Phenotypic and genomic characterization of two novel lytic bacteriophages targeting multidrug-resistant Pseudomonas aeruginosa.

Bacteriophages, which are natural bacterial predators, demonstrate potential as safe and effective biological control agents against drug-resistant infections. This study aims to characterize the biological properties of the novel lytic phage vB_SmaS_QH3 and comprehensively evaluate its efficacy in preventing and controlling clinically multidrug resistance Stenotrophomonas maltophilia infections using both in vivo and in vitro models. The phage was isolated from hospital sewage using the multidrug resistant S. maltophilia no. 3738 as the host. Transmission electron microscopy (TEM) was used to observe phage morphology, and the host range was determined via spot assays. Proliferation kinetics, including multiplicity of infection (MOI), adsorption rate, and one-step growth curves, were analyzed. Stability was assessed under various physicochemical conditions. Based on Illumina whole-genome sequencing data, bioinformatics tools were employed for gene annotation, functional prediction, and phylogenetic analysis. Antimicrobial activity was assessed using in vitro and in vivo models. A lytic phage vB_SmaS_QH3 was isolated from hospital sewage. TEM revealed that it belongs to the class Caudoviricetes, featuring an icosahedral head (62 ± 3 nm) and a non-contractile long tail (121 ± 5 nm). Although the phage has a narrow host range, it exhibits cross-genus infectivity, lysing S. maltophilia (11/81) and Pseudomonas aeruginosa (3/24). The optimal MOI for phage vB_SmaS_QH3 is 0.01, with an adsorption rate of 49.16% within 20 min, a latent period of 40 min, a lytic period of 50 min, and a burst size of 41.67 plaque-forming units/cell. The phage remained stable at 4-60°C, at pH 3-11, and in chloroform, but it was completely inactivated following 20-min exposure to UV irradiation. Genomic analysis showed a linear double-stranded DNA genome of 43,085 bp with a GC content of 54.2%, containing 54 predicted ORFs, and no virulence or antibiotic resistance genes were detected. In vitro, vB_SmaS_QH3 effectively inhibited bacterial growth within 9 h. In vivo, it significantly improved the survival rate of Galleria mellonella larvae infected with S. maltophilia, regardless of the treatment timing. vB_SmaS_QH3 is a narrow host range lytic phage with a safe genome and excellent stability. It exhibits significant antibacterial activity both in vitro and in vivo, making it a promising candidate for therapeutic applications.

Antimicrobial resistance (AMR) in Escherichia coli, particularly among animal-derived isolates, poses a major threat to public and veterinary health. With conventional antibiotics becoming increasingly ineffective against multidrug-resistant (MDR) strains, alternative solutions are urgently needed. Lytic bacteriophages, known for their host specificity and potent antibacterial activity, are promising therapeutic options. However, limited genomic data on phages from diverse ecological contexts hinder a comprehensive understanding of their diversity and functional potential. This study aimed to isolate, characterize, and assess the therapeutic potential of lytic bacteriophages targeting MDR E. coli isolated from livestock. Phages were enriched from the sewage samples using an MDR E. coli host. Plaque morphology was assessed for lytic characteristics, and Transmission Electron Microscopy (TEM) was used for morphological identification. The thermal and pH stabilities were assessed under controlled incubation conditions. The host range was determined using 20 MDR E. coli strains from cattle, buffalo, and goats. One-step growth experiments were performed to determine the latent period and burst size of the phages. Whole-genome sequencing and annotation were performed to determine the genetic features, taxonomic classification, safety, and phylogenetic relationships. Three lytic Escherichia phage BASUE2, BASUE7, and BASUE10, were isolated, producing clear plaques indicative of strong lytic activity. All phages remained viable between 25 °C and 42°C and within pH 6–9, but were inactivated at 80°C and at highly acidic or alkaline conditions. BASUE7 exhibited the broadest host range, lysing 75% of MDR E. coli isolates, followed by BASUE2 (65%) and BASUE10 (55%), while none of the phages were active against K. pneumoniae, S. aureus, or Salmonella spp. Genome analysis revealed double-stranded DNA genomes of approximately 157 kb (BASUE2), 50 kb (BASUE7), and 48 kb (BASUE10), with G + C contents ranging from 44.62% to 45.47%. Taxonomically, BASUE2 was classified under Ackermannviridae, whereas BASUE7 and BASUE10 belonged to Siphoviridae, all within Caudovirales. All phages were predicted to be strictly lytic and lacked genes associated with lysogeny, antibiotic resistance, or virulence. Phylogenetic analysis revealed a distinct cluster of BASUE2, suggesting a divergent evolutionary origin from BASUE7 and BASUE10. This study presents the isolation of genetically safe, environmentally stable, and broadly active lytic phages effective against MDR E. coli of animal origin. These findings highlight the potential of these phages for use in veterinary therapy. However, further in vivo validation and development of phage cocktails are necessary to advance their clinical applications. Graphical representation of isolation and characterization of E. coli bacteriophages from sewage samples.

