hvKP Isolates Research Articles

Characterizing the bacteriophage PKp-V1 as a potential treatment for ESBL-producing hypervirulent K1 Klebsiella pneumoniae ST258 isolated from veterinary specimens.

The dearth of new antibiotics necessitates alternative approaches for managing infections caused by resistant superbugs. This study aimed to evaluate the lytic potential of the purified bacteriophage PKp-V1 against extended-spectrum β-lactamase (ESBL) harboring hypervirulent Klebsiella pneumoniae (hvKp)-K1 recovered from veterinary specimens. A total of 50 samples were collected from various veterinary specimens to isolate K. pneumoniae, followed by antimicrobial susceptibility testing and molecular detection of various virulence and ESBL genes. Multilocus sequence typing of the isolates was performed to identify prevalent sequence types. The bacteriophages were isolated using the double-agar overlay method and characterized using transmission electron microscopy, spot tests, plaque assays, stability tests, and one-step growth curve assays. Among 17 (34%) confirmed K. pneumoniae isolates, 6 (35%) were hvKp, whereas 13 (76%) isolates belonging to the K1 type were positive for the wzy (K1) virulence gene. All (100%) hvKp isolates exhibited the allelic profile of ST258. Overall, PKp-V1 exhibited an 88 % (15/17; (p ≤ 0.05) host range, among which all (100 %; p ≤ 0.01) hvKp isolates were susceptible to PKp-V1. PKp-V1 exhibited a lytic phage titer of 2.4 × 108 plaque forming unit (PFU)/mL at temperatures ranging from 25°C to 37°C. The lytic phage titers of PKp-V1 at pH = 8 and 0.5% chloroform were 2.1 × 108 PFU/mL and 7.2 × 109 PFU/mL, respectively. Although the incidence of ESBL-infected K. pneumoniae in veterinary settings is worrisome, PKp-V1 phages showed considerable lytic action against the host bacterium, indicating the potential of PKp-V1 as a possible alternative therapeutic option against MDR K. pneumoniae.

In vivo evolution to hypermucoviscosity and ceftazidime/avibactam resistance in a liver abscess caused by Klebsiella pneumoniae sequence type 512.

Here we describe the evolution of KPC-3-producing ST512 K. pneumoniae isolated at three different times from the same patient of which the last one, from a biliary abscess, showed CZA resistance by KPC-31 production and manifested hmv colony phenotype. Hypervirulent Klebsiella pneumoniae (hv-Kp) isolates are increasingly reported worldwide. Their hypervirulent traits are associated with the presence of rmpA/A2 genes and an hmv. In this study, we identified an hmv-Kp that lacked the rmpA-D cluster but showed an amino acid substitution in the Wzc tyrosin kinase protein, involved in the capsular biosynthesis. This hmv-Kp strain emerged in vivo and evolved resistance to ceftazidime/avibactam resistance in a liver abscess of a patient. Our findings suggest that wzc mutations may be underreported, making it challenging to distinguish hv-Kp from "classic" K. pneumoniae with an hmv phenotype.

Genomic insights unveil the plasmid transfer mechanism and epidemiology of hypervirulent Klebsiella pneumoniae in Vietnam.

Hypervirulent Klebsiella pneumoniae (hvKp) is a significant cause of severe invasive infections in Vietnam, yet data on its epidemiology, population structure and dynamics are scarce. We screened hvKp isolates from patients with bloodstream infections (BSIs) at a tertiary infectious diseases hospital in Vietnam and healthy individuals, followed by whole genome sequencing and plasmid analysis. Among 700 BSI-causing Kp strains, 100 (14.3%) were hvKp. Thirteen hvKp isolates were identified from 350 rectal swabs of healthy adults; none from 500 rectal swabs of healthy children. The hvKp isolates were genetically diverse, encompassing 17 sequence types (STs), predominantly ST23, ST86 and ST65. Among the 113 hvKp isolates, 14 (12.6%) carried at least one antimicrobial resistance (AMR) gene, largely mediated by IncFII, IncR, and IncA/C plasmids. Notably, the acquisition of AMR conjugative plasmids facilitated horizontal transfer of the non-conjugative virulence plasmid between K. pneumoniae strains. Phylogenetic analysis demonstrated hvKp isolates from BSIs and human carriage clustered together, suggesting a significant role of intestinal carriage in hvKp transmission. Enhanced surveillance is crucial to understand the factors driving intestinal carriage and hvKp transmission dynamics for informing preventive measures. Furthermore, we advocate the clinical use of our molecular assay for diagnosing hvKp infections to guide effective management.

Molecular detection of Class 1, 2, and 3 integrons in hypervirulent and classic Klebsiella pneumoniae isolates: A cross-sectional study.

