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.

Opposite evolution of pathogenicity driven by in vivo wzc and wcaJ mutations in ST11-KL64 carbapenem-resistant Klebsiella pneumoniae.

AIMS: To investigate the in vivo evolution of the mucoid-phenotype of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from the same patients and gain insights into diverse evolution and biology of these strains. METHODS: Whole genome sequencing and bioinformatic analysis were used to determine the mutation involved in the mucoid phenotype of ST11-KL64 CRKP. Gene knockout, bacterial morphology and capsular polysaccharides (CPS) extraction were used to verify the role of wzc and wcaJ in the mucoid phenotypes. Antimicrobial susceptibility, growth assay, biofilm formation, host cell adhesion and virulence assay were used to investigate the pleiotropic role of CPS changes in ST11-KL64 CRKP strains. RESULTS: Mutation of wzc S682N led to hypermucoid phenotype, which had negative impact on bacterial fitness and resulted in reduced biofilm formation and epithelial cell adhesion; while enhanced resistance to macrophage phagocytosis and virulence. Mutations of wcaJ gene led to non-mucoid phenotype with increased biofilm formation and epithelial cell adhesion, but reduced resistance of macrophage phagocytosis and virulence. Using virulence gene knockout, we demonstrated that CPS, rather than the pLVPK-like virulence plasmid, has a greater effect on mucoid phenotypic changes. CPS could be used as a surrogate marker of virulence in ST11-KL64 CRKP strains. CONCLUSIONS: ST11-KL64 CRKP strains sacrifice certain advantages to develop pathogenicity by changing CPS with two opposite in vivo evolution strategies.

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.

Genetic diversity of siderophores and hypermucoviscosity phenotype in Klebsiella pneumoniae.

Siderophore is a group of low molecular weight compounds with high affinity for ferric iron, facilitating bacterial iron consumption, especially when competing with the host. Genetic diversity of the siderophore-encoding loci largely contributes to the intricacy of siderophore production and regulation in K. pneumoniae. We analyzed the genetic diversity of three acquired siderophores (salmochelin, yersiniabactin and aerobactin) in 272 K. pneumoniae strains from China, and revealed that hypervirulent and multidrug-resistant lineages had distinguished predominant siderophore loci. Strains producing more types of siderophores tend to present higher mucoviscosity levels, suggesting that siderophore production might synergistically interact with hypermucoviscosity phenotype.

Core Genome Allelic Profiles of Clinical Klebsiella pneumoniae Strains Using a Random Forest Algorithm Based on Multilocus Sequence Typing Scheme for Hypervirulence Analysis.

BACKGROUND: Hypervirulent Klebsiella pneumoniae (hvKP) infections can have high morbidity and mortality rates owing to their invasiveness and virulence. However, there are no effective tools or biomarkers to discriminate between hvKP and nonhypervirulent K. pneumoniae (nhvKP) strains. We aimed to use a random forest algorithm to predict hvKP based on core-genome data. METHODS: In total, 272 K. pneumoniae strains were collected from 20 tertiary hospitals in China and divided into hvKP and nhvKP groups according to clinical criteria. Clinical data comparisons, whole-genome sequencing, virulence profile analysis, and core genome multilocus sequence typing (cgMLST) were performed. We then established a random forest predictive model based on the cgMLST scheme to prospectively identify hvKP. The random forest is an ensemble learning method that generates multiple decision trees during the training process and each decision tree will output its own prediction results corresponding to the input. The predictive ability of the model was assessed by means of area under the receiver operating characteristic curve. RESULTS: Patients in the hvKP group were younger than those in the nhvKP group (median age, 58.0 and 68.0 years, respectively; P < .001). More patients in the hvKP group had underlying diabetes mellitus (43.1% vs 20.1%; P < .001). Clinically, carbapenem-resistant K. pneumoniae was less common in the hvKP group (4.1% vs 63.8%; P < .001), whereas the K1/K2 serotype, sequence type (ST) 23, and positive string tests were significantly higher in the hvKP group. A cgMLST-based minimal spanning tree revealed that hvKP strains were scattered sporadically within nhvKP clusters. ST23 showed greater genome diversification than did ST11, according to cgMLST-based allelic differences. Primary virulence factors (rmpA, iucA, positive string test result, and the presence of virulence plasmid pLVPK) were poor predictors of the hypervirulence phenotype. The random forest model based on the core genome allelic profile presented excellent predictive power, both in the training and validating sets (area under receiver operating characteristic curve, 0.987 and 0.999 in the training and validating sets, respectively). CONCLUSIONS: A random forest algorithm predictive model based on the core genome allelic profiles of K. pneumoniae was accurate to identify the hypervirulent isolates.

