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Objective: This study used whole-genome sequencing (WGS) to explore the genetic diversity, virulence factors, and antimicrobial resistance determinants of string test-positive Klebsiella pneumoniae (KP) over a 4-year surveillance period in Huzhou, China. Methods: In total, 632 clinical isolates were collected via hospital surveillance from 2020 to 2023; 100 were positive in the string test and these 100 strains were subjected to antimicrobial susceptibility testing using an agar dilution method followed by WGS. Results: The resistance rates to cefotaxime (77.0%), trimethoprimsulfamethoxazole (67.0%), and nalidixic acid (64.0%) were high. Multilocus sequence typing revealed high genetic diversity; there were 33 sequence types (STs) and 15 capsular serotypes. The most common ST was ST23 (16.0%) and the most common capsular serotype was K1 (22.5%). Virulome analysis revealed among-strain differences in virulence factors that affected bacterial adherence, efflux pump action, iron uptake, nutritional factors, metabolic regulation, the secretion system, and toxin production. The Kleborate strain-specific virulence scores of all 100 string test-positive KPs were derived: 28 strains scored 5, 28 scored 4, 21 scored 3, 12 scored 1, and 11 scored 0. All 77 strains with scores of 3 to 5 contained the iucA gene. The phylogeny based on whole-genome single nucleotide polymorphisms (wgSNPs) indicated high clonality; the string testpositive KP strains were grouped into six clades. Closely related isolates in each genetic cluster usually shared STs. Conclusion: The present study highlights the significance of the KP iucA gene in terms of hypervirulence and the diverse genotypes of string test-positive KP strains isolated in Huzhou hospitals. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as nonredundant complementary tools of the innate immune system. [ABSTRACT FROM AUTHOR]

Aims Hypervirulent Klebsiella pneumoniae (hvKp) causes invasive community-acquired infections in healthy individuals, and hypermucoviscosity (HMV) is the main phenotype associated with hvKp. This study investigates the impact of microaerobic environment availability on the mucoviscosity of K. pneumoniae. Methods and results By culturing 25 clinical strains under microaerobic and aerobic environments, we observed a notable reduction in mucoviscosity in microaerobic environments. RNA sequencing and qRT-PCR revealed downregulated expressions of capsule synthesis genes (galf, orf2, wzi, wza, wzb, wzc, wcaj, manC, manB , and ugd) and regulatory genes (rmpA, rmpD , and rmpC) under microaerobic conditions. Transmission electron microscopy and Indian ink staining analysis were performed, revealing that the capsular thickness of K. pneumoniae decreased by half in microaerobic conditions compared to aerobic conditions. Deletion of rmpD and rmpC caused the loss of the HMV phenotype in both aerobic and microaerobic conditions. However, compared to wild-type strain in microaerobic condition, only rmpD overexpression strain, and not rmpC overexpression strain, displayed a significant increase in capsule thickness in microaerobic conditions. Conclusions Microaerobic conditions can suppress the mucoviscosity of K. pneumoniae , but this suppression can be overcome by altering the expression of rmpD , indicating a specific function for rmpD in the oxygen environmental adaptation of K. pneumoniae. [ABSTRACT FROM AUTHOR]

Background: Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Methods: Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Results: Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. Conclusion: This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments. [ABSTRACT FROM AUTHOR]

This study aimed to screen antibiotic resistance and virulence genes in carbapenem‐resistant hypermucoviscous Klebsiella pneumoniae isolates from an Egyptian hospital. Among 38 previously confirmed carbapenem‐nonsusceptible K. pneumoniae isolates, a string test identified three isolates as positive for hypermucoviscosity. Phenotypic characterization and molecular detection of carbapenemase‐ and virulence‐encoding genes were performed. PCR‐based multilocus sequence typing and phylogenetics were used to determine the clonality and global epidemiology of the strains. The coexistence of virulence and resistance genes in the isolates was analyzed statistically using a chi‐square test. Three isolates showed the presence of carbapenemase‐encoding genes (blaNDM, blaVIM, and blaIMP), adhesion genes (fim‐H‐1 and mrkD), and siderophore genes (entB); the isolates belonged to sequence types (STs) 101, 1310, and 1626. The relatedness between these sequence types and the sequence types of globally detected hypermucoviscous K. pneumoniae that also harbor carbapenemases was determined. Our analysis showed that the resistance and virulence profiles were not homogenous. Phylogenetically, different clones clustered together. There was no significant association between the presence of resistance and virulence genes in the isolates. There is a need for periodic surveillance of the healthcare settings in Egypt and globally to understand the true epidemiology of carbapenem‐resistant, hypermucoviscous K. pneumoniae. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (KP) is defined according to hypermucoviscosity or various virulence factors and is clinically associated with community-acquired liver abscess (CLA). In this study, we investigated the clinical and microbiological characteristics of KP and significant factors associated with hypervirulence. The clinical characteristics, antimicrobial susceptibility, hypermucoviscosity, serotypes, hypervirulence-related genes, and biofilm formation of 414 KP isolates collected from the Keimyung University Dongsan Hospital between December 2013 and November 2015 were analyzed according to CLA. Significant risk factors for hypervirulent KP (HvKP) associated with CLA were investigated using logistic regression analysis. Notably, 155 (37.4%) isolates were hypermucoviscous, and 170 (41.1%) harbored aerobactin. CLA was present in 34 cases (8.2%). Epidemiology and treatment outcomes did not differ significantly between the CLA and non-CLA groups. The CLA group had significantly higher antibiotic susceptibility, K1/K2, rmpA, magA, allS, kfu, iutA, string test-positive result, and biofilm mass. Multivariate logistic regression revealed rmpA (OR, 5.67; 95% CI, 2.09–15.33; p = 0.001), magA (OR, 2.34; 95% CI, 1.01–5.40; p = 0.047), and biofilm mass >0.80 (OR, 2.13; 95% CI, 1.00–4.56; p = 0.050) as significant risk factors for CLA. rmpA was identified as the most significant risk factor for CLA among KP strains, implying that it is an important factor associated with HvKP. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.

ABSTRACT Hypermucoviscosity (HMV) is a phenotype that is commonly associated with hypervirulence in Klebsiella pneumoniae. The factors that contribute to the emergence of HMV subpopulations remain unclear. In this study, eight K. pneumoniae strains were recovered from an inpatient who had been hospitalized for 20 days. Three of the isolates exhibited a non-HMV phenotype, which was concomitant with higher biofilm formation than the other five HMV isolates. All eight isolates were highly susceptible to serum killing, albeit HMV strains were remarkably more infective than non-HMV counterparts in a mouse model of infection. Whole genome sequencing (WGS) showed that the eight isolates belonged to the K57-ST412 lineage. Average nucleotide identity (FastANIb) analysis indicated that eight isolates share 99.96% to 99.99% similarity and were confirmed to be the same clone. Through comparative genomics analysis, 12 non-synonymous mutations were found among these isolates, eight of which in the non-HMV variants, including rmpA (c.285delG) and wbaP (c.1305T > A), which are assumed to be associated with the non-HMV phenotype. Mutations in manB (c.1318G > A), dmsB (c.577C > T) and tkt (c.1928C > A) occurred in HMV isolates only. RNA-Seq revealed transcripts of genes involved in energy metabolism, carbohydrate metabolism and membrane transport, including cysP, cydA, narK, tktA, pduQ, aceB, metN, and lsrA, to be significantly dysregulated in the non-HMV strains, suggesting a contribution to HMV phenotype development. This study suggests that co-occurrence of HMV and non-HMV phenotypes in the same clonal population may be mediated by mutational mechanisms as well as by certain genes involved in membrane transport and central metabolism.IMPORTANCEK. pneumoniae with a hypermucoviscosity (HMV) phenotype is a community-acquired pathogen that is associated with increased invasiveness and pathogenicity, and underlying diseases are the most common comorbid risk factors inducing metastatic complications. HMV was earlier attributed to the overproduction of capsular polysaccharide, and more data point to the possibility of several causes contributing to this bacterial phenotype. Here, we describe a unique event in which the same clonal population showed both HMV and non-HMV characteristics. Studies have demonstrated that this process is influenced by mutational processes and genes related to transport and central metabolism. These findings provide fresh insight into the mechanisms behind co-occurrence of HMV and non-HMV phenotypes in monoclonal populations as well as potentially being critical in developing strategies to control the further spread of HMV K. pneumoniae.

