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Bactericidal, anti-biofilm, and anti-virulence activity of vitamin C against carbapenem-resistant hypervirulent . Xu Chen,Dong Ning,Chen Kaichao,Yang Xuemei,Zeng Ping,Hou Changshun,Chi Chan Edward Wai,Yao Xi,Chen Sheng iScience The emergence of carbapenem-resistant hypervirulent (CR-hvKP) causing high mortality in clinical patients infers the urgent need for developing therapeutic agents. Here, we demonstrated vitamin C (VC) exhibited strong bactericidal, anti-biofilm, and virulence-suppressing effects on CR-hvKP. Our results showed such a bactericidal effect is dose-dependent both and in the mouse infection model and is associated with induction of reactive oxygen species (ROS) generation. In addition, VC inhibited biofilm formation of CR-hvKP through suppressing the production of exopolysaccharide (EPS). In addition, VC acted as an efflux pump inhibitor at subminimum inhibitory concentration (sub-MIC) to disrupt transportation of EPS and capsular polysaccharide to bacterial cell surface, thereby further inhibiting biofilm and capsule formation. Furthermore, virulence-associated genes in CR-hvKP exposed to sub-MIC of VC were downregulated. Our findings indicated VC could be an effective and safe therapeutic agent to treat CR-hvKP infections in urgent cases when all current treatment options fail. 10.1016/j.isci.2022.103894
Outer membrane vesicles-transmitted virulence genes mediate the emergence of new antimicrobial-resistant hypervirulent . Emerging microbes & infections Hypervirulent (hvKp) is a notorious clinical pathogen that is more likely to cause severe primary and metastatic abscesses. The dissemination of antimicrobial-resistant hvKp isolates has been reported worldwide, posing a great challenge and severe clinical threat. However, the mechanisms of antimicrobial-resistant hvKp isolates prevalent worldwide are not well precise. Outer membrane vesicles (OMVs) secreted from gram-negative bacteria are an important vehicle for delivering effector molecules inter- and intra-species. To explore whether OMVs as the vector of virulence genes horizontal transfer among and to explain the potential mechanism for the development of antimicrobial-resistant hvKp isolates, we isolated OMVs from hvKp and classical (cKp) by sequential differential centrifugation, respectively. Then, the characteristics and contents of hvKp-OMVs and cKp-OMVs were analyzed. These hvKp-OMVs contain virulence genes, which could be transferred from hvKp horizontally to extended-spectrum beta lactamase (ESBL)-producing cKp, leading to the production of antimicrobial-resistant hypervirulent transformants. Further experiments confirmed the transformants exhibited antimicrobial resistance and hypervirulent phenotypes and . In short, this work demonstrated that hvKp-OMVs facilitated virulence genes transfer, allowing an increase in the virulence level of ESBL-producing cKp and providing a new mechanism for the emergence of antimicrobial-resistant hvKp isolates. 10.1080/22221751.2022.2065935
Prevalence of multidrug-resistant hypervirulent without defined hypervirulent biomarkers in Anhui, China: a new dimension of hypervirulence. Frontiers in microbiology is an opportunistic pathogen that mainly causes nosocomial infections and hospital-associated pneumonia in elderly and immunocompromised people. However, multidrug-resistant hypervirulent (MDR-hvKp) has emerged recently as a serious threat to global health that can infect both immunocompromised and healthy individuals. It is scientifically established that plasmid-mediated regulator of mucoid phenotype genes ( and ) and other virulence factors (aerobactin and salmochelin) are mainly responsible for this phenotype. In this study, we collected 23 MDR-hvKp isolates and performed molecular typing, whole genome sequencing, comparative genomic analysis, and phenotypic experiments, including the infection model, to reveal its genetic and phenotypic features. Meanwhile, we discovered two MDR-hvKp isolates (22122315 and 22091569) that showed a wide range of hypervirulence and hypermucoviscosity without and and any virulence factors. In phenotypic experiments, isolate 22122315 showed the highest hypervirulence (infection model) with significant mucoviscosity, and conversely, isolate 22091569 exhibited the highest