hvKP Strains Research Articles

Clinical and Microbiological Characteristics of Klebsiella pneumoniae Bloodstream Infection in a Chinese Hospital: Hypervirulent and Multiclonal.

PurposeHypervirulent Klebsiella pneumoniae (hvKP) is emerging globally and can cause various infections. This study aimed to investigate the clinical and microbiological characteristics of bloodstream infection (BSI) caused by hvKP.Patients and MethodsThe clinical data of hospitalized patients with K. pneumoniae BSI were retrospectively analyzed. The K. pneumoniae strains were collected and re-identified, and antimicrobial susceptibility testing was performed using the broth microdilution method. Capsular serotypes and virulence genes were detected using polymerase chain reaction, and hvKP was defined as aerobactin positive. Molecular typing was done by multilocus sequence typing. The hvKP and classic K. pneumoniae (cKP) subgroups were compared.ResultsOf the 66 nonrepetitive BSI K. pneumoniae strains included, 29 (43.9%) were hvKP. In these BSI hvKP strains, salmochelin and yersiniabactin accounted for 86.2% and 72.4%, respectively. The prevalence of rmpA, iroBCD cluster, ybtS, clbA, and allS was 89.7%, 86.2%, 72.4%, 51.7%, and 41.4%, respectively, which were all significantly different between the hvKP and cKP subgroups. Serotypes K1 and K2 were strongly associated with hypervirulence (P < 0.05). Nineteen sequence types were scattered in the 29 hvKP strains, and the most common was ST23 (24.1%). None of the hvKP strains were carbapenem resistant. Compared with cKP, hvKP was more capable of developing a liver abscess. However, the 30-day mortality rate was lower (13.8% vs 21.6%) in the hvKP subgroup than in the cKP subgroup.ConclusionThis study demonstrated a high proportion of hvKP in BSI K. pneumoniae, most of which were RmpA and siderophore producing, and of multiclonal origin.

The Anti-Virulence Effect of Sub-Minimal Inhibitory Concentrations of Levofloxacin on Hypervirulent Klebsiella pneumoniae.

BackgroundHypervirulent Klebsiella pneumoniae (hvKp) is best described as a virulent pathogen and generally associated with the hypermucoviscosity phenotype. Increased capsule and aerobactin production are established important hvKp-specific virulence factors. Although hvKp strains have been relatively susceptible to antimicrobials, given the high morbidity and mortality, there is a critical need for alternative strategies for the treatment of hvKp infections. Thus, the anti-virulence therapy has been targeted for the hvKp development of therapeutics.Materials and MethodsFour hvKp isolates with hypermucoviscous phenotype were used in our experiments. Mucoviscosity of the capsule can be assessed by low-speed centrifugation of cultures. CPS amount was determined by glucuronic acid content. The capsule thickness was measured under microscope after ink staining. The transcriptions of gene were measured by quantitative real-time PCR (qRT-PCR). The effect of levofloxacin on the resistance of K. pneumoniae to phagocytosis by macrophages and mouse lethality assay was observed.ResultsOur data revealed that sub-Minimal Inhibitory Concentrations (sub-MIC) of LVX reduce mucoviscosity and CPS production of hvKp. Microscopic observations demonstrated that the capsule of hvkp bacteria became thinned after treatment with LVX. qRT-PCR showed decreased transcript levels of rmpA, wzi, magA, iroN and icuA genes. Down-regulation of these virulence genes occurred leading to increased susceptibility to phagocytosis by macrophages. Mouse lethality assay revealed that the wild strain had the LD50 of 103 CFU, while the sub-MIC LVX-treated bacteria had the LD50 of 105 CFU.ConclusionOur data suggested that LVX may serve as a potential anti-virulence agent for refractory infection by hvKp.

