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Wenqing Wang,1,2,* Chuchu Ye,2,3,* Bing Zhao,1 Yingjie Zheng,4 Ge Zhang,5 Jinghua Su,1 Hong Huang,1 Lipeng Hao,1,2 Mingliang Chen6 1Department of Microbiology, Pudong New Area Center for Disease Control and Prevention, Shanghai, People’s Republic of China; 2Fudan University Pudong Institute of Preventive Medicine, Shanghai, People’s Republic of China; 3Department of Infectious Disease Control and Prevention, Pudong New Area Center for Disease Control and Prevention, Shanghai, People’s Republic of China; 4Department of Epidemiology/Key Laboratory for Health Technology Assessment, National Commission of Health and Family Planning/Key Laboratory of Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai, People’s Republic of China; 5School of Public Health, Dali University, Yunnan, People’s Republic of China; 6Research and Translational Laboratory of Acute Injury and Secondary Infection, and Department of Laboratory Medicine, Minhang Hospital, Fudan University, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Mingliang Chen; Lipeng Hao, Email cmlbright@126.com; hlpmail@126.comBackground: The occurrence and dissemination of hypermucoviscous and hypervirulent Klebsiella pneumoniae (hm-hvKp) isolates in clinical settings are a critical public health problem in the world. However, the data on these isolates in community populations are limited. This study aims to understand the prevalence and molecular characteristics of hm-hvKp isolates in community patients in Shanghai, China.Methods: In 2018, an active surveillance system focused on hm-hvKp in community diarrhoeal cases was implemented in Pudong New Area, Shanghai, China, involving 12 sentinel hospitals. The antimicrobial susceptibility of hm-hvKp isolates from fecal samples was tested, and whole-genome sequencing (WGS) was performed to predict the serotypes and sequence types and to identify antimicrobial resistance determinants, virulence determinants, and phylogenetic clusters.Results: The overall prevalence of hm K. pneumoniae isolates was 2.48% (31/1252), with the proportions of 1.76% (22/1252) for hm-hvKp and 0.72% (9/1252) for hm not hv K. pneumoniae. The prevalence of hm-hvKp isolates among different age groups and different months was statistically significant. All the 22 hm-hvKp isolates were susceptible to 20 antimicrobial agents and only carried blaSHV gene, and KL1 and KL2 accounted for eight (36.36%) cases and seven (31.82%) cases, respectively. The eight ST23/KL1 isolates belonged to the predominant CG23-I clade, which typically possessed the virulence determinants profile of rmpA/rmpA2-iro-iuc-ybt-irp-clb. The five ST86/KL2 isolates were assigned to the global clusters ST86/KL2-1 (n=2), ST86/KL2-2 (n=2), ST86/KL2-3 (n=1), all lack of the clb gene. Shanghai ST23/KL1 and ST86/KL2 isolates were closely related to the global isolates from liver abscesses, blood, and urine.Conclusion: Hm-hvKp is carried by the community population of Shanghai, with ST23/KL1 and ST86/KL2 isolates predominant. Hm-hvKp isolates of different continents, different sources, and different virulence levels were closely related. Ongoing surveillance of hm-hvKp isolates in the community population is warranted.Keywords: hypermucoviscous, hypervirulent, Klebsiella pneumoniae, drug resistance determinants, virulence determinants, whole-genome sequencing

Abstract Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection.

Abstract Klebsiella pneumoniae is a Gram-negative Enterobacteriaceae that is classified by the World Health Organisation (WHO) as a Priority One ESKAPE pathogen. South and Southeast Asian countries are regions where both healthcare associated infections (HAI) and community acquired infections (CAI) due to extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant K. pneumoniae (CRKp) are of concern. As K. pneumoniae can also exist as a harmless commensal, the spread of resistance genotypes requires epidemiological vigilance. However there has been no significant study of carriage isolates from healthy individuals, particularly in Southeast Asia, and specially Malaysia. Here we describe the genomic analysis of respiratory isolates of K. pneumoniae obtained from Orang Ulu and Orang Asli communities in Malaysian Borneo and Peninsular Malaysia respectively. The majority of isolates were K. pneumoniae species complex (KpSC) 1 K. pneumoniae (n = 53, 89.8%). Four Klebsiella variicola subsp. variicola (KpSC3) and two Klebsiella quasipneumoniae subsp. similipneumoniae (KpSC4) were also found. It was discovered that 30.2% (n = 16) of the KpSC1 isolates were ST23, 11.3% (n = 6) were of ST65, 7.5% (n = 4) were ST13, and 13.2% (n = 7) were ST86. Only eight of the KpSC1 isolates encoded ESBL, but importantly not carbapenemase. Thirteen of the KpSC1 isolates carried yersiniabactin, colibactin and aerobactin, all of which harboured the rmpADC locus and are therefore characterised as hypervirulent. Co-carriage of multiple strains was minimal. In conclusion, most isolates were KpSC1, ST23, one of the most common sequence types and previously found in cases of K. pneumoniae infection. A proportion were hypervirulent (hvKp) however antibiotic resistance was low.

