Characterization and in vitro testing of newly isolated lytic bacteriophages for the biocontrol of Pseudomonas aeruginosa

Aim: Two lytic phages were isolated using P. aeruginosa DSM19880 as host and fully characterized. Materials & methods: Phages were characterized physicochemically, biologically and genomically. Results & conclusion: Host range analysis revealed that the phages also infect some multidrug-resistant (MDR) P. aeruginosa clinical isolates. Increasing MOI from 1 to 1000 significantly increased phage efficiency and retarded bacteria regrowth, but phage ph0034 (reduction of 7.5 log CFU/ml) was more effective than phage ph0031 (reduction of 5.1 log CFU/ml) after 24 h. Both phages belong to Myoviridae family. Genome sequencing of phages ph0031 and ph0034 showed that they do not carry toxin, virulence, antibiotic resistance and integrase genes. The results obtained are highly relevant in the actual context of bacterial resistance to antibiotics.

Diverse Bacteriophages Infecting the Bacterial Striped Catfish Pathogen Edwardsiella ictaluri

Bacteriophages infecting Edwardsiella ictaluri have been less investigated, although the host bacterium is one of the most important fish pathogens causing enteric septicemia of catfish (ESC). We present here two distinctly novel bacteriophages vB_EiM_PVN06 and vB_EiA_PVN09 infecting Edwardsiella ictaluri E1, with their geographical origins from the Mekong Delta, Vietnam. Bacteriophage vB_EiM_PVN06 native to a mud sample reveals complete differences of biological properties with the phage vB_EiA_PVN09 originated from a viscus of a healthy catfish (Pangasianodon hypophthalmus) cultured in the same area. Morphological analyses combined with genomic data indicate that phage vB_EiM_PVN06 is classified to Myoviridae family and shares high similarity with E. ictaluri phage PEi21 genome, while vB_EiA_PVN09 is a member of Teseptimavirus genus, Autographiviridae family, and mostly closes to phage vB_EcoP_IME390. The vB_EiA_PVN09 is a T7-like bacteriophage, which has been firstly found infecting to E. ictaluri, and host range analysis also evidences for the cross-infection of this phage to Escherichia coli K12 and Escherichia coli DH5α. Together, our research highlights the diversity of bacteriophages infecting the pathogen E. ictaluri and suggests further explorations of lytic phages in environmental niches, to be exploited in feasible strategies of phage therapy in ESC disease control.

Characterization of a Lytic Bacteriophage against Pseudomonas syringae pv. actinidiae and Its Endolysin

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 ∘C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 ∘C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

A Novel Lytic Phage SZW_AS01 of Human Gut Bacteria Alistipes Shahii

Alisitipes phage SZW_AS01, a novel lytic phage that specifically infects Alistipes shahii, was isolated from wastewater samples in Shenzhen, China. The phage's genome consists of 45,392 bp, with a GC content of 47%. The genome encodes 56 putative open reading frames (ORFs) and 1 tRNA gene. Direct terminal repeats with a length of 55 bp are present at both ends of the genome. Phylogenetic analysis of the amino acid sequences of terminase large subunit shows that phage SZW_AS01 forms a distinct branch from the Siphoviridae family phages, but is far from the Podoviridae and Myoviridae family phages. Transmission electron microscopy confirmed that SZW_AS01 belongs to the Siphoviridae family. To the best of our knowledge, this is the first report of a lytic phage infecting bacteria in the Alistipes genus.

Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, Characterization and Remediation of Larval Oyster Mortalities

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortalities in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family and two were Siphoviridae. In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% at 6 days post-challenge with lethal doses of V. coralliilyticus. Larvae survival depended on the oyster species, the quantity of phages and vibrios applied, and the species and strain of Vibrio. A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phage vB_VcorM-GR7B; vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A and 28A). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by > 90% (P < 0.0001) over 6 days with phage treatment compared to untreated controls. Genomic sequencing of phages 7B, 11A and 28A revealed 207,758; 194,800; and 154,046 bp, linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries. IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of the hatcheries and commercial shellfish industry. Shellfish pathogens, like Vibrio coralliilyticus and Vibrio tubiashii, have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species, and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach in stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.

