99%. The blaKPC-2 carbapenemase gene and a tet(A) mutation were found in tigecycline-resistant isolate FK6768. Subsequent cloning experiments confirmed the contribution of a tet(A) variant to reduced tigecycline susceptibility. Conclusion: Here we report a K. pneumoniae isolate carrying both tet(A) mutation and the blaKPC-2 gene, which led to increased tigecycline resistance during tigecycline treatment. This is the first report describing tigecycline resistance of K. pneumoniae first increasing and subsequently decreasing in vivo.

ABSTRACT Generation of hybrid MDR plasmids accelerated the evolution and transmission of resistance genes. In this study, we characterized a blaKPC-2- and blaIMP-4-coharboring conjugative hybrid plasmid constituted of an IncHI5 plasmid-like region, an IncFII(Yp)/IncFIA plasmid-like region, and a KPN1344 chromosome-like region from a clinical ST852-KL18 Klebsiella quasipneumoniae strain. The blaIMP-4 gene was captured by a novel integron In1965, and the blaKPC-2 gene was located on a new non-Tn4401 group I NTEKPC element. Both blaKPC-2- and blaIMP-4-containing genetic architectures were distinguished from classical structures, highlighting the constant evolution of these genetic elements. IMPORTANCE The emergence of carbapenem-resistant Enterobacterales (CRE) that coexpress serine- and metallo-carbapenemases is a severe threat to the efficacy of ceftazidime-avibactam (CZA), which has been proven to be extremely effective against KPC-producing Enterobacterales strains. Our study described the cooccurrence of KPC-2, a serine β-lactamase, and IMP-4, a metallo-β-lactamase (MBL), on a conjugative hybrid plasmid from a clinical carbapenem-resistant K. quasipneumoniae strain, and it revealed an alternative route for IncHI5 plasmid to evolve by recombining with other plasmids to form a hybrid plasmid. Moreover, this hybrid plasmid can be transferred into other Klebsiella species and stably persist during passage. The propagation of two important carbapenemase genes with a new genetic background using well-evolved plasmids in the clinical setting promotes the emergence of superbugs that require careful monitoring.

ABSTRACT: Objectives: The aim of this study was to characterise the co-occurrence of blaKPC and blaNDM in a K64-ST11 carbapenem-resistant Klebsiella pneumoniae strain. Methods: Antimicrobial susceptibility was determined by the disk diffusion method. Whole-genome sequencing was performed using Illumina MiSeq and PacBio II sequencers. High-quality reads were de novo assembled using the SOAPdenovo package. Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP), and genome characteristics were analysed using bioinformatics methods. Results: Klebsiella pneumoniae strain KPWX136 was resistant to most of the tested antibiotics, being susceptible only to polymyxin B and tigecycline. The genome of strain KPWX136 is composed of a single chromosome (5 473 976 bp) and six plasmids including pA (191 359 bp), pB (134 972 bp), pC (117 844 bp), pD (87 095 bp), pE (11 970 bp) and pF (5596 bp). Complete sequence analysis revealed the resistome of isolate KPWX136, which included blaKPC-2 and blaNDM-5 together with 23 other resistance genes, of which 6 resistance genes were located on the chromosome and 19 on plasmids. Virulome analysis showed that KPWX136 carried a large number of virulence-associated genes. Meanwhile, 26 genomic islands and 6 prophages were predicted within the genome. Conclusion: Genetic characterisation of K. pneumoniae KPWX136 co-harbouring blaNDM-5 and blaKPC-2 showed that it carried not only 25 resistance genes and a large number virulence factors but also various mobile genetic elements (MGEs) such as plasmids and genomic islands. Therefore, we must be alert to the transmission of resistance genes and virulence determinants via MGEs.

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has received considerable attention. Typically, the genetic elements that confer virulence are harbored by nonconjugative plasmids. In this study, we report a CR-hvKP strain, CY814036, of high-risk sequence type 25 (ST25) and the K2 serotype, which is uncommon among K. pneumoniae isolates but caused serious lung infection in a tertiary teaching hospital in China. Whole-genome sequencing (WGS) revealed a rare conjugative plasmid, pCY814036-iucA, carrying a virulence-associated iuc operon (iucABCD-iutA) coding for aerobactin and determinants of multidrug resistance (MDR), coexisting with a conjugative blaKPC-2-bearing plasmid, pCY814036-KPC2, in the same strain. A conjugation assay showed that pCY814036-iucA and pCY814036-KPC2 could be efficiently cotransmitted from CY814036 to Escherichia coli EC600. Further phenotypic investigation, including antimicrobial susceptibility tests, serum resistance assays, and mouse infection models, confirmed that pCY814036-iucA was capable of cotransferring multidrug resistance and hypervirulence features to the recipient. pCY814036-KPC2 also conferred resistance to antibiotics, including β-lactams and aminoglycosides. Overall, the rare coexistence of a conjugative MDR-virulence plasmid and a blaKPC-2-bearing plasmid in a K. pneumoniae isolate offers a possible mechanism for the formation of CR-hvKP strains and the potential to significantly accelerate the propagation of high-risk phenotypes. IMPORTANCE The increased reporting of carbapenem-resistant hypervirulent K. pneumoniae is considered a worrisome concern to human health care and has restricted the choice of effective antibiotics for clinical treatment. Moreover, virulence plasmids with complete conjugation modules have been identified, which evolved via homologous recombination. Here, we characterize an ST25 CR-hvKP strain, CY814036, harboring both a conjugative MDR-virulence plasmid and a blaKPC-2-bearing plasmid in China. This study highlights that the cotransmission of drug resistance and virulence plasmids increases therapeutic difficulties and worsens clinical prognoses. Also, active surveillance of the conjugative MDR-virulence plasmid is necessary.