Prevalence and characteristics of pks gene cluster harbouring Klebsiella pneumoniae from bloodstream infection in China.

Bloodstream infection (BSI), caused by Klebsiella pneumoniae, is associated with high morbidity and mortality, where the pks gene cluster plays a major role in their occurrence and prevalence. Information on the prevalence and characteristics of this gene cluster in K. pneumoniae is currently limited in mainland China. We therefore undertook a multicentre longitudinal study which revealed the prevalence, overall, community-onset and hospital-acquired BSI to be 20.5%, 28.3% and 13.0%, respectively. Compared to pks-negative, pks-positive isolates were significantly more susceptible to antimicrobial agents with a low incidence (5.1%) of multidrug-resistance and with infrequent extended-spectrum beta-lactamase (ESBL) production. Among pks-positive isolates, ST23 (78/117) and ST65 (20/117) were the dominant sequence types, and the majority harboured virulence genes. Community-onset BSI patients infected with pks-positive isolates had a higher proportion of liver abscesses and a lower proportion of biliary obstructions (P < 0.05). The pks-positive isolates were mostly sporadic in the phylogenetic tree, with a 65.8 and 47.0 average allele difference between Clade 1 and Clade 2, respectively. We concluded that although pks-positive K. pneumoniae were generally susceptible to antimicrobials, the high prevalence of such isolates in community cases and the genotoxicity, merits further investigation.

The Characterization of OXA-232 Carbapenemase-Producing ST437 Klebsiella pneumoniae in China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) was epidemic around the world and become a global threat to public health. The most important carbapenem-resistant mechanism is producing carbapenemases, especially Klebsiella pneumoniae carbapenemase (KPC), which is prevalent in the international clonal complex CC11. The high-risk multidrug-resistant CC11 is widespread worldwide, and KPC-producing and (New Delhi metallo) NDM-producing strains had been reported in this clonal complex before; moreover, cases with the CC11 strain faced more severe forms of drug resistance and treatment challenges than other clonal complexes. In this study, we identified an OXA-232-producing ST437 Klebsiella pneumoniae isolate in China, which belonged to CC11. The isolate was resistant to ß-lactams, aminoglycosides, and fluoroquinolones but susceptible to fosfomycin, tigecycline, and colistin. The bla OXA-232 gene was located on a 6141 bp ColKP3-type nonconjugative plasmid, and the plasmid was transformed by chemical transformation successfully. This is the first report of OXA-232-producing ST437 K. pneumoniae in China, a new clone of high-risk multidrug-resistant CC11.

Diversity of virulence level phenotype of hypervirulent Klebsiella pneumoniae from different sequence type lineage.

BACKGROUND: Hypervirulent Klebsiella pneumoniae (hvKP) is emerging around the Asian-Pacific region and it is the major cause of the community-acquired pyogenic liver abscesses. Multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKP) isolates were reported in France, China and Taiwan. However, the international-ally agreed definition for hvKP and the virulence level of hvKP are not clear. RESULTS: In this study, 56 hvKP isolates were collected from March 2008 to June 2012 and investigated by string test, capsule serotyping, multilocus sequence typing (MLST), virulence gene detection and serum resistance assay. Among the 56 K. pneumoniae isolates, 64.3% had the hypermucoviscosity phenotype, meanwhile, 64.3% were the K1 serotype and 19.6% were the K2 serotype. Within the K1 serotype, 94.4% were ST23, and within the K2 serotype, ST65, ST86 and ST375 accounted for the same percentage 27.3%. The serum resistance showed statistically normal distribution. According to the 50% lethal dose of Galleria. mellonella infection model, hvKP isolates were divided into high virulence level group and moderate virulence level group. The ability of each method evaluating the virulence level of hvKP was assessed using the area under the receiver operating characteristic curve. CONCLUSIONS: K1 ST23 K. pneumoniae was the most prevalent clone of the hvKP. However, K1 ST23 K. pneumoniae was the dominant clone in the moderate virulence level group. MLST was a relatively reliable evaluation method to discriminate the virulence level of hvKP in our study.

