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Capsular serotype K2 of sequence type (ST) 65 has been recognized as a hypervirulent clone. Simultaneous presence of different genes has never been reported in this clone. In the present study, the genetic characteristics and virulence phenotype of a CTX-M-3 and CTX-M-14 coproducing ST65 human isolate, KP_06, that caused an intracranial infection, are evaluated. The potential virulence of KP_06 was assayed by in vitro and in vivo methods. The molecular biology and whole-genome sequencing technology were used to analyze the genomic features associated with the virulence of this strain. The KP_06 exhibited typical features of hypervirulent (hvKP), showing hypermucoviscosity phenotype and belonging to K2 and ST65. Apart from virulence genes linked to hvKP, including , , and cluster and genes encoding siderophores, it was found to harbor a ~170 kb pLVPK-like virulence plasmid. In contrast to most hvKP, KP_06 was resistant to cephalosporins and the coexistence of and was detected. Further experiments demonstrated that this strain was classified as a nonbiofilm producer and serum sensitivity (grade 1) and killed only 30% of inoculated with 1×10 colony-forming unit of the specimen within 48 hours, suggesting relatively low virulence. Comparative genomic analysis of KP_06 with five K2 hypermucoviscous (HMKP) revealed seven unique orthologies with varied function in this strain. Intriguingly, the virulence genes identified in KP-06 were unexpectedly more diverse than those observed in five other K2 HMKP strains. Our data support the notion that neither virulence-associated genes (clusters) nor the pLVPK-like virulence plasmid is sufficient for the hypervirulence of . Future studies aiming to explore the virulence of should take genome-based profile together with experimental work. The detailed mechanism involving in the impaired virulence of KP_06 remains to be further explored.

Klebsiella pneumoniae is among the leading bacteria that cause nosocomial infections. The capsule of this Gram-negative bacterium is a dominant virulence factor, with a prominent role in defense and biofilm formation. Bacteriophages, which are specific for one bacterial strain and its capsule type, can evoke the lysis of bacterial cells, aided by polysaccharide depolymerase enzymes. In this study, we isolated and characterized a bacteriophage against the nosocomial K. pneumoniae 52145 strain with K2 capsular serotype. The phage showed a narrow host range and stable lytic activity, even when exposed to different temperatures or detergents. Preventive effect of the phage in a nasal colonization model was investigated in vivo. Phlyogenetic analysis showed that the newly isolated Klebsiella phage B1 belongs to the Webervirus genus in Drexlerviridae family. We identified the location of the capsule depolymerase gene of the new phage, which was amplified, cloned, expressed, and purified. The efficacy of the recombinant B1dep depolymerase was tested by spotting on K. pneumoniae strains and it was confirmed that the extract lowers the thickness of the bacterium lawn as it degrades the protective capsule on bacterial cells. As K. pneumoniae strains possessing the K2 serotype have epidemiological importance, the B1 phage and its depolymerase are promising candidates for use as possible antimicrobial agents.

At present, invasive syndrome caused by hypervirulent Klebsiella pneumoniae (HvKp) is a widespread concern, and HvKp strains of different genotypes have been isolated. Here, we report a case of community- acquired liver abscess and endogenous endophthalmitis caused by a genotype ST25 serotype K2 (ST25-K2) HvKp strain in China. A 51-year-old man with diabetes was transferred to our hospital from a local community hospital with persistent fever for > 20 days and blurred vision in his left eye. A detailed examination revealed a liver abscess, endogenous endophthalmitis, and pneumonia. Bacterial cultures of pus from the liver abscess and the vitreous abscess of the left eye yielded Klebsiella pneumoniae (Kp), which was sensitive to the recommended drugs. In addition to positive string tests, a genetic analysis showed that the strain belonged to sequence type 25 (ST25) and serotype K2, and carried already-reported virulence genes, including iucA, rnipA2, rmpA, aerobactin, and entB. The pathogenic agent isolated from this patient was identified as HvKp. The patient's general condition improved after a combination of treatments, including antimicrobial therapy, abscess drainage, and nutritional support. Unfortunately, the patient lost the vision in his left eye and developed secondary glaucoma, resulting in inevitable enucleation. Sequence 25 serotype K2 HvKp strains have been previously associated with nosocomial infections, but none associated with community-acquired liver abscess combined with endogenous endophthalmitis has yet been reported. Clinicians must be alert to the possibility of genotype ST25-K2 HvKp infection in patients with community-acquired liver abscess combined with an invasive infection, such as ocular discomfort. Keywords: hypervirulent Klebsiella pneumoniae, liver abscess, endogenous endophthalmitis, sequence type 25, serotype K2