The "hypervirulent" variant of Klebsiella pneumoniae (hvKp) is an emerging pathogen that cause life-threatening infection. The present study was conducted to identify the prevalence of hvKp and to investigate the presence class 1, 2, and 3 integrons in these isolates. A cross-sectional study was conducted at three teaching hospitals, Ahvaz, South-west of Iran, from January 1, 2019 to December 31, 2020. Samples were collected from inpatients and included only the first samples collected from each patient. K. pneumoniae strains were isolated from different specimens using biochemical test and confirmed by targeting 16S-23S rDNA internal transcribed spacer. HvKp isolates were recovered using string test and were further characterized by detection virulence-associated genes (rmpA, iucA, and magA). Antibiotic susceptibility patterns of isolates were determined using the disc diffusion method. Isolates were screened for presence the integron genes (intI, intII, and intIII) and repetitive element sequence-based polymerase chain reaction (PCR) performed to determine strain relatedness. SPSS version 22 was used for the data analysis. Seventy-one (77%) of isolates showed multidrug-resistant (MDR) phenotype. HvKP accounted for 14% (13/92) of cKp isolated from blood (46%) and urinary tract infection (38%), and the great majority of them (61.5%; 8/13) exhibited MDR phenotype. Using the PCR assay, 29 of 92 isolates (31.5%) were found to have positive results for the presence of IntI. Three of the IntI-positive strains were hvKP. Class 2 integron was present in 8/92 cKp isolates. Integron Class 2 was found to coexist with Class 1 integron in 3/8 isolates. All integron-positive isolates (IntI and/or IntII) were resistant to at least three different classes of antibiotics and showed MDR phenotype. No Class 3 integrons were detected among the isolates. The results of our study revealed that considering the role of integrons in facilitating the acquisition and dissemination of resistance genes among bacteria, monitoring the emergence of hvKp, emphasizing on the mechanism of antimicrobial resistance, can prevent from the spread of carbapenemase-producing hvKp strains.

Hypervirulent Capsular Serotypes K1 and K2 Klebsiella pneumoniae Strains Demonstrate Resistance to Serum Bactericidal Activity and Galleria mellonella Lethality.

Hypervirulent Klebsiella pneumoniae (hvKp) is a variant that has been increasingly linked to severe, life-threatening infections including pyogenic liver abscess and bloodstream infections. HvKps belonging to the capsular serotypes K1 and K2 have been reported worldwide, however, very scarce studies are available on their genomics and virulence. In the current study, we report four hypermucoviscous extended-spectrum β-lactamase-producing hvKp clinical strains of capsular serotype K1 and K2 isolated from pus and urine of critically ill patients in tertiary care hospitals in Oman. These strains belong to diverse sequence types (STs), namely ST-23(K1), ST-231(K2), ST-881(K2), and ST-14(K2). To study their virulence, a Galleria mellonella model and resistance to human serum killing were used. The G. mellonella model revealed that the K1/ST-23 isolate was the most virulent, as 50% of the larvae died in the first day, followed by isolate K2/ST-231 and K2/ST-14, for which 75% and 50% of the larvae died in the second day, respectively. Resistance to human serum killing showed there was complete inhibition of bacterial growth of all four isolates by the end of the first hour and up to the third hour. Whole genome sequencing (WGS) revealed that hvKp strains display a unique genetic arrangement of k-loci. Whole-genome single-nucleotide polymorphism-based phylogenetic analysis revealed that these hvKp isolates were phylogenetically distinct, belonging to diverse clades, and belonged to different STs in comparison to global isolates. For ST-23(K1), ST-231(K2), ST-881(K2), and ST-14(K2), there was a gradual decrease in the number of colonies up to the second to third hour, which indicates neutralization of bacterial cells by the serum components. However, this was followed by a sudden increase of bacterial growth, indicating possible resistance of bacteria against human serum bactericidal activity. This is the first report from Oman detailing the WGS of hvKp clinical isolates and assessing their resistance and virulence genomics, which reinforce our understanding of their epidemiology and dissemination in clinical settings.

One-year surveillance for hypervirulent Klebsiella pneumoniae detected carbapenem-resistant superbugs.

