Icosahedral Head Research Articles

PluMu—A Mu-like Bacteriophage Infecting Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia, an economically important lung disease in pigs. In draft genomes of two Cypriot clinical A. pleuropneumoniae isolates (MIDG3457 and MIDG3459), we previously identified single genomic regions with homology to Mu-like bacteriophage and presented preliminary evidence of active phage. Here, updated Phastest genomic analysis identified two loci in both MIDG3457 and MIDG3459 that were predicted to encode proteins with high homology to, and whose organisation was characteristic of, Mu-like phages. Phylogenetically, the closest matches were with Mannheimia Vb and Glaesserella SuMu phages. Phastest scored the loci as “complete”, indicating they produced active phage. PCR amplification of the Mu-like phage c and tail genes from DNase-treated polyethylene glycol 8000 (PEG)-precipitated supernatants of MIDG3457 and MIDG3459 (grown in either Brain Heart Infusion-NAD or Grace’s Insect Medium-NAD broth) indicated the presence of intact virions. The phages from MIDG3457 and MIDG3459 were named PluMu 3457-1, 3457-2, and PluMu 3459-1 and PluMu 3459-2, respectively. Transmission electron microscopy (TEM) of the PEG-precipitated supernatants of broth-grown MIDG3459 identified virions with icosahedral heads and tails, consistent with other Mu-like phages. We conclude that MIDG3459 produces an active Mu-like phage.

Biological characteristics of the bacteriophage LDT325 and its potential application against the plant pathogen Pseudomonas syringae.

Bud blight disease caused by Pseudomonas syringae is a major bacterial disease of tea plants in China. Concerns regarding the emergence of bacterial resistance to conventional copper controls have indicated the need to devise new methods of disease biocontrol. Phage-based biocontrol may be a sustainable approach to combat bacterial pathogens. In this study, a P. syringae phage was isolated from soil samples. Based on morphological characteristics, bacteriophage vB_PsS_LDT325 belongs to the Siphoviridae family; it has an icosahedral head with a diameter of 53 ± 1 nm and nonretractable tails measuring 110 ± 1 nm. The latent period and burst size of the phage were 10 min and 17 plaque-forming units (PFU)/cell, respectively. Furthermore, an analysis of the biological traits showed that the optimal multiplicity of infection (MOI) of the phage was 0.01. When the temperature exceeded 60°C, the phage titer began to decrease. The phage exhibited tolerance to a wide range of pH (3-11) and maintained relatively stable pH tolerance. It showed a high tolerance to chloroform, but was sensitive to ultraviolet (UV) light. The effects of phage LDT325 in treating P. syringae infections in vivo were evaluated using a tea plant. Plants were inoculated with 2 × 107 colony-forming units (CFU)/mL P. syringae using the needle-prick method and air-dried. Subsequently, plants were inoculated with 2 × 107 PFU/mL LDT325 phage. Compared with control plants, the bacterial count was reduced by 1 log10/0.5 g after 4 days in potted tea plants inoculated with the phage. These results underscore the phage as a potential antibacterial agent for controlling P. syringae.

Characterization of ES10 lytic bacteriophage isolated from hospital waste against multidrug-resistant uropathogenic E. coli.

Escherichia coli is the major causative agent of urinary tract infections worldwide and the emergence of multi-drug resistant determinants among clinical isolates necessitates the development of novel therapeutic agents. Lytic bacteriophages efficiently kill specific bacteria and seems promising approach in controlling infections caused by multi-drug resistant pathogens. This study aimed the isolation and detailed characterization of lytic bacteriophage designated as ES10 capable of lysing multidrug-resistant uropathogenic E. coli. ES10 had icosahedral head and non-contractile tail and genome size was 48,315 base pairs long encoding 74 proteins. Antibiotics resistance, virulence and lysogenic cycle associated genes were not found in ES10 phage genome. Morphological and whole genome analysis of ES10 phage showed that ES10 is the member of Drexlerviridae. Latent time of ES10 was 30 min, burst size was 90, and optimal multiplicity of infection was 1. ES10 was stable in human blood and subsequently caused 99.34% reduction of host bacteria. Calcium chloride shortened the adsorption time and latency period of ES10 and significantly inhibited biofilm formation of host bacteria. ES10 caused 99.84% reduction of host bacteria from contaminated fomites. ES10 phage possesses potential to be utilized in standard phage therapy.