ABSTRACT: Objective: The main objectives were to describe two blaKPC-2 plasmids recovered from Pseudomonas aeruginosa isolates belonging to the ST654 and ST235 high-risk clones, and to compare with complete sequences of blaKPC-2 harbouring plasmids available in public databases. Methods: Antimicrobial susceptibility was determined according to CLSI (Clinical and Laboratory Standards Institute) guidelines. Genomes were sequenced using an Illumina MiSeq platform, and blaKPC-2 plasmid sequences were achieved using MinION platform. Sequences were analysed using Unicycler and RAST. In silico predictions of the isolates sequence type (ST), antimicrobial resistance genes, plasmid replicon typing and MOB relaxases were fulfilled using bioinformatics tools. Results: PA_2047 and PA_HdC isolates corresponded to the high-risk clones ST654 and ST235, respectively. The carbapenem resistance was mediated by KPC-2. Both blaKPC-2 harbouring plasmids, pPA_2047 and pPA_HdC, were different among them, non-conjugative and untypable by PlasmidFinder. pPA_2047 presented high identity with a Pae-13 plasmid, and these both located blaKPC-2 in Tn4401b isoform. pPA_HdC displayed a novel architecture, and the genetic context of blaKPC-2 was original. Besides the blaKPC-2 gene, resistance genes to aminoglycosides and quinolones were detected, including the novel phosphotransferase CrpP in PA_HdC. Conclusion: This study expands the limited knowledge about the molecular epidemiology of blaKPC-2 in P. aeruginosa from Latin America. Two novel plasmids harbouring blaKPC-2 were described that were untypable by their incompatibility group. The plasmid recovered from P. aeruginosa PA_HdC (ST235) displayed a novel architecture and an original context for blaKPC-2. On the other hand, the genetic platform carrying blaKPC-2 in P. aeruginosa PA_2047 (ST654) seems to a be a classical one.

Objectives: The aim of this study was to investigate the potential for transmission and heritability of carbapenemase gene (blaKPC-2 or blaNDM-1)-encoding or mcr-1-encoding plasmids in clinical Enterobacteriaceae strains. Methods: Potential for transmission of carbapenemase gene (blaKPC-2 or blaNDM-1)-encoding or mcr-1-encoding plasmids in clinical Enterobacteriaceae strains was tested in three conjugation models, namely filter-mating conjugation in laboratory conditions, a meat product model and the gastrointestinal (GI) tract of rats. Plasmid stability in Enterobacteriaceae strains was also determined. Results: We demonstrated that plasmids carrying a carbapenemase gene (blaKPC-2 or blaNDM-1) could be efficiently conjugated to strains carrying the mcr-1 gene and vice versa, and that these plasmids could stably co-exist in clinical Enterobacteriaceae strains. These findings suggest that Enterobacteriaceae can readily acquire phenotypic resistance to both carbapenems and colistin in natural environments such as food products and the GI tract of human and animals. Conclusion: Gene transfer events are common among members of the Enterobacteriaceae and serve as a key mechanism facilitating adaptation to new environments. Development of innovative strategies and surveillance measures to curtail the dissemination of multidrug resistance plasmids is necessary. Transmission and stable inheritance of these two types of plasmids would lead to the emergence of multidrug-resistant pathogens that are resistant to all currently available last-line antibiotics for Gram-negative bacterial infections.

According to the World Health Organization, carbapenem-resistant Enterobacteriaceae (CRE) belong to the highest priority group for the development of new antibiotics. Argentina-WHONET data showed that Gram-negative resistance frequencies to imipenem have been increasing since 2010 mostly in two CRE bacteria: Klebsiella pneumoniae and Enterobacter cloacae Complex (ECC). This scenario is mirrored in our hospital. It is known that K. pneumoniae and the ECC coexist in the human body, but little is known about the outcome of these species producing KPC, and colonizing or infecting a patient. We aimed to contribute to the understanding of the rise of the ECC in Argentina, taking as a biological model both a patient colonized with two KPC-producing strains (one Enterobacter hormaechei and one K. pneumoniae) and in vitro competition assays with prevalent KPC-producing ECC (KPC-ECC) versus KPC-producing K. pneumoniae (KPC-Kp) high-risk clones from our institution. A KPC-producing E. hormaechei and later a KPC-Kp strain that colonized a patient shared an identical novel conjugative IncM1 plasmid harboring blaKPC-2. In addition, a total of 19 KPC-ECC and 58 KPC-Kp strains isolated from nosocomial infections revealed that high-risk clones KPC-ECC ST66 and ST78 as well as KPC-Kp ST11 and ST258 were prevalent and selected for competition assays. The competition assays with KCP-ECC ST45, ST66, and ST78 versus KPC-Kp ST11, ST18, and ST258 strains analyzed here showed no statistically significant difference. These assays evidenced that high-risk clones of KPC-ECC and KPC-Kp can coexist in the same hospital environment including the same patient, which explains from an ecological point of view that both species can exchange and share plasmids. These findings offer hints to explain the worldwide rise of KPC-ECC strains based on the ability of some pandemic clones to compete and occupy a certain niche. Taken together, the presence of the same new plasmid and the fitness results that showed that both strains can coexist within the same patient suggest that horizontal genetic transfer of blaKPC-2 within the patient cannot be ruled out. These findings highlight the constant interaction that these two species can keep in the hospital environment, which, in turn, can be related to the spread of KPC.