Hypervirulent Klebsiella pneumoniae (hvKp) can cause infections in clinically healthy people, such as young and immunocompetent patients. Genes involved in the capsule synthesis or those encoding the siderophores have been adopted as predictors of hvKp. Certain sequence types, such as ST23 and ST86, have been associated with hvKp strains, too. The aim of this study was to investigate the presence of hvKp among 354 K. pneumoniae strains isolated from clinical samples of patients admitted to an Italian 900-bed hospital between 21 May 2021 and April 2022. All the isolates were screened by PCR for the amplification of virulence loci. Whole genome sequencing was performed in strains tested positive for at least one target gene. Thirteen out of 354 (3.7%) were hvKp. Five were wild type and belonged to the hypervirulent clones ST23, ST86, ST5, and ST375 and to the new clone ST6310. Six strains carried the blaKPC gene: three belonged to ST101, two to ST512, and one to ST395. Two isolates were ST147 and carried the blaNDM gene. Although hvKp isolation is not frequent, their presence should be systematically investigated to avoid the spreading of both virulent strains and strains with combined increase in virulence and resistance to antibiotics. PCR-based protocols are essential for surveillance of these strains, which do not always show a recognizable phenotype. Moreover, hvKp strains were isolated also from patients without history of recent foreign travels, indicating an increased spreading of these strains as well as an underestimated of their circulation so far.IMPORTANCEKlebsiella pneumoniae is a healthcare-associated pathogen frequently resistant to antibiotics. Hypervirulent strains of pneumoniae (hvKp) can spread from the primary site of infection to multiple sites causing life-threatening infections also in young otherwise healthy individuals. This study described the isolation of 13 isolates of K. pneumoniae with increased virulence in a large tertiary hospital over a 1-year period. Among them, eight strains were multidrug resistant and hypervirulent. Although these hypervirulent strains are still rare in Italy, their presence is particularly concerning since they can cause difficult-to-treat life-threatening infections. Moreover, not all the hypervirulent isolates were positive by the string test, so hvKp isolates were not always phenotypically detectable. Molecular biology techniques such as PCR amplification and next generation sequencing are therefore necessary for the detection of hvKp isolates, and surveillance programs exploiting molecular techniques are highly desirable.

Genotypic characterization of hypervirulent Klebsiella pneumoniae (hvKp) in a tertiary care Indian hospital.

HypervirulentKlebsiella pneumoniae(hvKp) is an emerging pathogen and causes endophthalmitis, liver abscess, osteomyelitis, meningitis, and necrotizing soft tissue infections in both immunodeficient and healthy people. The acquisition of the antibiotic resistance genes of hvKp has become an emerging concern throughout the globe. In this study, a total of 74K. pneumoniaeisolates were collected and identified by VITEK2 andblaSHVgene amplification. Out of these, 18.91% (14/74) isolates were identified as hvKp by both phenotypic string test and genotypiciucAPCR amplification. The antibiotic susceptibility revealed that 57.14% (8/14) isolates were multidrug-resistant (MDR) and 35.71% (5/14) isolates were extremely drug-resistant (XDR). All the isolates were resistant to β-lactam, β-lactamase + inhibitor groups of antibiotics, and the least resistance to colistin. Of 14 hvKp isolates, all isolates are positive for iroB(100%),followed byiutA(92.85%),peg344(85.71%), rmpA (57.14%), and magA(21.42%) genes. Among serotypes, K1 was the most prevalent serotype 21.4% (3/14), followed by K5 14.3% (2/14). The most common carbapenemase gene was blaOXA-48 (78.57%) followed by blaNDM (14.28%) and blaKPC (14.28%) which co-carried multiple resistance genes such as blaSHV (100%), blaCTX-M (92.85%), and blaTEM (78.57%). About 92.85% (13/14) of hvKp isolates were strong biofilm producers, while one isolate (hvKp 10) was the only moderate biofilm producer. The (GTG)5-PCR molecular typing method revealed high diversity among the hvKp isolates in the tertiary care hospital. Our findings suggest that MDR-hvKp is an emerging pathogen and a challenge for clinical practice. In order to avoid hvKp strain outbreaks in hospital settings, robust infection control and effective surveillance should be implemented.

Hypervirulent Klebsiella pneumoniae employs genomic island encoded toxins against bacterial competitors in the gut.

The hypervirulent lineages of Klebsiella pneumoniae (HvKp) cause invasive infections such as Klebsiella-liver abscess. Invasive infection often occurs after initial colonization of the host gastrointestinal tract by HvKp. Over 80% of HvKp isolates belong to the clonal group 23 sublineage I that has acquired genomic islands (GIs) GIE492 and ICEKp10. Our analysis of 12 361K. pneumoniae genomes revealed that GIs GIE492 and ICEKp10 are co-associated with the CG23-I and CG10118 HvKp lineages. GIE492 and ICEKp10 enable HvKp to make a functional bacteriocin microcin E492 (mccE492) and the genotoxin colibactin, respectively. We discovered that GIE492 and ICEKp10 play cooperative roles and enhance gastrointestinal colonization by HvKp. Colibactin is the primary driver of this effect, modifying gut microbiome diversity. Our in vitro assays demonstrate that colibactin and mccE492 kill or inhibit a range of Gram-negative Klebsiella species and Escherichia coli strains, including Gram-positive bacteria, sometimes cooperatively. Moreover, mccE492 and colibactin kill human anaerobic gut commensals that are similar to the taxa found altered by colibactin in the mouse intestines. Our findings suggest that GIs GIE492 and ICEKp10 enable HvKp to kill several commensal bacterial taxa during interspecies interactions in the gut. Thus, acquisition of GIE492 and ICEKp10 could enable better carriage in host populations and explain the dominance of the CG23-I HvKp lineage.