Abstract Background Acinetobacter baumannii (A. baumannii) is associated with both hospital-acquired infections (HAP) and community-acquired pneumonia (CAP). In this study, we present a novel CAP-associated A. baumannii (CAP-AB) strain causing severe pneumonia in an afore healthy male patient without underlying conditions. Subsequently, we investigated the pathogenicity and immunogenicity of this CAP-AB strain using a mice pneumonia model. Results A 58-year-old male patient with no underlying conditions experienced worsening symptoms of a productive cough, sputum, and fever that developed acutely, in just 24 h. The diagnosis was severe community-acquired pneumonia (CAP) and type-1 respiratory failure. An A. baumannii strain was isolated from his sputum and blood cultures. To gain a deeper understanding of the rapid progression of its pathology, we utilized the CAP-associated A. baumannii strain YC128, a previously obtained hospital-acquired pneumonia A. baumannii (HAP-AB) strain YC156, and a highly virulent A. baumannii control strain LAC-4 to construct a mouse pneumonia model, and subsequently compared the mortality rate of the three groups. Following inoculation with 107 CFU of A. baumannii, the mortality rate for the YC128, LAC-4, and YC156 groups was 60% (6/10), 30% (3/10), and 0%, respectively. The bacterial burden within the pulmonary, liver, and spleen tissues of mice in the YC128 group was significantly higher than that of the YC156 group, and slightly higher than that of the LAC-4 group. Pathological analysis of lung tissue using HE-staining revealed that the inflammatory pathological changes in mice from the YC128 group were significantly more severe than those in the YC156 group. Additionally, CT scan images displayed more pronounced inflammation in the lungs of mice from the YC128 group compared to the YC156 group. Local levels of cytokines/chemokines such as IL-1β, IL-6, TNF-α, and CXCL1 were assessed via RT-qPCR in lung tissues. In comparison with the YC156 strain, the highly virulent YC128 strain induced the expression of proinflammatory cytokines more rapidly and severely. Furthermore, we examined the in vitro anti-phagocytosis ability of YC128 and YC156 strains against mice peritoneal macrophages, revealing that the highly virulent YC128 isolate displayed greater resistance to macrophage uptake in contrast to YC156. Results from Whole Genome Sequencing (WGS) indicated that YC128 harbored a complete type VI secretion system (T6SS) gene cluster, while YC156 lacked the majority of genes within the T6SS gene cluster. The other virulence-related genes exhibited minimal differences between YC128 and YC156. Drawing from previous studies, we postulated that the T6SS is linked to the hypervirulence and robust anti-phagocytic ability of YC128. Conclusions This article reports on the isolation of a novel hypervirulent CAP-AB strain, YC128, from a severe CAP patient. The results demonstrate that this CAP-AB strain, YC128, is capable of inducing fatal pneumonia and extrapulmonary dissemination in a mouse pneumonia model. Moreover, this highly virulent CAP-AB strain exhibits significantly stronger anti-phagocytic abilities compared to the HAP-AB YC156 strain. Genome sequencing comparisons reveal that the heightened hypervirulence and enhanced anti-phagocytosis abilities observed in YC128 may be attributed to the presence of the T6SS.

Introduction: A distinct strain of Klebsiella pneumoniae (K. pneumoniae) referred to as hypervirulent (hvKp) is associated with invasive infections such as pyogenic liver abscess in young and healthy individuals. In South Africa, limited information about the prevalence and virulence of this hvKp strain is available. The aim of this study was to determine the prevalence of hvKp and virulence-associated factors in K. pneumoniae isolates from one of the largest tertiary hospitals in a South African province. Methods: A total of 74 K. pneumoniae isolates were received from Pelonomi Tertiary Hospital National Health Laboratory Service (NHLS), Bloemfontein. Virulence-associated genes (rmpA, capsule serotype K1/K2, iroB and irp2) were screened using Polymerase Chain Reaction (PCR). The iutA (aerobactin transporter) gene was used as a primary biomarker of hvKp. The extracted DNAs were sequenced using the next-generation sequencing pipeline and the curated sequences were used for phylogeny analyses using appropriate bioinformatic tools. The virulence of hvKp vs. classical Klebsiella pneumoniae (cKp) was investigated using the Caenorhabditis elegans nematode model. Results: Nine (12.2%) isolates were identified as hvKp. Moreover, hvKp was significantly (p < 0.05) more virulent in vivo in Caenorhabditis elegans relative to cKp. The virulence-associated genes [rmpA, iroB, hypermucoviscous phenotype (hmv) phenotype and capsule K1/K2] were significantly (p < 0.05) associated with hvKp. A homology search of the curated sequences revealed a high percentage of identity between 99.8 and 100% with other homologous iutA gene sequences of other hvKp in the GenBank. Conclusion: Findings from this study confirm the presence of hvKp in a large tertiary hospital in central South Africa. However, the low prevalence and mild to moderate clinical presentation of infected patients suggest a marginal threat to public health. Further studies in different settings are required to establish the true potential impact of hvKp in developing countries. [ABSTRACT FROM AUTHOR]

Background: The human microbiota, K.oxytoca, is resistant to multiple drugs, endangering the lives of patients. Hypervirulent strains evolved into multidrug-resistant strains due to the acquisition of mobile genetic elements. Objectives: To detect the antibiotic-resistant profile and the related virulence genes in the hypervirulent and non-hypervirulent strains isolated from clinical specimens. Materials and methods: A total of 136 clinical samples were collected from patients at Baghdad City Hospitals, Iraq. The isolates were identified, and an antibiotics sensitivity test was carried out. The polymerase chain reaction typing method was used to detect the virulence genes. Results: The most frequent source of K. oxytoca was urine samples (36.03%), then blood samples (15.44%), particularly inpatient samples. About 12.5% of isolates were positive for the hypervirulent test (the string test). Isolates showed variable levels of resistance towards antibiotic groups. The blaCTX-M and aac(6’)-Ib-cr genes were revealed in 88% of isolates, and the blaOXA-48 gene was in 44%. All the tested isolates were negative for the rmpA gene. Conclusion: K. oxytoca is recognized as one of the leading causes of hospital-acquired infections. The rapid identification of antibiotics-resistant, hypervirulent isolates that present a considerable threat to human health is highly recommended in the local hospitals. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is a Gram-negative Enterobacteriaceae that is classified by the World Health Organisation (WHO) as a Priority One ESKAPE pathogen. South and Southeast Asian countries are regions where both healthcare associated infections (HAI) and community acquired infections (CAI) due to extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant K. pneumoniae (CRKp) are of concern. As K. pneumoniae can also exist as a harmless commensal, the spread of resistance genotypes requires epidemiological vigilance. However there has been no significant study of carriage isolates from healthy individuals, particularly in Southeast Asia, and specially Malaysia. Here we describe the genomic analysis of respiratory isolates of K. pneumoniae obtained from Orang Ulu and Orang Asli communities in Malaysian Borneo and Peninsular Malaysia respectively. The majority of isolates were K. pneumoniae species complex (KpSC) 1 K. pneumoniae (n = 53, 89.8%). Four Klebsiella variicola subsp. variicola (KpSC3) and two Klebsiella quasipneumoniae subsp. similipneumoniae (KpSC4) were also found. It was discovered that 30.2% (n = 16) of the KpSC1 isolates were ST23, 11.3% (n = 6) were of ST65, 7.5% (n = 4) were ST13, and 13.2% (n = 7) were ST86. Only eight of the KpSC1 isolates encoded ESBL, but importantly not carbapenemase. Thirteen of the KpSC1 isolates carried yersiniabactin, colibactin and aerobactin, all of which harboured the rmpADC locus and are therefore characterised as hypervirulent. Co-carriage of multiple strains was minimal. In conclusion, most isolates were KpSC1, ST23, one of the most common sequence types and previously found in cases of K. pneumoniae infection. A proportion were hypervirulent (hvKp) however antibiotic resistance was low. [ABSTRACT FROM AUTHOR]

