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The rise of a new hypervirulent variant of Klebsiella pneumoniae (hvKp) was recently reported, mainly linked to the ST23 lineage. The hvKp variants can cause severe infections, including hepatic abscesses, bacteremia, and meningitis, with a particularly disconcerting propensity to cause community-acquired, life-threatening infection among young and otherwise healthy individuals. The present study aimed to report the clinical characteristics of a hypermucoviscous K. pneumoniae strain isolated in Italy and sustaining recurrent meningitis in a patient of Peruvian origin. A further objective was to retrospectively investigate, by means of whole-genome sequencing (WGS) analysis, the genomic features of such an isolate. The hypermucoviscosity phenotype of the strain (sk205y205t) was determined using the string test. Genomic information was obtained by WGS (Illumina) and bioinformatic analysis. Strain sk205y205t was susceptible to most antibiotics, despite the presence of some resistance genes, including blaSHV-11, blaSHV-67, fosA, and acrR. The isolate belonged to ST65 and serotype K2, and exhibited several virulence factors related to the hvKp variant. Among these, were the siderophore genes entB, irp2, iroN, iroB, and iucA; the capsule-regulating genes rmpA and rmpA2; and the type 1 and 3 fimbriae fimH27 and mrkD, respectively. A further operon, encoding the genotoxin colibactin (clbA-Q), was also identified. The virulence plasmids pK2044, pRJA166b, and pNDM. MAR were also detected. Phylogenetic investigation showed that this Italian strain is highly similar to a Chinese isolate, suggesting a hidden circulation of this hvKp ST65 K2 lineage.

Hypervirulent Klebsiella pneumoniae (Hv-Kp) strains have emerged as pathogens causing life-threatening, invasive disease even in immunocompetent hosts. Systemic dissemination usually occurs following perturbations of the gut microbiota and is facilitated by Hv-Kp resistance to phagocytosis and complement activity. Hv-Kp are usually associated with K1 or K2 capsular types, produce several iron uptake systems (e.g., aerobactin and salmochelin) and are often but not invariably, capsular material hyper-producers (hypermucoviscous phenotype: HMV). Whether Hv-Kp escape the immune response at mucosal site is unknown. In this work, we studied the effects of Hv-Kp on human dendritic cells (DCs), central players of the IL-23/IL-17 and IL-12/IFN-γ axis at mucosal sites, essential for pathogen clearance. Four Hv-Kp and HMV strains were selected and their activity on DC maturation and cytokine production was compared to that of non-virulent Kp strains with classic or HMV phenotypes. While the maturation process was equally induced by all Kp strains, significant differences between virulent and non-virulent strains were found in the expression of genes for cytokines involved in T-cell activation and differentiation. The non-virulent KP04C62 and the classic Kp, KPC157 induced high expression of TH1 (IL-12p70 and TNFα) and TH17 cytokines (IL-23, IL-1β and IL-6), while Hv-Kp poorly activated these cytokine genes. Moreover, conditioned media from DCs cultured with non-virulent Kp, either classical or hypercapsulated, induced the activation of IL-17 and IFN-γ genes in preactivated CD4+-cells suggesting their TH17/TH1 differentiation. Conditioned media from Hv-Kp poorly activated IL-17 and IFN-γ genes. In summary, our data indicate that Hv-Kp interfere with DC functions and T-cell differentiation and suggest that the escape from the IL-23/IL-17 and IL-12/IFN-γ axes may contribute to pathogen dissemination in immunocompetent hosts.

Abstract Background Aerobactin is a critical factor for the hypervirulent Klebsiella pneumoniae (hvKp), but data for the aerobactin-positive genotype of hvKp in elderly persons with ventilator-associated pneumonia (VAP) is limited. The purpose of this study is to understand the risk factors and characteristics of the hvKp genotype for elderly patients with VAP. Methods A retrospective study of 73 elderly patients with Kp was conducted from November 2008 to December 2017 in two tertiary hospitals. The clinical and microbiological data, including inflammatory reaction, nutritional status, antimicrobial susceptibility testing, string test, extended-spectrum-β-lactamase (ESBL) production, virulence-associated gene (capsular serotype-specific gene and rmpA/A2,magA,aerobaction) and multilocus sequence typing, of the hvKp group defined as aerobactin positive were compared with those of classic Kp strains. Results Of 73 Kp isolates, 46.6% were hvKp. ST23 is highly prevalent in two hospitals but is not highly associated with hvKp in different hospitals. Additionally, ST23, ST37 and ST2906 are more likely to induce lethal VAP. Most hvKp strains are sensitive to common antibiotics, but the number of multidrug-resistant (MDR) hvKp is increasing. Importantly, 38.2% of hvKp isolates produced ESBLs. Hypermucoviscosity and virulence-associated genes (K1,magA and rmpA/A2) were highly clustered in the hvKp group (P

Yuan-Pin Hung,1–3,* Pei-Jane Tsai,4,* Yuan-Ti Lee,5,6 Hung-Jen Tang,7 Hsiao-Ju Lin,1–3 Hsiu-Chuan Liu,1 Jen-Chieh Lee,2 Bo-Yang Tsai,4 Po-Ren Hsueh,8 Wen-Chien Ko2,9,10 1Department of Internal Medicine, Tainan Hospital, Ministry of Health and Welfare, Tainan, Taiwan; 2Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; 3Graduate Institute of Clinical Medicine, National Cheng Kung University, Tainan, Taiwan; 4Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 5Institute of Medicine and School of Medicine, Chung Shan Medical University, Taichung, Taiwan; 6Division of Infectious Diseases, Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan; 7Department of Internal Medicine, Chi-Mei Hospital, Tainan, Taiwan; 8Departments of Laboratory Medicine and Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan; 9Center of Infection Control, National Cheng Kung University Hospital, Tainan, Taiwan; 10Department of Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan *These authors contributed equally to this work Objectives: The information of antimicrobial susceptibility, toxin gene, and ribotype distribution of toxigenic Clostridium difficile isolates in Taiwan remain limited. Patients and methods: The study was conducted from January 2015 to December 2016 in 5 hospitals in Taiwan. Adults aged ≥20 years with a hospital stay for >5 days were included, and those with colectomy or intestinal infection due to other enteropathogens were excluded. Multiplex PCR was used to detect tcdA, tcdB, cdtA, cdtB, and tcdC deletions, and antimicrobial susceptibility for metronidazole, vancomycin, doxycycline, and tigecycline was investigated. Ribotypes of those isolates with tcdC deletion and tcdA+/tcdB+ were determined. Results: Of 1112 C. difficile isolates collected from adults at 5 hospitals, 842 were toxigenic, including 749 (89.0%) tcdA+/tcdB+ isolates and 93 (11.0%) tcdA−/tcdB+. Of the toxigenic isolates, 76 (9.0%) had a tcdC deletion and were cdtA+/cdtB+, indicative of hypervirulence, and RT078 lineage, including RT126, RT127, and RT078, predominated (n=53, 76.3%). Similar to the susceptibility data in Asia countries, metronidazole or vancomycin resistance was rare, noted in 1.2% or 2.1%, respectively. Reduced doxycycline susceptibility (minimum inhibitory concentration [MIC] of ≥8 mg/L) was more common among RT078 lineage than non-RT078 lineage (75.9%, 44/58 vs 6.0%, 47/784; P

Abstract Background Hypervirulent strains of Klebsiella pneumoniae are a recognized cause of a distinct invasive syndrome that results in pyogenic liver abscesses and metastatic complications, particularly in the Asia Pacific region. Reports of hypervirulent K.pneumoniae in Europe, the Americas and Australia indicate worldwide spread. We present a case of multi-focal osteomyelitis, a rarely described complication of hypervirulent K.pneumoniae in the medical literature. The prevalence of this condition in countries outside Asia may be expected to rise with increasing travel. Case presentation A 20-year-old Chinese man residing in Australia for 2 years presented with a 2-week history of gradually worsening leg pain preceded by 2 weeks of constitutional symptoms. Imaging with computerized axial tomography (CT) and other modalities revealed bilateral tibial lesions described as lattice-like linear lucencies involving the cortices with scalloping of the outer involved cortex. Cultures of tissue from a left tibial bone biopsy were positive cultures for K.pneumoniae. Whole-genome sequencing identified the isolate as K1 serotype ST23, a well-recognized hyper virulent strain capable of causing invasive disease. An abdominal CT revealed a 27x22mm liver abscess. The patient had no other metastatic manifestations of the disease, and responded to 6 weeks of intravenous ceftriaxone followed by 3 months of oral Ciprofloxacin. Conclusions Increased awareness of the manifestations and subsequent management of hyper virulent strains of K.pneumoniae by clinicians is important to assist early recognition and help minimize serious sequelae. Cases with overseas links, such as previous residence in the Asia Pacific area, are at higher risk for infection with the hyper virulent strain. This case highlights the need for clinicians to be able to recognize this important disease, especially in patients with the right epidemiological links, and to investigate and treat appropriately to prevent severe metastatic complications.

BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hypervirulent K. pneumoniae (hvKP), new variant of K. pneumoniae, can cause invasive infections in young healthy individuals as well as in the immunocompromised population. Hypervirulent strains frequently belong to capsular serotypes K1 or K2. Emergence of antimicrobial resistance in hvKP is a cause for concern. AIM AND OBJECTIVE: The present study was done to detect the K1 and K2 serotypes among clinical isolates of K. pneumoniae, spectrum of infections caused by them and presence of common beta-lactamases encoding genes in them. MATERIALS AND METHODS: A total of 370 isolates of K. pneumoniae, isolated from various clinical samples over a period of 1 year was included in this study. Antibiotic susceptibility testing to various classes of antimicrobials was done as per Clinical and Laboratory Standard Institute guidelines. The presence of K2A (specific to serotype K2), magA (specific to serotype K1), and rmpA genes was detected by multiplex polymerase chain reaction (PCR). Extended-spectrum beta-lactamases (TEM, SHV, and CTX-M), plasmid-mediated AmpCs (MOX, CIT, DHA, ACC, EBC, and FOX), and carbapenemase genes (IMP, VIM, NDM, KPC, and OXA-48) were also determined by PCR. RESULTS: Among the 370 isolates, 8 harbored K2A gene and one harbored magA. rmpA gene was detected in three isolates along with K1 or K2 serotypes. Seven K2A-positive isolates were resistant to one or more classes of antimicrobials. The studied ESBL genes were present in four isolates. Two isolates harbored carbapenemase genes (NDM-1, OXA-48) along with ESBLs. Conclusions: K2 serotype is more prevalent among hvKP isolates. They can harbor ESBLs and Carbapenemase genes. K1 serotype is rather uncommon in K. pneumoniae. Acquisition of multidrug-resistant genes by these strains adds to their virulence and limits the treatment options.

Chao Liu,1 Jiaojiao Shi,2 Jun Guo3,4 1Department of Respiratory Medicine, Peking Union Medical College, Chinese Academy of Medical Sciences, China-Japan Friendship Hospital, Beijing, China; 2Department of Respiratory Medicine, Peking University Third Hospital, Peking University, Beijing, China; 3Department of Respiratory Medicine, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, China; 4Department of Geriatric Respiratory Medicine, Chinese PLA General Hospital, Beijing, China Purpose: Aerobactin is a critical factor for hypervirulent Klebsiella pneumoniae (hvKp) in genetic backgrounds, but data based on the genotype for the elderly is limited. Materials and methods: A retrospective study was conducted on elderly patients from June 2008 to July 2017 in 2 teaching hospitals. The clinical and microbiological data, including antimicrobial susceptibility testing, string test, extended-spectrum β-lactamase (ESBL) production, virulence gene, and multilocus sequence typing, of the hvKp group defined as aerobactin positive were compared with those of classic K. pneumoniae isolates. Results: A total of 45.7% of 202 K. pneumoniae isolates were hvKp.ST23, which were predominant in 2 hospitals, but they were not highly associated with hvKp in different hospitals. Hypermucoviscosity, K1, K2, magA, and rmpA/A2 genes were highly related to hvKp (P=0.000). With regard to the host, invasive infections (P=0.000), liver abscess (P=0.000), abdominal infection (P=0.000), pneumonia (P=0.037), and septic shock (P=0.045) were significantly higher in the elderly with hvKp. In the hvKp group, patients with better nutritional status were associated with a more severe sequential organ failure assessment score and a more serious inflammation reaction. Patients with diabetes (odds ratio [OR]=2.566) are more likely to be infected with hvKp. Previous hvKp is associated with hypermucoviscosity (OR=15.249) are often paralleled with hvKp. Importantly, 26% of hvKp isolates produced ESBLs, and most of them showed a carbapenems-resistant (CR) phenotype. Multivariate analysis implied that patients with a history of surgery within the last 1 month (OR=15.999) is an independent risk factor for CR-hvKp infection. Conclusion: The prevalence of hvKP is high in the elderly. ESBL-hvKp, especially CR-hvKp, is emerging, which is a sign that clinical awareness and infection monitoring needs to improve. Keywords: Klebsiella pneumoniae, hypervirulent, aerobactin, risk factor, ESBL-hvKp, CR-hvKp

Clostridium difficile ; a group of spore forming, toxin forming, gram positive anerobel is implicated in hospital associated diarrhea and is the causative agent of infectious diarrhea. It is the most common hospital associated infection in Europe and North America, and is presumed to be as prevalent in the rest of the world. There has been emergence of new virulent strain of C. difficile, identified as BI, NAP1, and toxinotype III and ribotype 027 (subsequently known as BI/NAP1/027) by various typing method in recent years, implicated in dramatic increase in C. difficile infections. Diagnosis is established by presence of C. difficile toxin or C. difficile toxin gene in stool. Lab testing does not distinguish C. difficile infection and asymptomatic carriage. Clinical suspicion and positive stool study confirms a diagnosis. Clostridium Difficile infection, is most common health care associated infection in Europe and North America, and the available studies show it may have similar prevalence in Nepal. Literature review does not reveal any significant study being conducted in Nepal as of now. It warrants further study to exactly determine the incidence/prevalence and its impact in current health care in Nepal. Clinicians need increased awareness and prompt diagnosis to reduce morbidity and further prevention of transmission. Nepalese Medical Journal, vol.1, No. 1, 2018, page: 43-47

Abstract Background By using whole genome sequence data we aimed at describing a population snapshot of carbapenemase-producing K. pneumoniae isolated from hospitalized patients in Germany between 2008 and 2014. Methods We selected a representative subset of 107 carbapenemase-producing K. pneumoniae clinical isolates possessing the four most prevalent carbapenemase types in Germany (KPC-2, KPC-3, OXA-48, NDM-1). Isolates were processed via illumina NGS. Data were analysed using different SNP-based mapping and de-novo assembly approaches. Relevant information was extracted from NGS data (antibiotic resistance determinants, wzi gene/cps type, virulence genes). NGS data from the present study were also compared with 238 genome data from two previous international studies on K. pneumoniae. Results NGS-based analyses revealed a preferred prevalence of KPC-2-producing ST258 and KPC-3-producing ST512 isolates. OXA-48, being the most prevalent carbapenemase type in Germany, was associated with various K. pneumoniae strain types; most of them possessing IncL/M plasmid replicons suggesting a preferred dissemination of bla OXA-48 via this well-known plasmid type. Clusters ST15, ST147, ST258, and ST512 demonstrated an intermingled subset structure consisting of German and other European K. pneumoniae isolates. ST23 being the most frequent MLST type in Asia was found only once in Germany. This latter isolate contained an almost complete set of virulence genes and a K1 capsule suggesting occurrence of a hypervirulent ST23 strain producing OXA-48 in Germany. Conclusions Our study results suggest prevalence of “classical” K. pneumonaie strain types associated with widely distributed carbapenemase genes such as ST258/KPC-2 or ST512/KPC-3 also in Germany. The finding of a supposed hypervirulent and OXA-48-producing ST23 K. pneumoniae isolates outside Asia is highly worrisome and requires intense molecular surveillance.

