Epidemiology of carbapenem-resistant Klebsiella pneumoniae in China and the evolving trends of predominant clone ST11: a multicentre, genome-based study.

OBJECTIVES: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major nosocomial infectious pathogen with rapidly increasing prevalence. The genomic epidemiological characteristics of CRKP nationwide, especially the evolving trends within the predominant clones, should be evaluated clearly. METHODS: We collected 3415 K. pneumoniae strains from 28 hospitals across China. Antimicrobial susceptibility testing and WGS were performed. Subsequent genomic analyses, including sequence typing, K-locus (KL) identification, antimicrobial resistance gene screening, and virulence score assessment were performed. The phylogenetic relationship of clonal group 11 was determined based on core-genome analysis, and the presence of the pLVPK-like virulence plasmid in ST11 isolates was confirmed using plasmid core-gene analysis. Additionally, the trends of the ST11 lineage with different KL types on a global scale were investigated using Beast2. RESULTS: Of the K. pneumoniae strains, 708 were identified as CRKP isolates (20.7%), of which 97.7% were MDR. ST11 was the predominant clone, and KPC-2 was the prevalent carbapenemase in China, although the prevalence of specific clones and carbapenemases varied by geographic region. Among ST11 isolates, KL47 and KL64 were the predominant KL types, and KL64 gradually replaced KL47, with a higher percentage of KL64 isolates harbouring the pLVPK-like plasmid. Global genome data showed a significant increase in the effective population size of KL64 over the last 5 years. CONCLUSIONS: The prevalence of CRKP was very high in certain regions in China. The increasing convergence of virulence and resistance, particularly in ST11-KL64 isolates, should be given more attention and further investigation.

The Effect of ß-Lactam Antibiotics on the Evolution of Ceftazidime/Avibactam and Cefiderocol Resistance in KPC-Producing Klebsiella pneumoniae.

In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during ß-lactam antibiotic therapy. Five KPC-Kp isolates were collected from a single patient. Whole-genome sequencing and a comparative genomics analysis were performed on the isolates and all blaKPC-2-containing plasmids to predict the population evolution process. Growth competition and experimental evolution assays were conducted to reconstruct the evolutionary trajectory of the KPC-Kp population in vitro. Five KPC-Kp isolates (KPJCL-1 to KPJCL-5) were highly homologous, and all harbor an IncFII blaKPC-containing plasmid (pJCL-1 to pJCL-5). Although the genetic structures of these plasmids were almost identical, distinct copy numbers of the blaKPC-2 gene were detected. A single copy of blaKPC-2 was presented in pJCL-1, pJCL-2, and pJCL-5, two copies of blaKPC (blaKPC-2 and blaKPC-33) were presented in pJCL-3, and three copies of blaKPC-2 were presented in pJCL-4. The blaKPC-33-harboring KPJCL-3 isolate presented resistance to ceftazidime-avibactam and cefiderocol. The blaKPC-2 multicopy strain KPJCL-4 had an elevated ceftazidime-avibactam MIC. The patient had been exposed to ceftazidime, meropenem, and moxalactam, after which KPJCL-3 and KPJCL-4 were isolated, which both showed a significant competitive advantage under antimicrobial pressure in vitro. Experimental evolution assays revealed that blaKPC-2 multicopy-containing cells were increased in the original single-copy blaKPC-2-harboring KPJCL-2 population under selection with ceftazidime, meropenem, or moxalactam, generating a low-level ceftazidime-avibactam resistance phenotype. Moreover, blaKPC-2 mutants with a G532T substitution, G820 to C825 duplication, G532A substitution, G721 to G726 deletion, and A802 to C816 duplication increased in the blaKPC-2 multicopy-containing KPJCL-4 population, generating high-level ceftazidime-avibactam resistance and reduced cefiderocol susceptibility. Ceftazidime-avibactam and cefiderocol resistance can be selected by ß-lactam antibiotics other than ceftazidime-avibactam. Notably, blaKPC-2 gene amplification and mutation are important in KPC-Kp evolution under antibiotic selection.

Detection of an Escherichia coli Sequence Type 167 Strain with Two Tandem Copies of blaNDM-1 in the Chromosome.

