Ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae accompanied hypermucoviscosity acquisition.

BACKGROUND: Antimicrobial resistance and bacterial hypermucoviscosity, associated with escalating production of capsules, constitute major challenges for the clinical management of Klebsiella pneumoniae (K. pneumoniae) infections. This study investigates the association and underlying mechanism between ceftazidime-avibactam (CAZ-AVI) resistance and bacterial hypermucoviscosity in Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp). RESULTS: The proportion of CAZ-AVI-sensitive clinical isolates exhibiting the hypermucoviscous phenotype was significantly lower than that of the resistant strains (5.6% vs. 46.7%, P < 0.001). To further verify the correlation and molecular mechanism between CAZ-AVI resistance and hypermucoviscosity, 10 CAZ-AVI-resistant isolates were generated through in vitro resistance selection from CAZ-AVI-sensitive KPC-Kp. The results showed the same association as it showed in the clinical isolates, with four out of ten induced CAZ-AVI-resistant isolates transitioning from negative to positive in the string tests. Comparative genomic analysis identified diverse mutations in the wzc gene, crucial for capsule polysaccharide (CPS) synthesis, in all four CAZ-AVI-resistant hypermucoviscous KPC-Kp strains compared to the parent strains. However, these mutations were absent in the other six KPC-Kp strains that did not exhibit induced hypermucoviscosity. Cloning of the wzc gene variants and their expression in wild-type strains confirmed that mutations in the wzc gene can induce bacterial hypermucoviscosity and heightened virulence, however, they do not confer resistance to CAZ-AVI. CONCLUSIONS: These results indicated that resistance to CAZ-AVI in KPC-Kp isolates may be accompanied by the acquisition of hypermucoviscosity, with mutations in the wzc gene often involving in this process.

Microaerobic-mediated suppression of Klebsiella pneumoniae mucoviscosity is restored by rmpD overexpression.

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.

Klebsiella pneumoniae TolC contributes to antimicrobial resistance, exopolysaccharide production, and virulence.

Klebsiella pneumoniae is a Gram-negative bacterium that causes a variety of human diseases, ranging from pneumonia to urinary tract infections and invasive diseases. The emergence of K. pneumoniae strains that are resistant to multiple antibiotics has made treatment more complex and led to K. pneumoniae becoming a global health threat. Addressing this threat necessitates the development of new therapeutic strategies to combat this pathogen, including strategies to overcome antimicrobial resistance and therapeutics for novel targets such as antivirulence. Here, we investigated the function of TolC, an outer membrane protein essential for the function of tripartite transporters, in K. pneumoniae. Mutation of tolC rendered K. pneumoniae hypersensitive to multiple antibiotics. Moreover, the tolC mutation impaired capsule production and affected the expression of key capsule biosynthetic genes, indicating a regulatory role for TolC in capsule biosynthesis. Additionally, TolC was essential for growth under iron-limiting conditions, suggesting its involvement in iron acquisition. The tolC mutant exhibited increased adherence to human enterocytes and enhanced serum sensitivity. In the Galleria mellonella infection model, the tolC mutant displayed reduced virulence compared to the wild type. Our findings highlight the pleiotropic role of TolC in K. pneumoniae pathobiology, influencing antimicrobial resistance, capsule production, iron homeostasis, adherence to host cells, and virulence. Understanding the multifaceted role of TolC in K. pneumoniae may guide the development of new therapeutic strategies against this pathogen. .

Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35.

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.

Clinical characteristics and manifestations in patients with hypermucoviscous Klebsiella pneumoniae bacteremia from extra-hepatobiliary tract infection.

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.

Hypervirulent Klebsiella pneumoniae: An update on epidemiology, detection and antibiotic resistance.

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.

Characterization of hypervirulent Klebsiella pneumoniae isolates in Belgium.

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.

Multidrug-resistant and potentially pathogenic Enterobacteriaceae found in a tertiary hospital sewage in southeastern Brazil.

Hospital sewage is considered an environment with the potential to favor the spread and increase of multidrug-resistant bacteria (MDR). The increase in antimicrobial resistance is one of the greatest global threats today. Therefore, this study aimed to evaluate the profile of antimicrobial susceptibility and virulence factors in Enterobacteriaceae isolated from the sewage of a tertiary hospital located in southeastern Brazil. For bacterial isolation, membrane filtering, serial dilution, and spread-plate techniques were used. The bacterial isolates were identified using the MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) technique. Antimicrobial susceptibility profile was performed by disk-diffusion test. Virulence genes were screened by Polymerase Chain Reaction (PCR) and the hypermucoviscosity phenotype by string test. In total, 13 enterobacteria distributed in three species were identified (Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii) and 76.9% (n = 10) were classified as MDR. Two K. pneumoniae demonstrated the hypermucoviscosity phenotype. The virulence genes ycfM and entB were detected in all K. pneumoniae isolates (other genes found were fimH, mrkD, and kfu). The results indicated that the sewage from the analyzed hospital receives MDR bacteria and has the potential to contaminate and spread through the environment.