Abstract Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-β-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 108 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 108 CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

BackgroundConvergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307.MethodsHypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics.ResultsOur results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways.ConclusionThis study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

Klebsiella pneumoniae is a major human pathogen as it is responsible for various infections. In the past years hypervirulent K. pneumoniae (hvKP) emerged and disseminated worldwide. In this review a summary will be given about epidemiology, detection and antibiotic resistance of hypervirulent K. pneumoniae. A common feature of hypervirulent K. pneumoniae is a combined expression of several virulence factors. A mucoviscosus phenotype, certain capsulare serotypes (e.g.: K1, K2, K28, K47, K63) together with additional genetic markers namely, magA , rmpA or iucABCD, are needed in combinations to achieve the hypervirulent pathotype. Plasmid coded virulence determinants are also detected, that indicates horizontal gene transfer of hypervirulence factors in K. pneumoniae. Interestingly, infections caused by hypervirulent K. pneumoniae occur usually in the community in otherwise healthy people, and during these infections multiple infection sites are detected. Clinical pictures include both invasive infections and local abscess formation. Pyogenic liver abscess is the most frequently reported clinical manifestation and abscess formation in brain, spleen and lung are also diagnosed. Additionally, meningitis, endophthalmitis, trombophlebitis, pneumonia can also develop. In the early reports, hypervirulent K. pneumoniae strains exhibited enhanced virulence but these were susceptible to commonly used antibiotics. However, recently KPC, VIM, NDM and OXA-48 carbapenemase producing hypervirulent K. pneumoniae strains are increasingly reported, furthermore, well-known high-risk K. pneumoniae clones (e.g.: ST11, ST147, ST307) can develop hypervirulent pathotype, that poses an even more alarming challenge. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is an opportunistic pathogen that mainly causes nosocomial infections and hospital-associated pneumonia in elderly and immunocompromised people. However, multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKp) has emerged recently as a serious threat to global health that can infect both immunocompromised and healthy individuals. It is scientifically established that plasmid-mediated regulator of mucoid phenotype genes (rmpA and rmpA2) and other virulence factors (aerobactin and salmochelin) are mainly responsible for this phenotype. In this study, we collected 23 MDR-hvKp isolates and performed molecular typing, whole genome sequencing, comparative genomic analysis, and phenotypic experiments, including the Galleria mellonella infection model, to reveal its genetic and phenotypic features. Meanwhile, we discovered two MDR-hvKp isolates (22122315 and 22091569) that showed a wide range of hypervirulence and hypermucoviscosity without rmpA and rmpA2 and any virulence factors. In phenotypic experiments, isolate 22122315 showed the highest hypervirulence (infection model) with significant mucoviscosity, and conversely, isolate 22091569 exhibited the highest mucoviscosity (string test) with higher virulence compared to control. These two isolates carried carbapenemase (blaKPC − 2), β-lactamase (blaOXA − 1, blaTEM − 1B), extended-spectrum β-lactamase (ESBL) genes (blaCTX − M − 15, blaSHV − 106), outer membrane protein-coding genes (ompA), fimbriae encoding genes (ecpABCDER), and enterobactin coding genes (entAB, fepC). In addition, single nucleotide polymorphism analysis indicated that both isolates, 22122315 and 22091569, were found to have novel mutations in loci FEBNDAKP_03184 (c. 2084A > C, p. Asn695Thr), and EOFMAFIB_02276 (c. 1930C > A, p. Pro644Thr), respectively. Finally, NCBI blast analysis suggested these mutations are located in the wzc of the capsule polysaccharide (cps) region and are responsible for putative tyrosine kinase. This study would be a strong reference for enhancing the current understanding of identifying the MDR-hvKp isolates that lacked both mucoid regulators and virulence factors. [ABSTRACT FROM AUTHOR]

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are capable of causing various systemic infections in both humans and animals. In this study, we isolated and characterized 30 E. coli strains from the parenchymatic organs and brains of young (E. coli: three strains carried cnf1, encoding cytotoxic necrotizing factor type 1, the key virulence gene of necrotoxigenic E. coli (NTEC), and one carried eae encoding intimin, the key virulence gene of enteropathogenic E. coli (EPEC). An investigation of the integration sites of pathogenicity islands (PAIs) and the presence of prophage-related sequences showed that the strains carry diverse arrays of mobile genetic elements, which may contribute to their antimicrobial resistance and virulence patterns. Our work is the first to describe ExPEC strains from camels, and points to their veterinary pathogenic as well as zoonotic potential in this important domestic animal.

Hypervirulent Klebsiella pneumoniae (KP) is defined according to hypermucoviscosity or various virulence factors and is clinically associated with community-acquired liver abscess (CLA). In this study, we investigated the clinical and microbiological characteristics of KP and significant factors associated with hypervirulence. The clinical characteristics, antimicrobial susceptibility, hypermucoviscosity, serotypes, hypervirulence-related genes, and biofilm formation of 414 KP isolates collected from the Keimyung University Dongsan Hospital between December 2013 and November 2015 were analyzed according to CLA. Significant risk factors for hypervirulent KP (HvKP) associated with CLA were investigated using logistic regression analysis. Notably, 155 (37.4%) isolates were hypermucoviscous, and 170 (41.1%) harbored aerobactin. CLA was present in 34 cases (8.2%). Epidemiology and treatment outcomes did not differ significantly between the CLA and non-CLA groups. The CLA group had significantly higher antibiotic susceptibility, K1/K2, rmpA, magA, allS, kfu, iutA, string test-positive result, and biofilm mass. Multivariate logistic regression revealed rmpA (OR, 5.67; 95% CI, 2.09–15.33; p = 0.001), magA (OR, 2.34; 95% CI, 1.01–5.40; p = 0.047), and biofilm mass >0.80 (OR, 2.13; 95% CI, 1.00–4.56; p = 0.050) as significant risk factors for CLA. rmpA was identified as the most significant risk factor for CLA among KP strains, implying that it is an important factor associated with HvKP.

ABSTRACT Klebsiella pneumoniae is a hospital-associated pathogen primarily causing urinary tract infections (UTIs), pneumonia, and septicemia. Two challenging lineages include the hypervirulent strains, causing invasive community-acquired infections, and the carbapenem-resistant classical strains, most frequently isolated from UTIs. While hypervirulent strains are often characterized by a hypermucoid phenotype, classical strains usually present with low mucoidy. Since clinical UTI isolates tend to exhibit limited mucoidy, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract and select against mucoid isolates. We found that both hypervirulent K. pneumoniae and classical Klebsiella UTI isolates significantly suppressed mucoidy when cultured in urine without reducing capsule abundance. A genetic screen identified secondary mutations in the wzc tyrosine kinase that overcome urine-suppressed mucoidy. Over-expressing Wzc variants in trans was sufficient to boost mucoidy in both hypervirulent and classical Klebsiella UTI isolates. Wzc is a bacterial tyrosine kinase that regulates capsule polymerization and extrusion. Although some Wzc variants reduced Wzc phospho-status, urine did not alter Wzc phospho-status. Urine does, however, increase K. pneumoniae capsule chain length diversity and enhance cell-surface attachment. The identified Wzc variants counteract urine-mediated effects on capsule chain length and cell attachment. Combined, these data indicate that capsule chain length correlates with K. pneumoniae mucoidy and that this extracellular feature can be fine-tuned by spontaneous Wzc mutations, which alter host interactions. Spontaneous Wzc mutation represents a global mechanism that could fine-tune K. pneumoniae niche-specific fitness in both classical and hypervirulent isolates. IMPORTANCE Klebsiella pneumoniae is high-priority pathogen causing both hospital-associated infections, such as urinary tract infections, and community-acquired infections. Clinical isolates from community-acquired infection are often characterized by a tacky, hypermucoid phenotype, while urinary tract isolates are usually not mucoid. Historically, mucoidy was attributed to capsule overproduction; however, recent reports have demonstrated that K. pneumoniae capsule abundance and mucoidy are not always correlated. Here, we report that human urine suppresses K. pneumoniae mucoidy, diversifies capsule polysaccharide chain length, and increases cell surface association. Moreover, specific mutations in the capsule biosynthesis gene, wzc, are sufficient to overcome urine-mediated suppression of mucoidy. These Wzc variants cause constitutive production of more uniform capsular polysaccharide chains and increased release of capsule from the cell surface, even in urine. These data demonstrate that K. pneumoniae regulates capsule chain length and cell surface attachment in response host cues, which can alter bacteria-host interactions.

Klebsiella pneumoniae is an opportunistic pathogen that mainly causes nosocomial infections and hospital-associated pneumonia in elderly and immunocompromised people. However, multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKp) has emerged recently as a serious threat to global health that can infect both immunocompromised and healthy individuals. It is scientifically established that plasmid-mediated regulator of mucoid phenotype genes (rmpA and rmpA2) and other virulence factors (aerobactin and salmochelin) are mainly responsible for this phenotype. In this study, we collected 23 MDR-hvKp isolates and performed molecular typing, whole genome sequencing, comparative genomic analysis, and phenotypic experiments, including the Galleria mellonella infection model, to reveal its genetic and phenotypic features. Meanwhile, we discovered two MDR-hvKp isolates (22122315 and 22091569) that showed a wide range of hypervirulence and hypermucoviscosity without rmpA and rmpA2 and any virulence factors. In phenotypic experiments, isolate 22122315 showed the highest hypervirulence (infection model) with significant mucoviscosity, and conversely, isolate 22091569 exhibited the highest mucoviscosity (string test) with higher virulence compared to control. These two isolates carried carbapenemase (blaKPC − 2), β-lactamase (blaOXA − 1, blaTEM − 1B), extended-spectrum β-lactamase (ESBL) genes (blaCTX − M − 15, blaSHV − 106), outer membrane protein-coding genes (ompA), fimbriae encoding genes (ecpABCDER), and enterobactin coding genes (entAB, fepC). In addition, single nucleotide polymorphism analysis indicated that both isolates, 22122315 and 22091569, were found to have novel mutations in loci FEBNDAKP_03184 (c. 2084A > C, p. Asn695Thr), and EOFMAFIB_02276 (c. 1930C > A, p. Pro644Thr), respectively. Finally, NCBI blast analysis suggested these mutations are located in the wzc of the capsule polysaccharide (cps) region and are responsible for putative tyrosine kinase. This study would be a strong reference for enhancing the current understanding of identifying the MDR-hvKp isolates that lacked both mucoid regulators and virulence factors.

