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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]

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.

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.

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]

The convergence of carbapenem-resistance and hypervirulence genes in Klebsiella pneumoniae has led to the emergence of highly drug-resistant superbugs capable of causing invasive disease. We analyzed 556 carbapenem-resistant K. pneumoniae isolates from patients in Singapore hospitals during 2010-2015 and discovered 18 isolates from 7 patients also harbored hypervirulence features. All isolates contained a closely related plasmid (pKPC2) harboring blaKPC-2, a K. pneumoniae carbapenemase gene, and had a hypervirulent background of capsular serotypes K1, K2, and K20. In total, 5 of 7 first patient isolates were hypermucoviscous, and 6 were virulent in mice. The pKPC2 was highly transmissible and remarkably stable, maintained in bacteria within a patient with few changes for months in the absence of antimicrobial drug selection pressure. Intrapatient isolates were also able to acquire additional antimicrobial drug resistance genes when inside human bodies. Our results highlight the potential spread of carbapenem-resistant hypervirulent K. pneumoniae in Singapore. [ABSTRACT FROM AUTHOR]

Introduction: The rapid spread of Hypervirulent Klebsiella pneumoniae (hvKP) and Multi Drug Resistant (MDR) strains among the clinical isolates of K.pneumoniae is a great deal of concern. The drug resistance and limited treatment options are the major threats. However, the spread of drug resistant strains is through plasmid which may carry virulence determinants such as capsular antigens. Aim: To know the occurrence of hvKP; to study the occurrence of various capsular serotypes such as K1, K2, K3 and K4 among the isolates of K. pneumoniae using Co-agglutination (Co-A) test; to know the occurrence of beta lactamase genes (bla genes) among the K1, K2, K3 and K4 positive serotypes of K. pneumoniae. The study also tried to assess the reproducibility of Co-A test by comparing the result with PCR. Materials and Methods: A prospective study was carried with a total of 60 consecutive, non-duplicate isolates of K. pneumoniae recovered from various clinical specimens obtained from both outpatients and inpatients between April to December 2017. To determine the occurrence of hvKP by string test, the identification of various serotypes was done by Co-A test and PCR. The determination of beta lactamase genes such as blaTEM, blaSHV and blaCTX-M among the capsular serotypes was done by PCR study. Results: Rate of typability by Co-A was found to be 13(21.67%), 0(0%), 32(53.33%), 7(11.67%) for K1, K2, K3 and K4, respectively. Eight (13.33%) of them remained untypable. The rate of typability among the K. pneumoniae K1 serotype by Co-A test and PCR remained somewhat similar. Conclusion: Co-A test is a good method of serotyping and can be used not only for diagnostic purpose but also for epidemiological study.

Introduction: The rapid spread of Hypervirulent Klebsiella pneumoniae (hvKP) and Multi Drug Resistant (MDR) strains among the clinical isolates of K.pneumoniae is a great deal of concern. The drug resistance and limited treatment options are the major threats. However, the spread of drug resistant strains is through plasmid which may carry virulence determinants such as capsular antigens. Aim: To know the occurrence of hvKP; to study the occurrence of various capsular serotypes such as K1, K2, K3 and K4 among the isolates of K. pneumoniae using Co-agglutination (Co-A) test; to know the occurrence of beta lactamase genes (bla genes) among the K1, K2, K3 and K4 positive serotypes of K. pneumoniae. The study also tried to assess the reproducibility of Co-A test by comparing the result with PCR. Materials and Methods: A prospective study was carried with a total of 60 consecutive, non-duplicate isolates of K. pneumoniae recovered from various clinical specimens obtained from both outpatients and inpatients between April to December 2017. To determine the occurrence of hvKP by string test, the identification of various serotypes was done by Co-A test and PCR. The determination of beta lactamase genes such as blaTEM, blaSHV and blaCTX-M among the capsular serotypes was done by PCR study. Results: Rate of typability by Co-A was found to be 13(21.67%), 0(0%), 32(53.33%), 7(11.67%) for K1, K2, K3 and K4, respectively. Eight (13.33%) of them remained untypable. The rate of typability among the K. pneumoniae K1 serotype by Co-A test and PCR remained somewhat similar. Conclusion: Co-A test is a good method of serotyping and can be used not only for diagnostic purpose but also for epidemiological study. [ABSTRACT FROM AUTHOR]