Molecular Epidemiological Characteristics of Hypervirulent Klebsiella pneumoniae in Yakeshi City, Hulunbuir, China.

This study aimed to investigate the molecular epidemiological characteristics of hypervirulent Klebsiella pneumoniae (hvKp) in Yakeshi City, Hulunbuir, China, analyze the resistance of hvKp to commonly used antibiotics, explore independent risk factors for hvKp infection, and provide a research basis for anti-infection treatment. In total, 519 strains of K. pneumoniae, identified by the Inner Mongolia Forestry General Hospital from January 2020 to December 2022, were collected, and high-viscosity (HMV-Kp) and non-HMV-Kp strains were differentiated using string test. PCR and agarose gel electrophoresis were used to detect the rmpA, rmpA2, and iutA genes to identify hvKp strains. Sixty strains of hvKp were randomly selected for capsular serotyping by PCR and agarose gel electrophoresis. Sanger sequencing was used to sequence the housekeeping genes of 60 hvKp strains and perform ST analysis. A minimum spanning tree was drawn using capsule serotyping and ST typing. Significant differences in resistance to commonly used antibiotics between classical K. pneumoniae (cKp) and hvKp were analyzed by using the chi-squared test. Finally, the risk factors for hvKp infection were analyzed through binary logistic regression. The HMV-Kp detection rate was 39.69%, versus 37.19% for hvKp. HMV-Kp accounted for 84.97% of all hvKp isolates. The hvKp detection rate was highest in the general surgery department. In capsule serotyping, K1 was the main subtype, accounting for 63.33% of all isolates (38/60), followed by K2 (16.67%, 10/60). Through ST typing, 18 subtypes were detected, with ST23 being the most common (50.00%), followed by ST86 (8.33%), and the remaining subtypes were scattered throughout the distribution. Compared with cKp, hvKp strains exhibited higher sensitivity to commonly used antibiotics, excluding furantoin. Male gender (odds ratio (OR) = 1.977), liver abscess (OR = 15.019), and the use of macrolide antibiotics in the past 3 months (OR = 5.473) were independent risk factors for hvKp infection. The hvKp detection rate in the local area was 37.19%, and a strong correlation was noted between hvKp and HMV-Kp strains. K1-ST23 was the dominant subtype in this study. Compared with cKp, hvKp strains were more sensitive to commonly used antibiotics. Male gender, liver abscess, suppuration or infection of other tissues and organs, and recent macrolide antibiotic use were risk factors for hvKp infection.

Prevalence of Hypervirulent rmpA and magA Genes in Clinical Isolates of Klebsiella pneumoniae and their association with Drug Resistant Pattern: A Cross-sectional Study

Introduction: Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a pathogen of global concern that is hypermucoviscous, causing infections and rapidly acquiring Antimicrobial Resistance (AMR). HvKp is more virulent than classical K. pneumoniae (cKp) and frequently infects healthy individuals with community-acquired illnesses. Since hvKp commonly exists in the gastrointestinal tract, its communicable spread affects the general public and healthcare facilities. The study was necessary due to the rapid evolution of hvKp, the need to identify novel, reliable agents of hypervirulence and drug resistance, and more reliable targets for therapeutic intervention. Aim: To investigate the prevalence of hvKp strains (rmpA and magA genes) and drug-resistant patterns in clinical isolates of Klebsiella pneumoniae (K. pneumoniae) in a tertiary care hospital in Puducherry, India. Materials and Methods: The cross-sectional study included a total of 100 non duplicate consecutive isolates of K. pneumoniae collected from Mahatma Gandhi Medical College and Research Institute and Hospital, Puducherry, India. These isolates were recovered from various clinical specimens such as urine, sputum, pus, blood, and other miscellaneous specimens mainly obtained from both outpatients and inpatients between August 2021 and April 2022. The antibiotic susceptibility of cultured K. pneumoniae was determined by the disk-diffusion test following the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines. The rmpA and magA genes were detected using conventional Polymerase Chain Reaction (PCR). PCR products were sequenced using Sanger sequencing. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) statistics software for Windows, version 15.0 (available from SPSS Inc., Chicago, USA). A p-value <0.05 was considered statistically significant. Results: A total of 100 isolates were studied for phenotypic and genotypic properties. HvKp was identified phenotypically using the string test, and further, the rmpA and magA genes were identified genotypically using PCR. Out of 100 isolates, the phenotypic study showed 11 (11%) isolates as string test positive. Furthermore, genotypic results showed that 22 (22%) isolates expressed the rmpA gene, 11 (11%) isolates expressed the magA gene, and 7 (7%) isolates expressed both rmpA and magA. Most of the hvKp isolates were obtained from urine specimens of K. pneumoniae isolates and showed more resistance than cKp towards all classes of antibiotics. Conclusion: The high prevalence of rmpA and magA genes suggests their strong role and capability as genetic markers for the identification of hvKp in the laboratory. This study indicated the important role of genetic elements in the emergence of drug resistance in hypervirulent strains and showed that hvKp has a multidrug resistance pattern.