Liver abscesses containing hypervirulent Klebsiella pneumoniae have emerged during the past 2 decades, originally in Southeast Asia and then worldwide. We hypothesized that hypervirulent K. pneumoniae might also be emerging in France. In a retrospective, monocentric, cohort study, we analyzed characteristics and outcomes for 199 consecutive patients in Paris, France, with liver abscesses during 2010−2015. We focused on 31 patients with abscesses containing K. pneumoniae. This bacterium was present in most (14/27, 52%) cryptogenic liver abscesses. Cryptogenic K. pneumoniae abscesses were more frequently community-acquired (p

Abstract Background Hypervirulent K. pneumoniae (hvKp) causes severe community acquired infections, predominantly in Asia. Though initially isolated from liver abscesses, they are now prevalent among invasive infections such as bacteraemia. There have been no studies reported till date on the prevalence and characterisation of hvKp in India. The objective of this study is to characterise the hypervirulent strains isolated from bacteraemic patients for determination of various virulence genes and resistance genes and also to investigate the difference between healthcare associated and community acquired hvKp with respect to clinical profile, antibiogram, clinical outcome and molecular epidemiology. Results Seven isolates that were susceptible to all of the first and second line antimicrobials and phenotypically identified by positive string test were included in the study. They were then confirmed genotypically by presence of rmpA and rmpA2 by PCR. Among the study isolates, four were from patients with healthcare associated infections; none were fatal. All patients with community acquired infection possessed chronic liver disease with fatal outcome. Genes encoding for siderophores such as aerobactin, enterobactin, yersiniabactin, allantoin metabolism and iron uptake were identified by whole genome sequencing. Five isolates belonged to K1 capsular type including one K. quasipneumoniae. None belonged to K2 capsular type. Four isolates belonged to the international clone ST23 among which three were health-care associated and possessed increased virulence genes. Two novel sequence types were identified in the study; K. pneumoniae belonging to ST2319 and K. quasipneumoniae belonging to ST2320. Seventh isolate belonged to ST420. Conclusion This is the first report on whole genome analysis of hypervirulent K. pneumoniae from India. The novel sequence types described in this study indicate that these strains are evolving and hvKp is now spread across various clonal types. Studies to monitor the prevalence of hvKp is needed since there is a potential for the community acquired isolates to develop multidrug resistance in hospital environment and may pose a major challenge for clinical management.

Necrotizing fasciitis of the chest wall or upper torso is a rare clinical entity. Monomicrobial Klebsiella pneumoniae as a causative agent of necrotizing fasciitis is far less common than the polymicrobial etiology. Here, we report a case of community-onset pyogenic necrotizing fasciitis caused by Klebsiella pneumoniae in an immunocompetent male of tribal background from the rural area of Jharkhand, India. The hypermucoviscous phenotype of the bacterium causing necrotizing fasciitis has been infrequently reported from the Indian subcontinent to date. The existence of multidrug resistant trait in the hypervirulent pathotype poses a unique challenge in treatment in such a case and emerges as a critical community health problem requiring prompt attention of the public health stakeholders. Thus, there is a need for widespread awareness for proper protocols in antimicrobial usage, infection control, early diagnosis, and prompt treatment. [ABSTRACT FROM AUTHOR]

Multi-drug resistance (MDR) and hypervirulence (hv) were exhibited by different well-separated Klebsiella pneumoniae lineages in the past, but their convergence clones—MDR-hypervirulent K. pneumoniae (HvKPs)—both highly pathogenic and resistant to most available antibiotics, have increasingly been reported. In light of the clonal lineages and molecular characteristics of the studied MDR-HvKP strains found in the literature since 2014, this review discusses the epidemiology of MDR-HvKPs, in particular summarizing the three general aspects of plasmids-associated mechanisms underlying the formation of MDR-HvKPs clones: MDR-classic K. pneumoniae (cKPs) acquiring hv plasmids, hvKPs obtaining MDR plasmids, and the acquisition of hybrid plasmids harboring virulence and resistance determinants. A deeper understanding of epidemiological characteristics and possible formation mechanisms of MDR-HvKPs is greatly needed for the proper surveillance and management of this potential threat. [ABSTRACT FROM AUTHOR]

Aim: Clinical strains of Klebsiella quasipneumoniae subsp. similipneumoniae have been reported worldwide. Multidrug-resistant (MDR) hypermucoviscous (hm)/hypervirulent (hv) lineages have become a global problem for public health worldwide. Therefore, this study aimed to characterize by whole-genome sequencing a MDRhm/hv K. quasipneumoniae subsp. similipneumoniae SWT10 strain belonging to the new sequence type ST4417 isolated from a sewage treatment plant. Results: The SWT10 strain was recovered from a sewage treatment plant in Brazil and presented the hm and MDR phenotypes. Resistome analysis showed antimicrobial resistance genes associated with resistance to fluoroquinolones, b-lactams, tetracyclines, trimethoprim, aminoglycosides, sulfonamides, macrolides, and fosfomycin as well as several heavy metal resistance genes. Virulome analysis showed virulence factors related to hv lineages. Multilocus sequence typing analysis revealed the new ST4417, which was grouped in CC1584 by the minimum-spanning tree. Besides, five plasmid incompatibility groups, two prophage-related sequences, and 66 genomic islands were detected. Conclusion: This study reports for the first time the genome sequence of a MDR-hm/hv K. quasipneumoniae subsp. similipneumoniae recovered from the environment, which contributes to a better understanding about these lineages as well as for surveillance studies worldwide. [ABSTRACT FROM AUTHOR]

• Molecular characteristics of ESBL-producing Klebsiella pneumoniae were investigated. • CTX-M-15 was the predominant ESBL of K. pneumoniae in Japan. • ESBL-producing hypervirulent K. pneumoniae strains were isolated in Japan. • ESBL-producing hypervirulent K. pneumoniae strains comprised various clones. To provide data on the molecular characteristics of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae clinical isolates in Japan. A total of 100 clinical isolates of ESBL-producing K. pneumoniae collected throughout Japan between June and July 2018 were studied. ESBL genes were analyzed using PCR and DNA sequencing. Transferability of ESBL genes was investigated by conjugation experiments. Plasmid replicon types, virulence genes (rmpA , rmpA2 , iucA , iroB , and peg-344) associated with hypervirulent K. pneumoniae (hvKp), and capsule types were detected using PCR. Genotyping was performed using multilocus sequence typing. All ESBL-producing isolates carried bla CTX-M genes. The most predominant CTX-M-type identified was CTX-M-15 (n = 55). We identified 24 sequence types (STs) among the CTX-M-15 producers, with ST25 (n = 8) being the most common. Most of the transconjugants carrying bla CTX-M-15 contained the FIIk replicon. Of the 100 ESBL-producing isolates, 31 were hvKp defined by the presence of the virulence genes. These ESBL-producing hvKp isolates belonged to eight STs (STs 23, 25, 36, 65, 86, 268, 412, and 4492), with five capsule types (K1, K2, K20, K57, and undefined). CTX-M-15 was the predominant ESBL among K. pneumoniae isolates from Japan. This study shows that ESBL-producing hvKp strains comprising various clones are emerging in Japan. [ABSTRACT FROM AUTHOR]

