Antibiotic resistance rates in hypervirulent Klebsiella pneumoniae strains: A systematic review and meta-analysis.

OBJECTIVES: In response to the growing global concerns regarding antibiotic resistance, we conducted a meta-analysis to assess the prevalence of antibiotic resistance in hypervirulent Klebsiella pneumoniae (hvKp) strains. METHODS: We conducted a meta-analysis of antibiotic resistance in the hvKp strains. Eligible studies published in English until April 10, 2023, were identified through a systematic search of various databases. After removing duplicates, two authors independently assessed and analysed the relevant publications, and a third author resolved any discrepancies. Data extraction included publication details and key information on antibiotic resistance. Data synthesis employed a random-effects model to account for heterogeneity, and various statistical analyses were conducted using R and the metafor package. RESULTS: This meta-analysis of 77 studies from 17 countries revealed the prevalence of antibiotic resistance in hvKp strains. A high resistance rates have been observed against various classes of antibiotics. Ampicillin-sulbactam faced 45.3% resistance, respectively, rendering them largely ineffective. The first-generation cephalosporin cefazolin exhibited a resistance rate of 38.1%, whereas second-generation cefuroxime displayed 26.7% resistance. Third-generation cephalosporins, cefotaxime (65.8%) and ceftazidime (57.1%), and fourth-generation cephalosporins, cefepime (51.3%), showed substantial resistance. The last resort carbapenems, imipenem (45.7%), meropenem (51.0%) and ertapenem (40.6%), were not spared. CONCLUSION: This study emphasizes the growing issue of antibiotic resistance in hvKp strains, with notable resistance to both older and newer antibiotics, increasing resistance over time, regional disparities and methodological variations. Effective responses should involve international cooperation, standardized testing and tailored regional interventions.

Occurrence of carbapenem-resistant hypervirulent Klebsiella pneumoniae in oysters in Egypt: a significant public health issue.

BACKGROUND: The global dissemination of critical-priority carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) via food sources represents a significant public health concern. Epidemiological data on CR-hvKp in oysters in Egypt is limited. This study aimed to investigate the potential role of oysters sold in Egypt as a source for carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKp), and CR-hvKp and assess associated zoonotic risks. METHODS: A sample of 330 fresh oysters was randomly purchased from various retail fish markets in Egypt and divided into 33 pools. Bacteriological examination and the identification of Klebsiella pneumoniae were performed. Carbapenem resistance in K. pneumoniae isolates was determined by phenotypic and molecular methods. Additionally, the presence of hypervirulent K. pneumoniae was identified based on virulence gene markers (peg-344, rmpA, rmpA2, iucA, and iroB), followed by a string test. The clustering of CR-hvKp strains was carried out using R with the pheatmap package. RESULTS: The overall prevalence of K. pneumoniae was 48.5% (16 out of 33), with 13 isolates displaying carbapenem resistance, one intermediate resistance, and two sensitive. Both carbapenem-resistant K. pneumoniae and carbapenem-intermediate-resistant K. pneumoniae strains exhibited carbapenemase production, predominantly linked to the blaVIM gene (68.8%). HvKp strains were identified at a rate of 62.5% (10/16); notably, peg-344 was the most prevalent gene. Significantly, 10 of the 13 CRKP isolates possessed hypervirulence genes, contributing to the emergence of CR-hvKp. Moreover, cluster analysis revealed the clustering of two CR-hvKp isolates from the same retail fish market. CONCLUSION: This study provides the first insight into the emergence of CR-hvKp among oysters in Egypt. It underscores the potential role of oysters as a source for disseminating CR-hvKp within aquatic ecosystems, presenting a possible threat to public health.

Molecular Characterization of Klebsiella pneumoniae Clinical Isolates Through Whole-Genome Sequencing: A Comprehensive Analysis of Serotypes, Sequence Types, and Antimicrobial and Virulence Genes.