BackgroundVibrio parahaemolyticus is a marine bacterium causing seafood-associated gastrointestinal illness in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. Bacteriophages have emerged as promising biocontrol agents against V. parahaemolyticus. This study characterizes Vibrio phage VPK8, focusing on host specificity, efficiency of plating (EOP) variability across V. parahaemolyticus isolates from diverse sources and other Vibrio species, morphology, genomic features, and bacteriolytic potential.MethodsVibrio phage VPK8 was isolated from blood cockles in Thailand using a mixed-host approach and purified via the double-layer agar method. Host specificity was evaluated using spot assays and EOP measurements against 120 Vibrio strains, including AHPND-associated, clinical, and seafood isolates. Phage morphology was characterized by transmission electron microscopy (TEM), while genomic features were analyzed using next-generation sequencing. Lytic characteristics, including latent period and burst size, were determined through one-step growth curves, and bacterial growth reduction was evaluated over a 24-h.ResultsVibrio phage VPK8 is a lytic phage with a 42,866 bp linear double-stranded genome, G + C content of 49.4%, and 48 coding sequences. Phylogenetic analysis grouped it within the Autographiviridae family, showing 95.96% similarity to Vibrio phage vB_VpaP_MGD1. Viral proteomic analysis placed VPK8 within the Pseudomonadota host group. Spot assays indicated broad lytic activity, but EOP analysis revealed high infectivity in clinical and seafood V. parahaemolyticus isolates, as well as some V. cholerae and V. mimicus strains. TEM revealed an icosahedral head (~ 60 nm) and a short tail. At a multiplicity of infection of 0.01, VPK8 exhibited a latent period of 25 min, a burst size of 115, and effectively inhibited the reference host V. parahaemolyticus PSU5124 within 6 h, maintaining its lytic activity and stability for over 24 h.ConclusionsThis study provides a detailed characterization of Vibrio phage VPK8 which exhibits targeted infectivity with high EOP in clinical and seafood V. parahaemolyticus isolates, as well as selected Vibrio species. Its stable lytic performance, rapid replication, and genomic safety suggest its potential for phage-based applications. Further studies should explore its in vivo efficacy and the genetic features contributing to phage resistance mechanisms, enhancing its potential applicability in managing Vibrio-related diseases.