Purpose This study aimed to assess the resistance phenotype, virulence phenotype, and genetic characteristics of a bla NDM−1 and bla SHV−12 co-harboring ST65 K2 Klebsiella pneumoniae (KP114), which was isolated from General hospital of Ningxia Medical University. Methods Antibiotic susceptibility test was determined by Vitek 2 Compact system. Multilocus Sequence typing (MLST), antimicrobial resistance and virulence genes were examined by PCR and Sanger sequencing. The virulence of KP114 was evaluated through string test, macrophage phagocytosis assay, serum resistance assay, and mouse infection model. Whole-genome sequencing was performed for further analysis of genetic information. Results The presence of the bla NDM−1 and bla SHV−12 genes in KP114 confered resistance to multi-antibiotics. The hypervirulence of KP114 was demonstrated through various in vitro experiments and in vivo mouse infection model. KP114 was found to harbor two distinct plasmids: a drug-resistant plasmid (pKP114-NDM), classified as the IncX3 type, which contained various transfer elements including type IV coupling protein (T4CP) and type IV secretion system (T4SS), and a virulence plasmid (pKP114-vir) that exhibited a high sequence similarity with pLVPK. The results of the conjugation experiment showed that resistance and virulence traits were successfully transferred from KP114 to Escherichia coli EC600 and J53. Conclusions We reported a Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strain of ST65 K2 serotype carrying the bla NDM−1 and bla SHV−12 , which exhibited hypervirulence and drug resistance with potential for transmission. This finding allows improved clinical surveillance and control of this clone, thereby holding considerable value for clinical treatment.

Background Klebsiella pneumoniae is a zoonotic opportunistic pathogen, and also one of the common pathogenic bacteria causing mink pneumonia. The aim of this study was to get a better understanding of the whole-genome of multi-drug resistant Klebsiella pneumoniae with K2 serotype in China. This study for the first time to analyze Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, resistance and virulence genes of Klebsiella pneumoniae in mink. Results The isolate was Klebsiella pneumoniae with serotype K2 and ST6189 by PCR method. The string test was positive and showed high mucus phenotype. There was one plasmid with IncFIB replicons in the genome. The virulence factors including capsule, lipopolysaccharide, adhesin, iron uptake system, urease, secretory system, regulatory gene ( rcsA , rcsB ), determinants of pili adhesion, enolase and magnesium ion absorption related genes. The strain was multi-drug resistant. A total of 26  resistance genes, including beta-lactam, aminoglycosides, tetracycline, fluoroquinolones, sulfonamides, amide alcohols, macrolides, rifampicin, fosfomycin, vancomycin, diaminopyrimidines and polymyxin. Multidrug-resistant efflux protein AcrA, AcrB, TolC, were predicted in the strain. Conclusion It was the first to identify that serotype K2 K. pneumonia with ST6189 isolated from mink in China. The finding indicated that hypervirulent and multi-drug resistant K. pneumoniae was exist in Chinese mink. The whole-genome of K. pneumoniae isolates have importance in mink farming practice.

Klebsiella pneumoniae is a nosocomial pathogen. Among its virulence factors is the capsule with a prominent role in defense and biofilm formation. Bacteriophages (phages) can evoke the lysis of bacterial cells. Due to the mode of action of their polysaccharide depolymerase enzymes, phages are typically specific for one bacterial strain and its capsule type. In this study, we characterized a bacteriophage against the capsule-defective mutant of the nosocomial K. pneumoniae 52145 strain, which lacks K2 capsule. The phage showed a relatively narrow host range but evoked lysis on a few strains with capsular serotypes K33, K21, and K24. Phylogenetic analysis showed that the newly isolated Klebsiella phage 731 belongs to the Webervirus genus in the Drexlerviridae family; it has a 31.084 MDa double-stranded, linear DNA with a length of 50,306 base pairs and a G + C content of 50.9%. Out of the 79 open reading frames (ORFs), we performed the identification of orf22, coding for a trimeric tail fiber protein with putative capsule depolymerase activity, along with the mapping of other putative depolymerases of phage 731 and homologous phages. Efficacy of a previously described recombinant K2 depolymerase (B1dep) was tested by co-spotting phage 731 on K. pneumoniae strains, and it was demonstrated that the B1dep-phage 731 combination allows the lysis of the wild type 52145 strain, originally resistant to the phage 731. With phage 731, we showed that B1dep is a promising candidate for use as a possible antimicrobial agent, as it renders the virulent strain defenseless against other phages. Phage 731 alone is also important due to its efficacy on K. pneumoniae strains possessing epidemiologically important serotypes.

In the last few years, gold nanoparticle biosensors have been developed for rapid, precise, easy and inexpensive with high specificity and sensitivity detection of human, plant and animal pathogens. Klebsiella pneumoniae serotype K2 is one of the common gram-negative pathogens with high prevalence. Therefore, it is essential to provide the effective and exclusive method to detect the bacteria. Klebsiella pneumoniae serotype K2 strain ATCC9997 genomic DNA was applied to establish the detection protocol either with thiol-capped oligonucleotide probes and gold nanoparticles or polymerase chain reaction based on K2A gene sequence. In the presence of the genomic DNA and oligonucleotide probes, a change in the color of gold nanoparticles and maximum changes in wavelength at 550-650 nm was achieved. In addition, the result showed specificity of 15 × 105 CFU/mL and 9 pg/μL by gold nanoparticles probes. The lower limit of detection obtained by PCR method was 1 pg/μL. Moreover, results demonstrated a great specificity of the designed primers and probes for colorimetric detection assay and PCR. Colorimetric detection using gold nanoparticle probe with advantages such as the lower time required for detection and no need for expensive detection instrumentation compared to the biochemical and molecular methods could be introduced for rapid, accurate detection of the bacteria.

Background In Asia, serotype K1/K2 Klebsiella pneumoniae are the major capsular serotypes that cause liver abscess or bacteremia in patients. The purpose of this study was to compare novel immunochromatographic strips (ICSs), which can rapidly detect K. pneumoniae serotypes K1/K2 in clinical samples, to conventional capsular serotyping methods. Methods Pus drainage samples from 16 patients with a liver abscess caused by K. pneumoniae , blood samples from 112 positive flagged blood culture bottle and a subsequent single colony in the medium were tested with the ICS. The results were then compared to findings of capsular swelling tests. Samples subjected to the polymerase chain reaction (PCR) analysis were used as reference. Results The identification of K. pneumoniae via the traditional bacterial culture from pus samples took 3.4 days on average (ranging from 2.2 to 5.5 days). Further capsular serotyping of K. pneumoniae by the capsular swelling test of pure isolates lasted 5–10 min, and the PCR method took ~ 4 h. As for ICSs, the time for direct identification of the K. pneumoniae capsular serotype K1/K2 in pus was < 4 min (ranging from 2 to 4 min). The results of ICSs were consistent with capsular swelling tests and PCR methods. Testing of 112 blood culture samples and subsequent single colonies in the medium with ICSs yielded consistent results for most samples. Conclusions This study indicates that ICSs can rapidly detect K. pneumoniae serotypes K1 and K2 in pus or positive flagged blood culture broth samples within 5 min. Their accuracy is comparable to that of the conventional capsular serotyping methods such as a serum agglutination assay or PCR.

This study aimed to assess the resistance phenotype, virulence phenotype, and genetic characteristics of a bla and bla co-harboring ST65 K2 Klebsiella pneumoniae (KP114), which was isolated from General hospital of Ningxia Medical University. Antibiotic susceptibility test was determined by Vitek 2 Compact system. Multilocus Sequence typing (MLST), antimicrobial resistance and virulence genes were examined by PCR and Sanger sequencing. The virulence of KP114 was evaluated through string test, macrophage phagocytosis assay, serum resistance assay, and mouse infection model. Whole-genome sequencing was performed for further analysis of genetic information. The presence of the bla and bla genes in KP114 confered resistance to multi-antibiotics. The hypervirulence of KP114 was demonstrated through various in vitro experiments and in vivo mouse infection model. KP114 was found to harbor two distinct plasmids: a drug-resistant plasmid (pKP114-NDM), classified as the IncX3 type, which contained various transfer elements including type IV coupling protein (T4CP) and type IV secretion system (T4SS), and a virulence plasmid (pKP114-vir) that exhibited a high sequence similarity with pLVPK. The results of the conjugation experiment showed that resistance and virulence traits were successfully transferred from KP114 to Escherichia coli EC600 and J53. We reported a Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strain of ST65 K2 serotype carrying the bla and bla , which exhibited hypervirulence and drug resistance with potential for transmission. This finding allows improved clinical surveillance and control of this clone, thereby holding considerable value for clinical treatment.

Hypervirulent Klebsiella pneumoniae (HV-KP) typically causes pyogenic liver abscess and bacteremia with metastatic infections. Community-acquired pneumonia (CAP) due to HV-KP is uncommon and details of its clinical and microbiological features are limited. We report the first case of CAP due to capsular genotype K2-ST86 HV-KP in Okinawa, Japan and review infections caused by the K2-ST86 strain. A 79-year-old woman presenting with fever and productive cough persisting for the past three days was admitted to hospital. Her vital signs indicated septic shock. Lung examination by auscultation revealed holo-crackle and lobar pneumonia in chest radiography, and Streptococcus pneumoniae was suspected. However, sputum and blood cultures revealed Gram-negative coccus identified as K. pneumoniae. Genetic analysis identified the isolated strain as the K2 serotype harboring rmpA, iutA, entB, and mrkD. Therefore, we identified the isolated strain as hypervirulent. The isolate belonged to ST86 as determined by multilocus sequence typing. The case was not complicated by predisposing factors such as diabetes mellitus and malignancy related to HV-KP infection; thus, this CAP-causing HV-KP strain may differ from the typical HV-KP strain that induces liver abscess. A literature review identified only nine cases with CAP due to HV-KP. In all cases, the disease mainly occurred in older males with diabetes mellitus, which makes the present case unusual, and had high rates of septic shock and death. No case, including ours, was complicated by metastatic infection, suggesting that CAP due to HV-KP poses little distant metastasis risk, even in patients with bloodstream infection. In our review, consistent with our case, K2-ST86 was the most common strain of HV-KP in patients with CAP. Therefore, studies are needed to elucidate the clinical and microbiological features of HV-KP CAP, with a focus on the K2-ST86 strain. Physicians should always consider K. pneumoniae in cases of sepsis CAP with lobar pneumonia. Keywords: hypervirulent Klebsiella pneumoniae, community-acquired pneumonia, serotype K2, sequence type 86, lobar pneumonia, Streptococcus pneumoniae