We recovered 2 carbapenem-resistant K2-ST86 hypermucoviscous Klebsiella pneumoniae isolates from patients in France. The isolates had genetic attributes of hypervirulent K. pneumoniae but differed in ability to cause mouse lethality. Convergence of hypervirulent K. pneumoniae toward resistance could cause a health crisis because such strains could be responsible for severe and untreatable infections. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes (L. monocytogenes) is a ubiquitous foodborne pathogen that comprises 14 serotypes, of which serovar 4h is a novel serotype recently reported. Serovar 4h L. monocytogenes belonging to hybrid sub-lineage II exhibit hypervirulent features. Conventional biochemical tests and widely used PCR-based serogrouping schemes could not distinguish serovar 4h strains. In this study, we developed a new multiplex PCR assay for rapid detection of serotype 4h L. monocytogenes. Three primer pairs based on the target genes, LMxysn_1095, lmo1083 , and smcL , were designed. The multiplex PCR results showed that serovar 4h strains could be specifically identified from all tested strains, including various L. monocytogenes serovars, Listeria spp., and other species. The detection limits of the multiplex PCR were 291 fg/μL for genomic DNA and 5.5 × 106 CFU/mL for bacterial suspension. Furthermore, pork meat artificially contaminated with serovar 4h L. monocytogenes in a concentration of 1.8 × 103–1.8 × 100 CFU/10 g were successfully detected within 10–16 h. These results demonstrate that the multiplex PCR with high specificity and sensitivity is applicable for the rapid detection of L. monocytogenes serotype 4h strains. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKP) strains are associated with vigorous clinical presentation and relapses. Initially reported from Asia, these variants have spread globally and become an emerging agent of significant health threat. This study was carried out to identify hvKP strains in a previously uninvestigated region and to evaluate the impact of commonly-employed phenotypic and genotypic markers as diagnostic assays. A total of 111 blood culture isolates, collected at a tertiary care center was investigated. The hvKP strains were sought by a string test and the amplification of partial magA , rmpA , iucA and peg344. All products were characterized via sequencing. Evidence for hvKP was observed in 10.8% via iucA amplification (7.2%), string test (2.7%) and magA amplification (0.9%). Specific products were not produced by assays targeting rmpA and peg344 genes. Antibiotic susceptibility patterns compatible with possible extensive or pan-antimicrobial resistance was noted in 66.7% of the hvKP candidate strains. Capsule type in the magA positive strain was characterized as K5. We have detected hvKP in low prevalence at a region with no prior documentation. Targetting the aerobactin gene via iucA amplification provided the most accurate detection in this setting. The epidemiology of hvKP in Anatolia requires elucidation for effective control and management. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKP) causes Klebsiella‐induced liver abscess. Capsule is important for the pathogenesis of Klebsiella in systemic infection, but its role in gut colonisation is not well understood. By generating ΔwcaJ, Δwza and Δwzy capsule‐null mutants in a prototypical K1 hypervirulent isolate, we show that inactivation of wza (capsule exportase) and wzy (capsule polymerase) confer cell envelope defects in addition to capsule loss, making them susceptible to bile salts and detergent stress. Bile salt resistance is restored when the initial glycosyltransferase wcaJ was inactivated together with wzy, indicating that build‐up of capsule intermediates contribute to cell envelope defects. Mouse gut colonisation competition assays show that the capsule and its regulator RmpA were not required for hvKP to persist in the gut, although initial colonisation was decreased in the mutants. Both ΔrmpA and ΔwcaJ mutants gradually outcompeted the wild type in the gut, whereas Δwza and Δwzy mutants were less fit than wild type. Together, our results advise caution in using the right capsule‐null mutant for determination of capsule's role in bacterial pathogenesis. With the use of ΔwcaJ mutant, we found that although the capsule is important for bacterial survival outside the gut environment, it imposes a fitness cost in the gut. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a concerning global pathogen. hvKp is more virulent than classical K. pneumoniae (cKp) and capable of causing community-acquired infections, often in healthy individuals. hvKp is carried in the gastrointestinal tract, which contributes to its spread in the community and healthcare settings. First recognized in Asia, hvKp arose as a leading cause of pyogenic liver abscesses. In the decades since, hvKp has spread globally and causes a variety of infections. In addition to liver abscesses, hvKp is distinct from cKp in its ability to metastasize to distant sites, including most commonly the eye, lung and central nervous system (CNS). hvKp has also been implicated in primary extrahepatic infections including bacteremia, pneumonia and soft tissue infections. The genetic determinants of hypervirulence are often found on large virulence plasmids as well as chromosomal mobile genetic elements which can be used as biomarkers to distinguish hvKp from cKp clinical isolates. These distinct virulence determinants of hvKp include up to four siderophore systems for iron acquisition, increased capsule production, K1 and K2 capsule types, and the colibactin toxin. Additionally, hvKp strains demonstrate hypermucoviscosity, a phenotypic description of hvKp in laboratory conditions that has become a distinguishing feature of many hypervirulent isolates. Alarmingly, multidrug-resistant hypervirulent strains have emerged, creating a new challenge in combating this already dangerous pathogen. [ABSTRACT FROM AUTHOR]

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

Background: Klebsiella pneumoniae is a leading cause of bloodstream infection (BSI). Strains producing extended-spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial-resistant (AMR) K. pneumoniae and also for the characteristically antimicrobial-sensitive, community-acquired "hypervirulent" strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. Methods: We conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate, a K. pneumoniae-specific genomic typing tool. Results: K. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common among South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes among K. pneumoniae. Conclusions: K. pneumoniae BSI in South and Southeast Asia are caused by different STs from those predominating in other regions, and with higher frequency of acquired virulence determinants. K. pneumoniae carrying both iuc and AMR genes were also detected at higher rates than have been reported elsewhere. The study demonstrates how genomics-based surveillance—reporting full molecular profiles including STs, AMR, virulence and serotype locus information—can help standardise comparisons between sites and identify regional differences in pathogen populations. [ABSTRACT FROM AUTHOR]

We report a case of severe infection with liver abscess and endophthalmitis caused by a hypervirulent Klebsiella pneumoniae strain in an immunocompetent German male patient without travel history to Asia. Phenotypic and molecular characterization showed high similarity to the reference genome NTUH-K2044 isolated in Asia. The isolate was assigned as ST2398 (clonal complex 66). The findings underline global spread of hypervirulent Klebsiella pneumoniae strains to Europe.

ABSTRACT Clostridium difficile infection (CDI) is a leading cause of hospital-acquired diarrhea. In recent decades, the emergence of the “hypervirulent” BI/NAP1/027 strains of C. difficile significantly increased the morbidity and mortality of CDI. The pathogenesis of CDI is primarily mediated by the action of two toxins, TcdA and TcdB, with TcdB being the major virulent factor in humans. In this report, we describe the engineering of a panel of designed ankyrin repeat proteins (DARPins) that potently neutralize TcdB from the BI/NAP1/027 strains (e.g., TcdBUK1). The most effective DARPin, D16, inhibits TcdBUK1 with a 50% effective concentration (EC50) of 0.5 nM, which is >66-fold lower than that of the FDA-approved anti-TcdB antibody bezlotoxumab (EC50, ∼33 nM). Competitive enzyme-linked immunosorbent assays (ELISAs) showed that D16 blocks interactions between TcdB and its receptor, chondroitin sulfate proteoglycan 4 (CSPG4). The dimeric DARPin U3D16, which pairs D16 with DARPin U3, a disrupter of the interaction of TcdB with Frizzled 1/2/7 receptor, exhibits 10-fold-to-20-fold-enhanced neutralization potency against TcdB from C. difficile strains VPI 10463 (laboratory strain) and M68 (CF/NAP9/017) but identical activity against TcdBUK1 relative to D16. Subsequent ELISAs revealed that TcdBUK1 did not significantly interact with Frizzled 1/2/7. Computation modeling revealed 4 key differences at the Frizzled 1/2/7 binding interface which are likely responsible for the significantly reduced binding affinity. IMPORTANCE We report the engineering and characterization of designed ankyrin proteins as potent neutralizers of TcdB toxin secreted by a hypervirulent ribotype 027 strain of Clostridium difficile. We further show that although TcdB toxins from both ribotype 027 and VPI 10461 interact efficiently with TcdB receptors CSPG4 and Pvrl3, TcdB027 lacks significant ability to bind the only known physiologically relevant TcdB receptor, Frizzled 1/2/7.