Background: Acinetobacter baumannii (A. baumannii) is associated with both hospital-acquired infections (HAP) and community-acquired pneumonia (CAP). In this study, we present a novel CAP-associated A. baumannii (CAP-AB) strain causing severe pneumonia in an afore healthy male patient without underlying conditions. Subsequently, we investigated the pathogenicity and immunogenicity of this CAP-AB strain using a mice pneumonia model. Results: A 58-year-old male patient with no underlying conditions experienced worsening symptoms of a productive cough, sputum, and fever that developed acutely, in just 24 h. The diagnosis was severe community-acquired pneumonia (CAP) and type-1 respiratory failure. An A. baumannii strain was isolated from his sputum and blood cultures. To gain a deeper understanding of the rapid progression of its pathology, we utilized the CAP-associated A. baumannii strain YC128, a previously obtained hospital-acquired pneumonia A. baumannii (HAP-AB) strain YC156, and a highly virulent A. baumannii control strain LAC-4 to construct a mouse pneumonia model, and subsequently compared the mortality rate of the three groups. Following inoculation with 107 CFU of A. baumannii, the mortality rate for the YC128, LAC-4, and YC156 groups was 60% (6/10), 30% (3/10), and 0%, respectively. The bacterial burden within the pulmonary, liver, and spleen tissues of mice in the YC128 group was significantly higher than that of the YC156 group, and slightly higher than that of the LAC-4 group. Pathological analysis of lung tissue using HE-staining revealed that the inflammatory pathological changes in mice from the YC128 group were significantly more severe than those in the YC156 group. Additionally, CT scan images displayed more pronounced inflammation in the lungs of mice from the YC128 group compared to the YC156 group. Local levels of cytokines/chemokines such as IL-1β, IL-6, TNF-α, and CXCL1 were assessed via RT-qPCR in lung tissues. In comparison with the YC156 strain, the highly virulent YC128 strain induced the expression of proinflammatory cytokines more rapidly and severely. Furthermore, we examined the in vitro anti-phagocytosis ability of YC128 and YC156 strains against mice peritoneal macrophages, revealing that the highly virulent YC128 isolate displayed greater resistance to macrophage uptake in contrast to YC156. Results from Whole Genome Sequencing (WGS) indicated that YC128 harbored a complete type VI secretion system (T6SS) gene cluster, while YC156 lacked the majority of genes within the T6SS gene cluster. The other virulence-related genes exhibited minimal differences between YC128 and YC156. Drawing from previous studies, we postulated that the T6SS is linked to the hypervirulence and robust anti-phagocytic ability of YC128. Conclusions: This article reports on the isolation of a novel hypervirulent CAP-AB strain, YC128, from a severe CAP patient. The results demonstrate that this CAP-AB strain, YC128, is capable of inducing fatal pneumonia and extrapulmonary dissemination in a mouse pneumonia model. Moreover, this highly virulent CAP-AB strain exhibits significantly stronger anti-phagocytic abilities compared to the HAP-AB YC156 strain. Genome sequencing comparisons reveal that the heightened hypervirulence and enhanced anti-phagocytosis abilities observed in YC128 may be attributed to the presence of the T6SS. [ABSTRACT FROM AUTHOR]

Summary: 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 log10 LD50 (range 1.5–2.9)) were created to measure the effects on LD50 in a murine model of the hypervirulence-associated plasmid (pVir), iucA, prmpA, prmpA2 (truncated), irp2, and clbBC. Findings: Curing pVir had the greatest increase in survival (mean LD50 to 7.6 (range 7.0–9.0, p ≤ 0.0001), a dosage comparable to classical K. pneumoniae. Results also showed increased mean LD50s for ΔprmpA (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 LD50 increase of 1.3 compared to the pVir-encoded ΔprmpAΔiucA (p ≤ 0.01), suggesting presence of additional pVir-virulence genes. Truncated pRmpA2 did not contribute to virulence. Odd ratios in the absence of pVir/yersiniabactin, pVir, pRmpA/aerobactin, pRmpA, 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.

Background: 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. Methods: 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. Results: A. baumannii XH2146 strain belongs to ST10Pas and ST447Oxf. 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 blaADC-76, blaOXA-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 blaOXA-58; blaOXA-23 was identified in 11 isolates and three ST447 A. baumannii strains harbored blaNDM-1. Conclusions: Early detection of community-acquired hypervirulent Acinetobacter baumannii strains is recommended to prevent their extensive spread in hospitals.

Ceftazidime-avibactam resistance attributable to the blaKPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harboured the blaKPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The blaKPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the blaKPC-2-positive cloned strain, only the MIC of ceftazidime increased against blaKPC-135-positive K. pneumoniae and wasn’t inhibited by avibactam (MIC 32 μg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harboured resistance and virulence genes, posing an elevated challenge in clinical treatment.