Characterization of a new Pseudomonas aeruginosa Queuovirinae bacteriophage.

The ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.

Characterization of a novel high-efficiency cracking Burkholderia gladiolus phage vB_BglM_WTB and its application in black fungus

Burkholderia gladiolus (B. gladiolus) is foodborne pathogenic bacteria producing bongkrekic acid (BA), which causes food poisoning and has a mortality rate of up to 40 % or more. However, no drugs have been reported in the literature for the prevention and treatment of this infection. In this study, a phage was identified to control B. gladiolus. The novel phage vB_BglM_WTB (WTB), which lyse B. gladiolus with high efficiency, was isolated from sewage of Huaihe Road Throttle Well Sewage Treatment Plant in Hefei. Transmission electron microscopy showed that WTB had an icosahedral head (69 ± 2 nm) and a long retractable tail (108 ± 2 nm). Its optimal temperature and pH ranges to control B. gladiolus were 25 °C -65 °C and 3–11 respectively. The phage WTB was identified as a linear double-stranded DNA phage of 68, 541 bp with 60.04 % G + C content, with a long latent period of 60 min. Phylogenetic analysis and comparative genetic analysis indicated that phage WTB has low identity (<50 %) with other phages, with the highest similarity to Burkholderia phage Maja (25.7 %), which showed that it does not belong to any previous genera recognized by the International Committee on Taxonomy of Viruses (ICTV) and was a candidate for a new genus within the Caudoviricetes. We have submitted a new proposal to ICTV to create a new genus, Bglawtbvirus. No transfer RNA (tRNA), virulence associated and antibiotic resistance genes were detected in phage WTB. Experimental results indicated that WTB at 4 °C and 25 °C had excellent inhibition activity against B. gladiolus in the black fungus, with an inhibition efficiency of over 99 %. The amount of B. gladiolus in the black fungus was reduced to a minimum of 89 CFU/mL when treated by WTB at 25 °C for 2 h. The inhibition rate remained at 99.97 % even after 12 h. The findings showed that the phage WTB could be applied as a food-cleaning agent for enhancing food safety and contributed to our understanding of phage biology and diversity.

Development of carrageenan-immobilized lytic coliphage vB_Eco2571-YU1 hydrogel for topical delivery of bacteriophages in wound dressing applications

Bacteriophages are employed as cost-effective and efficient antibacterial agents to counter the emergence of antibiotic-resistant bacteria and other host bacteria in phage therapy. The increasing incidence of skin wounds is a significant concern in clinical practice, especially considering the limitations of antibiotic therapy. Furthermore, the lack of an effective delivery system that preserves the stability of bacteriophages hampers their clinical implementation. In recent years, there has been a growing amount of research on bacteriophage applications in veterinary and biomedical sciences. In our study, lytic coliphage vB_Eco2571-YU1 was isolated against pathogenic Escherichia coli host bacteria, and hydrogel wound dressing materials were fabricated with marine polysaccharide carrageenan (carr-vB_Eco2571-YU1) for their antibacterial activity. Transmission electron microscopy (TEM) morphology identified it as a Myoviridae coliphage with an icosahedral head length and width of approximately 60 and 56.8 nm, respectively, and a tail length of 119.7 nm. The one-step growth curve of coliphage revealed a latent period of 10 min, a rise period of 15 min, and a burst size of 120 virions per cell. The bacteriolytic activity of unimmobilized coliphages was observed within 2 h; however, strain-specific phage resistance was acquired after 9 h. In contrast, carr-vB_Eco2571-YU1 showed a sharp decline in the growth of bacteria in the log phase after 2 h and did not allow for the acquisition of phage resistance by the E. coli strain. The stability of coliphage under different pH, temperature, osmolarity, detergents, and organic solvents was evaluated. We also studied the long-term storage of carr-vB_Eco2571-YU1 hydrogels at 4 °C and found that the titer value decreased during a time-dependent period of 28 days. These hydrogels were also found to be hemocompatible using a hemolysis assay. The addition of plasticizer (0.6 % (w/v)) to the carrageenan (2 % (w/v)) to prepare carr-vB_Eco2571-YU1 hydrogels showed a decrease in compressive strength with enhanced elasticity. This phage therapy using polymeric immobilization of bacteriophages is a promising next-generation wound dressing biomaterial alternative to conventional wound and skin care management.