mucoviscosity (string test) with higher virulence compared to control. These two isolates carried carbapenemase (), β-lactamase (, ), extended-spectrum β-lactamase (ESBL) genes (, ), outer membrane protein-coding genes (), fimbriae encoding genes (), and enterobactin coding genes (, ). In addition, single nucleotide polymorphism analysis indicated that both isolates, 22122315 and 22091569, were found to have novel mutations in loci (c. 2084A > C, p. Asn695Thr), and (c. 1930C > A, p. Pro644Thr), respectively. Finally, NCBI blast analysis suggested these mutations are located in the of the capsule polysaccharide () region and are responsible for putative tyrosine kinase. This study would be a strong reference for enhancing the current understanding of identifying the MDR-hvKp isolates that lacked both mucoid regulators and virulence factors. 10.3389/fmicb.2023.1247091
RVFScan predicts virulence factor genes and hypervirulence of the clinical metagenome. Briefings in bioinformatics Bacterial infections often involve virulence factors that play a crucial role in the pathogenicity of bacteria. Accurate detection of virulence factor genes (VFGs) is essential for precise treatment and prognostic management of hypervirulent bacterial infections. However, there is a lack of rapid and accurate methods for VFG identification from the metagenomic data of clinical samples. Here, we developed a Reads-based Virulence Factors Scanner (RVFScan), an innovative user-friendly online tool that integrates a comprehensive VFG database with similarity matrix-based criteria for VFG prediction and annotation using metagenomic data without the need for assembly. RVFScan demonstrated superior performance compared to previous assembly-based and read-based VFG predictors, achieving a sensitivity of 97%, specificity of 98% and accuracy of 98%. We also conducted a large-scale analysis of 2425 clinical metagenomic datasets to investigate the utility of RVFScan, the species-specific VFG profiles and associations between VFGs and virulence phenotypes for 24 important pathogens were analyzed. By combining genomic comparisons and network analysis, we identified 53 VFGs with significantly higher abundances in hypervirulent Klebsiella pneumoniae (hvKp) than in classical K. pneumoniae. Furthermore, a cohort of 1256 samples suspected of K. pneumoniae infection demonstrated that RVFScan could identify hvKp with a sensitivity of 90%, specificity of 100% and accuracy of 98.73%, with 90% of hvKp samples consistent with clinical diagnosis (Cohen's kappa, 0.94). RVFScan has the potential to detect VFGs in low-biomass and high-complexity clinical samples using metagenomic reads without assembly. This capability facilitates the rapid identification and targeted treatment of hvKp infections and holds promise for application to other hypervirulent pathogens. 10.1093/bib/bbad403
Development of a quadruple qRT-PCR assay for simultaneous identification of hypervirulent and carbapenem-resistant . Microbiology spectrum IMPORTANCE:Globally, the increasing number of hypervirulent (hvKp) and carbapenem-resistant Kp (CR-Kp) infections poses a huge public health challenge with high morbidity and mortality. Worrisomely, due to the mobility of elements carrying virulence and drug-resistance genes, the increasing prevalence of CR-hvKp has also been found with an overwhelming mortality rate in recent years. However, the current detection methods for hvKp and CR-Kp have many disadvantages, such as long turnaround time, complex operation, low sensitivity, and specificity. Herein, a more sensitive, rapid, single-reaction, and multiplex quantitative real-time PCR was developed and validated to differentiate the circulating lineages of Kp with excellent performance in sensitivity and specificity, providing a useful tool for the differential diagnosis and the surveillance of the circulating Kp. 10.1128/spectrum.00719-23
Emergence of OXA-48-producing hypervirulent Klebsiella pneumoniae strains in Taiwan. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology The OXA-48-producing hypervirulent Klebsiella pneumoniae (hvKP) strains were rarely reported. In this study, we characterized three carbapenem-resistant hvKP strains (KP2185, NCRE61, and KP2683-1) isolated from renal abscess, scrotal abscess, and blood samples in a Taiwan hospital. The three strains belonged to two different clones: ST23 K1 (KP2683-1) and ST11 KL64 (KP2185 and NCRE61). KP2683-1 exhibited the highest virulence in an in vivo model. Whole-genome sequencing analysis showed that KP2185 and NCRE61 acquired IncFIB type plasmids containing a set of virulence genes (iroBCDN, iucABCD, rmpA, rmpA2, and iutA), while KP2683-1 acquired an IncL type plasmid harboring bla. 10.1007/s10096-023-04733-3