Introduction Antimicrobial resistance (AMR) has become a menace, spreading among bacterial species globally. AMR is now recognized as a silent pandemic responsible for treatment failures. Therefore, an effective surveillance mechanism is warranted to understand the bacterial species isolated from human clinical specimens. The present study employed next-generation sequencing (NGS) or whole-genome sequencing (WGS) to identify the resistance and virulence genes, sequence type, and serotypes. Methods This study included 18 multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) isolates obtained from patients suffering from different infections attending the Prathima Institute of Medical Sciences, Karimnagar, India. All isolates were identified, and antimicrobial susceptibility profiles were determined through conventional microbiological techniques and confirmed by automated systems. All the isolates were investigated using NGS or WGS to identify the genes coding for resistance, such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases, and virulence genes. Multilocus sequence typing (MLST) was conducted to identify the sequence types, and Kleborate analysis was performed to confirm the species, genes for AMR, and virulence and evaluate the capsular polysaccharide (KL) and cell wall/lipopolysaccharide (O) serotypes carried by the isolates. Results The mean age of the patients was 46.11±20.35 years. Among the patients included, 12 (66.66%) were males and 6 (33.33%) were females. A high percentage (>50%) of hypervirulent K. pneumoniae (hvKp) strains that had genes coding for AMR and plasmids having the potential to carry blaNDM and resistance genes were observed. Among the isolates, 16 (88.88%) revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance. There was a high prevalence of blaSHV (17/18; 94.44%) and blaCTX-M-15 (16/18; 88.88%) AMR genes. Other AMR genes identified included blaTEM (83.33%; 15/18) and blaOXA (14/18; 77.77%). Two (11.11%) strains each showed the presence of blaNDM-1 and blaNDM-5 genes. The virulence genes identified included gapA, infB, mdh, pgi, phoE, rpoB, tonB, and ybt. The most frequent K. pneumoniae serotypes found were KL51:O1v2 (3/18, 16.66%), KL17:O1v1 (3/18, 16.66%), and KL64:O2v1 (3/18, 16.66%). KL64 (4/18; 22.22%) was the most common capsular serotype identified among the isolates. The most frequent MLST-based sequence type (ST) identified included ST-147 (5/18, 27.77%), followed by ST-231 (3/18, 16.66%) and ST-101 (2/18, 11.11%). Conclusions The molecular analysis of K. pneumoniae isolates revealed multiple AMR, plasmid, and virulence genes. Additionally, many global STs were noticed by MLST. The results noted a high prevalence of hvKp strains. Molecular characterization of bacterial strains using NGS/WGS is important to understand the epidemiology of bacterial strains and the antibiotic resistance and virulence genes they are potentially carrying. The data obtained from this study may be utilized to devise careful antibiotic-prescribing approaches and improve patient management practices.

Host immunity involvement in the outcome of phage therapy against hypervirulent Klebsiella pneumoniae infections.

Highly encapsulated hypervirulent Klebsiella pneumoniae (hvKp) causes severe infections. Bacteriophage therapy, an antibiotic alternative, effectively treats bacterial infections. Phage φFK1979 encoding polysaccharide depolymerases can target and disarm the capsule of hvKp FK1979, showing promise against FK1979 infection. Resistant strains induced by φFK1979 are possibly eliminated by host immunity and new phage phiR3 targeting them. We constructed varied immunocompromised FK1979 infection mouse models to assess the therapy efficacy of φFK1979 alone or in combination with phiR3. Survival rates, bacterial loads, histopathology, inflammation, and immune cell distribution of mice were studied. Prompt and adequate administration of φFK1979, rather than phiR3, significantly improved survival rates in mice with different immune statuses. However, immunocompromised mice showed lower efficacy due to reduced tolerance to low-virulence φFK1979-resistant bacteria compared to immunocompetent mice. Adding phiR3 sequentially greatly enhanced therapy efficacy for them, leading to increased survival rates and notable improvements in pathology and inflammation. Immunocompetent mice exhibited the most favorable response to φFK1979 monotherapy, as their immune system cleared φFK1979-resistant bacteria while avoiding a robust response to phiR3 combating φFK1979-resistant bacteria. This study revealed host immunity involvement in the outcome of phage therapy against infections and introduced, for the first time, personalized phage therapy strategies for hvKp-infected mice with varying immune statuses.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp), with high capsular polysaccharide production, can cause severe invasive infections. Capsule-targeting phage poses the potential to fight against hvKp. We previously elucidated that the capsule-targeting phage induces resistance in hvKp, while phage-resistant strains exhibit sensitivity to host innate immunity and new phages targeting them. This indicated that phage-resistant strains can be eliminated by the immune system in immunocompetent patients, whereas they may require treatment with phages targeting resistant bacteria in immunocompromised patients. HvKp can infect individuals with varying immune statuses, including both immunocompetent and immunocompromised/deficient patients. This study, for the first time, developed personalized phage therapy strategies for hvKp-infected mice with different immune statuses, optimizing phage therapy against hvKp infections. This research is expected to provide a theoretical foundation and novel insights for clinical phage therapy against hvKp infections, offering significant societal benefits and clinical value.