Introduction: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains combining virulence and multidrug resistance (MDR) features pose a great public health concern. The aimof this study is to explore the evolutionary characteristics of virulence in CR-HvKP by investigating the genetic features of resistance and virulence hybrid plasmids. Methods: The resistance and virulence phenotypes were determined by using antimicrobial susceptibility testing and the mouse bacteremia infection model, respectively. Plasmid profiles were investigated by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting, conjugation assay, and whole genome sequencing (WGS). Bioinformatics tools were used to uncover the genetic features of the resistance and virulence hybrid plasmids. Results: Two ST11-KL64 CRKP clinical isolates (KP18-3-8 and KP18-2079), which exhibited enhanced virulence compared with the classic CRKP, were detected positive for blaKPC-2 and rmpA2. The virulence level of the hypermucoviscous strain KP18-3-8 was higher than that of KP18-2079. S1-PFGE, Southern hybridization and WGS analysis identified two novel hybrid virulence plasmids in KP18-3-8 (pKP1838-KPC-vir, 228,158 bp) and KP18-2079 (pKP1838-KPC-vir, 182,326 bp), respectively. The IncHI1B/repB-type plasmid pKP1838-KPC-vir co-harboring blaKPC-2 and virulence genes (rmpA2, iucABCD and iutA) but lacking type IV secretion system could transfer into non-hypervirulent ST11 K. pneumoniae with the assistance of a helper plasmid in conjugation. The IncFII/IncR-type virulence plasmid pKP18-2079-vir may have been generated as a result of recombination between a typical pLVPK-like virulence plasmid and an MDR plasmid. Conclusion: Our studies further highlight co-evolution of the virulence and resistance plasmids in ST11-CRKP isolates. Close surveillance of such hybrid virulence plasmids in clinical K. pneumoniae should be performed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae is an emerging pathogen that has gained attention due to its increased ability to cause infections even in healthy individuals. The aim of this study is to investigate virulence factors in K. pneumoniae strains isolated from clinical specimens and their association with carbapenem resistance. The study was conducted on 260 isolates identified between 2018 and 2023 at the Mohammed V Military Teaching Hospital in Rabat, Morocco. The isolates were categorized based on their susceptibility to antibiotics. The hypermucoviscosity was determined by a string test, while the presence of capsular serotypes and virulence genes were identified by PCR. Among our strains, 6.2% (n = 16) exhibited hypervirulent characteristics, 56% were resistant to carbapenem. Notably, 5.7% (n = 6) of carbapenem-resistant isolates expressed the hypermucoviscous phenotype, while 1.5% (n = 2) of carbapenem-susceptible K. pneumoniae isolates exhibited the same trait. In our study, we found that a total of 10 isolates (3.8%) had virulent capsular serotypes, with K2 being the most prevalent 40% (n = 4) and K20 in 30% (n = 3). Furthermore, we detected the presence of the Aerobactin gene in 1.5% (n = 4) of the isolates examined. Based on our findings, it appears that there was no correlation between the presence of virulence factors and carbapenem resistance. In conclusion, identifying hypervirulent K. pneumoniae in clinical specimens and assessing their antibiotic resistance profiles are crucial to ensure effective therapy and to prevent outbreaks. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKP) causes multisite infections and abscesses. However, endocarditis is a rare presentation of hvKP infection. Herein, we report a case of K. pneumoniae native valve infective endocarditis secondary to community-acquired liver and prostate abscesses. The patient developed papillary muscle rupture, leading to mitral regurgitation, and underwent emergent mitral valve replacement. The diagnosis of endocarditis was confirmed microbiologically and histologically. The causative strain belonged to the hypermucoid K1 capsular genotype and possessed the rmpA gene. The genome sequence was deposited in GenBank under the accession number JAQZBZ000000000. [ABSTRACT FROM AUTHOR]

IntroductionA distinct strain of Klebsiella pneumoniae (K. pneumoniae) referred to as hypervirulent (hvKp) is associated with invasive infections such as pyogenic liver abscess in young and healthy individuals. In South Africa, limited information about the prevalence and virulence of this hvKp strain is available. The aim of this study was to determine the prevalence of hvKp and virulence-associated factors in K. pneumoniae isolates from one of the largest tertiary hospitals in a South African province.MethodsA total of 74 K. pneumoniae isolates were received from Pelonomi Tertiary Hospital National Health Laboratory Service (NHLS), Bloemfontein. Virulence-associated genes (rmpA, capsule serotype K1/K2, iroB and irp2) were screened using Polymerase Chain Reaction (PCR). The iutA (aerobactin transporter) gene was used as a primary biomarker of hvKp. The extracted DNAs were sequenced using the next-generation sequencing pipeline and the curated sequences were used for phylogeny analyses using appropriate bioinformatic tools. The virulence of hvKp vs. classical Klebsiella pneumoniae (cKp) was investigated using the Caenorhabditis elegans nematode model.ResultsNine (12.2%) isolates were identified as hvKp. Moreover, hvKp was significantly (p < 0.05) more virulent in vivo in Caenorhabditis elegans relative to cKp. The virulence-associated genes [rmpA, iroB, hypermucoviscous phenotype (hmv) phenotype and capsule K1/K2] were significantly (p < 0.05) associated with hvKp. A homology search of the curated sequences revealed a high percentage of identity between 99.8 and 100% with other homologous iutA gene sequences of other hvKp in the GenBank.ConclusionFindings from this study confirm the presence of hvKp in a large tertiary hospital in central South Africa. However, the low prevalence and mild to moderate clinical presentation of infected patients suggest a marginal threat to public health. Further studies in different settings are required to establish the true potential impact of hvKp in developing countries.

Klebsiella pneumoniae is a highly prevalent pathogen among nosocomial and community-acquired infections, including urinary tract infections (UTIs), pneumonia, surgical site infections, and bloodstream infections.One hundred (100) isolates were obtained from different sources, including urine, burns, sputum, blood, and wound. All isolates were bacteriologically identified as Klebsiella pneumoniae using phenotypic and biochemical tests. The antimicrobial susceptibility test was done by using the disc diffusion method for thirteen different antimicrobial agents. String test was used to evaluate the hypermucoviscousity of the isolates and siderophore production was estimated by CAS agar and colorimetric methods. Two genes, iucA and iroB, were detected by PCR and their correlation with phenotypic markers was evaluated. The results showed that 51% of the isolates were positive for the string test and 49 % were negative. The results showed that the siderophores production ranged between 14 to 74 psu. Genotyping results showed that 23% of the isolates contained iucA gene and 17 % contained iroB. There was a high correlation between the two genes and the hypermucoviscousity. Also, siderophore production was highly associated with having one of the genes or both with more than 60 psu of siderophores production.

ABSTRACT: 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 blaNDM-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.

ABSTRACT: 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.