Drug Resistance and Molecular Characteristics of Carbapenem-Resistant OXA-48-Producing Strains in Hainan, China. Microorganisms BACKGROUND:The emergence and global spread of carbapenem-resistant hypervirulent (CR-hvKP) are of great concern to health services worldwide. These β-lactamases hydrolyze almost all β-lactams, are plasmid-encoded, and are easily transferable among bacterial species. They are mostly of the KPC types in CR-hvKp. OXA-48-producing hvKP strains have been rarely reported in the literature. METHODS:OXA-48-producing hvKP strains were collected from clinical specimens at the First Affiliated Hospital of Hainan Medical University from January 2022 to March 2023. Hypervirulent strains were tested for virulence in a mouse lethality study and underwent whole genome sequencing to identify genomic features. RESULTS:A total of 42 unique OXA-48-bearing strains were identified, including three CR-hvKP strains (KP2683-1, NCRE61, and KP2185), which were isolated from bacteremia, pulmonary abscess, and liver abscess separately. The three CR-hvKP strains belonged to two different clones of ST11 KL64 (KP2185 and NCRE61) and ST23 K1 (KP2683-1). The KP2683-1 strain had the highest virulence. Whole genome sequencing analysis indicated that NCRE61 and KP2185 acquired IncFIB-type plasmids with a set of virulence genes (, , , , and ), while KP2683-1 acquired an IncL-type -harboring plasmid. Consecutive cultures showed that the -harboring plasmids were highly stable in the three hvKP strains and could be transmitted to J53 by conjugation. The drug susceptibility testing results show that Ceftazidime/avibactam is sensitive for OXA-48-producing hvKP. CONCLUSIONS:Our study highlighted the two evolutionary pathways of OXA-48-producing hvKP strains and confirmed their virulence through in vivo testing. Ceftazidime/avibactam may be a viable option for treating OXA-48-producing hvKP strains. 10.3390/microorganisms12010049
Emergence of Extensively Drug-Resistant and Hypervirulent KL2-ST65 Klebsiella pneumoniae Harboring in Beijing, China. Microbiology spectrum Multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKp) has been emerging worldwide. However, the clinical, microbiological, and genomic characteristics of newly emerged MDR sequence type 65 (ST65) hvKp are unclear. We conducted active longitudinal genomic surveillance of in the hospital starting in 2017. Clinical characteristics, including demographic data, infection type, and outcomes, were collected. Whole-genome sequencing was performed to clarify phylogenetic and plasmid features, and phenotype determined by growth curves, plasmid transferability and stability, hypermucoviscosity, biofilm formation, and serum survival were analyzed to microbiologically characterize ST65 in depth. Ten ST65 (1.4%, 10/720) isolates were detected from 720 isolates in total. Nine patients (90%, 9/10) were older than 60 years and had multiple underlying diseases. All ST65 isolates harbored , , , , and and were identified as hvKp. Surprisingly, two MDR-hvKp isolates that grew slowly were observed. Isolate PEKP4222 harbored a pLVPK-like plasmid and a conjunctive MDR plasmid. Isolate P1 harbored in a new plasmid, pP1-54, resulting in an extensively drug-resistant (XDR) phenotype; this isolate, which might have evolved from a strain harboring , resulted in fatal infection. The pP1-54 plasmid could not be transferred to Escherichia coli by conjugation but could be stably inherited vertically. Interestingly, P1 also carried the pLVPK-like plasmid and acquired various antimicrobial resistance genes, and was detected in the IncB/O/K/Z plasmid. The convergence of XDR and hypervirulence within classical ST65 hvKp is emerging, highlighting the need for enhanced genomic surveillance. XDR-hvKp poses a great challenge to public health. ST65, a classical hvKp subtype, mostly presented with hypermucoviscosity, which restricts antimicrobial resistance