Carbapenem-resistant, hypervirulent Klebsiella pneumoniae (CR-hvKP) has recently emerged as a significant threat to public health. In this study, 29 K. pneumoniae isolates were isolated from eight patients admitted to the intensive care unit (ICU) of a comprehensive teaching hospital located in China from March 2017 to January 2018. Clinical information of patients was the basis for the further analyses of the isolates including antimicrobial susceptibility tests, identification of antibiotic resistance and virulence gene determinants, multilocus sequence typing (MLST), XbaI-macrorestriction by pulsed-field gel electrophoresis (PFGE). Selected isolates representing distinct resistance profiles and virulence phenotypes were screened for hypervirulence in a Galleria mellonella larvae infection model. In the course of the outbreak, the overall mortality rate of patients was 100% (n = 8) attributed to complications arising from CR-hvKP infections. All isolates except one (28/29, 96.6%) were resistant to multiple antimicrobial agents, and harbored diverse resistance determinants that included the globally prevalent carbapenemase blaKPC−2. Most isolates had hypervirulent genotypes being positive for 19 virulence-associated genes, including iutA (25/29, 86.2%), rmpA (27/29, 93.1%), ybtA (27/29, 93.1%), entB (29/29, 100%), fimH (29/29, 100%), and mrkD (29/29, 100%). MLST revealed ST11 for the majority of isolates (26/29, 89,7%). Infection assays demonstrated high mortality in the Galleria mellonella model with the highest LD50 values for three isolates (

ABSTRACT The polysaccharide capsule is an essential virulence factor for Klebsiella pneumoniae in both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulent K. pneumoniae strains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S: rcsB and rmpA mutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene near rmpA. The ΔrmpC strain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of the rmpA promoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of the rmpC and rmpA mutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains. IMPORTANCE Klebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.

The emergence of hypervirulent Klebsiella pneumoniae (hvKp) poses a significant public health threat, particularly regarding its carriage in the healthy population. However, the genomic epidemiological characteristics and population dynamics of hvKp within a single patient across distinct infection episodes remain largely unknown. This study aimed to investigate the clonal replacement of hvKp K2-ST881 and K54-ST29 lineage strains in a single patient experiencing multiple-site infections during two independent episodes. Two strains, designated EDhvKp-1 and EDhvKp-2, were obtained from blood and cerebrospinal fluid during the first admission, and the strain isolated from blood on the second admission was named EDhvKp-3. Whole-genome sequencing, utilizing both short-read Illumina and long-read Oxford Nanopore platforms, was conducted. In silico multilocus sequence typing (MLST), identification of antimicrobial resistance and virulence genes, and the phylogenetic relationship between our strains and other K. pneumoniae ST881 and ST29 genomes retrieved from the public database were performed. Virulence potentials were assessed through a mouse lethality assay. Our study indicated that the strains were highly susceptible to multiple antimicrobial agents. Plasmid sequence analysis confirmed that both virulence plasmids, pEDhvKp-1 (166,008 bp) and pEDhvKp-3 (210,948 bp), belonged to IncFIB type. Multiple virulence genes, including rmpA , rmpA2 , rmpC , rmpD , iroBCDN , iucABCD , and iutA , were identified. EDhvKp-1 and EDhvKp-2 showed the closest relationship to strain 502 (differing by 51 SNPs), while EDhvKp-3 exhibited 69 SNPs differences compared to strain TAKPN-1, which all recovered from Chinese patients in 2020. In the mouse infection experiment, both ST881 EDhvKp-1 and ST29 EDhvKp-3 displayed similar virulence traits, causing 90 and 100% of the mice to die within 72 h after intraperitoneal infection, respectively. Our study expands the spectrum of hvKp lineages and highlights genomic alterations associated with clonal switching between two distinct lineages of hvKP that successively replaced each other in vivo . The development of novel strategies for the surveillance, diagnosis, and treatment of high-risk hvKp is urgently needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Lou, Huang, Chen, Li, Zhang, Hong, Cao and Xiao.)

Distinguishing hypervirulent (hvKp) from classical Klebsiella pneumoniae (cKp) strains is important for clinical care, surveillance, and research. Some combinations of iucA, iroB, peg-344, rmpA, and rmpA2 are most commonly used, but it is unclear what combination of genotypic or phenotypic markers (e.g., siderophore concentration, mucoviscosity) most accurately predicts the hypervirulent phenotype. Furthermore, acquisition of antimicrobial resistance may affect virulence and confound identification. Therefore, 49 K . pneumoniae strains that possessed some combinations of iucA, iroB, peg-344, rmpA, and rmpA2 and had acquired resistance were assembled and categorized as hypervirulent hvKp (hvKp) ( N = 16) or cKp ( N = 33) via a murine infection model. Biomarker number, siderophore production, mucoviscosity, virulence plasmid's Mash/Jaccard distances to the canonical pLVPK, and Kleborate virulence score were measured and evaluated to accurately differentiate these pathotypes. Both stepwise logistic regression and a CART model were used to determine which variable was most predictive of the strain cohorts. The biomarker count alone was the strongest predictor for both analyses. For logistic regression, the area under the curve for biomarker count was 0.962 ( P = 0.004). The CART model generated the classification rule that a biomarker count = 5 would classify the strain as hvKP, resulting in a sensitivity for predicting hvKP of 94% (15/16), a specificity of 94% (31/33), and an overall accuracy of 94% (46/49). Although a count of ≥4 was 100% (16/16) sensitive for predicting hvKP, the specificity and accuracy decreased to 76% (25/33) and 84% (41/49), respectively. These findings can be used to inform the identification of hvKp.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a concerning pathogen that can cause life-threatening infections in otherwise healthy individuals. Importantly, although strains of hvKp have been acquiring antimicrobial resistance, the effect on virulence is unclear. Therefore, it is of critical importance to determine whether a given antimicrobial resistant K. pneumoniae isolate is hypervirulent. This report determined which combination of genotypic and phenotypic markers could most accurately identify hvKp strains with acquired resistance. Both logistic regression and a machine-learning prediction model demonstrated that biomarker count alone was the strongest predictor. The presence of all five of the biomarkers iucA, iroB, peg-344, rmpA, and rmpA2 was most accurate (94%); the presence of ≥4 of these biomarkers was most sensitive (100%). Accurately identifying hvKp is vital for surveillance and research, and the availability of biomarker data could alert the clinician that hvKp is a consideration, which, in turn, would assist in optimizing patient care., Competing Interests: The authors declare no conflict of interest.

The emergence of Klebsiella pneumoniae carbapenemase-2 (KPC-2) and New Delhi metallo-β-lactamase (NDM)-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae (KPC-2-NDM-hv-CRKP) poses a certain threat to public health. Currently, only a few sporadic reports of such double-positive hv-CRKPs were available. In this study, we isolated two KPC-2-NDM-5-hv-CRKPs from elderly patients with serious underlying diseases and poor prognoses. We found both FK3122 and FK3127 were typical multidrug-resistant (MDR) isolates, exhibiting high-level resistance to both carbapenems and novel β-lactamase inhibitors ceftazidime/avibactam. Notably, FK3122 is even resistant to cefiderocol due to multiple bla NDM-5 elements. Besides the MDR phenotype, A549 human lung epithelial cells and Galleria mellonella infection model all indicated that FK3122 and FK3127 were highly pathogenic. According to the whole-genome sequencing analysis, we observed over 10 resistant elements, and the uncommon co-existence of bla , and virulence plasmids in both two isolates. Both virulence plasmids identified in FK3122 and FK3127 shared a high identity with classical virulence plasmid pK2044, harboring specific hypervirulent factors: KPC-2 , bla NDM-5 , and virulence plasmids in both two isolates. Both virulence plasmids identified in FK3122 and FK3127 shared a high identity with classical virulence plasmid pK2044, harboring specific hypervirulent factors: rmpA drug resistance has increased gradually with the emergence of carbapenem-resistant hypervirulent iuc operon. We also found that the resistance and virulence plasmids in FK3127 could not only be transferred to Escherichia coli EC600 independently but also together as a co-transfer, which was additionally confirmed by the S1-pulsed-field gel electrophoresis plasmid profile. Moreover, polymorphic mobile genetic elements were found surrounding resistance genes, which may stimulate the mobilization of resistance genes and result in the duplication of these elements. Considering the combination of high pathogenicity, limited therapy options, and easy transmission of KPC-2-NDM-5-hv-CRKP, our study emphasizes the need for underscores the imperative for ongoing surveillance of these pathogens.IMPORTANCEHypervirulent Klebsiella pneumoniae drug resistance has increased gradually with the emergence of carbapenem-resistant hypervirulent K. pneumoniae (hv-CRKP). However, little information is available on the virulence characteristics of the New Delhi metallo-β-lactamase (NDM) and Klebsiella pneumoniae carbapenemase-2 (KPC-2) co-producing K. pneumoniae strains. In this study, we obtained two KPC-2-NDM-hv-CRKPs from elderly patients, each with distinct capsule types and sequence types: ST11-KL64 and ST15-KL24; these ST-type lineages are recognized as classical multidrug-resistant (MDR) K. pneumoniae . We found these KPC-2-NDM-hv-CRKPs were not only typical MDR isolates, including resistance to ceftazidime/avibactam and cefiderocol, but also displayed exceptionally high levels of pathogenicity. In addition, these high-risk factors can also be transferred to other isolates. Consequently, our study underscores the need for ongoing surveillance of these isolates due to their heightened pathogenicity, limited therapeutic options, and potential for easy transmission., Competing Interests: The authors declare no conflict of interest.