Abstract Background The development of tigecycline resistance in hypervirulent Klebsiella pneumoniae strains has resulted in decreased virulence that is associated with reduced production of capsular polysaccharides (CPS). In this study, we investigated the mechanisms that link tigecycline susceptibility to decreased virulence. Methods We compared transcriptomes from tigecycline-susceptible wild-type strains and tigecycline-resistant mutants using mRNA sequencing. ompR-overexpressed and ompR-deleted mutants were constructed from wild-type strains and tigecycline-resistant mutants, respectively. Antibiotic susceptibility tests were performed, and string tests and precipitation assays were conducted to identify phenotypic changes related to tigecycline susceptibility and ompR expression. Bacterial virulence was assessed by serum resistance and Galleria mellonella infection assays. Results Transcriptomic analyses demonstrated a significant decrease in the expression of ompK35 in the tigecycline-resistant mutants. We observed that tigecycline-resistant mutants overexpressed ompR, and that the expression of ompK35 was regulated negatively by ompR. While tigecycline-resistant mutants and ompR-overexpressed mutants exhibited reduced hypermucoviscosity and virulence, deletion of ompR from tigecycline-resistant mutants restored their hypermucoviscosity and virulence. Conclusions In hypervirulent K. pneumoniae strains, ompR expression, which is regulated by exposure to tigecycline, may affect the production of CPS, leading to bacterial virulence.

Human infections induced by Klebsiella pneumoniae (K. pneumoniae) include pneumonia; urinary tract infections, liver abscesses, bacteremia, and others. The introduction and spread of the hypervirulent K. pneumoniae (hvKp) strains have raised the number of persons who are already susceptible to infections, including those who are healthy or immune-compromised. Infections can occur worldwide; however, they are particularly prevalent in the Asia-Pacific area. Virulence plasmids as well as other conjugal components contain the genetic material that gives hvKp its hypervirulence phenotype. Although the vast majority of hvKp isolates are antibiotic-susceptible, the incidence of virulent as well as resistant isolates, such as carbapenem-resistant hvKp isolates, is continuously growing. Multidrug resistance (MDR) and increased virulence of these strains may be the cause of the subsequent clinical crisis. This study aimed to review and analyse the epidemiology, the factors associated with hypervirulence, and the mechanisms of antibiotic resistance of the hvKp strains in order to provide a better understanding of the basic biology of these strains.

Binzhi Dan,* Heping Dai,* Dangui Zhou, Hongfang Tong, Mei Zhu Department of Clinical Laboratory, the Affiliated Chaohu Hospital of Anhui Medical University, Chaohu, Anhui, People’s Republic of China*These authors contributed equally to this workCorrespondence: Mei Zhu, Tel +86 551 8232 4254, Email meizhu532@sina.comObjective: To conduct epidemiological analysis of Klebsiella pneumoniae (K. pneumoniae) with hypervirulence, and to investigate its drug resistance phenotype, Extended-spectrum β-lactamase (ESBLs) gene, virulence factor, capsular serotype and biofilm formation, so as to provide theoretical basis for further understanding of the drug resistance mechanism of K. pneumoniae with hypervirulence.Methods: K. Pneumoniae were isolated from clinical samples collected from inpatients. All strains were identified by VITEK2 Compact using fully automatic microbial analyzer, the minimal inhibitory concentration (MIC) of antibiotics was determined by microbroth dilution test. The double disk diffusion method was used to detect the production of ESBLs, modified carbapenem inactivation method (mCIM) was used to detect the production of carbapenemase, and hypermucoviscosity phenotype was detected by wire drawing test. PCR was used to detect ESBLs gene, virulence factor and capsular serotype. Crystal violet staining was used to detect the ability of biofilm formation.Results: The ESBLs genes detected in this study included strains blaTEM 35 (36.5%), blaSHV 51 (53.1%), and blaCTX-M 49 (51.0%). Most strains carried multiple ESBLs genes, but not all of them produce ESBLs. K1 and K2 accounted for 14.6% and 11.5% respectively. Most (91.7%) strains carried the fimH gene, and the other virulence genes were ybtS (53.1%), entB (46.9%), rmpA (41.7%), aerobactin (32.3%), allS (15.6%), kfu (15.6%). Of all the Klebsiella pneumoniae strains, 33 (34.4%) exhibited ESBLs phenotype, 16 (16.7%) were carbapenemase-producing, and 20 (20.8%) with ESBLs phenotype tested were resistant to all four drugs. The correlation between ESBLs-producing strains and biofilm formation was significantly increased compared to strains without ESBLs phenotype (P=0.035).Conclusion: Compared to hypervirulent Klebsiella pneumoniae (hvKP), classical Klebsiella pneumoniae (cKP) has a tendency to acquire antibiotic resistance. Our study showed that genes encoding rmpA, K1 or K2, and kfu were highly associated with hvKP.Keywords: Klebsiella pneumoniae, antimicrobial resistance, hypermucoviscosity, virulence factors, epidemiology

Purpose: Hypermucoviscous strains of Klebsiella pneumoniae (KP) are associated with invasive liver abscess syndrome. However, little is known about the characteristics of this phenotype in non-hepatobiliary infections. In this study, we investigated the clinical characteristics of patients with hypermucoviscous Kp (hmvKp) bacteremia from non-hepatobiliary tract infection. Methods: This retrospective cohort study was implemented at Samsung Changwon Hospital. From March 2018 to December 2019, adult patients (≥ 18 years) with KP bacteremia of the extra-hepatobiliary system were enrolled. Hypermucoviscosity was defined by the string test. Clinical characteristics and 30-day all-cause mortality between patients with hmvKp and non-hmvKp bacteremia were compared. Results: Among 179 cases of non-hepatobiliary KP bacteremia, 67 (37.4%) and 112 (62.6%) isolates were classified as hmvKp and non-hmvKp, respectively. In the hmvKp group, metastatic infection (9.0 vs. 1.8%, P = 0.054) and purulent or necrotizing infection (31.3 vs. 9.8%, P < 0.001) were more frequently observed. Additionally, non-hmvKp had more frequent resistance to cefotaxime (11.9 vs. 38.4%, P < 0.001). Thirty-day all-cause mortality was similar in the hmvKp (41.8%) and non-hmvKp (39.3%) groups (P = 0.643). In multivariable analysis, septic shock (adjusted hazard ratio [aHR] = 3.05, 95% confidence interval [CI]: 1.22–7.63) and Pitt bacteremia score (aHR = 1.23 per 1 point, 95% CI 1.14–1.33) were associated with increased mortality in patients with Kp bacteremia, while urinary-tract infection (aHR = 0.38, 95% CI 0.18–0.76) was associated with decreased mortality. Conclusion: hmvKp was associated with less frequent drug resistance and metastatic-purulent presentation in non-hepatobiliary infection like in hepatobiliary infection. However, hmvKp was not associated with clinical outcomes. [ABSTRACT FROM AUTHOR]

Mengxin Xu,1 Changrui Qian,2 Huaiyu Jia,1 Luozhu Feng,3 Shiyi Shi,1 Ying Zhang,3 Lingbo Wang,1 Jianming Cao,3 Tieli Zhou,1 Cui Zhou1 1Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou, People’s Republic of China; 2School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou, People’s Republic of China; 3Department of Medical Laboratory Science, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, People’s Republic of ChinaCorrespondence: Tieli Zhou; Cui Zhou, Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University; Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou, 325035, People’s Republic of China, Tel +86 0577 8668 9885, Email wyztli@163.com; ZHOUCUI0826@163.comIntroduction: Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to human public health. Ceftazidime–avibactam (CZA) is currently one of the few effective antibiotics for carbapenem-resistant Enterobacteriaceae (CRE).Methods and Results: Here, we analyzed two longitudinal Klebsiella pneumoniae clinical isolates (FK8578, FK8695) that were isolated from an ICU patient during antimicrobial treatment. Broth microdilution method, whole-genome sequencing (WGS) and comparative genomic analysis were used to elucidate the dynamics and mechanisms of antibiotic resistance. String test, quantification of capsule, biofilm inhibition test and Galleria mellonella (G. mellonella) infection model were used to explore the changes in virulence of the two clinical isolates. During antibiotic treatment, CRKP FK8578 underwent a series of drug resistance and virulence changes, including CZA resistance, carbapenem susceptibility and virulence attenuation. The results of WGS showed that mutation of blaKPC-2 to blaKPC-33 was responsible for the change of drug resistance phenotype between FK8578 and FK8695. pLVPK-like virulence plasmid without siderophore synthesis operon was identified in the two strains. On the other hand, the loss of hypermucoviscosity phenotype in the FK8695 strain may be related to a single nucleotide deletion of the rmpA gene, which would further lead to a decrease in virulence. Virulence results showed that compared with FK8578, FK8695 was negative in the string test, with decreased capsular production, smaller amounts of biofilm formation and higher survival rate of G. mellonella.Conclusion: This is the first report of CZA resistance and decreased virulence in ST11 CRKP strains during antimicrobial treatment. It is urgent to monitor CZA resistance and timely adjust anti-infective treatment strategies.Keywords: ceftazidime–avibactam, carbapenem-resistantKlebsiella pneumoniae, whole-genome sequencing, hypermucoviscosity, pLVPK-like plasmid, virulence attenuation