Characterisation of clinical carbapenem-resistant K1 Klebsiella quasipneumoniae subsp. similipneumoniae strains harbouring a virulence plasmid

The continuous emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) poses a great challenge to human health owing to the associated extremely high morbidity and mortality. Klebsiella quasipneumoniae is a newly described bacterial species that is often misidentified as K. pneumoniae. Clinical K. quasipneumoniae strains have been reported worldwide, among which multidrug-resistant lineages have become a severe health problem, while less has been understood on this important pathogen. In this study, we characterised three clinical carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae isolates belonging to sequence type 367 (ST367) and capsular type K1 and containing several virulence genes, including salmochelin (iroBCDN), aerobactin (iucABCDiutA) and regulator of mucoid phenotype (rmpA/A2), as well as some resistance genes, including blaKPC-2, blaTEM-1, blaOKP-B-9 and oqxAB. These carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae strains containing virulence genes exhibited a higher level of virulence and serum resistance than a classical K. pneumoniae strain, while their virulence levels were slightly lower compared with typical ST11 CR-hvKP and ST23 K1 hvKP strains. This study reports for the first time the genetic and virulence characterisation of clinical K. quasipneumoniae subsp. similipneumoniae strains that simultaneously contained blaKPC-2 and virulence genes, contributing to a better understanding of their resistance and pathogenicity as well as for epidemic surveillance worldwide.

Genomic Analysis of KPC-2-Producing Klebsiella pneumoniae ST11 Isolates at the Respiratory Department of a Tertiary Care Hospital in Beijing, China.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is an important pathogen causing hospital-associated outbreaks worldwide. The spread of K. pneumoniae carbapenemase-2 (KPC-2)-producing CRKP is primarily associated with sequence type (ST) 11.MethodsA total of 152 KPC-2-producing K. pneumoniae ST11 isolates were collected from the respiratory department of a tertiary care hospital in Beijing, China between 2009 and 2018. The genome sequencing of these isolates was performed on the HiSeq X Ten sequencer. Multilocus sequence typing (MLST), capsular type, plasmid replicon types and resistance genes were identified. Fifteen isolates were selected for the subsequent single-molecule real-time (SMRT) sequencing on the PacBio RS II. Alignment of the complete sequences of the plasmids carrying blaKPC–2 and/or virulence genes was performed by using BRIG and Easyfig.ResultsFrom 2012 to 2018, the detection rate of the blaKPC–2-carrying CRKP rose rapidly from 3.3 to 28.1%. KPC-2-producing K. pneumoniae ST11 isolates were dominant in CRKP, which emerged in 2012 and caused several outbreaks. Most isolates exhibited multidrug-resistant to commonly used antibiotics, while all the isolates remained susceptible to tigecycline and polymyxin B. The single nucleotide polymorphism (SNP) analysis showed that all these 152 KPC-2-producing K. pneumoniae ST11 isolates could be divided into three genetically distinct clades (A, B, and C) and eleven subclades (A1–A9 and B1–B2). The majority belonged to clade A with KL47 serotype (n = 117, 77.0%), while KL64 and KL16 were identified in clades B and C, respectively. The blaKPC–2-carrying plasmids exhibited diverse types, namely, IncFII (pHN7A8)/IncR(6/15), IncFII (pHN7A8)/IncpA1763–KPC (5/15), IncFII (pHN7A8) (1/15), IncR (1/15), and IncpA1763–KPC (1/15). The genetic environment of blaKPC–2 showed nine IS26-based composite transposons, which had a basic core structure ISKpn27-blaKPC–2-ΔISKpn6. About 27.6% (42/152) isolates co-carried 2 to 4 virulence marker genes (namely, peg344, iucA, iroB, rmpA, and rmpA2) for hvKp strains. At least three isolates were identified to harbor virulence gene-carrying plasmids.ConclusionKPC-2-producing K. pneumoniae ST11 was highly heterogeneous in our hospital. Transmission of these strains was mainly mediated by twelve high-risk clones. The blaKPC–2-carrying plasmids and genetic environment of blaKPC–2 genes exhibited active evolution in K. pneumoniae ST11. More attention should be paid to the tendency of KPC-2-ST11 to acquire hypervirulent plasmids.

