Wei Zhang

Background: The global rise in hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses a severe threat to public health. However, the evolutionary mechanisms underlying hypervirulence in carbapenem-resistant strains remain incompletely understood. Here, we investigated the genetic basis of hypervirulence in a prevalent carbapenem-resistant lineage. Methods: Whole-genome sequencing and comparative genomic analyses were performed on 112 locally collected carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates collected from 2020 to 2022 and 4,636 publicly available K. pneumoniae genomes, including 557 hv-CRKP. Results: Among 66 hv-CRKP identified locally, the ST11-KL64 lineage was dominant, accounting for 87.9%. As against the classical fusion-plasmid model, these isolates harbored candidate chromosomal virulence-locus configurations, supported by convergent short-read sequencing evidence, with insertion sequences identified at or near the inferred breakpoint regions. IS-positive hv-CRKP isolates included in the phenotypic comparison showed significantly greater virulence in Galleria mellonella infection models than the IS-negative hv-CRKP comparator isolates (log-rank test, p < 0.0001). Global phylogenetic analysis showed that strains carrying related candidate chromosomal virulence-locus configurations clustered within a distinct lineage, with the currently available genomes in this group predominantly originating from China. Conclusion: This study identified recurrent candidate chromosome-associated virulence-locus configurations in ST11 hv-CRKP that are distinct from canonical fusion-plasmid architectures. This evidence extends the current models beyond plasmid fusion and underscores the value of genomic surveillance strategies capable of detecting chromosomal virulence, which may be overlooked when surveillance focuses solely on virulence plasmids. Defining this evolutionary trajectory provides a foundation for infection prevention and control, as well as for designing targeted interventions to limit the spread of these high-risk pathogens. Using multiple lines of genomic evidence, we established a framework for assessing candidate chromosomal localizations and identifying breakpoint-associated read signatures.

Purpose: This study aims to investigate the prevalence of resistance genes, efflux pump genes, and biofilm-forming genes in carbapenem-resistant Acinetobacter baumannii, as well as the clinical characteristics of patients infected with carbapenem-resistant Acinetobacter baumannii, in order to provide guidance for rational clinical prevention and control. Methods: This study included 244 patients with CRAB infections admitted to a tertiary hospital in Zhangjiakou, China, between June 2021 and December 2024. Statistical analysis was performed using SPSS 27.0 software to investigate potential risk factors for mortality associated with CRAB. Conventional PCR amplification was used to qualitatively detect resistance genes, efflux pump genes, and biofilm-related genes in isolated CRAB strains. Biofilm formation capacity was assessed using the crystal violet staining method. The study analyzed the association between the aforementioned genetic and phenotypic characteristics, biofilm formation capacity, and antibiotic resistance. Results: A total of 244 CRAB isolates were collected in this study, primarily from sputum (55.33%), followed by bronchoalveolar lavage fluid (26.23%). The majority of isolates originated from the ICU (48.77%), followed by the Department of Respiratory Medicine (31.15%). PCR results showed that the predominant resistance genes in CRAB isolates from this hospital were blaOXA-51 and blaOXA-23, at 89.3% and 87.3%, respectively. The detection rates for blaNDM, blaOXA-58, and blaOXA-24 were 1.6%, 1.2%, and 0.4%, respectively. The efflux pump genes adeB, adeR, adeS, and adeJ all showed high detection rates, while the adeG gene was not detected. The biofilm-related genes with generally high detection rates were bap (240/244, 98.4%), abaI (227/244, 93.0%), and ompA (223/244, 91.4%); the detection rates of the remaining genes ranged between 84.8% and 90.6%. Of the 244 CRAB strains tested in this study, the vast majority (97.95%) possessed biofilm-forming ability. The OD values of the strains were determined using the crystal violet method, with weak biofilm-forming strains (47.54%) and moderate biofilm-forming strains (44.26%) predominating; strong biofilm-forming strains accounted for 6.15%, while strains incapable of biofilm formation accounted for only 2.05%. Antimicrobial susceptibility testing revealed that 99.58% of the 244 CRAB strains were resistant to piperacillin/tazobactam, and 99.15% were resistant to imipenem and meropenem. Resistance rates for amoxicillin/clavulanate, cefepime, ceftazidime, ceftiofur, ciprofloxacin, and levofloxacin, all exceeded 95%, while 3.08% were resistant to tigecycline. No resistance to polymyxin was observed. Results of multivariate logistic regression analysis showed that age (OR = 1.044, 95% CI: 1.009– 1.079, P < 0.05) and white blood cell count (OR = 1.106, 95% CI: 1.029– 1.188, P < 0.05) were independent risk factors for mortality in CRAB infections. Conclusion: In this study, CRAB strains were primarily isolated from the lungs (81.56%) and mainly originated from the ICU and respiratory department. CRAB strains exhibited extremely high resistance rates to most commonly used antimicrobial agents; specifically, resistance rates to 9 antimicrobial agents, including piperacillin/tazobactam, imipenem, and meropenem, were all > 90%. Only tigecycline showed a relatively low resistance rate (3.08%), and no resistance to polymyxin was detected. The CRAB strains in our hospital primarily harbor the blaOXA-23 and blaOXA-51 resistance genes and commonly carry efflux pump and biofilm formation genes, suggesting that their resistance may be closely associated with carbapenemases, efflux pump systems, and biofilm formation.

