Phage-antibiotic synergy (PAS) represents a superior treatment strategy for pathogen infections with less probability of resistance development. Here, we aim to understand the molecular mechanism by which PAS suppresses resistance in terms of population evolution. A novel hypervirulent Klebsiella pneumoniae (KP) phage H5 was genetically and structurally characterized. The combination of H5 and ceftazidime (CAZ) showed a robust synergistic effect in suppressing resistance emergence. Single-cell Raman analysis showed that the phage-CAZ combination suppressed bacterial metabolic activities, contrasting with the upregulation observed with phage alone. The altered population evolutionary trajectory was found to be responsible for the contrasting metabolic activities under different selective pressures, resulting in pleiotropic effects. A pre-existing wcaJ point mutation (wcaJG949A) was exclusively selected by H5, conferring a fitness advantage and up-regulated activity of carbohydrate metabolism, but also causing a trade-off between phage resistance and collateral sensitivity to CAZ. The wcaJ point mutation was counter-selected by H5-CAZ, inducing various mutations in galU that imposed evolutionary disadvantages with higher fitness costs, and suppressed carbohydrate metabolic activity. H5 and H5-CAZ treatments resulted in opposite effects on the transcriptional activity of the phosphotransferase system and the ascorbate and aldarate metabolism pathway, suggesting potential targets for phage resistance suppression. Our study reveals a novel mechanism of resistance suppression by PAS, highlighting how the complexity of bacterial adaptation to selective pressures drives treatment outcomes.
OBJECTIVE: Phage-antibiotic synergy (PAS) has been recently proposed as a superior strategy for the treatment of multidrug-resistant pathogens to effectively reduce bacterial load and slow down both phage and antibiotic resistance. However, the underlying mechanisms of resistance suppression by PAS have been poorly and rarely been studied. In this study, we tried to understand how PAS suppresses the emergence of resistance using a hypervirulent Klebsiella pneumoniae (KP) strain and a novel phage H5 in combination with ceftazidime (CAZ) as a model. Our study reveals a novel mechanism by which PAS drives altered evolutionary trajectory of bacterial populations, leading to suppressed emergence of resistance. The findings advance our understanding of how PAS suppresses the emergence of resistance, and are imperative for optimizing the efficacy of phage-antibiotic therapy to further improve clinical outcomes.

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

The evolution of hypervirulent and carbapenem-resistant Klebsiella pneumoniae can be categorized into three main patterns: the evolution of KL1/KL2-hvKp strains into CR-hvKp, the evolution of carbapenem-resistant K. pneumoniae (CRKp) strains into hv-CRKp, and the acquisition of hybrid plasmids carrying carbapenem resistance and virulence genes by classical K. pneumoniae (cKp). These strains are characterized by multi-drug resistance, high virulence, and high infectivity. Currently, there are no effective methods for treating and surveillance this pathogen. In addition, the continuous horizontal transfer and clonal spread of these bacteria under the pressure of hospital antibiotics have led to the emergence of more drug-resistant strains. This review discusses the evolution and distribution characteristics of hypervirulent and carbapenem-resistant K. pneumoniae, the mechanisms of carbapenem resistance and hypervirulence, risk factors for susceptibility, infection syndromes, treatment regimens, real-time surveillance and preventive control measures. It also outlines the resistance mechanisms of antimicrobial drugs used to treat this pathogen, providing insights for developing new drugs, combination therapies, and a \"One Health\" approach. Narrowing the scope of surveillance but intensifying implementation efforts is a viable solution. Monitoring of strains can be focused primarily on hospitals and urban wastewater treatment plants.