New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Enterobacteriaceae has disseminated rapidly throughout the world and poses an urgent threat to public health. Previous studies confirmed that the blaNDM-1 gene is typically carried in plasmids but rarely in chromosome. We discovered a multidrug-resistant Escherichia coli strain Y5, originating from a urine sample and containing the blaNDM-1 gene, which did not transfer by either conjugation or electrotransformation. We confirmed the possibility of its chromosome location by S1-pulsed-field gel electrophoresis (PFGE) and XbaI-PFGE, followed by Southern blotting. To determine the genomic background of blaNDM-1, the genome of Y5 was completely sequenced and compared to other reference genomes. The results of our study revealed that this isolate consists of a 4.8-Mbp chromosome and three plasmids, it is an epidemic clone of sequence type (ST) 167, and it shows 99% identity with Escherichia coli 6409 (GenBank accession no. CP010371), which lacks the same blaNDM-1 gene-surrounding structure as Y5. The blaNDM-1 gene is embedded in the chromosome along with two tandem copies of an insertion sequence common region 1 (ISCR1) element (sul1-ARR-3-cat-blaNDM-1-bleo-ISCR1), which appears intact in the plasmid from Proteus mirabilis (GenBank accession no. KP662515). The genomic context indicates that the ISCR1 element mediated the blaNDM-1 transposition from a single source plasmid to the chromosome. Our study is the first report of an Enterobacteriaceae strain harboring a chromosomally integrated blaNDM-1, which directly reveals the vertical spreading pattern of the gene. Close surveillance is urgently needed to monitor the emergence and potential spread of ST167 strains that harbor blaNDM-1.

High prevalence of ESBL-producing Escherichia coli and Klebsiella pneumoniae in community-onset bloodstream infections in China.

OBJECTIVES: Community-onset bloodstream infections (COBSIs) caused by ESBL-producing Escherichia coli (ESBL-EC) and ESBL-producing Klebsiella pneumoniae (ESBL-KP) are increasing globally. This study aimed to investigate the epidemiology and risk factors of ESBL-EC and ESBL-KP in COBSIs in China. METHODS: A prospective, multicentre study was performed in 28 tertiary hospitals from September 2013 to November 2014. All isolates and ESBLs were microbiologically characterized. A statistical analysis of risk factors was performed using binary logistic regression. The trial was registered with ClinicalTrials.gov (NCT01961206). RESULTS: A total of 919 consecutive episodes of COBSIs were reported and 640 E. coli and 279 K. pneumoniae isolates (non-duplicate) were collected. According to the criteria, 662 (72.0%) cases were classified as having community-acquired bloodstream infections, while the remaining 257 (28.0%) were classified as having healthcare-associated bloodstream infections. The proportions of ESBL producers were 55.5% (355/640) among E. coli isolates and 16.5% (46/279) among K. pneumoniae isolates, respectively. Healthcare-associated infections, obstructive urinary tract disease, previous surgical history and use of a cephalosporin antibiotic within 3 months were independent predictors of COBSIs caused by ESBL-EC. Heart failure was the only independent risk factor for COBSIs due to ESBL-KP. Age was not independently associated with infections caused by ESBL producers. CTX-M-14 was the most common ESBL genotype and was widespread throughout the country. CONCLUSIONS: ESBL producers are highly prevalent in COBSIs in China, especially among cases caused by E. coli. For these resistant pathogens, clinicians should consider adequate empirical therapy, and different risk factors for prediction should be used in this country.

The correlation between intestinal colonization and infection of carbapenem-resistant Klebsiella pneumoniae: a systemactic review.

As a widely spread Gram-negative bacteria, klebsiella pneumoniae mainly causes acquired infections in hospitals, such as lung infections, urinary tract infections, bloodstream infections, etc. In recent years, the number of multidrug-resistant K. pneumoniae strains has increased dramatically, posing a great threat to human health. Carbapenem-resistant Klebsiella pneumoniae (CRKP) can be colonized in human body, especially in gastrointestinal tract, and some colonized patients can be infected during hospitalization, among which invasive operation, underlying disease, admission to intensive care unit, antibiotic use, severity of the primary disease, advanced age, operation, coma and renal failure are common risk factors for secondary infection. Active screening and preventive measures can effectively prevent the occurrence of CRKP infection. Based on the epidemiological status, this study aims to discuss the correlation between colonization and secondary infection induced by carbapenem-resistant Klebsiella pneumoniae and risk factors for their happening, and provide some reference for nosocomial infection prevention and control.

Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaIMP-4 induce resistance transmission and fitness variation.

To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of blaIMP-4 and blaNDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-ß-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that blaIMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of blaIMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that blaIMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, blaIMP-4 in different locations did not affect the gene stability. The blaNDM-1-harbouring plasmid contributed to the increased resistance to ß-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.

Tigecycline treatment of infection caused by KPC-producing Escherichia coli in a pediatric patient.

Tigecycline shows great antimicrobial activity against both Gram-positive and Gram-negative bacteria, and has been considered to be an appropriate choice in controlling infection caused by multi-drug resistant (MDR) pathogens, such as carbapenemase-producing Enterobacteriaceae (CPE). Although many clinical trials evaluate the efficacy and safety of tigecycline on adults, rare reports recommend tigecycline to treat pediatric patient. In this study, we presented a clinical case with tigecycline as an anti-infectious agent on a 14-year-old child who was suffering from infection of intraperitoneal abscess caused by Klebsiella pneumoniae carbapenemases (KPC)-producing Escherichia coli with extreme drug resistant profile. By accessing the clinical outcome and efficacy of the patient, and the side effects of tigecycline, our research explored the documented experience of tigecycline on controlling infection caused by CPE isolate in children.

Development of a Novel Typing Scheme Based on the Genetic Diversity of Heme/Hemin Uptake System Hmu in Klebsiella pneumoniae Species Complex.

Iron is essential for the survival and reproduction of Klebsiella pneumoniae. Although K. pneumoniae employs multiple types of siderophores to scavenge iron during infections, the majority of host iron is retained within erythrocytes and carried by hemoglobin that is inaccessible to siderophores. HmuRSTUV is a bacterial hemin/hemoprotein uptake system. However, the genetic background and function of HmuRSTUV in K. pneumoniae remain unknown. We collected 2,242 K. pneumoniae genomes, of which 2,218 (98.9%) had complete hmuRSTUV loci. Based on the 2,218 complete hmuRSTUV sequences, we established a novel typing scheme of K. pneumoniae named hmST, and 446 nonrepetitive hmSTs were identified. In hypervirulent lineages, hmST was diversely distributed and hmST1 mainly existed in ST23 strains. In contrast, hmST was less diversely distributed among multidrug-resistant strains. hmST demonstrated greater genetic diversity in hypervirulent lineages and community-acquired and bloodstream-sourced strains. In vitro and in vivo experiments revealed that an intact hmuRSTUV was essential for hemin uptake, playing an important role in bloodstream infections. This study established a novel typing scheme of hmST based on hmuRSTUV providing new insights into identifying and monitoring the emergence of novel virulence evolution in K. pneumoniae. IMPORTANCE Siderophore is a group of low molecular weight compounds with high affinity for ferric iron, which could facilitate bacterial iron consumption. Similarly, hemin/heme scavenged by the hemin uptake system HmuRSTUV usually act as another critical iron source for K. pneumoniae. This study proved that Hmu system significantly promoted the growth of K. pneumoniae in the presence of hemin and played an important role in bloodstream infections. A novel typing scheme named hmST was established, and the genetic diversity of hmuRSTUV loci was analyzed based on a large number of genomes. This study provides new insights into identifying and monitoring the emergence of novel virulence evolution in K. pneumoniae.

A Novel CMY Variant Confers Transferable High-Level Resistance to Ceftazidime-Avibactam in Multidrug-Resistant Escherichia coli.