ABSTRACT The objective of this study is to characterize the molecular mechanism of a clinical carbapenem-resistant Citrobacter portucalensis strain K218, which coproduces KPC and NDM carbapenemases. K218 was isolated from a patient's blood sample in a Chinese tertiary hospital. Carbapenemases were detected by the immunocolloidal gold technique. The MIC values were determined by VITEK2. Whole-genome sequencing was performed on K218 and sequence data were analyzed using phylogenetics and extensive genomic comparison. This study reveals that K218 contains a single 5.08 Mb chromosome (51.8% GC content) and four plasmids, pK218-KPC (106 Kb), pK218-NDM (111 Kb), pK218-SHV (191 Kb), and pK218-NR (5 Kb). Twenty-nine types of antibiotic resistance genes were carried on K218, including blaKPC-2 harbored on pK218-KPC and blaNDM-1 harbored on pK218-NDM. Detailed comparison of related plasmids of pK218-KPC, pK218-NDM, and pK218-SHV showed that they shared similar conserved backbone regions, respectively. Comprehensive annotation revealed large accessory modules were recombined on the genome of K218. Further analysis speculated that mobile genetic elements bearing abundant resistance genes facilitated the formation of these accessory modules. In conclusion, this study provides an in-depth understanding of the genomic characterization of K218, an extensively drug-resistant C. portucalensis strain coproducing NDM and KPC carbapenemase. To the best of our knowledge, this is the first report of C. portucalensis strain coharboring blaKPC-2 and blaNDM-1 from the clinical setting. IMPORTANCE This is the first report of extensively drug-resistant C. portucalensis harboring both blaKPC-2 and blaNDM-1. This study will not only extend the understanding of the structural dissection of plasmids and chromosomes carried in C. portucalensis, but also expand knowledge of the genetic environment of the blaKPC-2 and blaNDM-1 genes. blaKPC-2 and blaNDM-1 genes have been suggested to facilitate the propagation and persistence of their host bacteria under different antimicrobial selection pressures. Large accessory regions carrying blaKPC-2 and blaNDM-1 genes have become hot spots for transposition and integration, and their structural variation and evolution should receive attention. The multidrug-resistant plasmids pK218-KPC, pK218-NDM, and pK218-SHV with several multidrug resistance regions and the chromosome cK218 with two novel transposons Tn7410 and Tn7411 contribute to the formation of extensively drug-resistant C. portucalensis.

ABSTRACT Ceftazidime/avibactam (CAZ/AVI) is regarded as an effective alternative antibiotic for the clinical treatment of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates. As resistance has been reported in some strains, it is critical to understand the key mechanisms contributing to the acquired resistance to CAZ/AVI. From January 2018 to April 2020, 127 KPC-producing carbapenem-resistant Klebsiella pneumoniae strains (CRKPs) were isolated at a university hospital in Chongqing, China, and 25 strains showed reduced susceptibility to CAZ/AVI. All reduced-susceptibility CRKPs were deficient in Ompk35 and Ompk36 porins, and 24 strains had a premature termination at amino acid position 63 in Ompk35 and 134 to 135 glycine and aspartic acid (GD) insertion in OmpK36, while the blaKPC-2 expression level showed no significant difference compared to that of strain BAA-1705. Four reduced-susceptibility strains evolved resistance under selective pressure of CAZ/AVI with the blaKPC-2 expression level increased, and two of these strains had mutations in the Ω-loop. The study found a strain of CRKP55 with changes in the resistance phenotype during conjugation, evolving from reduced sensitivity to high-level resistance to CAZ/AVI. Through plasmid sequencing and reverse transcription-quantitative PCR, it was speculated that insertion sequence (IS)26-mediated blaKPC-2 gene amplification caused the MIC value change in the conjugant JKP55. Our findings illustrated the potential of CAZ/AVI resistance under antibiotic stress and demonstrated that IS26 may mediate blaKPC-2 replication transposition, leading to high-level resistance during horizontal gene transfer. Investigation of CAZ/AVI resistance mechanisms may offer a unique opportunity to study the horizontal evolutionary trajectories of K. pneumoniae high-risk clones. IMPORTANCE Klebsiella pneumoniae carbapenemase (KPC) production is the most common mechanism of K. pneumoniae resistance to carbapenems in China. Currently, CAZ/AVI is considered a potential alternative therapeutic option for infections caused by these isolates. However, there have been increasing reports of resistant or reduced-sensitivity strains since the approval of this agent. In this study, resistance to CAZ/AVI was induced under drug-selective pressure and was caused by blaKPC-2 overexpression and/or substitutions in the Ω-loop of KPC. Additionally, it was demonstrated that a conjugative plasmid carrying blaKPC-2 could transfer horizontally between species, and perhaps, IS26-derived tandem amplification of blaKPC-2 during this period led to high-level resistance to CAZ/AVI. Our research suggests that IS26-mediated resistance evolution may have important implications in guiding clinical antibiotic use.

Multidrug-resistant (MDR) Proteus, especially those strains producing extended-spectrum β-lactamases (ESBL) and carbapenemases, represents a major public health concern. In the present work, we characterized 27 MDR Proteus clinical isolates, including 23 Proteus mirabilis, three Proteus terrae, and one Proteus faecis, by whole-genome analysis. Among the 27 isolates analyzed, SXT/R391 ICEs were detected in 14 strains, and the complete sequences of nine ICEs were obtained. These ICEs share a common backbone structure but also have different gene contents in hotspots and variable regions. Among them, ICEPmiChn2826, ICEPmiChn2833, ICEPmiChn3105, and ICEPmiChn3725 contain abundant antibiotic resistance genes, including the ESBL gene blaCTX-M-65. The core gene phylogenetic analysis of ICEs showed their genetic diversity, and revealed the cryptic dissemination of them in Proteus strains from food animals and humans on a China-wide scale. One of the isolates, FZP3105, acquired an NDM-1-producing MDR plasmid, designated pNDM_FZP3105, which is a self-transmissible type 1/2 hybrid IncC plasmid. Analysis of the genetic organization showed that pNDM_FZP3105 has two novel antibiotic resistance islands bearing abundant antibiotic resistance genes, among which blaNDM-1 is located in a 9.0 kb ΔTn125 bracketed by two copies of IS26 in the same direction. In isolates FZP2936 and FZP3115, blaKPC-2 was detected on an IncN plasmid, which is identical to the previously reported pT211 in Zhejiang province of China. Besides, a MDR genomic island PmGRI1, a variant of PmGRI1-YN9 from chicken in China, was identified on their chromosome. In conclusion, this study demonstrates abundant genetic diversity of mobile genetic elements carrying antibiotic resistance genes, especially ESBL and carbapenemase genes, in clinical Proteus isolates, and highlights that the continuous monitoring on their transmission and further evolution is needed.

