A novel virulent bacteriophage, vB_PmuP_PHB02 (phage PHB02), infecting Pasteurella multocida capsular type A strains, was isolated from wastewater from a swine farm in China. Phage PHB02 has a linear double-stranded DNA genome consisting of 38,670 base pairs (bp), with a G+C content of 40.8% and a 127-bp terminal redundancy. Forty-eight putative open reading frames were identified, and no transfer RNA-encoding genes were detected. The morphology and genomic structure of phage PHB02 resemble those of T7-like phages belonging to the family Podoviridae, of the order Caudovirales. Phage PHB02 was stable over a wide range of temperatures (4-50 °C) and pH values (5.0-9.0), and lysed 30 of the 31 capsular-type-A P. multocida strains tested. Phage PHB02 had no effect on other bacterial species or on P. multocida strains belonging to capsular types D or F.

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Phage PHB02 specifically infects Pasteurella multocida capsular serogroup A strains. In this study, we found that capsule deletion mutants were not lysed by PHB02, suggesting that the capsule of P. multocida serogroup A strains might be the primary receptor. Based on sequence analysis, a gene encoding a phage-associated putative depolymerase was identified. The corresponding recombinant depolymerase demonstrated specific activity against capsular serogroup A strains but did not strip capsule deletion mutants. In vivo experiments showed that PHB02 was retained at detectable levels in the liver, spleen, kidneys, lung, and blood, at 24 h post-administration in mice. Depolymerase plus serum significantly reduced the number of viable wild-type P. multocida strain HB03 cells (3.5–4.5 log decrease in colony-forming units). Moreover, treatment with phage or purified depolymerase resulted in significantly increased survival of mice infected with P. multocida HB03, and an absence of increase of eosinophils and basophils or other pathological changes when compared with the control group. These results show that phage PHB02 and its putative depolymerase represent a novel strategy for controlling P. multocida serogroup A strains.

A lytic bacteriophage PHB01 specific for Pasteurella multocida type D was isolated from the sewage water collected from a pig farm. This phage had the typical morphology of the family Podoviridae, order Caudovirales, presenting an isometric polyhedral head and a short noncontractile tail. PHB01 was able to infect most of the non-toxigenic P. multocida type D strains tested, but not toxigenic type D strains and those belonging to other capsular types. Phage PHB01, the first lytic phage specific for P. multocida type D sequenced thus far, presents a 37,287-bp double-stranded DNA genome with a 223-bp terminal redundancy. The PHB01 genome showed the highest homology with that of PHB02, a lytic phage specific for P. multocida type A. Phylogenetic analysis showed that PHB01 and PHB02 were composed of a genus that was close to the T7-virus genus. In vivo tests using mouse models showed that the administration of PHB01 was safe to the mice and had a good effect on treating the mice infected with different P. multocida type D strains including virulent strain HN05. These findings suggest that PHB01 has a potential use in therapy against infections caused by P. multocida type D.

Salmonella is a common and widely distributed foodborne pathogen that is frequently implicated in gastrointestinal infections. The emergence and spread of Salmonella strains resistant to multiple antibiotics poses a significant health threat, highlighting the urgent need for early and effective therapeutic strategies. We isolated a total of 32 phages from water samples and anal swabs from pigs. Of these, three phages that produced large, clear plaques were selected for further study using the following methods: electron microscopy, analysis of the life cycle parameters, genetic analysis, inhibition of bacterial growth, and activity against biofilms. The three Salmonella phages (vB_SenS_CSP01, vB_SenS_PHB06, and vB_SenS_PHB07) were assigned to the family Siphoviridae on the basis of their morphology. All showed polyvalent infectivity, and individual phages or phage cocktails could inhibit the growth of host Salmonella enterica serovar Enteritidis strains or reduce biofilm formation by Salmonella enterica serovar Typhimurium. In summary, these three phages merit further research as biocontrol agents for Salmonella infection.

Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel Bordetella bronchiseptica temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of B. bronchiseptica. Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain B. bronchiseptica Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing B. bronchiseptica strain completely protected mice against lethal challenge with wild-type virulent B. bronchiseptica, indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines.

Enterococcus faecalis is an opportunistic pathogen that causes illnesses ranging from urinary tract infections to sepsis in humans and animals. However, the overuse of antibiotics has increased rates of drug resistance among E. faecalis isolates. Bacteriophages and their derivatives have recently been identified as good candidates for the treatment of drug-resistant bacterial infections. Here, we isolated a virulent E. faecalis phage, PHB08, using the double-layer plate method. The bioactivity of the phage was determined via one-step growth curve testing and bacterial killing assays, and whole-genome sequencing was performed using the Illumina HiSeq platform. In addition, protein expression and antibiofilm assays were performed to investigate the activity of the phage lysin. Results showed that PHB08 has a 55,244-bp linear double-stranded DNA genome encoding 91 putative coding sequences. PHB08 inhibited the growth of host strain EF3964 at 37 °C in tryptic soy broth (TSB) medium, while in vegetable models, PHB08 caused a 4.69-log decrease in viable E. faecalis cells after 24 h. Both PHB08 and its endolysin lys08 showed antibiofilm activity against E. faecalis biofilms, which was enhanced by Mn2+ ions. Thus, virulent phage PHB08 and endolysin lys08 may be good candidates for reducing and/or eradicating E. faecalis infections.

A novel virulent bacteriophage, vB_BbrM_PHB04, infecting Bordetella bronchiseptica was isolated from wastewater collected at a swine farm in China. Phage vB_BbrM_PHB04 exhibited growth over a wide range of temperature and pH conditions and showed different efficiency of plating values and lytic spectra within the same strains at 25 °C and 37 °C. High-throughput sequencing revealed that vB_BbrM_PHB04 has a linear double-stranded DNA genome with 124 putative open reading frames. Overall, the genome of vB_BbrM_PHB04 showed very low similarity (the highest nucleotide identity 82%, 1% coverage) to other phage sequences in the GenBank database. Phylogenetic analysis indicated that vB_BbrM_PHB04 is a new member of the family Myoviridae. In addition, polymerase chain reaction-based detection of phage genes in phage-resistant B. bronchiseptica variants revealed no evidence of lysogenic activity of phage vB_BbrM_PHB04.