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Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a great public health problem and is associated with many disease outbreaks and high mortality rates. Alarmingly, K. pneumoniae has been isolated from food in several recent studies. This study aimed to investigate the prevalence and characteristics of CRKP in food samples from Egypt. A total of 311 food samples (including 116 minced meat, 92 chicken meat, 75 diced meat, and 28 mutton) were collected from local markets in Egypt and were screened for CRKP with the determination of their antimicrobial resistance profiles. The whole genome sequence was done for 23 CRKP isolates to clarify the relationship between CRKP from food and human cases in Egypt using the SNP core genome. The conjugation probability of the bla NDM-5 harboring plasmid was identified using oriTfinder CRKP was isolated from 11% (35/311) of the samples, with 45.71% (16/35) of them showing resistance to colistin, one of the last-resort options for treating CRKP-mediated infections. In addition to the carbapenem and colistin resistance, the CRKP isolates frequently exhibited resistance to multiple antimicrobials including β-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and chloramphenicol. In addition, most of the CRKP were potentially hypervirulent K. pneumoniae (HvKP) identified as phylogroup Kp1 and of high-risk groups as detected in STs reported in many human outbreaks globally, such as ST383 and ST147. The core-genome phylogeny showed similarities between the isolates from this study and those previously isolated from clinical human samples in Egypt. In addition, analysis of the plasmid on which bla NDM is encoded revealed that several antimicrobial resistance genes such as bla OXA-9 , bla CTX-M-15 , aac(6′)-Ib, qnrS1, and several virulence genes are encoded on the same plasmid. This study is significant for food safety and public health and is important to further identify the change in the epidemiology of CRKP infections, especially the consumption of contaminated food products. [ABSTRACT FROM AUTHOR]

• The virulence genes in ST11-KL47 CRKP in China have evolved towards chromosomal vertical inheritance, low pathogenicity, and high fitness. • The intermediate stage of isolates in this evolutionary process, where virulence genes carried by pNDM-Mar-like plasmids, exhibit significantly increased virulence despite decreased adaptability. • The conjugative pNDM-Mar-like plasmids have been identified in strains from diverse sources worldwide. • Global attention and precise control measures to pNDM-Mar-like plasmids within a one health perspective are needed, and we suggest incorporating the two replicon nucleotide sequences of pNDM-Mar into basic screening amplification. ST11-KL47 is a hypervirulent carbapenem-resistant Klebsiella pneumoniae (CRKP) that is highly prevalent in China and poses a major public health risk. To investigate the evolutionary dynamics of virulence genes in this subclone, we analysed 78 sequenced isolates obtained from a long-term study across 29 centres from 17 cities in China. Virulence genes were located in large hybrid pNDM-Mar-like plasmids (length: ∼266 kilobases) rather than in classical pK2044-like plasmids. These hybrid plasmids, derived from the fusion of pK2044 and pNDM-Mar plasmids mediated by insertion sequence (IS) elements (such as IS Kpn28 and IS 26), integrated virulence gene fragments into the chromosome. Analysis of 217 sequences containing the special IncFIB (pNDM-Mar) replicon using public databases indicated that these plasmids typically contained T4SS-related and multiple antimicrobial resistance genes, were present in 24 countries, and were found in humans, animals, and the environment. Notably, the chromosomal integration of virulence genes was observed in strains across five countries across two continents. In vivo and in vitro models showed that the large hybrid plasmid increased the host fitness cost while increasing virulence. Conversely, virulence genes transferred to chromosomes resulted in increased fitness and lower virulence. In conclusion, virulence genes in the plasmids of ST11-KL47 CRKP are evolving, driven by adaptive negative selection, to enable vertical chromosomal inheritance along with conferring a survival advantage and low pathogenicity. [Display omitted] [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes high morbidity and mortality worldwide. The purpose of the study was to assess the synergistic activity of fosfomycin in combination with other antimicrobial agents against CRKP isolated from patients in Songklanagarind Hospital, Thailand. A total of 35 K. pneumoniae isolates were obtained from patients in Songklanagarind Hospital. The MICs of imipenem and meropenem were determined in all isolates by broth microdilution. In all CRKP isolates, the presence of carbapenemase and extended-spectrum β-lactamase (ESBL) genes was investigated by PCR, while the production of these enzymes was determined by combined disk test. In the carbapenemase-genes-negative CRKP isolates, the porin loss and efflux pump were characterized by SDS-PAGE and broth microdilution, respectively. Finally, the synergistic effects of fosfomycin and other antimicrobial agents were evaluated by checkerboard analysis. Twenty-one of 35 K. pneumoniae isolates were classified as CRKP. Most of CRKP isolates carried bla NDM-1 (n = 18), bla SHV (n = 21), bla CTX-M (n = 21), and bla TEM (n = 16). In fosfomycin-based combination, the result showed that the highest synergistic activity in this study was observed in the combination of fosfomycin and gentamicin (61.9%). These findings suggested that the fosfomycin and gentamicin combination might be useful as a possible treatment option for CRKP infection. [ABSTRACT FROM AUTHOR]