Phenotypic and genetic characterization of hypervirulent Klebsiella pneumoniae in patients with liver abscess and ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) and pyogenic liver abscess (PLA) due to Klebsiella pneumoniae infection can trigger life-threatening malignant consequences, however, there are few studies on the strain-associated clinical pathogenic mechanisms between VAP and PLA. A total of 266 patients consist of 129 VAP and 137 PLA were included for analysis in this study. We conducted a comprehensive survey for the two groups of K. pneumoniae isolates, including phenotypic experiments, clinical epidemiology, genomic analysis, and instrumental analysis, i.e., to obtain the genomic differential profile of K. pneumoniae strains responsible for two distinct infection outcomes. We found that PLA group had a propensity for specific underlying diseases, especially diabetes and cholelithiasis. The resistance level of VAP was significantly higher than that of PLA (78.57% vs. 36%, P < 0.001), while the virulence results were opposite. There were also some differences in key signaling pathways of biochemical processes between the two groups. The combination of iucA, rmpA, hypermucoviscous phenotype, and ST23 presented in K. pneumoniae infection is more important and highly prudent for timely treatment. The present study may contribute a benchmark for the K. pneumoniae clinical screening, epidemiological surveillance, and effective therapeutic strategies.

Polyclonal Multidrug ESBL-Producing Klebsiella pneumoniae and Emergence of Susceptible Hypervirulent Klebsiella pneumoniae ST23 Isolates in Mozambique.

Globally, antibiotic-resistant Klebsiella spp. cause healthcare-associated infections with high mortality rates, and the rise of hypervirulent Klebsiella pneumoniae (hvKp) poses a significant threat to human health linked to community-acquired infections and increasing non-susceptibility. We investigated the phenotypic and genetic features of 36 Klebsiella isolates recovered from invasive infections at Hospital Central of Maputo in Mozambique during one year. The majority of the isolates displayed multidrug resistance (MDR) (29/36) to cephalosporins, gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole but retained susceptibility to amikacin, carbapenems, and colistin. Most isolates were ESBLs-producing (28/36), predominantly carrying the blaCTX-M-15 and other beta-lactamase genes (blaSHV, blaTEM-1, and blaOXA-1). Among the 16 genomes sequenced, multiple resistance genes from different antibiotic classes were identified, with blaCTX-M-15, mostly in the ISEcp1-blaCTX-M-15-orf477 genetic environment, co-existing with blaTEM-1 and aac(3)-IIa in five isolates. Our results highlight the presence of polyclonal MDR ESBL-producing K. pneumoniae from eight sequence types (ST), mostly harbouring distinct yersiniabactin within the conjugative integrative element (ICE). Further, we identified susceptible hvKp ST23, O1-K1-type isolates carrying yersiniabactin (ybt1/ICEKp10), colibactin, salmochelin, aerobactin, and hypermucoid locus (rmpADC), associated with severe infections in humans. These findings are worrying and underline the importance of implementing surveillance strategies to avoid the risk of the emergence of the most threatening MDR hvKp.