Acinetobacter baumannii is an opportunistic pathogen and a major causative agent of hospital-acquired infections. This pathogen can acquire various antibiotic resistance genes, including those conferring resistance to last-resort antibiotics such as carbapenems. MDR A. baumannii is known to cause several infections, including pneumonia and urinary tract infections. Consequently, there is an urgent need to explore alternative therapies, and bacteriophage (phage) has emerged as a promising therapeutic approach for combating multidrug-resistant (MDR) infections. This study investigates the therapeutic potential of specific bacteriophages against MDR, particularly carbapenem-resistant A. baumannii, and evaluates lytic activity against 41 clinical isolates of MDR A. baumannii. The phages morphotypes were identified by transmission electron microscope. The stability of these phages was assessed under different conditions, including pH (2, 3, 4, 7, and 10-12), temperature (-80, -20, 4, 37, 50, 60, 70, and 80 oC), UV exposure (15, 30, 45, 60. 75, 90). Their antibacterial activity was also evaluated using a time-killing assay. Bacteriophage Insensitive Mutants (BIM) was assessed by MOI of 100. Genomic characterization was performed to predict protein-coding genes, life cycle, and suitability for therapeutic applications. Additionally, the safety and therapeutic efficacy of the phage were assessed using a cell viability MTT assay on adenocarcinomic human alveolar basal epithelial (A549) cells to evaluate the ability to rescue the lung cells from infection. Two phages, vB_AbaP_ZC2 (ΦZC2) and vB_AbaM_ZC3 (ΦZC3), were isolated from hospital wastewater in Egypt. The phages demonstrated lytic activity against 24.3% (n = 10) and 31.7% (n = 13) of the isolates, respectively. Phage ΦZC2 demonstrated high EOP values (0.75-1) against AB23 and AB26, moderate activity on AB34 and AB35 (EOP = 0.19), and low or no activity on AB10, AB24, and AB31. Similarly, phage ΦZC3 exhibited high EOP on AB24 (EOP = 1), moderate levels on AB12, AB29, and AB38, while showing low or no efficacy against the remaining tested isolates. The morphotypes of ΦZC2 and ΦZC3 are podovirus and myovirus, respectively. The two phages were amplified using a bioreactor and reached titers of approximately 10¹⁰ PFU/ml in 2L.ΦZC2 was stable at a pH range from 3 to 12 approximately 108 PFU/ml, while ΦZC3 was stable at a pH range from 3 to 11 approximately 109 PFU/ml compared to pH 7. ΦZC2 was stable at -80, 37, and 50°C approximately 108 PFU/ml, while ΦZC3 was stable at -80, 37,50, 60, and 70°C with approximately 109 PFU/ml compared to 4°C. Additionally, the ΦZC2 phage exhibited stability at 90min, while ΦZC3 phage exhibited stability at 75min of exposure to UV light. The optimum MOI at which the ΦZC2 and ΦZC3 significantly reduced bacterial growth 0.1 and 0.01, respectively. The BIM frequency was higher for phage ΦZC3 compared to ΦZC2, indicating a slightly greater emergence of phage-resistant mutants with ΦZC3. Whole genome sequencing and annotation did not identify markers for lysogeny or antibiotic resistance. Phylogenetic analysis classified ΦZC2 and ΦZC3 within the genera of Obolenskvirus and Friunavirus, respectively. ΦZC3 was selected for its broad host range to be evaluated for rescuing A549 cells from MDR A. baumannii infection. ΦZC3 phage was not cytotoxic to A549 cells and rescued lung cells cocultured, reducing the concentration of bacteria by approximately 5 logs with different MOIs, after 6h of incubation. In this study, the two lytic phages have antibacterial activity against MDR A. baumannii. particularly, ΦZC3 can be a potential therapy for pulmonary infections.

