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Objective:To investigate whether the hypervirulent Klebsiella pneumoniae (hvKP) induces liver abscess through activating NLRP3 inflammasome. Methods:K1-hvKP and K35-non-hvKP bacterial suspensions were intraperitoneally injected into C57BL/6 mice to establish the models of liver abscess. Human peripheral blood neutrophils were sorted by immunomagnetic beads with CD45 + and Gr-1 + , and the purity was detected by flow cytometry. The concentrations of capsular polysaccharide of K1-hvKP and K35-non-hvKP were detected by total carbohydrate assay kit. The expression of IL-18 and IL-33 by neutrophils at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and ELISA, respectively. The activation of NLRP3 inflammasome in neutrophils was detected by Western blot. Neutrophil extracellular trap formation (NETosis) was observed under confocal laser scanning microscope. Results:The C57BL/6 mice with K1-hvKP infection had significantly serious liver abscess as compared with the K35-non-hvKP-infected mice. The purity of human neutrophils was more than 95%. The concentration of capsular polysaccharide in K1-hvKP was significantly higher than that in K35-non-hvKP. Compared with K35-non-hvKP, K1-hvKP significantly promoted the neutrophils to express IL-18 and IL-33 at both mRNA and protein levels, enhanced the activation of NLRP3 and induced NETosis.Conclusions:This study suggested that hvKP could promote NETosis by activating NLRP3 inflammasome to cause liver abscess.
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Objective:To construct a mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 with hfq gene deletion and to analyze its biological characteristics. Methods:The hfq gene of NTUH-K2044 was knocked out by homologous recombination technology to construct △ hfq mutant strain. Its biological characteristics including growth rate, environmental stress tolerance, biofilm formation, capsular polysaccharide synthesis, resistance to neutrophil phagocytosis and lethality to Galleria mellonella larvae were analyzed by comparing with the wild-type strain using phenotypic experiments. Results:The △ hfq mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 was successfully constructed. Phenotypic experiments showed that the △ hfq mutant strain had significantly slower growth rate, smaller colonies and decreased hypermucoviscosity. Its growth was significantly inhibited under different environmental stress conditions such as pH9, pH5.5, 0.7 mmol/L SDS, 5% NaCl, 0.1% H 2O 2 and high temperature of 50℃. In terms of virulence and pathogenicity, the △ hfq mutant strain showed decreased ability to form biofilm and capsule, significantly down-regulated expression of magA and rmpA genes required for capsule synthesis, lower survival rate in the neutrophil bactericidal test and obviously reduced lethality to Galleria mellonella larvae. Conclusions:As a RNA chaperone, Hfq protein could participate in post-transcriptional regulation and play an important role in regulating the physiology, environmental adaptability and virulence of hypervirulent Klebsiella pneumoniae. This study provided reference for further study on hypervirulent Klebsiella pneumoniae.
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Objective To describe the clinical characteristics of hypervirulent Klebsiella pneumoniae (hvKP) infection.To analyze the antibiotic susceptibility of hvKP to provide the empiric antibiotic options.To investigate capsule serotype and sequence type (ST) of hvKP and their correlation with clinical profiles.Methods hvKP was defined as bacteria isolated from patients with community-acquired pyogenic liver abscess (CA-PLA) with co-infection sites outside liver or a bloodstream infection in a host without underlying biliary tract diseases.Patients with CA-PLA hospitalized in the First Hospital of China Medical University were retrospectively analyzed from January 2011 to December 2017.Antibiotic susceptibility was detected by automatic bacterial identification and antibiotic susceptibility analysis system in vitro.Polymerase chain reaction method and gene sequencing were used to detect the main capsule serotype and ST.Results A total of 140 cases with hvKP infection were enrolled.The co-infections outside liver abscess included 98 bloodstream infections,53 pneumonia,11 perianal abscess,10 urinary system infections,3 subphrenic abscess,3 endophthalmitis,2 spleen abscess,and other miscellaneous infections including 1 peritonitis,1 skin and soft tissue infection,1 myelitis,1 colitis,1 psoas major abscess and 1 myocardial abscess.Among the 140 cases,106 presented with single co-infection site,32 with 2 sites,and 2 with 3 sites.HvKP manifested high antibiotic susceptibility up to 80％ for most commonly used antibiotics.Capsule serotyping of 4,3 revived isolates indicated that K1 serotype accounted for 53.49％ (23/43),K2 34.88 (15/43),K54 2.33％ (1/43),K57 2.33％ (1/43),and other serotypes 6.98％(3/43).There was no significant distribution among K1,K2,K54,and K57 of hvKP capsule serotypes in patients with or without diabetes mellitus (P＞0.05).Multilocus sequence typing (MLST) suggested that ST23 and ST65 were predominant accounting for 39.53％ (17/4.3) and 25.58％ (11/4.3) respectively.No serotype or ST predominance was seen in any of the clinical infections.Conclusion HvKP is related to a wide spectrum of infectious diseases,including multiple extrahepatic sites and bloodstream infections besides CA-PLA with high antibiotic susceptibility.K1 and K2 are the predominant capsule serotypes,and ST 23 and ST65 are the predominant sequence types.