Characterization of Pseudomonas aeruginosa bacteriophages and control hemorrhagic pneumonia on a mice model.

Pseudomonas aeruginosa is one of the most common pathogens causing hemorrhagic pneumonia in Chinese forest musk deer. Multidrug-resistant P. aeruginosa is frequently isolated from the lungs of affected musk deer in Shaanxi Province, China. With the increasing bacterial drug resistance, commonly used antibiotics have shown limited efficacy against drug-resistant P. aeruginosa. Therefore, phages have garnered attention as a promising alternative to antibiotics among researchers. In this study, phages vB_PaeP_YL1 and vB_PaeP_YL2 (respectively referred to as YL1 and YL2) were isolated from mixed sewage samples from a farm. YL1 and YL2 exhibit an icosahedral head and a non-contractile short tail, belonging to the Podoviridae family. Identification results demonstrate good tolerance to low temperatures and pH levels, with minimal variation in potency within 30 min of UV irradiation. The MOI for both YL1 and YL2 was 0.1, and their one-step growth curve latent periods were 10 min and 20 min, respectively. Moreover, both single phage and phage cocktail effectively inhibited the growth of the host bacteria in vitro, with the phage cocktail showing superior inhibitory effects compared to the single phage. YL1 and YL2 possess double-stranded DNA genomes, with YL1 having a genome size of 72,187 bp and a total G + C content of 55.02%, while YL2 has a genome size of 72,060 bp and a total G + C content of 54.98%. YL1 and YL2 are predicted to have 93 and 92 open reading frames (ORFs), respectively, and no ORFs related to drug resistance or lysogeny were found in both phages. Genome annotation and phylogenetic analysis revealed that YL1 is closely related to vB_PaeP_FBPa1 (ON857943), while YL2 is closely related to vB_PaeP_FBPa1 (ON857943) and Phage26 (NC041907). In a mouse model of hemorrhagic pneumonia, phage cocktail treatment showed better control of the disease and significantly reduced lung bacterial load compared to single phage treatment. Therefore, YL1 and YL2 have the potential for the prevention and treatment of multidrug-resistant P. aeruginosa infections.

Characterization and genomic analysis of a lytic Stenotrophomonas maltophilia short-tailed phage A1432 revealed a new genus of the family Mesyanzhinovviridae.

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.

Characterization of the novel temperate Escherichia coli phage phiStx2k.

A novel temperate phage, phiStx2k, was induced from a clinical Escherichia coli isolate producing Shiga toxin (Stx) 2k. The phage particles have an icosahedral head (50 nm in diameter) and a long non-contractile tail (149 nm long). The phage genome consists of 46,647 bp of double-stranded DNA with an average G + C content of 51%. Genome sequence comparisons suggested that phiStx2k represents a new genus in the class Caudoviricetes. phiStx2k was capable of converting non-Stx-producing E. coli strains to Stx producers. These results expand our knowledge on the characteristics of Stx phages and highlight the potential risks of the emergence of Stx-producing strains or novel pathogens via horizontal gene transfer.

Complete genome sequence of Xanthomonas phage M29, a new member of Foxunavirus isolated in the Czech Republic.

The newly discovered Xanthomonas phage M29 (Xp M29) is the first lytic phage infecting Xanthomonas campestris pv. campestris (Xcc) that was isolated from cabbage leaves in the Czech Republic. The phage consists of icosahedral head approximately 60nm in diameter and a probably contractile tail of 170nm. The complete genome size was 42891bp, with a G + C content of 59.6%, and 69 ORFs were predicted on both strands. Pairwise nucleotide comparison showed the highest similarity with the recently described Xanthomonas phage FoX3 (91.2%). Bacteriophage Xp M29 has a narrow host range infecting 5 out of 21 isolates of Xcc. Xp M29 is a novel species in a newly formed genus Foxunavirus assigned directly to the class Caudoviricetes.

Characterization of a Vibriophage Infecting Pathogenic Vibrio harveyi.