Prevalence and characteristics of ertapenem-mono-resistant isolates among carbapenem-resistant Enterobacterales in China.

Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the optimal therapy options remain uncertain. To investigate the prevalence and characteristics of ETP-mono-resistant CRE, CRE strains were systematically collected from 102 hospitals across China between 2018 and 2021. A 1:1 randomized matching study was conducted with ETP-mono-resistant strains to meropenem- and/or imipenem-resistant (MEM/IPM-resistant) strains. Antimicrobial susceptibility testing, whole-genome sequencing, carbapenem-hydrolysing activity and the expression of carbapenemase genes were determined. In total, 18.8% of CRE strains were ETP-mono-resistant, with relatively low ertapenem MIC values. ETP-mono-resistant strains exhibited enhanced susceptibility to ß-lactams, ß-lactam/ß-lactamase inhibitor combinations, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains (P < 0.05). Phylogenetic analysis revealed high genetic diversity among ETP-mono-resistant strains. Extended-spectrum ß-lactamases (ESBLs) and/or AmpC, as well as porin mutations, were identified as potential major mechanisms mediating ETP-mono-resistance, while the presence of carbapenemases was found to be the key factor distinguishing the carbapenem-resistant phenotypes between the two groups (P < 0.001). Compared with the MEM/IPM-resistant group, limited carbapenemase-producing CRE (CP-CRE) strains in the ETP-mono-resistant group showed a significantly lower prevalence of ESBLs and porin mutations, along with reduced expression of carbapenemase. Remarkably, spot assays combined with modified carbapenem inactivation method indicated that ETP-mono-resistant CP-CRE isolates grew at meropenem concentrations eightfold above their corresponding MIC values, accompanied by rapidly enhanced carbapenem-hydrolysing ability. These findings illustrate that ETP-mono-resistant CRE strains are relatively prevalent and that caution should be exercised when using meropenem alone for treatment. The detection of carbapenemase should be prioritized.

Assessment of microorganisms in drinking water disinfected by catalytic ozonation with fluorinated ceramic honeycomb and NaClO disinfectants under laboratory and pilot conditions.

While sodium hypochlorite (NaClO) has long been used to disinfect drinking water, concerns have risen over its use due to causing potentially hazardous byproducts. Catalytic ozonation with metal-free catalysts has attracted increasing attention to eliminate the risk of secondary pollution of byproducts in water treatment. Here, we compared the disinfection efficiency and microbial community of catalytic ozone with a type of metal-free catalyst fluorinated ceramic honeycomb (FCH) and NaClO disinfectants under laboratory- and pilot-scale conditions. Under laboratory conditions, the disinfection rate of catalytic ozonation was 3∼6-fold that of ozone when the concentration of Escherichia coli was 1 × 106 CFU/ml, and all E. coli were killed within 15 s. However, 0.65 mg/L NaClO retained E. coli after 30 min using the traditional culturable approach. The microorganism inactivation results of raw reservoir water disinfected by catalytic ozonation and ozonation within 15 s were incomparable based on the cultural method. In pilot-scale testing, catalytic ozonation inactivated all environmental bacteria within 4 min, while 0.65 mg/L NaClO could not achieve this success. Both catalytic ozonation and NaClO-disinfected methods significantly reduced the number of microorganisms but did not change the relative abundances of different species, i.e., bacteria, viruses, eukaryotes, and archaea, based on metagenomic analyses. The abundance of virulence factors (VFs) and antimicrobial resistance genes (ARGs) was detected few in catalytic ozonation, as determined by metagenomic sequencing. Some VFs or ARGs, such as virulence gene 'FAS-II' which was hosted by Mycobacterium_tuberculosis, were detected solely by the NaClO-disinfected method. The enriched genes and pathways of cataO3-disinfected methods exhibited an opposite trend, especially in human disease, compared with NaClO disinfection. These results indicated that the disinfection effect of catalytic ozone is superior to NaClO, this finding contributed to the large-scale application of catalytic ozonation with FCH in practical water treatment.