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[Display omitted] Although hypervirulent Klebsiella pneumoniae (hvKP) can produce community-acquired infections that are fatal in young and adult hosts, such as pyogenic liver abscess, endophthalmitis, and meningitis, it has historically been susceptible to antibiotics. Carbapenem-resistant K. pneumoniae (CRKP) is usually associated with urinary tract infections acquired in hospitals, pneumonia, septicemias, and soft tissue infections. Outbreaks and quick spread of CRKP in hospitals have become a major challenge in public health due to the lack of effective antibacterial treatments. In the early stages of K. pneumoniae development, HvKP and CRKP first appear as distinct routes. However, the lines dividing the two pathotypes are vanishing currently, and the advent of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) is devastating as it is simultaneously multidrug-resistant, hypervirulent, and highly transmissible. Most CR-hvKP cases have been reported in Asian clinical settings, particularly in China. Typically, CR-hvKP develops when hvKP or CRKP acquires plasmids that carry either the carbapenem-resistance gene or the virulence gene. Alternatively, classic K. pneumoniae (cKP) may acquire a hybrid plasmid carrying both genes. In this review, we provide an overview of the key antimicrobial resistance mechanisms, virulence factors, clinical presentations, and outcomes associated with CR-hvKP infection. Additionally, we discuss the possible evolutionary processes and prevalence of CR-hvKP in China. Given the wide occurrence of CR-hvKP, continued surveillance and control measures of such organisms should be assigned a higher priority. [ABSTRACT FROM AUTHOR]

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 blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-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. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to identify hypervirulent clinical Klebsiella pneumoniae isolates from Anbar (Iraq) and differentiate them from classical isolates using molecular markers and capsule serotyping. Methodology and results: Between June and December 2022, we gathered a total of 100 K. pneumoniae isolates from patients with hospital-acquired infections in four prominent hospitals located in Anbar province. We performed the identification of all isolates using biochemical tests and the VITEK2 compact system. Hypervirulent genes were detected using PCR and multiplex PCR. Five virulent genes, namely, iucA, iroB, rmpA, rmpA2 and peg344, were considered in the study. Each isolate was serotyped using six capsular serotypes, K1, K2, K5, K20, K54 and K57. Three genes, iucA, iroB and rmpA were detected by PCR. The most prevalent gene identified was iucA (23%). Serotyping results identified four capsular types among the 100 isolates, with K2 (71.2%) being the most predominant type. Of the 100 isolates, 18 (18%) K. pneumonia isolates were identified as hypervirulent. According to the presence of the virulent genes, four virulent types (VP) were determined, namely, VP1, VP2, VP3 and VP4, with VP3 being the most common among the four types. Conclusion, significance and impact of study: Eighteen hypervirulent K. pneumoniae strains were identified using molecular markers. The results indicate that hypervirulent K. pneumoniae infections are emerging in Anbar province and need active monitoring to minimize spreading through the region. [ABSTRACT FROM AUTHOR]

Updating data on Clostridioides difficile antibiotic resistance is important for treatment improvement of C. difficile infections (CDIs). Results from 20 countries were included. The mean resistance to 2 mg/l vancomycin, 2 mg/l metronidazole, 4 mg/l moxifloxacin, and 4 mg/l clindamycin was 4.7% (0 to ≥ 26% in two studies), 2.6% (0 to ≥ 40% in 3 studies), 34.9% (6.6->80%), and 61.0% (30->90%), respectively. Resistance to erythromycin (>60–88%), rifampin (>23–55.0%), imipenem (0.6 to > 78% in a clone), tigecycline (0-<5.0%), and fidaxomicin (0–2%) was also found. Resistance to ≥ 5 antibiotics of different classes was reported in some countries. High resistance and multidrug resistance were observed in hypervirulent and epidemic strains. Although only 1% of COVID-19 patients had CDIs, the proportion might be underestimated. C. difficile antimicrobial susceptibility varied by country/region, study period, and circulating ribotypes. For CDI treatment, fidaxomicin (preferably) or vancomycin is recommended, while metronidazole is suitable for mild infections. New approaches, including biotherapeutics (Rebyota), strains, antibiotics (ridinilazole and ibezapolstat), and monoclonal antibodies/cocktails merit further evaluation. Because of the resistance rate variations, C. difficile antibiotic susceptibility should be regularly monitored. Post-COVID-19 resistance should be separately presented. Some discrepancies between vancomycin and metronidazole results need to be clarified. Clostridioides difficile can cause mild to dangerous diarrhea in people following antibiotic use. Many antibacterial agents can cause diseases. However, treatment is limited to three antibiotics. The study of resistance to them is important for improving the treatment of infections. The study of resistance to other antibiotics helps to understand the spread and risks of infections. We discussed data about C. difficile antibiotic resistance from 20 countries according to recent publications. For the treatment of C. difficileinfections, fidaxomicin is the drug of choice with 0–2% resistance to it. Resistance to the two other antibiotics used to treat infections is less than 5% of isolates. Much higher resistance was found to antibiotics that can cause C. difficile infections such as ciprofloxacin, clindamycin, ceftriaxone, erythromycin, and others. The resistance varies according to the country, patients' groups, years of study, and circulating strains. The resistance was high in hypervirulent and epidemic strains. In some studies, there was resistance to 5 and 6 antibiotics of different classes. Antibiotic use and incidence of infections during the COVID-19 pandemic varied. However, the evolution of C. difficile infection during the pandemic has yet to be determined. [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

Hypervirulent Klebsiella pneumoniae strains are the major cause of liver abscesses ,diabetes foot ulcers and other infections throughout Iraq, and these strains are usually antibiotics susceptible. Recently, hypervirulent K. pneumoniae isolates have emerged due to acquiring antimicrobial resistance determinants or the transfer of a virulence plasmid into a classic isolates ,.Klebsiella pneumoniae is responsible for hospital- and community-acquired infections. This study aimed to determine the prevalence of hyper irulence K. pneumoniae and investigate the antibiotics resistance profile among clinical specimens at Al Najaf Hospitals in/ Iraq, and detect the genes for molecular identification of K. pneumoniae in comparison with phenotypic and biochemical methods. In total, 100 clinical specimens were collected from patients infected with K. pneumoniae, between January 2023 and May 2023 from both sexes males (38) and Females (62) with age ranged between (20-70) years. The Hypervirulent Klebsiella pneumoniae isolates were cultured on general and selective media as (Blood agar and MacConkey agar and CHROM agar), and confirmed by using Vitek-2 system. The isolates were reported with high resistance towards various types of antibiotics, especially penicillins and cephalosporins. In contrast, K. pneumoniae showed very low resistance to imipenem and amikacin. According to the results the range of multidrug-resistant K. pneumoniae isolates in this study was estimated at 100%.