Non-typhoidal Salmonella (NTS) typically cause self-limiting enterocolitis, but can lead to life-threatening invasive diseases, particularly in sub-Saharan Africa. In Kenya, multidrug-resistant (MDR) NTS strains with increasing non-susceptibility to third-generation cephalosporins pose a growing public health threat. As traditional antimicrobial treatments become less effective, bacteriophages are emerging as a potential alternative. This study aimed to isolate and characterize bacteriophages targeting MDR and extended spectrum-β-lactamase (ESBL)-producing non-typhoidal Salmonella (NTS). Environmental samples were collected from seven sites in Nairobi City County, Kenya. Four NTS bacterial strains were used for phage enrichment, screening, and purification via spot tests and plaque assays. Phage efficacy was assessed in vitro by testing host range and efficiency of plating (EOP) against 12 Salmonella strains isolated in Kenya over different years. Ten selected broad-host-range phages were evaluated for thermal and pH stability and their ability to disrupt pre-formed NTS biofilms. Phage genomes were sequenced using the Illumina sequencing platform, and analyzed with bioinformatics tools to screen for antimicrobial resistance (AMR), lysogeny, virulence, and allergenic genes. The morphological characteristics of four representative phages were examined using Transmission Electron Microscopy. Thirty-one phages were isolated, with host ranges varying from lysing one strains to all 12 strains. Ten phages lysed more than 80% of the Salmonella strains and were selected for further characterization. Most phages exhibited high production EOP on at least one bacterial strain, except KE26 and KE28. All phages were stable from - 80°C to 40°C and pH 5 to 11, with noticeable but statistically insignificant biofilm disruption. Genome sizes ranged from 23,215bp to 159,981bp, and were free of known AMR, lysogeny, or virulence genes. Allergenicity screening identified no allergenic hits across most phages, with exception of KE23, which showed potential allergenic regions in its tail fiber and endolysin proteins. All phages belonged to class Caudoviricetes, with KE23, KE26, and KE28 exhibiting a myovirus-like morphotype, and KE15 displaying a siphovirus morphotype. This study identified phages with desirable safety and stability profiles for potential usage against MDR and ESBL-producing NTS infections. Further in vivo studies are recommended to evaluate their therapeutic potential. Non-typhoidal Salmonella (NTS) typically cause self-limiting enterocolitis but can lead to life-threatening invasive diseases. In Kenya, multidrug-resistant (MDR) NTS strains with increasing nonsusceptibility to third-generation cephalosporins have been reported, posing a significant public health concern that requires urgent attention. Bacteriophages are increasingly being considered as an alternative treatment for MDR bacterial infections because of the growing ineffectiveness of conventional antibiotics. Our study reports the isolation and characterization of lytic Salmonella phages devoid of detectable antimicrobial resistance (AMR) genes, lysogeny potential, allergens or virulence factors. These attributes position them as promising candidates for therapeutic interventions against MDR NTS infections. These findings highlight the potential of our study phages as a therapy for drug-resistant NTS and underscore the need for further investigation into their clinical application against MDR strains.

Multidrug-resistant Shigella flexneri (MDR S. flexneri) serotype 2a is the predominant cause of shigellosis in China, presenting a major public health challenge amid escalating antibiotic resistance and limited treatment options. Bacteriophages are gradually emerging as a highly promising alternative to antibiotics because of its highly specific bactericidal ability. However, only 113 Shigella phage genomes are available in NCBI as of August 2025, highlighting the need for novel lytic phages targeting prevalent MDR strains. A novel lytic phage, vB_SflP_DSF2 (DSF2), was isolated from untreated sewage at the 305 Hospital of PLA using MDR S. flexneri 2a strain 301 as host. Morphology was examined by transmission electron microscopy. Host range and efficiency of plating were determined against 41 bacterial strains (33 Shigella, 6 Escherichia coli, and others) using double-layer agar spot assays. One-step growth curves, pH and thermal stability, and biological properties were assessed using standard plaque assays. The complete genome was sequenced via Illumina NovaSeq, with comparative genomic and phylogenetic analyses performed using VIRIDIC, TerL phylogeny, AlphaFold structural predictions, and Swiss-Model for protein structure comparisons. The DSF2 is a Schitoviridae phage with an elongated prolate head, short non-contractile tail. It produces haloed 1-2 mm plaques indicating depolymerase activity, with a 60-min latent period and 115 PFU/cell burst size. The DSF2 remains stable from 4 °C to 50 °C and active at pH 4-10, selectively lysing all S. flexneri serotype 2/X strains. Genomic analysis revealed that DSF2 possesses a 72,532 bp dsDNA genome with a G+C content of 44.89%, containing 89 predicted open reading frames. The DSF2 harbors no virulence or antibiotic resistance genes. Closest relative Shigella virus Moo19 shares 94.1% identity, defining the DSF2 as a new species. The prolate head of DSF2 closely resembles that of Escherichia coli phage PH444, driven by divergent Hoc-like head decoration, despite the conservation of capsid and portal proteins when compared to Shigella virus Moo19. The DSF2 represents a novel Schitoviridae species that expands the limited Shigella phage repertoire, offering precision biocontrol against MDR S. flexneri serotype 2/X with minimal microbiome disruption. Hoc-like head decoration likely drives DSF2's unique prolate morpholog through intercapsomer angular constraints.