Detection of hypervirulent and classical type of Klebsiella pneumoniae and screening their resistant properties in Erbil city

Background and objective: Klebsiella pneumoniaei s an opportunistic pathogenic bacterium and is considered one of the main causes of nosocomial infection. Hypervirulent K. pneumoniae (hvKp) has emerged as a pathogen of global importance. The current study aimed to determine Extended-spectrum beta-lactamase, carbapenem resistance, and serum bactericidal effect among the clinical isolates and to find the relationship of the above features with antimicrobial resistance. Methods: A total of 90 K. pneumoniae isolates were collected from different clinical specimens. Isolates were diagnosed using routine bacteriological methods and VITEK 2 compact system. Several phenotypic tests including string test, serum resistant, ESBL test, and Modified Carbapenem inactivation method were performed. The antibiotic resistance pattern was compared among ESBL-positive, carbapenem-resistant in both Hypervirulent (hvKp) and classical K. pneumoniae (cKp) isolates. Results: The results revealed that among 90 isolates, 56.7% of the isolates were of classical K. pneumoniae (cKp) type within which 70.6% of them were ESBL positive, 37.3% of them were resistant to carbapenem, and 51% were resistant to serum bactericidal activity. On the other hand, 43.3% were of hvKp type within which 61.5% of them were ESBL positive, 30.8% were carbapenem-resistant and all of the hvKp were resistant to human serum. The rate of antibiotic-resistant among cKp was higher than hvKp isolates. Conclusion: In this study, classical strains were more resistant to antibiotics and the rates of ESBL and Carbapenem resistance were higher compared to hvKp strains. but they were killed by serum bactericidal activities more rapidly.

Distinct epigenetic signatures of classical and hypervirulent Klebsiella pneumoniae.

Emergence and spread of the hypervirulent pathotype of Klebsiella pneumoniae have significantly increased infection rates in community as well as healthcare settings. There is an increasing interest to identify discriminating features between classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) to facilitate our understanding of the rapid emergence and dissemination of the hypervirulent pathotype. Here, we sought to identify unique epigenetic signatures of hvKp pathotype that differ from its classical counterpart using single-base resolution methylome analysis of native DNA sequencing on the Oxford Nanopore Technologies platform. The overall global adenine methylation in GATC motifs (i.e., Dam methylation motif) and cytosine methylation in CCWGG motifs (i.e., Dcm methylation motif) were significantly higher in hvKp isolates compared to that in cKp isolates, irrespective of their position in chromosomes or putative extra-chromosomal genetic elements. Notably, we observed significant enrichment of hypermethylated GATC and CCWGG motifs in the virulome of hvKp compared to hvKp genes not directly associated with virulence. We also observed increased methylation of GATC and CCWGG motifs in the capsule synthesis locus of hvKp isolates compared to cKp isolates. Furthermore, we identified several differentially methylated genes (DMGs) between the two pathotypes; interestingly, these DMGs include metal ion transporters, multidrug efflux pumps, transcriptional regulators of stress response, and genes associated with biofilm formation. Our results highlight hypermethylation of GATC and CCWGG motifs as unique epigenetic signatures of hvKp isolates.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a more virulent and rapidly evolving hypermucoviscous pathotype of classical K. pneumoniae (cKp). The hypervirulent pathotype is a major public health concern and is associated with high infection rates in community as well as hospital settings. With the recent emergence of multidrug-resistant hvKp, it has become imperative to investigate non-classical mechanisms such as epigenetics in addition to canonical biochemical and genetic mechanisms that delineate and differentiate the hypervirulent pathotype from its classical counterpart. Here, we identify genome-wide differences in adenine and cytosine methylation marks at well-characterized motifs between the two pathotypes. Overall, significantly higher levels of methylation were observed across chromosomal DNA and extrachromosomal elements in hvKp compared to cKp. Among hvKp isolates, the genes associated with virulence are particularly enriched for methylation marks. Our findings shed light on how epigenetic signatures may help distinguish the pathogenic potential of bacteria.