Comparative in silico characterization of Klebsiella pneumoniae hypervirulent plasmids and their antimicrobial resistance genes

BackgroundThe hypervirulent pathotype of Klebsiella pneumoniae (hvKp) is mainly mediated by large virulent plasmids. It seems that these hypervirulent plasmids (HVPs) are accumulating antimicrobial resistance genes (ARGs) and are turning quickly into drug-resistant hypervirulent hybrids. Therefore, molecular mechanisms involved in this convergence needs to be investigated to control their global spread.MethodsIn this study, the complete sequence of 79 non-redundant hypervirulent plasmids were retrieved from GenBank and their genetic features, hypervirulence and antimicrobial resistance patterns (AMR) as well as their putative transmission capability were compared using bioinformatics tools.ResultsThe majority of HVPs belonged to clonal complex (CC)23, and sequence type (ST)11. IncFIB and IncHI1B were the most prevalent plasmid replicon types. Out of 79 plasmids, 78 were positive for iutA and iucA. The iucC, iucB and iucD genes were found in 77 plasmids. Almost 26% of the HVPs were potentially conjugative of which 71% carried AGRs. ARGs against beta-lactams, carbapenems, quinolones, aminoglycosides, chloramphenicols, tetracyclines and macrolides were detected in 30% of HVPs. Class 1 integron and prophage structures harboring multiple ARGs were found in eight plasmids. Insertion sequences (IS)6, IS110 and IS1380 appeared to be important genetic elements in transmission of ARGs.ConclusionsThe high prevalence of iucA and iutA suggests their strong capability for rapid and accurate genetic markers for discrimination of hvKp in the laboratory. This study indicated the important role of mobile genetic elements (MGEs) in the emergence of drug-resistance in hypervirulent strains. The high prevalence of putative conjugative hybrids implies higher incidence of multidrug-resistant (MDR)-hvKp strains in near future.

Epidemiological Features of Klebsiella pneumoniae Infection in the Hepatobiliary System of Patients in Yantai, China, Based on Clinical and Genetic Analyses.

PurposeTo investigate the epidemiological features of Klebsiella pneumoniae infection of the hepatobiliary system of patients in Yantai, China.MethodsThis retrospective study was conducted from January to December 2019 in Yantai Yuhuangding Hospital. Patients for whom K. pneumoniae was isolated from the hepatobiliary system were considered for inclusion. The clinical features and genetic analyses were conducted to explore the epidemiological characteristics.ResultsA total of 88 cases were enrolled, including 69 cases of hypervirulent K. pneumoniae (hvKP) and 19 cases of classical K. pneumoniae (cKP). Community-acquired infections, fever, liver abscess, and C-reactive protein (CRP) and procalcitonin (PCT) levels were significantly higher, while biliary tract disease was lower in the hvKP group compared with the cKP group. Among the 69 hvKP infections, 61 developed a liver abscess. Community-acquired infections, fever, and CRP and PCT levels were higher, whereas biliary tract disease and malignancy were lower in the liver abscess group compared with the non-liver abscess group. All strains were susceptible to the majority of antibiotics tested. All hvKP strains possessed the blaSHV, oqxA, oqxB and fosA resistance genes. K1 and K2 accounted for 78% of hvKP strains. K1 strains belonged to sequence types ST23 and ST700, whereas K2 strains belonged to ST65, ST86 and ST5212. K1 isolates possessed the most virulence determinants, followed by K2 and non-K1/K2 isolates. K2 isolates lacked the allS gene, which was rare in non K1/K2 isolates, but present in most K1 isolates. The mceG gene was only detected in K1 isolates. AllS and virulence determinants were significantly more prevalent in the liver abscess group than in the non-liver abscess group.ConclusionThe prevalence of hvKP among K. pneumoniae infections of the hepatobiliary system is high in Yantai, China. Greater vigilance of hvKP infection is required in clinical and microbiological laboratories.

Prevalence of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae and Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae in China Determined via Mouse Lethality Tests.

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 bla KPC(+)-HvKP and Hv-bla KPC(+)-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, bla KPC(+)-KP, bla KPC(+)-HvKP and Hv-bla KPC(+)-KP. Mouse lethality test confirmed 2 bla KPC(+)-HvKP strains (JS184 and TZ20) and no Hv-bla KPC(+)-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.

Genomic Analysis of Multidrug-Resistant Hypervirulent (Hypermucoviscous) Klebsiella pneumoniae Strain Lacking the Hypermucoviscous Regulators (rmpA/rmpA2).