Here, our objective was to explore the molecular mechanism underlying ceftazidime-avibactam resistance in a novel CMY-178 variant produced by the clinical Escherichia coli strain AR13438. The antibiotic susceptibility of the clinical isolate, its transconjugants, and its transformants harboring transferable blaCMY were determined by the agar dilution method. S1-PFGE, cloning experiments, and whole-genome sequencing (WGS) were performed to investigate the molecular characteristics of ceftazidime-avibactam resistance genes. Kinetic parameters were compared among the purified CMY variants. Structural modeling and molecular docking were performed to assess the affinity between the CMYs and drugs. The horizontal transferability of the plasmid was evaluated by a conjugation experiment. The fitness cost of the plasmid was analyzed by determining the maximal growth rate, the maximum optical density at 600 nm (OD600), and the duration of the lag phase. AR13438, a sequence type 457 E. coli strain, was resistant to multiple cephalosporins, piperacillin-tazobactam, and ceftazidime-avibactam at high levels and was susceptible to carbapenems. WGS and cloning experiments indicated that a novel CMY gene, blaCMY-178, was responsible for ceftazidime-avibactam resistance. Compared with the closely related CMY-172, CMY-178 had a nonsynonymous amino acid substitution at position 70 (Asn70Thr). CMY-178 increased the MICs of multiple cephalosporins and ceftazidime-avibactam compared with CMY-172. The kinetic constant Ki values of CMY-172 and CMY-178 against tazobactam were 2.12 ± 0.34 and 2.49 ± 0.51 µM, respectively. Structural modeling and molecular docking indicated a narrowing of the CMY-178 ligand-binding pocket and its entrance and a stronger positive charge at the pocket entrance compared with those observed with CMY-172. blaCMY-178 was located in a 96.9-kb IncI1-type plasmid, designated pAR13438_2, which exhibited high transfer frequency without a significant fitness cost. In conclusion, CMY-178 is a novel CMY variant that mediates high-level resistance to ceftazidime-avibactam by enhancing the ability to hydrolyze ceftazidime and reducing the affinity for avibactam. Notably, blaCMY-178 could be transferred horizontally at high frequency without fitness costs. IMPORTANCE Ceftazidime-avibactam is a novel ß-lactam-ß-lactamase inhibitor (BLBLI) combination with powerful activity against Enterobacterales isolates producing AmpC, such as CMY-like cephalosporinase. However, in recent years, CMY variants have been reported to confer ceftazidime-avibactam resistance. We reported a novel CMY variant, CMY-178, that confers high-level ceftazidime-avibactam resistance with potent transferability. Therefore, this resistance gene is a tremendous potential menace to public health and needs attention of clinicians.

Epidemiological characteristics and genetic structure of blaNDM-1 in non-baumannii Acinetobacter spp. in China.

OBJECTIVES: The goal of this study was to investigate the epidemiological characteristics and the surrounding genetic structure of bla(NDM-1) in non-baumannii Acinetobacter spp. in China. METHODS: Non-baumannii Acinetobacter spp. were collected from 28 provinces in China and were screened for the presence of bla(NDM-1) using PCR. The following four methods were used to classify the Acinetobacter isolates: the Vitek 2 system, 16S-23S rRNA gene intergenic spacer sequencing, amplified rDNA restriction analysis and partial rpoB sequence analysis. An S1-PFGE assay and Southern blot hybridization were performed to determine the plasmid location of bla(NDM-1). The transferability of bla(NDM-1)-harbouring plasmids was confirmed by conjugation experiments and electrotransformation. The surrounding genetic structure of the bla(NDM-1) gene was analysed using a restriction endonuclease-based cloning approach and primer walking. RESULTS: Among 726 non-baumannii Acinetobacter spp., nine isolates collected from six different provinces and assigned to seven different Acinetobacter spp. contained the bla(NDM-1) gene. None of these isolates was directly infectious to the patients or demonstrated an epidemiological importation from abroad. These bla(NDM-1) genes were located on plasmids that could be transferred to Escherichia coli J53 by conjugation and Acinetobacter baylyi ADP1 by electrotransformation. Seven of the nine strains shared a common genetic structure in which bla(NDM-1) was flanked by two copies of ISAba125. CONCLUSIONS: The clinical challenge posed by bla(NDM-1) is currently minimal in China; however, more attention should be devoted to monitoring the dissemination of this gene due to its potential transferability via the ISAba125-associated transposon.

The Value of Neutrophil-To-Lymphocyte Ratio for Evaluating Blood Stream Infection Caused by Carbapenem-Resistant Klebsiella pneumoniae: A Retrospective Cohort Study.