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.

Detection and characterization of putative hypervirulent Klebsiella pneumoniae isolates in microbiological diagnostics

Hypervirulent Klebsiella pneumoniae strains (hvKp) can cause invasive community-acquired infections in healthy patients of all ages. In this study, the prevalence of putative hvKp in a German tertiary center was investigated and hvKp were characterized by phenotypic and molecular assays. All K. pneumoniae isolates in routine microbiological diagnostics from a single center were screened by string-testing over a period of 6 months. String-test positive (≥ 0.5 mm) isolates were re-evaluated on different media and under various conditions (aerobe, anaerobe). For string-test positive isolates, genes (magA, iutA, rmpA and rmpA2) associated with hypermucoviscosity and hypervirulence were amplified by multiplex PCR. PCR-positive isolates were subjected to whole-genome sequencing and sedimentation and biofilm formation assays. From 1310 screened K. pneumoniae isolates in clinical routine 100 isolates (7.6%) were string test positive. From these, 9% (n = 9) were defined as putative hvKp (string-test+/PCR+). Highest rate of string-test-positive isolates was observed on MacConkey agar under aerobic conditions. Amongst these nine putative hvKp isolates, the international lineage ST23 carrying hvKp-plasmid pKpVP-1 was the most common, but also a rare ST86 with pKpVP-2 was identified. All nine isolates showed hypermucoviscosity and weak biofilm formation. In conclusion, 9% of string-positive, respectively 0.69% of all K. pneumoniae isolates from routine were defined as putative hypervirulent. MacConkey agar was the best medium for hvKp screening.

Evaluating the incidence of ampC-lactamase genes, biofilm formation, and antibiotic resistance among hypervirulent and classical Klebsiella pneumoniae strains.

Both immunocompetent and healthy individuals can become life-threateningly ill when exposed to the hypervirulent (hvKp) strains of Klebsiella pneumoniae (Kp). The main objectives of this study were to evaluate the presence of ampC-lactamase genes, biofilm formation, and antibiotic resistance in clinical strains of hvKp and cKp (classical K. pneumoniae). Kp strains were collected from patients referred to Shahidzadeh Hospital in Behbahan city, Khuzestan Province, Iran. Several techniques were used to identify hvKp. The hypermucoviscosity phenotype was determined using the string test. Isolates that developed dark colonies on tellurite agar were assumed to be hvKp strains. If any of the iucA, iutA, or peg-344 genes were detected, the isolates were classified as hvKp. Phenotypic and genotypic detection of AmpC β-lactamases of hvKp strains was performed by combined disk method and PCR, respectively. In addition, crystal violet staining was used to determine the biofilm formation of these isolates. For this study, 76 non-duplicative isolates of Kp were collected. Overall, 22 (28.94%) strains had positive string test results, and 31 (40.78%) isolates were grown in tellurite-containing medium. The genes iucA and iutA or peg-344 were found in 23.68% of all Kp strains and in 50% of tellurite-resistant isolates, respectively. The most effective antibiotics against hvKp isolates were tetracycline (85.52%) and chloramphenicol (63.15%). Using the cefoxitin disc diffusion method, we observed that 56.57% (43/76) of the strains were AmpC producer. A total of 30.26% (n=23/76) of the isolates tested positive for at least one ampC gene, including blaDHA (52.63%, n=40), blaCIT (40.78%, n=31), blaACC (19.76%, n=15), blaMOX (25%, n=19), and blaFOX (43.42%, n=33). Biofilm formation analysis revealed that most hvKp isolates were weak (n=6, 40%) and moderate (n=5, 33.33%) biofilm producers. Healthcare practitioners should consider the possibility of the existence and acquisition of hvKp everywhere. The exact mechanisms of bacterial acquisition are also unknown, and it is unclear whether the occurrence of infections is related to healthcare or not. Thus, there are still many questions about hvKp that need to be investigated.

Interaction of multidrug-resistant hypervirulent Klebsiella pneumoniae with components of human innate host defense.

Klebsiella pneumoniae strains with a combination of multidrug resistance and hypervirulence genotypes (MDR hvKp) have emerged as a cause of human infections. The ability of these microbes to avoid killing by the innate immune system remains to be tested fully. To that end, we compared the ability of a global collection of hvKp and MDR hvKp clinical isolates to survive in human blood and resist phagocytic killing by human neutrophils. The two MDR hvKp clinical isolates tested (ST11 and ST147) were killed in human blood and by human neutrophils in vitro, whereas phagocytic killing of hvKp clinical isolates (ST23 and ST86) required specific antisera. Although the data were varied and often isolate specific, they are an important first step toward gaining an enhanced understanding of host defense against MDR hvKp.