Hypervirulent K. pneumoniae (hvKP) strains possess distinct characteristics such as hypermucoviscosity, unique serotypes, and virulence factors associated with high pathogenicity. To better understand the genomic characteristics and virulence profile of the isolated hvKP strain, genomic data were compared to the genomes of the hypervirulent and typical K. pneumoniae strains. The K. pneumoniae strain was isolated from a patient with a recurrent urinary tract infection, and then the string test was used for the detection of the hypermucoviscosity phenotype. Whole-genome sequencing was conducted using Illumina, and bioinformatics analysis was performed for the prediction of the isolate resistome, virulome, and phylogenetic analysis. The isolate was identified as hypermucoviscous, type 2 (K2) capsular polysaccharide, ST14, and multidrug-resistant (MDR), showing resistance to ciprofloxacin, ceftazidime, cefotaxime, trimethoprim-sulfamethoxazole, cephalexin, and nitrofurantoin. The isolate possessed four antimicrobial resistance plasmids (pKPN3-307_type B, pECW602, pMDR, and p3K157) that carried antimicrobial resistance genes (ARGs) (blaOXA-1, blaCTX-M-15, sul2, APH(3″)-Ib, APH(6)-Id, and AAC(6′)-Ib-cr6). Moreover, two chromosomally mediated ARGs (fosA6 and SHV-28) were identified. Virulome prediction revealed the presence of 19 fimbrial proteins, one aerobactin (iutA) and two salmochelin (iroE and iroN). Four secretion systems (T6SS-I (13), T6SS-II (9), T6SS-III (12), and Sci-I T6SS (1)) were identified. Interestingly, the isolate lacked the known hypermucoviscous regulators (rmpA/rmpA2) but showed the presence of other RcsAB capsule regulators (rcsA and rcsB). This study documented the presence of a rare MDR hvKP with hypermucoviscous regulators and lacking the common capsule regulators, which needs more focus to highlight their epidemiological role.

Hypervirulent Klebsiella Pneumoniae, an Emerging Cause of Endogenous Endophthalmitis in A French Center: A Comparative Cohort Study

ABSTRACT Introduction Klebsiella pneumoniae (KP) is the most common cause of endogenous endophthalmitis (EE) in Asia, but data in Europe are scarce. We describe eight cases of KP EE compared to a cohort of EE in a French center. Methods EE cases were retrospectively studied between January 2014 and January 2021. KP EE cases were analyzed to assess clinical, microbiological features, and outcome. Results Among the 33 EE cases identified, the first causative agent (24%, n = 8) was KP, mainly (7/8) with hypervirulent phenotype (hvKP). All but one of these cases occurred from December 2019 to January 2021. Contrary to non-KP patients, KP patients had multiple extraocular infective foci (p = .006), all presented with liver abscesses (p < .001), 50% had cerebral involvement (p = .13). Visual outcome was poor in both groups. Conclusion KP is an emerging cause of EE in a French center, consistently associated with liver abscesses, frequent cerebral involvement, and predominance of hvKP strains.

The Role of the Two-Component QseBC Signaling System in Biofilm Formation and Virulence of Hypervirulent Klebsiella pneumoniae ATCC43816.

Hypervirulent Klebsiella pneumoniae (hvKP) is an evolving infectious pathogen associated with high mortality. The convergence of hypervirulence and multidrug resistance further challenges the clinical treatment options for K. pneumoniae infections. The QseBC two-component system (TCS) is a component of quorum-sensing regulatory cascade and functions as a global regulator of biofilm growth, bacterial motility, and virulence in Escherichia coli. However, the functional mechanisms of QseBC in hvKP have not been reported, and we aim to examine the role of QseBC in regulating virulence in hvKP strain ATCC43816. The CRISPR-Cas9 system was used to construct qseB, qseC, and qseBC knockout in ATCC43816. No significant alterations in the growth and antibiotic susceptibility were detected between wild-type and mutants. The deletion of qseC led to an increase of biofilm formation, resistance to serum killing, and high mortality in the G. mellonella model. RNAseq differential gene expression analysis exhibited that gene-associated biofilm formation (glgC, glgP, glgA, gcvA, bcsA, ydaM, paaF, ptsG), bacterial type VI secretion system (virB4, virB6, virB10, vgrG, hcp), and biosynthesis of siderophore (entC, entD, entE) were significantly upregulated in comparison with the wild-type control. In addition, qseB, ygiW (encode OB-family protein), and AraC family transcriptional regulator IT767_23090 genes showed highest expressions in the absence of QseC, which might be related to increased virulence. The study provided new insights into the functional importance of QseBC in regulating the virulence of hvKP.