We investigated the implications of hypervirulence determinants on clinical features of 48 adult patients with bacteremia caused by extended-spectrum β-lactamase-producing Klebsiella pneumoniae. Isolates in the hypervirulence group included any of the following virulence determinants: K1/K2 capsule serotypes, hypermucoviscosity phenotype, rmpA gene, or rmpA2 gene. Nonhypervirulence group isolates were negative for all of the above virulence factors. In this study, all isolates used were non-K1/K2 strains. Statistically significant differences were observed in clinical features of patients between the two groups. The hypervirulent isolates ( n = 19), including 11 isolates with the hypermucoviscosity phenotype, 15 with the rmpA gene, and 16 with the rmpA2 gene, were more commonly recovered from diabetic patients and mainly manifested as secondary bacteremia (such as pneumonia, urinary tract infections, or other localized infections). The nonhypervirulent isolates ( n = 29) were more commonly recovered from patients after prolonged hospital stays (>30 days) and mostly manifested as primary bacteremia. The overall in-hospital mortality was 56.3%. Hazard ratio (HR) analysis revealed the following positive predictors for mortality: nosocomial infection, stay in an intensive care unit, no removal of the central venous catheter, Charlson comorbidity score, and APACHE II score (≧15). The negative predictors were initial appropriate antibiotic therapy (HR 0.42) and urinary tract infection (HR 0.19). Charlson score was an independent confounder based on multivariate analysis (HR 1.43, 95% confidence interval 1.04-1.99). In conclusion, hypervirulence determinants played a role in causing secondary infections in diabetic patients; however, the presence of morbidity cofactors could themselves influence mortality, despite the absence of hypervirulence determinants. [ABSTRACT FROM AUTHOR]

Background:Klebsiella pneumoniaewith hypermucoviscosity (HM) phenotype is generally more virulent than HM-negative strains. The aim of this study was to investigate the prevalence of HM phenotype among urinary isolates and to compare the virulence factors, antimicrobial susceptibility patterns and clinical characteristics of HM-positive and -negativeK. pneumoniaeisolated from urine of hospitalized patients. Methods:From June to October 2013, a total of 81 non-repetitiveK. pneumoniaestrains were isolated from urine. HM phenotype was determined by a string test. The K1 and K2 genotypes, theallS,kfu,rmpA,rmpA2andwabG, aerobactin gene were detected by polymerase chain reaction. Results:Of the 81K. pneumoniaeisolates, 12.3% produced a positive string test. The aerobactin (80.0%[8/10] vs. 15.5%[11/71],p = .0001),allS(40.0%[4/10] vs. 9.9%[7/71],p = .009),rmpA(70.0%[7/10] vs. 14.1%[10/71],p = .0001) andrmpA2(60.0%[6/10] vs. 16.9%[12/71],p = .002) genes were more prevalent in HM positive than in HM negative strains. The K1 (20.0%[2/10) vs. 8.5%[6/71]] and K2 (30.0%[3/10] vs. 4.2%[3/71]) capsular serotypes were more common in HM strains than in non-HM strains (p = .0001). HM-positiveK. pneumoniaeisolates were more susceptible to amoxicillin/clavulanic acid (p = .02), cefazolin (p = .03), cefotaxime (p = .02) and ciprofloxacin (p = .03) than HM-negative isolates. Multivariate analysis showed that HM phenotype (OR, 23.87; 95% CI, 3.91–145.4,p < .01) and age >60 years (OR, 8.33; 95% CI, 1.25–55.31,p = .03) were significant risk factors for concurrent bacteraemia. Conclusion:Klebsiella pneumoniaewith expression of HM phenotype isolated from urine were more likely to be associated with concurrent bacteraemia than isolates without the HM phenotype, and were more susceptible to antibiotics. Physicians have to be aware of the possibility of bacteraemia in patients withK. pneumoniaebacteriuria, especially if strains are HM positive and the patient is >60 years old. [ABSTRACT FROM PUBLISHER]

Abstract: Hypermucoviscosity, rmpA (regulator of mucoid phenotype), aerobactin (an iron siderophore), kfu (an iron uptake system), allS (associated with allantoin metabolism), and K1/K2 capsules are important virulence determinants in Klebsiella pneumoniae for liver abscesses. We determined the prevalence of these virulence factors of 50 nonrepeat K. pneumoniae isolates recovered from patients with primary liver abscesses who were treated at 2 medical centers in Taiwan. Virulence genes were surveyed by polymerase chain reaction analysis. The prevalence of hypermucoviscosity phenotype, plasmid-born rmpA, aerobactin, kfu, and allS genes revealed 96%, 100%, 100%, 100%, and 100% in 26 capsular K1 isolates; 90%, 100%, 100%, 0%, and 0% in 10 K2 isolates; and 79%, 86%, 93%, 50%, and 0% in 14 non-K1/K2 isolates; respectively. When injected into mice intraperitoneally, regardless of any capsule K serotype, K. pneumoniae isolates with hypermucoviscosity phenotype as well as presence of rmpA and aerobactin genes exhibited high virulence for mouse lethality (LD50, <102 CFU). Without significant difference in the prevalence of expressing hypermucoviscosity phenotype and carriage of rmpA and aerobactin genes, these virulent non-K1/K2 isolates are as capable as K1/K2 isolates of causing primary liver abscesses. [Copyright &y& Elsevier]