Objectives: The aim of this study was to explore the genomic content of a blaKPC-2- and mcr-1-harbouring Escherichia coli strain and to clarify the molecular mechanism for the transmission of multidrug resistance. Methods: Antimicrobial susceptibility testing was conducted by the broth microdilution method to determine the resistance profile. Filter-mating conjugation assays were performed to confirm the plasmid transfer ability. Whole-genome sequence data were acquired by a combination of Illumina paired-end reads and Nanopore long-read sequencing. Results: Escherichia coli strain QE11−421 was an mcr-1-positive colistin-resistant isolate that co-harboured the blaKPC-2 gene conferring carbapenem resistance. Genome sequence data confirmed QE11−421 as a sequence type 48 (ST48) E. coli that harboured five large conjugative plasmids encoding several multidrug resistance genes. The blaKPC-2 gene was located on a Tn3–Tn4401 composite transposon, which is part of a 65-kb multidrug-resistant IncN plasmid. The mcr-1 gene was harboured on another 33-kb IncX4 plasmid that was more conserved and presented few antimicrobial resistance determinants. No copies of insertion sequence ISApl1 were found flanking the mcr-1 gene, decreasing the mobility of mcr-1 based on its original Tn6330 transposon. Conclusions: Horizontal transfer of multidrug resistance plasmids or resistance-related transposon units was responsible for the emergence of this notorious superbug. The coexistence of blaKPC-2–IncN and mcr-1–IncX4 plasmids in a ST48 E. coli strain in humans poses a great threat.

ABSTRACT The spread of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales is a public health concern. KPC-encoding blaKPC is predominantly spread by strains of a particular phylogenetic lineage, clonal group 258, but can also be spread by horizontal transfer of blaKPC-carrying plasmids. Here, we report the transfer of a blaKPC-2-harboring plasmid via mobilization from K. pneumoniae to Citrobacter freundii complex and Morganella morganii strains in a single patient. We performed draft whole-genome sequencing to analyze 20 carbapenemase-producing Enterobacterales strains (15 of K. pneumoniae, two of C. freundii complex, and three of M. morganii) and all K. pneumoniae strains using MiSeq and/or MinION isolated from a patient who was hospitalized in New York and Montreal before returning to Japan. All strains harbored blaKPC-2-containing Tn4401a. The 15 K. pneumoniae strains each belonged to sequence type 258 and harbored a Tn4401a-carrying multireplicon-type plasmid, IncN and IncR (IncN+R). Three of these K. pneumoniae strains also possessed a Tn4401a-carrying ColRNAI plasmid, suggesting that Tn4401a underwent interplasmid transposition. Of these three ColRNAI plasmids, two and one were identical to plasmids harbored by two Citrobacter europaeus and three M. morganii strains, respectively. The Tn4401a-carrying ColRNAI plasmids were each 23,753 bp long and incapable of conjugal transfer via their own genes alone, but they mobilized during the conjugal transfer of Tn4401a-carrying IncN+R plasmids in K. pneumoniae. Interplasmid transposition of Tn4401a from an IncN+R plasmid to a ColRNAI plasmid in K. pneumoniae and mobilization of Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of blaKPC-2 in C. europaeus and M. morganii. IMPORTANCE Plasmid transfer plays an important role in the interspecies spread of carbapenemase genes, including the Klebsiella pneumoniae carbapenemase (KPC)-coding gene, blaKPC. We conducted whole-genome sequencing (WGS) analysis and transmission experiments to analyze blaKPC-2-carrying mobile genetic elements (MGEs) between the blaKPC-2-harboring K. pneumoniae, Citrobacter europaeus, and Morganella morganii strains isolated from a single patient. blaKPC-2 was contained within an MGE, Tn4401a. WGS of blaKPC-2-carrying K. pneumoniae, C. europaeus, and M. morganii strains isolated from one patient revealed that Tn4401a-carrying ColRNAI plasmids were generated by plasmid-to-plasmid transfer of Tn4401a from a multireplicon-type IncN and IncR (IncN+R) plasmid in K. pneumoniae strains. Tn4401a-carrying ColRNAI plasmids were incapable of conjugal transfer in C. europaeus and M. morganii but mobilized from K. pneumoniae to a recipient Escherichia coli strain during the conjugal transfer of Tn4401a-carrying IncN+R plasmid. Therefore, Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of blaKPC-2 in C. europaeus and M. morganii.

Treatment strategies of infection by carbapenem-resistant Klebsiella pneumoniae (CRKP) are limited. Fosfomycin, a broad-spectrum antibiotic, has attracted renewed interest in combination therapy to fight K. pneumoniae infections. However, reports on fosfomycin-resistant K. pneumoniae are increasing. Among the 57 CRKP strains, 40 (70.2%) were resistant to fosfomycin. Thus, whole-genome sequencing and bioinformatics analysis were conducted to reveal molecular characteristics of fosfomycin-resistant K. pneumoniae. Twenty-three isolates coharbored fosAkp and fosA3, with K. pneumoniae carbapenemase (KPC)-producing ST11-KL64-wzi64-O2 (n = 13) and ST11-KL47-wzi209-OL101 (n = 8), the predominating clonal groups, while fosA3 was not detected in isolates carrying class B carbapenemase genes. Twenty-two (out of 26) ST11-KL64 strains were positive for rmpA2, of which 12 carried fosA3. Four of the 23 fosA3-positive isolates could successfully transfer their fosfomycin-resistant determinants to Escherichia coli J53AziR. All four strains belonged to ST11-KL47 with the same pulsed-field gel electrophoresis profile, and their transconjugants acquired fosfomycin, carbapenem, and aminoglycoside resistance. A 127-kb conjugative pCT-KPC-like hybrid plasmid (pJNKPN52_KPC_fosA) coharboring fosA3, blaKPC–2, blaCTX–M–65, blaSHV–12, rmtB, and blaTEM–1 was identified. ST11-KL64 and ST11-KL47 K. pneumoniae, with higher resistance and virulence, should be critically monitored to prevent the future dissemination of resistance.