Polyclonal Multidrug ESBL-Producing Klebsiella pneumoniae and Emergence of Susceptible Hypervirulent Klebsiella pneumoniae ST23 Isolates in Mozambique

Globally, antibiotic-resistant Klebsiella spp. cause healthcare-associated infections with high mortality rates, and the rise of hypervirulent Klebsiella pneumoniae (hvKp) poses a significant threat to human health linked to community-acquired infections and increasing non-susceptibility. We investigated the phenotypic and genetic features of 36 Klebsiella isolates recovered from invasive infections at Hospital Central of Maputo in Mozambique during one year. The majority of the isolates displayed multidrug resistance (MDR) (29/36) to cephalosporins, gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole but retained susceptibility to amikacin, carbapenems, and colistin. Most isolates were ESBLs-producing (28/36), predominantly carrying the blaCTX-M-15 and other beta-lactamase genes (blaSHV, blaTEM-1, and blaOXA-1). Among the 16 genomes sequenced, multiple resistance genes from different antibiotic classes were identified, with blaCTX-M-15, mostly in the ISEcp1-blaCTX-M-15-orf477 genetic environment, co-existing with blaTEM-1 and aac(3)-IIa in five isolates. Our results highlight the presence of polyclonal MDR ESBL-producing K. pneumoniae from eight sequence types (ST), mostly harbouring distinct yersiniabactin within the conjugative integrative element (ICE). Further, we identified susceptible hvKp ST23, O1-K1-type isolates carrying yersiniabactin (ybt1/ICEKp10), colibactin, salmochelin, aerobactin, and hypermucoid locus (rmpADC), associated with severe infections in humans. These findings are worrying and underline the importance of implementing surveillance strategies to avoid the risk of the emergence of the most threatening MDR hvKp.

Molecular characteristics and virulence factors of carbapenem-resistant Klebsiella pneumoniae among pediatric patients in Shanghai, China.

We retrospectively investigated CRKP isolates among 92 pediatric patients (32 neonates and 60 non­neonates) in 2019 and 2020 (59 and 33 isolates, respectively) to investigate the molecular characteristics and virulence factors of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from pediatric patients,. All the CRKP isolates were subjected to antimicrobial susceptibility testing, string testing, molecular typing of virulence and carbapenemase genes, and multilocus sequence typing. Hypervirulent K. pneumoniae (Hvkp) was defined based on the detection of the regulator of mucoid phenotype A (rmpA).Sequence type 11 (ST11) accounted for the majority of infections in both neonates (37.5%) and non­neonates (43.3%) (P > 0.05), whereas it increased from 30.5% (18/59) in 2019 to 60.6% (20/33) in 2020 (P < 0.05). Carbapenemase gene KPC-2 was predominant in both neonates and non­neonates (46.9% vs. 51.7%, respectively), followed by New Delhi metallo-beta-lactamase 1 (NDM-1) (34.4% vs. 28.3%, respectively) (all P > 0.05). Compared to 2019, the proportion of blaNDM-1 decreased (44.1% vs. 6.1%) (P < 0.001), while that of blaKPC-2 increased (40.7% vs. 66.7%) (P = 0.017) in 2020. ybtS and iutA had a higher positivity rate in KPC-2 and ST11 producers (all P < 0.05); the KPC-2-, ybtS-, and iutA-positive isolates showed relatively higher resistance to fluoroquinolones and aminoglycosides, nitrofurantoin, and piperacillin/tazobactam, respectively. Furthermore, the combined expression (95.7%, 88/92) of carbapenemase and virulence-associated genes was detected, with the carbapenemase genes blaKPC-2 and blaTEM-1 combined with virulence-associated genes entB, mrkD, and ybtS accounting for the highest percentage (20.7%).Carbapenemase gene mutations in the CRKP strain from 2019 to 2020 highlight the importance of dynamic monitoring. The spread of hypervirulence-associated genes in CRKP strains and the high positivity rates of ybtS and iutA in KPC-2- and ST11-producing ones signify their high virulence potential in pediatric patients.