Bacterial diseases caused by Vibrio spp. are prevalent in aquaculture and can lead to high mortality rates among aquatic species and significant economic losses. With the increasing emergence of multidrug-resistant Vibrio strains, phage therapy is being explored as a potential alternative to antibiotics for biocontrol of infectious diseases. Here, a new lytic phage named vB_VhaS_R21Y (R21Y) was isolated against Vibrio harveyi BVH1 obtained from seawater from a scallop-farming area in Rongcheng, China. Its morphology, infection cycle, lytic profile, phage stability, and genetic features were characterized. Transmission electronic microscopy indicated that R21Y is siphovirus-like, comprising an icosahedral head (diameter 73.31 ± 2.09 nm) and long noncontractile tail (205.55 ± 0.75 nm). In a one-step growth experiment, R21Y had a 40-min latent period and a burst size of 35 phage particles per infected cell. R21Y was highly species-specific in the host range test and was relatively stable at pH 4-10 and 4-55 °C. Genomic analysis showed that R21Y is a double-stranded DNA virus with a genome size of 82,795 bp and GC content of 47.48%. Its high tolerance and lytic activity indicated that R21Y may be a candidate for phage therapy in controlling vibriosis in aquacultural systems.

Genomic Analysis of Two Cold-Active Pseudoalteromonas Phages Isolated from the Continental Shelf in the Arctic Ocean.

Cold-active bacteriophages are bacterial viruses that infect and replicate at low temperatures (≤4 °C). Understanding remains limited of how cold-active phage-host systems sustain high viral abundance despite the persistently low temperatures in pelagic sediments in polar seas. In this study, two Pseudoalteromonas phages, ACA1 and ACA2, were isolated from sediment core samples of the continental shelf in the western Arctic Ocean. These phages exhibited successful propagation at a low temperature of 1 °C and displayed typical myovirus morphology with isometric icosahedral heads and contractile tails. The complete genome sequences of phages ACA1 and ACA2 were 36,825 bp and 36,826 bp in size, respectively, sharing almost the same gene content. These are temperate phages encoding lysogeny-related proteins such as anti-repressor, immunity repressor and integrase. The absence of cross-infection between the host strains, which were genomically distinct Pseudoalteromonas species, can likely be attributed to heavy divergence in the anti-receptor apparently mediated by an associated diversity-generating retroelement. HHpred searching identified genes for all of the structural components of a P2-like phage (family Peduoviridae), although the whole of the Peduoviridae family appeared to be divided between two anciently diverged tail modules. In contrast, Blast matching and whole genome tree analysis are dominated by a nonstructural gene module sharing high similarity with Pseudoalteromonas phage C5a (founder of genus Catalunyavirus). This study expands the knowledge of diversity of P2-like phages known to inhabit Peudoalteromonas and demonstrates their presence in the Arctic niche.

A novel Vibrio alginolyticus phage and its therapy application in Portunus trituberculatus larvae

Ubiquitous pathogenic marine Vibrio alginolyticus is a very important pathogenic factor causing the outbreaks of sever vibriosis in marine-cultured animals broadly, an opportunistic pathogen for human, and a major pathogen associated with seafood. Antimicrobial drug resistances increased considerably, making the development of alternative therapeutics urgently needed. Isolation and characterization of V. alginolyticus phage vB_Va_Val-yong3 was performed. vB_Va_Val-yong3 contains an icosahedral head and a flexible tail. The diameter of the head is 62 ± 4 nm. The length and width of the tail is 110 ± 9.5 nm and 8 ± 1 nm, respectively. Its burst size is 87.5 PFU/cell. vB_Va_Val-yong3 is euryhaline, chloroform-tolerant, relatively UV-tolerant, stable at 4–50 °C and pH 5–12. Its genome size and GC content are 42,534 bp and 45.6%. It shared the highest nucleotide sequence similarity of 29.67% and the highest intergenomic similarity of 16.3% with all the other phages in PASC scanning and VIRIDIC scanning. Both the values were far below the threshold (> 70%) for genus definition, which illustrated that vB_Va_Val-yong3 revealed a novel phage genus. In the proteomic tree, vB_Va_Val-yong3 and three unclassified Vibrio phages (HY01, ST2-1pr and Seahorse) formed a clade, which clustered with two other clades representing the Mesyanzhionvvidriae family and the Casjensvidriae family respectively. VirClust analysis revealed that 27.7% of genes were shared between Vibrio phages vB_Va_Val-yong3, HY01 and ST2-1pr, which was higher than the family demarcation criteria (> 17%). Far <17% of genes shared between the three Vibrio phages and other viruses (including phage Seahorse and the phages of Mesyanzhionvvidriae and Casjensvidriae family). We advocate the establishment of a new phage family harboring Vibrio phages vB_Va_Val-yong3, HY01 and ST2-1pr. Phage therapy tests on swimming crab Portunus trituberculatus larvae were performed via immersion. vB_Va_Val-yong3 increased the survival rate of the larvae from 33.33% to 51.67% (p < 0.01). Very satisfactorily, the survivors of the infected larvae treated with vB_Va_Val-yong3 could molt and grow normally, while the survivors of the untreated larvae infected with V. alginolyticus all had the problems of molting and slow growth. This brings the very valuable results: all the survivors of the control groups were dwarf, while that of the experimental groups were normal. Food processing tests were also performed using vB_Va_Val-yong3 to eliminate V. alginolyticus in sashimi. The relative bacteriostatic rate of vB_Va_Val-yong3 in sashimi was 97.58% (p < 0.01). Results suggest that vB_Va_Val-yong3 has good potential to be developed as therapeutic agent against V. alginolyticus. This study enriches knowledge about Vibrio phages and lays a foundation for the application of the V. alginolyticus phage.