Background: The neutrophil-to-lymphocyte ratio (NLR) is a useful marker of inflammation. However, the prognostic function of the NLR in patients with carbapenem-resistant Klebsiella pneumoniae (CRKP) blood stream infection (BSI) remains largely unknown. The aim of this study was to explore the potential relationship between the NLR and mortality in these patients. Methods: We performed a retrospective cohort study based on data retrieved from the computerized patient record system in a tertiary hospital from 1 January 2017 to 31 October, 2020. A total of 134 inpatients with CRKP BSI were enrolled in this study, including 54 fatal cases and 80 survival cases, 28 days after the onset of CRKP BSI. A logistic analysis was performed to assess the association between the NLR on the 4th day and 28-day mortality. Multivariate analyses were used to control for the confounders. Results: The overall 28-day mortality rate of patients with a CRKP BSI episode was 40.3% (54/134). We conducted a multivariate analysis of the data of 134 patients and found that the NLR on the 4th day [odds ratio (OR) 1.148, 95% confidence interval (CI) 1.076-1.225, p < 0.001] and antibiotic exposure before BSI onset (OR 3.847, 95% CI 1.322-11.196, p = 0.013) were independent risk factors for 28-day mortality of patients with CRKP BSI, while appropriate initial therapy (AIT, OR 0.073, 95% CI 0.017-0.307, p < 0.001) was an independent protective factor. Among patients treated with AITs, the Cox proportional hazards regression analysis revealed a significant difference in prognosis (p = 0.006) between the ceftazidime/avibactam contained (CAZ) group and non CAZ-AVI groups. After dividing the non CAZ-AVI group into the tigecycline (TGC), colistin (COL), and TGC + COL groups, there were no differences between the CAZ-AVI group and the TGC group (p = 0.093), but CAZ-AVI group showed lower 28-day mortality than COL (p = 0.002) and TGC + COL (p = 0.002) groups. Meanwhile, there was no difference in NLR on the 1st day (p = 0.958) of patients in different groups but significant difference in NLR on the 4th day (p = 0.047). Conclusions: The NLR on the 4th day is a readily available and independent prognostic biomarker for patients with CRKP BSI. This marker may have the potential for use in evaluating the efficacy of different anti-infection therapy strategies at an early stage.

Phenotypic and Genotypic Characterization of a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae ST17-KL38 Clinical Isolate Harboring the Carbapenemase IMP-4.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is a threat to global public health. We characterized a sequence type 17 (ST17) K. pneumoniae clinical isolate that was resistant to carbapenems and belonged to serotype KL38/O2. Its complete genome is comprised of a 5.1-Mb chromosome and two conjugative plasmids. The 52,578-bp N-type plasmid pXH210-IMP contains the blaIMP-4 carbapenemase gene and the quinolone resistance gene qnrS1. The 272,742-bp FII(K)-9:FIB(K)-10 plasmid pXH210-AMV carries an array of genes that confer resistance to aminoglycosides, chloramphenicol, quinolones, tetracycline, sulfonamides, trimethoprim, arsenic, copper, and silver. However, the XH210 genome otherwise lacks the genes that are considered characteristic markers of hypervirulence in K. pneumoniae. The virulence potential of XH210 was assessed using a random forest algorithm predictive model, as well as Galleria mellonella and mouse infection models. The results of these were concordant and suggested that XH210 is hypervirulent and therefore a CR-hvKP strain. This worrying convergence of virulence and clinically significant antibiotic resistance is particularly concerning given the absence of typical hypervirulence markers. Further investigations are required to understand the virulence mechanisms of XH210 and to improve the diagnostics of hypervirulent K. pneumoniae. IMPORTANCE The combination of drug resistance and hypervirulence significantly limits the available treatment options for life-threatening infections caused by multidrug-resistant hvKP, especially CR-hvKP. To date, research on IMP-producing CR-hvKP is extremely scarce, and the virulence mechanisms of CR-hvKP are far more complicated and diverse than has been described in the literature so far. In this study, we characterized the tigecycline-resistant and IMP-4 carbapenemase-producing ST17 K. pneumoniae isolate XH210 from a human blood sample. Importantly, XH210 exhibits hypervirulence but does not possess traits that are frequently associated with the phenotype, highlighting the urgent need to improve identification of potentially hypervirulent isolates and enhance active surveillance of CR-hvKP strains to prevent their dissemination.

High prevalence of colistin resistance and mcr-9/10 genes in Enterobacter spp. in a tertiary hospital over a decade.