This study aimed to isolate and characterize biological and genomic features of a phage infecting Enterococcus faecalis. The phage was isolated from environmental water and temperature and pH stability, one-step growth curve, and multiplicity of infection (MOI) were determined. Whole genome sequencing(WGS) and structural and functional annotations were performed. Its antibiofilm activity was also evaluated. The optimal MOI was 0.01, the latency period was 5min, and the burst size was 202 plaque forming unit (PFU). High phage survival rates were observed at between pH 4-10 and temperatures between 4-50°C. WGS andTransmission electron microscopy (TEM) showed that it was an Efquatrovirus representing siphovirus morphotyperespectively. It was named as Enterococcus phage Ef212 and has a linear 40,690bp double-stranded DNA with 45.3% G + C content (GenBank accession number: OR052631). BACPHLIP tool demonstrated that Enterococcus phage Ef212 is a lytic phage (88%). A total of 80 open reading frames (ORFs) were found and there were no antibiotic resistance genes, pathogenicity, virulence genes, or tRNAs in the phage genome. It was diverged from the most similar phages (identity, 88.35%; coverage, 89%) by phylogenetic analysis. Phage Ef212 shared a large part of its genome (60/80) with several other phages, yet some unique parts were found in their genomes. Host range analysis showed that phage Ef212 showed lytic activity against vancomycin-resistant and vancomycin-susceptible E. faecalis clinical isolates. This novel phage Ef212 showed the ability to inhibit and reduce the biofilm formation by around 42% and 38%, respectively. The biological and genomic features indicate that having an effective antibacterial activity, phage Ef212 seemed a promising therapeutic and biocontrol agent.

Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide. It can produce a range of debilitating infections, have a propensity for developing antimicrobial resistance, and present with a variety of potent virulence factors. This study investigated the sequence types (ST), phenotypic antimicrobial susceptibility profiles, and resistance and virulence genes among clinical isolates from urinary tract and skin and soft tissue infections. Fifty-six P. aeruginosa clinical isolates were obtained from six medical centers across five counties in Kenya between 2015 and 2020. Whole-genome sequencing (WGS) was performed to conduct genomic characterization, sequence typing, and phylogenetic analysis of the isolates. Results showed the presence of globally distributed high-risk clones (ST244 and ST357), local high-risk clones (ST2025, ST455, and ST233), and a novel multidrug-resistant (MDR) clone carrying virulence genes (ST3674). Furthermore, 31% of the study isolates were found to be MDR with phenotypic resistance to a variety of antibiotics, including piperacillin (79%), ticarcillin-clavulanic acid (57%), meropenem (34%), levofloxacin (70%), and cefepime (32%). Several resistance genes were identified, including carbapenemases VIM-6 (ST1203) and NDM-1 (ST357), fluoroquinolone genes, crpP, and qnrVCi, while 14 and 22 different chromosomal mutations were detected in the gyrA and parC genes, respectively. All isolates contained at least three virulence genes. Among the virulence genes identified, phzB1 was the most abundant (50/56, 89%). About 21% (12/56) of the isolates had the exoU+/exoS- genotype, while 73% (41/56) of the isolates had the exoS+/exoU- genotype. This study also discovered 12 novel lineages of P. aeruginosa, of which one (ST3674) demonstrated both extensive antimicrobial resistance and the highest number of virulence genes (236/242, 98%). Although most high-risk clones were detected in Nairobi County, high-risk and clones of interest were found throughout the country, indicating the local spread of global epidemic clones and the emergence of new strains. Thus, this study illustrates the urgent need for coordinated local, regional, and international antimicrobial resistance surveillance efforts.