ABSTRACT The increasing prevalence of hypervirulent (hv) and carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) highlights the importance of timely and accurate differential diagnosis for epidemiological investigation and clinical management. A multiplex quantitative real-time PCR (qRT-PCR) assay for the simultaneous identification of hvKp and CR-Kp was developed and validated with excellent performance in sensitivity and specificity. Generally, the gltA gene for Kp, the iucA, rmpA and rmpA2 genes for hvKp, and the Klebsiella pneumoniae carbapenemases (KPC) gene for CR-Kp were included in the qRT-PCR assay. The detection limits for classic Kp (cKp), CR-cKp, hvKp, and CR-hvKp strains could all reach 50 genome equivalent copies and 20 CFUs per reaction with high accuracy (R 2 > 0.99) and reliability (CV values < 3%). Detection results from 84 Kp positive clinical samples showed 31 hvKp with 8 CR-hvKp and 53 cKp with 1 CR-cKp strains. The presence of virulence-associated factors for the identified hvKp and KPC genes for CR-Kp was confirmed by previously developed conventional PCR and antimicrobial susceptibility tests, respectively. Furthermore, the qRT-PCR identified hvKp strains showed mortality rates of ≥40% in the outbred murine infection model, while no death for the identified cKp strains. These results indicated that our multiplex qRT-PCR assay could accurately identify hvKp and CR-Kp strains, which will be of great use for the rapid and accurate diagnosis in a clinical setting and the surveillance of the circulating Kp. 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.

Shuang Song,1,2,&ast; Shulong Zhao,2,&ast; Wei Wang,1 Fei Jiang,2 Jingfang Sun,2 Ping Ma,1,2 Haiquan Kang2 1Medical Technology School, Xuzhou Medical University, Xuzhou, People’s Republic of China; 2Department of Clinical Laboratory, Affiliated Hospital of Xuzhou Medical University, Xuzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Haiquan Kang, Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, 221000, People’s Republic of China, Tel +86-516 85802759, Email hqk811029@163.com Ping Ma, Medical Technology School, Xuzhou Medical University, Department of Laboratory Medicine, Xuzhou, People’s Republic of China, Tel +86-516 85807017, Email pingm62@aliyun.comBackground: The prevalence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (hv-CRKP) is a serious public threat globally. Here, we performed clinical, molecular, and phenotypic monitoring of hv-CRKP strains isolated from the intensive care unit (ICU) to offer evidence for prevention and control in hospitals.Methods: Data analysis of ICU patients suffering from ventilator-associated pneumonia (VAP) because of hv-CRKP infection, admitted at the Chinese Teaching Hospital between March 2019 and September 2021 was performed. Patients’ antibiotic-resistance genes, virulence-associated genes, and capsular serotypes of these isolates were detected. Homology analysis of the strains was performed by MLST and PFGE. Six different strains were tested for their virulence traits using the serum killing test and the Galleria mellonella infection assay. For whole genome sequencing, KP3 was selected as a representative strain.Results: Clinical data of 19 hv-CRKP-VAP patients were collected and their hv-CRKP were isolated, including 10 of ST11-KL64, 4 of ST15-KL112, 2 of ST11-KL47, 1 of ST15-KL19, 1 of ST17-KL140, and 1 of ST48-KL62. Four ST15 and 8 ST11 isolates revealed high homology, respectively. Most strains carried the carbapenemase gene blaKPC-2 (14/19, 73.68%), followed by blaOXA-232 (4/19, 21.05%). All strains were resistant to almost all the antibiotics except polymyxin and tigacycline. Ten patients were treated with polymyxin or tigacycline based on their susceptibility results, and unfortunately 6 patients died. All strains exhibited a hyper-viscous phenotype, and the majority (17/19, 89.47%) of them contained rmpA and rmpA2. The serum killing test showed that KP9 was resistant to normal healthy serum, others were intermediately or highly sensitive. G. mellonella larvae infection assay suggested that the strains in this study were hypervirulent.Conclusion: This study highlights the dominant strain and molecular epidemiology of hv-CRKP in a hospital in China. We should pay more attention to the effect of hv-CRKP on VAP, strengthen monitoring and control transmission.Keywords: Klebsiella pneumoniae, hypervirulent, carbapenem-resistant, ventilator-associated pneumonia

Abstract Background Severe infections caused by β- lactamase producers, hypervirulent Klebsiella pneumoniae (BhvKp) with K2 serotype, highlight emergency need for new therapeutic strategies against this pathogen. We aimed to assess the efficacy of a novel phage, PSKP16, in the treating of pneumonia induced by BhvKp in mice models. Method Genome sequences of PSKP16 were analyzed, and associated information can be found in NCBI. We applied treatment in two ways: by using mice for immediate and delayed treatments. Moreover, acute pneumonia obtained by BhvKp with intranasal method, was characterized in terms of histopathology of pulmonary lesions, biomarkers of inflammation level, leukocytes cells infiltration extent in mice, and was assessed treatment of them with PSKP16 multiplicity of infection (MOI: 10), either individually or in combination with gentamicin. Assessment of the ability of PSKP16 to inhibit BhvKp biofilm was studied. Results PSKP16 was associated with the Drexlerviridae family, and had a genome size of 46,712 bp, and 67 predicted ORFs. Herein, prompt phage administration’s efficacy to decrease bacterial load and improve the survival rate in pneumonia models was faster than the synergism model with delay, but both almost displayed similar endpoints. The distribution of BhvKp strains in the lung was consistent with the histopathological findings, simultaneous inflammation, and level of serum tumor necrosis factor-α (TNF α). The phage treatment presented a lack of severe lesions and alveolar edema, reduction of inflammatory cell infiltration, which not only was it not associated with an over-inflammation but also provided a faster correction of blood cell count abnormalities compared to gentamicin. Phage with a high concentration in in vitro model effectively eliminated biofilms. Conclusion It is essential to raise clinical awareness and management of BhvKp infections, signaled as the next superbug in waiting. The results of our study underscore the importance of PSKP16 as a phage with promising therapeutic potential in treating BhvKp-induced pneumonia.