Within-patient horizontal transfer of pOXA-48 from a hypervirulent Klebsiella pneumoniae SL218 to Serratia marcescens following spread of the K. pneumoniae isolate among hospitalised patients, Denmark, 2021.

A hypervirulent Klebsiella pneumoniae SL218 (ST23-KL57), phylogenetically distinct from the classical hypervirulent SL23 (ST23-KL1) lineage, was transmitted between hospitalised patients in Denmark in 2021. The isolate carried a hybrid resistance and virulence plasmid containing bla NDM-1 and a plasmid containing bla OXA-48 (pOXA-48); the latter plasmid was horizontally transferred within-patient to Serratia marcescens. The convergence of drug resistance and virulence factors in single plasmids and in different lineages of K. pneumoniae is concerning and requires surveillance.

Presence of Polyketide Synthase (PKS) Gene and Counterpart Virulence Determinants in Klebsiella pneumoniae Strains Enhances Colorectal Cancer Progression In-Vitro.

Klebsiella pneumoniae (K. pneumoniae) colonizes the human gut and is a causative factor of pyogenic liver abscess (PLA). Retrospective studies conducted on K. pneumoniae PLA patients revealed subsequent CRC development in later years of their life with increasing prevalence of these strains harbouring polyketide synthase (PKS) genes. To our knowledge there are no known studies directly implicating K. pneumoniae with CRC to date. Our aims are to characterize K. pneumoniae isolates from CRC patients and investigate its effects on cell proliferation in vitro. K. pneumoniae isolates were characterized by screening virulence genes including polyketide synthase (PKS), biofilm assay, antibiotic susceptibility, and string test to determine hypervirulent (hvKp) strains. Solubilised antigens of selected K. pneumoniae isolates were co-cultured with primary colon cell lines and CRC cell lines (Stage I-IV) for 48 h. The enhancement of proliferation was measured through MTT and ECIS assay. Twenty-five percent of K. pneumoniae isolates were PKS-positive out of which 50% were hvKp strains. The majority of the isolates were from the more virulent serotype of K1 (30%) and K2 (50%). PKS-positive K. pneumoniae isolates did not possess genes to confer carbapenem resistance but instead were more highly associated with siderophore genes (aerobactin, enterobactin, and yersiniabactin) and allantoin metabolism genes (allS, allS2). Cell proliferation in primary colon, SW1116 (Stage I), and SW480 (Stage II) CRC cell lines were enhanced when co-cultured with PKS-positive K. pneumoniae antigens. ECIS revealed enhanced cell proliferation upon recurrent antigen exposure. This demonstrates the possible role that PKS-positive K. pneumoniae has in exacerbating CRC progression.

Molecular and clinical characterization of hypervirulent Klebsiella pneumoniae isolates from individuals with urinary tract infections.

Despite being a significant public health concern, hypervirulent Klebsiella pneumoniae (hvKP) has rarely been investigated in urinary tract infections (UTIs). To investigate the molecular and clinical characterization of hvKP in UTIs, we collected K. pneumoniae strains and clinical data from patients with UTIs. HvKP was confirmed by virulence-related genes and the Galleria mellonella model and sequenced by next-generation sequencing. Our data showed that 30/121 isolates were hvKP [17 carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP), 12 hvKP, and 1 extended-spectrum ß-lactamase-producing hvKP]; these had higher resistance to most antimicrobials and were more likely to cause complicated UTIs (cUTIs). Notably, the mucoid phenotype-regulating genes prmpA and prmpA2 were truncated in 3 and 19 hvKP, respectively. Eight serotypes were detected and divided into three groups: K64 (n = 17), K1/K2 (n = 6), and others (n = 7). Furthermore, 16/17 K64 hvKP isolates were CR-hvKP but with a lower mortality rate of G. mellonella as the truncated prmpA/prmpA2 incurred high fitness cost to the isolates. In addition, all K64 isolates belonged to ST11 with the same cluster, and in two of these strains (KP88 and KP92) bla KPC-2 gene was successfully transferred to EC600. Genetic environment analysis showed that IS26-tnpR-ISKpn27-bla KPC-2-ISKpn6 may be the core structure in the horizontal transfer of bla KPC-2. The highest mortality rate among the infected G. mellonella was observed in the K1/K2 group. In conclusion, hvKP had a higher resistance rate and was more likely to lead to cUTIs. Convergence of hypervirulence and carbapenem resistance in a transmissible ST11 clone of K64 K. pneumoniae was mediated by a plasmid in UTIs. Therefore, surveillance of hvKP in UTIs should be strengthened.