Importance: Globally, the increasing number of hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant Kp (CR-Kp) infections poses a huge public health challenge with high morbidity and mortality. Worrisomely, due to the mobility of elements carrying virulence and drug-resistance genes, the increasing prevalence of CR-hvKp has also been found with an overwhelming mortality rate in recent years. However, the current detection methods for hvKp and CR-Kp have many disadvantages, such as long turnaround time, complex operation, low sensitivity, and specificity. Herein, a more sensitive, rapid, single-reaction, and multiplex quantitative real-time PCR was developed and validated to differentiate the circulating lineages of Kp with excellent performance in sensitivity and specificity, providing a useful tool for the differential diagnosis and the surveillance of the circulating Kp., Competing Interests: The authors declare no conflict of interest.

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 12 361 K. 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., (© The Author(s) [2024]. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Objectives: 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., Methods: 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., Results: 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., Conclusion: Clonal structures were revealed for hvKp and hvKv clinical isolates with their AMR status in northern Japan., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Hypervirulent strains of Klebsiella pneumoniae (hvKP) can cause atypical multilocular infections in otherwise healthy patients. Diagnosis of infection caused by hvKP is based mainly on clinical findings and laboratory results, including detection of virulence genes. It typically manifests as hepatic abscess with metastatic spread. Treatment is based on surgical intervention in combination with targeted antimicrobial therapy. The occurrence of hvKP infection is relatively common in Asia, and while still rare in Europe, incidence is increasing. The article aims to provide a short overview of the issue and increase awareness of the possible occurrence of hvKP infections.

Emerging mobile colistin resistance ( mcr ) genes pose a significant threat to public health for colistin was used as the last resort to treat multidrug-resistant (MDR) pathogenic bacterial infections. Hypervirulent Klebsiella pneumoniae (hvKP) is a clinically significant pathogen resulting in highly invasive infections, often complicated by devastating dissemination. Worryingly, the untreatable and severe infections caused by mcr -harbouring hvKP leave the selection of antibiotics for clinical anti-infective treatment in a dilemma. Herein, we screened 3,461 isolates from a tertiary teaching hospital from November 2018 to March 2021, and an mcr-8.2 -harbouring hvKP FAHZZU2591 with a conjugative plasmid was identified from paediatric sepsis. This is the first report of MCR-8-producing hvKP from paediatric sepsis to our best knowledge. The susceptibility, genetic features, and plasmid profiles of the isolate were investigated. Further, we assessed the virulence potential of FAHZZU2591 and verified its pathogenicity and invasive capacity using a mouse model. The phylogenetic analysis of mcr-8 -bearing K. pneumoniae revealed that China is the predominant reservoir of the mcr-8 gene, and the clinic is the primary source. Our work highlights the risk for the spread of mcr -positive hvKP in clinical, especially in paediatric sepsis, and the persistent surveillance of colistin-resistance hvKP is urgent.

Objectives: To characterize the evolution and interspecies transfer of plasmids between Klebsiella pneumoniae and Escherichia coli within a single patient., Methods: Minimum inhibitory concentrations were measured using broth microdilution assays. Conjugation assays, string tests, and Galleria mellonella infection model experiments were also conducted. Whole-genome sequencing was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants, insertion sequences, and virulence factors were identified using ABRicate/ResFinder database, ISFinder, and virulence factor database. Wzi and capsular polysaccharide (KL) were typed using Kleborate and Kaptive. Multi-locus sequence typing (MLST), replicon typing, and single nucleotide polymorphism analyses were conducted using the BacWGSTdb server., Results: The carbapenem-resistant K. pneumoniae 2111KP was characterized as ST11, wzi64, and KL64, with a positive string test result and a relatively high virulence phenotype. Analysis of the 2111KP genome revealed that bla NDM-1 was located in a 268,400-bp IncFIB/IncHI1B/IncX3 conjugative plasmid (p2111KP-1), regulated by IS26, IS5, and ISKox3. p2111KP-1 was also a rmpA2-associated virulence plasmid with an iutA-iucABCD gene cluster and a IS26-mediated multidrug-resistant fusion plasmid, which contained 8-bp (AGCTGCAC or GGCCTTTG) target site duplications. Segments flanked by IS26 of p2111KP-1 were 99.99% identical to a 49,016-bp E. coli plasmid., Conclusions: This study provided direct evidence of plasmid fusion via IS26 between two different bacterial species within one patient and revealed the process by which genetic elements conferring carbapenem resistance and virulence were simultaneously transferred between these species. It highlights the need for strategic antibiotic use and rigorous monitoring to prevent the plasmid-mediated fusion and transmission of drug-resistance/virulence factors., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Hypervirulent Klebsiella pneumoniae have been rarely described in Latin America. This work describes the characterization of hypervirulent K. pneumoniae isolates capsular serotype K2 belonging to sequence types 86 and 380. The assays showed the hypervirulent K. pneumoniae highly virulent, which is determined by the plasmid borne virulence genes. At this time, the hypervirulent K. pneumoniae clinical isolates in Mexico are extensively susceptible to antibiotics. [ABSTRACT FROM AUTHOR]

In 2017, we identified a Clostridium difficile strain HKCD4 that caused community-acquired fulminant colitis in a previously healthy child. Phylogenetically, it belonged to clade 2, sequence type 67 and was resistant to fluoroquinolone and tetracycline. The strain was pathogenicity locus and binary toxin positive. It has a mutation in the trehalose repressor treR leading to the L172I substitution that was previously reported in the epidemic ribotype 027 lineage. HKCD4 has a tcdB sequence that shared very high identities with 3 highly virulent reference strains. It has a CpG depleted genome that is characteristic of hypervirulent C. difficile. The emergence of ST67 lineage with molecular feature of hypervirulence in the community is concerning and emphasizes the need for full characterization of strains causing severe disease in patients without classical risk factors. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae (CR-HMKP) poses a significant public health challenge. We investigated its epidemiology and molecular characteristics in a tertiary care hospital in eastern China. Methods: CR-HMKP were identified among 106 non-duplicated carbapenem-resistant K. pneumoniae isolates (from June 2013 to September 2017) using the string test. The pulsotype (PT) and sequence type (ST) of CR-HMKP isolates were determined using pulsedfield gel electrophoresis and multilocus sequence typing. Resistance determinants, capsular serotypes, and virulence genes were detected by PCR and sequencing. Representative isolates from each PT were selected, and their virulence phenotypes were established using the serum killing and Galleria mellonella lethality assays. Results: Of the 106 isolates, 13 (12.3%) were CR-HMKP. Seven were positive for blaNDM-1 and shared the same genotype (PT5/ST1764); the others were positive for blaKPC-2, belonged to ST11, and were divided into four different PTs. The serotype of all blaNDM-1-positive isolates was K64, while that of blaKPC-2-positive isolates were K47 (N=4) and K64 (N=2). The NDM-1-producing HMKP isolates were positive for aerobactin, exhibited high serum resistance, and elicited significantly increased larval mortality compared with the other isolates. All patients had received invasive treatment prior to infection by NDM-1-producing HMKP. The infections occurred between July and August 2016 and were hospital-acquired. Conclusions: NDM-1-producing HMKP ST1764 isolates were identified; this is the first report worldwide on an outbreak of nosocomial infection caused by these isolates. Effective surveillance and strict infection control strategies should be implemented to prevent CRHMKP dissemination. [ABSTRACT FROM AUTHOR]

