Early Response of Antimicrobial Resistance and Virulence Genes Expression in Classical, Hypervirulent, and Hybrid hvKp-MDR Klebsiella pneumoniae on Antimicrobial Stress.
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SUBMITTER: Fursova AD
PROVIDER: S-EPMC8773033 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundHypervirulent 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.MethodsNine 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.ResultsAmong the nine isolates, seven were carbapenem-resistant, two of which carried bla NDM-5 on an IncFII plasmid and five carried bla OXA-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, bla OXA-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.ConclusionThe 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.
| S-EPMC9094440 | biostudies-literature
Project description:Hypervirulent Klebsiella pneumoniae (hvKp) is a major human pathogen associated with liver abscess, pneumonia, meningitis, and endophthalmitis. It is challenging to differentiate hvKp from classical Klebsiella pneumoniae (cKp) using conventional methods, necessitating the development of a rapid, sensitive, and convenient assay for hvKp detection. In this study, we constructed a recombinase-aided amplification (RAA) method targeting hvKp genes peg344 and rmpA, and also analyzed the pathogenic characteristics of hvKp. We optimized the reaction temperature and system, and evaluated its sensitivity, specificity, and clinical application. The primer and probe sets peg344-set1 and rmpA-set2 delivered significant fluorescent signals at 39°C with the shortest gene amplification times (sensitivity: 20 copies/reaction). This RAA assay showed no cross-reactivity with 15 other common pathogenic bacteria. Its applicability was confirmed by the evaluation of 208 clinical specimens, of which 45 were confirmed to be hvKp. The sensitivity and specificity of the RAA assay were both 100% compared with real-time PCR as the reference standard. To verify the assay, we also assessed the diversity of molecular characteristics among the hvKp isolates and identified serotype K1 and sequence type ST23 as the dominant clone. Virulence factors iroN and iutA were highly associated with virulence level. In conclusion, our novel RAA assay is a powerful tool for early diagnosis and epidemiological surveillance of hvKp. IMPORTANCEKlebsiella pneumoniae is the most common opportunistic bacterial species and a major threat to public health. Since the 1990s, hvKp has received increasing attention from public health officials and infectious disease specialists. Hypervirulent strains differ from classical strains in terms of phenotypic features and clinical outcomes. It is hard to identify hvKp from cKp using the conventional methods including colony morphology analysis, serum killing assays, mouse lethality assays, string tests, and real-time PCR. In this study, we established a rapid, sensitive and convenient recombinase-aided amplification assay for hvKp detection targeting virulence genes peg344 and rmpA. Our RAA assay provides an important tool for the rapid diagnosis of infectious diseases caused by hvKp, particularly in primary laboratories.
| S-EPMC10100362 | biostudies-literature
Project description: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 Klebsiella pneumoniae (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 K. pneumoniae 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 phoPQ, contributed more to colistin resistance. mgrB mutation was the most common molecular mechanism of colistin resistance, caused by either nonsense mutations or insertion sequences, which drove the overexpression of phoPQ system. This study also identified three novel point mutations in pmrAB system, in which D313N mutation in pmrB 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 mrkA. In conclusion, our result revealed the emergence of colistin-resistant and hypervirulent MDR K. pneumoniae, which is a noticeable superbug and could cause a severe challenge to public health.
| S-EPMC8896207 | biostudies-literature
Project description:ObjectivesTo establish a rapid molecular diagnostics of hvKp using the peg-344 loop-mediated isothermal amplification technique (LAMP).MethodsIn all, 28 K. pneumoniae strains isolated from the blood of patients were used for the peg-344 LAMP. K. pneumoniae NTUH-K2044 and K. pneumoniae ATCC700603 were used as positive control and negative control, respectively. For comparison, all the results were detected in a polymerase chain reaction (PCR), which was considered the gold standard for the detection of the gene. Mouse lethality assay, and Serum killing assay were also used to determine the virulence phenotype of K. pneumoniae.ResultsWe determined the specificity and sensitivity of the primers for peg-344 detection in the LAMP reactions. This LAMP assay was able to specifically differentiate hvKp from classical K. pneumoniae (cKp) at 65°C, which was 100-fold more sensitive than a PCR assay for peg-344 detection. The virulence phenotype of K. pneumoniae detected by LAMP was as precise as by Mouse lethality assay and Serum killing assay.ConclusionThe LAMP assay is easy to perform and rapid. Therefore, it can be routinely applied to differentiate hvKp from cKp in the clinical laboratory.