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Pseudomonas aeruginosa (KPC-PA) has been reported sporadically. However, epidemiological and antimicrobial susceptibility data specific for KPC-PA are lacking. We collected 374 carbapenem-resistant P. aeruginosa (CRPA) isolates from seven hospitals in China from June 2016 to February 2019 and identified the blaKPC-2 gene in 40.4% (n = 151/374) of the isolates. Approximately one-half of all KPC-PA isolates (n = 76/151; 50.3%) were resistant to ceftazidime-avibactam (CAZ-AVI). Combining Kraken2 taxonomy identification and Nanopore sequencing, we identified eight plasmid types, five of which carried blaKPC-2, and 13 combination patterns of these plasmid types. In addition, we identified IS26-ΔTn6296 and Tn1403-like–ΔTn6296 as the two mobile genetic elements that mediated blaKPC-2 transmission. blaKPC-2 plasmid curing in 28 strains restored CAZ-AVI susceptibility, suggesting that blaKPC-2 was the mediator of CAZ-AVI resistance. Furthermore, the blaKPC-2 copy number was found to correlate with KPC expression and, therefore, CAZ-AVI resistance. Taken together, our results suggest that KPC-PA is becoming a clinical threat and that using CAZ-AVI to treat this specific pathogen should be done with caution. IMPORTANCE Previous research has reported several cases of KPC-PA strains and three KPC-encoding P. aeruginosa plasmid types in China. However, the prevalence and clinical significance of KPC-PA are not available. In addition, the susceptibility of the strains to CAZ-AVI remains unknown. Samples in this study were collected from seven tertiary hospitals prior to CAZ-AVI clinical approval in China. Therefore, our results represent a retrospective study establishing the baseline efficacy of the novel β-lactam/β-lactamase combination agent for treating KPC-PA infections. The observed correlation between the blaKPC copy number and CAZ-AVI resistance suggests that close monitoring of the susceptibility of the strain during treatment is required. It would also be beneficial to screen for the blaKPC gene in CRPA strains for antimicrobial surveillance purposes.

The emergence of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) was regarded as an emerging threat in clinical settings. Here, we investigated the prevalence of CRKP strains among inpatients in a new hospital over 1 year since its inception with various techniques, and carried out a WGS-based phylogenetic study to dissect the genomic background of these isolates. The genomes of three representative blaNDM–1-positive strains and the plasmids of four blaKPC–2-positive strains were selected for Nanopore long-read sequencing to resolve the complicated MDR structures. Thirty-five CRKP strains were identified from 193 K. pneumoniae isolates, among which 30 strains (85.7%) harbored blaKPC–2, whereas the remaining five strains (14.3%) were positive for blaNDM–1. The antimicrobial resistance profiles of blaNDM–1-positive isolates were narrower than that of blaKPC–2-positive isolates. Five isolates including two blaNDM–1-positive isolates and three blaKPC–2-positive strains could successfully transfer the carbapenem resistance phenotype by conjugation. All CRKP strains were categorized into six known multilocus sequence types, with ST11 being the most prevalent type. Phylogenetic analysis demonstrated that the clonal spread of ST11 blaKPC–2-positive isolates and local polyclonal spread of blaNDM–1-positive isolates have existed in the hospital. The blaNDM–1 gene was located on IncX3, IncFIB/IncHI1B, and IncHI5-like plasmids, of which IncFIB/IncHI1B plasmid has a novel structure. By contrast, all ST11 isolates shared the similar blaKPC–2-bearing plasmid backbone, and 11 of them possessed pLVPK-like plasmids. In addition, in silico virulome analysis, Galleria mellonella larvae infection assay, and siderophore secretion revealed the hypervirulence potential of most blaKPC–2-positive strains. Given that these isolates also had remarkable environmental adaptability, targeted measures should be implemented to prevent the grave consequences caused by hv-CRKP strains in nosocomial settings.

