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IntroductionHypervirulent Klebsiella pneumoniae produce an increased amount of capsular substance and are associated with a hypermucoviscous phenotype. Capsule production is regulated by capsular regulatory genes and capsular gene cluster variations. In the present study, we focus on the effect of rmpA and wcaJon capsule biosynthesis.MethodsPhylogenetic trees were constructed to analyze wcaJ and rmpA sequence diversity in different serotypes hypervirulent strains. Then mutant strains (K2044ΔwcaJ, K2044K1wcaJ, K2044K2wcaJand K2044K64wcaJ) were used to verify the effects of wcaJ and its diversity on capsule synthesis and strain virulence. Furthmore, the role of rmpA in capsular synthesis and its mechanisms were detected in K2044ΔrmpA strain.ResultsRmpA sequences are conversed in different serotypes. And rmpA promoted the production of hypercapsules by simultaneously acting on three promoters in cps cluster. Whereas wcaJ, its sequences are different in different serotypes, and its loss result in the termination of capsular synthesis. Moreover, the results verified that K2 wcaJ could form hypercapsule in K2044 strains (K1 serotype), but K64 wcaJ could not.DiscussionThe interaction of multiple factors is involved in capsule synthesis, including wcaJ and rmpA. RmpA, an known conserved capsular regulator gene, acts on cps cluster promoters to promote the production of the hypercapsule. WcaJ as initiating enzyme of CPS biosynthesis, its presence determines the synthesis of capsule. Besides, different from rmpA, wcaJ sequence consistency is limited to the same serotype, which cause wcaJ functioning in different serotype strains with sequence recognition specificity.

PurposeTo investigate the mechanisms of Klebsiella pneumoniae-induced pyogenic liver abscess (PLA).MethodsForty-three K. pneumoniae strains from PLAs and 436 from non-PLAs were collected. Their differences were compared for virulence genes and factors, sequence types, and serotypes. Virulence genes wzi, wzy-K1, and wzi+wzy-K1 were deleted in K. pneumoniae NTUH-K2044. Various analyses, such as transmission electron microscopy, neutrophil killing tests, and mouse lethality tests, were used to confirm the consequent changes.ResultsDifferences were found between K. pneumoniae strains from PLA and non-PLA samples for virulence genes and factors, including metabolism genes (allS and peg-344), capsular polysaccharide (CPS)-synthesis channel gene (wzy-K1), CPS-regulating genes (p-rmpA, p-rmpA2, and c-rmpA), and siderophore genes (iucA and iroN). When wzy-K1 was positive, the difference between PLA and non-PLA samples was only observed with c-rmpA. Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 strains reverted to hypovirulence. In the Kupffer cell stimulation assay, interleukin (IL)-6, IL-12, IL-10, and transforming growth factor-β secretions were found to be equivalent in NTUH-K2044, Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 groups. Lower IL-1β and higher tumor necrosis factor-α secretions were observed for Δwzi, Δwzy-K1, and ΔwziΔwzy-K1.ConclusionsHypercapsule production is the cornerstone of hypervirulence, regardless of exopolysaccharides. K1 K. pneumoniae-induced PLA may decrease core inflammatory cytokines rather than increase anti-inflammatory cytokines. Exopolysaccharides could also attenuate the inflammatory response to aid in the immune escape of K. pneumoniae.

ObjectiveTo investigate the epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP).MethodsTotally 436 K. pneumoniae strains were collected from 7 hospitals in mainland China between 2017.01 and 2018.02. Sequence types, serotypes, antimicrobial-resistance and virulence genes were analyzed. Additionally, string test, capsule stain, Periodic Acid Schiff stain, fitness analysis, quantitative real-time PCR and mouse lethality test were also performed. Molecular combinations were used to screen putative blaKPC(+)-HvKP and Hv-blaKPC(+)-KP, followed by the confirmation of mouse lethality test.ResultsDiverse detection rates were found for the virulence genes, ranging from c-rmpA (0.0%) to entB (100.0%). According to the molecular criteria, 127, 186, 9 and 26 strains were putatively denoted as HvKP, blaKPC(+)-KP, blaKPC(+)-HvKP and Hv-blaKPC(+)-KP. Mouse lethality test confirmed 2 blaKPC(+)-HvKP strains (JS184 and TZ20) and no Hv-blaKPC(+)-KP. JS184 showed K2 serotype, thin capsule, positive exopolysaccharid and string test. TZ20 presented K20 serotype, thin capsule, negative exopolysaccharide and string test. Compared with the positive control NTUH-K2044, equal galF expression and growth curves were confirmed for JS184 and TZ20.ConclusionsMolecular determination of CR-HvKP and Hv-CRKP brings remarkable bias compared with mouse lethality test. The exact prevalence of CR-HvKP is less than 1.0%, which of Hv-CRKP is much lower.

The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

ObjectiveTo investigate the overall distributions of key virulence genes in Klebsiella pneumoniae, especially the hypervirulent blaKPC-positive K. pneumoniae (Hv-blaKPC(+)-KP).MethodsA total of 521 complete genomes of K. pneumoniae from GenBank were collected and analyzed. Multilocus sequence typing, molecular serotyping, antibiotic-resistance, virulence genes and plasmid replicon typing were investigated.ResultsPositive rates of virulence genes highly varied, ranging from 2.9 (c-rmpA/A2) to 99.6% (entB). Totally 207 strains presented positive fimH, mrkD, entB and wzi and 190 showed positive fimH, mrkD, entB, irp2 and wzi, which were the two primary modes. A total of 94, 165 and 29 strains were denoted as hypervirulent K. pneumoniae (HvKP), blaKPC(+)-KP and Hv-blaKPC(+)-KP. ST11 accounted for 17 among the 29 Hv-blaKPC(+)-KP strains; Genes iucA, p-rmpA2 and p-rmpA were positive in 28, 26 and 18 Hv-blaKPC(+)-KP strains respectively. Among the 29 Hv-blaKPC(+)-KP strains exhibiting four super clusters from GenBank, IncHI1B plasmids carrying virulence genes and IncFII ones with blaKPC were responsible for both 23 strains respectively.ConclusionsPositive rates of virulence genes vary remarkably in K. pneumoniae. Genes iucA, p-rmpA2 and p-rmpA were primary ones inducing Hv-blaKPC(+)-KP. IncHI1B plasmids carrying virulence genes and IncFII ones with blaKPC constitute the primary combination responsible for Hv-blaKPC(+)-KP. The making of Hv-blaKPC(+)-KP is mostly via blaKPC(+)-KP acquiring another plasmid harboring virulence genes.