Risk factors and molecular epidemiology of intestinal colonization by carbapenem-resistant Gram-negative bacteria in patients with hematological diseases: a multicenter case‒control study.

Patients with hematological diseases are considered to be at high risk for intestinal colonization by carbapenem-resistant Gram-negative bacteria (CR-GNB). However, the epidemiological data regarding risk factors and molecular characteristics of intestinal colonized CR-GNB isolates in this population are insufficient in China. A multicenter case‒control study involving 4,641 adult patients with hematological diseases from 92 hospitals across China was conducted. Following culture of collected rectal swabs, mass spectrometry and antimicrobial susceptibility tests were performed to identify GNB species and CR phenotype. Risk factors were assessed through retrospective clinical information. Whole-genome sequencing was used to analyze the molecular characteristics of CR-GNB isolates. This trial is registered with ClinicalTrials.gov as NCT05002582. Our results demonstrated that among 4,641 adult patients, 10.8% had intestinal colonization by CR-GNB. Of these, 8.1% were colonized by carbapenem-resistant Enterobacterales (CRE), 2.6% were colonized by carbapenem-resistant Pseudomonas aeruginosa (CRPA), and 0.3% were colonized by carbapenem-resistant Acinetobacter baumannii (CRAB). The risk factors for CR-GNB colonization include male gender, acute leukemia, hematopoietic stem cell transplantation, ß-lactam antibiotic usage, and the presence of non-perianal infections within 1 week. Compared with CRPA-colonized patients, patients using carbapenems were more likely to be colonized with CRE. NDM was the predominant carbapenemase in colonized CRE. This study revealed a high CR-GNB intestinal colonization rate among adult patients with hematological diseases in China, with CRE being the predominant one. Notably, a significant proportion of CRE exhibited metallo-ß-lactamase production, indicating a concerning trend. These findings emphasize the importance of active screening for CR-GNB colonization in patients with hematological diseases.IMPORTANCECarbapenem-resistant Gram-negative bacteria (CR-GNB) has emerged as a significant threat to public health. Patients with hematological diseases are at high risk of CR-GNB infections due to their immunosuppressed state. CR-GNB colonization is an independent risk factor for subsequent infection. Understanding the risk factors and molecular characteristics of CR-GNB associated with intestinal colonization in patients with hematological diseases is crucial for empirical treatment, particularly in patients with febrile neutropenia. However, the epidemiology data are still insufficient, and our study aims to determine the intestinal colonization rate of CR-GNB, identify colonization risk factors, and analyze the molecular characteristics of colonized CR-GNB isolates.

Clinical and laboratory insights into the threat of hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKP) typically causes severe invasive infections affecting multiple sites in healthy individuals. In the past, hvKP was characterized by a hypermucoviscosity phenotype, susceptibility to antimicrobial agents, and its tendency to cause invasive infections in healthy individuals within the community. However, there has been an alarming increase in reports of multidrug-resistant hvKP, particularly carbapenem-resistant strains, causing nosocomial infections in critically ill or immunocompromised patients. This presents a significant challenge for clinical treatment. Early identification of hvKP is crucial for timely infection control. Notably, identifying hvKP has become confusing due to its prevalence in nosocomial settings and the limited predictive specificity of the hypermucoviscosity phenotype. Novel virulence predictors for hvKP have been discovered through animal models or machine learning algorithms, while standardization of identification criteria is still necessary. Timely source control and antibiotic therapy have been widely employed for the treatment of hvKP infections. Additionally, phage therapy is a promising alternative approach due to escalating antibiotic resistance. In summary, this narrative review highlights the latest research progress in the development, virulence factors, identification, epidemiology of hvKP, and treatment options available for hvKP infection.