Klebsiella spp. cause severe and fatal disease in Mozambican children: antimicrobial resistance profile and molecular characterization.

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 ß-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.

Acquisition of Plasmid with Carbapenem-Resistance Gene blaKPC2 in Hypervirulent Klebsiella pneumoniae, Singapore.

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.

Clinical and molecular characteristics of Klebsiella pneumoniae ventilator-associated pneumonia in mainland China.

BACKGROUND: Klebsiella pneumoniae is a prominent nosocomial pathogen that accounts for up to 10 % of all hospital-acquired infections. It is a frequent cause of ventilator-associated pneumonia (VAP). The purpose of this study was to investigate the clinical characteristics of K. pneumoniae-associated VAP and the molecular characteristics of K. pneumoniae strains. METHODS: We retrospectively reviewed 70 mechanically ventilated patients with K. pneumoniae isolated. All K. pneumoniae strains were examined to determine hypermucoviscosity (HV) phenotype, capsular serotypes, virulence genes, multilocus sequence typing and antimicrobial susceptibility. RESULTS: Hypermucoviscosity was found in 14 of 70 (20 %) isolates of K. pneumoniae. Among the 70 patients, 43 cases (61.4 %) developed VAP. Furthermore, VAP was more frequently induced by HV-positive K. pneumoniae (14/14, 100 %) than by HV-negative strains (29/56, 51.7 %). HV-positive K. pneumoniae-associated VAP patients were more inclined to develop bacteremia and had a higher mortality rate than HV-negative strains VAP patients. Antibiotic resistance was more frequent in HV- negative strains- than in HV- positive strains-infected patients. The prevalence of rmpA and aerobactin genes were 85.7 % and 85.7 % respectively, and serotypes K1 and K2 accounted for 14.3 % and 28.6 % of the hypermucoviscosity strains, respectively. Strains carrying rmpA and aerobactin genes were significantly associated with HV-phenotype, and rmpA and aerobactin coexisted in HV-positive strains. Multilocus sequence typing analysis identified 24 different sequence types from K. pneumoniae VAP samples. CONCLUSIONS: HV-phenotype is the major virulence determinant for mechanically ventilated patients. There was a specific sequence typing (ST) distribution between HV-positive and HV-negative strains.

Pediatric hypervirulent Klebsiella pneumoniae septic arthritis.

Cases of infection with hypervirulent Klebsiella pneumoniae are gradually increasing in number, and cause life-threatening community-acquired infection even in immunocompetent patients. A 14-year-old boy developed septic hip arthritis due to hypervirulent K. pneumoniae (sequence type 23, serotype K1, magA positive). The patient initially seemed to have been successfully treated with antibiotics and surgical intervention, but septic arthritis developed into osteomyelitis of the femoral head and myositis, which required long-term antibiotic therapy and additional surgical intervention. This is the first pediatric case of hypervirulent K. pneumoniae septic hip arthritis. Treatment plans should mainly consist of antibiotic therapy and surgical intervention. Clinicians, even pediatricians, in developed countries should be aware of the increasing incidence of hypervirulent Klebsiella pneumoniae infection.

Comprehensive study reveals phenotypic heterogeneity in Klebsiella pneumoniae species complex isolates.

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-ß-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.

Comparative analysis of multidrug-resistant Klebsiella pneumoniae strains of food and human origin reveals overlapping populations.

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 ß-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.

Clinical and Microbiologic Analysis of Klebsiella pneumoniae Infection: Hypermucoviscosity, Virulence Factor, Genotype, and Antimicrobial Susceptibility.

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.

Molecular characterization of hypermucoviscous carbapenemase-encoding Klebsiella pneumoniae isolates from an Egyptian hospital.

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.

Co-occurrence of ST412 Klebsiella pneumoniae isolates with hypermucoviscous and non-mucoviscous phenotypes in a short-term hospitalized patient.

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.

Hypervirulent Klebsiella pneumoniae with a hypermucoviscosity phenotype challenges strategies of water disinfection for its capsular polysaccharides.

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.