Asymmetric Structure of Podophage GP4 Reveals a Novel Architecture of Three Types of Tail Fibers

Bacteriophage tail fibers (or called tail spikes) play a critical role in the early stage of infection by binding to the bacterial surface. Podophages with known structures usually possess one or two types of fibers. Here, we resolved an asymmetric structure of the podophage GP4 to near-atomic resolution by cryo-EM. Our structure revealed a symmetry-mismatch relationship between the components of the GP4 tail with previously unseen topologies. In detail, two dodecameric adaptors (adaptors I and II), a hexameric nozzle, and a tail needle form a conserved tail body connected to a dodecameric portal occupying a unique vertex of the icosahedral head. However, five chain-like extended fibers (fiber I) and five tulip-like short fibers (fiber II) are anchored to a 15-fold symmetric fiber-tail adaptor, encircling the adaptor I, and six bamboo-like trimeric fibers (fiber III) are connected to the nozzle. Five fibers I, each composed of five dimers of the protein gp80 linked by an elongated rope protein, are attached to the five edges of the tail vertex of the icosahedral head. In this study, we identified a new structure of the podophage with three types of tail fibers, and such phages with different types of fibers may have a broad host range and/or infect host cells with considerably high efficiency, providing evolutionary advantages in harsh environments.

Isolation and Characterization of Lytic Bacteriophages against Aeromonas dhakensis Isolated from Water in Thailand

Phage therapy has emerged as a potential solution to the issue of multidrug-resistant bacteria. In this study, a novel bacteriophage vB_AdhS_M4 , which infects Aeromonas dhakensis, was isolated. A. dhakensis strains were isolated from water samples in Thailand and identified by biochemical and 16S rDNA sequence analysis. Four isolates, namely AM, NGP8, AK3, and SBKN4 were identified as A. dhakensis and used as hosts for phage isolation. Only 1 phage, vB_AdhS_M4, was obtained using A. dhakensis AM as host. Phage vB_AdhS_M4 morphology, host range, one-step growth curve, pH and temperature stability and genome were investigated. Transmission electron microscopy revealed that vB_AdhS_M4 has an icosahedral head of 64.8 ± 0.51 nm and a long tail of 185.4 ± 0.48 nm, suggesting that it belongs to the Siphoviridae family. Phage vB_AdhS_M4 had a latent period of 50 mins and a mean burst size of approximately 48 phage particles per infected cell. Our study on host-range determination demonstrated that this phage was specific for infecting A. dhakensis. Phage vB_AdhS_M4 was stable within a pH range of 4-7 and at temperatures ranging from 4 to 45 °C. The complete genome of Aeromonas phage vB_AdhS_M4 is 61,429 bp in length, with a G+C content of 61.7% and 77 open reading frames. In vitro A. dhakensis growth inhibition were observed with vB_AdhS_M4 at various MOIs. The greatest reduction in cell count occurred during the first 6-12 h of incubation with the phage, compared to the uninfected bacterial control, at all MOIs tested. Although the bacteria regrew after 24 h of incubation with the phage, the bacterial count remained lower than that of the control throughout the entire 48-h period. Based on these findings, it appears that vB_AdhS_M4 could be a valuable tool for biocontrol of A. dhakensis in aquaculture.