This study compared the epidemiology of carbapenem-resistant (CRKP) and carbapenem-sensitive (CSKP) K. pneumoniae bloodstream infections (BSIs), and assessed risk factors for 28-day mortality of patients with K. pneumoniae BSIs. A retrospective cohort study was conducted in a 2000-bed tertiary teaching hospital of Beijing between Jan 1st 2013 to Dec 31st, 2019. All patients with K. pneumoniae BSI were identified through the Hospital Information System. The endpoints included incidence rate, mortality and risk factors for mortality of patients with K. pneumoniae BSIs. 496 patients with K. pneumoniae BSIs were included in the analysis, with 108 CRKP BSIs. The incidence rate of K. pneumoniae BSI was 10.6 (CI: 9.7, 11.6) per 100 000 patient-days, with the rate for CRKP BSI was 2.3 (95% CI: 1.9, 2.8). The 28-day mortality was 38.0% for CRKP BSI and 8.8% for CSKP BSI, respectively. Logistic analysis showed, higher Charlson Comorbidity Index score (OR = 1.26, 95%CI 1.12–1.43, p < 0.001), respiratory failure (OR = 2.73, 95%CI1.28−5.84, p = 0.010), renal failure (OR = 4.13, 95%CI1.93−8.83, p < 0.001), septic shock (OR = 8.77, 95%CI3.60−21.32, p < 0.001), mechanical ventilation (OR = 4.41, 95%CI1.59−12.25, p = 0.004) and CRKP infection (OR = 3.04, 95%CI1.28−7.22, p = 0.012) were independently associated with 28-day mortality. Considerable incidence rate and remarkable mortality of patients with K. pneumoniae (especially CRKP) BSI was declared in the study. Patient conditions before (higher CCI) and after presentation (respiratory failure, renal failure, septic shock), and healthcare factors (mechanical ventilation and CRKP infection) were independently associated with 28-day mortality. Understanding these risks helps better establishment of infection control strategies. [ABSTRACT FROM AUTHOR]

Abstract Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a major cause of nosocomial infections and posed challenges on clinical treatments. The main objective of this study was to determinate the genetic characteristics of the NDM-19–producing CRKP strain SCM96. From 2015 to 2017, 18 CRKP strains were recovered from sputum samples of patients in respiratory medicine in 6 hospitals from 5 provinces and cities in China. Polymerase chain reaction results for carbapenem resistance genes detection showed strain SCM96 carried bla NDM-19. Three types of transconjugants harboring different plasmids were selected by conjugation experiment. The Whole Genome Sequencing (WGS) was performed using the PacBio RS platform. The genome size of SCM96 was 5,579,775 bp and composed of chromosomal DNA (5,398,745 bp) and 2 plasmids, IncFII type plasmid pSCM96-1 (134,869 bp) and IncX3 type plasmid pSCM96-2 (46,161 bp). SCM96 belonged to ST15 and K28. In addition to the 4 antibiotic resistance genes located in the chromosome, pSCM96-1 carried a complex resistance region containing 17 resistance genes and several mobile genetic elements (MGEs) like △Tn 6029 , In4-like integron, and Tn 3 , and pSCM96-2 had only 1 bla NDM-19 gene. As far as we know, this was the first description of bla NDM-19 in K. pneumoniae. Up to 22 antibiotic resistance genes, several important MGEs, and transferable plasmids might increase the possibility of co-spreading of bla NDM-19 with other resistance genes. Highlights • This was the first report of bla NDM-19 in K. pneumoniae strain SCM96. • K. pneumoniae ST15 was identified in IncX3-positive NDM producers in China for the first time. • A 45-kb complex resistance region was identified, which carried 17 resistance genes, a △Tn 6029 , an In4-like integron, and a Tn 3. [ABSTRACT FROM AUTHOR]

