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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. 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 .

Klebsiella variicola is a Gram‐negative bacillus belonging to Enterobacteriaceae recognized as an emerging human pathogen in the last years. The species is frequently misidentified as Klebsiella pneumoniae by conventional microbiological tests, raising questions about its real prevalence and clinical impact. K. variicola virulence is mediated by traits that are expressed in human body niches containing different nutrients, including carbohydrates, that influence their expression. In this way, we analyzed the effect of different carbohydrates on the expression of virulence traits by K. variicola isolates. For this approach, three classical strains (cKv15, cKv35, and cKv57) and one hypermucoviscous (HMV) were submitted to growth curve characterization, biofilm production and serum survival assays, siderophores, and mrk A (encoding MRK adhesin) RNA quantification. The strains were cultivated in broth containing specific carbohydrates as the sole carbon source to perform the assays. Among all carbohydrates tested, sorbitol, galactose, and maltose were the most effective in promoting biomass production in biofilm for the K. variicola classical strains (cKv). Additionally, bacterial incubation in these carbohydrates resulted in the production of siderophores by all strains (cKv and HMV). Notably, cKvs cultivated in all carbohydrates tested survived and proliferated in human serum, while also producing high concentrations of siderophores and biofilm biomass. Except for siderophore production, HMV did not present any virulence trait tested (biofilm production, serum survival, and mrk A expression). However, its growth in media supplemented with galactose promoted serum survival. These observations indicate cKv isolates were able to use sorbitol, galactose, and maltose for rapid proliferation and to express determinants associated with bacterial colonization (as biofilm production, siderophores, and serum survival). The hypermucoviscosity of HMV did not promote biofilm and siderophore production. For this strain, galactose promoted survival in human serum. Altogether, the results highlight the role of galactose in promoting virulence in K. variicola.

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 × 10 8 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 10 8 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.

Klebsiella pneumoniae is widely recognized as a pathogen with a propensity for acquiring antibiotic resistance. It is capable of causing a range of hospital-acquired infections (urinary tract infections [UTI], pneumonia, sepsis) and community-acquired invasive infections. The genetic heterogeneity of K. pneumoniae isolates complicates our ability to understand the virulence of K. pneumoniae . Characterization of virulence factors conserved between strains as well as strain-specific factors will improve our understanding of this important pathogen. The MarR family of regulatory proteins is widely distributed in bacteria and regulates cellular processes such as antibiotic resistance and the expression of virulence factors. Klebsiella encodes numerous MarR-like proteins, and they likely contribute to the ability of K. pneumoniae to respond to and survive under a wide variety of environmental conditions, including those present in the human body. We tested loss-of-function mutations in all the marR homologues in a murine pneumonia model and found that two ( kvrA and kvrB ) significantly impacted the virulence of K1 and K2 capsule type hypervirulent ( hv ) strains and that kvrA affected the virulence of a sequence type 258 (ST258) classical strain. In the hv strains, kvrA and kvrB mutants displayed phenotypes associated with reduced capsule production, mucoviscosity, and transcription from galF and manC promoters that drive expression of capsule synthesis genes. In contrast, kvrA and kvrB mutants in the ST258 strain had no effect on capsule gene expression or capsule-related phenotypes. Thus, KvrA and KvrB affect virulence in classical and hv strains but the effect on virulence may not be exclusively due to effects on capsule production. IMPORTANCE In addition to having a reputation as the causative agent for hospital-acquired infections as well as community-acquired invasive infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. Due to the rapid emergence of carbapenem resistance among K. pneumoniae strains, a better understanding of virulence mechanisms and identification of new potential drug targets are needed. This study identified two novel regulators (KvrA and KvrB) of virulence in K. pneumoniae and demonstrated that their effect on virulence in invasive strains is likely due in part to effects on capsule production (a major virulence determinant) and hypermucoviscosity. KvrA also impacts the virulence of classical strains but does not appear to affect capsule gene expression in this strain. KvrA and KvrB are conserved among K. pneumoniae strains and thus could regulate capsule expression and virulence in diverse strains regardless of capsule type. In addition to having a reputation as the causative agent for hospital-acquired infections as well as community-acquired invasive infections, Klebsiella pneumoniae has gained widespread attention as a pathogen with a propensity for acquiring antibiotic resistance. Due to the rapid emergence of carbapenem resistance among K. pneumoniae strains, a better understanding of virulence mechanisms and identification of new potential drug targets are needed. This study identified two novel regulators (KvrA and KvrB) of virulence in K. pneumoniae and demonstrated that their effect on virulence in invasive strains is likely due in part to effects on capsule production (a major virulence determinant) and hypermucoviscosity. KvrA also impacts the virulence of classical strains but does not appear to affect capsule gene expression in this strain. KvrA and KvrB are conserved among K. pneumoniae strains and thus could regulate capsule expression and virulence in diverse strains regardless of capsule type.