Enterobacter spp. are members of the 'ESKAPE' group of pathogens, which which are recognised as the leading cause of multidrug-resistant (MDR) hospital-acquired infections. Colistin is usually regarded as a last-line therapeutic option for MDR Gram-negative bacilli infections. However, colistin-resistant Enterobacter spp. have emerged in the last decade. Here we investigated the prevalence of colistin resistance and mcr genes in Enterobacter spp. of clinical origin between 2011 and 2020 in a tertiary hospital in China. Colistin resistance rates ranged between 17.1% and 34.5%, with an overall prevalence of 22.2% (190/854). No mcr-1 to mcr-8 genes were identified in the colistin-resistant Enterobacter spp. isolates, while mcr-9 and mcr-10 were detected at rates of 8.4% (16/190) and 12.6% (24/190), respectively. All of the mcr-9/10-positive Enterobacter isolates belonged to the Enterobacter cloacae complex (ECC). Meanwhile, 14.8% (98/664) and 6.0% (40/664) of non-colistin-resistant Enterobacter spp. isolates carried mcr-9 and mcr-10 genes, respectively. For the 40 mcr-9/10-positive colistin-resistant ECC isolates, mcr-9-positive ECC isolates usually co-produced extended-spectrum ß-lactamases (ESBLs) or carbapenemases, while mcr-10-positive ECC isolates produced neither. Most mcr-9/10 genes were located on plasmids. The backbone of mcr-9-harbouring plasmids was conserved, while that of mcr-10-harbouring plasmids was diverse. Our findings revealed a high prevalence of colistin resistance and a silent distribution of mcr-9/10 genes in clinical Enterobacter spp. isolates in China. It is urgent to take steps and interventions to control the prevalence of colistin resistance and prevent the dissemination of mcr-9/10 genes.

The Genetic Characteristics and Carbapenem Resistance Mechanism of ST307 Klebsiella pneumoniae Coharbouring blaCMY-6, blaOXA-48, and a Truncated blaNDM-1.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a common nosocomial pathogen causing severe infectious diseases, and ST307 CRKP is an emerging clone. In this study, we collected five ST307 CRKP isolates, evaluated their antimicrobial susceptibility using microbroth dilution, and their clonality and population structure by PFGE, cgMLST, and SNP-based phylogenetic analysis. Then, the genome characteristics, such as antimicrobial resistance genes and plasmid profiles, were studied by subsequent genomic analysis. The plasmid transfer ability was evaluated by conjugation, and the carbapenem resistance mechanism was elucidated by gene cloning. The results showed that all five ST307 CRKP isolates harboured blaCMY-6, blaOXA-48, and blaNDM-1; however, the end of the blaNDM-1 signal peptide was interrupted and truncated by an IS10 element, resulting in the deactivation of carbapenemase. The ST307 isolates were closely related, and belonged to the globally disseminated clade. blaOXA-48 and blaNDM-1 were located on the different mobilisable IncL/M- and IncA/C2-type plasmids, respectively, and either the pOXA-48 or pNDM-1 transconjugants were ertapenem resistant. Gene cloning showed that blaCMY-6 could elevate the MICs of carbapenems up to 64-fold and was located on the same plasmid as blaNDM-1. In summary, ST307 is a high-risk clone type, and its prevalence should be given additional attention.

Risk factors for infection and mortality caused by carbapenem-resistant Klebsiella pneumoniae: A large multicentre case-control and cohort study.

OBJECTIVES: To elucidate the predictors of carbapenem-resistant Klebsiella pneumoniae (CRKP) infection and help clinicians better identify CRKP infection at an early stage. METHODS: We conducted a multicentre case-control study of 422 patients with CRKP infection and 948 with carbapenem-susceptible K. pneumoniae (CSKP) infection from March to July 2017. Binary logistic regression was used to identify risk factors for CRKP infection. The subgroups of CRKP respiratory infection, intra-abdominal infection, and bloodstream infection were also evaluated. Patients were followed up for 28 days. Independent risk factors for 28-day crude mortality of CRKP infection were analysed using Cox proportional hazards regression models. RESULTS: Longer stay of hospitalization, stay in the intensive care unit (ICU), previous exposure to antibacterial agents (especially carbapenems, quinolones, aminoglycosides, and tigecycline), invasive procedures, intravascular catheter use, tracheotomy, and admission to ICU in the preceding 90 days were risk factors for CRKP infection. Carbapenem exposure was the only common predictor of different types of CRKP infection. The 28-day crude mortality of CRKP infection was 24.2% and was independently associated with sex, admitted unit, and type of infection. CONCLUSIONS: Strict policies for antibiotic use, cautious decisions regarding the implementation of invasive procedures, and careful management of patients with catheters, especially intravascular catheters, are necessary to handle CRKP infection.