A Novel Acinetobacter baumannii Bacteriophage Endolysin LysAB54 With High Antibacterial Activity Against Multiple Gram-Negative Microbes

The rapid spread and emergence of multidrug-resistant Acinetobacter baumannii and other pathogenic Gram-negative bacteria spurred scientists and clinicians to look for alternative therapeutic agents to conventional antibiotics. In the present study, an A. baumannii bacteriophage p54 was isolated and characterized. Morphological and genome analysis revealed that bacteriophage p54 belongs to Myoviridae family with a genome size of 165,813 bps. A novel endolysin, namely LysAB54, showing low similarity with other well-known related endolysins, was cloned, expressed, and characterized from the bacteriophage p54. LysAB54 showed significant bactericidal activity against multidrug-resistant A. baumannii and other Gram-negative bacteria, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli, in the absence of outer membrane permeabilizers. Based on all those observations, LysAB54 could represent a potential agent for the treatment of multidrug-resistant Gram-negative superbugs.

First Description of a Temperate Bacteriophage (vB_FhiM_KIRK) of Francisella hispaniensis Strain 3523

Here we present the characterization of a Francisella bacteriophage (vB_FhiM_KIRK) including the morphology, the genome sequence and the induction of the prophage. The prophage sequence (FhaGI-1) has previously been identified in F. hispaniensis strain 3523. UV radiation induced the prophage to assemble phage particles consisting of an icosahedral head (~52 nm in diameter), a tail of up to 97 nm in length and a mean width of 9 nm. The double stranded genome of vB_FhiM_KIRK contains 51 open reading frames and is 34,259 bp in length. The genotypic and phylogenetic analysis indicated that this phage seems to belong to the Myoviridae family of bacteriophages. Under the conditions tested here, host cell (Francisella hispaniensis 3523) lysis activity of KIRK was very low, and the phage particles seem to be defective for infecting new bacterial cells. Nevertheless, recombinant KIRK DNA was able to integrate site-specifically into the genome of different Francisella species after DNA transformation.

Biodiversity of new lytic bacteriophages infecting Shigella spp. in fresh water environment

Background Bacteriophages, viruses that infects and replicates within prokaryotic cells are the most abundant life forms in the environment, yet the vast majority of them have not been properly reported or even discovered. Almost all reported bacteriophages infecting the Enterobacteriaceae family, with E. coli being the major subject of the study, have been isolated from wastewater, sewage, and effluent resources. In the present study we focused on the distribution and biodiversity of Shigella phages in an aquatic ecosystem. Results While no Shigella bacteria was recovered from the Yangtze River, three lytic phages were isolated from this ecosystem and were subjected to biological, morphological, and genomic characteristics. Comparative genomics and phylogenetic analyses demonstrated that vB _SflM_004 isolate belongs to Myoviridae family, Felixounavirus genus of Ounavirinae subfamily, vB_SdyM_006 was classified under the same family, however, it is suggested to be in a new genus under Tevenvirinae subfamily with some other related bacteriophages. vB_SsoS_008 phage belongs to the Siphoviridae family, Tunavirus genus, Tunavirinae subfamily. The phages did not harbor any genes involved in the lysogenic cycles, and showed a high temperature and pH stability. Conclusions It can be concluded that isolation of bacteriophages could be independent of their bacterial host presence in the isolation environment.

Haemophilus influenzae HP1 Bacteriophage Encodes a Lytic Cassette with a Pinholin and a Signal-Arrest-Release Endolysin

HP1 is a temperate bacteriophage, belonging to the Myoviridae family and infecting Haemophilus influenzae Rd. By in silico analysis and molecular cloning, we characterized lys and hol gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence of the lys gene product revealed the presence of signal-arrest-release (SAR) and muraminidase domains, characteristic for some endolysins. HP1 endolysin was able to induce lysis on its own when cloned and expressed in Escherichia coli, but the new phage release from infected H. influenzae cells was suppressed by inhibition of the secretion (sec) pathway. Protein encoded by hol gene is a transmembrane protein, with unusual C-out and N-in topology, when overexpressed/activated. Its overexpression in E. coli did not allow the formation of large pores (lack of leakage of β-galactosidase), but caused cell death (decrease in viable cell count) without lysis (turbidity remained constant). These data suggest that lys gene encodes a SAR-endolysin and that the hol gene product is a pinholin. HP1 SAR-endolysin is responsible for cell lysis and HP1 pinholin seems to regulate the cell lysis and the phage progeny release from H. influenzae cells, as new phage release from the natural host was inhibited by deletion of the hol gene.