Morganella psychrotolerans is a histamine-producing bacterium that causes histamine poisoning. In this study, we isolated and characterized a novel phage, MopsHU1, that infects M. psychrotolerans. MopsHU1 is a podovirus with a limited host spectrum. Genomic analysis showed that MopsHU1 belongs to the family Autographiviridae, subfamily Studiervirinae, and genus Kayfunavirus. Comparative analysis revealed that the MopsHU1 genome is similar to those of Citrobacter phage SH3 and Cronobacter phage Dev2. Moreover, the Escherichia coli phage K1F genome is also similar, except for its tailspike gene sequence. These results expand our understanding of the Kayfunavirus phages that infect Morganella spp. Note: The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank database under the accession number LC799501.

Double-layer agar (DLA) overlay plaque assay is the gold standard for phage enumeration. However, it is cumbersome and time-consuming. Given the great interest in phage therapy, we explored alternative assays for phage quantitation. A total of 16 different phages belonging to Myoviridae, Siphoviridae, and Podoviridae families were quantitated with five K. pneumoniae, eight P. aeruginosa, and three A. baumannii host isolates. Phages were quantitated with the standard DLA assay (10 mL of LB soft agar 0.7% on LB hard agar 1.5%) and the new single-layer agar (SLA) assay (10 mL of LB soft agar 0.7%) with phages spread (spread) into or spotted (spot) onto soft agar. Phage concentrations with each assay were correlated with the standard assay, and the relative and absolute differences between each assay and the standard double-layer agar spread were calculated. Phage concentrations 1 × 104-8.3 x1012 PFU/mL with the standard DLA assay were quantitated with SLA-spread, SLA-spot, and DLA-spot assays, and the median (range) relative and absolute differences were <10% and <0.98 log10PFU/mL, respectively, for all phage/bacterial species (ANOVA P = 0.1-0.43), and they were highly correlated (r > 0.77, P < 0.01). Moreover, plaques could be quantified at 37°C after 4-h incubation for K. pneumoniae phages and 6-h incubation for P. aeruginosa and A. baumannii phages, and estimated concentrations remained the same over 24 hours. Compared to DLA assay, the SLA-spot assay required less media, it was 10 times faster, and generated same-day results. The SLA-spot assay was cheaper, faster, easier to perform, and generated similar phage concentrations as the standard DLA-spread assay.

Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages.
OBJECTIVE: Phage resistance is a direct threat to phage therapy, and understanding phage-host interactions, especially how bacteria block phage infection, is essential for developing successful phage therapy. In the present study, we demonstrate for the first time that Vibrio alginolyticus uses quorum sensing (QS) to promote capsular polysaccharide (CPS)-specific phage infection by upregulating ugd expression, which is necessary for the synthesis of Autographiviridae phage receptor CPS. Although increased CPS-specific phage susceptibility is a novel trade-off mediated by QS, it results in the upregulation of virulence factors, promoting biofilm development and enhanced capsular polysaccharide production in V. alginolyticus. This suggests that inhibiting QS may improve the effectiveness of antibiotic treatment, but it may also reduce the efficacy of phage therapy.

Multidrug-resistant pathogens are now thought to be the primary global causes of disease and death. Therefore, it is imperative to develop new effective bioactive compounds from microbial sources, such as Streptomyces species. Nevertheless, the pharmaceutical industry suffered financial losses and low-quality end products as a result of Streptomyces bacteriophage contamination. To reduce the likelihood of phage-induced issues in the medical industry, it is crucial to develop a method for finding phage-resistant strains. Hence, we aimed to isolate and characterize Streptomyces spp. and Streptomyces phages from various rhizospheric soil samples in Egypt and to investigate their antibacterial activities. Moreover, we targeted development of a Streptomyces phage-resistant strain to extract its active metabolites and further testing its antibacterial activity. Herein, the antibacterial activities of the isolated 58 Streptomyces isolates showed that 10 (17.2 %) Streptomyces isolates had antibacterial activities against the tested bacteria including Listeria monocytogenes, E. coli O157, Acinetobacter baumannii, methicillin resistant-vancomycin-intermediate Staphylococcus aureus (MRSA-VISA) and Micrococcus luteus. Three lytic bacteriophages (ϕPRSC1, ϕPRSC2, and ϕPRSC4) belonging to the families Siphoviridae and Podoviridae were obtained from the rhizospheric soil samples using the most potent S. abietis isolate as the host strain. The three isolated Streptomyces phages were thermostable, ultraviolet stable, infectious, and had a wide range of hosts against the 10 tested Streptomyces isolates with antibacterial activities. The DNA of the ϕPRSC1 and ϕPRSC4 phages were resistant to digestion by EcoRI and HindIII, but the DNA of ϕPRSC2 was resistant to digestion by EcoRI and sensitive to digestion by HindIII. Of note, we developed a S. abietis strain resistant to the three isolated phages and its antibacterial activities were twice that of the wild strain. Finally, telomycin was recognized as an antibacterial metabolite extracted from phage-resistant S. abietis strain, which was potent against the tested Gram-positive bacteria including L. monocytogenes, MRSA-VISA, and M. luteus. Thus, our findings open new horizons for researching substitute antimicrobial medications for both existing and reemerging illnesses.