acquisition. However, few studies have demonstrated the clinical, microbiological, and genomic characteristics of ST65, especially MDR-ST65 hvKp. Here, we first reported that ST65 hvKp acquired and then conferred the XDR-hvKp phenotype. Genomic context analysis concluded that the gene might have evolved from . Additionally, the pLVPK-like plasmid seemed to acquire more resistance genes, and located in the IncB/O/K/Z plasmid was observed. The XDR-hvKp phenotype could be stably inherited vertically, indicating that strains harboring and pLVPK-like plasmids could persistently exist in hospital settings. These data suggest that genomic adaptation is rapid and that enhanced surveillance is essential. 10.1128/spectrum.03044-22
Isolation of Hv-CRKP with co-production of three carbapenemases (, or , and ) and a virulence plasmid: a study from a Chinese tertiary hospital. Frontiers in microbiology Background:The worldwide dissemination of isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of isolates that carry both virulence plasmids and genes in a tertiary hospital in China. Methods:A total of 217 clinical isolates of carbapenem-resistant (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (, , , , and ), ESBLs genes (, , ), and virulence plasmid pLVPK-borne genes (, , , , and ) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Results:Of the 217 CRKP clinical isolates collected, 23% were identified as carrying genes. All isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be and . MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay () and infection model () indicated hypervirulence. PBRT showed that the and producing hypervirulent carbapenem-resistant (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (, , and ). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. Conclusion:In our investigation, we have observed the emergence of hv-CRKP carrying genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent and . Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (, , and ) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission. 10.3389/fmicb.2023.1182870
Emergence of colistin-resistant hypervirulent (CoR-HvKp) in China. Liu Xiaoyu,Wu Yarong,Zhu Ying,Jia Peiyao,Li Xue,Jia Xinmiao,Yu Wei,Cui Yujun,Yang Ruifu,Xia Wei,Xu Yingchun,Yang Qiwen Emerging microbes & infections Colistin is regarded as a last-resort agent to combat infections caused by multidrug-resistant (MDR) Gram-negative bacteria, especially carbapenem-resistant isolates. In recent years, reports of colistin-resistant (CoRKp) are increasing. However, the molecular mechanism and relevance of colistin resistance and virulence remain unclear. Fourteen CoRKp strains were retrospectively screened from 1884 clinical isolates during 2017-2018 in China. Six CoRKp strains belonging to ST11 were MDR strains. Plasmid-mediated mobile colistin-resistance genes had a low prevalence in CoRKp. Our results revealed that up-regulated expression of two-component systems, especially contributed more to colistin resistance. mutation was the most common molecular mechanism of colistin resistance, caused by either nonsense mutations or insertion sequences, which drove the overexpression of . This study also identified three novel point mutations in system, in which D313N mutation in was proved to increase the MIC to colistin by 16-fold. In addition, 6 out of 14 CoRKP strains independently carried hypervirulence genes. All six strains showed medium-to-high virulence phenotype compared with NTUH-K2044 strain in mice intraperitoneal challenge models. We found that 4 strains were biofilm strong producers and transcriptome analysis revealed that three of them significantly up-regulated expression of type III fimbrial shaft gene . In conclusion, our result revealed the emergence of colistin-resistant and hypervirulent MDR which is a noticeable superbug and could cause a severe challenge to public health. 10.1080/22221751.2022.2036078