Distribution of virulence genes and capsule types in Klebsiella pneumoniae among bloodstream isolates from patients with hematological malignancies.

The hypervirulent Klebsiella pneumoniae (hv-K. pneumoniae) pathotype has been spreading over during the last years. We evaluated the distribution of virulence genes (iucA, rmpA or rmpA2) and capsule types in K. pneumoniae isolates. A total of 572 K. pneumoniae were evaluated; of those 114 (20%) were carbapenemase-producing K. pneumoniae (CP-K.pneumoniae); 285 (49.8%) - extended-spectrum ß-lactamase-producing K. pneumoniae (ESBL-K. pneumoniae); and 173 (30.2%) - non-CP- and non-ESBL. Among CP-K. pneumoniae the prevalent sequence type was ST395 (37.7%), followed by ST23 (16.7%). A total of 138 (24.1%) hv-K. pneumoniae were detected. The rate of hv-K. pneumoniae (55.3%) was higher among CP-K. pneumoniae compared to ESBL-K. pneumoniae (17.3%) and non-CP- and non-ESBL (15.8%).The iucA and rmpA2 genes were detected in 89.5% of ST23 and 58.1% of ST395. The K57 capsule type was detected in all ST23; K2 was found in 55.8% of ST395. The hv-K. pneumoniae were common in bloodstream isolates, with a significantly higher rate among CP-K. pneumoniae. Most of them belonged to ST23/K57 and ST395/K2.

Dissemination of virulence and resistance genes among Klebsiella pneumoniae via outer membrane vesicle: An important plasmid transfer mechanism to promote the emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae.

Klebsiella pneumoniae is well-known opportunistic enterobacteria involved in complex clinical infections in humans and animals. The domestic animals might be a source of the multidrug-resistant virulent K. pneumoniae to humans. K. pneumoniae infections in domestic animals are considered as an emergent global concern. The horizontal gene transfer plays essential roles in bacterial genome evolution by spread of virulence and resistance determinants. However, the virulence genes can be transferred horizontally via K. pneumoniae-derived outer membrane vesicles (OMVs) remains to be unreported. In this study, we performed complete genome sequencing of two K. pneumoniae HvK2115 and CRK3022 with hypervirulent or carbapenem-resistant traits. OMVs from K. pneumoniae HvK2115 and CRK3022 were purified and observed. The carriage of virulence or resistance genes in K. pneumoniae OMVs was identified. The influence of OMVs on the horizontal transfer of virulence-related or drug-resistant plasmids among K. pneumoniae strains was evaluated thoroughly. The plasmid transfer to recipient bacteria through OMVs was identified by polymerase chain reaction, pulsed field gel electrophoresis and Southern blot. This study revealed that OMVs could mediate the intraspecific and interspecific horizontal transfer of the virulence plasmid phvK2115. OMVs could simultaneously transfer two resistance plasmids into K. pneumoniae and Escherichia coli recipient strains. OMVs-mediated horizontal transfer of virulence plasmid phvK2115 could significantly enhance the pathogenicity of human carbapenem-resistant K. pneumoniae CRK3022. The CRK3022 acquired the virulence plasmid phvK2115 could become a CR-hvKp strain. It was critically important that OMVs-mediated horizontal transfer of phvK2115 lead to the coexistence of virulence and carbapenem-resistance genes in K. pneumoniae, resulting in the emerging of carbapenem-resistant hypervirulent K. pneumoniae.

A Siderophore-Encoding Plasmid Encodes High-Level Virulence in Escherichia coli.