Genome sequencing unveils blaKPC-2-harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae.

The threat posed by Klebsiella pneumoniae in healthcare settings has worsened due to the evolutionary advantages conferred by blaKPC-2-harboring plasmids (pKPC-2). However, the specific evolutionary pathway of nosocomial K. pneumoniae carrying pKPC-2 and its transmission between patients and healthcare environments are not yet well understood. Between 1 August and 31 December 2019, 237 ST11 KPC-2-producing-carbapenem-resistant K. pneumoniae (CRKP) (KPC-2-CRKP) were collected from patient or ward environments in an intensive care unit and subjected to Illumina sequencing, of which 32 strains were additionally selected for Nanopore sequencing to obtain complete plasmid sequences. Bioinformatics analysis, conjugation experiments, antimicrobial susceptibility tests, and virulence assays were performed to identify the evolutionary characteristics of pKPC-2. The pKPC-2 plasmids were divided into three subgroups with distinct evolutionary events, including Tn3-mediated plasmid homologous recombination, IS26-mediated horizontal gene transfer, and dynamic duplications of antibiotic resistance genes (ARGs). Surprisingly, the incidence rates of multicopy blaKPC-2, blaSHV-12, and blaCTX-M-65 were quite high (ranging from 27.43% to 67.01%), and strains negative for extended-spectrum ß-lactamase tended to develop multicopy blaKPC-2. Notably, the presence of multicopy blaSHV-12 reduced sensitivity to ceftazidime/avibactam (CZA), and the relative expression level of blaSHV-12 in the CZA-resistant group was 6.12 times higher than that in the sensitive group. Furthermore, a novel hybrid pKPC-2 was identified, presenting enhanced virulence levels and decreased susceptibility to CZA. This study emphasizes the notable prevalence of multicopy ARGs and provides a comprehensive insight into the intricate and diverse evolutionary pathways of resistant plasmids that disseminate among patients and healthcare environments.IMPORTANCEThis study is based on a CRKP screening program between patients and ward environments in an intensive care unit, describing the pKPC-2 (blaKPC-2-harboring plasmids) population structure and evolutionary characteristics in clinical settings. Long-read sequencing was performed in genetically closely related strains, enabling the high-resolution analysis of evolution pathway between or within pKPC-2 subgroups. We revealed the extremely high rates of multicopy antibiotic resistance genes (ARGs) in clinical settings and its effect on resistance profile toward novel ß-lactam/ß-lactamase inhibitor combinations, which belongs to the last line treatment choices toward CRKP infection. A novel hybrid pKPC-2 carrying CRKP with enhanced resistance and virulence level was captured during its clonal spread between patients and ward environment. These evidences highlight the threat of pKPC-2 to CRKP treatment and control. Thus, surveillance and timely disinfection in clinical settings should be practiced to prevent transmission of CRKP carrying threatful pKPC-2. And rational use of antibiotics should be called for to prevent inducing of pKPC-2 evolution, especially the multicopy ARGs.

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.

Investigating Possible Interspecies Communication of Plasmids Associated with Transfer of Third-Generation Cephalosporin, Quinolone, and Colistin Resistance Between Simultaneously Isolated Escherichia Coli and Klebsiella Pneumoniae.