Prevalence, clonal diversity, and antimicrobial resistance of hypervirulent Klebsiella pneumoniae and Klebsiella variicola clinical isolates in northern Japan

Hypervirulent Klebsiella pneumoniae (hvKp) and Klebsiella variicola (hvKv) cause hospital/community-acquired infections, often associated with antimicrobial resistance (AMR). This study aimed to investigate the molecular epidemiology of hvKp and hvKv in northern Japan. A total of 500 K. pneumoniae and 421 K. variicola clinical isolates collected from August to December 2021 were studied. Prevalence of virulence factor-encoding genes, wzi sequence and associated K/KL type, sequence type (ST), and beta-lactamases and their types were characterized. Any virulence gene (rmpA, rmpA2, peg-344, iucA, iutA, and iroB) and/or magA was detected in 25% (n=125) of K. pneumoniae and 1% (n=5) of K. variicola. Among these hvKp/hvKv, 22 wzi types (18 and 4 types, respectively) and 24 STs (20 and 4 STs, respectively) were identified. Sequence types of hvKp were classified into some clonal groups (CGs), among which CG35, including six STs, was the most common (n=59; 47%), followed by CG23, and CG65. ST268 (CG35) associated with wzi95-K20 or wzi720 was the dominant lineage (n=43, 34%), while K1:ST23/ST249 and K2:ST65/ST86 accounted for 26% and 13% of hvKp, respectively. Extended-spectrum beta-lactamase (ESBL) genes (blaCTX-M-2, blaCTX-M-3, blaCTX-M-15, and blaCTX-M-27) were detected in only ST23 and CG35 (ST268 and ST412) hvKp. No isolate was resistant to carbapenems, without detection of the ESBL gene in K. variicola. Phylogenetically, wzi was differentiated into two main clusters of K. pneumoniae and K. variicola. A major clonal group CG347 was identified in K. variicola. Clonal structures were revealed for hvKp and hvKv clinical isolates with their AMR status in northern Japan.

Comparative Studies on Clinical Isolates of Hypervirulent and Classical Klebsiella pneumoniae

Hypervirulent Klebsiella pneumonia (hvKP), typically linked with the hypermucoviscous phenotype, has evolved as a clinically significant pathogen responsible for catastrophic disseminated infections including osteomyelitis, pyogenic liver abscesses (PLA), and endophthalmitis in an otherwise younger and healthier population. Methods: Mansoura University Hospitals, Egypt have utilised VITEK® 2 to identify clinical K. pneumoniae isolates. The Kirby Bauer's disc diffusion method was employed to asses sensitivity to antimicrobial. String test for detecting microvasculitis, Serotyping, and Bio-typing are among the phenotypic methods employed. Polymerase chain reaction (PCR) was performed to amplify capsular serotype-specific genes (K1 and K2) as well as virulence-associated genes (rmpA and magA). Results: Over two-years, 100 hypermucoviscous phenotype K. pneumoniae and 100 classical K. pneumoniae strains were isolated and screened by string test. Hypermucoviscosity Serotyping revealed that 76 isolates had K1-type capsules, 19 had K2-type capsules, and 5 were non-K1/K2 isolates. Among the positive hypermucoviscusisolates, the prevalence of rmpA gene was 79 %, and magA gene was 40 %. Although, the prevalence of these two genes in classical Klebsiella pneumoniae (cKP) isolates was very low 11% and 7%, respectively. Aside from that, the occurrence of K capsular genes in hvKP isolates were; K1 gene 66% and K2 gene 63%.However, the occurrence of these genes in cKP isolates were limited. Conclusion: In our geographical area, hypervirulent Klebsiella pneumoniae hvKP isolates were infrequent among K. pneumoniae isolates (El-Mansoura, Egypt). This study summarized and concentrated on the epidemiology, hypervirulence-associated variables, and antibiotic resistance mechanisms of hvKP strains. This was done to let the microbiologist or clinician know that the infecting strain could be hvKP, which is more dangerous than cKP. As a result, detecting these hypervirulent strains, which harbor resistance variants, should be a top goal priority.

Clinical and Molecular Characteristics and Antibacterial Strategies of Klebsiella pneumoniae in Pyogenic Infection.