A plasmid that harbored the virulence factors highly like those of the virulence plasmid commonly found in clinical hypervirulent Klebsiella pneumoniae strains was detected in a foodborne Escherichia coli strain EC1108 and designated p1108-IncFIB. This virulent-like plasmid was found to be common in E. coli from various sources. To understand the contribution of this plasmid to the virulence of E. coli, plasmid p1108-IncFIB in strain EC1108 was first cured to generate strain EC1108-PC. The virulence plasmid p15WZ-82_Vir in Klebsiella pneumoniae strain 15WZ-82 was then transmitted to EC1108-PC to produce the transconjugant, EC1108-PC-TC to assess the contribution of this virulence plasmid to the virulence level of E. coli. During the process of conjugation, p15WZ-82_Vir was found to be evolved into p15WZ-82_int, which underwent homologous recombination with a plasmid encoding a carbapenemase gene, blaNDM-1, p1108-NDM, in EC1108-PC. Comparison between the level of virulence in the EC1108, EC1108-PC-TC, and EC1108-PC through serum and macrophage resistance assay, as well as animal experiments, confirmed that plasmid p1108-IncFIB encoded a high level of virulence in E. coli, yet the fusion plasmid derived from p15WZ-82_Vir did not encode virulence but instead imposed a high fitness cost in the E. coli strain EC1108-PC-TC. These findings indicate that E. coli strains carrying the virulence plasmid p1108-IncFIB in multidrug-resistant (MDR) strains may also impose serious public health threats like that of hypervirulent Klebsiella pneumoniae strains harboring the p15WZ-82_Vir plasmid. IMPORTANCE Acquisition of pLVPK-like virulence plasmid by Klebsiella pneumoniae converts it to hypervirulent K. pneumoniae (HvKP), which has become one of the most important clinical bacterial pathogens. The potential of transmission of this virulence plasmid and its contribution to the virulence of other Enterobacteriaceae, such as E. coli, are not clear yet. In this study, we showed that pLVPK-like virulence plasmid exhibited fitness costs and did not contribute to the virulence in E. coli. However, we identified a novel virulence plasmid, p1108-IncFIB, that encodes similar siderophore genes as those of pLVPK from a foodborne E. coli strain and showed that p1108-IncFIB encoded a high level of virulence in E. coli. BLAST of E. coli genomes from GenBank showed that these siderophore genes were widespread in clinical E. coli strains. Further studies are warranted to understand the impact of this plasmid in the control of clinical infections caused by E. coli.

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

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

Association of fluoroquinolone resistance and ESBL production in hypervirulent Klebsiella pneumoniae ST11 and ST893 in Iran.

The spread of multidrug resistance in Klebsiella pneumoniae is a serious threat to the public health. In this study, the prevalence of fluoroquinolone resistance and virulence determinants among ESBL-producing K. pneumoniae isolates was investigated. A total of 50 third-generation cephalosporin resistant K. pneumoniae strains were collected from patients' clinical cultures between September 1st, 2019 and February 30th, 2020. Clonal relatedness of clinical isolates was determined by multilocus sequence typing. All 50 isolates were multidrug-resistant (MDR) and carried at least one of the ESBL resistance determinants. The bla CTX-M-15 gene was the major ESBL determinant found in K. pneumoniae (88%), followed by bla SHV (86%) and bla TEM (78%). PMQR was detected in 96% of the isolates and aac(6')-Ib-cr was the most common (78%) as well as multiple mutations in gyrA (S83I, D87G) and parC (S80I) were found. Selected isolates were assigned to seven sequence types (STs) (ST11, ST893, ST147, ST16, ST377, ST13, and ST392). Overall, hypervirulent phenotypes were identified in 26 (52%) of the isolates. Among the 50 isolates, 28 (56%) were positive for ybt, 23 (46%) for rmpA, 17 (34%) for iroB, 15 (30%) for magA, 4 (8%) for alls and 3 (6%) for iucA genes. The K1 capsular type was the most prevalent (11/50; 22%) among isolates. The emergence of hypervirulent K. pneumoniae (hvKp) ST11 and ST893, which co-carried ESBL, PMQR determinants and different virulence genes has become a threat to the treatment of inpatients in the clinical setting.