| S-EPMC7325879 | biostudies-literature
Project description:A hypervirulent Klebsiella pneumoniae (hvKp) pathotype is undergoing global dissemination. In contrast to the usual health care-associated epidemiology of classical K. pneumoniae (cKp) infections, hvKp causes tissue-invasive infections in otherwise healthy individuals from the community, often involving multiple sites. An accurate test to identify hvKp strains is needed for improved patient care and epidemiologic studies. To fill this knowledge gap, clinical criteria or random blood isolates from North American and United Kingdom strain collections were used to assemble hvKp-rich (n = 85) and cKp-rich (n = 90) strain cohorts, respectively. The isolates were then assessed for multiple candidate biomarkers hypothesized to accurately differentiate the two cohorts. The genes peg-344, iroB, iucA, plasmid-borne rmpA gene ( prmpA), and prmpA2 all demonstrated >0.95 diagnostic accuracy for identifying strains in the hvKp-rich cohort. Next, to validate this epidemiological analysis, all strains were assessed experimentally in a murine sepsis model. peg-344, iroB, iucA, prmpA, and prmpA2 were all associated with a hazard ratio of >25 for severe illness or death, additionally supporting their utility for identifying hvKp strains. Quantitative siderophore production of ≥30 μg/ml also strongly predicted strains as members of the hvKp-rich cohort (accuracy, 0.96) and exhibited a hazard ratio of 31.7 for severe illness or death. The string test, a widely used marker for hvKp strains, performed less well, achieving an accuracy of only 0.90. Last, using the most accurate biomarkers to define hvKp, prevalence studies were performed on two Western strain collections. These data strongly support the utility of several laboratory markers for identifying hvKp strains with a high degree of accuracy.
| S-EPMC6113484 | biostudies-literature
Project description:Emergence and spread of the hypervirulent pathotype of Klebsiella pneumoniae have significantly increased infection rates in community as well as healthcare settings. There is an increasing interest to identify discriminating features between classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) to facilitate our understanding of the rapid emergence and dissemination of the hypervirulent pathotype. Here, we sought to identify unique epigenetic signatures of hvKp pathotype that differ from its classical counterpart using single-base resolution methylome analysis of native DNA sequencing on the Oxford Nanopore Technologies platform. The overall global adenine methylation in GATC motifs (i.e., Dam methylation motif) and cytosine methylation in CCWGG motifs (i.e., Dcm methylation motif) were significantly higher in hvKp isolates compared to that in cKp isolates, irrespective of their position in chromosomes or putative extra-chromosomal genetic elements. Notably, we observed significant enrichment of hypermethylated GATC and CCWGG motifs in the virulome of hvKp compared to hvKp genes not directly associated with virulence. We also observed increased methylation of GATC and CCWGG motifs in the capsule synthesis locus of hvKp isolates compared to cKp isolates. Furthermore, we identified several differentially methylated genes (DMGs) between the two pathotypes; interestingly, these DMGs include metal ion transporters, multidrug efflux pumps, transcriptional regulators of stress response, and genes associated with biofilm formation. Our results highlight hypermethylation of GATC and CCWGG motifs as unique epigenetic signatures of hvKp isolates.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a more virulent and rapidly evolving hypermucoviscous pathotype of classical K. pneumoniae (cKp). The hypervirulent pathotype is a major public health concern and is associated with high infection rates in community as well as hospital settings. With the recent emergence of multidrug-resistant hvKp, it has become imperative to investigate non-classical mechanisms such as epigenetics in addition to canonical biochemical and genetic mechanisms that delineate and differentiate the hypervirulent pathotype from its classical counterpart. Here, we identify genome-wide differences in adenine and cytosine methylation marks at well-characterized motifs between the two pathotypes. Overall, significantly higher levels of methylation were observed across chromosomal DNA and extrachromosomal elements in hvKp compared to cKp. Among hvKp isolates, the genes associated with virulence are particularly enriched for methylation marks. Our findings shed light on how epigenetic signatures may help distinguish the pathogenic potential of bacteria.