The characterization and genome analysis of a novel phage phiA034 targeting multiple species of Aeromonas

Aeromonas is one of the most serious pathogens in freshwater aquaculture. Overuse of antibiotics for the treatment of fish diseases has led to the frequent occurrence of drug-resistant strains. Phage therapy is an alternative approach to overcoming the multi-drug resistance associated with antibiotics. In this study, a novel phage phiA034 targeting the host A. veronii A034 was isolated. The phage could infect 14 strains of 4 species Aeromonas. The phage phiA034 displayed head-tail structure with an icosahedral head in the TEM image. At the optimal MOI of 1, it had a latent period of nearly 20 minutes and a burst size of 286 PFU/cell. Besides, the phage phiA034 exhibited high tolerance to a wide range of temperature (30–70 °C) and acid-base (pH 6.0–10.0). The whole genome of phage phiA034 was sequenced with a size of 61,443 bp and annotated with 82 ORFs, mainly related to structure, DNA replication, and lysis. Based on the analysis and comparison of the genomes and proteomes, phage phiA034 could be classified as a novel species of an existing genus Duplodnaviria Heunggongvirae, Uroviricota, Caudoviricetes, Casjensviridae, Sharonstreetvirus. These findings have expanded the species bank and genomes library of bacterial virus and will promote the application of phage therapy in Aeromonas disease.

Therapeutics and prophylactic efficacy of novel lytic Escherichia phage vB_EcoS_PJ16 against multidrug-resistant avian pathogenic E. coli using in vivo study.

Avian pathogenic Escherichia coli (APEC) is the causative agent of avian colibacillosis, which causes significant economic losses to the poultry industry. The growing resistance of bacteria to antibiotics is a major global public health concern. However, there is limited data on the efficacy of phage therapy in effectively controlling and treating APEC infections. In this study, a novel lytic Escherichia phage, vB_EcoS_PJ16, was isolated from poultry farm wastewater and characterized in both in vitro and in vivo conditions. Transmission electron microscopy analysis revealed the presence of an icosahedral head and a long non-contractile tail, classifying the phage under the Caudoviricetes class. Host range determination showed that Escherichia phage vB_EcoS_PJ16 exhibited lytic activity against multiple strains of pathogenic E. coli, while no significant signs of lysis for Klebsiella pneumoniae, Salmonella Typhimurium, Listeria monocytogenes, and Staphylococcus aureus. Biophysical characterization revealed that the isolated phage was sturdy, as it remained viable for up to 300days at temperatures of 30°C, 37°C, and 42°C and for up to 24h at pH 5 to 11, with only minor changes in titer. Kinetic analysis at multiplicity of infection (MOI) 0.1 showed a latency period of about 20min and a burst size of 26.5 phage particles per infected cell for phage vB_EcoS_PJ16. Whole genome sequencing unveiled that the phage vB_EcoS_PJ16 genome consists of a double-stranded linear DNA molecule with 57,756bp and a GC content of 43.58%. The Escherichia phage vB_EcoS_PJ16 genome consisted of 98 predicted putative ORFs, with no transfer RNA identified in the genome. Among these 98 genes, 34 genes were predicted to have known functions. A significant reduction in APEC viability was observed at MOI 100 during in vitro bacterial challenge tests conducted at different MOIs (0.01, 1, and 100). In vivo oral evaluation of the isolated phage to limit E. coli infections in day-old chicks indicated a decrease in mortality within both the therapeutic (20%) and prophylactic (30%) groups, when compared to the control group. The findings of this study contribute to our current knowledge of Escherichia phages and suggest a potentially effective role of phages in the therapeutic and prophylactic control of antibiotic-resistant APEC strains.