Objectives: The Klebsiella pneumoniae carbapenemase (blaKPC) gene is one of the most widespread carbapenemase genes in the world. However, there are few reports on KPC-producing bacteria in Japan. The aim of this study was therefore to investigate KPC-producing K. pneumoniae in Japan. Methods: A KPC-2-producingK. pneumoniae strain (KAM260) was isolated from hospital sewage water in Japan in 2018. The complete genome was determined by whole-genome sequencing. Subsequent comparative sequence analysis of the blaKPC-2-carrying plasmid was performed. Results: Klebsiella pneumoniae KAM260, belonging to sequence type 3026 (ST3026), harboured the blaKPC-2 gene in 114.6-kbp plasmid pKAM260_2 with IncFIB(pQIL) and IncFII(K) replicons. pKAM260_2 was highly identical to pKpQIL-like plasmids, which carry blaKPC genes and have spread worldwide. pKAM260_2 had functional conjugation-associated genes and was transferable to Escherichia coli. Conclusion: pKAM260_2, the self-transmissible plasmid carrying theblaKPC-2 gene, was detected from hospital sewage water in Japan and was characterised as a pKpQIL-like plasmid. This plasmid needs to be monitored in Japan in the future owing to its high diffusivity.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with an increase in the frequency of infections caused by multidrug resistant (MDR) and extensively drug resistant (XDR) strains, limiting the available therapeutic options. The most troublesome resistance is the acquisition and production of carbapenemases such as Verona integron-encoded metallo-β-lactamases (VIM), the most frequent and widespread, and the Klebsiella pneumoniae carbapenemases (KPC), which has continuously spread in the last decade. Its dissemination is linked to their location on mobile genetic elements (MGEs). In Colombia, VIM and KPC have been increasing in its frequency showing major successful dissemination. In this article, we molecularly characterized and analyzed the genetic context of blaVIM and blaKPC in carbapenem-resistant P. aeruginosa (CRPA) isolates from infected and colonized patients in two tertiary-care hospitals, one in Medellín and the other in a municipality close to Medellín, both areas with high carbapenemase endemicity in Colombia (2013–2015). Using whole-genome sequencing (WGS), we identified a remarkable variety of genetic backgrounds in these MDR P. aeruginosa isolates carrying blaKPC–2 and blaVIM–2. There were a diversity of class 1 integron and variations in the gene cassettes associated to blaVIM–2, as well as a possible event of spread of blaKPC–2 mediated by a plasmid that contained part of Tn4401b in one infection case. The dissemination of blaVIM–2 and blaKPC–2 in P. aeruginosa in this area in Colombia has been strongly influenced by successful international clones, carrying these genes and additional determinants of resistance on MGEs, accompanied by gene rearrangement under an antimicrobial selection pressure. These findings emphasize the need to implement control strategies based on rational antibiotic use.

Objectives: The resistance rate of Klebsiella pneumoniae to commonly used antibiotics has been increasing rapidly, causing serious concern among clinicians and microbiologists. Resistance to carbapenems in K. pneumoniae is increasing dramatically in Chinese hospitals. Here we report the genome sequence of an NDM-1 and KPC-2 co-producing K. pneumoniae (CRKP380) isolated from a bloodstream infection in Hangzhou, China. Methods: The whole genome sequence of strain CRKP380 was determined using an Illumina NovaSeq 6000 platform. Antimicrobial resistance genes (ARGs) were identified using the BacWGSTdb server. The phylogenetic relationship between strain CRKP380 and other K. pneumoniae strains isolated from Hangzhou currently deposited in the NCBI GenBank database was analysed using a core genome-based single nucleotide polymorphism strategy. Results: Klebsiella pneumoniae CRKP380 was resistant to all antibiotics tested except tigecycline and colistin. The genome sequence of K. pneumoniae CRKP380 consisted of 75 contigs comprising 5 590 460 bp. According to the Pasteur multilocus sequence typing (MLST) scheme, CRKP380 belongs to ST11. Eight ARGs were identified in CRKP380, including two carbapenemase genes (blaKPC-2 and blaNDM-1). Ten phylogenetically-related K. pneumoniae strains from Hangzhou were identified with identical ARGs and the same capsular serotype KL105, but none of these strains carried the blaNDM gene. Conclusion: Here we report the genome sequence of a K. pneumoniae ST11 clinical strain co-carrying blaNDM-1 and blaKPC-2 from Hangzhou, China. The genome sequence of CRKP380 can be used as a reference sequence for future comparative genomic analysis, including the acquisition and mobilisation of carbapenem resistance genes.

ABSTRACTThe incidence and transmission of Klebsiella pneumoniae carbapenemase (KPC) producing plasmids have been well documented. However, the evolutionary dynamics of KPC plasmids and their fitness costs are not well characterized. Here, two carbapenemase-producing plasmids from Proteus mirabilis, pT18 and pT211 (both carrying blaKPC-2), were characterized through whole genome sequencing. pT211 is a 24.2 kbp N-type plasmid that contains blaKPC-2 and a single copy of the IS6-family insertion sequence IS26. pT18 is a 59 kbp cointegrate plasmid comprised of sequences derived from three different plasmids: a close relative of pT211 (containing blaKPC-2), an FII-33 plasmid (blaTEM-1B, blaCTX-M-65, rmtB and fosA3) and a rolling-circle plasmid. The segments of pT18 derived from each of the different plasmids are separated by copies of IS26, and sequence analysis indicated that pT18 was likely generated by both conservative and replicative IS26-mediated cointegrate formation. pT18 and pT211 were transferred into Escherichia coli DH5α separately to assess the impact of plasmids on host fitness. Only DH5α harbouring pT18 grew slower than the wild type in antibiotic-free media. However, in sub-inhibitory concentrations of fosfomycin and amikacin, cells containing pT18 grew faster than the wild type, and the minimum concentrations of fosfomycin and amikacin required to observe an advantage for plasmid-carrying cells were 1/3 and 1/20 the DH5α MIC, respectively. This study highlights the importance of the role of cointegrate plasmids in the dissemination of antibiotic resistance genes between pathogenic bacterial species, and highlights the importance of sub-inhibitory concentrations of antibiotics to the persistence of such plasmids.

Escherichia coli sequence type 131 (ST131) is well known for its multidrug resistance profile. Carbapenems have been considered the treatment of choice for E. coli ST131 infections, and resistance to carbapenems is emerging due to the acquisition of carbapenemase-encoding genes. In this study, 45 carbapenem-resistant E. coli strains were collected in a hospital. The resistance mechanisms, plasmid profiles, and genetic relatedness of these strains were determined. Phylogenetic relationships between these strains were assessed by molecular profiling and aligned with patient clinical details. The genetic context of blaKPC–2 was analyzed to trace the potential dissemination of blaKPC–2. The 45 carbapenem-resistant E. coli ST131 strains were closely related. Initially prevalent only in a single ward, ST131 subsequently spread to other ward, resulting in a respiratory infection outbreak of carbapenem-resistant E. coli ST131. Eight of the 30 patients died within 28 days of the first isolation of E. coli ST131. The blaKPC–2-positive plasmid profiles suggest that the carbapenem resistance was due to the acquisition by E. coli ST131 of transmissible plasmids pE0272_KPC and pE0171_KPC carrying blaKPC–2. Additionally, diverse multidrug resistance elements were transferred and rearranged between these plasmids mediated by IS26. Our research indicates that clinical attention should be paid to the importance of E. coli ST131 in respiratory infections and the spread of blaKPC-carrying E. coli ST131.

