Broad-spectrum lytic potential of endolysins derived from prophages of clinical Salmonella isolates

Genomic and functional characterization of novel therapeutic lytic bacteriophages targeting multidrug-resistant Enterobacter cloacae

The alarming rates at which extensively drug-resistant (XDR) and pandrug-resistant (PDR) Enterobacter cloacae in hospitals are increasing has begun to severely limit treatment options, and thus the urgency for alternative interventions, including bacteriophage therapy. The purpose of the study was to isolate and molecularly characterize phages that can infect E. cloacae , and, furthermore, to assess the antimicrobial efficacy of the four novel lytic bacteriophages (MMRP1, MMRP2, MMRP3, and MMRP4) against antimicrobial-resistant E. cloacae isolates and to evaluate their potential as alternative therapeutic strategies. These novel phages were characterized by plaque morphology, transmission electron microscopy (TEM), host range testing, thermal and chloroform stability assays, bacterial reduction assays, and whole-genome sequencing (WGS). Among 27 clinical isolates, MDR, XDR, and PDR phenotypes were observed in 20 (74.1%), six (22.2%), and one (3.7%) isolates, respectively. All four phages produced clear lytic plaques (0.5–3.0 mm) with titers reaching up to 6 × 10 10 PFU/mL, and the phage cocktail lysed 81.4% (22 of 27 isolates) of clinical isolates with high host specificity. TEM revealed that all four E. cloacae -infecting phages (MMRP1, MMRP2, MMRP3, and MMRP4) belong to the class Caudoviricetes , exhibiting icosahedral capsids, tailed morphology, and double-stranded DNA genomes, consistent with current ICTV classification criteria. Whole genome sequencing and comparative phylogenetic analysis further resolved the taxonomic placement of these phages at the family level, positioning MMRP1 within the family Demerecviridae and MMRP4 within the family Straboviridae . All phages were stable from −20 to 40 °C and were unaffected by exposure to chloroform. Phage cocktail reduced bacterial OD₆₀₀ to ≤ 0.3 within 4 h in the bacterial reduction test. WGS revealed large circular dsDNA genomes of ~132 kbp (MMRP1) and ~149 kbp (MMRP4), GC content of 38%, and modular architectures encoding structural, lytic, and replication gene modules. The most striking and highlighted suggestion that in vitro evaluation of MMRP1 and MMRP4 are highly recommended to more deeper future experimental studies to combat MDR E. cloacae nosocomial infections supported by genomic foundation and eventually, the possibility to be suitable for phage-engineering applications in clinical settings.

Prophage landscapes in clinical MRSA: safety profiling and discovery of Lys81, a broad-spectrum bacteriolytic enzyme

Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat to global healthcare, requiring novel therapeutic strategies. Prophages, latent phage genomes integrated into bacterial chromosomes, are important resources for antimicrobial development due to their genomic stability and genetic engineering potential. In this study, we performed genomewide sequencing on 329 MRSA isolates to predict prophage sequences, followed by analyses of these prophages—including examinations of virulence genes, antibiotic resistance genes, homologous proteins of pathogenic MRSA phages, and functional predictions of these homologous proteins—to evaluate their safety and value as genetic engineering scaffolds and to screen for novel broadspectrum bacteriolytic enzymes. Our data indicate that 85.7% (282/329) of strains carried complete prophage sequences; 64 strains lacked virulence factors or genes, meeting the core criteria for safe vectors. Resistance screening found only 6 prophages carried msrA , confirming the biosafety of the remaining strains. A significant correlation existed between prophage virulence gene capacity and genomic structure (R 2 = 0.99986684, p = 3.64e-69). High-virulence clusters (>10 factors) showed high structural similarity; 10 characteristic sequences linked to S. aureus phages and their prevalence patterns were identified via conserved motif analysis. Collinearity analysis with reference to virulent MRSA phages and 3D structural predictions of orthologous proteins identified two lysozymes and a host-recognition device. Notably, Lys81, an N-acetylmuramoyl-L-alanine amidase ortholog, was prioritized and characterized as a broad-spectrum lytic enzyme. Our data show Lys81 has key properties: (1) Broad-spectrum antibacterial activity, lysing 52.3% (23/44) of clinical S. aureus strains and cross-acting against Gram-positive bacteria such as Pseudomonas aeruginosa and Listeria ; (2) Excellent environmental adaptability, maintaining activity at pH 5.0 and 0°C, with 25 mM Na + and Ca 2 + enhancing function; (3) Potent biofilm clearance, achieving 83% MRSA biofilm reduction at 50 μg/mL; and (4) Favorable in vivo safety/efficacy, eradicating MRSA infections in lung organoid models with minimal cytotoxicity. This study establishes a theoretical foundation for the clinical translation of MRSA prophages, positioning Lys81 as a novel candidate for treating drug-resistant bacterial infections.