Characterization and genomic analysis of a novel Pseudoalteromonas phage PS_L5

A pathogen of significance in the aquaculture sector, the Gram-negative marinebacterium Vibrio parahaemolyticus causes gastroenteritis associated with consumption of improperly prepared seafood. This bacterium can be controlled using lytic bacteriophages as an alternative to antibiotics.ϕVP-1 is alytic phage of V. parahaemolyticus that was isolated from an aquafarm water sample with the aim of assessing its potential as a bio-control agent and determining its physicochemical properties and genomic sequence. Morphological analysis by transmission electron microscopy and phylogenetic analysis based on the large terminase subunit gene showed that this phage belongs to the family Myoviridae. It could infect multiple-drug-resistant (MDR) V. parahaemolyticus and V. alginolyticus strains of mangrove and seafood origin. With a maximum adsorption time of 30 min, ϕVP-1 has a short latent period of 10 min with burst size of 44 particles/cell. Whole-genome sequencing was done using the Illumina platform, and annotation was done using GeneMarkS and Prodigal.The 150,764bp genome with an overall G+C content of 41.84% had 203 putative protein-encoding open reading frames, one tRNA gene, and 66 predicted promoters. A number of putative DNA replication and regulation, DNA packaging and structure, and host lysis genes were identified. Comparison ofthe ϕVP-1 genome sequence to those of knownVibriophages indicated littlediscernible DNA sequence similarity, suggesting that ϕVP-1 is a novelVibriophage.Sequence analysis revealed the presence of 64 potential ORFs with a T4-like genomic organization. In silico analysis suggested an obligate lytic life cycle and showedthe absence of lysogeny or virulence genes. The complete sequence ofϕVP-1 was annotated and deposited in the GenBank database (accession no. MH363700). The genetic features of this novel phage suggest that it might be applicable for phage therapy against pathogenic strains ofV. parahaemolyticus.

BackgroundStenotrophomonas maltophilia, an important opportunistic pathogen resistant to multiple antibiotics, necessitates alternative therapies. Phages, with their high specificity and bacteriolytic ability, are emerging as promising antibiotic alternatives. This study aimed to isolate and characterize a novel lytic phage targeting S. maltophilia and to evaluate its antibacterial potential.MethodsA novel lytic phage, vB_SmaS_QH16, was isolated from hospital sewage using S. maltophilia no.981 as the host. Phage morphology was analyzed using transmission electron microscopy (TEM), and genome sequencing and annotation were performed. Host range, efficiency of lysis (EOP), optimal multiplicity of infection (MOI), one-step growth curves, and physicochemical stability were also determined. Biofilm inhibition and eradication were assessed using crystal violet staining, MTT assays, and acridine orange fluorescence microscopy. Using Galleria mellonella and mouse infection models, the in vivo anti-infective effects of phages were evaluated.ResultsPhage vB_SmaS_QH16, a member of the class Caudoviricetes, has a 43,500 bp genome with 64 open reading frames (ORFs) and no virulence, antibiotic resistance, or lysogeny-related genes. It exhibits a broad host range, lysing 47.95% (35/73) of tested S. maltophilia strains. The optimal MOI was 0.01, with an average burst size of 37.69 PFU/cell. The phage is stable at 4–50 °C and pH 3.0–11.0 but is highly sensitive to UV light. It effectively inhibits biofilm formation and eradicates mature biofilms in a concentration-dependent manner. In vitro, the phage significantly suppresses bacterial growth, though resistant mutants emerge over time. In vivo, vB_SmaS_QH16 increases the survival rates of larvae and mice, with a higher MOI offering better protection.ConclusionsPhage vB_SmaS_QH16 shows therapeutic potential against S. maltophilia infections, characterized by a broad host range, efficient lytic capability, and biofilm-disrupting activity. Its stability and safety further support its clinical application potential. Future research should explore its biofilm disruption mechanisms and monitor resistance development. Additionally, since its efficacy has been validated in mammalian models, further studies can focus on advancing its clinical translation.