Horizontal gene transfer OMVs co-carrying virulence and antimicrobial-resistant genes is a novel way for the dissemination of carbapenem-resistant hypervirulent . Frontiers in microbiology Introduction:The rapidly increased isolation rate of CR-HvKP worldwide has brought great difficulties in controlling clinical infection. Moreover, it has been demonstrated that the transmission of drug-resistant genes among bacteria can be mediated by outer membrane vesicles (OMVs), which is a new way of horizontal gene transfer (HGT). The transmission of virulence genes among bacteria has also been well studied; however, it remains unclear whether virulence and drug-resistant genes can be co-transmitted simultaneously. Co-transmission of virulence and drug-resistant genes is essential for the formation and prevalence of CR-HvKP. Methods:First, we isolated OMVs from CR-HvKP by cushioned-density gradient ultracentrifugation (C-DGUC). TEM and DLS were used to examine the morphology and size of bacterial OMVs. OMV-mediated gene transfer in liquid cultures and the acquisition of the carbapenem gene and virulence gene was confirmed using colony-PCR. Antimicrobial susceptibility testing, mCIM and eCIM were conducted for the resistance of transformant. Serum killing assay, assessment of the anti-biofilm effect and galleria mellonella infection model, mucoviscosity assay, extraction and quantification of capsules were verified the virulence of transformant. Pulsed-field gel electrophoresis (PFGE), S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting hybridization confirmed the plasmid of transformant. Results:Firstly, OMVs were isolated from CR-HvKP NUHL30457 (K2, ST86). TEM and DLS analyses revealed the spherical morphology of the vesicles. Secondly, our study demonstrated that CR-HvKP delivered genetic material, incorporated DNA within the OMVs, and protected it from degradation by extracellular exonucleases. Thirdly, the vesicular lumen DNA was delivered to the recipient cells after determining the presence of virulence and carbapenem-resistant genes in the CR-HvKP OMVs. Importantly, S1-PFGE and Southern hybridization analysis of the 700603 transformant strain showed that the transformant contained both drug-resistant and virulence plasmids. Discussion:In the present study, we aimed to clarify the role of CRHvKP-OMVs in transmitting CR-HvKP among . Collectively, our findings provided valuable insights into the evolution of CR-HvKP. 10.3389/fmicb.2022.945972
Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence. Nature communications Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections. 10.1038/s41467-023-43802-1
Characterization difference of typical KL1, KL2 and ST11-KL64 hypervirulent and carbapenem-resistant Klebsiella pneumoniae. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy Almost all the formation of hypervirulent and carbapenem-resistant Klebsiella pneumoniae follow two major patterns: KL1/KL2 hvKP strains acquire carbapenem-resistance plasmids (CR-hvKP), and carbapenem-resistant Klebsiella pneumoniae (CRKP) strains obtain virulence plasmids (hv-CRKP). These two patterns may pose different phenotypes. In this study, three typical resistance and hypervirulent K. pneumoniae (KL1, KL2, and ST11-KL64), isolating from poor prognosis patients, were selected. Compared with ST11-KL64 hv-CRKP, KL1/KL2 hypervirulent lineages harbor significantly fewer resistance determinants and exhibited lower-level resistance to antibiotics. Notably, though the bla gene could be detected in all these isolates, KL1/KL2 hvKP strain did not exhibit corresponding high-level carbapenem resistance. Unlike the resistance features, we did not observe significant virulence differences between the three strains. The ST11-KL64 hv-CRKP (1403) in this study, showed similar mucoviscosity, siderophores production, and biofilm production compared with KL1 and KL2 hvKP. Moreover, the hypervirulent of ST11-KL64 hvKP also verified with the human lung epithelial cells infection and G. mellonella infection models. Moreover, we found the pLVPK-like virulence plasmid and IncF bla plasmid was crucial for the formation of hypervirulent and carbapenem-resistant K. pneumoniae. The conservation of origin of transfer site (oriT) in these virulence and bla plasmids, indicated the virulence plasmids could transfer to CRKP with the help of bla plasmids. The co-existence of virulence plasmid and bla plasmid facilitate the formation of ST11-KL64 hv-CPKP, which then become nosocomial epidemic under the antibiotic stress. The ST11-KL64 hv-CPKP may poses a substantial threat to healthcare networks, urgent measures were needed to prevent further dissemination in nosocomial settings. 10.1016/j.drup.2023.100918