A novel lytic phage exhibiting a remarkable in vivo therapeutic potential and higher antibiofilm activity against Pseudomonas aeruginosa

BackgroundPseudomonas aeruginosa is a nosocomial bacterium responsible for variety of infections. Inappropriate use of antibiotics could lead to emergence of multidrug-resistant (MDR) P. aeruginosa strains. Herein, a virulent phage; vB_PaeM_PS3 was isolated and tested for its application as alternative to antibiotics for controlling P. aeruginosa infections.MethodsPhage morphology was observed using transmission electron microscopy (TEM). The phage host range and efficiency of plating (EOP) in addition to phage stability were analyzed. One-step growth curve was performed to detect phage growth kinetics. The impact of isolated phage on planktonic cells and biofilms was assessed. The phage genome was sequenced. Finally, the therapeutic potential of vB_PaeM_PS3 was determined in vivo.ResultsIsolated phage has an icosahedral head and a contractile tail and was assigned to the family Myoviridae. The phage vB_PaeM_PS3 displayed a broad host range, strong bacteriolytic ability, and higher environmental stability. Isolated phage showed a short latent period and large burst size. Importantly, the phage vB_PaeM_PS3 effectively eradicated bacterial biofilms. The genome of vB_PaeM_PS3 consists of 93,922 bp of dsDNA with 49.39% G + C content. It contains 171 predicted open reading frames (ORFs) and 14 genes as tRNA. Interestingly, the phage vB_PaeM_PS3 significantly attenuated P. aeruginosa virulence in host where the survival of bacteria-infected mice was markedly enhanced following phage treatment. Moreover, the colonizing capability of P. aeruginosa was markedly impaired in phage-treated mice as compared to untreated infected mice.ConclusionBased on these findings, isolated phage vB_PaeM_PS3 could be potentially considered for treating of P. aeruginosa infections.

Isolation, biological and whole genome characteristics of a Proteus mirabilis bacteriophage strain

Proteus mirabilis, a naturally resistant zoonotic bacterium belonging to the Enterobacteriaceae family, has exhibited an alarming increase in drug resistance. Consequently, there is an urgent need to explore alternative antimicrobial agents. Bacteriophages, viruses that selectively target bacteria, are abundant in the natural environment and have demonstrated potential as a promising alternative to antibiotics. In this study, we successfully isolated four strains of Proteus mirabilis phages from sewage obtained from a chicken farm in Sichuan, China. Subsequently, we characterized one of the most potent lytic phages, Q29, by examining its biological and genomic features. Comparative genomic analysis revealed the functional genes and phylogenetic evolution of Q29 phages. Our findings revealed that Proteus mirabilis bacteriophage Q29 possesses an icosahedral symmetrical head with a diameter of 95 nm and a tail length of 240 nm. Moreover, phage Q29 exhibited stability within a temperature range of 37 ℃ to 55 ℃ and under pH conditions ranging from 4 to 9. The optimal multiplicity of infection (MOI) for this phage was determined to be 0.001. Furthermore, the one-step growth curve results indicated an incubation period of approximately 15 min, an outbreak period of approximately 35 min, and an average cleavage quantity of approximately 60 plaque-forming units (PFU) per cell. The genome of phage Q29 was found to have a total length of 58,664 base pairs and encoded 335 open reading frames (ORFs) without carrying any antibiotic resistance genes. Additionally, genetic evolutionary analysis classified phage Q29 within the family Caudalidae and the genus Myotail. This study provides valuable research material for further development of Proteus mirabilis bacteriophage biologics as promising alternatives to antibiotics, particularly in light of the growing challenge of antibiotic resistance posed by this bacterium.

Structure of Vibrio Phage XM1, a Simple Contractile DNA Injection Machine.

Antibiotic resistance poses a growing risk to public health, requiring new tools to combat pathogenic bacteria. Contractile injection systems, including bacteriophage tails, pyocins, and bacterial type VI secretion systems, can efficiently penetrate cell envelopes and become potential antibacterial agents. Bacteriophage XM1 is a dsDNA virus belonging to the Myoviridae family and infecting Vibrio bacteria. The XM1 virion, made of 18 different proteins, consists of an icosahedral head and a contractile tail, terminated with a baseplate. Here, we report cryo-EM reconstructions of all components of the XM1 virion and describe the atomic structures of 14 XM1 proteins. The XM1 baseplate is composed of a central hub surrounded by six wedge modules to which twelve spikes are attached. The XM1 tail contains a fewer number of smaller proteins compared to other reported phage baseplates, depicting the minimum requirements for building an effective cell-envelope-penetrating machine. We describe the tail sheath structure in the pre-infection and post-infection states and its conformational changes during infection. In addition, we report, for the first time, the in situ structure of the phage neck region to near-atomic resolution. Based on these structures, we propose mechanisms of virus assembly and infection.