Background: The development of tigecycline resistance in hypervirulent Klebsiella pneumoniae strains has resulted in decreased virulence that is associated with reduced production of capsular polysaccharides (CPS). In this study, we investigated the mechanisms that link tigecycline susceptibility to decreased virulence. Methods: We compared transcriptomes from tigecycline-susceptible wild-type strains and tigecycline-resistant mutants using mRNA sequencing. ompR-overexpressed and ompR-deleted mutants were constructed from wild-type strains and tigecycline-resistant mutants, respectively. Antibiotic susceptibility tests were performed, and string tests and precipitation assays were conducted to identify phenotypic changes related to tigecycline susceptibility and ompR expression. Bacterial virulence was assessed by serum resistance and Galleria mellonella infection assays. Results: Transcriptomic analyses demonstrated a significant decrease in the expression of ompK35 in the tigecycline-resistant mutants. We observed that tigecycline-resistant mutants overexpressed ompR, and that the expression of ompK35 was regulated negatively by ompR. While tigecycline-resistant mutants and ompR-overexpressed mutants exhibited reduced hypermucoviscosity and virulence, deletion of ompR from tigecycline-resistant mutants restored their hypermucoviscosity and virulence. Conclusions: In hypervirulent K. pneumoniae strains, ompR expression, which is regulated by exposure to tigecycline, may affect the production of CPS, leading to bacterial virulence. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKp) is emerging worldwide. Hypermucoviscousity is the characteristic trait that distinguishes it from classic K. pneumoniae (cKp), which enables Kp to cause severe invasive infections. This research aimed to investigate the hypermucoviscous Kp (hmvKp) phenotype among gut commensal Kp isolated from healthy individuals and attempted to characterize the genes encoding virulence factors that may regulate the hypermucoviscosity trait. Using the string test, 50 identified Kp isolates from healthy individuals' stool samples were examined for hypermucoviscosity and investigated by transmission electron microscopy (TEM). Antimicrobial susceptibility profiles of Kp isolates were determined using the Kirby Bauer disc method. Kp isolates were tested for genes encoding different virulence factors by PCR. Biofilm formation was assayed by the microtiter plate method. All Kp isolates were multidrug-resistant (MDR). Phenotypically, 42% of isolates were hmvKp. PCR-based genotypic testing revealed the hmvKp isolates belonged to capsular serotype K2. All study Kp isolates harbored more than one virulence gene. The genes magA and rmpA were not detected, while the terW gene was present in all isolates. The siderophores encoding genes entB and irp2 were most prevalent in hmvKp isolates (90.5%) and non-hmvKp (96.6%), respectively. hmvKp isolates harbored the genes wabG and uge with rates of 90.5% and 85.7%, respectively. The outcomes of this research highlight the potential health risk of commensal Kp to cause severe invasive diseases, owing to being hmvKp and MDR, and harboring multiple virulence genes. The absence of essential genes related to hypermucoviscosity such as magA and rmpA in hmvKp phenotypes suggests the multifactorial complexity of the hypermucoviscosity or hypervirulence traits. Thus, further studies are warranted to verify the hypermucoviscosity-related virulence factors among pathogenic and commensal Kp in different colonization niches. [ABSTRACT FROM AUTHOR]

Background: Hypervirulent Klebsiella pneumoniae (hvKP) is a virulent subtype of K. Pneumoniae. It commonly causes a serious community acquired infections, but it can also cause hospital acquired (nosocomial) infections. Emergence of antimicrobial resistance in hvKP ia a cause of concern. Objective: Phenotypic and molecular characterization and differentiation of classical and hypervirulent klebsiella pneumoniae isolated from Benha University Hospital. Subjects and Methods: Klebsiella pneumoniae was isolated by cultivation on Mac-Conkey agar media. Identified phenotypically and subjected to antibiotic sensitivity test. Conventional PCR was performed for detection of RmpA and RmpA2 virulence genes. Results: Eight strains out of 70 (11.4%) K.Pneumoniae were identified as probable hvKP by detection of RmpA2 that was detected in all hvKP while rmpA was detected in 2 out of 8 hvKP isolates. Both cKP and hvKP isolates exhibited high resistance rates for most of the tested antibiotics. Conclusion: Particularly in the presence of antibiotic resistance, HvKP pose a new hazard. Both Rmp A and Rmp A2 virulence genes were strongly associated with hvKP. [ABSTRACT FROM AUTHOR]

Introduction: Hypermucoviscous Klebsiella pneumoniae (HmKp) poses an emerging and highly pathogenic global health threat. This study aimed to investigate the clinical and genomic characteristics of HmKp isolates to better understand the virulence mechanisms of the hypermucoviscous (HMV) phenotype. Methods: From May 2018 to August 2021, 203 non-repeat K. pneumoniae isolates causing invasive infections were collected from a hospital in Beijing, China. Isolates were divided into HmKp (n=90, 44.3%) and non-HmKp (n=113, 55.7%) groups according to string test results. Results: Multivariate regression showed that diabetes mellitus (odds ratio [OR]= 2.20, 95% confidence interval (CI): 1.20-4.05, p=0.010) and liver abscess (OR=2.93, CI 95%:1.29-7.03, p=0.012) were associated with HmKp infections. K. pneumoniae was highly diverse, comprising 87 sequence types (STs) and 54 serotypes. Among HmKp isolates, ST23 was the most frequent ST (25/90, 27.8%), and the most prevalent serotypes were KL2 (31/90, 34.4%) and KL1 (27/90, 30.0%). Thirteen virulence genes were located on the capsular polysaccharide synthesis region of KL1 strains. HmKp isolates were sensitive to multiple antibiotics but carried more SHV-type extended spectrumb-lactamase (ESBL) resistance genes (p<0.05), suggesting that the emergence of ESBL-mediated multidrug resistance in HmKp should be monitored carefully during treatment. Phylogenetic analysis disclosed that HmKp isolates were highly diverse. Comparative genomic analysis confirmed that the HMV phenotype is a plasmid-encoded virulence factor. Seventeen HmKp genes were highly associated with HmKp, and included rmpAC, 7 iron-acquisitionrelated genes, and pagO, which may promote liver abscess formation. Discussion: This investigation provides insight into the mechanisms producing the HMV phenotype. [ABSTRACT FROM AUTHOR]

ABSTRACT Klebsiella pneumoniae is a leading cause of nosocomial infections, including pneumonia, bacteremia, and urinary tract infections. Treatment options are increasingly restricted by the high prevalence of resistance to frontline antibiotics, including carbapenems, and the recently identified plasmid-conferred colistin resistance. The classical pathotype (cKp) is responsible for most of the nosocomial infections observed globally, and these isolates are often multidrug resistant. The hypervirulent pathotype (hvKp) is a primary pathogen capable of causing community-acquired infections in immunocompetent hosts. The hypermucoviscosity (HMV) phenotype is strongly associated with the increased virulence of hvKp isolates. Recent studies demonstrated that HMV requires capsule (CPS) synthesis and the small protein RmpD but is not dependent on the increased amount of capsule associated with hvKp. Here, we identified the structure of the capsular and extracellular polysaccharide isolated from hvKp strain KPPR1S (serotype K2) with and without RmpD. We found that the polymer repeat unit structure is the same in both strains and that it is identical to the K2 capsule. However, the chain length of CPS produced by strains expressing rmpD demonstrates more uniform length. This property was reconstituted in CPS from Escherichia coli isolates that possess the same CPS biosynthesis pathway as K. pneumoniae but naturally lack rmpD. Furthermore, we demonstrate that RmpD binds Wzc, a conserved capsule biosynthesis protein required for CPS polymerization and export. Based on these observations, we present a model for how the interaction of RmpD with Wzc could impact CPS chain length and HMV. IMPORTANCE Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD, is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain. Here, we demonstrate that RmpD regulates capsule chain length and interacts with Wzc, a part of the capsule polymerization and export machinery shared by many pathogens. We further show that RmpD confers HMV and regulates capsule chain length in a heterologous host (E. coli). As Wzc is a conserved protein found in many pathogens, it is possible that RmpD-mediated HMV and increased virulence may not be restricted to K. pneumoniae.