Hypervirulent Klebsiella pneumoniae (hvKP) typically causes severe invasive infections affecting multiple sites in healthy individuals. In the past, hvKP was characterized by a hypermucoviscosity phenotype, susceptibility to antimicrobial agents, and its tendency to cause invasive infections in healthy individuals within the community. However, there has been an alarming increase in reports of multidrug-resistant hvKP, particularly carbapenem-resistant strains, causing nosocomial infections in critically ill or immunocompromised patients. This presents a significant challenge for clinical treatment. Early identification of hvKP is crucial for timely infection control. Notably, identifying hvKP has become confusing due to its prevalence in nosocomial settings and the limited predictive specificity of the hypermucoviscosity phenotype. Novel virulence predictors for hvKP have been discovered through animal models or machine learning algorithms, while standardization of identification criteria is still necessary. Timely source control and antibiotic therapy have been widely employed for the treatment of hvKP infections. Additionally, phage therapy is a promising alternative approach due to escalating antibiotic resistance. In summary, this narrative review highlights the latest research progress in the development, virulence factors, identification, epidemiology of hvKP, and treatment options available for hvKP infection.

Invasive infection caused by hypervirulent Klebsiella pneumoniae (HvKP) has been reported worldwide. Most of the patients are community population, related to diabetes mellitus (DM), chronic liver disease and other basic diseases, which prone to systemic migratory infection. In this study, we collected 377 patients with community acquired Klebsiella pneumoniae liver abscess in our hospital from January 2013 to December 2018, 65.8% of whom were male, and 49.6% had DM. Patients with DM are prone to eye and central nervous system (CNS) infection, which need continuous local abscess drainage during treatment. Among them, patients with poor blood glucose control have a higher rate of blood stream infections (BSI). 219 strains of HvKP were obtained, with K1/K2 Serotype accounted for 81.7%. The incidence of BSI in K2 patients was higher than that in K1 patients. The PCR results indicate that the carrying rate of virulence genes (rmpA、areo、kfu、allS、iroN、magA、uge、wcaG) in K1/K2 type strains is significantly higher than that in non K1/K2 type strains. ST23 and ST65 are the most common multilocus sequence typing (MLST), which belong to K1 and K2 Serotype respectively. All of HvKP strains showed high sensitivity to commonly used clinical antibiotics other than ampicillin, with 54.3% of the strains exhibiting high viscosity characteristics. Meanwhile, 35 classic Klebsiella pneumoniae (cKP) strains were collected, and their serum typing is mainly non K1/K2. The carrying rate of virulence genes and viscosity degree in HvKP are significantly higher than those in cKP. Primary liver abscess caused by HvKP is prone to multiple tissue and organ infections, but it shows higher sensitivity to most commonly used antibiotics in clinical practice except for ampicillin. After effective treatment, the overall prognosis of patients is better. This study analyzes the pathogenic characteristics of HvKP and elaborates on the clinical characteristics of patients, which can provide reference for clinical and scientific research work.
高毒力肺炎克雷伯菌(hypervirulent Klebsiella pneumoniae，HvKP)造成侵袭性感染已在全球范围内被广泛报道，其感染者主要集中在患有糖尿病(diabetes mellitus，DM)、慢性肝病等基础疾病的社区人群，且容易发生全身迁徙性感染。本研究收集了本院2013年1月~2018年12月社区获得性肺炎克雷伯菌肝脓肿患者377名，男性占65.8%，其中49.6%有DM。DM患者易发生眼部及中枢神经系统(central nervous system，CNS)感染，治疗过程中更需要持续的局部脓肿引流，其中血糖控制差的患者继发血流感染(bloodstream infections，BSI)的比率更高。共获得HvKP菌株219株，K1/K2血清型占总数81.7%，K2型患者发生BSI的比率高于K1型。PCR检测结果表明，毒力基因(rmpA、areo、kfu、allS、iroN、magA、uge、wcaG)在K1/K2型菌株的携带率明显高于non-K1/K2型，ST23和ST65是最常见的多位点序列分型(multilocus sequence typing, MLST)，分别属于K1及K2血清型。另外收集35株经典肺炎克雷伯菌(classic Klebsiella pneumoniae，cKP)，其血清分型主要以非K1/K2型为主。HvKP的毒力基因携带率及黏性程度明显高于cKP，前者造成的原发性肝脓肿患者易出现多组织器官感染，但对除氨苄西林以外的临床常用抗菌药物表现出更高敏感性，经过有效的治疗，患者的总体预后较好。本研究对社区获得性高毒肺炎克雷伯菌的病原学特征进行分析，并结合患者临床特征进行阐述，可对临床及科研工作起到一定参考价值。.