Wan He,1,&ast; Changbu Wu,2,3,&ast; Guanping Chen,2– 4 Guili Zhang,2,3 Zihan Zhao,2,3 Shu’an Wen,2,3 Yuan Zhou,2,3 Xue Deng,2,3 Yu Feng,2,3 Lan-Lan Zhong,2,3 Guo-Bao Tian,2– 4 Min Dai1 1School of Laboratory Medicine, Chengdu Medical College, Chengdu, 610500, People’s Republic of China; 2Department of Immunology and Microbiology, Advanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China; 3Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, People’s Republic of China; 4Department of Immunology, School of Medicine, Sun Yat-Sen University, Shenzhen, 518107, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Min Dai, School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan Province, People’s Republic of China, Tel +86 28-62739128, Email daimin1015@163.com Guo-Bao Tian, Department of Immunology and Microbiology, Advanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, People’s Republic of China, Tel/Fax +86 20 87335387, Email tiangb@mail.sysu.edu.cnBackground: The emergence and spread of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is a potential epidemiological threat that needs to be monitored. However, the transmission and pathogenic characteristics of hv-CRKP in China remain unclear. We investigated the epidemiological characteristics of gut colonized hv-CRKP in a hospital in Guangdong Province, China.Methods: A total of 46 gut colonized hv-CRKP isolates were collected from Sun Yat-Sen Memorial Hospital (Guangzhou, China) from August 31st to December 31st, 2021. Minimum inhibitory concentrations (MICs) were obtained for 15 antibiotics for 46 hv-CRKP isolates. BALB/C mice infection model and mucoviscosity assay was used to evaluate the virulence of the isolates. The characteristics of genome, phylogenetic relationship and the structure of the plasmid of 46 gut colonized hv-CRKP isolates were compared with pathogenic isolates from GeneBank based on whole-genome data.Results: The hv-CRKP isolation rate of all gut colonized carbapenem-resistant Klebsiella pneumoniae was 17% (46/270), and the intestinal colonization rate of hv-CRKP was irrelevant to the sex, age, department of hospitalization, and history of antibiotic use of the host. The gut colonized hv-CRKP showed pandrug resistance and hypervirulence. The gut colonized hv-CRKP and pathogenic hv-CRKP prevalent in China were mainly ST11 hv-CRKP and had two major epidemic clades. The similarities in genomic characteristics between gut colonized hv-CRKP and pathogenic hv-CRKP were consistent. The gut colonized hv-CRKP carried an incomplete structure pK2044 virulence plasmid from hypervirulent K. pneumoniae NTUH-K2044 by analyzing the virulence plasmid structure.Conclusion: Our results suggest that the gut colonized ST11 hv-CRKP may serve as a reservoir for the clinical pathogenic ST11 HV-CRKP. It is necessary to further strengthen the monitoring of gut colonized hv-CRKP and research the potential mechanism of infection caused by gut colonized hv-CRKP.Keywords: K. pneumoniae, intestinal colonization, hypervirulent, genomic characterization, carbapenemase

Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging pathotype in addition to classical Klebsiella pneumoniae, with its ability to cause life-threatening, community-acquired metastatic infections even in healthy individuals. We presented a case of cerebral abscess preceded by otitis media in a 10-year-old child caused by hvKp. The isolates from blood pus aspirate were later identified as K. pneumoniae capsular serotype K2 and closely related to sequence type (ST65), with multiple hypervirulent genes detected (rmpA, rmpA2, iucA and peg344). She succumbed to death despite surgical drainage and susceptible antibiotic therapy. Clinicians should be cognizant of the rising incidence of hvKp infections in pediatric populations.

The World Health Organization (WHO)’s list of neglected tropical diseases (NTDs) highlights conditions that are responsible for devastating health, social and economic consequences, and yet, they are overlooked and poorly resourced. The NTD list does not include conditions caused by Gram-negative bacilli (GNB). Infections due to GNB cause significant morbidity and mortality and are prevalent worldwide. Southeast Asia is a WHO region of low- and middle-income countries carrying the largest burden of NTDs. Two significant health threats in Southeast Asia are Burkholderia pseudomallei (causing melioidosis) and hypervirulent Klebsiella pneumoniae (HvKp). Both diseases have high mortality and increasing prevalence, yet both suffer from a lack of awareness, significant under-resourcing, incomplete epidemiological data, limited diagnostics, and a lack of evidence-based treatment. Emerging evidence shows that both melioidosis and HvKp are spreading globally, including in high-income countries, highlighting the potential future global threat they pose. In this article, we review both conditions, identifying current trends and challenges in Southeast Asia and areas for future research. We also argue that melioidosis and HvKp merit inclusion as NTDs, and that mandatory global surveillance and reporting systems should be established, and we make an urgent call for research to better understand, detect, and treat these neglected diseases.

Abstract 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.

ABSTRACTEmerging 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.

