Emergence of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Coharboring a bla(NDM-1)-Carrying Virulent Plasmid and a bla(KPC-2)-Carrying Plasmid in an Egyptian Hospital.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly blaNDM-1 and blaKPC-2 Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes blaNDM-1 and blaKPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the blaNDM-1 and virulence genes and a blaKPC-2-carrying plasmid.IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

Ahmed MAEE ，Yang Y ，Yang Y ，Yan B ，Chen G ，Hassan RM ，Zhong LL ，Chen Y ，Roberts AP ，Wu Y ，He R ，Liang X ，Qin M ，Dai M ，Zhang L ，Li H ，Yang F ，Xu L ，Tian GB ... -  《mSphere》

Molecular characteristics and pathogenic mechanisms of KPC-3 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (ST23-K1).

This study aimed to comprehensively investigate hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) in the Ningbo region. Importantly, we sought to elucidate its molecular characteristics and pathogenic mechanisms. This information will provide evidence-based insights for preventing and controlling nosocomial infections and facilitate improved clinical diagnosis and treatment in this region. 96 carbapenem-resistant Klebsiella pneumoniae strains were collected from the Ningbo region between January 2021 and December 2022. Whole genome sequencing and bioinformatic methods were employed to identify and characterize CR-hvKP strains at the molecular level. The minimum inhibitory concentrations (MICs) of common clinical antibiotics were determined using the VITEK-2 Compact automatic microbiological analyzer. Plasmid conjugation experiments evaluated the transferability of resistance plasmids. Finally, mouse virulence assays were conducted to explore the pathogenic mechanisms. Among the 96 strains, a single CR-hvKP strain, designated CR-hvKP57, was identified, with an isolation frequency of 1.04%. Whole-genome sequencing revealed the strain to be ST23 serotype with a K1 capsule. This strain harbored three plasmids. Plasmid 1, a pLVPK-like virulence plasmid, carried multiple virulence genes, including rmpA, rmpA2, iroB, iucA, and terB. Plasmid 2 contained transposable element sequences such as IS15 and IS26. Plasmid 3, classified as a resistance plasmid, harbored the bla KPC-3 carbapenem resistance gene. Mouse virulence assays demonstrated a high mortality rate associated with CR-hvKP57 infection. Additionally, there was a significant increase in IL-1β, IL-6, and TNF-α levels in response to CR-hvKP57 infection, indicating varying degrees of inflammatory response. Western blot experiments further suggested that the pathogenic mechanism involves activation of the NF-κB signaling pathway. This study confirms the emergence of hypervirulent CR-hvKP in the Ningbo region, which likely resulted from the acquisition of a pLVPK-like virulence plasmid and a bla KPC-3 resistance plasmid by the ST23-K1 type Klebsiella pneumoniae. Our findings highlight the urgent need for more judicious use of antibiotics to limit the emergence of resistance. Additionally, strengthening infection prevention and control measures is crucial to minimize the spread of virulence and resistance plasmids.

Tu Y ，Gao H ，Zhao R ，Yan J ，Wu X ... -  《Frontiers in Cellular and Infection Microbiology》

Genomic insights into the evolution and mechanisms of carbapenem-resistant hypervirulent Klebsiella pneumoniae co-harboring bla(KPC) and bla(NDM): implications for public health threat mitigation.

Wang Q ，Liu Y ，Chen R ，Zhang M ，Si Z ，Wang Y ，Jin Y ，Bai Y ，Song Z ，Lu X ，Hao M ，Hao Y ... -  《Annals of Clinical Microbiology and Antimicrobials》

Carbapenem-resistant hypermucoviscous Klebsiella pneumoniae clinical isolates from a tertiary hospital in China: Antimicrobial susceptibility, resistance phenotype, epidemiological characteristics, microbial virulence, and risk factors.

Hypervirulent and multidrug-resistant Klebsiella pneumoniae poses a significant threat to public health. We aimed to determine the common carbapenemase genotypes and the carriage patterns, main antibiotic resistance mechanisms, and in vitro susceptibility of clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) to ceftazidime/avibactam (CZA) for the reasonable selection of antimicrobial agents and determine whether hypermucoviscous (HMV) phenotype and virulence-associated genes are key factors for CRKP colonization and persistence. Antibiotics susceptibility of clinical CRKP isolates and carbapenemase types were detected. CRKP isolates were identified as hypermucoviscous K. pneumoniae (HMKP) using the string test, and detection of virulence gene was performed using capsular serotyping. The bla KPC-2, bla NDM, bla IMP, and/or bla OXA-48-like were detected in 96.4% (402/417) of the isolates, and the bla KPC-2 (64.7%, 260/402) was significantly higher (P<0.05) than those of bla NDM (25.1%), bla OXA-48-like (10.4%), and bla IMP (4.2%). Carriage of a single carbapenemase gene was observed in 96.3% of the isolates, making it the dominant antibiotic resistance genotype carriage pattern (P < 0.05). Approximately 3.7% of the isolates carried two or more carbapenemase genotypes, with bla KPC-2 + bla NDM and bla NDM + bla IMP being the dominant multiple antibiotic resistance genotype. In addition, 43 CRKP isolates were identified as HMKP, with a prevalence of 10.3% and 2.7% among CRKP and all K. pneumoniae isolates, respectively. Most clinical CRKP isolates were isolated from elderly patients, and carbapenemase production was the main mechanism of drug resistance. Tigecycline and polymyxin B exhibited exceptional antimicrobial activity against CRKP isolates in vitro. Furthermore, bla KPC-2, bla NDM, and bla OXA-48-like were the main carbapenemase genes carried by the CRKP isolates. CZA demonstrated excellent antimicrobial activity against isolates carrying the single bla KPC-2 or bla OXA-48-like genotype. Capsular serotype K2 was the main capsular serotype of the carbapenem-resistant HMKP isolates. Survival rates of Galleria mellonella injected with K. pneumoniae 1-7 were 20.0, 16.7, 6.7, 23.3, 16.7, 3.3, and 13.3, respectively. Therefore, worldwide surveillance of these novel CRKP isolates and carbapenem-resistant HMKP isolates as well as the implementation of stricter control measures are needed to prevent further dissemination in hospital settings.

Wang Q ，Chen M ，Ou Q ，Zheng L ，Chen X ，Mao G ，Fang J ，Jin D ，Tang X ... -  《Frontiers in Cellular and Infection Microbiology》

Hybrid Plasmids Encoding Antimicrobial Resistance and Virulence Traits Among Hypervirulent Klebsiella pneumoniae ST2096 in India.

Hypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes. Nine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment. Among the nine isolates, seven were carbapenem-resistant, two of which carried blaNDM-5 on an IncFII plasmid and five carried blaOXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB-IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, blaOXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition. The convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.

Shankar C ，Vasudevan K ，Jacob JJ ，Baker S ，Isaac BJ ，Neeravi AR ，Sethuvel DPM ，George B ，Veeraraghavan B ... -  《Frontiers in Cellular and Infection Microbiology》