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Objective To understand the microbiological and clinical features of bloodstream infection(BSI) with high virulent Klebsiella pneumoniae(hvKP).Methods The strains and clinical data of 159 patients with Klebsiella pneumoniae (K.pneumoniae) BSI at the Second Hospital of Dalian Medical University from April 2013 to March 2016 were collected.Statistical analysis was performed using SPSS 19.0 software.Results 35.22％(56/159)of patients were with hvKP BSI,K1 and K2 serotypes in hvKP BSI accounted for 51.79％ and 26.79％ respectively.The main source of hvKP BSI was liver abscess(n =26,46.43 ％),the classic type of K.pneumoniae (cKP) BSI was primary bacteremia(n =41,39.81 ％).Difference in different types of infection between two groups of patients was statistically significant(x2 =57.782,P＜0.001),89.29 ％ of hvKP BSI was community-associated infection(CAI),and 73.79％ of cKP BSI was healthcare-associated infection(HAI).Difference in underlying diseases between two groups of patients was statistically significant(x2 =36.532,P＜0.001),50.00％ of hvKP BSI patients had diabetes,45.63％ of cKP BSI patients had malignant tumor.Icidence of septic shock in hvKP BSI patients was higher than that of cKP BSI patients(32.14％ vs 8.74％;x2 =14.096,P＜0.001).The proportion of ESBLs-producing of hvKP and cKP were 5.36％ (3/56)and 47.57％ (49/103)respectively,difference was statistically significant(x2 =29.375,P＜0.001).Klebsiella pneumoniae carbapemase(KPC)-producing hvKP was not found.Resistance rates of hvKP to ceftazidime,ceftriaxone,cefepime,aztreonam,gentamicin,levofloxacin,and compound sulfamethoxazole were all lower than cKP(all P＜0.05).Conclusion hvKP BSI is common in CAI,infection sources and underlying diseases are difference from cKP BSI,hvKP BSI is prone to cause septic shock.Both laboratories and clinicians should pay attention to hvKP infection and the change trend of antimicrobial resistance.
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Objective To evaluate the feasibility of using neutrophil bactericidal activity assay for analyzing the anti-bactericidal ability of hypervirulent Klebsiella pneumoniae ( hvKP) strains that harbored the virulence genes of rmpA and rmpA2 and were positive for string test .Methods A total of 150 non-duplicate blood-borne Klebsiella pneumoniae strains were collected from Zhejiang Province from January 2016 to July 2017.PCR was performed to detect carbapenem resistance genes (blaKPC, blaNDM, blaIMP-1, blaIMP-2), cap-sule genotypes (K1, K2, K5, K20, K54 and K57) and virulence genes (rmpA, rmpA2, iucA and iroN). Klebsiella pneumoniae strains that were positive for string test and harbored rmpA and rmpA2 genes were iden-tified as hvKP strains, while classic Klebsiella pneumoniae (cKP) strains were negative for string test, rmpA or rmpA2 gene.Neutrophil bactericidal activity assay was performed to analyze the virulence of Klebsiella pneumoniae strains and the survival rate was determined by using the following equation: the number of colony-forming units ( CFUs) in experimental group divided by the number of CFUs in control group .Re-sults Of the 150 Klebsiella pneumoniae strains, 43.3％ (65/150) harbored the rmpA2 gene and among them, strains positive for genes of rmpA, iroN and blaKPC and K2 respectively accounted for 73.8％, 80.0％, 75.4％and 40.0％.Twenty-four (36.9％) rmpA2 gene-positive strains showed positive result of string test.The survival rates of hvKP and cKP groups were respectively 0.866±0.056 and 0.368±0.058 and the difference between them was statistically significant (P<0.001).Conclusion Most of the hvKP strains that carry rmpA and rmpA2 genes and are positive for string test in Zhejiang Province survive the neu-trophil treatment , which indicates that the neutrophil bactericidal activity assay is an effective and simple method for identifying the virulence of Klebsiella pneumoniae.