| S-EPMC10826340 | biostudies-literature
Project description:Hypervirulent K. pneumoniae (hvKp) is an evolving pathotype that is more virulent than classical K. pneumoniae (cKp). hvKp usually infects individuals from the community, who are often healthy. Infections are more common in the Asian Pacific Rim but are occurring globally. hvKp infection frequently presents at multiple sites or subsequently metastatically spreads, often requiring source control. hvKp has an increased ability to cause central nervous system infection and endophthalmitis, which require rapid recognition and site-specific treatment. The genetic factors that confer hvKp's hypervirulent phenotype are present on a large virulence plasmid and perhaps integrative conjugal elements. Increased capsule production and aerobactin production are established hvKp-specific virulence factors. Similar to cKp, hvKp strains are becoming increasingly resistant to antimicrobials via acquisition of mobile elements carrying resistance determinants, and new hvKp strains emerge when extensively drug-resistant cKp strains acquire hvKp-specific virulence determinants, resulting in nosocomial infection. Presently, clinical laboratories are unable to differentiate cKp from hvKp, but recently, several biomarkers and quantitative siderophore production have been shown to accurately predict hvKp strains, which could lead to the development of a diagnostic test for use by clinical laboratories for optimal patient care and for use in epidemiologic surveillance and research studies.
| S-EPMC6589860 | biostudies-literature
Project description:BackgroundKlebsiella pneumoniae, as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear.MethodsA total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by XbaI and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids.ResultsA nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site.ConclusionsThe nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.
| S-EPMC8299605 | biostudies-literature
Project description:BACKGROUND:MDR and hypervirulence (hv) are typically observed in separate Klebsiella pneumoniae populations. However, convergent strains with both properties have been documented and potentially pose a high risk to public health in the form of invasive infections with limited treatment options. OBJECTIVES:Our aim was to characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15. METHODS:The complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing. RESULTS:Both isolates carried large mosaic plasmids in which AMR and virulence loci have converged within the same vector. These closely related mosaic hv-MDR plasmids include sequences typical of the K. pneumoniae virulence plasmid 1 (KpVP-1; including aerobactin synthesis locus iuc) fused with sequences typical of IncFIIK conjugative AMR plasmids. One hv-MDR plasmid carried three MDR elements encoding the ESBL gene blaCTX-M-15 and seven other AMR genes (blaTEM, aac3'-IIa, dfrA1, satA2, blaSHV, sul1 and aadA1). The other carried remnants of these elements encoding blaTEM and aac3'-IIa, and blaCTX-M-15 was located in a second plasmid in this isolate. The two isolates originated from patients hospitalized in Norway but have epidemiological and genomic links to Romania. CONCLUSIONS:The presence of both virulence and AMR determinants on a single vector enables simultaneous transfer in a single event and potentially rapid emergence of hv-MDR K. pneumoniae clones. This highlights the importance of monitoring for such convergence events with stringent genomic surveillance.
| S-EPMC6477991 | biostudies-literature
Project description:Hypervirulent Klebsiella pneumoniae (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 Klebsiella pneumoniae and to explain the potential mechanism for the development of antimicrobial-resistant hvKp isolates, we isolated OMVs from hvKp and classical Klebsiella pneumoniae (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 in vitro and in vivo. 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.
| S-EPMC9132476 | biostudies-literature