Emergence of polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae from an individual in the community with asymptomatic bacteriuria.

BACKGROUND: The heteroresistance of polymyxin B, a last-resort antibiotic used to treat many serious bacterial infections, may lead to antibiotic treatment failure. However, polymyxin B-heteroresistant isolates are rare in individuals living in the community. We report a polymyxin B-heteroresistant hypervirulent Klebsiella pneumoniae (hvKP) isolate from an individual in the community with asymptomatic bacteriuria. RESULTS: The NYTJ35 isolate had multiple virulence genes that encoded a mucoid phenotype regulator (rmpA), aerobactin (iucABCD-iutA), salmochelin (iroBCDN), yersiniabactin (irp1-2 and ybtAEPQSTUX), and a truncated rmpA2. Infection of galleria mellonella larvae indicated the isolate was hypervirulent. Antimicrobial susceptibility testing showed it was susceptible to all tested antibiotics except polymyxin B. The proportion of surviving bacteria was 1.2 × 10- 7 based on the population analysis profile (PAP) method, suggesting the presence of polymyxin B heteroresistance. The isolate was not hypermucoviscous, but it was a strong biofilm producer. It had capsular serotype K1 and belonged to sequence type 23 (ST23). The isolate also had the D150G substitution in phoQ, which is known to confer polymyxin B resistance. CONCLUSIONS: We identified the co-occurrence of hypervirulence and polymyxin B heteroresistance in a K. pneumoniae isolate from an individual with asymptomatic bacteriuria. We suggest the use of increased screening for hvKP in individuals living in the community.

A Case of Forearm Soft Tissue Infection Caused by Hypervirulent K. pneumoniae in an Otherwise Healthy 24-Year-Old Woman.

In recent years, hypervirulent Klebsiella pneumoniae (hvKp) has received greater attention. It mainly infects diabetic patients and typically causes a hepatic abscess. Here, we report a case of hvKp that caused forearm muscle and soft tissue infection in addition to bacteremia, hepatic and pulmonary abscess, and hyperglycemia. The patient's condition stabilized after comprehensive treatment. She eventually recovered and was discharged after several debridement and flap operations. At 9 months of follow-up, no signs of infectious recurrence were noted, and the hyperglycemia resolved. Here, we detail important clinical features of a severe hvKp case diagnosed in an otherwise healthy individual. This report underscores the potential of hvKp to cause deep tissue infection and present with clinical symptoms similar to gas gangrene. Symptom onset in the setting of hvKp infection is usually gradual and misdiagnosis is common. The diagnosis of hvKp should be routinely considered in the clinical setting, and be strongly suspected when presenting with characteristic epidemiological, clinical and laboratory features. Although diabetes is a predisposing factor to hvKp infection, hyperglycemia appeared to manifest as a consequence of hvKp infection in this patient.

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

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

Phage Selective Pressure Reduces Virulence of Hypervirulent Klebsiella pneumoniae Through Mutation of the wzc Gene.

Hypervirulent Klebsiella pneumoniae (hvKp), one of the major community-acquired pathogens, can cause invasive infections such as liver abscess. In recent years, bacteriophages have been used in the treatment of K. pneumoniae, but the characteristics of the phage-resistant bacteria produced in the process of phage therapy need to be evaluated. In this study, two Podoviridae phages, hvKpP1 and hvKpP2, were isolated and characterized. In vitro and in vivo experiments demonstrated that the virulence of the resistant bacteria was significantly reduced compared with that of the wild type. Comparative genomic analysis of monoclonal sequencing showed that nucleotide deletion mutations of wzc and wcaJ genes led to phage resistance, and the electron microscopy and mucoviscosity results showed that mutations led to the loss of the capsule. Meanwhile, animal assay indicated that loss of capsule reduced the virulence of hvKp. These findings contribute to a better understanding of bacteriophage therapy, which not only can kill bacteria directly but also can reduce the virulence of bacteria by phage screening.