ObjectivesCarbapenemase-producing Klebsiella pneumoniae (CP-Kp) is a major cause of infections in transplanted patients and has been associated with high mortality rates in this group. There is a lack of information about the Brazilian structure population of CP-Kp isolated from transplanted patients. By whole-genome sequencing (WGS), we analyzed phylogeny, resistome, virulome of CP-Kp isolates, and the structure of plasmids encoding blaKPC–2 and blaNDM–1 genes.MethodsOne K. pneumoniae isolated from each selected transplanted patient colonized or infected by CP-Kp over a 16-month period in a hospital complex in Porto Alegre (Brazil) was submitted for WGS. The total number of strains sequenced was 80. The hospital complex in Porto Alegre comprised seven different hospitals. High-resolution SNP typing, core genome multilocus sequence typing (cgMLST), resistance and virulence genes inference, and plasmid reconstruction were performed in 80 CP-Kp.ResultsThe mortality rate of CP-Kp colonized or infected transplanted inpatients was 21.3% (17/80). Four CP-Kp epidemic clones were described: ST11/KPC-2, ST16/KPC-2, and ST15/NDM-1, all responsible for interhospital outbreaks; and ST437/KPC-2 affecting a single hospital. The average number of acquired resistance and virulence genes was 9 (range = 2–14) and 27 (range = 6–36), respectively. Two plasmids carrying the blaKPC–2 were constructed and belonged to IncN and IncM types. Additionally, an IncFIB plasmid carrying the blaNDM–1 was described.ConclusionWe detected intrahospital and interhospital spread of mobile structures and international K. pneumoniae clones as ST11, ST16, and ST15 among transplanted patients, which carry a significant range of acquired resistance and virulence genes and keep spreading across the world.

This study aimed to investigate the dissemination and characteristics of blaKPC, blaNDM, blaOXA-48-like, blaIMP, and blaVIM among the carbapenem-resistant Enterobacteriaceae (CRE) strains isolated from adult and children patients. A total of 935 non-duplicate CRE strains were collected from 36 hospitals in 24 provinces or cities across China from 2016 to 2018. Antimicrobial susceptibility testing was performed by broth microdilution method and carbapenemase genes blaKPC, blaNDM, blaOXA-48-like, blaIMP, and blaVIM were screened by PCR and confirmed by DNA sequencing. Overall, carbapenemases were produced in 97.4% (911/935) of CRE strains, including KPC-2 (51.6%, 482/935), NDM (35.7%, 334/935), and OXA-48-like carbapenemases (7.3%, 68/935). Overall, the most prevalent carbapenemase gene was blaKPC-2 among Klebsiella pneumoniae (64.6%, 457/709) and the CRE strains isolated from adult patients (70.3%, 307/437), and blaNDM among Escherichia coli (96.0%, 143/149) and the CRE strains from children (49.0%, 247/498). The blaOXA-232-positive carbapenem-resistant K. pneumoniae (9.3%, 66/709) were all isolated from children. Sixteen strains were positive for blaIMP and 9 strains produced multiple carbapenemases. No strain was positive for blaVIM. Most of the CRE strains (>90%) were resistant to cephalosporins and carbapenems, more than half (>50%) were resistant to aminoglycosides and fluoroquinolones, but the majority (95.8 and 98.4%) were susceptible to polymyxin B and tigecycline. Ceftazidime-avibactam showed excellent in vitro activity against blaKPC-2 and blaOXA-48-like positive strains (100% susceptible). In China, KPC-2, NDM, and OXA-48-like carbapenemases were predominant among CRE clinical isolates. The most prevalent carbapenemase gene was blaKPC-2 among K. pneumoniae isolates from adult patients, and blaNDM among E. coli isolates from children.

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become widespread in hospitals and the environment. Here, we describe a blaKPC-2-carrying plasmid called pCRE3-KPC, which was recovered from a clinical multidrug-resistant Citrobacter braakii CRE3 strain in China. The complete nucleotide sequence of pCRE3-KPC was determined by combining MiSeq and MinION sequencing and then compared with those of three related plasmids. Plasmid conjugal transfer and electroporation tests, modified carbapenem inactivation method, and bacterial antimicrobial susceptibility test were carried out. We compared this plasmid with three related plasmids to verify that the backbone of pCRE3-KPC was composed of the backbones of the IncR plasmid and IncP6 plasmid. Further bioinformatics analysis showed that pCRE3-KPC carried two resistance-related regions (the blaKPC-2 gene cluster and the aacC2-tmrB-related region). The aacC2-tmrB-related region included two novel insertion sequences (ISCfr28 and ISCfr16). IMPORTANCE Reports of human-pathogenic C. braakii strains, especially of strains showing resistance to carbapenems, are rare. To the best of our knowledge, our results represent the first detection of carbapenemase gene blaKPC-2 in C. braakii strains. In addition, we have studied detailed genetic characteristics of the novel IncR/IncP6 hybrid plasmid pCRE3-KPC, which was isolated from a clinical multidrug-resistant Citrobacter braakii CRE3 strain. Our results may provide further insight into the horizontal transfer of multidrug resistance genes in bacteria and into the genomic diversity and molecular evolution of plasmids.