Antimicrobial resistance (AMR) has become a global public health concern, particularly in developing countries where antibiotics are often overused and misused. In Bolivia, the indiscriminate use of antibiotics, including (fluoro-)quinolones, has led to the proliferation of multidrug-resistant (MDR) Campylobacter spp., increasing the risk of resistance gene dissemination to other bacteria, and further deepening the AMR problem. To help mitigate the proliferation of MDR bacteria, bacteriophages can be a valuable complementary treatment to antibiotics. In the present study, we isolated and characterized three novel lytic bacteriophages with activity against (fluoro-)quinolone-resistant Campylobacter isolates and C. jejuni strains. The isolated bacteriophages, BMBo_CjP_006, BMBo_CjP_007, and BMBo_CjP_009, belong to the class Caudoviricetes and possess a linear double-stranded DNA genome. Their genome size ranges from 59 to 77 kb, with a GC-content between 42 to 46%. The 90, 144, and 146 predicted coding sequences (CDSs) of the different bacteriophages did not encode any antibiotic resistance, virulence, or lysogenic-associated genes, confirming their genetic safety and lytic nature. The isolated bacteriophages showed a narrow host range and lytic activity against nine (fluoro-)quinolone-resistant Campylobacter spp., including C. jejuni, with lytic activity varying at MOIs from 0.1 to 100, dependent on bacteriophage and host isolate. In addition, the bacteriophages were stable across a pH range of 4 to 10 and a temperature range of −20 °C to 70 °C. These characteristics make them promising for biotechnological applications due to their lytic activity, lack of resistance and virulence genes, and potential utility for product preservation.

Stenotrophomonas maltophilia (S. maltophilia) is an emerging opportunistic pathogen that exhibits resistant to a majority of commonly used antibiotics. Phages have the potential to serve as an alternative treatment for S. maltophilia infections. In this study, a lytic phage, A1432, infecting S. maltophilia YCR3A-1, was isolated and characterized from a karst cave. Transmission electron microscopy revealed that phage A1432 possesses an icosahedral head and a shorter tail. Phage A1432 demonstrated a narrow host range, with an optimal multiplicity of infection of 0.1. The one-step growth curve indicated a latent time of 10 min, a lysis period of 90 min, a burst size of 43.2 plaque-forming units per cell. In vitro bacteriolytic activity test showed that phage A1432 was capable to inhibit the growth of S. maltophilia YCR3A-1 in an MOI-dependent manner after 2 h of co-culture. BLASTn analysis showed that phage A1432 genome shares the highest similarity (81.46%) with Xanthomonas phage Xoo-sp2 in the NCBI database, while the query coverage was only 37%. The phage contains double-stranded DNA with a genome length of 61,660 bp and a GC content of 61.92%. It is predicted to have 79 open reading frames and one tRNA, with no virulence or antibiotic resistance genes. Phylogenetic analysis using terminase large subunit and DNA polymerase indicated that phage A1432 clustered with members of the Bradleyvirinae subfamily but diverged into a distinct branch. Further phylogenetic comparison analysis using Average Nucleotide Identity, proteomic phylogenetic analysis, genomic network analysis confirmed that phage A1432 belongs to a novel genus within the Bradleyvirinae subfamily, Mesyanzhinovviridae family. Additionally, phylogenetic analysis of the so far isolated S. maltophilia phages revealed significant genetic diversity among these phages. The results of this research will contribute valuable information for further studies on their morphological and genetic diversity, will aid in elucidating the evolutionary mechanisms that give rise to them.