Treatment of Klebsiella pneumoniae causing pyogenic infections is challenging. The clinical and molecular characteristics of Klebsiella pneumoniae causing pyogenic infections are poorly understood, and antibacterial treatment strategies are limited. We analyzed the clinical and molecular characteristics of K. pneumoniae from patients with pyogenic infections and used time-kill assays to reveal the bactericidal kinetics of antimicrobial agents against hypervirulent K. pneumoniae (hvKp). A total of 54 K. pneumoniae isolates were included, comprising 33 hvKp and 21 classic K. pneumoniae (cKp) isolates, and the hvKp and cKp isolates were identified using five genes (iroB, iucA, rmpA, rmpA2, and peg-344) that have been applied as hvKp strain markers. The median age of all cases was 54 years (25th and 75th percentiles, 50.5 to 70), 62.96% of individuals had diabetes, and 22.22% of isolates were sourced from individuals without underlying disease. The ratios of white blood cells/procalcitonin and C-reactive protein/procalcitonin were potential clinical markers for the identification of suppurative infection caused by hvKp and cKp. The 54 K. pneumoniae isolates were classified into 8 sequence type 11 (ST11) and 46 non-ST11 strains. ST11 strains carrying multiple drug resistance genes have a multidrug resistance phenotype, while non-ST11 strains carrying only intrinsic resistance genes are generally susceptible to antibiotics. Bactericidal kinetics revealed that hvKp isolates were not easily killed by antimicrobials at susceptible breakpoint concentrations compared with cKp. Given the varied clinical and molecular features and the catastrophic pathogenicity of K. pneumoniae, it is critical to determine the characteristics of such isolates for optimal management and effective treatment of K. pneumoniae causing pyogenic infections. IMPORTANCE Klebsiella pneumoniae may cause pyogenic infections, which are potentially life-threatening and bring great challenges for clinical management. However, the clinical and molecular characteristics of K. pneumoniae are poorly understood, and effective antibacterial treatment strategies are limited. We analyzed the clinical and molecular features of 54 isolates from patients with various pyogenic infections. We found that most patients with pyogenic infections had underlying diseases, such as diabetes. The ratio of white blood cells to procalcitonin and the ratio of C-reactive protein to procalcitonin were potential clinical markers for differentiating hypervirulent K. pneumoniae strains from classical K. pneumoniae strains that cause pyogenic infections. K. pneumoniae isolates of ST11 were generally more resistant to antibiotics than non-ST11 isolates. Most importantly, hypervirulent K. pneumoniae strains were more tolerant to antibiotics than classic K. pneumoniae isolates.

Detection of multiple extensively-drug resistant hypervirulent Klebsiella pneumoniae clones from patients with ventilator-associated pneumonia in Egypt.

Introduction. Hypervirulent-K. pneumoniae (hvKP) is an evolving pathotype that is more virulent than the classical-K. pneumoniae (cKP) and causes serious fatal illnesses.Hypothesis/Gap Statement. Although there are few reports on hvKP isolated from Egyptian patients, the molecular characteristics and clonal relatedness of MDR-hvKP have not been adequately investigated.Aim. To investigate the microbiological and genetic characteristics as well as the epidemiology of hvKP induced ventilator-associated pneumonia (VAP).Methodology. A retrospective study of 59 K. pneumoniae inducing VAP was conducted at Assiut University Hospitals from November 2017 to January 2019. All K. pneumoniae were tested for resistance phenotype, capsular genotype (K1 and K2), virulence gene profile (c-rmpA, p-rmpA, iucA, kfu, iroB, iroN), and the presence of resistance genes (blaNDM-1, blaCTX-M-3-like, blaCTX-M-14-like). Clonal relatedness was assessed by Pulsed field gel electrophoresis (PFGE).Result. HvKP accounted for 89.8 % (53/59) of K. pneumoniae isolates with ~95 % exhibiting extensively-drug resistant (XDR) phenotype. Hypermucoviscous phenotype was detected in 19 (35.8 %) hvKP and K2 capsular gene was identified in 18 (33.9 %) of hvKP. Regarding the virulence genotype of hvKP strains, iucA was the most prevalent virulence gene (98.1%), while p-rmpA and kfu were detected in 75.4 and 52.8 % of hvKP strains, respectively. Resistance genes were highly prevalent in both cKP and hvKP with blaCTX-M-3-like being more prevalent in hvKP (100 % vs 94.3 % for blaNDM-1, 50 % vs 62.2 % for blaCTX-M-3-like and 83.3 % vs 69.8 % for blaCTX-M-14 -like, respectively). PFGE typing of 29 representative K. pneumoniae revealed 15 pulsotypes, with identical hvKP pulsotypes isolated from different ICUs at different times and several hvKP and cKP isolates belonged to the same pulsotype.Conclusion. This study highlights the dominance and clonal spread of XDR-hvKP strains at Assiut University Hospital in Egypt. Physicians should be aware of the increased risk of hvKP induced-VAP and support further epidemiologic studies.