Transmission of ST45 and ST2407 extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in neonatal intensive care units, associated with contaminated environments.

OBJECTIVES: Given the increasing frequency of infections due to extended-spectrum ß-lactamase (EBSL)-producing Klebsiella pneumoniae in humans over recent decades, infection control against this pathogen is of high importance. METHODS: In this study, the transmission mode of ESBL-producing K. pneumoniae in neonatal intensive care units (NICU) was investigated. We collected K. pneumoniae isolates from patients admitted to the NICU and performed environmental screening of the NICU and nearby obstetrics department. All isolates were analysed using antimicrobial susceptibility testing, whole-genome sequencing, molecular typing, and antimicrobial and virulence determinant screening. The phylogenetic relationships of all the isolates were analysed using core-genome multi-locus sequence type and single-nucleotide polymorphism-based analysis, and their plasmids harbouring antimicrobial resistance genes in ST2407 were compared. RESULTS: Eighteen K. pneumoniae isolates were collected, of which 10 isolates from patients belonged to ST45 and ST2407, and eight isolates from the environment belonged to various other clones. Although 80% and 100% of isolates from patients were ESBL-positive (blaCTX-M-14 and blaCTX-M-55) and possessed siderophores, respectively; fewer environmental isolates harboured antimicrobial resistance and virulence genes. For both ST45 and ST2407 isolates, the phylogenetic assessment revealed a close relationship between clinical and environmental isolates, indicating that bloodstream infections were associated with the contaminated environments. CONCLUSIONS: Based on these results, the environmental prevalence of K. pneumoniae should be considered given its pathogenicity in humans. Early and active infection control measures could decrease the spread of multidrug-resistant K. pneumoniae.

Pharmacokinetics/pharmacodynamics of polymyxin B in patients with bloodstream infection caused by carbapenem-resistant Klebsiella pneumoniae.

Introduction: Polymyxin B is a last-line therapy for carbapenem-resistant microorganisms. However, a lack of clinical pharmacokinetic/pharmacodynamic (PK/PD) data has substantially hindered dose optimization and breakpoint setting. Methods: A prospective, multi-center clinical trial was undertaken with polymyxin B [2.5 mg/kg loading dose (3-h infusion), 1.25 mg/kg/12 h maintenance dose (2-h infusion)] for treatment of carbapenem-resistant K. pneumoniae (CRKP) bloodstream infections (BSI). Safety, clinical and microbiological efficacy were evaluated. A validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was applied to determine the concentrations of polymyxin B in blood samples. Population pharmacokinetic (PK) modeling and Monte Carlo simulations were conducted to examine the susceptibility breakpoint for polymyxin B against BSI caused by CRKP. Results: Nine patients were enrolled and evaluated for safety. Neurotoxicity (5/9), nephrotoxicity (5/9), and hyperpigmentation (1/9) were recorded. Blood cultures were negative within 3 days of commencing therapy in all 8 patients evaluated for microbiological efficacy, and clinical cure or improvement occurred in 6 of 8 patients. Cmax and Cmin following the loading dose were 5.53 ± 1.80 and 1.62 ± 0.41 mg/L, respectively. With maintenance dosing, AUCss,24 h was 79.6 ± 25.0 mg h/L and Css,avg 3.35 ± 1.06 mg/L. Monte Carlo simulations indicated that a 1 mg/kg/12-hourly maintenance dose could achieve >90% probability of target attainment (PTA) for isolates with minimum inhibitory concentration (MIC) ≤1 mg/L. PTA dropped substantially for MICs ≥2 mg/L, even with a maximally recommended daily dose of 1.5 mg/kg/12-hourly. Conclusion: This is the first clinical PK/PD study evaluating polymyxin B for BSI. These results will assist to optimize polymyxin B therapy and establish its breakpoints for CRKP BSI.