ABSTRACT Expression of the hypermucoviscosity (HMV) phenotype and capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae were reported to be encoded by genes located in the chromosomal rmp locus. However, the functions of the rmp locus in the virulence plasmid remained unclear, and most of the rmp loci in clinical K. pneumoniae are plasmid carried. In this study, we investigated the functional characteristics of plasmid-borne rmp homologues in clinical hypervirulent K. pneumoniae (hvKP) strains by cloning and introducing such gene homologues into K. pneumoniae strains of different capsule types, followed by the evaluation of phenotypic changes in these strains. Acquisition of the plasmid-borne prmpADC and prmpA2D2 loci were found to result in an increase in mucoviscosity and CPS production in K1 and K2 K. pneumoniae, while only the prmpA2D2 locus contributed to phenotypic changes in the ST11/KL64 strain. Consistently, both rmpD and rmpD2 increased HMV in K1 and K2 K. pneumoniae, while only rmpD2 contributed to HMV in the ST11/KL64 strain; rmpC contributed to CPS overproduction in K1 and K2 strains but not in the ST11/KL64 strain. Furthermore, we proposed a logistic molecular basis of the HMV phenotype of K. pneumoniae on which prmpD2-mediated HMV is attributed to the increase of cell-free CPS production. Our data confirm that the rmp homologues carried by the virulence plasmid play a key role in virulence expression in K. pneumoniae, but the phenotype is highly dependent on the genetic background of the host strain and explained why most of the clinical ST11 strains carry only the prmpA2D2 locus. IMPORTANCE Klebsiella pneumoniae has become the most frequently isolated bacterial pathogen in hospital settings, with a very high mortality rate worldwide. Factors contributing to the virulence of K. pneumoniae are the overproduction of capsular polysaccharide (CPS) as well as the hypermucoviscosity (HMV) phenotype. These two phenotypes were reported to be regulated by rmpA/A2 homologues, which are often carried by virulence plasmids. Here, we determined the functional role of two plasmid-borne rmpA in mediating expression of the HMV phenotype and CPS production in K. pneumoniae. Different capsule types exhibited differences in the expression of HMV and CPS production although they harbored an identical plasmid-borne rmpA or rmpA2 locus, indicating that these virulence-related phenotypes are strongly related to the genetic background of the host strains. Our study provides a novel understanding of the regulation of virulence-related phenotypes and clinical management of K. pneumoniae infections.

ABSTRACT Hypermucoviscosity is a hallmark of hypervirulent Klebsiella pneumoniae (hvKP). However, the molecular basis of its regulation is largely unknown. We hypothesize that hypermucoviscosity is modulated via two-component signal transduction systems (TCSs). In-frame deletion mutants of all 33 response regulators of hvKP ATCC43816 were generated using CRISPR/CAS and evaluated for their impacts on hypermucoviscosity. The response regulator OmpR is required for hypermucoviscosity in vitro and virulence in vivo in a mouse pneumonia model. The ΔompR mutant lost its mucoidy but retained its capsule level and comparable rmpADC expression, so transcriptomic analysis by RNA-Seq was performed to identify differentially expressed genes (DEGs) in ΔompR mutant. The top 20 Gene Ontology terms of 273 DEGs belong to purine ribonucleotide triphosphate biosynthetic and metabolic process, transmembrane transport, and amino acid metabolism. Among the overexpressed genes in the ΔompR mutant, the atp operon encoding F-type ATP synthase and the gcvTHP encoding glycine cleavage system were characterized further as overexpression of either operon reduced the mucoviscosity and increased the production of ATP. Furthermore, OmpR directly bound the promoter region of the atp operon, not the gcvTHP, suggesting that OmpR regulates the expression of the atp operon directly and gcvTHP indirectly. Hence, the loss of OmpR led to the overexpression of F-type ATP synthase and glycine cleavage system, which altered the energetic status of ΔompR cells and contributed to the subsequent reduction in the mucoviscosity. Our study has uncovered a previously unknown regulation of bacterial metabolism by OmpR and its influence on hypermucoviscosity. IMPORTANCE Hypermucoviscosity is a critical virulent factor for Klebsiella pneumoniae infections, and its regulation remains poorly understood at the molecular level. This study aims to address this knowledge gap by investigating the role of response regulators in mediating hypermucoviscosity in K. pneumoniae. We screened 33 response regulators and found that OmpR is essential for hypermucoviscosity and virulence of K. pneumoniae in a mouse pneumonia model. Transcriptomic analysis uncovered that genes involved in energy production and metabolism are highly upregulated in the ΔompR mutant, suggesting a potential link between bacterial energy status and hypermucoviscosity. Overexpression of those genes increased production of ATP and reduced mucoviscosity, recapitulating the ΔompR mutant phenotype. Our findings provide new insights into the regulation of K. pneumoniae hypermucoviscosity by a two-component signal transduction system, highlighting the previously unknown role of OmpR in regulating bacterial energy status and its influence on hypermucoviscosity.

Hypervirulent Klebsiella pneumoniae (HvKP), which causes highly fatal infections, is a new threat to human health. In an attempt to investigate the underlying mechanisms of resistance to neutrophil-mediated killing and hence expression of high-level virulence by HvKP, we tested the binding affinity of HvKP strains to various types of human cells. Our data showed that HvKP exhibited weaker binding to both lung epithelial cells, intestinal Caco-2 cells and macrophages when compared to the classic, non-hypervirulent strains (cKP). Consistently, transconjugants that have acquired a rmpA or rmpA2-bearing plasmid were found to exhibit decreased adhesion to various types of human cells, and hence higher survival rate upon exposure to neutrophil cells. We further found that over production of hypermucoviscosity (HMV), but not capsular polysaccharide (CPS), contributed to the reduced binding and phagocytosis. The effect of hypermucoviscosity on enhancing HvKP virulence was further shown in human serum survival assays and animal experiments. Findings in this study therefore confirmed that rmpA/A2-mediated hypermucoviscosity in HvKP plays a key role in the pathogenesis of this organism through conferring the ability to evade neutrophil binding and phagocytosis.

Due to the strong pathogenicity of hypervirulent Klebsiella pneumoniae (hvKP), its performance against disinfectants in water should be understood to protect public health and ecological environment. Unfortunately, the disinfectant tolerance of hvKP with a hypermucoviscosity (HMV) phenotype is a critical underexplored area. Here, the tolerance of K. pneumoniae isolates to common disinfectants was evaluated, and its underlying mechanisms were clarified. Results showed that hvKP strains with HMV exhibited remarkable tolerance to triclosan (TCS), sodium hypochlorite (NaClO), and benzalkonium bromide (BB), surpassing that of low-virulent K. pneumoniae (lvKP) and Escherichia coli, which is the microbial indicator of drinking water quality. Ct value of NaClO reached 4.41 mg/L·min to kill 4-log hvKP, while the values were 2.52 and 2.28 mg/L·min to achieve 4-log killing of lvKP and E. coli, respectively. The curing of the virulence plasmid from hvKP strain K2044 revealed that capsular polysaccharide (CPS) synthesis, driven by the virulence plasmids, helped mitigate cell membrane injury and bacterial inactivation under NaClO stress; consequently, it provided a protective advantage to hvKP. Enhancing the antioxidative stress system to reduce ROS production and mitigate oxidative stress caused by NaClO further improved the disinfectant resistance of hvKP strains with HMV. This study emphasized that hvKP strains with HMV posed a considerable challenge to disinfection procedure of water treatment. It also revealed that an improved dosage of NaClO ensures bacteria killing, indicating the optimization of the design of water treatment processes involving disinfection strategies and technical parameters should be considered., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: This study used whole-genome sequencing (WGS) to explore the genetic diversity, virulence factors, and antimicrobial resistance determinants of string test-positive Klebsiella pneumoniae (KP) over a 4-year surveillance period in Huzhou, China., Methods: In total, 632 clinical isolates were collected via hospital surveillance from 2020 to 2023; 100 were positive in the string test and these 100 strains were subjected to antimicrobial susceptibility testing using an agar dilution method followed by WGS., Results: The resistance rates to cefotaxime (77.0%), trimethoprim-sulfamethoxazole (67.0%), and nalidixic acid (64.0%) were high. Multilocus sequence typing revealed high genetic diversity; there were 33 sequence types (STs) and 15 capsular serotypes. The most common ST was ST23 (16.0%) and the most common capsular serotype was K1 (22.5%). Virulome analysis revealed among-strain differences in virulence factors that affected bacterial adherence, efflux pump action, iron uptake, nutritional factors, metabolic regulation, the secretion system, and toxin production. The Kleborate strain-specific virulence scores of all 100 string test-positive KPs were derived: 28 strains scored 5, 28 scored 4, 21 scored 3, 12 scored 1, and 11 scored 0. All 77 strains with scores of 3 to 5 contained the iucA gene. The phylogeny based on whole-genome single nucleotide polymorphisms (wgSNPs) indicated high clonality; the string test-positive KP strains were grouped into six clades. Closely related isolates in each genetic cluster usually shared STs., Conclusion: The present study highlights the significance of the KP iuc A gene in terms of hypervirulence and the diverse genotypes of string test-positive KP strains isolated in Huzhou hospitals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yan, Xu, Shen, Dong and Ji.)

Hypermucoviscosity (HMV) is a phenotype that is commonly associated with hypervirulence in Klebsiella pneumoniae . The factors that contribute to the emergence of HMV subpopulations remain unclear. In this study, eight K. pneumoniae strains were recovered from an inpatient who had been hospitalized for 20 days. Three of the isolates exhibited a non-HMV phenotype, which was concomitant with higher biofilm formation than the other five HMV isolates. All eight isolates were highly susceptible to serum killing, albeit HMV strains were remarkably more infective than non-HMV counterparts in a mouse model of infection. Whole genome sequencing (WGS) showed that the eight isolates belonged to the K57-ST412 lineage. Average nucleotide identity (FastANIb) analysis indicated that eight isolates share 99.96% to 99.99% similarity and were confirmed to be the same clone. Through comparative genomics analysis, 12 non-synonymous mutations were found among these isolates, eight of which in the non-HMV variants, including rmpA (c.285delG) and wbaP (c.1305T > A), which are assumed to be associated with the non-HMV phenotype. Mutations in manB (c.1318G > A), dmsB (c.577C > T) and tkt (c.1928C > A) occurred in HMV isolates only. RNA-Seq revealed transcripts of genes involved in energy metabolism, carbohydrate metabolism and membrane transport, including cysP , cydA , narK , tktA , pduQ , aceB , metN, and lsrA , to be significantly dysregulated in the non-HMV strains, suggesting a contribution to HMV phenotype development. This study suggests that co-occurrence of HMV and non-HMV phenotypes in the same clonal population may be mediated by mutational mechanisms as well as by certain genes involved in membrane transport and central metabolism., Importance: K. pneumoniae with a hypermucoviscosity (HMV) phenotype is a community-acquired pathogen that is associated with increased invasiveness and pathogenicity, and underlying diseases are the most common comorbid risk factors inducing metastatic complications. HMV was earlier attributed to the overproduction of capsular polysaccharide, and more data point to the possibility of several causes contributing to this bacterial phenotype. Here, we describe a unique event in which the same clonal population showed both HMV and non-HMV characteristics. Studies have demonstrated that this process is influenced by mutational processes and genes related to transport and central metabolism. These findings provide fresh insight into the mechanisms behind co-occurrence of HMV and non-HMV phenotypes in monoclonal populations as well as potentially being critical in developing strategies to control the further spread of HMV K. pneumoniae ., Competing Interests: The authors declare no conflict of interest.

