Genomic Characterization and Therapeutic Potential of the Lytic Bacteriophage Curly against Klebsiella pneumoniae in Human Innate Immune Cells and a Murine Pneumonia Model
Findings identify Curly as a promising bacteriophage candidate against K. pneumoniae and support further evaluation of its host range, resistance profile, and therapeutic potential.
Klebsiella pneumoniae is an important zoonotic opportunistic pathogen. Of particular concern is the emergence of multidrug‐resistant hypervirulent K. pneumoniae (MDR‐hvKP), which represents a serious public health threat. Bacteriophage (phage) therapy has emerged as a promising solution to combat antibiotic‐resistant K. pneumoniae infections. Here, we characterize a novel lytic phage, vB_Kp_H122, that specifically targets K57 capsular‐type MDR‐hvKP. vB_Kp_H122 exhibited the characteristic morphology of a siphovirus and demonstrated efficient infection kinetics, with an optimal multiplicity of infection (MOI) of 0.001 and a latent period of ~5 min. It also demonstrated considerable stability across a pH range of 4–11 and at temperatures from 4°C to 50°C, as well as potent activity against K. pneumoniae biofilms. The phage has a linear double‐stranded DNA genome of 46,077 bp with a G + C content of 47.61% and belongs to a novel species within the genus Roufvirus. Its genome contained no identifiable genes associated with lysogeny, virulence, or antibiotic resistance, supporting its therapeutic safety. In a mouse infection model, a single dose of vB_Kp_H122 at 2 × 106 PFU significantly reduced bacterial loads in organs, alleviated pathological damage, and provided complete protection against lethal challenge with K57 MDR‐hvKP. These findings suggest that vB_Kp_H122 may have potential as an antibacterial candidate against K57 MDR‐hvKP isolates.
Cuilong Fan, Qiu Xu, S. Schwarz et al.· Transboundary and Emerging D...· 0 citations
Introduction The increasing prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has posed a major challenge to clinical infection management. Phage therapy represents a promising alternative against multidrug-resistant bacterial infections; however, its application is limited by the scarcity of effective therapeutic phages. Therefore, the identification and characterization of novel phages are urgently needed. Methods A novel Klebsiella pneumoniae phage was isolated and purified from hospital wastewater using the double-layer agar plate method with K. pneumoniae ATCC 700603 as the host strain. A total of 95 bacterial strains were used to evaluate the host range and lytic activity of the phage. The biological characteristics of the phage, including optimal multiplicity of infection (MOI), one-step growth curve, and stability under different environmental conditions (temperature, pH, ethanol, and ultraviolet exposure), were systematically investigated. Morphological characterization was performed by transmission electron microscopy. Whole-genome sequencing and bioinformatics analyses were conducted to determine genomic characteristics, taxonomic classification, and the presence of antibiotic resistance genes, virulence factors, or lysogeny-related genes to assess the therapeutic potential and biosafety of the phage. Results The novel phage vB_KpnD_A2 exhibited potent lytic activity against multidrug-resistant Klebsiella pneumoniae strains. Host range analysis showed that vB_KpnD_A2 lysed 57.1% of carbapenem-resistant K. pneumoniae isolates and 63.6% of extended-spectrum β-lactamase-producing strains, while displaying specificity toward K. pneumoniae. The phage showed excellent biological properties, with an optimal MOI of 10-⁶, a latent period of 40 min, and a burst size of 8.6×10⁴PFU/cell. In addition, vB_KpnD_A2 maintained infectivity over a broad temperature range (4-70°C) and pH range (pH 2-13). Whole-genome analysis identified vB_KpnD_A2 as a strictly lytic Webervirus phage within the family Drexlerviridae, without detectable antibiotic resistance genes, virulence factors, or lysogeny-associated genes, supporting its therapeutic potential and biosafety. Discussion The newly characterized phage vB_KpnD_A2 exhibited potent lytic activity against selected multidrug-resistant Klebsiella pneumoniae isolates, favorable genomic safety, and good environmental stability. These findings support its potential as a therapeutic phage candidate for CRKP control and expand anti-Klebsiella phage resources for future phage-based interventions against antimicrobial-resistant bacterial infections.
Haipeng Zhang, Qianwen Cheng, Xiaohong Nie et al.· Frontiers in Microbiology· 0 citations
Klebsiella pneumoniae (K. pneumoniae) is a critical pathogen responsible for a wide range of severe infections. Its escalating resistance to frontline antimicrobials, particularly carbapenems, severely compromises existing therapeutic options and undermines current treatment strategies. This clinical challenge is further exacerbated by the emergence of multidrug-resistant (MDR) strains and hypervirulent lineages, which substantially complicate infection management and worsen patient outcomes. Ultimately, this alarming convergence of resistance and virulence presents a severe public health threat that necessitates the urgent development of novel therapeutic approaches; in this regard, phage therapy is emerging as a promising strategy. Bacteriophages and their derivatives have garnered increasing recognition as targeted antibacterial agents, demonstrating potent activity against carbapenemase producing and MDR K. pneumoniae in vitro models. They effectively lyse MDR strains, disrupt biofilm formation, and have shown promising therapeutic efficacy across various in vivo models. To ensure safety in clinical applications, genome sequencing is essential to identify and exclude undesirable genes, thereby determining therapeutic suitability. Here, we have sequenced and analyzed the genomic characteristics of seven lytic bacteriophages targeting carbapenemase-producing MDR K. pneumoniae. Genomic evaluation confirmed that these phages are strictly lytic and revealed the presence of distinct lysis modules across the genomes. Phylogenetic analysis showed that these phages cluster within four genera of Caudoviricetes: Taipeivirus, Drulisvirus, Przondovirus, and Webervirus. The phage genomes ranged from 15,773 to 166,437 base pairs (bp) in length and encoded core structural and replication modules. Furthermore, distinct lytic modules predicted to have function in bacterial capsule degradation were identified across the genomes. Importantly, the absence of known virulence or toxin genes guarantees therapeutic safety and satisfies rigorous genomic criteria for clinical translation. These findings underscore the potential of these phages as a promising alternative for combating MDR and hypervirulent K. pneumoniae infections. Future in vivo investigations and clinical trials are essential to validate their therapeutic efficacy, safety profile, and optimal treatment protocols.