Abstract A 14-year-old otherwise healthy boy presented with right-sided back pain following high fever. Abdominal computed tomography scan showed a large liver abscess. Klebsiella pneumoniae (KP) was rapidly identified from peripheral blood using the melting temperature mapping (Tm) method, which enables identification of pathogenic microorganisms within four hours after patient sample collection. He was diagnosed with pyogenic liver abscess (PLA) caused by KP on the day of admission. The KP was the hypervirulent (hv) clinical variant (string test positive, serotype K1, sequence type 23, rmpA and magA positive). After intravenous antibiotic therapy and drainage of the abscess, his condition resolved. The highlights of this case report are a healthy child with hypervirulent Klebsiella pneumoniae liver abscess in Japan and the new Tm mapping method for rapid and accurate identification of the pathogenic microorganism. [ABSTRACT FROM AUTHOR]

This work is aimed to elucidate the prevalence and characteristics of antimicrobial resistance, virulence, and molecular typing in Klebsiella pneumoniae from clinical companion animals in Beijing, China. In total, 105 K. pneumoniae (2.0%) isolates were recovered from 5359 samples (dogs, n = 3356; cats, n = 2003). All tested isolates exhibited high resistance to amoxicillin-clavulanate (74.3%). Moreover, resistance rates in dog isolates (2.1%) were significantly higher than in cat isolates (0.9%); however, the rate of multidrug-resistance (MDR) was 57.1% and the MDR prevalence in cats was significantly higher than dogs. Whole-genome sequencing demonstrated plasmids IncX4 and IncFIA (HI1)/FII(K) carried mcr-1 (n = 1) and mcr-8 (n = 1), but blaOXA-181 (n = 1) and blaNDM-5 (n = 4) were harbored in IncX3-type plasmids, and the above genes were in different isolates. The most prevalent sequence types (STs) in companion animals were ST1 (n = 9) and ST37 (n = 9). Compared to National Center for Biotechnology Information (NCBI) data on human K. pneumoniae, resistance genes blaCTX-M and blaTEM were more prevalent in human isolates; however, aac(6′)-Ib-cr and oqxAB showed a higher prevalence in companion animals. Hypermucoviscosity was reported in 9 (8.6%) isolates, whereas 64 isolates (61.0%) were hypervirulent K. pneumoniae (hvKP) via the Galleria mellonella. These findings validate the high risk of K. pneumonia and necessitate its relevant control in pet clinics.

The effectiveness of ceftolozane/tazobactam for the treatment of infections in neutropenic patients caused by hypervirulent multidrug-resistant (MDR) Pseudomonas aeruginosa has not been previously reported. We identified seven cases of MDR P. aeruginosa infection in neutropenic patients over a four-month period within the same hematology ward. Four cases were associated with rapid progression despite piperacillin-tazobactam or meropenem therapy, and three patients developed sepsis or extensive skin/soft tissue necrosis. In three of the four cases, patients were empirically switched from meropenem to ceftolozane/avibactam before carbapenem susceptibility test results were available, and all four patients underwent extensive surgical debridement or amputation of affected tissues and survived. Further investigation revealed a common bathroom source of MDR P. aeruginosa clonal subtypes ST175 and ST235 that harbored genes for type III secretion system expression and elaboration of ExoU or ExoS exotoxin. We conclude that ceftolozane/tazobactam plus early source control was critical for control of rapidly progressing skin and soft infection in these neutropenic patients caused by highly virulent ST175 and ST235 clones of MDR P. aeruginosa.

Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging pathogen and it has more virulence factors than classical Klebsiella pneumoniae strains. Carbapenem-resistant hvKp (CR-hvKp) is a dangerous bacteria that has both high virulence and antibiotic resistance and poses a global public health problem worldwide. The current study was carried out to investigate the occurrence of hvKp as well as carbapenem-resistant Klebsiella pneumoniae (CRKP) and CR-hvKp among diarrheic farm animals. For this purpose, rectal swabs from 165 farm animals (45 cattle, 66 sheep, and 54 goats) were collected. Samples were processed for the isolation and identification of Klebsiella pneumoniae. Moreover, hvKp was detected using molecular techniques by amplification of biomarker virulence genes (rmpA , rmpA2 , iucA , iroB , and peg-344), followed by a string test. On the other hand, all K. pneumoniae isolates were examined for carbapenem resistance by both phenotypic and molecular methods. The phylogenetic analysis of peg- 344 sequences was carried out. The overall prevalence rates of K. pneumoniae , hvKp, CRKP, and CR-hvKp were 24.2%, 7.9%, 16.4%, and 6.1% respectively. HvKp and CR-hvKp were detected among all examined farm animal species. On a Molecular basis, all biomarker virulence genes were identified except iroB, but rmpA is the most prevalent one. The phylogenetic analysis of peg-344 sequences obtained from the study points out their genetic relatedness to those circulated among humans. In conclusion, the emergence of hvKp and CR-hvKp among diarrheic farm animals confers a great public health implication and thus, the possible animal reservoirs for such hypervirulent-antimicrobial resistant strains cannot be ruled out. • The study highlights the emergence of hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant hvKp (CR-hvKp) among diarrheic farm animals. • The overall prevalence rates of Klebsiella pneumoniae , hvKp, carbapenem-resistant Klebsiella pneumoniae (CRKP), and CR-hvKp were determined. Remarkably, both hvKp and CR-hvKp were detected across all three examined farm animal species. • The study employed molecular techniques to detect hvKp by amplifying biomarker virulence genes such as rmpA, rmpA2, iucA, iroB , and peg-344. • The finding suggests that the hvKp strains in farm animals may share genes with human-infecting ones, raising concerns about animal-to-human transmission, and emphasizing surveillance and control. [ABSTRACT FROM AUTHOR]

We describe a hypervirulent clone of group B Streptococcus serotype III, subtype 4, sequence type 283, that caused invasive disease with a predilection for meningitis in Hong Kong during 1993–2012. The organism is associated with high mortality and increased summer prevalence and is linked to diseased fish from freshwater fish farms.

Background: Development of improved therapeutics against tuberculosis (TB) is hindered by an inadequate understanding of the relationship between disease severity and genetic diversity of its causative agent, Mycobacterium tuberculosis. We previously isolated a hypervirulent M. tuberculosis strain H112 from an HIV-negative patient with an aggressive disease progression from pulmonary TB to tuberculous meningitis—the most severe manifestation of tuberculosis. Human macrophage challenge experiment demonstrated that the strain H112 exhibited significantly better intracellular survivability and induced lower level of TNF-α than the reference virulent strain H37Rv and other 123 clinical isolates.Aim: The present study aimed to identify the potential genetic determinants of mycobacterial virulence that were common to strain H112 and hypervirulent M. tuberculosis strains of the same phylogenetic clade isolated in other global regions.Methods: A low-virulent M. tuberculosis strain H54 which belonged to the same phylogenetic lineage (L2) as strain H112 was selected from a collection of 115 clinical isolates. Both H112 and H54 were whole-genome-sequenced using PacBio sequencing technology. A comparative genomics approach was adopted to identify mutations present in strain H112 but absent in strain H54. Subsequently, an extensive phylogenetic analysis was conducted by including all publically available M. tuberculosis genomes. Single-nucleotide-polymorphisms (SNPs) and structural variations (SVs) common to hypervirulent strains in the global collection of genomes were considered as potential genetic determinants of hypervirulence.Results:Sequencing data revealed that both H112 and H54 were identified as members of the same sub-lineage L2.2.1. After excluding the lineage-related mutations shared between H112 and H54, we analyzed the phylogenetic relatedness of H112 with global collection of M. tuberculosis genomes (n = 4,338), and identified a novel phylogenetic clade in which four hypervirulent strains isolated from geographically diverse regions were clustered together. All hypervirulent strains in the clade shared 12 SNPs and 5 SVs with H112, including those affecting key virulence-associated loci, notably, a deleterious SNP (rv0178 p. D150E) within mce1 operon and an intergenic deletion (854259_ 854261delCC) in close-proximity to phoP.Conclusion: The present study identified common genetic factors in a novel phylogenetic clade of hypervirulent M. tuberculosis. The causative role of these mutations in mycobacterial virulence should be validated in future study.