Genomic and Phenotypic Characterization of Two Novel Enterobacter Phages With EDTA-Enhanced Antibiofilm Activity.

Multidrug-resistant members of the Enterobacter cloacae complex (ECC) are increasingly linked to difficult-to-treat infections and biofilm-mediated antimicrobial tolerance. Here, two lytic phages, vB_EhoIP_HHH and vB_EluM_RZH, displaying podovirus-like and myovirus-like morphology, respectively, were isolated from the River Chelt. HHH has a 39,582 bp genome (51.2% GC, 63 ORFs), while RZH has a 174,197 bp genome (39.4% GC, 314 ORFs), with neither genome carrying antimicrobial resistance, virulence or lysogeny-associated genes. VIRIDIC and VICTOR analyses placed HHH within Kayfunavirus and RZH within Karamvirus, supporting their classification as distinct species. Both phages demonstrated rapid adsorption, short latent periods and stability across physiological pH and temperature ranges. A phage cocktail targeting MDR ECC strain was evaluated with EDTA against established biofilms. Crystal violet assays showed the greatest biomass reduction at MOI 10 with 0.5-0.75 mM EDTA. Bliss independence analysis revealed localized synergy within this window but significant overall antagonism at higher EDTA concentrations. CFU enumeration confirmed greater activity against 24 h than 48 h biofilms. The optimized combination also reduced recoverable bacteria in a fibroblast infection model while maintaining low LDH release. These findings identify two novel lytic Enterobacter phages and support a narrow EDTA concentration window for enhanced phage-mediated antibiofilm activity.

Characterizing the pangenome-based virulome and resistome of uropathogenic Escherichia coli from Ecuador

ABSTRACT Uropathogenic Escherichia coli (UPEC) is one of the leading causes of bacterial Urinary Tract Infections (UTIs) worldwide. In this study, we characterized the UPEC pangenome in Ecuador, focusing on the virulome and resistome of 142 genomes sequenced using whole-genome sequencing (WGS). Our analysis revealed a structure of 16,732 genes, including a conserved core of 3,385 genes and a dynamic accessory genome of 13,347 genes. The ST131 lineage was predominant, accounting for 42.3 % of the total, whereas the O25:H4 serotype was present in 42.96 % of the cases. The resistome was associated with a large accessory gene repertoire, with 3,712 significant associations identified across 29 antimicrobial resistance genes (ARGs). The virulome was organized into 23 main profiles, with adhesion and siderophore genes being the most abundant. These findings demonstrate the presence of high-risk variants in terms of virulence and resistance circulating in Ecuador, which requires the establishment of improved health control strategies in the region.

Identification of KL29 type acinetobacter baumannii phage of obolenskvirus subfamily and tail-associated depolymerase activity

Interactions of antiphage defense systems in the ESKAPE pathogen plasmids.

BACKGROUND The global rise of multidrug resistant (MDR) ESKAPE pathogens represents a serious threat to antimicrobial therapy. While phage therapy has re-emerged as a promising alternative, its effectiveness may be compromised by bacterial defense systems, particularly those encoded on plasmids. Comprehensive surveillance of the distribution, diversity, and mobilome context of plasmid-encoded defense systems in ESKAPE pathogens remains key to the design of effective phage therapies. RESULTS We analyzed 7,330 dereplicated plasmids from ESKAPE pathogens to characterize the prevalence, diversity, and co-occurrence of plasmid-encoded antiphage defense systems. Conjugative plasmids, especially from Enterobacter spp. and K. pneumoniae, harbored the highest prevalence and diversity of defense systems. Defense-positive plasmids showed larger sizes, higher GC content, and frequent co-occurrence of resistance genes, especially from β-lactam, aminoglycoside, and sulfonamide classes, along with transposable elements such as IS6, IS3, and Tn3. Random forest and correlation analyses confirmed TEs and ARGs as dominant predictors of defense system occurrence. Network analysis revealed structured and partially conserved interactions among defense genes, TEs, and ARGs. RM and CBASS systems were frequently linked to beta-lactam and aminoglycoside resistance genes, as well as TEs such as IS6 and IS3. Recurrent associations such as RM-IS6, RM-IS1380, CBASS-IS3 and RM-OXA suggest shared horizontal transfer mechanisms. CONCLUSIONS Plasmid-encoded antiphage defense systems in ESKAPE pathogens are widespread, structured, and linked to ARGs and mobile genetic elements. These findings highlight the contribution of plasmids to the dissemination of phage-resistance traits, underscore the importance of the mobilome in shaping phage-resistance landscapes in multidrug-resistant pathogens, and support the incorporation of plasmid defense profiling into phage therapy design.