The ability of Klebsiella pneumoniae to form biofilm renders the pathogen recalcitrant to various antibiotics. The difficulty in managing K. pneumoniae related chronic infections is due to its biofilm-forming ability and associated virulence factors, necessitating the development of efficient strategies to control virulence factors. This study aimed at evaluating the inhibitory potential of selected phytochemical compounds on biofilm-associated virulence factors in K. pneumoniae, as well as authenticating their antibiofilm activity. Five phytochemical compounds (alpha-terpinene, camphene, fisetin, glycitein and phytol) were evaluated for their antibacterial and anti-biofilm-associated virulence factors such as exopolysaccharides, curli fibers, and hypermucoviscosity against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae strains. The antibiofilm potential of these compounds was evaluated at initial cell attachment, microcolony formation and mature biofilm formation, then validated by in situ visualization using scanning electron microscopy (SEM). Exopolysaccharide surface topography was characterized using atomic force microscopy (AFM). The antibacterial activity of the compounds confirmed fisetin as the best anti-carbapenem-resistant K. pneumoniae, demonstrating a minimum inhibitory concentration (MIC) value of 0.0625 mg/mL. Phytol, glycitein and α-terpinene showed MIC values of 0.125 mg/mL for both strains. The assessment of the compounds for anti-virulence activity (exopolysaccharide reduction) revealed an up to 65.91% reduction in phytol and camphene. Atomic force microscopy detected marked differences between the topographies of untreated and treated (camphene and phytol) exopolysaccharides. Curli expression was inhibited at both 0.5 and 1.0 mg/mL by phytol, glycitein, fisetin and quercetin. The hypermucoviscosity was reduced by phytol, glycitein, and fisetin to the shortest mucoid string (1 mm) at 1 mg/mL. Phytol showed the highest antiadhesion activity against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae (54.71% and 50.05%), respectively. Scanning electron microscopy correlated the in vitro findings, with phytol significantly altering the biofilm architecture. Phytol has antibiofilm and antivirulence potential against the highly virulent K. pneumoniae strains, revealing it as a potential lead compound for the management of K. pneumoniae-associated infections. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKp) raised concern worldwide. We studied 22 hvKp clinical invasive isolates referred to the Belgian national reference laboratory between 2014 and 2020. Sixty-four percent of the isolates expressed K2 capsular serotype and belonged to 7 different MLST lineages, while 32% expressed K1 (all belonging to ST23) and were associated with liver abscesses. Primary extra-hepatic infections were reported in 36% and sepsis for 95% of the patients with 30% of deaths. Improved clinical and microbiological diagnostics are required as hvKp may represent an underestimated cause of community-acquired invasive infections in Belgium. [ABSTRACT FROM AUTHOR]

Abstract Background Klebsiella spp. are important pathogens associated with bacteremia among admitted children and is among the leading cause of death in children

ABSTRACT Klebsiella pneumoniae can be broadly classified into classical strains that cause drug-resistant, hospital-associated infections and hypervirulent strains that cause invasive, community-acquired, drug-susceptible infections. Hypermucoviscosity in Klebsiella pneumoniae has been associated with immune evasion and hypervirulence. A string-test-positive, hypermucoviscous strain of Klebsiella pneumoniae, P34, was isolated from the cystic lesion of a patient who reported to a tertiary care hospital in Jodhpur, Rajasthan, India. Given the antibiotic-susceptible and hypermucoviscous nature of the isolate, it was suspected to belong to the hypervirulent lineage of Klebsiella pneumoniae. However, P34 did not overproduce capsular polysaccharides and also remained susceptible to the antimicrobial effects of human serum when tested alongside strains that were non-hypermucoviscous. Sequencing of the genome of P34 revealed the absence of any large virulence plasmids or integrative conjugative elements that usually carry hypermucoviscosity- and hypervirulence-associated genes. P34 also lacked key virulence determinants such as aerobactin, yersiniabactin, and salmochelin biosynthesis clusters. In addition, P34 lacked homologs for genes associated with enhanced capsule synthesis and hypermucoviscosity, such as rmpA, rmpA2, rmpC, and rmpD (regulator of mucoid phenotype). These observations suggest that P34 may harbor novel genetic determinants of hypermucoviscosity independent of the indirectly acting rmpA and the recently described rmpD. IMPORTANCE Hypermucoviscosity is a characteristic of hypervirulent Klebsiella pneumoniae strains, which are capable of causing invasive disease in community settings. This study reports phenotyping and genomic analysis of an unusual clinical isolate of Klebsiella pneumoniae, P34, which exhibits hypermucoviscosity and yet does not harbor rmp (regulator of mucoid phenotype) genes, which are known determinants of hypermucoviscosity (rmpA and rmpD). Similar clinical isolates belonging to the K. pneumoniae complex that are hypermucoviscous but do not harbor the rmp loci have been reported from India and abroad, indicating the prevalence of unknown determinants contributing to hypermucoviscosity. Therefore, strains like P34 will serve as model systems to mechanistically study potentially novel determinants of hypermucoviscosity in the K. pneumoniae complex.

Hypervirulent Klebsiella pneumoniae (hvKp) is emerging worldwide. Hypermucoviscousity is the characteristic trait that distinguishes it from classic K. pneumoniae (cKp), which enables Kp to cause severe invasive infections. This research aimed to investigate the hypermucoviscous Kp (hmvKp) phenotype among gut commensal Kp isolated from healthy individuals and attempted to characterize the genes encoding virulence factors that may regulate the hypermucoviscosity trait. Using the string test, 50 identified Kp isolates from healthy individuals’ stool samples were examined for hypermucoviscosity and investigated by transmission electron microscopy (TEM). Antimicrobial susceptibility profiles of Kp isolates were determined using the Kirby Bauer disc method. Kp isolates were tested for genes encoding different virulence factors by PCR. Biofilm formation was assayed by the microtiter plate method. All Kp isolates were multidrug-resistant (MDR). Phenotypically, 42% of isolates were hmvKp. PCR-based genotypic testing revealed the hmvKp isolates belonged to capsular serotype K2. All study Kp isolates harbored more than one virulence gene. The genes magA and rmpA were not detected, while the terW gene was present in all isolates. The siderophores encoding genes entB and irp2 were most prevalent in hmvKp isolates (90.5%) and non-hmvKp (96.6%), respectively. hmvKp isolates harbored the genes wabG and uge with rates of 90.5% and 85.7%, respectively. The outcomes of this research highlight the potential health risk of commensal Kp to cause severe invasive diseases, owing to being hmvKp and MDR, and harboring multiple virulence genes. The absence of essential genes related to hypermucoviscosity such as magA and rmpA in hmvKp phenotypes suggests the multifactorial complexity of the hypermucoviscosity or hypervirulence traits. Thus, further studies are warranted to verify the hypermucoviscosity-related virulence factors among pathogenic and commensal Kp in different colonization niches.