This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp).
Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted.
Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105.
In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

Hypervirulent Klebsiella pneumoniae (hvKP) has emerged as a novel variant of K. pneumoniae, exhibiting distinct phenotypic and genotypic characteristics that confer increased virulence and pathogenicity. It is not only responsible for nosocomial infections but also community-acquired infections, including liver abscesses, endophthalmitis, and meningitis, leading to significant morbidity and mortality. HvKP has been reported all over the world, but it is mainly prevalent in Asia Pacific, especially China. Moreover, hvKP can acquire carbapenemase genes resulting in the emergence of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP), which possesses both high virulence and drug resistance capabilities. Consequently, CR-hvKP poses substantial challenges to infection control and presents serious threats to global public health. In this paper, we provide a comprehensive summary of the epidemiological characteristics, virulence factors, and mechanisms underlying carbapenem resistance in hvKP strains with the aim of offering valuable insights for practical prevention strategies as well as future research.

UNASSIGNED: Respiratory infections are a major global health concern, with Klebsiella pneumoniae standing out due to its evolving antibiotic resistance. This study compares the resistance profiles of hypervirulent Klebsiella pneumoniae (hvKP) and classical Klebsiella pneumoniae (cKP), aiming to shed light on their clinical implications.
UNASSIGNED: We analyzed 86 cases, comprising 42 hvKP and 44 cKP strains, using comprehensive antimicrobial susceptibility testing and clinical data evaluation to assess antibiotic tolerance and resistance mechanisms.
UNASSIGNED: Our findings reveal distinct resistance patterns between hvKP and cKP, highlighting the role of chromosomal mutations and plasmid-mediated gene transfer in conferring antibiotic resistance. Notably, hvKP strains exhibited unique resistance trends, including the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases, differing from those of cKP.
UNASSIGNED: This research underscores the importance of continuous surveillance and the development of targeted therapies against antibiotic-resistant Klebsiella pneumoniae. It emphasizes the critical need for judicious antibiotic use and novel therapeutic approaches to combat respiratory infections caused by these increasingly resistant pathogens.

BACKGROUND: The occurrence of multidrug-resistant and hypervirulent Klebsiella pneumoniae (MDR-hvKp) worldwide poses a great challenge for public health. Few studies have focused on ST218 MDR-hvKp.
METHODS: Retrospective genomic surveillance was conducted at the Peking University Third Hospital from 2017 and clinical information was obtained. To understand genomic and microbiological characteristics, antimicrobial susceptibility testing, plasmid conjugation and stability, biofilm formation, serum killing, growth curves and whole-genome sequencing were performed. We also assessed the clinical and microbiological characteristics of ST218 compared with ST23.
RESULTS: A total of eleven ST218 Kp isolates were included. The most common infection type was lower respiratory tract infection (72.7%, 8/11) in our hospital, whereas ST23 hvKp (72.7%, 8/11) was closely associated with bloodstream infection. Notably, nosocomial infections caused by ST218 (54.5%, 6/11) was slightly higher than ST23 (36.4%, 4/11). All of the ST218 and ST23 strains presented with the virulence genes combination of iucA + iroB + peg344 + rmpA + rmpA2. Interestingly, the virulence score of ST218 was lower than ST23, whereas one ST218 strain (pPEKP3107) exhibited resistance to carbapenems, cephalosporins, β-lactamase/inhibitors and quinolones and harbored an ~ 59-kb IncN type MDR plasmid carrying resistance genes including blaNDM-1, dfrA14 and qnrS1. Importantly, blaNDM-1 and qnrS1 were flanked with IS26 located within the plasmid that could successfully transfer into E. coli J53. Additionally, PEKP2044 harbored an ~ 41-kb resistance plasmid located within tetA indicating resistance to doxycycline.
CONCLUSIONS: The emergence of blaNDM-1 revealed that there is great potential for ST218 Kp to become a high-risk clone for MDR-hvKp, indicating the urgent need for enhanced genomic surveillance.

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