Klebsiella pneumoniae is a Gram-negative pathogen that has a large accessory genome of plasmids and chromosomal gene loci. This accessory genome divides K. pneumoniae strains into opportunistic, hypervirulent, and multidrug-resistant groups and separates K. pneumoniae from two closely related species, Klebsiella variicola and Klebsiella quasipneumoniae. Some strains of K. pneumoniae act as opportunistic pathogens, infecting critically ill and immunocompromised patients. These K. pneumoniae are a common cause of health-care associated infections including pneumonia, urinary tract infections (UTIs), and bloodstream infections. K. variicola and K. quasipneumoniae are often clinically indistinguishable from opportunistic K. pneumoniae. Other strains of K. pneumoniae are hypervirulent, infecting healthy people in community settings and causing severe infections including pyogenic liver abscess, endophthalmitis, and meningitis. A third group of K. pneumoniae encode carbapenemases, making them highly antibiotic-resistant. These strains act as opportunists but are exceedingly difficult to treat. All of these groups of K. pneumoniae and related species can colonize the gastrointestinal tract, and the accessory genome may determine if a colonizing strain remains asymptomatic or progresses to cause disease. This review will explore the associations between colonization and infection with opportunistic, antibiotic-resistant, and hypervirulent K. pneumoniae strains and the role of the accessory genome in distinguishing these groups and related species. As K. pneumoniae infections become progressively more difficult to treat in the face of antibiotic resistance and hypervirulent strains, an increased understanding of the epidemiology and pathogenesis of these bacteria is vital.

Objectives: To provide the clinical and molecular characteristics of carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae (cr-hvKP) in a tertiary hospital in Beijing, China. Methods: The clinical characteristics of four patients with cr-hvKP isolates and 29 patients with carbapenem-resistant classic K. pneumoniae (cr-cKP) infections were analyzed retrospectively. The molecular characteristics of cr-hvKP and cr-cKP isolates were compared. Results: The KPC-2 gene was detected in all cr-hvKPs except for cr-hvKP6. The cr-hvKPs belonged to three sequence types (STs; ST25, ST65, and ST11), with three pulsed-field gel electrophoresis patterns (I, II, and III) and two capsular serotypes (K2 and non-typeable). Although cr-hvKP1–7 did not cause invasive clinical syndromes such as community-acquired liver abscess with or without extrahepatic complications, they were all nosocomially acquired; cr-hvKP1–5 were clones disseminated between patients A and B. Compared with cr-cKPs, pLVPK-related loci, repA, iroN, and K2 capsular serotype were more prevalent in cr-hvKPs, although no significant difference was found in clinical characteristics between patients with cr-hvKP and cr-cKP infection. Conclusions: The hypervirulent ST65 and ST25 K. pneumoniae, along with carbapenem-resistant clonal populations ST11, appear to have evolved into cr-hvKP strains. The evidence of bi-directional evolution and emergence of hospital-acquired multi-clone cr-hvKP indicates a confluence of virulence and carbapenem resistance, which might pose major problems in the management of K. pneumoniae infection.

Aim: 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)., Materials and Methods: 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 the combined disk method and polymerase chain reaction, respectively. In addition, crystal violet staining was used to determine the biofilm formation of these isolates., Results: 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., Conclusion: 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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Background: Colistin has emerged as a last-resort therapeutic against antibiotic-resistant bacterial infections, particularly those attributed to carbapenem-resistant Enterobacteriaceae (CRE) like CRKP. Yet, alarmingly, approximately 45% of multidrug-resistant Klebsiella pneumoniae strains now manifest resistance to colistin. Through our study, we discerned that the synergy between carbapenemase and IS elements amplifies resistance in Klebsiella pneumoniae , thereby narrowing the existing therapeutic avenues. This underscores the instrumental role of IS elements in enhancing colistin resistance through mgrB disruption., Methods: From 2021 to 2023, 127 colistin-resistant Klebsiella pneumoniae isolates underwent meticulous examination. We embarked on an exhaustive genetic probe, targeting genes associated with both plasmid-mediated mobile resistance-encompassing bla KPC, bla NDM, bla IMP, bla VIM, bla OXA-48-like, and mcr-1 to mcr-8 -and chromosome-mediated resistance systems, including PhoP/Q , PmrA/B , and mgrB . PCR amplification revealed the presence of virulence-associated genes from the pLVPK plasmid, such as rmpA , rmpA2 , iucA , iroB , and peg344 . mgrB sequencing was delegated to Sangon Biotech, Shanghai, and the sequences procured were validated using BLAST. Our search for IS elements was navigated through the IS finder portal. Phenotypically, we harnessed broth microdilution (BMD) to ascertain the MICs of colistin. To sketch the clonal lineage of mgrB-mutated CoR-Kp isolates, sophisticated methodologies like MLST and PFGE were deployed. S1-PFGE unraveled the intrinsic plasmids in these isolates. Our battery of virulence assessment techniques ranged from the string test and capsular serotyping to the serum killing assay and the Galleria mellonella larval infection model., Results: Among the 127 analyzed isolates, 20 showed an enlarged mgrB PCR amplicon compared to wild-type strains. These emerged over a three-year period: three in 2021, thirteen in 2022, and four in 2023. Antimicrobial susceptibility tests revealed that these isolates consistently resisted several drugs, notably TCC, TZP, CAZ, and COL. Additionally, 85% resisted both DOX and TOB. The MICs for colistin across these strains ranged between 16 to 64 mg/L, with a median of 40 mg/L. From a genetic perspective, MLST unanimously categorized these mgrB -mutated CoR-hvKp isolates as ST11. PFGE further delineated them into six distinct clusters, with clusters A and D being predominant. This distribution suggests potential horizontal and clonal genetic transmission. Intriguingly, every mgrB-mutated CoR-hvKP isolate possessed at least two virulence genes akin to the pLVPK-like virulence plasmid, with iroB and rmpA2 standing out. Their virulence was empirically validated both in vitro and in vivo . A pivotal discovery was the identification of three distinct insertion sequence (IS) elements within or near the mgrB gene. These were:ISKpn26 in eleven isolates, mainly in cluster A, with various insertion sites including +74, +125, and an upstream -35.ISKpn14 in four isolates with insertions at +93, -35, and two upstream at -60.IS903B present in five isolates, marking positions like +74, +125, +116, and -35 in the promoter region. These diverse insertions, spanning six unique locations in or near the mgrB gene, underscore its remarkable adaptability., Conclusion: Our exploration spotlights the ISKpn element's paramount role in fostering mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae . Employing MLST and PFGE, we unearthed two primary genetic conduits: clonal and horizontal. A striking observation was the ubiquitous presence of the KPC carbapenemase gene in all the evaluated ST11 hypervirulent colistin-resistant Klebsiella pneumoniae strains, with a majority also harboring the NDM gene. The myriad mgrB gene insertion locales accentuate its flexibility and the overarching influence of IS elements, notably the pervasive IS5-like variants ISKpn26 and IS903B. Our revelations illuminate the escalating role of IS elements in antibiotic resistance within ST11 hypervirulent colistin-resistant Klebsiella pneumoniae , advocating for innovative interventions to counteract these burgeoning resistance paradigms given their profound ramifications for prevailing treatment modalities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhu, Li, Zhang, He, Tao, Ji, Yang, Zhu, Luo, Liao, Chen, Liu and Zhang.)