Hypervirulent Klebsiella pneumoniae (HvKP), which causes highly fatal infections, is a new threat to human health. In an attempt to investigate the underlying mechanisms of resistance to neutrophil-mediated killing and hence expression of high-level virulence by HvKP, we tested the binding affinity of HvKP strains to various types of human cells. Our data showed that HvKP exhibited weaker binding to both lung epithelial cells, intestinal Caco-2 cells and macrophages when compared to the classic, non-hypervirulent strains (cKP). Consistently, transconjugants that have acquired a rmpA or rmpA2-bearing plasmid were found to exhibit decreased adhesion to various types of human cells, and hence higher survival rate upon exposure to neutrophil cells. We further found that over production of hypermucoviscosity (HMV), but not capsular polysaccharide (CPS), contributed to the reduced binding and phagocytosis. The effect of hypermucoviscosity on enhancing HvKP virulence was further shown in human serum survival assays and animal experiments. Findings in this study therefore confirmed that rmpA/A2-mediated hypermucoviscosity in HvKP plays a key role in the pathogenesis of this organism through conferring the ability to evade neutrophil binding and phagocytosis. [ABSTRACT FROM AUTHOR]

Abstract Background Hypervirulent Klebsiella pneumoniae (HVKp) infections have distinct clinical manifestations from classical K. pneumoniae infections. The hallmark of HVKp infections are liver abscess formation and metastatic infections. Due to the severe sequelae of these complications, method to identify patients at-risk of HVKp infections should be developed. Results A retrospective cohort study of 222 patients with K. pneumoniae bloodstream infections (BSIs) was performed. Patient demographics, clinical manifestations, and bacterial characteristics were investigated. Ten cases of liver abscesses were identified. Characteristics such as community-onset BSIs, hypermucoviscosity phenotype, and capsular serotype K1 were identified as risk factors for HVKp infections. A scoring system was developed based on the risk factors. The area under the receiver operating characteristic curve for the scoring system was 0.90. A score of ≥ 2 points provided sensitivity and specificity of 0.70 and 0.94, respectively. Conclusions Simple scoring system was developed for the diagnosis of HVKp infections. The system allows early identification of patients with K. pneumoniae BSIs in whom hypervirulent infections should be evaluated. Prospective evaluation is expected.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are capable of causing various systemic infections in both humans and animals. In this study, we isolated and characterized 30 E. coli strains from the parenchymatic organs and brains of young (<3 months of age) camel calves which died in septicemia. Six of the strains showed hypermucoviscous phenotype. Based on minimum inhibitory concentration (MIC) values, seven of the strains were potentially multidrug resistant, with two additional showing colistin resistance. Four strains showed mixed pathotypes, as they carried characteristic virulence genes for intestinal pathotypes of E. coli : three strains carried cnf1, encoding cytotoxic necrotizing factor type 1, the key virulence gene of necrotoxigenic E. coli (NTEC), and one carried eae encoding intimin, the key virulence gene of enteropathogenic E. coli (EPEC). An investigation of the integration sites of pathogenicity islands (PAIs) and the presence of prophage-related sequences showed that the strains carry diverse arrays of mobile genetic elements, which may contribute to their antimicrobial resistance and virulence patterns. Our work is the first to describe ExPEC strains from camels, and points to their veterinary pathogenic as well as zoonotic potential in this important domestic animal.

Introduction Hypermucoviscous Klebsiella pneumoniae  (hvKP) is related to invasive infections; however, there have been very few comprehensive reports on the clinical features and prognosis of critically ill patients with the infection. Methods We conducted a retrospective case series in a general intensive care unit in Japan. Patients with positive blood cultures for KP between January 1, 2020 and December 31, 2022 were included. hvKP was defined by the positivity in the string test. We analyzed the patient's characteristics at baseline, including comorbidities, abscess formation, Sequential Organ Failure Assessment (SOFA) score, Acute Physiology and Chronic Health Evaluation (APACHE) II score, septic shock, duration of hospitalization, 30-day mortality, and infection site. Results A total of 24 patients had a positive blood culture for KP; nine patients (37.5%) were positive for the string test (hvKP) while 15 (62.5%) were negative (non-hvKP). In both groups, the patients were old (mean age, hvKP 80.4 vs. non-hvKP 75.7 years) and more often male (five patients (55.6%) vs. 12 patients (80.0%)). No statistically significant difference was found between the two groups in terms of comorbidities, such as diabetes mellitus, chronic obstructive pulmonary disease, chronic kidney disease, and malignancy. No statistical difference was seen in abscess formation (two patients [22.2%] vs. one patient (6.7%)), SOFA score (5.2±4.8 vs. 4.7±3.4), APACHE II score (19.6 (15.0-20.0) vs. 17.0 (11.2-20.8)), septic shock (five patients (55.6%) vs. four patient (26.7%)), duration of hospitalization (37.2 (12.0-51.0) vs. 32.3 (9.5-21.0)), and 30-day mortality (two patients (22.2%) vs. two patients (13.3%)). Two cases with hvKP died within 24 h. No significant difference was seen in the infection sources; respiratory infection (2 (22.2%) vs. 1 (6.7%)), hepatobiliary infection (2 (22.2%) vs. 7 (46.7%)), and genitourinary infection (1 (11.1%) vs. 5 (33.3%)). Conclusions Critically ill patients with hvKP infection showed characteristics similar to those reported previously. However, the disease could rapidly become severe and have a poor prognostic outcome., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Yomogida et al.)

Background:Klebsiella pneumoniae (K. pneumoniae) is one of the most clinically important opportunistic pathogens involved in both community-(CAIs) and hospitalacquired infections (HAIs).The hypervirulent K.pneumoniae (hvKp) responsible for disseminated infections in healthy and immunosuppressed individuals has emerged with considerable ability to get antibiotic resistance as well. We aimed to characterize the virulence genetic profile and resistance phenotypes of the clinical K.pneumoniae isolates at Menoufia University Hospitals (MUHs) by phenotypic and molecular methods.Methods: 84 K.pneumoniae isolates were collected and classified as classic (cKp) or hypermucoviscous (hmvKp) phenotypes by string test. Antimicrobial resistance patterns were determined phenotypically and multiplex PCR verified the existence of some of the suspected virulence genes.Results: Out of 84 K.pneumoniae isolates, 27 (32.1%) had a positive string test and identified as hmvKp. The remaining 57 isolates (67.9%) were string negative and reported as cKp. Higher resistance rates associated with ESβL, AmpC and carbapenemase production were observed in cKp compared to hmvKp particularly those of hospital origin with a significant statistical difference (p<0.05). RmpA and iutA genes were strongly associated with hmvKp than cKp. The prevalence of blaKPC-2 gene was significantly higher in cKp (33.3%) than hmvKp (7.7%). 80.8% of the isolated hmvKp isolates proved to be hvKp (positive for both rmpA and iutA genes).Conclusions: HmvKp strains are isolated from patients with increasing frequency and constitute a significant proportion of clinical K. pneumoniae isolates. The emergence of blaKPC-producing hvKp strains in the hospital settings confirms the importance of epidemiologic surveillance and clinical awareness of this pathogen. [ABSTRACT FROM AUTHOR]

Background: Klebsiella spp. are important pathogens associated with bacteremia among admitted children and is among the leading cause of death in children < 5 years in postmortem studies, supporting a larger role than previously considered in childhood mortality. Herein, we compared the antimicrobial susceptibility, mechanisms of resistance, and the virulence profile of Klebsiella spp. from admitted and postmortem children.Methods: Antimicrobial susceptibility and virulence factors of Klebsiella spp. recovered from blood samples collected upon admission to the hospital (n = 88) and postmortem blood (n = 23) from children < 5 years were assessed by disk diffusion and multiplex PCR.Results: Klebsiella isolates from postmortem blood were likely to be ceftriaxone resistant (69.6%, 16/23 vs. 48.9%, 43/88, p = 0.045) or extended-spectrum β-lactamase (ESBL) producers (60.9%, 14/23 vs. 25%, 22/88, p = 0.001) compared to those from admitted children. blaCTX-M-15 was the most frequent ESBL gene: 65.3%, 9/14 in postmortem isolates and 22.7% (5/22) from admitted children. We found higher frequency of genes associated with hypermucoviscosity phenotype and invasin in postmortem isolates than those from admitted children: rmpA (30.4%; 7/23 vs. 9.1%, 8/88, p = 0.011), wzi-K1 (34.7%; 8/23 vs. 8%; 7/88, p = 0.002) and traT (60.8%; 14/23 vs. 10.2%; 9/88, p < 0.0001), respectively. Additionally, serine protease auto-transporters of Enterobacteriaceae were detected from 1.8% (pic) to 12.6% (pet) among all isolates. Klebsiella case fatality rate was 30.7% (23/75).Conclusion: Multidrug resistant Klebsiella spp. harboring genes associated with hypermucoviscosity phenotype has emerged in Mozambique causing invasive fatal disease in children; highlighting the urgent need for prompt diagnosis, appropriate treatment and effective preventive measures for infection control. [ABSTRACT FROM AUTHOR]

Given the increasing incidence of multidrug-resistant (MDR) Klebsiella pneumoniae infections, it is of great interest to investigate the risk of transmission associated with the prevalence of this pathogen. Some studies have described fresh raw poultry meat as a reservoir of MDR K. pneumoniae , including clinically relevant sequence types (ST) and extended-spectrum β-lactamase (ESBL) strains, indicating possible consumer exposure. This study compared 47 MDR strains of K. pneumoniae from poultry meat and human clinical isolates to assess similarities, including analysis of antimicrobial resistance profiles and virulence factors involved in infection. In addition, several biofilm culture methods were evaluated for reproducible assessment of biofilm formation in K. pneumoniae strains. Globally, no association between strain origin and STs, hypermucoviscosity, biofilm formation or serum resistance could be found between isolates of food and clinical origin, nor an associated AMR pattern, suggesting overlapping populations. We found that LB supplemented with glucose in microaerobiosis was the best discrimination condition for biofilm formation in the active attachment biofilm cultivation model. The biofilm formation capacity was strongly dependent on culture conditions, with a strain-specific response, but only a minor increase in biofilm levels was recorded in clinical K. pneumoniae populations. Our results suggest that a similar risk of zoonosis transmission from potentially virulent foodborne strains previously observed in E. coli is also present in this high-priority pathogen. This study further confirms that foodborne isolates of K. pneumoniae pose a risk to consumers and therefore this pathogen should be included in the surveillance of foodborne pathogens with high risk of MDR infections and therapeutic failure. • The zoonotic potential of foodborne isolates of K. pneumoniae is confirmed. • AMR-profile analysis revealed overlapping clinical and foodborne populations. • Culture conditions strongly affect biofilm formation and hypermucoviscosity. • No correlation was found between strain origin or ST and phenotypic traits related to virulence. • K. pneumoniae should be included in the surveillance of MDR foodborne pathogens. [ABSTRACT FROM AUTHOR]