Molecular Epidemiology of Hypervirulent K. pneumoniae and Problems of Health-Care Associated Infections.

The review describes virulence factors of hypervirulent K. pneumoniae (hvKp) including genes determining its virulence and discusses their role in the development of health-care associated infections. The contribution of individual virulence factors and their combination to the development of the hypervirulence and the prospects of using these factors as biomarkers and therapeutic targets are described. Virulence factors of hvKp and “classical” K. pneumoniae strains (cKp) with no hypervirulence genes were compared. The mechanisms of biofilm formation by hvKp and high incidence of its antibiotic resistance are of particular importance for in health care institutions. Therefore, the development of methods for hvKp identification allowing early prevention of severe hvKp infection and novel approaches to abrogate its spreading are new challenges for epidemiology, infection diseases, and critical care medicine. New technologies including bacteriological and molecular studies make it possible to develop innovative strategies to diagnose and treat infection caused by hvKp. These include monitoring of both genetic biomarkers of hvKp and resistance plasmid that carry of virulence genes and antibiotic resistance genes, creation of immunological agents for the prevention and therapy of hvKp (vaccines, monoclonal antibodies) as well as personalized hvKp-specific phage therapies and pharmaceuticals enhancing the effect of antibiotics. A variety of approaches can reliably prepare our medicine for a new challenge: spreading of life-threatening health-care associated infections caused by antibiotic-resistant hvKp strains.

Occurrence of Hypervirulent Klebsiella pneumoniae in Clinical Settings and Lytic Potential of Bacteriophages Against the Isolates

Background: Antibiotic resistance is a major health hazard around the globe. Hypervirulent Klebsiella Pneumoniae (hvKp) is associated with hospital-acquired and community-acquired infections. Since there is a lack of new antibiotics against multidrug-resistant (MDR) pathogens, phage therapy might provide an alternative approach to confer antibiotic resistance. Objectives: This study aimed to estimate the occurrence of hvKp and characterize the bacteriophage against the hvKp prevalence in clinical settings, which might be used as an alternative to antibiotics. Methods: Different clinical samples (n = 50) were collected to isolate K. pneumoniae, and the assessment of multidrug resistance was carried out based on the Clinical and Laboratory Standards Institute guidelines (2020). The bacteriophage was isolated from hospital waste, and the double agar overlay method was used for phage purification and propagation. Spot test and one-step curve were performed to determine host-phage interactions. For the evaluation of phage stability in environmental conditions, the phage was incubated at various ranges of temperature, pH, and chloroform. Results: Out of the collected samples, 22 (44%) isolates were confirmed as K. pneumoniae. Among confirmed K. pneumoniae isolates, a total of 11 (50%) isolates were detected as hvKp. Moreover, 14 (64%) isolates were detected as MDR, out of which 5 (35%) isolates were among hvKp phenotypes. Maximum resistance was observed against ampicillin (86%) followed by ceftriaxone (81%) which was the highest among cephalosporins. The isolated bacteriophage showed a broad host range, short latent period, and stability. Overall, 16 isolates (85%) of K. pneumoniae were susceptible to phage infection, among which 12 isolates were MDR (75%); however, all 5 (100%) hvKp isolates were susceptible to phage infection. One-step growth analysis revealed a burst size of 190 phages/host bacterial cells with a short latent period of 24 minutes. Conclusions: Altogether, the significant prevalence of hvKp was estimated in clinical settings, and the isolated bacteriophage showed significant lytic activity as it killed all the hvKp strains. Phage therapy might be exploited and used as a potential alternative therapeutic approach against infections caused by this resistant pathogen.

Hypervirulent Klebsiella pneumoniae Strains Modulate Human Dendritic Cell Functions and Affect TH1/TH17 Response.