Assefa Asnakew Abebe, A. Birhanu, Molelegne Bite et al.· BMC Microbiology· 0 citations
Klebsiella aerogenes is an opportunistic pathogen increasingly associated with healthcare-associated infections and severe respiratory complications. However, it's in vivo adaptation during host colonization remains poorly understood. We characterized three clonal K. aerogenes ST93 isolates sequentially recovered on days 4, 14, and 19 of hospitalization from a patient with fatal viral pneumonia and concurrent abdominal sepsis. While initially identified generically as Klebsiella spp. by automated clinical systems, Whole-Genome Sequencing and Average Nucleotide Identity confirmed their identity at the species level. Genomic analysis revealed an in vivo plasmid curing event characterized by the loss of a 6.1 kb blaOXA-232-carrying plasmid in the final isolate (18281). Despite this plasmid loss, isolate 18281 sustained persistent carbapenem resistance due to a conserved ompK36 chromosomal mutation. Phenotypically, 18281 exhibited significantly higher lethality in Galleria mellonella larvae and induced progressive body weight loss in dexamethasone-treated mice compared to the other isolates. This enhanced virulence strongly correlated with a tissue-specific, compartmentalized transcriptional reprogramming, characterized by a marked 15.66-fold up-regulation of the yersiniabactin gene irp1 within hepatic tissues, whereas the colibactin gene clbA was consistently downregulated in vivo across both isolates and organs. Our findings demonstrate that intra-host microevolution of K. aerogenes involves structural plasmid instability and targeted transcriptional remodeling during host adaptation. This localized adaptive behavior underscores the clinical threat posed by transitional clonal variants in critically ill patients undergoing viral-mediated immune dysregulation.
Luis Duarte-Zambrano, Neli Nava-Domínguez, J. P. Ramírez-Hinojosa et al.· International Microbiology· 0 citations
The rapid rise of multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) poses a serious threat in healthcare settings, as these bacteria can cause infections that are untreatable with conventional antibiotics. This crisis has driven the search for alternative therapies, one of which is bacteriophages. In this study, we isolated a novel bacteriophage, vB_AbaM_EA1 (E7 for short), from wastewater and evaluated its potential activity against clinical MDR A. baumannii strains. Phage E7 successfully infects (27.5%) of the 40 clinical MDR A. baumannii isolates we tested. Laboratory characterization suggested that E7 is a highly efficient lytic phage, rapidly attaching to bacterial cells and producing 1000 new viral particles. A key finding was its potent activity against bacterial biofilms, a major source of persistent infections. Not only did Phage E7 effectively prevent the formation of new biofilms, but more importantly disrupted mature ones, which typically confer higher resistance to antibiotics. Genome sequencing confirmed that Phage E7 is a new member of the Obolenskvirus genus and that its genome lacks genes encoding bacterial toxins, antibiotic resistance, or lysogenic factors, indicating a mandatory safety profile for therapeutic use. Furthermore, E7 remained stable across a broad temperature range (-20 to 45 °C) and pH values (5–9) that it might encounter during storage and administration. In conclusion, Phage E7 is a stable, safe, and highly effective candidate for phage therapy against infections by MDR A. baumannii. Its ability to target both planktonic bacteria and biofilms makes it a promising candidate for future development into clinical applications.
Ethar M. Amin, R. Aziz, Fathia E. Murad et al.· BMC Microbiology· 0 citations
Introduction Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with intrinsic and acquired resistance to multiple classes of antibiotics. Phage therapy has emerged as a promising approach to combat multidrug-resistant bacterial infections. Methods In this study, a lytic P. aeruginosa phage, WEN7, was isolated from hospital wastewater using PAO1-SZ1 as the host. We systematically characterized its morphology, growth kinetics, stability, host range, whole-genome sequence and bactericidal performance. Results WEN7 formed clear plaques. Transmission electron microscopy revealed an icosahedral 66 nm head attached to a contractile tail, with intact virions captured in both fully extended (140 nm total tail length) and sheath-contracted (60 nm) states—this classic myoviral morphology places the phage within the class Caudoviricetes. One-step growth curve analysis revealed a latent period of approximately 25 min and an average burst size of about 165 PFU/cell. WEN7 remained stable under various temperatures, pH values, UV exposure, and ethanol concentrations. The phage displayed a broad host range, lysing 19 of the 29 clinical P. aeruginosa isolates tested (65.5%). Its genome is a circular double-stranded DNA molecule of 66,379 bp with a GC content of 55.62%. Notably, no known antibiotic resistance genes or virulence factors were annotated. WEN7 shares the highest genomic similarity with the Pseudomonas phage PCCM_PaP004 (genus Pbunavirus). At low multiplicities of infection, WEN7 showed strong bactericidal activity in both milk and hospital wastewater. Discussion Together, these results indicate that phage WEN7 is a promising candidate for phage therapy against multidrug-resistant P. aeruginosa infections.
Chang Wen, Xiao-Hong Xiao, Jin-Yi Chen et al.· Frontiers in Microbiology· 0 citations