The ability of Klebsiella pneumoniae to form biofilm renders the pathogen recalcitrant to various antibiotics. The difficulty in managing K. pneumoniae related chronic infections is due to its biofilm-forming ability and associated virulence factors, necessitating the development of efficient strategies to control virulence factors. This study aimed at evaluating the inhibitory potential of selected phytochemical compounds on biofilm-associated virulence factors in K. pneumoniae, as well as authenticating their antibiofilm activity. Five phytochemical compounds (alpha-terpinene, camphene, fisetin, glycitein and phytol) were evaluated for their antibacterial and anti-biofilm-associated virulence factors such as exopolysaccharides, curli fibers, and hypermucoviscosity against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae strains. The antibiofilm potential of these compounds was evaluated at initial cell attachment, microcolony formation and mature biofilm formation, then validated by in situ visualization using scanning electron microscopy (SEM). Exopolysaccharide surface topography was characterized using atomic force microscopy (AFM). The antibacterial activity of the compounds confirmed fisetin as the best anti-carbapenem-resistant K. pneumoniae, demonstrating a minimum inhibitory concentration (MIC) value of 0.0625 mg/mL. Phytol, glycitein and α-terpinene showed MIC values of 0.125 mg/mL for both strains. The assessment of the compounds for anti-virulence activity (exopolysaccharide reduction) revealed an up to 65.91% reduction in phytol and camphene. Atomic force microscopy detected marked differences between the topographies of untreated and treated (camphene and phytol) exopolysaccharides. Curli expression was inhibited at both 0.5 and 1.0 mg/mL by phytol, glycitein, fisetin and quercetin. The hypermucoviscosity was reduced by phytol, glycitein, and fisetin to the shortest mucoid string (1 mm) at 1 mg/mL. Phytol showed the highest antiadhesion activity against carbapenem-resistant and extended-spectrum beta-lactamase-positive K. pneumoniae (54.71% and 50.05%), respectively. Scanning electron microscopy correlated the in vitro findings, with phytol significantly altering the biofilm architecture. Phytol has antibiofilm and antivirulence potential against the highly virulent K. pneumoniae strains, revealing it as a potential lead compound for the management of K. pneumoniae-associated infections.

Hypermucoviscosity (hmv) is a capsule-associated phenotype usually linked with hypervirulent Klebsiella pneumoniae strains. The key components of this phenotype are the RmpADC proteins contained in non-transmissible plasmids identified and studied in K. pneumoniae. Klebsiella variicola is closely related to K. pneumoniae and recently has been identified as an emergent human pathogen. K. variicola normally contains plasmids, some of them carrying antibiotic resistance and virulence genes. Previously, we described a K. variicola clinical isolate showing an hmv-like phenotype that harbors a 343-kb pKV8917 plasmid. Here, we investigated whether pKV8917 plasmid carried by K. variicola 8917 is linked with the hmv-like phenotype and its contribution to virulence. We found that curing the 343-kb pKV8917 plasmid caused the loss of hmv, a reduction in capsular polysaccharide (P < 0.001) and virulence. In addition, pKV8917 was successfully transferred to Escherichia coli and K. variicola strains via conjugation. Notably, when pKV8917 was transferred to K. variicola, the transconjugants displayed an hmv-like phenotype, and capsule production and virulence increased; these phenotypes were not observed in the E. coli transconjugants. These data suggest that the pKV8917 plasmid carries novel hmv and capsule determinants. Whole-plasmid sequencing and analysis revealed that pKV8917 does not contain rmpADC/rmpA2 genes; thus, an alternative mechanism was searched. The 343-kb plasmid contains an IncFIB backbone and shares a region of ∼150 kb with a 99% identity and 49% coverage with a virulence plasmid from hypervirulent K. variicola and multidrug-resistant K. pneumoniae. The pKV8917-unique region harbors a cellulose biosynthesis cluster (bcs), fructose- and sucrose-specific (fru/scr) phosphotransferase systems, and the transcriptional regulators araC and iclR, respectively, involved in membrane permeability. The hmv-like phenotype has been identified more frequently, and recent evidence supports the existence of rmpADC/rmpA2-independent hmv-like pathways in this bacterial genus.

Hypermucoviscosity (hmv) is a capsule-associated phenotype usually linked with hypervirulent Klebsiella pneumoniae strains. The key components of this phenotype are the RmpADC proteins contained in non-transmissible plasmids identified and studied in K. pneumoniae. Klebsiella variicola is closely related to K. pneumoniae and recently has been identified as an emergent human pathogen. K. variicol a normally contains plasmids, some of them carrying antibiotic resistance and virulence genes. Previously, we described a K. variicola clinical isolate showing an hmv-like phenotype that harbors a 343-kb pKV8917 plasmid. Here, we investigated whether pKV8917 plasmid carried by K. variicola 8917 is linked with the hmv-like phenotype and its contribution to virulence. We found that curing the 343-kb pKV8917 plasmid caused the loss of hmv, a reduction in capsular polysaccharide (P < 0.001) and virulence. In addition, pKV8917 was successfully transferred to Escherichia coli and K. variicola strains via conjugation. Notably, when pKV8917 was transferred to K. variicola , the transconjugants displayed an hmv-like phenotype, and capsule production and virulence increased; these phenotypes were not observed in the E. coli transconjugants. These data suggest that the pKV8917 plasmid carries novel hmv and capsule determinants. Whole-plasmid sequencing and analysis revealed that pKV8917 does not contain rmpADC / rmpA2 genes; thus, an alternative mechanism was searched. The 343-kb plasmid contains an IncFIB backbone and shares a region of ∼150 kb with a 99% identity and 49% coverage with a virulence plasmid from hypervirulent K. variicola and multidrug-resistant K. pneumoniae. The pKV8917-unique region harbors a cellulose biosynthesis cluster (bcs), fructose- and sucrose-specific (fru/scr) phosphotransferase systems, and the transcriptional regulators araC and iclR , respectively, involved in membrane permeability. The hmv-like phenotype has been identified more frequently, and recent evidence supports the existence of rmpADC / rmpA2 -independent hmv-like pathways in this bacterial genus. [ABSTRACT FROM AUTHOR]

Mahoko Ikeda,1,2,* Miyuki Mizoguchi,1,* Yukie Oshida,3 Keita Tatsuno,1 Ryoichi Saito,4 Mitsuhiro Okazaki,5 Shu Okugawa,2 Kyoji Moriya1,2 1Department of Infection Control and Prevention, The University of Tokyo Hospital, Hongo, Bunkyo-ku, Tokyo, Japan; 2Department of Infectious Diseases, The University of Tokyo Hospital, Hongo, Bunkyo-ku, Tokyo, Japan; 3Division of Infectious Diseases, Shizuoka Cancer Center, Simonagakubo, Nagaizumi-chou, Suntou-gun, Shizuoka, Japan; 4Department of Microbiology and Immunity, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan; 5Department of Medical Technology, School of Health Sciences, Tokyo University of Technology, Kamata, Ota-ku, Tokyo, Japan *These authors contributed equally to this work Purpose: Klebsiella pneumoniae is a pathogen that causes pneumonia and urinary tract infection. Hypervirulent K. pneumoniae strains often show hypermucoviscosity, are of the K1 or K2 serotype, and harbor the rmpA and magA genes. However, the differences in the prevalence of K. pneumoniae with these hypervirulent characteristics between the infection and colonization status are not well understood. Therefore, in this study, we compared the clinical and microbiological characteristics of K. pneumoniae isolated from urine or sputum samples of cases of infection and colonization. Patients and methods: This retrospective study was conducted at a tertiary care teaching hospital in Tokyo, Japan. Patients whose sputum or urine tested positive for the presence of K. pneumoniae isolates were randomly included in the study. Clinical and microbiological data were collected from medical records. Results: Of the 130 cases investigated, 68 and 62 cases showed the presence of K. pneumoniae in the sputum and urine, respectively. There were 49 infection cases, including 21 in the sputum group and 28 in the urine group. The infections were not accompanied by liver abscess. Of the 130 K. pneumoniae isolates, 25 (19.2%) showed capsular serotype K1 or K2, whereas 33 (25.4%) showed hypermucoviscosity. The prevalence of virulence genes magA, allS, rmpA, mrkD, uge, kfu-BC, and wabG was 10% (all in K1), 13.1%, 16.9%, 85.4%, 79.2%, 36.9%, and 91.5%, respectively. In both the sputum and urine groups, there was no difference in the characteristics of patients with infection and those with colonization. Analysis of microbiological characteristics revealed that only rmpA was significantly more frequent in the infection cases than in the colonization/asymptomatic cases in both the sputum and urine groups. Conclusion: The rmpA-positive K. pneumoniae isolates were dominant in the infection cases compared with those in the colonization/asymptomatic cases, suggesting that rmpA may play a crucial role in the development of urinary tract infection and pneumonia. Keywords: Klebsiella pneumoniae, hypermucoviscosity, rmpA, capsular polysaccharide serotype, infection, colonization, pneumonia, urinary tract infection