A distinctive syndrome caused by hypermucoviscous (HMKP) including pyogenic liver abscess (PLA) is now becoming a globally emerging disease. In the present study, 22.8% (84/369) of clinical isolates associated with various types of invasive infections were identified as HMKP, with 45.2% associated with PLA. Multivariate regression analysis showed that male patients with 41-50 years, PLA, diabetes mellitus, and hypertension were independent risk factors for HMKP infections. K2 (42.9%, 36/84) was the most common capsular serotype among HMKP isolates, followed by K1 (23.8%, 20/84). Seventy-five percentage of K1 HMKP isolates were associated with PLA, while K2 HMKP isolates accounted for more types of invasive infections. The positive rates of , and among HMKP isolates were significantly higher than those among non-HMKP isolates ( < 0.05). There was a correlation between , and and K1 isolates. Interestingly, was exclusively detected among HMKP (32.1%, 27/84) and K2 isolates (65.9%, 27/41). All K1 and K2 HMKP and non-HMKP isolates were positive for . was found among 95.0 and 97.5% of K1 and K2 isolates. ST23 was found to be the most prevalent ST among 69 HMKP isolates with K1, K2, K5, K20, and K57 (27.5%, 19/69) and was only found among K1 isolates. ST65 was the second most prevalent ST (26.1%, 18/69) and was also only found among K2 isolates. ST23-K1 HMKP isolates (84.2%, 16/19) were associated with PLA, while ST65-K2 isolates were correlated with more types of infections relative to ST23-K1 isolates. PFGE results showed that the homology of 84 HMKP isolates was diverse. Only five PFGE clusters with more than 75% similarity accounted for more than three isolates. These five PFGE clusters only accounted for 35 (41.7%, 35/84) isolates. In conclusion, our study first found that hypertension and male patients with 41-50 years old were independent risk factors. The composition of ST types and PFGE clusters among K2 isolates was more diverse than K1 isolates. K1 and K2 HMKP isolates had respective specific profiles of virulence-associated genes.

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.

To conduct epidemiological analysis of ( ) with , and to investigate its drug resistance phenotype, ( ) gene, and biofilm formation, so as to provide theoretical basis for further understanding of the drug resistance mechanism of with . were isolated from clinical samples collected from inpatients. All strains were identified by Compact using fully automatic microbial analyzer, the minimal inhibitory concentration ( ) of antibiotics was determined by microbroth dilution test. The double disk diffusion method was used to detect the production of , modified carbapenem inactivation method ( ) was used to detect the production of , and phenotype was detected by wire drawing test. was used to detect gene, virulence factor and capsular serotype. Crystal violet staining was used to detect the ability of biofilm formation. The detected in this study included strains 35 (36.5%), 51 (53.1%), and 49 (51.0%). Most strains carried multiple , but not all of them produce and accounted for 14.6% and 11.5% respectively. Most (91.7%) strains carried the gene, and the other virulence genes were (53.1%), (46.9%), (41.7%), (32.3%), (15.6%), (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 strains and biofilm formation was significantly increased compared to strains without phenotype ( =0.035). Compared to ( ), classical ( ) has a tendency to acquire antibiotic resistance. Our study showed that genes encoding or , and were highly associated with .

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.

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.

Background Klebsiella pneumoniae is an opportunistic pathogen and a leading cause of healthcare-associated infections in hospitals, which are frequently antimicrobial resistant (AMR). Exacerbating the public health threat posed by K. pneumoniae , some strains also harbour additional hypervirulence determinants typically acquired via mobile genetic elements such as the well-characterised large virulence plasmid KpVP-1. The rmpADC locus is considered a key virulence feature of K. pneumoniae and is associated with upregulated capsule expression and the hypermucoid phenotype, which can enhance virulence by contributing to serum resistance. Typically such strains have been susceptible to all antimicrobials besides ampicillin; however, the recent emergence of AMR hypermucoid strains is concerning. Methods Here, we investigate the genetic diversity, evolution, mobilisation and prevalence of rmpADC , in a dataset of 14,000 genomes from isolates of the Klebsiella pneumoniae species complex, and describe the RmST virulence typing scheme for tracking rmpADC variants for the purposes of genomic surveillance. Additionally, we examine the functionality of representatives for variants of rmpADC introduced into a mutant strain lacking its native rmpADC locus. Results The rmpADC locus was detected in 7% of the dataset, mostly from genomes of K. pneumoniae and a very small number of K. variicola and K. quasipneumoniae . Sequence variants of rmpADC grouped into five distinct lineages ( rmp1 , rmp2 , rmp2A , rmp3 and rmp4 ) that corresponded to unique mobile elements, and were differentially distributed across different populations (i.e. clonal groups) of K. pneumoniae . All variants were demonstrated to produce enhanced capsule production and hypermucoviscosity. Conclusions These results provide an overview of the diversity and evolution of a prominent K. pneumoniae virulence factor and support the idea that screening for rmpADC in K. pneumoniae isolates and genomes is valuable to monitor the emergence and spread of hypermucoid K. pneumoniae , including AMR strains.