BackgroundThe presence of antimicrobial resistance and virulence genes in Escherichia coli allows them to survive and cause infections. The close contact between humans and pets can reinforce the risk of transmitting resistant and virulent bacteria between them.ObjectivesThis study aims to compare the patterns of the presence of tetracycline and streptomycin resistance genes, as well as important virulence genes in E. coli isolated from faeces of healthy dogs and their owners.MethodsPolymerase chain reactions were performed for detection of antimicrobial resistance (tetA, tetB, tetC, tetD, strA and strB) and virulence (fimH, iss, sitA and malX) genes in 144 faecal E. coli isolates from 28 dog–owner pairs and 16 humans who did not keep any pets as controls.ResultsAmong the investigated antimicrobial resistance and virulence genes, tetA (52.1%) and fimH (86.8%) genes had the highest prevalence. No statistically significant difference was found between the prevalence of antimicrobial resistance and virulence genes in isolates of dogs and their owners. In total, 46.4% of dog–owner pairs had the same patterns of presence or absence of six antimicrobial resistance genes, 50.0% had the same patterns of presence or absence of four virulence genes and 25.0% had the same patterns of presence or absence of all 10 tested genes.ConclusionThe presence of antimicrobial‐resistant virulent E. coli in humans and pets may predispose them to infections that are hard to cure with conventional antibiotics. Notable frequency of dogs’ and their owners’ E. coli isolates with similar patterns of antimicrobial resistance and virulence genes may indicate the possibility of sharing virulent antimicrobial resistant E. coli between them.

Escherichia coli is a major cause of hospital-acquired infections in China, including urinary tract, bloodstream, and intestinal infections. Given the rising prevalence of antibiotic-resistant E. coli, phages are increasingly regarded as promising alternatives to conventional antibiotics. Henuyfy11N was isolated using the double-layer agar method and characterized via transmission electron microscopy in this study. Biological assays included stability under varying pH and temperature, UV sensitivity, host range, optimal multiplicity of infection, adsorption rate, and one-step growth curve. In vitro lytic activity against extended-spectrum β-lactamase (ESBL)-producing E. coli and biofilm eradication capacity was assessed. Whole-genome sequencing enabled phylogenetic, synteny (the analysis of conserved blocks of genetic sequence between different genomes), and functional annotation analyses. In vivo, the therapeutic efficacy was evaluated in a mouse infection model. Phage Henuyfy11N has not yet been classified. It demonstrated high lytic activity, a short latent period, and a burst size of 57.1 PFU/cell. The phage remained stable across a broad pH range (3-11) and temperatures up to 70℃. Its circular double-stranded DNA genome (41,103 bp, G + C% 50.88) contains 54 open reading frames, with no tRNA, virulence, or antibiotic resistance genes. Genomic and phylogenetic analyses revealed close relatedness to phage BUCT789. Henuyfy11N effectively lysed ESBL-producing E. coli, disrupted biofilms, and significantly improved survival in the mouse infection model. Henuyfy11N shows high host specificity, efficient lytic activity, rapid replication, and a safe genomic profile, demonstrating some potential as a therapeutic agent against ESBL-producing E. coli infections.IMPORTANCEThe widespread use of antibiotics has led to increasing antibiotic resistance, which is a growing global health concern. Therefore, the development of novel antimicrobial therapy that can cure drug-resistant bacteria-induced infections is imperative. Phages are of increasing interest as natural enemies of bacteria, with clear advantages in antibacterial applications. In this study, by using extended-spectrum β-lactamase (ESBL)-producing Escherichia coli 2025011N as a host, we successfully isolated and purified Escherichia phage Henuyfy11N and conducted a series of experiments to verify its genomic character and biological character. Our findings revealed that the phage exhibited excellent tolerance to a broad spectrum of pH and wide temperature range. Phage Henuyfy11N was effective in disrupting mature biofilm, and no genes for virulence, lysogenic, integrase, or AMRs were found in the genome. Besides, Henuyfy11N showed promising antibacterial effects in vivo and in vitro, indicating potential as a therapeutic agent against ESBL-producing E. coli infections.