We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The Acinetobacter baumannii satellite podovirus Aci01-2-Phanie (short name Phanie) uses a phage phi29-like DNA replication and packaging mode. Its linear 11,885 bp dsDNA genome bears 171 bp inverted terminal repeats (ITR). Phanie is related to phage DU-PP-III from Pectobacterium and to members of the Astrithrvirus from Salmonella enterica. Together, they form a new clade of phages with 27% to 30% identity over the whole genome. Detailed 3D protein structure prediction and mass spectrometry analyses demonstrate that Phanie encodes its capsid structural genes and genes necessary to form a short tail. However, our study reveals that Phanie virions are non-infectious unless they associate with the contractile tail of an unrelated phage, Aci01-1, to produce chimeric myoviruses. Following the coinfection of Phanie with myovirus Aci01-1, hybrid viral particles composed of Phanie capsids and Aci01-1 contractile tails are assembled together with Phanie and Aci01-1 particles.IMPORTANCEThere are few reported cases of satellite-helper phage interactions but many more may be yet undiscovered. Here we describe a new mode of satellite phage dependence on a helper phage. Phanie, like phage phi29, replicates its linear dsDNA by a protein primed-mechanism and protects it inside podovirus-like particles. However, these particles are defective, requiring the acquisition of the tail from a myovirus helper for production of infectious virions. The formation of chimeras between a phi29-like podovirus and a helper contractile tail reveals an unexpected association between very different bacterial viruses.

Vibrio parahaemolyticus is a common foodborne pathogenic bacterium. With the overuse of antibiotics, an increasing proportion of drug-resistant strains are emerging, which puts enormous pressure on public health. In this study, a V. parahaemolyticus-specific phage, VP41s3, was isolated. The head length, width, and tail length of the phage were 77.7 nm, 72.2 nm, and 17.5 nm, respectively. It remained active in the temperature range of 30-50°C and pH range of 4-11. The lytic curve of phage VP41s3 showed that the host bacteria did not grow until 11 h under phage treatment at MOI of 1000, indicating that the phage had good bacteriostatic ability. When it was added to shellfish contaminated with V. parahaemolyticus (15°C, 48 h), the number of bacteria in the experimental group was 2.11 log10 CFU/mL lower than that in the control group at 24 h. Furthermore, genomic characterization and phylogenetic analysis indicated that phage VP41s3 was a new member of the Podoviridae family. The genome contained 50 open reading frames (ORFs), in which the ORF19 (thymidine kinase) was an enzyme involved in the pyrimidine salvage pathway, which might lead to the accelerated DNA synthesis efficiency after phage entered into host cells. This study not only contributed to the improvement of phage database and the development of beneficial phage resources but also revealed the potential application of phage VP41s3 in food hygiene and safety.

Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%-1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications.

Multiple pathogenic types or serotypes restrict treatment for colibacillosis. In addition, rising antibiotic resistance has heightened public awareness to prevent and control pathogenic Escherichia coli. The bacteriophage is a viable technique to treat colibacillosis as an alternative to antibiotics. In this study, PH444, a relatively broad-spectrum obligate lytic phage, was screened from 48 Shiga toxin-producing Escherichia coli (STEC) phages isolated from farm manure samples and sewage samples in order to conduct genome-wide analysis, biological characterization, and a bacterial challenge experiment in milk. The results demonstrated that PH444 was a T7-like phage with a double-stranded DNA of 115,111 bp that belongs to the Kuravirus and was stable at temperatures between 4 and 50 °C and a pH range of 3 to 11. After adding PH444, the bacterial load in milk could be reduced from 3 × 103 PFU/ mL to zero within 1 h. In consideration of the biological properties of phage PH444, it was, therefore, demonstrated that PH444 has the potential to be used in phage biocontrol.

Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonas spp. In this study, 12 Pseudomonas phages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30 °C and 4 °C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonas spp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescens and P. putida at 30 °C, 8 °C, and 4 °C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.

Infections by multidrug resistant bacteria (MDR) are becoming increasingly difficult to treat and alternative approaches like phage therapy, which is unhindered by drug resistance, are urgently needed to tackle MDR bacterial infections. During phage therapy phage cocktails targeting different receptors are likely to be more effective than monophages. In the present study, phages targeting carbapenem resistant clinical isolate of E. coli U1007 was isolated from Ganges River (U1G), Cooum River (CR) and Hospital waste water (M). Capsid architecture discerned using TEM identified the phage families as Podoviridae for U1G, Myoviridae for CR and Siphoviridae for M phage. Genome sequencing showed the phage genomes varied in size U1G (73,275 bp) CR (45,236 bp) and M (45,294 bp). All three genomes lacked genes encoding tRNA sequence, antibiotic resistant or virulent genes. A machine learning (ML) based multi-class classification model using Random Forest, Logistic Regression, and Decision Tree were employed to predict the host receptor targeted by receptor binding protein of all 3 phages and the best performing algorithm Random Forest predicted LPS O antigen, LamB or OmpC for U1G; FhuA, OmpC for CR phage; and FhuA, LamB, TonB or OmpF for the M phage. OmpC was validated as receptor for U1G by physiological experiments. In vivo intramuscular infection study in zebrafish showed that cocktail of dual phages (U1G + M) along with colsitin resulted in a significant 3.5 log decline in cell counts. Our study highlights the potential of ML tool to predict host receptor and proves the utility of phage cocktail to restrict E. coli U1007 in vivo.