Background: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) represents a critical public health threat in China, characterized by the convergence of multidrug resistance and hypervirulence. The emergence of ceftazidime/avibactam (CZA) resistance further complicates clinical management. This study aimed to elucidate the molecular epidemiology, risk factors, and resistance mechanisms of CZA resistance in CR-hvKP across China, providing evidence for targeted interventions. Methods: A single-center molecular epidemiological analysis was conducted on 81 Carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates collected. All isolates underwent whole-genome sequencing for MultiLocus Sequence Typing, capsule typing, and identification of resistance genes ( bla KPC-2 and blaNDM-1 ) and virulence factors ( iucA , iroB , rmpA , rmpA 2, and peg- 344). CZA resistance mechanisms were investigated through broth microdilution minimum inhibitory concentration (MIC) testing and bioinformatics analysis. Galleria mellonella infection models were employed to assess virulence potential. Risk factors were analyzed using multivariate regression of clinical variables. Phylogenetic reconstruction employed single-nucleotide polymorphism-based analysis. Results: ST11 accounted for 96.15% (50/52) of CR-hvKP isolates, with K64 being the predominant capsule type (92.31%, 48/52). Additionally, 98.77% (80/81) of CRKP carried ≥1 virulence gene; 64.2% (52/81) of isolates with all five virulence genes exhibited lethality. Galleria mellonella revealed that the survival rate of CR-hvKP was lower than that of carbapenem-resistant non-hypervirulent Klebsiella pneumoniae (p128 mg/L) without compromising carbapenem resistance, facilitated by a unique genetic context (TnpR_Tn3-ISKpn27- bla KPC-2-ISKpn6). Conclusion: China faces a rapid dissemination of ST11 CR-hvKP clones carrying diversified CZA resistance mechanisms. The convergence of hypervirulence and resistance in ST11 lineages—accelerated by invasive procedures and international transmission—demands enhanced genomic surveillance. CZA resistance arises through multiple pathways, necessitating combination therapies and stewardship programs limiting prolonged CZA use. Our findings underscore an urgent need for rapid diagnostics targeting emergent resistance determinants and infection control measures to contain high-risk clones.

Carbapenem-resistant Klebsiella pneumoniae (CRKP), a major nosocomial pathogen, poses an increasingly serious threat to human health. In the quest for innovative therapeutic modalities to treat CRKP infections, a newly discovered phage, vB_KpnM_JYSS3, which can specifically infect and lyse K2-type CRKP, was isolated and subjected to an array of comprehensive characterizations and genome sequencing. Furthermore, an analysis of the expression of the tail protein (ORF17) and the detection of its functions were carried out. Double-layer agar plates containing CRKP were used to isolate phages from hospital sewage samples. The phage's optimal multiplicity of infection (MOI), lytic spectrum, bactericidal activity, stability, and one-step growth curve were tested. The biofilm development ability of the CRKP strain 21AA2216 and the phage antibiofilm activity were determined using a 96-well microtitration plate. vB_KpnM_JYSS3 prevented biofilm generation and broke down mature biofilms. The whole-genome sequencing results revealed that vB_KpnM_JYSS3 is a toxic phage (nontemperate). The genome size of vB_KpnM_JYSS3 is 47,244 bp, and the G + C content is 48.48 mol%. A total of 71 coding sequences (CDSs) were predicted. Protein function prediction revealed that the tail protein (ORF17) of vB_KpnM_JYSS3 may possess depolymerase activity, consistent with the halo phenomenon around the plaque. ORF17 was expressed, purified, and named Dep17. Dep17 can degrade the capsular polysaccharide (CPS) surrounding K2-type CRKP, and it can also enhance the activity of polymyxin B against K2-type CRKP biofilms. Phage vB_KpnM_JYSS3 and its novel depolymerase Dep17 may represent promising strategies for combating infections resulting from biofilm-forming and drug-resistant K2-type CRKP.