Hypervirulent Klebsiella pneumoniae (Hv-Kp) strains have emerged as pathogens causing life-threatening, invasive disease even in immunocompetent hosts. Systemic dissemination usually occurs following perturbations of the gut microbiota and is facilitated by Hv-Kp resistance to phagocytosis and complement activity. Hv-Kp are usually associated with K1 or K2 capsular types, produce several iron uptake systems (e.g., aerobactin and salmochelin) and are often but not invariably, capsular material hyper-producers (hypermucoviscous phenotype: HMV). Whether Hv-Kp escape the immune response at mucosal site is unknown. In this work, we studied the effects of Hv-Kp on human dendritic cells (DCs), central players of the IL-23/IL-17 and IL-12/IFN-γ axis at mucosal sites, essential for pathogen clearance. Four Hv-Kp and HMV strains were selected and their activity on DC maturation and cytokine production was compared to that of non-virulent Kp strains with classic or HMV phenotypes. While the maturation process was equally induced by all Kp strains, significant differences between virulent and non-virulent strains were found in the expression of genes for cytokines involved in T-cell activation and differentiation. The non-virulent KP04C62 and the classic Kp, KPC157 induced high expression of TH1 (IL-12p70 and TNFα) and TH17 cytokines (IL-23, IL-1β and IL-6), while Hv-Kp poorly activated these cytokine genes. Moreover, conditioned media from DCs cultured with non-virulent Kp, either classical or hypercapsulated, induced the activation of IL-17 and IFN-γ genes in preactivated CD4+-cells suggesting their TH17/TH1 differentiation. Conditioned media from Hv-Kp poorly activated IL-17 and IFN-γ genes. In summary, our data indicate that Hv-Kp interfere with DC functions and T-cell differentiation and suggest that the escape from the IL-23/IL-17 and IL-12/IFN-γ axes may contribute to pathogen dissemination in immunocompetent hosts.

Distribution, characterization, and antibiotic resistance of hypervirulent Klebsiella pneumoniae isolates in a Chinese population with asymptomatic bacteriuria

BackgroundAsymptomatic bacteriuria (ASB) frequently occurs among all ages and may develop into urinary tract infections (UTIs). Hypervirulent Klebsiella pneumoniae (hvKP) has become a new threat to human health. In our study, we aimed to investigate the epidemiological characteristics of hvKP in population with ASB.ResultsA total of 61 K. pneumoniae isolates were collected from 7530 urine samples between October and December 2020. The strains were sensitive to most of the antimicrobial agents tested, but a polymyxin resistant strain was found (MIC>16 μg/mL). Three serotypes were detected, including K1 (16.4%, 10/61), K5 (1.6%, 1/61) and K57 (3.2%, 2/61). Four strains (KPNY9, KPNY31, KPNY40, and KPNY42) carried a combination of two or more hypervirulent markers (peg-344, iroB, iucA, prmpA, and prmpA2), and their survival rates after Galleria mellonella infection were lower than those of the other strains (40.0 vs. 70.0%), suggesting that they were hvKP. These hvKP strains with lower biofilm forming ability than classical K. pneumoniae (0.2625 ± 0.0579 vs. 0.6686 ± 0.0661, P = 0.033) were identified as belonging to K2-ST65, K2-ST86, K57-ST592, and K2-ST5559 (a new ST type). KPNY31 (ST5559) shared a close genetic relationship with KPNY42 (ST86) and other ST86 isolates, which have been detected in both nosocomial and community-acquired infections.ConclusionsThe hvKP with relatively weak biofilm formation was detected in a population with ASB, which was more likely to cause bacteremia and serious consequences. A novel sequence type (ST5559) hvKP derived from ST86 was found. Therefore, hvKP should be monitored in the population with ASB.

High Prevalence and Fitness of IncFrepB Carrying qnrS1 in Hypervirulent Klebsiella pneumoniae Isolates.

Objective: This study aimed to reveal the prevalence and fitness of qnrS1-carrying plasmids in hypervirulent Klebsiella pneumoniae (hvKP) isolates. Materials and Methods: Two hundred ninety-nine hvKP strains carrying qnrS1 were collected and screened for resistance genes using PCR and sequencing. The location of qnrS1 and rmpA2 was identified by Southern blotting. The transferability and fitness of qnrS1-carrying plasmids were analyzed by conjugation experiments and plasmid stability assay. Result: In 299 hvKP isolates, the most frequently detected capsular serotype was K64 (81.9%, 245/299), followed by K1 (4.7%, 14/299) and K2 (3.7%, 11/299). All K64-hvKP were sequence type (ST) 11. The qnrS1 and rmpA2 gene mainly was located on the ∼70-210 kb IncFrepB and ∼170-220 kb IncFIB plasmid, respectively. QnrS1-carrying plasmids could be transferred into Escherichia coli J53. However, the plasmid was transferred at a low rate of 13.4% (40/299). The 40 donor isolates belong to 4 STs-ST11, ST700, ST592, and ST86, and none contains the CRISPR-Cas loci. CRISPR-Cas loci were mainly found in ST23 K. pneumoniae. The relative fitness (RF) of qnrS1-carrying plasmids in ST86 and ST11 (cotransfer with blaTEM-1 genes) was more than one and enhanced during cultivation, especially in ST86. However, the RF of qnrS1-carrying plasmids in ST592 and ST700 showed a high fitness cost. Whole-genome sequencing showed that the qnrS1-carrying plasmids in ST86 harbored more maintenance modules (SOS inhibitor protein psiB, parA, and parB partition systems) and insertion sequence (IS) elements (IS91, IS481-like, IS1380), indicating that the qnrS1-carrying plasmid in ST86 is more stable than the other types of qnrS1-carrying plasmids. Conclusion: QnrS1-carrying IncFrepB plasmids were highly prevalent and show polymorphism in hvKP strains. The qnrS1-carrying IncFrepB plasmid in ST86 hvKP should be highlighted due to its remarkable adaptability advantages.

Ruptured Emphysematous Prostatic Abscess Caused by K1-ST23 Hypervirulent Klebsiella pneumoniae Presenting as Brain Abscesses: A Case Report and Literature Review

Emphysematous prostatic abscess (EPA) is an extremely rare but potentially fatal urinary tract infection (UTI). Here, we describe a case (a 69-year-old male with prediabetes) of ruptured EPA caused by a hypervirulent Klebsiella pneumoniae (hvKp) K1-ST23 strain, presenting as motor aphasia. Our patient presented with ruptured EPA concurrent with various severe systemic pyogenic complications (e.g., urethro-prostatic fistula, ascending UTIs, epididymal and scrotal abscesses, and liver, lung, and brain abscesses). Whole-body computed tomography (CT) and next-generation sequencing (NGS) were useful for the detection of ruptured EPA and its systemic complications, and for identification of K1-ST23 hvKp strains, respectively. Subsequently, the infections were successfully treated with aggressive antimicrobial therapy and multiple surgical procedures. This case highlights the significance of awareness of this rare entity, the clinical importance of CT for the early diagnosis of EPA and the detection of its systemic complications in view of hvKp being an important causative organism of severe community-acquired UTI, and the usefulness of NGS to identify hvKp strains.

Clinical and Imaging Characteristics of Primary Severe Community-Acquired Pneumonia Caused by Hypervirulent Klebsiella Pneumoniae.

To investigate the CT imaging features and microbial phenotypes of primary severe community-acquired pneumonia caused by hypervirulent Klebsiella pneumoniae (hvKp). Patients diagnosed with primary hvKp pneumonia were included, and their clinical data were analyzed, including the baseline characteristics and CT imaging results. After hypermucoviscosity phenotyping, the strains, serological types, and virulence genes of hvKp were identified using multiplex PCR. Twelve patients with primary hvKp pneumonia were included (11 males, 1 female). All patients were infected via respiratory tract inhalation. Ten patients were long-term drinkers. Four patients (33.3%), who were long-term alcohol abusers, died within 30 days after diagnosis. No extrapulmonary metastatic infection was found in any patient. The imaging of lung lesions at the early disease stage exhibited an extensive consolidation in the lungs. As the disease progressed, the most common imaging features were pleural effusion (9/12), cavitation and necrosis (8/12), and pneumothorax (3/12). The serological typing of the capsular polysaccharides on hvKp strains were K1 (6/12) and K2 (6/12). Furthermore, the virulence genotyping showed rmpA (11/12), magA (11/12), ureA (12/12), mrkD (12/12), fim-1 (12/12), wabG (12/12), ybtS (12/12), and iucB (11/12). Primary severe community-acquired hvKp-associated pneumonia is more common in men, especially those with a long-term history of alcohol consumption. CT scanning at the early disease stage mostly showed extensive pulmonary consolidation, which was prone to be combined with cavitation, necrosis, and pleural effusion. K1 and K2 serotypes were identified among the hvKp strains, which were not prone to form extrapulmonary metastasis via the bloodstream.