Background: Epidemiology of fosfomycin susceptibility and the plasmid-mediated fosfomycinase genes of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Taiwan remain unclear. Methods: 642 CRKP clinical isolates were collected from a nation-wide surveillance study (16 hospitals) in Taiwan in 2012–2013. Antimicrobial susceptibilities were determined. PFGE and MLST determined the clonal relatedness. Carbapenemases and fosfomycinases genes were detected by PCR, and their flanking regions were determined by PCR and sequencing. Synergistic activity of meropenem with fosfomycin was examined by the checkerboard method. Results: In total, 36.4% (234/642) of CRKP isolates in Taiwan were resistant to fosfomycin. Among 234 fosfomycin-resistant CRKP isolates, PFGE analysis revealed 81 pulsotypes. Pulsotype XXIII (n = 63) was predominant and belonged to ST11. 71 had carbapnemases (65 blaKPC-2-positive, 1 blaVIM-1-positive and 5 blaIMP-8-positive) and 62 had fosfomycinases (35 fosA3-positive and 27 foskp96-positive). Only 18.5% (5/27) of foskp96-positive isolates carried foskp96 and blaKPC-2, while 71.4% (25/35) of fosA3-positive isolates contained fosA3 and blaKPC-2. There were five types of flanking sequences for fosA3, and 85.7% (30/35) of fosA3 genes were flanked by IS26, suggesting possible horizontal gene transfer. Synergistic effect of fosfomycin and meropenem was observed in all 25 randomly selected pulsotype XXIII strains (100%; 25/25), even those containing fosfomycinase (48%, 12/25) or carbapnemase (96%, 24/25). Conclusions: A clone (pulsotype XXIII, ST11) has been found to be prevailing among fosfomycin-resistant CRKP in Taiwan. According to the in vitro data, the combination of fosfomycin and meropenem is a potentially alternative choice.

The dissemination of Klebsiella pneumoniae carbapenemases (KPCs) among Gram-negative bacteria is an important threat to global health. However, KPC-producing bacteria from environmental samples are rarely reported. This study aimed to elucidate the underlying resistance mechanisms of three carbapenem-resistant Aeromonas taiwanensis isolates recovered from river sediment samples. Pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) analysis indicated a close evolutionary relationship among A. taiwanensis isolates. S1-PFGE, Southern blot and conjugation assays confirmed the presence of blaKPC–2 and qnrS2 genes on a non-conjugative plasmid in these isolates. Plasmid analysis further showed that pKPC-1713 is an IncP-6 plasmid with a length of 53,205 bp, which can be transformed into DH5α strain and mediated carbapenems and quinolones resistance. The plasmid backbone of p1713-KPC demonstrated 99% sequence identity to that of IncP-6-type plasmid pKPC-cd17 from Aeromonas spp. and IncP-6-type plasmid: 1 from Citrobacter freundii at 74% coverage. A 14,808 bp insertion sequence was observed between merT gene and hypothetical protein in p1713-KPC, which include the quinolone resistance qnrS2 gene. Emergence of plasmid-borned blaKPC and qnrS2 genes from A. taiwanensis isolates highlights their possible dissemination into the environment. Therefore, potential detection of such plasmids from clinical isolates should be closely monitored.

ABSTRACT Antimicrobial resistance genes (ARGs) and the bacteria that harbor them are widely distributed in the environment, especially in surface water, sewage treatment plant effluent, soil, and animal waste. In this study, we isolated a KPC-2-producing Klebsiella pneumoniae strain (GSU10-3) from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP) and determined its complete genome sequence. Strain GSU10-3 is resistant to most β-lactam antibiotics and other antimicrobial agents (quinolones and aminoglycosides). This strain is classified as sequence type 11 (ST11), and a core genome phylogenetic analysis indicated that strain GSU10-3 is closely related to KPC-2-positive Chinese clinical isolates from 2011 to 2017 and is clearly distinct from strains isolated from the European Union (EU), United States, and other Asian countries. Strain GSU10-3 harbors four plasmids, including a blaKPC-2-positive plasmid, pGSU10-3-3 (66.2 kb), which is smaller than other blaKPC-2-positive plasmids and notably carries dual replicons (IncFII [pHN7A8] and IncN). Such downsizing and the presence of dual replicons may promote its maintenance and stable replication, contributing to its broad host range with low fitness costs. A second plasmid, pGSU10-3-1 (159.0 kb), an IncA/C2 replicon, carries a class 1 integron (containing intI1, dfrA12, aadA2, qacEΔ1, and sul1) with a high degree of similarity to a broad-host-range plasmid present in the family Enterobacteriaceae. The plasmid pGSU10-3-2 (134.8 kb), an IncFII(K) replicon, carries the IS26-mediated ARGs [aac(6')Ib-cr , blaOXA-1, catB4 (truncated), and aac(3)-IId], tet(A), and a copper/arsenate resistance locus. GSU10-3 is the first nonclinical KPC-2-producing environmental Enterobacteriaceae isolate from Japan for which the whole genome has been sequenced. IMPORTANCE We isolated and determined the complete genome sequence of a KPC-2-producing K. pneumoniae strain from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP). In Japan, the KPC type has been very rarely detected, while IMP is the most predominant type of carbapenemase in clinical carbapenemase-producing Enterobacteriaceae (CPE) isolates. Although laboratory testing thus far suggested that Japan may be virtually free of KPC-producing Enterobacteriaceae, we have detected it from effluent from a WWTP. Antimicrobial resistance (AMR) monitoring of WWTP effluent may contribute to the early detection of future AMR bacterial dissemination in clinical settings and communities; indeed, it will help illuminate the whole picture in which environmental contamination through WWTP effluent plays a part.

Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniae in clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/µl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene blaKPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain blaKPC-2 and blaNDM-1 genes simultaneously in the isolate. Our data showed the high prevalence of blaKPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis.