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PurposeAcinetobacter baumannii is associated with both hospital-acquired infections and community-acquired pneumonia (CAP). Here, we describe a novel strain of A. baumannii in a case of CAP in a previously healthy rural villager from Central Eastern China.Materials and methodsA. baumannii isolated from the patient (LS01) was compared to well-characterized pathogenic strain (AB5075), nosocomial circulating strain in China (ZJ06), and wild-type strain (ATCC17978). Growth rate studies were conducted under different environmental stressors, and virulence studies were performed using Galleria mellonella larvae. Whole genome sequencing (WGS) was performed using MinIon and MiSeq. Center for Genomic Epidemiology, CLCbio, Geneious, and Virulence Factors of Pathogenic Bacteria database were used for genomic analysis.ResultsLS01 grew significantly faster at 37°C and 42°C and in the presence of zinc compared to other strains. LS01 was more virulent in G. mellonella, killing all larvae within 8 h. Although WGS revealed 44 virulence genes, these genes were also present in the other strains. While two chromosomally encoded β-lactamases were identified, there were no plasmids identified and LS01 was pan-susceptible to all antibiotics tested. Phylogenetic analysis revealed that the closest related strains were only 72.552% identical, supporting a novel strain.ConclusionLS01 is a novel strain of hypervirulent yet pan-drug susceptible A. baumannii isolated from a patient with no prior hospitalizations, sick contacts, or any of the typical risk factors. This raises concerns for an emerging pathogen, and more epidemiological studies should be conducted to assess the prevalence of this A. baumannii strain.

Qingping Zhou,1,* Chunyang Wu,2,* Peiyao Zhou,3 Ji Zhang,1 Zhanghua Xiong,4 Ying Zhou,5 Fangyou Yu5 1Department of Neurology, Jiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, 330006, People’s Republic of China; 2Department of Respiratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People’s Republic of China; 3Department of Laboratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People’s Republic of China; 4Department of Clinical Laboratory Medicine, Jiangxi Provincial People’s Hospital, The First Affiliated Hospital of Nanchang Medical College, Nanchang, 330006, People’s Republic of China; 5Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200082, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ying Zhou, Email 18702195157@163.comBackground: Patients with neurological disorders were easier to develop severe intracranial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae, leading to a distressing clinical outcome. In this study, eight hv-CRKP were isolated from neurological patients, to clarify the resistant and virulent features.Methods: We tested the susceptibility of common antibiotics in these isolates to feature the antibiotic-resistant phenotypes. We also detected the key virulence factors, including mucoviscosity, siderophores production, biofilm formation in vitro, and further evaluated the virulence potential with serum killing model. We also used whole-genome sequencing (WGS) to investigate the molecular mechanisms.Results: We observed that ST11-KL64 hv-CRKP (6/8) has an overwhelming epidemic dominance in these hypervirulent and carbapenem-resistant K. pneumoniae. Though the acquirement of virulence plasmid made no influence to the maintain of multidrug-resistant phenotype of these isolates, only the ST11-KL64 strains fully exhibited the hypervirulent features. Compared with ST11-KL47 and ST15-KL24 strains, ST11-KL64 hv-CRKP were more advantages in productions of capsule polysaccharide, biofilm, and siderophores. The virulence potential of ST11-KL64 hv-CRKP was further confirmed by using serum killing model. Previous studies have demonstrated that IncFII plasmid could act as a helper plasmid to mobile the non-conjugative IncFIB/IncHIB virulence plasmids. We could only observe the co-existence of IncFII resistance plasmid and IncFIB/IncHIB virulence plasmids in ST11-KL64 isolates. The co-existence of such two plasmids facilitated the formation of ST11-KL64 hv-CPKP, which then become nosocomial epidemic under the antibiotic stress.Conclusion: Overall, we observed the ST11-KL64 hv-CRKP dominated in the isolates from neurological patients, and required most clinical attention.Keywords: Klebsiella pneumoniae, KPC-2, virulence plasmid, carbapenem resistance, hypervirulent

K1/K2 hvKP strains acquire carbapenem-resistance plasmids, known as CR-hvKp, and carbapenem-resistant Klebsiella pneumoniae (CRKP) strains obtain virulence plasmids, recognized as hv-CRKP. The two different evolution patterns of hypervirulent combined carbapenem-resistant Klebsiella pneumoniae may lead to their different prevalence in hospitals. Our study aimed to investigate the prevalence of hv-CRKP and CR-hvKp strains and to analyze factors influencing their evolution and prevalence. We collected 890 K. pneumoniae genomes from GenBank and 530 clinical K. pneumoniae isolates from nine hospitals. Our study found that hv-CRKP strains were more prevalent than CR-hvKp strains and both were dominated by blaKPC-2 gene. The blaKPC-2-carrying plasmids could mobilize non-conjugative virulence plasmids from hvKp strains to CRKP strains. The conserved oriT of virulence plasmids and the widespread of conjugative helper plasmids were potential factors for the mobilization of non-conjugative virulence plasmids. HvKp strains with KPC plasmid could hardly simultaneously exhibit hypervirulence and carbapenem resistance as CRKP strains with virulence plasmid, and we found that rfaH mutation reduced capsular synthesis and increased carbapenem resistance of the CR-hvKp strain. In summary, this study revealed that hv-CRKP strains were more suitable for survival in hospital settings than CR-hvKp strains and the widespread conjugative KPC-producing plasmids contributed to the emergence and prevalence of hv-CRKP strains.

Purpose: The type VI system (T6SS) has the potential to be a new virulence factor for hypervirulent Klebsiella pneumoniae (hvKp) strains. This study aimed to characterize the molecular and clinical features of T6SS-positive and T6SSnegative K. pneumoniae isolates that cause abscesses. Patients and methods: A total of 169 non-duplicate K. pneumoniae strains were isolated from patients with abscesses in a tertiary hospital in China from January 2018 to June 2022, and clinical data were collected. For all isolates, capsular serotypes, T6SS genes, virulence, and drug resistance genes, antimicrobial susceptibility testing, and biofilm formation assays were assessed. Multilocus sequence typing was used to analyze the genotypes of hvKp. T6SS-positive hvKp, T6SS-negative hvKp, T6SS-positive cKP, and T6SS-negative cKP (n = 4 strains for each group) were chosen for the in vivo Galleria mellonella infection model and in vitro competition experiments to further explore the microbiological characteristics of T6SS-positive K. pneumoniae isolates. Results: The positive detection rate for T6SS was 36.1%. The rates of hvKp, seven virulence genes, K1 capsular serotype, and ST23 in T6SS-positive strains were all higher than those in T6SS-negative strains (p < 0.05). Multivariate logistic regression analysis indicated that the carriage of aerobactin (OR 0.01) and wcaG (OR 33.53) were independent risk factors for T6SS-positive strains (p < 0.05). The T6SS-positive strains had a stronger biofilm-forming ability than T6SS-negative strains (p < 0.05). The T6SS-positive and T6SS-negative strains showed no significant differences in competitive ability (p = 0.06). In the in vivo G. mellonella infection model, the T6SS(+)/hvKP group had the worst prognosis. Except for cefazolin and tegacyclin, T6SS-positive isolates displayed a lower rate of antimicrobial resistance to other drugs (p < 0.05). The T6SS-positive isolates were more likely to be acquired from community infections (p < 0.05). Conclusion: Klebsiella pneumoniae isolates causing abscesses have a high prevalence of T6SS genes. T6SS-positive K. pneumoniae isolates are associated with virulence, and the T6SS genes may be involved in the hvKp virulence mechanism. [ABSTRACT FROM AUTHOR]

Background: Klebsiella pneumoniae capsular types K1, K2, K5, K20, K54, and K57 are prevalent hypervirulent types associated with community infections, and worrisomely, hypervirulent strains that acquired drug resistance have been found. In the search for alternative therapeutics, studies have been conducted on phages that infect K. pneumoniae K1, K2, K5, and K57-type strains and their phage-encoded depolymerases. However, phages targeting K. pneumoniae K20-type strains and capsule depolymerases capable of digesting K20-type capsules have rarely been reported. In this study, we characterized a phage that can infect K. pneumoniae K20-type strains, phage vB_KpnM‐20. Methods: A phage was isolated from sewage water in Taipei, Taiwan, its genome was analyzed, and its predicted capsule depolymerases were expressed and purified. The host specificity and capsule-digesting activity of the capsule depolymerases were determined. The therapeutic effect of the depolymerase targeting K. pneumoniae K20-type strains was analyzed in a mouse infection model. Results: The isolated Klebsiella phage, vB_KpnM‐20, infects K. pneumoniae K7, K20, and K27-type strains. Three capsule depolymerases, K7dep, K20dep, and K27dep, encoded by the phage were specific to K7, K20, and K27-type capsules, respectively. K20dep also recognized Escherichia coli K30-type capsule, which is highly similar to K. pneumoniae K20-type. The survival of K. pneumoniae K20-type-infected mice was increased following administration of K20dep. Conclusions: The potential of capsule depolymerase K20dep for the treatment of K. pneumoniae infections was revealed using an in vivo infection model. In addition, K7dep, K20dep, and K27dep capsule depolymerases could be used for K. pneumoniae capsular typing. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKp) infections confer notable morbidity and mortality. Differential diagnosis to determine whether the infections are caused by either the hvKp or classical K. pneumoniae (cKp) strain is particularly important for undertaking optimal clinical care and infection control efforts. To identify and assess the potential predictors of hvKp infections. PubMed, Web of Science, and Cochrane Library databases were searched for all relevant publications from January 2000 to March 2022. The search terms included a combination of the following terms: (i) Klebsiella pneumoniae or K. pneumoniae and (ii) hypervirulent or hypervirulence. A meta-analysis of factors for which risk ratio was reported in three or more studies was conducted, and at least one statistically significant association was identified. In this systematic review of 11 observational studies, a total of 1392 patients with K. pneumoniae infection and 596 (42.8%) with hvKp strains were evaluated. In the meta-analysis, diabetes mellitus and liver abscess (pooled risk ratio: 2.61 (95% confidence interval: 1.79–3.80) and 9.04 (2.58–31.72), respectively; all P < 0.001) were predictors of hvKp infections. For patients with a history of the abovementioned predictors, prudent management, including the search for multiple sites of infection and/or metastatic spread and the enforcement of an early and appropriate source control procedure, should be initiated in consideration of the potential presence of hvKp. We believe that this research highlights the urgent need for increasing clinical awareness of the management of hvKp infections. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKp) is emerging worldwide. Hypermucoviscousity is the characteristic trait that distinguishes it from classic K. pneumoniae (cKp), which enables Kp to cause severe invasive infections. This research aimed to investigate the hypermucoviscous Kp (hmvKp) phenotype among gut commensal Kp isolated from healthy individuals and attempted to characterize the genes encoding virulence factors that may regulate the hypermucoviscosity trait. Using the string test, 50 identified Kp isolates from healthy individuals' stool samples were examined for hypermucoviscosity and investigated by transmission electron microscopy (TEM). Antimicrobial susceptibility profiles of Kp isolates were determined using the Kirby Bauer disc method. Kp isolates were tested for genes encoding different virulence factors by PCR. Biofilm formation was assayed by the microtiter plate method. All Kp isolates were multidrug-resistant (MDR). Phenotypically, 42% of isolates were hmvKp. PCR-based genotypic testing revealed the hmvKp isolates belonged to capsular serotype K2. All study Kp isolates harbored more than one virulence gene. The genes magA and rmpA were not detected, while the terW gene was present in all isolates. The siderophores encoding genes entB and irp2 were most prevalent in hmvKp isolates (90.5%) and non-hmvKp (96.6%), respectively. hmvKp isolates harbored the genes wabG and uge with rates of 90.5% and 85.7%, respectively. The outcomes of this research highlight the potential health risk of commensal Kp to cause severe invasive diseases, owing to being hmvKp and MDR, and harboring multiple virulence genes. The absence of essential genes related to hypermucoviscosity such as magA and rmpA in hmvKp phenotypes suggests the multifactorial complexity of the hypermucoviscosity or hypervirulence traits. Thus, further studies are warranted to verify the hypermucoviscosity-related virulence factors among pathogenic and commensal Kp in different colonization niches. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKp) is a new emerging variant of K. pneumoniae that is increasingly reported worldwide. The variant hvKp is known to cause severe invasive community-acquired infections such as metastatic meningitis, pyogenic liver abscesses (PLA) and endophthalmitis, but its role in hospital-acquired infections (HAIs) is little known. The aim of this study was to evaluate the prevalence of hvKp among hospital-acquired (HA) K. pneumoniae infections in the intensive care unit (ICU) and to compare between hvKp and classical K. pneumoniae (cKP) regarding antimicrobial resistance pattern, virulence and molecular characteristics. The study was cross-sectional and included 120 ICU patients suffering from HA K. pneumoniae infections between January and September 2022. K. pneumoniae isolates were subjected to antimicrobial susceptibility testing and detection of extended-spectrum-β-lactamase (ESBL) production by the Phoenix 100 automated microbiology system, string test, biofilm formation, serum resistance assay, and detection of virulence-associated genes (rmpA, rmpA2, magA, iucA) and capsular serotype-specific genes (K1, K2, K5, K20, K57) by polymerase chain reaction (PCR). Of 120 K. pneumoniae isolates, 19 (15.8%) were hvKp. The hypermucoviscous phenotype was more significantly detected in the hvKp group than in the cKP group (100% vs. 7.9%, p ≤ 0.001). The rate of resistance to different antimicrobial agents was significantly higher in the cKP group than that in the hvKp group. Fifty-three strains were identified as ESBL-producing strains, which was more frequent in the cKP group than in the hvKp group (48/101 [47.5%] vs. 5/19 [26.3%], respectively, p ≤ 0.001). The hvKP isolates were highly associated with moderate and strong biofilm formation than cKP isolates (p = 0.018 and p = 0.043 respectively). Moreover, the hvKP isolates were highly associated with intermediate sensitivity and re sistance to serum in the serum resistance assay (p = 0.043 and p = 0.016 respectively). K1, K2, rmpA, rmpA2, magA and iucA genes were significantly associated with hvKp (p ≤ 0.001, 0.004, <0.001, <0.001, 0.037 and <0.001, respectively). However, K5, K20 and K57 were not associated with hvKp. The hvKp strains have emerged as a new threat to ICU patients because of their ability to cause more severe and life-threatening infections than cKP. The string test alone as a laboratory test for screening of hvKp has become insufficient. Recently, hvKp was defined as hypermucoviscous- and aerobactin-positive. It is important to improve the awareness towards the diagnosis and management of hvKp infections. [ABSTRACT FROM AUTHOR]

ABSTRACT Klebsiella pneumoniae is a leading cause of nosocomial infections, including pneumonia, bacteremia, and urinary tract infections. Treatment options are increasingly restricted by the high prevalence of resistance to frontline antibiotics, including carbapenems, and the recently identified plasmid-conferred colistin resistance. The classical pathotype (cKp) is responsible for most of the nosocomial infections observed globally, and these isolates are often multidrug resistant. The hypervirulent pathotype (hvKp) is a primary pathogen capable of causing community-acquired infections in immunocompetent hosts. The hypermucoviscosity (HMV) phenotype is strongly associated with the increased virulence of hvKp isolates. Recent studies demonstrated that HMV requires capsule (CPS) synthesis and the small protein RmpD but is not dependent on the increased amount of capsule associated with hvKp. Here, we identified the structure of the capsular and extracellular polysaccharide isolated from hvKp strain KPPR1S (serotype K2) with and without RmpD. We found that the polymer repeat unit structure is the same in both strains and that it is identical to the K2 capsule. However, the chain length of CPS produced by strains expressing rmpD demonstrates more uniform length. This property was reconstituted in CPS from Escherichia coli isolates that possess the same CPS biosynthesis pathway as K. pneumoniae but naturally lack rmpD. Furthermore, we demonstrate that RmpD binds Wzc, a conserved capsule biosynthesis protein required for CPS polymerization and export. Based on these observations, we present a model for how the interaction of RmpD with Wzc could impact CPS chain length and HMV. IMPORTANCE Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD, is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain. Here, we demonstrate that RmpD regulates capsule chain length and interacts with Wzc, a part of the capsule polymerization and export machinery shared by many pathogens. We further show that RmpD confers HMV and regulates capsule chain length in a heterologous host (E. coli). As Wzc is a conserved protein found in many pathogens, it is possible that RmpD-mediated HMV and increased virulence may not be restricted to K. pneumoniae.

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.

Dissemination of carbapenemase-encoding plasmids by horizontal gene transfer in multidrug-resistant bacteria is the major driver of rising carbapenem-resistance, but the conjugative mechanics and evolution of clinically relevant plasmids are not yet clear. We performed whole-genome sequencing on 1,215 clinical Enterobacterales isolates collected in Singapore during 2010–2015. We identified 1,126 carbapenemase-encoding plasmids and discovered pKPC2 is becoming the dominant plasmid in Singapore, overtaking an earlier dominant plasmid, pNDM1. pKPC2 frequently conjugates with many Enterobacterales species, including hypervirulent Klebsiella pneumoniae, and maintains stability in vitro without selection pressure and minimal adaptive sequence changes. Furthermore, capsule and decreasing taxonomic relatedness between donor and recipient pairs are greater conjugation barriers for pNDM1 than pKPC2. The low fitness costs pKPC2 exerts in Enterobacterales species indicate previously undetected carriage selection in other ecological settings. The ease of conjugation and stability of pKPC2 in hypervirulent K. pneumoniae could fuel spread into the community.

Hypervirulent Klebsiella pneumoniae (hvKp) is an important pathotype with enhanced virulence features compared with classical K. pneumoniae (cKp). hvKp usually causes life-threatening infections in the community, often affecting young and healthy individuals. During the past few decades, hvKp-induced liver abscess has been increasingly reported in Asia and is emerging as a global disease. To better comprehend the molecular characteristics of hvKp-induced liver abscess and recognize the global dissemination of hypervirulent strains with resistance determinants, we sequenced the whole genome of 26 K. pneumoniae strains from patients with liver abscess (KLA) and investigated the clinical factors related to different phenotype groups. The epidemiology, virulence-related factors, and antimicrobial resistance determinants were also discussed. The age, gender, and whether being hospitalized showed no differences among the string-positive and -negative groups were also studied. The assembly and annotation suggested that most of the 26 new liver abscess-causing hvKp strains were ST23-K1 or ST86-K2, and only one of the strains exhibited multidrug resistance. Compared with the existing 36 global liver abscess genome sequences, higher sequence type and virulence gene diversity were found in the new genomes. The clinical characteristics and genomic data of the isolated strains will enrich our knowledge for comparative genomic studies, allowing the better understanding of hvKp characteristics and evolution. [ABSTRACT FROM AUTHOR]

Background: Hypervirulent Klebsiella pneumoniae (hvKP) is a virulent subtype of K. Pneumoniae. It commonly causes a serious community acquired infections, but it can also cause hospital acquired (nosocomial) infections. Emergence of antimicrobial resistance in hvKP ia a cause of concern. Objective: Phenotypic and molecular characterization and differentiation of classical and hypervirulent klebsiella pneumoniae isolated from Benha University Hospital. Subjects and Methods: Klebsiella pneumoniae was isolated by cultivation on Mac-Conkey agar media. Identified phenotypically and subjected to antibiotic sensitivity test. Conventional PCR was performed for detection of RmpA and RmpA2 virulence genes. Results: Eight strains out of 70 (11.4%) K.Pneumoniae were identified as probable hvKP by detection of RmpA2 that was detected in all hvKP while rmpA was detected in 2 out of 8 hvKP isolates. Both cKP and hvKP isolates exhibited high resistance rates for most of the tested antibiotics. Conclusion: Particularly in the presence of antibiotic resistance, HvKP pose a new hazard. Both Rmp A and Rmp A2 virulence genes were strongly associated with hvKP. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Klebsiella pneumoniae are distributed worldwide. This study aimed to characterize a hypervirulent tigecycline-resistant and carbapenem-resistant Klebsiella pneumoniae strain, XJ-K2, collected from a patient's blood. We tested antimicrobial susceptibility, virulence, and whole-genome sequencing (WGS) on strain XJ-K2. WGS data were used to identify virulence and resistance genes and to perform multilocus sequence typing (MLST) and phylogenetic analysis. Three novel plasmids, including a pLVPK-like virulence plasmid (pXJ-K2-p1) and two multiple resistance plasmids (pXJ-K2-KPC-2 and pXJ-K2-p3), were discovered in strain XJ-K2. The IncFII(pCRY) plasmid pXJ-K2-p3 carried the dfrA14, sul2, qnrS1, blaLAP-2, and tet(A) resistance genes. The IncFII(pHN7A8)/IncR plasmid pXJ-K2-KPC-2 also carried a range of resistance elements, containing rmtB, blaKPC-2, blaTEM-1, blaCTX-M-65, and fosA3. MLST analysis revealed that strain XJ-K2 belonged to sequence type 11 (ST11). Seven complete phage sequences and many virulence genes were found in strain XJ-K2. Meanwhile, antimicrobial susceptibility tests and G. mellonella larval infection models confirmed the extensively drug resistance (XDR) and hypervirulence of KJ-K2. To our knowledge, this is the first observation and description of the ST11 hypervirulent tigecycline- and carbapenem-resistant K. pneumoniae strain co-carrying blaKPC-2 and the tet(A) in a patient's blood in China. Further investigation is needed to understand the resistance and virulence mechanisms of this significant hypervirulent tigecycline- and carbapenem-resistant strain. [ABSTRACT FROM AUTHOR]

The pks genotoxic K. pneumoniae has recently triggered a widespread alarm. DNA damage and higher virulence have been linked to colibactin, a genotoxin expressed by the pks genomic island. Little is known about its molecular epidemiology in clinical isolates from Egypt. Therefore, this study was conducted to determine the prevalence and the microbiological and clinical features of pks harboring hospital-acquired K. pneumoniae isolates from Egypt. Eighty-seven hospital-acquired K. pneumoniae isolates from various specimen types were screened for pks colibactin island markers clbB, clbQ, clbA, and clbN by PCR. The pks-positive hvKp isolates were classified to one of the capsular types K1 and K2 using multiplex-PCR targeting K-serotype wzi and rmpA genes. The prevalence of pks+ strains was 27.6% (24/87). K1 capsular type, phenotypic, and genotypic hypervirulent isolates were significantly higher among pks+ strains than pks− strains (P < 0.001), while pks+ K. pneumoniae strains were found to be significantly less resistant to 8 of the antibiotic compounds tested than pks− strains. Carriage of K1 capsular type and mucoviscosity-associated rmp A gene and diabetes mellitus were identified to remain independent risk factors having a substantial association to pks-positivity by multivariate regression analysis. In conclusion, Hospital-acquired K. pneumoniae isolates in Egypt had an increased prevalence of the pks colibactin genotoxin. The significant occurrence of hypervirulent determinants in pks+ K. pneumoniae highlighted the genotoxin's possible pathogenicity combined with its distribution in several specimen types, which necessitates clinical attention and epidemic tracking.

The combination of carbapenem resistance and hypervirulence in Klebsiella pneumoniae is an emerging and urgent threat due to its potential to resist common antibiotics and cause life-threatening infections in healthy hosts. This study aimed to evaluate the activity of clinically relevant antibiotic regimens against carbapenem-resistant K. pneumoniae with hypervirulence plasmids and to identify pathways associated with antibiotic tolerance using transcriptomics. We studied two carbapenem-resistant K. pneumoniae isolates, CDI694 and CDI231, both harboring hypervirulence plasmids. Time-kill and dynamic one-compartment pharmacokinetic/pharmacodynamic assays were used to assess ceftazidime/avibactam-based therapies. RNAseq was performed following 48 h of antibiotic exposure. Closed genomes of CDI694 and CDI231 were obtained; each isolate harbored carbapenem-resistance and hypervirulence (containing rmpA/rmpA2 and iut genes) plasmids. Ceftazidime/avibactam-based regimens were bactericidal, though both isolates continued to grow in the presence of antibiotics despite no shifts in MIC. Transcriptomic analyses suggested that perturbations to cell respiration, carbohydrate transport, and stress-response pathways contributed to the antibiotic tolerance in CDI231. Genes associated with hypervirulence and antibiotic resistance were not strongly impacted by drug exposure except for ompW, which was significantly downregulated. Treatment of carbapenem-resistant K. pneumoniae harboring hypervirulence plasmids with ceftazidime/avibactam-based regimens may yield a tolerant population due to altered transcription of multiple key pathways.

The rise in infections caused by the hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is an emergent threat to public health. We assessed the effects of chlorogenic acid (CA), a natural phenolic compound, on antibacterial, antivirulence, and anti-quorum sensing (QS) of hv-CRKP. Five hv-CRKP were selected for antimicrobial susceptibility test and confirmed to carry virulence genes and carbapenem-resistant genes by polymerase chain reaction (PCR). Subsequently, a series of time-kill assay, determinations of protease activity and capsule content, biofilm-related experiment, scanning electron microscopy (SEM) and transmission electron microscope (TEM) observation, G. mellonella infection model, quantitative real-time PCR (qRT-PCR) of QS-related genes and biofilm formation genes, as well as AI-2 binding test were conduct to verify the effect of CA on hv-CRKP. Five CRKP strains showed varying degrees of resistance to antibacterial agents. All strains carried the blaKPC–2 gene, primarily carrying rmpA2, iucA, and peg-344. CA showed no effect on CRKP growth at the 1/2 minimum inhibitory concentration (MIC), 1/4 MIC, and 1/8 MIC, CA could reduce the production of extracellular protease and capsular polysaccharides, and improve the survival rate of larvae in Galleria mellonella (G. mellonella) infection model. By means of crystal violet staining and scanning electron microscopy experiments, we observed that CA can inhibit the formation of CRKP biofilm. On the quantitative real-time PCR analysis, the expression of the luxS, mrkA and wbbm genes in most CRKP strains appeared downregulated because of the CA treatment. Besides, CA significantly inhibited the effect of AI-2 activity of BB170. Our study suggests that CA can be an effective antimicrobial, antivirulent compound that can target QS in hv-CRKP infections, thus providing a new therapeutic direction for treating bacterial infections.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), a type of Klebsiella pneumoniae (KP) that exhibits hypervirulence and carbapenem resistance phenotypes, can cause severe infections, both hospital- and community-acquired infections. CR-hvKP has brought great challenges to global public health and is associated with significant morbidity and mortality. There are many mechanisms responsible for the evolution of the hypervirulence and carbapenem resistance phenotypes, such as the horizontal transfer of the plasmid carrying the carbapenem resistance gene to hypervirulent Klebsiella pneumoniae (hvKP) or carbapenemase-producing Klebsiella pneumoniae (CRKP) acquiring a hypervirulence plasmid carrying a virulence-encoding gene. Notably, KP can evolve into CR-hvKP by acquiring a hybrid plasmid carrying both the carbapenem resistance and hypervirulence genes. In this review, we summarize the evolutionary mechanisms of resistance and plasmid-borne virulence as well as the prevalence of CR-hvKP. [ABSTRACT FROM AUTHOR]

Extensively drug-resistant and hypervirulent Klebsiella pneumoniae (XDR-hvKp) is a new problem for patients in Intensive Care Unit (ICU) and can become an even more severe threat if resistant to tigecycline, considered one of the 'last lines of defense' drugs. This study collected seven non-replicated tigecycline-resistant XDR-hvKp from seven patients and performed genome analysis and epidemiological investigation using whole genome equencing (WGS) and other methods. All strains in this study were identified as ST11-KL64 and showed high resistance to antibiotics such as β-lactams, aminoglycosides, quinolones, and tigecycline, and one strain was also resistant to colistin. All strains were determined to be hvKp by the results of serum resistance assay and Galleria mellonella infection models. All strains had resistance genes blaCTX-M- 65,b/aKPC-2,b/aLAP-2,b/aTEM-1B, rmtB, and qnrSl and virulence factors such as rmpA, rmpA2, and aerobactin (iucABCD, iutA). The expression of the AcrAB- TolC efflux pump was upregulated in all strains, and the expression levels of the gene pmrK was significantly upregulated in colistin-resistant strain DP compared to colistin-sensitive strain WT in this study. In conclusion, we described an outbreak caused by tigecycline-resistant XDR-hvKp in the ICU of a teaching hospital in southwest China. The spread of these superbugs poses a great threat to patients and therefore requires us to closely monitor these XDR-hvKp and develop relevant strategies to combat them. [ABSTRACT FROM AUTHOR]

Objectives: Individuals infected with extensively drug-resistant (XDR) Acinetobacter baumannii are difficult to cure and have a high mortality rate. In this study, we compared the genomic and phenotypic differences between XDR and non-multidrug-resistant (MDR) A. baumannii and further characterized hypervirulent XDR A. baumannii., Methods: A total of 1,403 Acinetobacter isolates were collected from patients with bacteremia between 1997 and 2015. Antimicrobial susceptibility test was performed to categorize isolates into non-MDR, MDR, and XDR groups. The presence of selected virulence-associated genes was determined by PCR. Bacterial phenotypes, including iron acquisition, biofilm formation, capsule production, and virulence to larvae and mice, were determined., Results: Multilocus sequence types revealed the high prevalence of ST2 (81.6%) and ST129 (18.4%) among 49 XDR isolates and sequence types of 18 non-MDR isolates were more diverse. Virulence-associated phenotypic assays showed that XDR isolates had higher iron acquisition ability and capsule production and higher virulence to Galleria mellonella larvae. However, their biofilm formation ability was lower compared to that of non-MDR isolates. XDR isolates contained more virulence genes, tonB, hemO, abaI, and ptk, while non-MDR isolates contained more pld and ompA. Twenty-one XDR isolates caused <20% larvae survival rate after 7 days post-infection were defined as hypervirulent XDR isolates. Among them, isolates 1677 (ST129) and 929-1 (ST2) also caused the death of all infected mice within two days., Conclusion: Some subpopulations of highly drug-resistant ST2 also exhibit high virulence. Therefore, it is of utmost importance to continue monitoring the spread of hypervirulent XDR A. baumannii isolates., Competing Interests: Declaration of competing interest The authors report that there are no competing interests to declare., (Copyright © 2024. Published by Elsevier Ltd.)

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is a significant pathogen causing major public health problems worldwide. This study characterized a novel sequence type 6417 (ST6471) CR-hvKP strain recovered from the blood of a male patient with septicemia. Strain CR2021 is not susceptible to carbapenems, cephalosporin, sulfonamides, quinolones, or levofloxacin and is susceptible to amikacin and tigecycline. Molecular typing indicated that ST6417 is derived from the most dominant hypervirulent K. pneumoniae (hvKP) clone in China, ST23, with a single-locus variation in tonB . The genomic characterization of CR2021, which contains three plasmids, was performed through whole-genome sequencing. The plasmid pCR2021&#95;IncFII contains 12 antibiotic resistance genes [ bla CTX-M-3 , bla TEM-1B , bla DHA-1 , aac (3)-Ild , aadA16 , sul1 , sul2 , qnrB4 , ARR-3 , dfrA27 , qacE , merACDE ], all of which are associated with genetic elements. The plasmid pCR2021&#95;IncFIB carries crucial virulence-related genes, while the plasmid pCR2021&#95;IncX3 only harbors the bla NDM-5 resistance gene and exhibits 99% similarity with two other bla NDM-5 -carrying IncX3 plasmids (pSHX180-NDM5, pNDM-K725), with coverage of 87% and 100%, respectively. The bla NDM-5 genetic region contains an additional IS26-Tn3 genetic module. Serum killing and anti-human neutrophil phagocytosis tests indicated that CR2021 exhibits high virulence, which was further confirmed in a Galleria mellonella larvae infection model. CR-hvKP is becoming more prevalent in China; however, the majority have evolved from the multidrug resistance clone ST11 and its variants by acquiring virulence factors. Conversely, CR-hvKP derived from hvKP, such as the clone ST23, remains relatively rare. Therefore, the discovery of ST6417 underscores the need for further research into the genetic characteristics and evolution of bacteria., Importance: ST11 and its variants, which often exhibit drug resistance, represent popular clones of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) in China, often leading to high morbidity and mortality rates owing to their high virulence and robust drug resistance. Conversely, CR-hvKP, originating from the high-virulence sequence type ST23, remains rarely reported. In this study, we identified a novel ST6417 CR-hvKP strain derived from ST23, carrying bla NDM-5 on an IncX3 plasmid conferring resistance to carbapenems. In addition, we elucidate its virulence, resistance to drugs, and genetic characteristics. The discovery of ST6417 highlights the diverse pathways in the evolution of CR-hvKP, warranting increased attention., Competing Interests: The authors declare no conflict of interest.

Background: Quantitating the contribution of phenotype-responsible elements in hypervirulent Klebsiella pneumoniae is needed., Methods: Isogenic mutants of four hypervirulent clinical isolates that produced K1 (ST23), K2 (ST86), K20 (ST1544), or K54 (ST29) capsules (mean 2.2 log 10 LD 50 (range 1.5-2.9)) were created to measure the effects on LD 50 in a murine model of the hypervirulence-associated plasmid (pVir), iucA, p rmpA, p rmpA2 (truncated), irp2, and clbBC., Findings: Curing pVir had the greatest increase in survival (mean LD 50 to 7.6 (range 7.0-9.0, p ≤ 0.0001), a dosage comparable to classical K. pneumoniae. Results also showed increased mean LD 50 s for Δ p rmpA (5.9, p ≤ 0.0001), ΔiucA (3.6, p ≤ 0.0001), Δirp2 (3.4), ΔrmpAΔiucA (6.3, p ≤ 0.0001), and ΔpVirΔirp2 (8.7, p ≤ 0.0001). Notably ΔpVir had an additional mean LD 50 increase of 1.3 compared to the pVir-encoded Δ p rmpAΔiucA (p ≤ 0.01), suggesting presence of additional pVir-virulence genes. Truncated p RmpA2 did not contribute to virulence. Odd ratios in the absence of pVir/yersiniabactin, pVir, p RmpA/aerobactin, p RmpA, aerobactin, yersiniabactin, and colibactin demonstrated a 250-fold, 67-fold, 20-fold, 16.7-fold, 9.6-fold, and 1.7-fold decrease in lethality respectively., Interpretation: These data can guide countermeasure development., Funding: This work was supported by NIH R21 AI123558-01 and 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo). This study was also partially funded by the U.S. Defense Health Program (DHP) Operations and Maintenance., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Incidence of hypervirulent Klebsiella pneumoniae (hvKp) infection has been steadily increasing in the Asia-Pacific rim. The characteristic of hvKp infection is its ability to cause multiple site infections and unpredictable metastatic spread in the community. We describe the first case of mycotic aneurysm caused by hvKp serotype K1 in a previously healthy man and review the literature. Of a total of 13 cases, including our case, three cases were related to hvKp. Among patients with hvKp, the level of mycotic aneurysm in most patients was the infrarenal aorta, and they underwent an aortic graft or coil embolization. All strains were susceptible to most antimicrobial agents, except ampicillin. Early detection of hvKp can help to prevent the metastatic spread of pathogens and be useful for optimal patient care and epidemiologic research.

ABSTRACT CG23-I lineage constitutes the majority of hypervirulent Klebsiella pneumoniae. A diabetic patient suffered six episodes of infections caused by CG23-I K. pneumoniae. A total of nine isolates were collected in 2020. We performed whole-genome sequencing to elucidate the within-patient evolution of CG23-I K. pneumoniae. The maximum pairwise difference among the nine longitudinally collected isolates was five single nucleotide polymorphisms. One of the mutations was at the Asp87 position of GyrA. Four indels were identified, including an initiator tRNAfMet duplication, a tRNAArg deletion, a 7-bp insertion, and a 22-bp deletion. All 9 isolates had the genomic features of CG23-I K. pneumoniae, a chromosome-borne ICEKp10, and a large virulence plasmid. The carriage of a complete set of genes for the biosynthesis of colibactin by ICEKp10 gave the nine isolates an ability to cause DNA damage to RAW264.7 cells. Compared with the initial isolate, the last isolate with an additional copy of initiator tRNAfMet grew faster in a nutrient-limiting condition and exhibited enhanced virulence in BALB/c mice. Collectively, we characterized the within-patient microevolution of CG23-I K. pneumoniae through an in-depth comparison of genome sequences. Using the in vitro experiments and mouse models, we also demonstrated that these genomic alterations endowed the isolates with advantages to pass through in vivo selection. IMPORTANCE CG23-I is a significant lineage of hypervirulent Klebsiella pneumoniae. This study characterizes the within-patient microevolution of CG23-I K. pneumoniae. Selective pressures from continuous use of antibiotics favored point mutations contributing to bacterial resistance to antibiotics. The duplication of an initiator tRNAfMet gene helped CG23-I K. pneumoniae proliferate to reach a maximal population size during infections. For longer persistence inside a human host, the large virulence plasmid evolved with more flexible control of replication through duplication of the iteron-1 region. With the genomic alterations, the last isolate had a growth advantage over the initial isolate and exhibited enhanced virulence in BALB/c mice. This study gives us a deeper understanding of the genome evolution during the within-patient pathoadaptation of CG23-I K. pneumoniae.

Infections with hypervirulent Klebsiella pneumoniae (hvKp) commonly presents with primary liver infection, bacteremia, and metastatic abscesses. Here, we present 2 cases of severe community-acquired pulmonary infections by hvKp in patients in the Netherlands without recent travel history. Both bacterial isolates are closely related to an archetype ST23 hvKp reference isolate. Based on these findings, surveillance programs on hvKp may consider to include isolates from community-acquired pneumonia by K. pneumoniae. [ABSTRACT FROM AUTHOR]

Reports on multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKP) in recent years indicate the wide-spreading trend of MDR-hvKP. The co-occurrence of hypervirulence and drug resistance poses a great challenge to clinical treatment. In this study, molecular characteristics of an MDR strain hvKP247 and 30 clinically isolated hvKP strains were characterized. The whole genome of hvKP247 belonging to sequence type (ST) 5214 and capsule serotype K1 was sequenced and analyzed. Conjugation experiments were performed to evaluate transferability of the plasmids in hvKP247. We found two new STs among our isolates, ST5570 and ST5571. The ST5214 hvKP247 contained two transferable plasmids: a hybrid virulence plasmid (pHvKP247-vir) carrying transfer-related modules that had self-transferable ability, and a drug-resistant plasmid (pHvKP247-MDR) that could be indirectly transferred with the help of pHvKP247-vir. The virulence-related genes were located on the pHvKP247-vir and chromosomal ICEKp1 mobile genetic element. In conclusion, the hybrid virulence plasmid and the drug-resistant plasmid are co-transferred, which emphasizes the importance of raising public awareness of the simultaneous spread of virulence and resistance genes of MDR-hvKP strains. [ABSTRACT FROM AUTHOR]

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Klebsiella pneumoniae has been the predominant pathogen of liver abscess, but ST11-K47 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has rarely been studied as the causative organism. We identified an ST11-K47 CR-hvKP (HvKp-su1) from the drainage fluid of a liver abscess in a Chinese man who was diagnosed with liver abscess combined with diabetes, pneumonia, pleural infection, abdominal abscess, and splenic abscess. HvKp-su1 was non-hypermucoviscous and lacked the magA and rmpA genes and pLVPK plasmid but exhibited high virulence, with a high mortality rate (90%) to wax moth larvae (G. mellonella), similar to the hypervirulent Klebsiella pneumoniae ATCC43816 (91.67%). Whole-genome sequencing and bioinformatics analysis indicated that HvKp-su1 possesses a plasmid similar to a type of pLVPK-like plasmid (JX-CR-hvKP-2-P2), which is an uncommon plasmid in CR-hvKP. HvKp-su1 carried multiple resistance genes, including blaKPC-2. blaTEM-1, blaSHV-55, and blaCTX-M-65; hypervirulence genes such as aerobactin (iutA), salmochelin (iroEN), and yersiniabactin (ybtAEPQSTUX); and the type 3 fimbriae-encoding system (mrkACDF). Moreover, v_5377 and v_5429 (cofT, CFA/III (CS8)) located on plasmid 1 were simultaneously predicted to be virulence genes. After the long-term combination use of antibiotics, the patient successfully recovered. In summary, our study clarified the clinical and molecular characteristics of a rare ST11-K47 CR-hvKP (HvKp-su1), raising great concerns about the emergence of ST11-K47 CR-hvKP with multidrug resistance and hypervirulence, and providing insights into the control and treatment of liver abscess caused by ST11-K47 CR-hvKP. [ABSTRACT FROM AUTHOR]

Cong Zhou,1 Qiang Wu,1 Leqi He,1 Hui Zhang,1 Maosuo Xu,1 Baoyu Yuan,2 Zhi Jin,3 Fang Shen1 1Department of Clinical Laboratory Medicine, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, People’s Republic of China; 2Department of Clinical Laboratory, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; 3Department of Neurology, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, People’s Republic of ChinaCorrespondence: Fang ShenDepartment of Clinical Laboratory Medicine, Shanghai Fifth People’s Hospital, Fudan University, No. 128, Ruili Road, Minhang District, Shanghai, 200240, People’s Republic of ChinaTel +86 18021073261Email shenfang5th@aliyun.comBackground: The convergence of carbapenem-resistance and hypervirulence in Klebsiella pneumoniae has led to a significant public health challenge. In recent years, there have been more and more reports on carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) isolates.Materials and Methods: Clinical data of patients infected with CR-hvKP from January 2019 to December 2020 in a tertiary hospital were retrospectively evaluated. The number of isolates of Klebsiella pneumoniae, hypermucoviscous Klebsiella pneumoniae (hmKP), carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-hmKP) and carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) collected during the period of 2 years was calculated. The antimicrobial resistance gene, virulence-associated gene, capsular serotype gene and multilocus sequence typing (MLST) of CR-hvKP isolates were detected by PCR.Results: During the study period, a total of 1081 isolates of non-repeat Klebsiella pneumoniae were isolated, including 392 isolates of hypermucoviscous Klebsiella pneumoniae (36.3%), 39 isolates of CR-hmKP (3.6%), and 16 isolates of CR-hvKP (1.5%). About 31.2% (5/16) of CR-hvKP were isolated from 2019, and 68.8% (11/16) of CR-hvKP were isolated from 2020. Among the 16 isolates of CR-hvKP, 13 isolates were ST11 and serotype K64, 1 isolate was ST11 and serotype K47, 1 isolate was ST23 and serotype K1, and 1 isolate was ST86 and serotype K2. The virulence-associated genes entB, fimH, rmpA2, iutA, iucA were present in all of 16 CR-hvKP isolates, followed by mrkD (n=14), rmpA (n=13), aerobactin (n=2), allS (n=1). Sixteen CR-hvKP isolates all carry carbapenemase gene blaKPC-2 and extended-spectrum β-lactamase gene blaSHV. ERIC-PCR DNA fingerprinting results showed that 16 CR-hvKP isolates were highly polymorphic, and there were significant differences in bands among the isolates, presenting a sporadic state.Conclusion: Although CR-hvKP was sporadically distributed, it showed an increasing trend year by year. Therefore, clinical attention should be paid, and necessary measures should be taken to avoid the cloning and transmission of superbacterium CR-hvKP.Keywords: Klebsiella pneumoniae, carbapenem-resistant, hypervirulent, hypermucoviscous, epidemiology

ABSTRACT Importance Hypervirulent variants of Klebsiella pneumoniae (hvKp) are capable of causing life‐threatening pyogenic liver abscesses (PLAs), but hvKp caused PLAs was seldom reported in pediatric populations. Hence, there is an urgent need to raise our awareness of this phenomenon in pediatric populations. Objective This study aimed to report the clinical characteristics of hvKp that caused fatal PLA complicated by bacteremia in an adolescent and further identify the microbiological and genomic features of the causative strain. Methods A 14‐year‐old boy with diabetes mellitus was admitted to our hospital with a diagnosis of PLA complicated by bacteremia. A hypermucoviscous hvKp strain, KPN_19‐106, was isolated from the drainage fluid present within the liver abscess cavity and blood. The hypermucoviscosity phenotype of the causative strain was determined by string test. Its virulence was measured using serum resistance assay and Galleria mellonella larvae‐killing assay. Antimicrobial susceptibility was determined by broth microdilution method. Genetic information was obtained by whole‐genome sequencing and bioinformatics analysis. Results KPN_19‐106 belonged to sequence type 380 and serotype K2 and exhibited stronger serum resistance and higher in vivo lethality than the well‐characterized hvKp NTUH‐K2044 strain. Although KPN_19‐106 is susceptible to most antibiotics, no sign of improvement was observed during treatment with such drugs. Whole‐genome sequencing revealed that the isolate had integrated multiple mobile genetic elements related to virulence. Interpretation Antibiotic‐susceptible hvKp can cause fatal PLA complicated by bacteremia in adolescents, with no improvement during antimicrobial therapy. The causative strain in this case had integrated multiple virulence genes and thus exhibited higher virulence both in vitro and in vivo when compared with NTUH‐K2044.

Abstract Background This case report describes a neck abscess caused by a strain of Hypervirulent Klebsiella pneumoniae in a middle aged man with diabetes without a history of travel to East and South East Asia. This case report is of notable significance as Hypervirulent Klebsiella pneumoniae neck abscesses are rarely seen in the UK and are very infrequently documented in individuals who have not first travelled to the high prevalence areas of East and South East Asia. Case presentation This case report describes a 53 year old diabetic man who contracted a Hypervirulent Klebsiella pneumoniae neck abscess which led to the development of sepsis. Klebsiella pneumoniae was cultured from blood cultures and fluid aspirated from the abscess grew the pathogen with same antimicrobial susceptibility. Hypervirulence was demonstrated after the samples were analysed, at the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit Public Health England Colindale, and found to contain the K20 (rmp)A and rmpA2 virulence genes. Discussion Hypervirulent Klebsiella pneumoniae is a Gram-negative, encapsulated, non-motile bacillus notable for its ability to metastatically spread and cause potentially life threatening infections in otherwise healthy adults, but especially in those with diabetes. Genes responsible for the production of hyperviscous mucoid polysaccharide capsules and siderophores, such as those isolated in this case, enable the bacteria to more efficiently evade the hosts immune system and disseminate and invade surrounding and distant tissues. Data from Public Health England shows Hypervirulent Klebsiella pneumoniae are rare in the UK. A review of current literature also showed Hypervirulent Klebsiella pneumoniae almost exclusively occur in those who have traveled to East and South East Asia. Conclusions This case reported a rare Hypervirulent Klebsiella pneumoniae neck abscess outside of, and without travel to, East and South East Asia. This raises concerns about future, potentially life threatening, Hypervirulent Klebsiella pneumoniae infections becoming more widespread without the need for endemic travel. This concern is further exacerbated by the growing global challenge of antimicrobial resistance.

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.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including blaSHV−67, blaSHV−11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

ABSTRACT Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that has become a leading causative agent of nosocomial infections, mainly infecting patients with immunosuppressive diseases. Capsular (K) serotypes K1, K2, K47, and K64 are commonly associated with higher virulence (hypervirulent Klebsiella pneumoniae), and more threateningly, isolates belonging to the last two K serotypes are also frequently associated with resistance to carbapenem (hypervirulent carbapenem-resistant Klebsiella pneumoniae). The prevalence of these isolates has posed significant threats to human health, and there are no appropriate therapies available against them. Therefore, in this study, a method combining immunoinformatics and pangenome analysis was applied for contriving a multiepitope subunit vaccine against these four threatening serotypes. To obtain cross-protection, 12 predicted conserved antigens were screened from the core genome of 274 complete Klebsiella pneumoniae genomes (KL1, KL2, KL47, and KL64), from which the epitopes of T and B cells were extracted for vaccine construction. In addition, the immunological properties, the interaction with Toll-like receptors, and the stability in a simulative humoral environment were evaluated by immunoinformatics methods, molecular docking, and molecular dynamics simulation. All of these evaluations indicated the potency of this constructed vaccine to be an effective therapeutic agent. Lastly, the cDNA of the designed vaccine was optimized and ligated to pET-28a(+) for expression vector construction. Overall, our research provides a newly cross-protective control strategy against these troublesome pathogens and paves the way for the development of a safe and effective vaccine. IMPORTANCE Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that has become a leading causative agent of nosocomial infections. Among the numerous capsular serotypes, K1, K2, K47, and K64 are commonly associated with higher virulence (hypervirulent K. pneumoniae). More threateningly, the last two serotypes are frequently associated with resistance to carbapenem (hypervirulent carbapenem-resistant K. pneumoniae). However, there is currently no therapeutic agent or vaccine specifically against these isolates. Therefore, development of a vaccine against these pathogens is very essential. In this study, for the first time, a method combining pangenome analysis, reverse vaccinology, and immunoinformatics was applied for contriving a multiepitope subunit vaccine against K. pneumoniae isolates of K1, K2, K47, and K64. Also, the immunological properties of the constructed vaccine were evaluated and its high potency was revealed. Overall, our research will pave the way for the vaccine development against these four threatening capsular serotypes of K. pneumoniae.

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has been increasingly reported worldwide. Here, we report an outbreak caused by sequence type 859-K19 (ST859-K19) CR-hvKP isolates in a teaching hospital in China. Interestingly, K. pneumoniae ST859 was a single-locus variant of ST11 but has never been reported before. A total of 11 nonrepetitive ST859 CR-hvKP isolates were collected from 11 patients, 3 of which died of severe CR-hvKP infection. Antimicrobial susceptibility assay results showed that all the 11 CR-hvKP isolates exhibited high-level resistance to commonly used antibiotics, only remaining susceptible to colistin, tigecycline, and ceftazidime/avibactam. Whole-genome sequencing (WGS) was performed using the Illumina platform for the 11 CR-hvKP isolates, and RJ9299 was further sequenced using the PacBio platform. A phylogram tree using WGS data revealed that all the 11 CR-hvKP isolates were clustered in 1 clade, which probably indicated clone transmission. Determinants of resistance and virulence gene analysis using WGS data confirmed the 11 isolates had almost identical resistance gene profiles (blaKPC-2, blaTEM-1B, blaSHV-187, rmtB, fosA6) and virulence gene (rmpA, rmpA2, iucABCDiutA) profiles, which hint at clone spread again. The complete genome size of RJ9299 was 5,875 kb, including a 5,445-kb chromosome, a 215-kb virulence plasmid (pVir-CR-hvKP-RJ9299), a 109-kb blaKPC-2-harboring plasmid (pKPC-2-RJ9299), and three circular plasmids. Comparative genomics showed pVir-RJ9299 (IncHI1B type) and pKPC-2-RJ9299 (IncFII-IncR) possessed over 99% similarity to pLVPK and pKPC-CR-hvKP-C789, respectively. Serum resistance assays and a Galleria mellonella infection model showed the 11 isolates exhibited different levels of virulence. This is the first report of an outbreak caused by ST859 CR-hvKP isolates. IMPORTANCE The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) in China has posed a great threat to public health, especially in the highly transmissible ST11 clone. With the transmission of virulence and resistance determinants, CR-hvKP isolates have been reported in an increasing number of sequence types (STs), including ST23, ST65, ST1797, ST43, ST231, ST147, ST15, ST383, ST268, ST595, ST375, ST48, and ST307. Here, we report an outbreak caused by ST859-K19 CR-hvKP isolates in a teaching hospital in China. ST859 is a single-locus variant of ST11. There is no literature on ST859 K. pneumoniae in public databases, let alone ST859 CR-hvKP isolates. To our knowledge, this is the first report to depict the molecular and genomic characteristics of ST859 CR-hvKP isolates. Active surveillance approaches should be implemented to promptly find the spread of CR-hvKP isolates in health care settings.

[Display omitted] • A simple and user-friendly diagnostics to detect the hypervirulent K. pneumoniae. • CRISPR-assisted point-of-need tool that stands worthy of the ASSURED criteria. • Species-specific detection of solely the test-strains of K. pneumoniae. • No cross-reactivity observed when tested on different genera besides the test strains. • Instrument-free readout to extend the reach of the technology to remote locations. Klebsiella pneumoniae , a prevalent healthcare-associated pathogen, poses a significant challenge to diagnosis and treatment due to its virulence and antimicrobial resistance. The demand for more efficient, precise and accessible diagnostic methods is imperative, as current approaches are labor-intensive and resource-dependent. In this study, a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based diagnostic tool for rapid detection of hypervirulent K. pneumoniae infections was proposed. We integrated recombinase polymerase amplification (RPA) coupled with a lateral flow assay and Cas13a (CRISPR associated protein 13a) to target the housekeeping rpoB gene for species-specific detection and the capsular polysaccharide regulating gene rmpA for identification of hypervirulent strains of K. pneumoniae. Tested on 18 K. pneumoniae strains, the devised tool successfully detected hypervirulent strains K. pneumoniae M59 and K. pneumoniae KP109 showing presence of rmpA. This study allows to develop an instrument-free platform for routine diagnosis of K. pneumoniae from serum, urine, and saliva samples that would empower healthcare personnel to facilitate proper and timely treatment of infections caused by the K. pneumoniae. [ABSTRACT FROM AUTHOR]

A. baumannii is an important and common clinical pathogen, especially in the intensive care unit (ICU). This study aimed to characterize one hypervirulent A. baumannii strain in a patient with community-acquired pneumonia and herpes simplex type 1 virus infection. Minimum inhibitory concentrations (MICs) were determined using the Kirby-Bauer (K-B) and broth microdilution methods. Galleria mellonella infection model experiment was conducted. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The resistance and virulence determinants were identified using the ABRicate program with ResFinder and the VFDB database. The capsular polysaccharide locus (K locus) and lipooligosaccharide outer core locus (OC locus) were identified using Kleborate with Kaptive. Phylogenetic analyses were conducted using the BacWGSTdb server. A. baumannii XH2146 strain belongs to ST10 Pas and ST447 Oxf. The strain was resistant to cefazolin, ciprofloxacin, and trimethoprim/sulfamethoxazole (TMP-SMX). Bautype and Kaptive analyses showed that XH2146 contains OCL2 and KL49. WGS analysis revealed that the strain harbored bla ADC-76 , bla OXA-68 , ant(3′')-IIa , tet (B), and sul2. Notably, tet (B) and sul2 , both were located within a 114,700-bp plasmid (designated pXH2146–1). Virulence assay revealed A. baumannii XH2146 possessed higher virulence than A. baumannii AB5075 at 12 h. Comparative genomic analysis showed that A. baumannii ST447 strains were mainly isolated from the USA and exhibited a relatively close genetic relationship. Importantly, 11 strains were observed to carry bla OXA-58 ; bla OXA-23 was identified in 11 isolates and three ST447 A. baumannii strains harbored bla NDM-1. Early detection of community-acquired hypervirulent Acinetobacter baumannii strains is recommended to prevent their extensive spread in hospitals. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKp) is a new emerging variant of K. pneumoniae that is increasingly reported worldwide. The variant hvKp is known to cause severe invasive community-acquired infections such as metastatic meningitis, pyogenic liver abscesses (PLA) and endophthalmitis, but its role in hospital-acquired infections (HAIs) is little known. The aim of this study was to evaluate the prevalence of hvKp among hospital-acquired (HA) K. pneumoniae infections in the intensive care unit (ICU) and to compare between hvKp and classical K. pneumoniae (cKP) regarding antimicrobial resistance pattern, virulence and molecular characteristics. The study was cross-sectional and included 120 ICU patients suffering from HA K. pneumoniae infections between January and September 2022. K. pneumoniae isolates were subjected to antimicrobial susceptibility testing and detection of extended-spectrum-β-lactamase (ESBL) production by the Phoenix 100 automated microbiology system, string test, biofilm formation, serum resistance assay, and detection of virulence-associated genes (rmpA, rmpA2, magA, iucA) and capsular serotype-specific genes (K1, K2, K5, K20, K57) by polymerase chain reaction (PCR). Of 120 K. pneumoniae isolates, 19 (15.8%) were hvKp. The hypermucoviscous phenotype was more significantly detected in the hvKp group than in the cKP group (100% vs. 7.9%, p ≤ 0.001). The rate of resistance to different antimicrobial agents was significantly higher in the cKP group than that in the hvKp group. Fifty-three strains were identified as ESBL-producing strains, which was more frequent in the cKP group than in the hvKp group (48/101 [47.5%] vs. 5/19 [26.3%], respectively, p ≤ 0.001). The hvKP isolates were highly associated with moderate and strong biofilm formation than cKP isolates (p = 0.018 and p = 0.043 respectively). Moreover, the hvKP isolates were highly associated with intermediate sensitivity and re sistance to serum in the serum resistance assay (p = 0.043 and p = 0.016 respectively). K1, K2, rmpA, rmpA2, magA and iucA genes were significantly associated with hvKp (p ≤ 0.001, 0.004, K5, K20 and K57 were not associated with hvKp. The hvKp strains have emerged as a new threat to ICU patients because of their ability to cause more severe and life-threatening infections than cKP. The string test alone as a laboratory test for screening of hvKp has become insufficient. Recently, hvKp was defined as hypermucoviscous- and aerobactin-positive. It is important to improve the awareness towards the diagnosis and management of hvKp infections.

Hypervirulent Klebsiella pneumoniae (hvKp) is emerging worldwide. Hypermucoviscousity is the characteristic trait that distinguishes it from classic K. pneumoniae (cKp), which enables Kp to cause severe invasive infections. This research aimed to investigate the hypermucoviscous Kp (hmvKp) phenotype among gut commensal Kp isolated from healthy individuals and attempted to characterize the genes encoding virulence factors that may regulate the hypermucoviscosity trait. Using the string test, 50 identified Kp isolates from healthy individuals’ stool samples were examined for hypermucoviscosity and investigated by transmission electron microscopy (TEM). Antimicrobial susceptibility profiles of Kp isolates were determined using the Kirby Bauer disc method. Kp isolates were tested for genes encoding different virulence factors by PCR. Biofilm formation was assayed by the microtiter plate method. All Kp isolates were multidrug-resistant (MDR). Phenotypically, 42% of isolates were hmvKp. PCR-based genotypic testing revealed the hmvKp isolates belonged to capsular serotype K2. All study Kp isolates harbored more than one virulence gene. The genes magA and rmpA were not detected, while the terW gene was present in all isolates. The siderophores encoding genes entB and irp2 were most prevalent in hmvKp isolates (90.5%) and non-hmvKp (96.6%), respectively. hmvKp isolates harbored the genes wabG and uge with rates of 90.5% and 85.7%, respectively. The outcomes of this research highlight the potential health risk of commensal Kp to cause severe invasive diseases, owing to being hmvKp and MDR, and harboring multiple virulence genes. The absence of essential genes related to hypermucoviscosity such as magA and rmpA in hmvKp phenotypes suggests the multifactorial complexity of the hypermucoviscosity or hypervirulence traits. Thus, further studies are warranted to verify the hypermucoviscosity-related virulence factors among pathogenic and commensal Kp in different colonization niches.

Worldwide, hypervirulent Klebsiella pneumoniae (hvKp) is one of the leading causes of multisystem infection. Serotype K54 has also been considered as one of the hvKp-associated capsular types that are rarely reported. In this study, we reported a K54-ST29 hvKp isolated from a 58-year-old male patient with diabetes in a teaching hospital in China. The patient rapidly developed sepsis and brain abscess, with a lethal multiple-organ-system failure due to K54 hvKp infection. This K54 hvKp isolate showed high level of toxicity in a mouse infection model and was susceptible to all the tested antibiotics. The isolate was fully sequenced, and its genome was compared with the available K54 K. pneumoniae genome. We predicted 133 virulence and pathogen-related genes, including those involved in fimbriae synthesis, iron transport, and enterobactin synthesis. Sequence alignment revealed >90% similarity among seven K54 K. pneumoniae strains. Our data suggest that community-acquired infection caused by hypervirulent K54 K. pneumoniae in patients with diabetes is a concern in East Asia. [ABSTRACT FROM AUTHOR]

Emergence of bla NDM–1 and bla KPC–2 co-producing Klebsiella pneumoniae strains is currently attracting widespread attention, but little information is available about their tigecycline resistance, virulence, and prevalence in Southwest China. In July 2021, an extensively drug-resistant K. pneumoniae strain AHSWKP25 whose genome contained both bla NDM–1 and bla KPC–2 genes was isolated from the blood of a patient with the malignant hematological disease in Luzhou, China. We investigated the resistance profiles of AHSWKP25 using microbroth dilution, agar dilution, modified carbapenemase inactivation (mCIM), and EDTA-modified carbapenemase inactivation methods (eCIM). The virulence of AHSWKP25 was assessed through string tests, serum killing assays, and a Galleria mellonella larval infection model. Conjugation and plasmid stability experiments were conducted to determine the horizontal transfer capacity of plasmids. And efflux pump phenotype test and real-time quantitative reverse transcription-PCR (RT-PCR) were used to determine its efflux pump activity. Sequencing of AHSWKP25 determined that AHSWKP25 belonged to ST464, which is resistant to antibiotics such as carbapenems, tetracycline, fluoroquinolones, tigecycline, and fosfomycin. The efflux pump phenotype tests and RT-PCR results demonstrated that efflux pumps were overexpressed in the AHSWKP25, which promoted the tigecycline resistance of the bacteria. AHSWKP25 also showed hypervirulence and serum resistance in vitro model. AHSWKP25 carried several different plasmids that contained bla NDM–1, bla KPC–2, and mutated tet(A) genes. Sequence alignment revealed that the plasmids carrying bla NDM–1 and bla KPC–2 underwent recombination and insertion events, respectively. We demonstrated that an X3 plasmid carrying bla NDM–1 was transferred from pSW25NDM1 to E. coli J53. We also identified missense mutations in the ramR , rcsA , lon , and csrD genes of AHSWKP25. Our results highlighted the potential of bla NDM–1 and bla KPC–2 co-producing K. pneumoniae strains to further develop antimicrobial resistance and hypervirulent phenotypes, but measures should be taken to closely monitor and control the spread of superbugs with multidrug-resistant phenotypes and hypervirulence. [ABSTRACT FROM AUTHOR]

Background: Hypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes. Methods: Nine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment. Results: Among the nine isolates, seven were carbapenem-resistant, two of which carried bla NDM-5 on an IncFII plasmid and five carried bla OXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2 , iutA , and iucABCD) carried on a large (~320 kbp) IncFIB–IncHI1B co-integrate. This hybrid plasmid also carried the aadA2 , armA , bla OXA-1, msrE , mphE , sul1 , and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition. Conclusion: The convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKP) is an evolving infectious pathogen associated with high mortality. The convergence of hypervirulence and multidrug resistance further challenges the clinical treatment options for K. pneumoniae infections. The QseBC two-component system (TCS) is a component of quorum-sensing regulatory cascade and functions as a global regulator of biofilm growth, bacterial motility, and virulence in Escherichia coli. However, the functional mechanisms of QseBC in hvKP have not been reported, and we aim to examine the role of QseBC in regulating virulence in hvKP strain ATCC43816. The CRISPR-Cas9 system was used to construct qseB , qseC , and qseBC knockout in ATCC43816. No significant alterations in the growth and antibiotic susceptibility were detected between wild-type and mutants. The deletion of qseC led to an increase of biofilm formation, resistance to serum killing, and high mortality in the G. mellonella model. RNAseq differential gene expression analysis exhibited that gene-associated biofilm formation (glgC, glgP, glgA, gcvA, bcsA, ydaM, paaF, ptsG), bacterial type VI secretion system (virB4, virB6, virB10, vgrG, hcp), and biosynthesis of siderophore (entC, entD, entE) were significantly upregulated in comparison with the wild-type control. In addition, qseB , ygiW (encode OB-family protein), and AraC family transcriptional regulator IT767_23090 genes showed highest expressions in the absence of QseC, which might be related to increased virulence. The study provided new insights into the functional importance of QseBC in regulating the virulence of hvKP. [ABSTRACT FROM AUTHOR]

Introduction: Infections caused by multidrug-resistant (MDR) hypervirulent Klebsiella pneumoniae are difficult to treat and associated with high mortality rates. Hence, this study was conducted to determine the antibiotic resistance pattern along with the distribution of virulence genes among isolated string test positive and negative strains. Materials and Methods: A total of 44 K. pneumoniae strains were isolated following standard microbiological methods from 350 different clinical samples from patients admitted to Dhaka Medical College Hospital, Bangladesh. String test was done to detect the hypermucoid phenotype. Antimicrobial resistance (AMR) pattern was determined by dichlorodiphenyltrichloroethane (except colistin and fosfomycin) among all isolates. Polymerase chain reaction was done to detect the hypervirulence genes (magA, rmpA, rmpA2 iutA, iroN). Results: In this study, 21/44 (47.73%) of the isolated K. pneumoniae were string test positive and distribution of the virulence genes except rmpA2 was higher among them. A total of 15/44 (34.09%) of the isolated K. pneumoniae were MDR, 10/44 (22.73%) were extensively drug resistant, 1/44 (2.27%) was pan drug resistant, and 14/44 (31.82%) were colistin resistant. Isolated organisms were highly resistant to third-generation cephalosporins and most sensitive to fosfomycin in this study. Although all the string test positive strains showed higher resistance rates than the string test negative ones toward most of the tested antibiotics, only the differences of resistance rates to amoxiclav and tigecycline among the two phenotypes were statistically significant. Conclusion: Our findings highlight the importance of surveillance of the AMR pattern of hypervirulent K. pneumoniae in clinical samples. Therefore, a response to check the global dissemination of this hypervirulent K. pneumoniae with resistance determinants is urgently needed. [ABSTRACT FROM AUTHOR]

Objectives: A single carbapenem-resistant, hypervirulent Klebsiella pneumoniae strain has attracted major public concern. The aim of the present study was to better understand the antimicrobial resistance and genetic characteristics of Klebsiella pneumoniae strain XJ-K1. Methods: Klebsiella pneumoniae strain XJ-K1 was isolated from a urine specimen of a 69-year-old male patient in a teaching hospital in Shanghai, China, in January 2018. Antimicrobial susceptibility testing, string test, whole-genome sequencing, bioinformatics analysis and phylogenetic analysis were performed in this study. Results: Klebsiella pneumoniae XJ-K1 was an extensively drug-resistant (XDR) hypervirulent strain that showed high-level resistance to antibacterial agents. Three novel plasmids were discovered in strain XJ-K1, including a 207,409-bp IncHI1B-type rmpA2-bearing pLVPK-like virulence plasmid, a 130,628-bp Col156/IncFIB/IncFII-type aadA2-, sul1-, mph(A)- and dfrA12-bearing MDR plasmid, and a 99,408-bp IncFII/IncR-type blaKPC-2-, blaTEM-1-, blaCTX-M-65-, blaSHV-12-, rmtB- and fosA3-bearing MDR plasmid. Sequence analysis of the chromosome revealed that the aadA2, fosA and sul1 genes were harboured by XJ-K1. Multilocus sequence typing (MSLT) showed that XJ-K1 was ST11. Conclusions: A large number of resistance genes and a pLVPK-like virulence plasmid carried by Klebsiella pneumoniae strain XJ-K1 might be the main reasons leading to the XDR and hypervirulent phenotype. To the best of our knowledge, this is the first report in China on the co-occurrence of a pLVPK-like virulence plasmid and two MDR plasmids in a single ST11 XDR and hypervirulent Klebsiella pneumoniae isolated from patient urine, which is a serious concern for its further spread.

Worldwide, hypervirulent Klebsiella pneumoniae (hvKp) is one of the leading causes of multisystem infection. Serotype K54 has also been considered as one of the hvKp-associated capsular types that are rarely reported. In this study, we reported a K54-ST29 hvKp isolated from a 58-year-old male patient with diabetes in a teaching hospital in China. The patient rapidly developed sepsis and brain abscess, with a lethal multiple-organ-system failure due to K54 hvKp infection. This K54 hvKp isolate showed high level of toxicity in a mouse infection model and was susceptible to all the tested antibiotics. The isolate was fully sequenced, and its genome was compared with the available K54 K. pneumoniae genome. We predicted 133 virulence and pathogen-related genes, including those involved in fimbriae synthesis, iron transport, and enterobactin synthesis. Sequence alignment revealed >90% similarity among seven K54 K. pneumoniae strains. Our data suggest that community-acquired infection caused by hypervirulent K54 K. pneumoniae in patients with diabetes is a concern in East Asia.

BackgroundHypervirulent variants of Klebsiella pneumoniae (HvKp) were typically associated with a broadly antimicrobial susceptible clone of sequence type (ST) 23 at the time of its emergence. Concerningly, HvKp is now also emerging within multidrug-resistant (MDR) clones, including ST11, ST15, and ST147. MDR-HvKp either carry both the virulence and resistance plasmids or carry a large hybrid plasmid coding for both virulence and resistance determinants. Here, we aimed to genetically characterize a collection of MDR-HvKp ST2096 isolates haboring hybrid plasmids carrying both antimicrobial resistance (AMR) and virulence genes.MethodsNine K. pneumoniae ST2096 isolated over 1 year from the blood sample of hospitalized patients in southern India that were MDR and suspected to be HvKp were selected. All nine isolates were subjected to short-read whole-genome sequencing; a subset (n = 4) was additionally subjected to long-read sequencing to obtain complete genomes for characterization. Mucoviscosity assay was also performed for phenotypic assessment.ResultsAmong the nine isolates, seven were carbapenem-resistant, two of which carried blaNDM-5 on an IncFII plasmid and five carried blaOXA-232 on a ColKP3 plasmid. The organisms were confirmed as HvKp, with characteristic virulence genes (rmpA2, iutA, and iucABCD) carried on a large (~320 kbp) IncFIB–IncHI1B co-integrate. This hybrid plasmid also carried the aadA2, armA, blaOXA-1, msrE, mphE, sul1, and dfrA14 AMR genes in addition to the heavy-metal resistance genes. The hybrid plasmid showed about 60% similarity to the IncHI1B virulence plasmid of K. pneumoniae SGH10 and ~70% sequence identity with the first identified IncHI1B pNDM-MAR plasmid. Notably, the hybrid plasmid carried its type IV-A3 CRISPR-Cas system which harbored spacer regions against traL of IncF plasmids, thereby preventing their acquisition.ConclusionThe convergence of virulence and AMR is clinically concerning in K. pneumoniae. Our data highlight the role of hybrid plasmids carrying both AMR and virulence genes in K. pneumoniae ST2096, suggesting that MDR-HvKp is not confined to selected clones; we highlight the continued emergence of such genotypes across the species. The convergence is occurring globally amidst several clones and is of great concern to public health.

Emergence of blaNDM–1 and blaKPC–2 co-producing Klebsiella pneumoniae strains is currently attracting widespread attention, but little information is available about their tigecycline resistance, virulence, and prevalence in Southwest China. In July 2021, an extensively drug-resistant K. pneumoniae strain AHSWKP25 whose genome contained both blaNDM–1 and blaKPC–2 genes was isolated from the blood of a patient with the malignant hematological disease in Luzhou, China. We investigated the resistance profiles of AHSWKP25 using microbroth dilution, agar dilution, modified carbapenemase inactivation (mCIM), and EDTA-modified carbapenemase inactivation methods (eCIM). The virulence of AHSWKP25 was assessed through string tests, serum killing assays, and a Galleria mellonella larval infection model. Conjugation and plasmid stability experiments were conducted to determine the horizontal transfer capacity of plasmids. And efflux pump phenotype test and real-time quantitative reverse transcription-PCR (RT-PCR) were used to determine its efflux pump activity. Sequencing of AHSWKP25 determined that AHSWKP25 belonged to ST464, which is resistant to antibiotics such as carbapenems, tetracycline, fluoroquinolones, tigecycline, and fosfomycin. The efflux pump phenotype tests and RT-PCR results demonstrated that efflux pumps were overexpressed in the AHSWKP25, which promoted the tigecycline resistance of the bacteria. AHSWKP25 also showed hypervirulence and serum resistance in vitro model. AHSWKP25 carried several different plasmids that contained blaNDM–1, blaKPC–2, and mutated tet(A) genes. Sequence alignment revealed that the plasmids carrying blaNDM–1 and blaKPC–2 underwent recombination and insertion events, respectively. We demonstrated that an X3 plasmid carrying blaNDM–1 was transferred from pSW25NDM1 to E. coli J53. We also identified missense mutations in the ramR, rcsA, lon, and csrD genes of AHSWKP25. Our results highlighted the potential of blaNDM–1 and blaKPC–2 co-producing K. pneumoniae strains to further develop antimicrobial resistance and hypervirulent phenotypes, but measures should be taken to closely monitor and control the spread of superbugs with multidrug-resistant phenotypes and hypervirulence.

Hypervirulent Klebsiella pneumoniae (hvKP) is an evolving infectious pathogen associated with high mortality. The convergence of hypervirulence and multidrug resistance further challenges the clinical treatment options for K. pneumoniae infections. The QseBC two-component system (TCS) is a component of quorum-sensing regulatory cascade and functions as a global regulator of biofilm growth, bacterial motility, and virulence in Escherichia coli. However, the functional mechanisms of QseBC in hvKP have not been reported, and we aim to examine the role of QseBC in regulating virulence in hvKP strain ATCC43816. The CRISPR-Cas9 system was used to construct qseB, qseC, and qseBC knockout in ATCC43816. No significant alterations in the growth and antibiotic susceptibility were detected between wild-type and mutants. The deletion of qseC led to an increase of biofilm formation, resistance to serum killing, and high mortality in the G. mellonella model. RNAseq differential gene expression analysis exhibited that gene-associated biofilm formation (glgC, glgP, glgA, gcvA, bcsA, ydaM, paaF, ptsG), bacterial type VI secretion system (virB4, virB6, virB10, vgrG, hcp), and biosynthesis of siderophore (entC, entD, entE) were significantly upregulated in comparison with the wild-type control. In addition, qseB, ygiW (encode OB-family protein), and AraC family transcriptional regulator IT767_23090 genes showed highest expressions in the absence of QseC, which might be related to increased virulence. The study provided new insights into the functional importance of QseBC in regulating the virulence of hvKP.