De novo identification of bacterial antigens of a clinical isolate by combining use of proteosurfaceomics, secretomics, and BacScan technologies.

Background: Emerging infectious diseases pose a significant threat to both human and animal populations. Rapid de novo identification of protective antigens from a clinical isolate and development of an antigen-matched vaccine is a golden strategy to prevent the spread of emerging novel pathogens. Methods: Here, we focused on Actinobacillus pleuropneumoniae, which poses a serious threat to the pig industry, and developed a general workflow by integrating proteosurfaceomics, secretomics, and BacScan technologies for the rapid de novo identification of bacterial protective proteins from a clinical isolate. Results: As a proof of concept, we identified 3 novel protective proteins of A. pleuropneumoniae. Using the protective protein HBS1_14 and toxin proteins, we have developed a promising multivalent subunit vaccine against A. pleuropneumoniae. Discussion: We believe that our strategy can be applied to any bacterial pathogen and has the potential to significantly accelerate the development of antigen-matched vaccines to prevent the spread of an emerging novel bacterial pathogen.

Landscape of New Nuclease-Containing Antiphage Systems in Escherichia coli and the Counterdefense Roles of Bacteriophage T4 Genome Modifications.

Many phages, such as T4, protect their genomes against the nucleases of bacterial restriction-modification (R-M) and CRISPR-Cas systems through covalent modification of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, raising the question of the role of phage genome modifications in countering these systems. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli and demonstrated the roles of T4 genome modifications in countering these systems. Our analysis identified at least 17 nuclease-containing defense systems in E. coli, with type III Druantia being the most abundant system, followed by Zorya, Septu, Gabija, AVAST type 4, and qatABCD. Of these, 8 nuclease-containing systems were found to be active against phage T4 infection. During T4 replication in E. coli, 5-hydroxymethyl dCTP is incorporated into the newly synthesized DNA instead of dCTP. The 5-hydroxymethylcytosines (hmCs) are further modified by glycosylation to form glucosyl-5-hydroxymethylcytosine (ghmC). Our data showed that the ghmC modification of the T4 genome abolished the defense activities of Gabija, Shedu, Restriction-like, type III Druantia, and qatABCD systems. The anti-phage T4 activities of the last two systems can also be counteracted by hmC modification. Interestingly, the Restriction-like system specifically restricts phage T4 containing an hmC-modified genome. The ghmC modification cannot abolish the anti-phage T4 activities of Septu, SspBCDE, and mzaABCDE, although it reduces their efficiency. Our study reveals the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of T4 genomic modification in countering these defense systems. IMPORTANCE Cleavage of foreign DNA is a well-known mechanism used by bacteria to protect themselves from phage infections. Two well-known bacterial defense systems, R-M and CRISPR-Cas, both contain nucleases that cleave the phage genomes through specific mechanisms. However, phages have evolved different strategies to modify their genomes to prevent cleavage. Recent studies have revealed many novel nuclease-containing antiphage systems from various bacteria and archaea. However, no studies have systematically investigated the nuclease-containing antiphage systems of a specific bacterial species. In addition, the role of phage genome modifications in countering these systems remains unknown. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli using all 2,289 genomes available in NCBI. Our studies reveal the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of genomic modification of phage T4 in countering these defense systems.

A temperate Siphoviridae bacteriophage isolate from Siberian tiger enhances the virulence of methicillin-resistant Staphylococcus aureus through distinct mechanisms.

The emergence and worldwide spread of Methicillin-resistant Staphylococcus aureus (MRSA) pose a threat to human health. While bacteriophages are recognized as an effective alternative to treat infections caused by drug resistant pathogens, some bacteriophages in particular the temperate bacteriophage may also influence the virulence of the host bacteria in distinct ways. In this study, we isolated a bacteriophage vB_Saus_PHB21 from an epidermal sample of Siberian tiger (Panthera tigris altaica) using an MRSA strain SA14 as the indicator. Our following laboratory tests and whole genome sequencing analyses revealed that vB_Saus_PHB21 was a temperate bacteriophage belonging to the Siphoviridae family, and this bacteriophage did not contain any virulence genes. However, the integration of PHB21 genome into the host MRSA increased the bacterial capacities of cell adhesion, anti-phagocytosis, and biofilm formation. Challenge of the lysogenic strain (SA14+) caused severe mortalities in both Galleria mellonella and mouse models. Mice challenged with SA14+ showed more serious organ lesions and produced higher inflammatory cytokines (IL-8, IFN-γ and TNF-α) compared to those challenged with SA14. In mechanism, we found the integration of PHB21 genome caused the upregulated expression of many genes encoding products involved in bacterial biofilm formation, adherence to host cells, anti-phagocytosis, and virulence. This study may provide novel knowledge of "bacteria-phage-interactions" in MRSA.

Characterization of a Lytic Bacteriophage vB_EfaS_PHB08 Harboring Endolysin Lys08 Against Enterococcus faecalis Biofilms.

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.

Specific Integration of Temperate Phage Decreases the Pathogenicity of Host Bacteria.

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.

Isolation of a T7-Like Lytic Pasteurella Bacteriophage vB_PmuP_PHB01 and Its Potential Use in Therapy against Pasteurella multocida Infections.

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.

Characterisation of a newly detected bacteriophage infecting Bordetella bronchiseptica in swine.

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.

Therapeutic Application of Bacteriophage PHB02 and Its Putative Depolymerase Against Pasteurella multocida Capsular Type A in Mice.

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.

Three Salmonella enterica serovar Enteritidis bacteriophages from the Siphoviridae family are promising candidates for phage therapy.

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.

Complete Genome Sequence of a Novel T7-Like Bacteriophage from a Pasteurella multocida Capsular Type A Isolate.

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.

Novel pseudorabies virus variant with defects in TK, gE and gI protects growing pigs against lethal challenge.

One of the distinct features of the emerging Chinese pseudorabies virus (PRV) variant is its ability to cause severe neurological signs and high mortality in growing pigs in Bartha-K61-vaccinated pig farms. Either single- or multiple-gene-deleted live vaccine candidates have been developed; however, none was evaluated thoroughly in growing pigs. Here, we generated rSMXΔgI/gEΔTK, an attenuated PRV variant with defects in TK, gI and gE genes. The growth kinetics of the attenuated virus was similar to the wild type (wt) strain. It was safe for 1-day-old piglets. Twenty one-day-old weaned pigs were immunized intramuscularly either with 10(6.0) TCID50 of rSMXΔgI/gEΔTK or one dose of commercial Bartha-K61 vaccine, or with DMEM, and were challenged intranasally with 10(7.0) TCID50 wt virus at 28 days post vaccination. rSMXΔgI/gEΔTK elicited higher level neutralization antibody against both PRV variant SMX and Bartha-K61 strain, while Bartha-K61 vaccine elicited lower neutralization activity of antibody against SMX. After challenge, all pigs in rSMXΔgI/gEΔTK group survived without any clinical signs, while unvaccinated group showed 100% mortality, and Bartha-K61 group showed severe respiratory symptoms and 3 out of 5 pigs exhibited severe neurological signs. Pigs in rSMXΔgI/gEΔTK group gained significantly higher body weight and diminished viral excretion titer and period, compared with Bartha-K61 group. Furthermore, the safety and efficacy of rSMXΔgI/gEΔTK was also evaluated in sheep and compared with local vaccine in growing pigs. These data suggest that the attenuated strain rSMXΔgI/gEΔTK is a promising live marker vaccine candidate for PR control in the context of emerging PRV variants.