Introduction: Hypervirulent and multidrug-resistant Klebsiella pneumoniae (hvKP and MDR-KP) are significant public health threats. This study aimed to isolate a lytic bacteriophage targeting these high-risk strains, systematically characterize its biological properties, genomic features, and therapeutic efficacy, and establish a foundation for clinical phage therapy and novel antimicrobial development.Methods: The phage vB_Kp_XP4 was isolated from river water using the double-layer agar plate method with the clinically isolated strain P4 as the host. Morphology was analyzed via transmission electron microscopy (TEM). Host range, pH, and thermal stability were assessed using spot assays and OD₆₃₀ measurements. One-step growth curves determined the latent period and burst size. Whole-genome sequencing and phylogenetic analysis were performed. Therapeutic efficacy and safety were evaluated in a Galleria mellonella infection model.Results: TEM revealed Phage vB_Kp_XP4 as a tailed phage with an icosahedral head and a long, flexible tail. It lysed an hvKP strain (carrying rmp, peg, iuc, iro genes) and an MDR-KP strain (resistant to carbapenems, fluoroquinolones, etc.), with an optimal MOI of 0.1 and latent period Drulisvirus, family Autographiviridae. In the Galleria mellonella model, vB_Kp_XP4 prolonged survival of P4-infected larvae (P < 0.001)Conclusion: Phage vB_Kp_XP4 exhibits high stability, specificity, potent lytic activity, and no undesirable genes, demonstrating effective in vivo therapeutic efficacy, suggest its potential for clinical applications against Klebsiella pneumoniae infections. The presence of multiple halos during plaque formation further enhances its research value. The complete genome sequence has been submitted to GenBank under accession number PP663283.

The high morbidity and mortality rates associated with carbapenem-resistant hypervirulent Klebsiella pneumoniae have become a global public health problem. We analyzed the whole genome sequence and epidemiological data of 81 carbapenem-resistant Klebsiella pneumoniae strains and conducted sensitivity experiments to explore the optimal concentration of carbapenem-resistant Klebsiella pneumoniae . We verified the resistance and virulence genes of carbapenem-resistant Klebsiella pneumoniae using polymerase chain reaction and compared virulence between carbapenem-resistant hypervirulent Klebsiella pneumoniae a ndcarbapenem-resistant-non-hypervirulent Klebsiella pneumoniae through Galleria mellonella assays.The prevalent types of multilocus sequence typing in our carbapenem-resistant Klebsiella pneumoniae strains were ST11 (69.14%, 56/81). ST11-K64-O1/O2v1 has become an epidemic type in our hospital, with drug-resistance genes dominated by bla _KPC-2 (90.12%, 73/81). Ceftazidime/avibactam has better antibacterial effects on carbapenem-resistant Klebsiella pneumoniae , and its resistance is mainly mediated by a resistance plasmid containing the bla _KPC-2 gene. 52 carbapenem-resistant hypervirulent Klebsiella pneumoniae strains carry the bla _KPC-2 , with a high detection rate of siderophore. Further research has shown that the genetic environment of the bla _KPC-2 contains TnpR_Tn3 and ISKpn27 upstream and ISKpn6 insertion sequences downstream. Galleria mellonella revealed that the survival rate of carbapenem-resistant hypervirulent Klebsiella pneumoniae was lower than that of carbapenem-resistant-non-hypervirulent Klebsiella pneumoniae , and the survival rate of carbapenem-resistant hypervirulent Klebsiella pneumoniae -K64 was lower than that of carbapenem-resistant hypervirulent Klebsiella pneumoniae -K47.The spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae strains is alarming, necessitating molecular monitoring of hypervirulent strains and strengthening the nosocomial infection prevention and control measures. Antibiotics should be used reasonably based on patients’ infection status and local epidemiological data. Importance: Carbapenem-resistant Klebsiella pneumonia is a global public health concern. We conducted a molecular epidemiological analysis of 81 carbapenem-resistant hypervirulent Klebsiella pneumoniae strains from our hospital and explored the resistance mechanism of ceftazidime/avibactam. We identified the bla _KPC-2 gene to be primarily responsible for carbapenem resistance in our hospital, and ST11-K64 was the dominant clone. A high-risk clone, ST307-K102, was detected, necessitating strengthened surveillance to prevent its spread. This study’s findings contribute to the optimization of antimicrobial management and indicate that genomic research can assist with the tracking of hospital infection outbreaks and the improvement of infection-control measures.