Background: Hypervirulent Klebsiella pneumoniae (K. pneumoniae), generally associated with the hypermucoviscous phenotype has emerged as a clinically significant pathogen responsible for serious disseminated infections, such as pyogenic liver abscesses, osteomyelitis, and endophthalmitis, in a generally younger and healthier population. The aim of this study was to investigate the molecular characterization of hypervirulent (hypermucoviscous) K. pneumoniae strains, which mainly emerged in EGYPT. Methods: Bacterial species were identified by standard methods with a VITEK 2 compact system. The serotyping, biotyping and the String test for phenotypic detection of mucoviscosity were used. Antimicrobial susceptibility to several commonly used antibiotics were determined by the Kirby-Bauer's disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI, 2018). Polymerase chain reaction (PCR) was used to amplify virulence-associated genes (rmpA, magA, entB, and iucA). Results: One-hundred isolates of each hypervirulent K. pneumoniae (hvKP) and classical K. pneumoniae (cKP) were screened over a 2-year period. The serotyping showed that 76 isolates had K1-type while 19 had K2-type of capsule and 5 non-K1/K2 isolates of hvKP while for cKP, only 3% was K1, 1% K2 and 96% were non k1/K2. Among the hvKP isolates, the prevalence of virulence associated-genes as rmpA gene was 93% and for magA gene was 40% and for iucA 92%. Otherwards, the prevalence of these genes in cKP isolates was very low 11%, 7% and 0%, respectively. However, there was no significant difference for entB (89 % and 92 %). Conclusion: The hypervirulent isolates were infrequent among K. pneumoniae isolates causing bacteremia in our geographical area (El-Mansoura, Egypt). For good understanding the basic biology of hypervirulent K. pneumoniae, this study summarized and focused on epidemiology, hypervirulence-associated factors, and antimicrobial resistance mechanisms of such hypervirulent strains. This epidemiological study give alarm about the dissemination of hypervirulent K. pneumoniae strains. Therefore, an immediate response to recognize these hypervirulent strains with resistance determinants is an urgent priority. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae strains are the major cause of liver abscesses,diabetes foot ulcers and other infections throughout Iraq, and these strains are usually antibiotics susceptible. Recently, hypervirulent K. pneumoniae isolates have emerged due to acquiring antimicrobial resistance determinants or the transfer of a virulence plasmid into a classic isolates,.Klebsiella pneumoniae is responsible for hospital- and community-acquired infections. This study aimed to determine the prevalence of hyper irulence K. pneumoniae and investigate the antibiotics resistance profile among clinical specimens at Al Najaf Hospitals in/ Iraq, and detect the genes for molecular identification of K. pneumoniae in comparison with phenotypic and biochemical methods. In total, 100 clinical specimens were collected from patients infected with K. pneumoniae, between January 2023 and May 2023 from both sexes males (38) and Females (62) with age ranged between (20-70) years. The Hypervirulent Klebsiella pneumoniae isolates were cultured on general and selective media as (Blood agar and MacConkey agar and CHROM agar), and confirmed by using Vitek-2 system. The isolates were reported with high resistance towards various types of antibiotics, especially penicillins and cephalosporins. In contrast, K. pneumoniae showed very low resistance to imipenem and amikacin. According to the results the range of multidrug-resistant K. pneumoniae isolates in this study was estimated at 100%. [ABSTRACT FROM AUTHOR]

Background: Severe infections caused by β- lactamase producers, hypervirulent Klebsiella pneumoniae (BhvKp) with K2 serotype, highlight emergency need for new therapeutic strategies against this pathogen. We aimed to assess the efficacy of a novel phage, PSKP16, in the treating of pneumonia induced by BhvKp in mice models. Method: Genome sequences of PSKP16 were analyzed, and associated information can be found in NCBI. We applied treatment in two ways: by using mice for immediate and delayed treatments. Moreover, acute pneumonia obtained by BhvKp with intranasal method, was characterized in terms of histopathology of pulmonary lesions, biomarkers of inflammation level, leukocytes cells infiltration extent in mice, and was assessed treatment of them with PSKP16 multiplicity of infection (MOI: 10), either individually or in combination with gentamicin. Assessment of the ability of PSKP16 to inhibit BhvKp biofilm was studied. Results: PSKP16 was associated with the Drexlerviridae family, and had a genome size of 46,712 bp, and 67 predicted ORFs. Herein, prompt phage administration's efficacy to decrease bacterial load and improve the survival rate in pneumonia models was faster than the synergism model with delay, but both almost displayed similar endpoints. The distribution of BhvKp strains in the lung was consistent with the histopathological findings, simultaneous inflammation, and level of serum tumor necrosis factor-α (TNF α). The phage treatment presented a lack of severe lesions and alveolar edema, reduction of inflammatory cell infiltration, which not only was it not associated with an over-inflammation but also provided a faster correction of blood cell count abnormalities compared to gentamicin. Phage with a high concentration in in vitro model effectively eliminated biofilms. Conclusion: It is essential to raise clinical awareness and management of BhvKp infections, signaled as the next superbug in waiting. The results of our study underscore the importance of PSKP16 as a phage with promising therapeutic potential in treating BhvKp-induced pneumonia. [ABSTRACT FROM AUTHOR]

Introduction: Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a clinically significant global pathogen in the last decade. However, the host immune responses of the macrophages during hvKp infection are largely unknown. In the present study, we aimed to compare the cytotoxic effects of hvKp and classical K. pneumoniae (cKp) in murine macrophages. Results: We found that the activation of caspase-1 -dependent pyroptosis was higher in cKp-infected macrophages compared with that in hvKp-infected macrophages. In Caspase-1 deficiency macrophages, pyroptosis diminished during infection. Both hvKp and cKp strains led to nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3) inflammasome formation and lysosomal cathepsin B activation, thus resulting in pyroptosis. Compared with the cKp strain, the hvKp strain inhibited these phenomena in murine macrophages. Conclusion: HvKp infection resulted in different levels of pyroptosis via the activation of cathepsin B-NLRP3-caspase-1 in murine macrophages. Therefore, the manipulation of pyroptotic cell death is a potential target for host response during hvKp infection in macrophages. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae (KP) is the most common cause of endogenous endophthalmitis (EE) in Asia, but data in Europe are scarce. We describe eight cases of KP EE compared to a cohort of EE in a French center. EE cases were retrospectively studied between January 2014 and January 2021. KP EE cases were analyzed to assess clinical, microbiological features, and outcome. Among the 33 EE cases identified, the first causative agent (24%, n = 8) was KP, mainly (7/8) with hypervirulent phenotype (hvKP). All but one of these cases occurred from December 2019 to January 2021. Contrary to non-KP patients, KP patients had multiple extraocular infective foci (p =.006), all presented with liver abscesses (p <.001), 50% had cerebral involvement (p =.13). Visual outcome was poor in both groups. KP is an emerging cause of EE in a French center, consistently associated with liver abscesses, frequent cerebral involvement, and predominance of hvKP strains. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging pathotype in addition to classical Klebsiella pneumoniae, with its ability to cause life-threatening, community-acquired metastatic infections even in healthy individuals. We presented a case of cerebral abscess preceded by otitis media in a 10-year-old child caused by hvKp. The isolates from blood pus aspirate were later identified as K. pneumoniae capsular serotype K2 and closely related to sequence type (ST65), with multiple hypervirulent genes detected (rmpA, rmpA2 , iucA and peg344). She succumbed to death despite surgical drainage and susceptible antibiotic therapy. Clinicians should be cognizant of the rising incidence of hvKp infections in pediatric populations. [ABSTRACT FROM AUTHOR]

Qingping Zhou,1,&ast; Chunyang Wu,2,&ast; 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&ast;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

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.

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]