The coinfection process producing multiple species of pathogens provides a specific ecological niche for the exchange of genetic materials between pathogens, in which plasmids play a vital role in horizontal gene transfer, especially for drug resistance, but the underlying transfer pathway remains unclear. Interspecies communication of the plasmids associated with the transfer of third-generation cephalosporins, quinolones, and colistin resistance has been observed in simultaneously isolated Escherichia coli and Klebsiella pneumoniae from abdominal drainage following surgery. The MICs of antimicrobial agents were determined by the broth microdilution method. The complete chromosome and plasmid sequences were obtained by combining Illumina paired-end short reads and MinION long reads. S1-PFGE, southern blot analysis and conjugation assay confirmed the transferability of the mcr-1-harboring plasmid. Both the E. coli isolate EC15255 and K. pneumoniae isolate KP15255 from the same specimen presented multidrug resistance. Each of them harbored one chromosome and three plasmids, and two plasmids and their mediated resistance could be transferred to the recipient by conjugation. Comparison of their genome sequences suggested that several genetic communication events occurred between species, especially among their plasmids, such as whole-plasmid transfer, insertion, deletion, amplification, or inversion. Exchange of plasmids or the genetic elements they harbor plays a critical role in antimicrobial resistance gene transmission and poses a substantial threat to nosocomial infection control, necessitating the continued surveillance of multidrug resistant pathogens, especially during coinfection. IMPORTANCE The genome sequence of bacterial pathogens commonly provides a detailed clue of genetic communication among clones or even distinct species. The intestinal microecological environment is a representative ecological niche for genetic communication. However, it is still difficult to describe the details of horizontal gene transfer or other genetic events within them because the evidence in the genome sequence is incomplete and limited. In this study, the simultaneously isolated Escherichia coli and Klebsiella pneumoniae from a coinfection process provided an excellent example for observation of interspecies communication between the two genomes and the plasmids they harbor. A complete genome sequence acquired by combining the Illumina and MinION sequencing platforms facilitated the understanding of genetic communication events, such as whole-plasmid transfer, insertion, deletion, amplification, or inversion, which contribute to antimicrobial resistance gene transmission and are a substantial threat to nosocomial infection control.

Prevalence and molecular characteristics of colistin-resistant isolates among clinically isolated carbapenem-resistant Klebsiella pneumoniae in China.

Colistin resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) poses health challenges. To investigate the prevalence and molecular characteristics of colistin-resistant CRKP, 708 isolates were collected consecutively from 28 tertiary hospitals in China from 2018 to 2019, and 14 colistin-resistant CRKP were identified. Two-component systems (TCSs) related to colistin resistance (PmrA/B, PhoP/Q, and CrrA/B), the negative regulator mgrB gene and mcr genes, were analysed using genomic sequencing. The relative expression of TCSs genes along with their downstream pmrC and pmrK genes was determined using quantitative real-time PCR (qRT‒PCR). A novel point mutation in PhoQ was confirmed by site-directed mutagenesis, and the subsequent transcriptome changes were analysed by RNA sequencing (RNA-Seq). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect modifications in lipid A. The results showed that only one isolate carried the mcr-8.1 gene, nine exhibited MgrB inactivation or absence, and three exhibited mutations in PmrB. One novel point mutation, L247P, in PhoQ was found to lead to a 64-fold increase in the minimum inhibitory concentration (MIC) of colistin. qRT‒PCR revealed overexpression of phoP/Q and pmrK in isolates with or without MgrB inactivation, and pmrB mutation resulted in overexpression of pmrA and pmrC. Furthermore, transcriptome analysis revealed that the PhoQ L247P novel point mutation caused upregulated expression of phoP/Q and its downstream operon pmrHFIJKLM. Meanwhile, the pmrA/B regulatory pathway did not evolve colistin resistance. Mass spectrometry analysis showed the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A in colistin-resistant isolates with absence of MgrB. These findings illustrate that the molecular mechanisms of colistin resistance in CRKP isolates are complex, and that MgrB inactivation or absence is the predominant molecular mechanism. Interventions should be initiated to monitor and control colistin resistance.

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.

Epidemiological Characteristics of OXA-232-Producing Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated during Nosocomial Clonal Spread Associated with Environmental Colonization.

Here, a program was designed to surveil the colonization and associated infection of OXA-232-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) (OXA-232-CRKP) in an intensive care unit (ICU) and to describe the epidemiological characteristics during surveillance. Samples were sourced from patient and environment colonization sites in the ICU from August to December 2019. During the surveillance, 106 OXA-232-CRKP strains were isolated from 8,656 samples of colonization sites, with an average positive rate of 1.22%. The rate from patient colonization sites was 3.59% (60/1,672 samples), over 5 times higher than that of the environment (0.66% [46/6,984 samples]). Rectal swabs and ventilator-related sites had the highest positive rates among patient and environment colonization sites, respectively. Six of the 15 patients who had OXA-232-CRKP at colonization sites suffered from OXA-232-CRKP-related infections. Patients could obtain OXA-232-CRKP from the environment, while long-term patient colonization was mostly accompanied by environmental colonization with subsequent infection. Antimicrobial susceptibility testing presented similar resistance profiles, in which all isolates were resistant to ertapenem but showed different levels of resistance to meropenem and imipenem. Whole-genome sequencing and single-nucleotide polymorphism (SNP) analysis suggested that all OXA-232-CRKP isolates belonged to the sequence type 15 (ST15) clone and were divided into two clades with 0 to 45 SNPs, sharing similar resistance genes, virulence genes, and plasmid types, indicating that the wide dissemination of OXA-232-CRKP between the environment and patients was due to clonal spread. The strains all contained ß-lactam resistance genes, including blaOXA-232, blaCTX-M-15, and blaSHV-106, and 75.21% additionally carried blaTEM-1. In brief, wide ST15 clonal spread and long-term colonization of OXA-232-CRKP between patients and the environment were observed, with microevolution and subsequent infection. IMPORTANCE OXA-232 is a variant of OXA-48 carbapenemase, which has been increasingly reported in nosocomial outbreaks in ICUs. However, the OXA-232-CRKP transmission relationship between the environment and patients in ICUs was still not clear. Our study demonstrated the long-term colonization of OXA-232-CRKP in the ICU environment, declared that the colonization was a potential risk to ICU patients, and revealed the possible threat that this OXA-232-CRKP clone would bring to public health. The wide dissemination of OXA-232-CRKP between the environment and patients was due to ST15 clonal spread, which presented a multidrug-resistant profile and carried disinfectant resistance genes and virulence clusters, posing a challenge to infection control. The study provided a basis for environmental disinfection, including revealing common environmental colonization sites of OXA-232-CRKP and suggesting appropriate usage of disinfectants to prevent the development of disinfectant resistance.

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.

Epidemiology, evolution and cryptic susceptibility of methicillin-resistant Staphylococcus aureus in China: a whole-genome-based survey.

OBJECTIVES: The aim of this study was to investigate the genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in China to identify predominant lineages and their associations with clinical data and antimicrobial resistance profiles. METHODS: We performed a national prevalence study of patients with S. aureus infections in 22 tertiary hospitals in China from 2015 to 2017. Clinical data from patients and the antimicrobial phenotypes were collected for each isolate. Genome sequencing was performed on a proportion of isolates and a phylogenetic analysis was undertaken. Genotypic and phenotypic ß-lactam susceptibilities were compared. RESULTS: A total of 1900 patients with S. aureus infections were included, of which 40% involved MRSA. Community-associated MRSA (CA-MRSA) infections were 24% of the total isolates. Genomic data showed that more than three-quarters of the MRSA were from three dominant lineages CC239 (25%, 116/471), CC5 (21%, 96/471) and CC59 (33%, 154/471) with CC59 accounting for more than half of the CA-MRSA isolates. Penicillin susceptibility genomic features were observed in 53% (251/470) of MRSA, including almost all of the CC59 (152/154) lineage, and 96% (242/251) of these isolates demonstrated in vitro susceptibility to penicillin or amoxicillin combined with clavulanic acid. Phylogenetic analysis indicated that the CC59 lineage can be divided into six lineages with all Asian CC59 isolates likely arising from an ancestral Mainland China lineage. CONCLUSIONS: This study showed a high prevalence of CA-MRSA in China, largely due to the widespread presence of CC59. As almost all isolates in this lineage possess genetic variants leading to increased ß-lactam susceptibility, we suggest that to improve antibiotic stewardship combinations of penicillins and ß-lactamase inhibitors should be included in the antibiotic susceptibility testing panels used to inform treatment decisions and research undertaken on this combination therapy.