Purpose: This single-center study aims to investigate the epidemiological characteristics of clinically isolated Escherichia coli from 2014 to 2022. Methods: In vitro drug sensitivity of E. coli to 20 antibiotics was examined using the microbroth dilution method. A total of 7580 clinical E. coli strains were isolated from 2014 to 2022, among which 56.9% were identified as extended spectrum beta-lactamase-producing strains. The data were analyzed using the software WHONET5.6 and the R language platform. Results: Over the study period, carbapenem resistance rates increased by more than 50% (2022 [1.34%] vs 2014 [0.8%]) and the annual number of isolates showed an upward trend (1264 in 2022 vs 501 in 2014). Drug resistance rates were the highest for penicillin (75– 85%) and lowest for imipenem (1%). The resistance rate of strains isolated from male patients and sputum was found to be higher than that of female patients and urine, except for quinolones (p < 0.05). The drug resistance rates from high to low were penicillins (75– 85%), tetracycline (64%), quinolones (64– 67%), sulfamethoxazole (59.3%), cephalosporins (22– 72%), aztreonam (34%), chloramphenicol (21%), amikacin (2.8%), colistin (1.4%), meropenem (1.1%), and imipenem (1%). Urine, sputum, and blood accounted for 51%, 16.6%, and 10.6% of the samples, respectively. A greater number of female patients were included more than male patients (4798[63.3%] vs 2782[26.7%]). Patients aged 50– 80 accounted for 64.2% of those surveyed. Conclusion: Carbapenems remain the optimal choice for treating extended spectrum beta-lactamase-producing E. coli infections (sensitivity rate: 98%). Colistin (87.7%) and amikacin (87%) exhibited good antibacterial activities against carbapenem-resistant E. coli. Long-term and continuous epidemiological surveillance of E. coli can facilitate the development of preventive strategies and control policies.

Introduction. The Candida parapsilosis complex can be divided into C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis subtypes. It is uncommon for drug sensitivity tests to type them. Gap Statement. In routine susceptibility reports, drug susceptibility of C. parapsilosis complex subtypes is lacking. Aim. The aim of this study is to investigate the antifungal susceptibility and clinical distribution characteristics of the C. parapsilosis complex subtypes causing deep infection in patients. Methodology. Non-repetitive strains of C. parapsilosis complex isolated from deep infection from 2017 to 2019 were collected. Species-level identification was performed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer and confirmed using ITS gene sequencing, when necessary. Antifungal susceptibility testing was performed using the Sensititre YeastOne system method. Results. A total of 244 cases were included in the study, including 176 males (72.13 %, 60.69±13.43 years) and 68 females (27.87 %, 60.21±10.59 years). The primary diseases were cancer (43.44 %), cardiovascular disease (25.00 %), digestive system diseases, (18.44 %), infection (6.97 %), and nephropathy (6.15 %). Strains were isolated from the bloodstream (63.11 %), central venous catheters (15.16 %), pus (6.56 %), ascites (5.74 %), sterile body fluid (5.33 %), and bronchoalveolar lavage fluid (BALF, 4.09 %). Of the 244 C. parapsilosis complex strains, 179 (73.26 %) were identified as C. parapsilosis sensu stricto, 62 (25.41 %) were C. orthopsilosis, and three (1.23 %) were C. metapsilosis. Only one C. parapsilosis sensu stricto strain was resistant to anidulafungin, micafungin, caspofungin, and voriconazole, and it was non-wild-type (NWT) to amphotericin B. Furthermore, six C. parapsilosis sensu stricto strains were resistant to fluconazole, and one was dose-dependent susceptible. Five C. parapsilosis sensu stricto strains were NWT to posaconazole. Only one C. orthopsilosis strain was NWT for anidulafungin, micafungin, caspofungin, fluconazole, voriconazole, amphotericin B, and posaconazole, while the rest of the strains were wild-type. Conclusion. C. parapsilosis sensu stricto was the main clinical isolate from the C. parapsilosis complex in our hospital. Most strains were isolated from the bloodstream. The susceptibility rate to commonly used antifungal drugs was more than 96 %. Furthermore, most of the infected patients were elderly male cancer patients.

Response to Letter in regards to Long-Term Characteristic of Clinical Distribution and Resistance Trends of Carbapenem-Resistant and Extended-Spectrum β-Lactamase Klebsiella pneumoniae Infections: 2014–2022 [Letter]