• The colistin resistance rate of CRKP isolates in China from 2018 to 2019 was 2.0% (14/708). • MgrB inactivation or absence was the predominant mechanism in colistin-resistant CRKP isolates. • A novel mutation L247P in PhoQ was determined in a clinically isolated colistin-resistant CRKP. • The prevalence of the mcr gene in colistin-resistant CRKP isolates was low, as only one isolate carried mcr-8.1. 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. [ABSTRACT FROM AUTHOR]

Whole genome sequencing (WGS) was compared to pulse-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA, as methods by which to evaluate a potential transmission of carbapenem-resistant Klebsiella pneumoniae between 2 hospital inpatients. PFGE result demonstrated only 1-band difference between the isolates, suggesting probable relatedness. In contrast, while WGS data demonstrated the same sequence type and very similar chromosomal sequences, over 20 single nucleotide variants were identified between the isolates, bringing into question whether there was a transmission event. WGS also identified an additional plasmid, with an XbaI restriction site in the isolates of the second patient that was not identified by PFGE. While WGS provided additional information that was not available by PFGE, in this study, neither method could definitively conclude the relatedness between the isolates. [ABSTRACT FROM AUTHOR]

Abstract: Carbapenem resistant Klebsiella pneumoniae (CRKP) are isolated with increasing frequency, especially from immunocompromized patients. The capsular polysaccharide (CPS) types of CPKP were not determined. Investigation of two CRKP isolates from a 2011 outbreak at the Clinical Center, the National Institutes of Health, identified a new capsular type shared by the two isolates, similar to K. pneumonia K19 and K34 but structurally different than any published K. pneumoniae CPS repeating unit: Display Omitted The LPS of the two isolates was found to have no O-specific polysaccharide and the chemical structure of the core oligosaccharides agreed with the published data. If this structure type will be prevalent among CPKP isolates, our findings could facilitate rapid diagnosis and help to develop new therapeutic solutions to this antibiotic resistant pathogen. [Copyright &y& Elsevier]

To analyze the drug-resistant phenotype and genetic characteristics of Carbapenem resistant Klebsiella pneumoniae (CRKP) in this region, and to study its different expression profiles in RNA level under the pressure of low levels of antibiotics. Trace dilution method and PCR method were used to detect the antibiotic resistance phenotype and antibiotic resistance gene carrying of CRKP strain, simulate the antibiotic stress process, and RNAseq was used to analyze the transcriptomic changes of CRKP strain. 37 CRKP strains, 27 Carbapenem sensitive Klebsiella pneumoniae (CSKP) CSKP strains and 42 sensitive strains were detected. The antibiotic resistance rate of CRKP strain was significantly higher than that of other drug-resistant strains, and there were many kinds of antibiotic resistance genes. Transcriptomic analysis showed that CRKP strain showed compensatory rise under meropenem stress at low concentration, and the expression of genes related to biofilm formation, pressure induction, pressure tolerance and transcriptional regulation was significantly changed. It was speculated that mrkAB , fimDH , phoHP and pspABCD clusters significantly altered their expression under the antibiotics stress response in CRKP strain. The detection rate of CRKP strain is high in this area. Under low levels of antibiotic stress, CRKP strain can not only survive by synthesizing antibiotic modified enzyme, but also respond by transcriptional regulation and biofilm changes, resulting in stress compensation. The discovery of this phenomenon explains the failure of treatment due to improper use of higher-order antibiotics from the perspective of genetic interaction. [Display omitted] [ABSTRACT FROM AUTHOR]