Clinical carbapenem-resistant Acinetobacter baylyi strain coharboring blaSIM-1 and blaOXA-23 from China.

bla(SIM-1) and bla(OXA-23) were codetected in clinical carbapenem-resistant Acinetobacter baylyi strain NB09A30. Both of carbapenemase genes were located on a large plasmid (ca. 360 kb). bla(SIM-1) was found as a gene cassette inserted into a class 1 integron identical to that determined in Acinetobacter sp. isolates from South Korea. The genetic structure of bla(OXA-23) in NB09A30 was different from that in the prevalent Acinetobacter baumannii of clonal complex 92 (CC92) from the same hospital.

ST11, the dominant clone of KPC-producing Klebsiella pneumoniae in China.

OBJECTIVES: Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae has spread rapidly in China. In this study, we aimed to investigate the molecular epidemiology of KPC-producing K. pneumoniae isolates in China. METHODS: Ninety-five carbapenem-resistant K. pneumoniae isolates from 13 hospitals in nine cities covering five provinces in China were analysed. Antibiotic susceptibility was determined by the Etest. Multilocus sequence typing (MLST) and PFGE were used for epidemiological analysis. The genetic structure around bla(KPC) and the encoding genes of extended-spectrum ß-lactamases and plasmid-mediated AmpC enzymes were detected by PCR and sequencing. Plasmids were analysed by transformation, restriction and Southern blot. RESULTS: All isolates harboured the bla(KPC-2) gene. Seven sequence types (STs) were obtained. The dominant clone was ST11 (61/95), which was identified in isolates from Zhejiang province (four hospitals in Hangzhou and one hospital in Ningbo), Jiangsu province (one hospital in Nanjing) and Anhui province (one hospital in Hefei). Isolates with ST11 showed >80% similarity in PFGE patterns. Plasmids from 14 selected transformants, their original isolates representing different STs and/or regions, had a diversity of HindIII restriction maps. CONCLUSIONS: The dominant clone of KPC-producing K. pneumoniae in China is ST11, which is closely related to ST258, which has been reported worldwide.

In vitro Effect of the Combination of Aztreonam and Amoxicillin/Clavulanic Acid Against Carbapenem-Resistant Gram-Negative Organisms Producing Metallo-ß-Lactamase.

INTRODUCTION: Antibiotics for treating infectious diseases caused by carbapenem-resistant Gram-negative pathogens (CR-GNOs) are very limited in clinical practice. We aim to provide supportive evidence by revealing the combined effect of aztreonam (ATM) and amoxicillin/clavulanic acid (AMC) against GNOs with carbapenem resistance mediated by metallo-ß-lactamase (MBL). METHODS: All isolates were identified by the VITEK system and EDTA inhibitory assays. PCR followed by sequencing was conducted to confirm the genotypes of MBL and extended spectrum ß-lactamase (ESBL). Time kill assay was performed to clarify the bactericidal effect of drug combination. RESULTS: A total of 59 MBL-producing CR-GNOs (33 Enterobacteriaceae spp. isolates and 26 Pseudomonadales isolates) were identified and there found three MBL genes, namely, bla IMP, bla NDM and bla VIM, with ratios of 76.2%, 11.8% and 11.8%, respectively. The Enterobacteriaceae spp. isolates were commonly positive for the ESBL genes, including bla TEM (18 isolates), bla SHV (20 isolates) and bla CTX-M-1 (8 isolates), while the P. aeruginosa isolates were positive for bla OXA-10 (11 isolates). The checkerboard microdilution assay was used to detect combination effect of ATM and AMC, which showed synergy (97.0%) and partial synergy (3.0%) in Enterobacteriaceae spp. isolates, and partial synergy (42.3%) and indifference (34.6%) in the Pseudomonadales isolates. Four Enterobacteriaceae spp. isolates were selected for a time-kill assay, and rapid bactericidal effects were observed in the combination groups compared to the control and mono-ATM groups; these effects began in the first hour and continued to the sixth hour, yielding a 5- to 7-fold reduction in Log10 CFU/mL. DISCUSSION: The combination of ATM and AMC would be an available option to control infections caused by MBL-producing CR-GNOs, especially Enterobacteriaceae spp. isolates that coproduce ESBLs, and exhibit significant synergic effects in vitro.