High Prevalence of Klebsiella pneumoniae Infections in AnHui Province: Clinical Characteristic and Antimicrobial Resistance.

BackgroundKlebsiella pneumoniae (K. pneumoniae) causes community-acquired and hospital-acquired pneumonia. The mortality rates of invasive infections caused by hypervirulent K. pneumoniae (HvKP) are extremely high. However, the microbiological characteristics and clinical manifestations of K. pneumoniae in AnHui province still remain unclear.PurposeTo show the high prevalence of HvKP infections regarding clinical characteristics and antimicrobial resistance in Anhui province.Patients and MethodsA retrospective analysis was conducted to study the clinical data of 115 strains of K. pneumoniae from July 2019 to March 2020 in The First Affiliated Hospital of AnHui Medical University. The virulence genes, capsular types, carbapenemase genes, and molecular subtypes of these hypervirulent isolates were detected.ResultsOverall, 59.1% (68/115) cases were HvKP infections, mainly from the department of intensive care unit (ICU, n=14, 20.6%) and the department of respiratory and critical care (n=13, 19.1%). K2 was the most prevalent capsular serotype (n=26), followed by K1 (n=21). The results of MLST identification of 68 strains showed that ST23 (n=15, 22.1%) was the most common type of ST, followed by ST11 and ST65 (n=12, 17.6%), ST86 (n=9, 13.2%), and ST412 (n=6, 8.8%). Among 68 hvKP strains, 12 isolates were carbapenem resistant, and all except two harboured KPC.ConclusionThe high incidence of carbapenemase producing HvKP in the Anhui province, especially the higher mortality of HvKP, should be paid more attention. Meanwhile, epidemiological surveillance and clinical treatment strategies should be continuously determined and implemented.

Interaction of Klebsiella pneumoniae with tissue macrophages in a mouse infection model and ex-vivo pig organ perfusions: an exploratory investigation

SummaryBackgroundHypervirulent Klebsiella pneumoniae (hvKp) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated with relapsing infections. Other K pneumoniae strains (non-hvKp), including lineages that have acquired carbapenem resistance, do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanism underpinning abscess formation in hvKp infections.MethodsIn this exploratory investigation, to study the pathophysiology of abscess formation, mice were intravenously infected with 106 colony forming units (CFU) of either hvKp isolates (six strains) or non-hvKp isolates (seven strains). Intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy of sliced cryopreserved organs of mice collected 30 min, 6 h, and 24 h after infection with the aim to provide data of bacterial association to Kupffer cells in the liver and to the different tissue macrophages in the spleen. Microbiological and microscopy analysis of an ex-vivo model of pig liver and spleen infection were used to confirm within-macrophage replication. Pig organs were perfused with heparinised, autologous pig's blood and injected with 6·5 × 107 CFU of hvKp K2 sequence type 25 strain GMR151. Blood and tissue biopsies collected before infection and 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h after infection were used to measure bacterial counts and to identify the subcellular localisation of bacteria by immunohistochemistry analysis.FindingsWe show that hvKp resisted phagocyte-mediated clearance and replicated in mouse liver macrophages to form clusters 6 h after infection, with a mean of 7·0 bacteria per Kupffer cell (SD 6·2); however, non-hvKp were efficiently cleared (mean 1·5 bacteria per cell [SD 1·1]). HvKp infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation as early as 24 h post-infection. Experiments in pig organs which share a high functional and anatomical resemblance to human organs, provided strong evidence for the propensity of hvKp to replicate within the hepatic macrophages.InterpretationThese findings show subversion of innate immune processes in the liver by K pneumoniae and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hvKp strains to cause hepatic abscesses.FundingUniversity of Oxford and a Royal Society Wolfson grant funded biosafety facility.

Mobilization of the nonconjugative virulence plasmid from hypervirulent Klebsiella pneumoniae

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.