Bacteriophages (phages) are viruses that infect bacteria and are the most abundant biological entity on the planet. Phages have gained popularity as an alternative to antibiotics due to their specificity and ability to efficiently lyse antimicrobial resistant bacterial pathogens. Before using phages, they must be isolated from the environment and tested to ensure purity and lytic ability against various hosts. This protocol walks through the entire multi-day procedure of enriching and processing raw environmental samples (seawater, primary sludge, and soil), testing for lytic activity, selecting and picking potential phage plaques, verifying phage purity, and finally, propagation (liquid and solid) of phages to obtain high-titer crude phage lysates.

Vibrio species are naturally found in estuarine and marine ecosystems, but are also recognized as significant human enteropathogens, often linked to seafood-related illnesses. In aquaculture settings, Vibrio poses a substantial risk of infectious diseases, resulting in considerable stock losses and prompting the use of antimicrobials. However, this practice contributes to the proliferation of antimicrobial-resistant (AMR) bacteria and resistance genes. Our investigation aimed to explore the potential of biological agents such as bacteriophage CH20 and endolysin LysVPp1 in reducing Vibrio bacterial loads in both rotifer and fish larvae. LysVPp1\'s lytic activity was assessed by measuring absorbance reduction against various pathogenic Vibrio strains. Phage CH20 exhibited a limited host range, affecting only Vibrio alginolyticus GV09, a highly pathogenic strain. Both CH20 and LysVPp1 were evaluated for their effectiveness in reducing Vibrio load in rotifers or fish larvae through short-setting bioassays. Our results demonstrated the significant lytic effect of endolysin LysVPp1 on strains of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio splendidus. Furthermore, we have showcased the feasibility of reducing the load of pathogenic Vibrio in live feed and fish larvae by using a non-antibiotic-based approach, such as lytic phage and endolysin LysVPp1, thus contributing to the progress of a sustainable aquaculture from a One Health perspective.

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.

Phage therapy has recently been revitalized in the West with many successful applications against multi-drug-resistant bacterial infections. However, the lack of geographically diverse bacteriophage (phage) genomes has constrained our understanding of phage diversity and its genetics underpinning host specificity, lytic capability, and phage-bacteria co-evolution. This study aims to locally isolate virulent phages against uropathogenic Escherichia coli (E. coli) and study its phenotypic and genomic features. Three obligately virulent Escherichia phages (øEc_Makalu_001, øEc_Makalu_002, and øEc_Makalu_003) that could infect uropathogenic E. coli were isolated and characterized. All three phages belonged to Krischvirus genus. One-step growth curve showed that the latent period of the phages ranged from 15 to 20 min, the outbreak period ~ 50 min, and the burst size ranged between 74 and 127 PFU/bacterium. Moreover, the phages could tolerate a pH range of 6 to 9 and a temperature range of 25-37 °C for up to 180 min without significant loss of phage viability. All phages showed a broad host spectrum and could lyse up to 30% of the 35 tested E. coli isolates. Genomes of all phages were approximately ~ 163 kb with a gene density of 1.73 gene/kbp and an average gene length of ~ 951 bp. The coding density in all phages was approximately 95%. Putative lysin, holin, endolysin, and spanin genes were found in the genomes of all three phages. All phages were strictly virulent with functional lysis modules and lacked any known virulence or toxin genes and antimicrobial resistance genes. Pre-clinical experimental and genomic analysis suggest these phages may be suitable candidates for therapeutic applications.

Campylobacter is a major cause of bacterial foodborne diarrhea worldwide. Consumption of raw or undercooked chicken meat contaminated with Campylobacter is the most common causative agent of human infections. Given the high prevalence of contamination in poultry meat and the recent rise of multi-drug-resistant (MDR) Campylobacter strains, an effective intervention method of reducing bird colonization is needed. In this study, the Campylobacter-specific lytic phage CP6 was isolated from chicken feces. Phage CP6 exhibited a broad host range against different MDR Campylobacter isolates (97.4% of strains were infected). Some biological characteristics were observed, such as a good pH (3-9) stability and moderate temperature tolerance (<50 ℃). The complete genome sequence revealed a linear double-stranded DNA (178,350 bp, group II Campylobacter phage) with 27.51% GC content, including 209 predicted open reading frames, among which only 54 were annotated with known functions. Phylogenetic analysis of the phage major capsid protein demonstrated that phage CP6 was closely related to Campylobacter phage CPt10, CP21, CP20, IBB35, and CP220. CP6 phage exerted good antimicrobial effects on MDR Campylobacter in vitro culture and reduced CFUs of the host cells by up to 1-log compared with the control in artificially contaminated chicken breast meat. Our findings suggested the potential of CP6 phage as a promising antimicrobial agent for combating MDR Campylobacter in food processing.

The consumption of fresh produce has led to increase in antibiotic-resistant (AR) Salmonella outbreaks. In this study, indigenous Salmonella was isolated from a total of two hundred-two samples including fresh produce and agricultural environmental samples in Korea. After biochemical confirmation using the Indole, Methyl Red, Voges-Proskauer, Citrate tests, presumable Salmonella isolates were identified by 16S rRNA sequencing. Identified Salmonella isolates were evaluated for antibiotic susceptibility against twenty-two antibiotics. The specificity and the efficiency of plating (EOP) of vB_SalS_KFSSM were evaluated against fifty-three bacterial strains. Twenty-five suspected Salmonella were isolated and confirmed by the positive result for methyl red and citrate, of which ten were identified as Salmonella spp. through 16S rRNA gene sequencing. Eight Salmonella isolates (4.0%, n = 8/202) were resistant to at least one antibiotic, among which five were multi-drug resistant. As a lytic phage against Salmonella spp. CMGS-1, vB_SalS_KFSSM was isolated from cow manure. The phage was observed as a tailed phage belonging to the class Caudoviricetes. It exhibited an intra-broad specificity against four indigenous AR Salmonella isolates, two indigenous Salmonella isolates, and five other Salmonella serotypes with great efficiencies (EOP ≥ 0.75). Thus, this study suggested the potential of vB_SalS_KFSSM to combat indigenous AR Salmonella.

The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has driven us to explore alternative treatments for the limitation of antimicrobial agents. Lytic phages are considered a promising alternative treatment for CR-hvKP infection. In this study, we reported three novel lytic phages, vB_KpnA_SCNJ1-Z, vB_KpnS_SCNJ1-C, and vB_KpnM_SCNJ1-Y, against a CR-hvKP strain SCNJ1, and they possess genomes of double-stranded DNA with a size of 43,428 bp, 46,039 bp, and 50,360 bp, respectively. Phylogenetic analysis demonstrated that vB_KpnA_SCNJ1-Z belongs to the family Autographiviridae within the class Caudoviricetes, while vB_KpnS_SCNJ1-C and vB_KpnM_SCNJ1-Y are unclassified Caudoviricetes. The phages showed a narrow host range only lysing 1 of 50 tested clinical bacterial strains. The one-step growth curves and stability results showed that the phages displayed relatively short latency periods, with broad pH (pH 3-14) and thermal stabilities (20-60°C). The phages showed significant inhibition of the biofilm formation by SCNJ1 and strong antibacterial activity in vitro. In the mouse model, we demonstrated that administration of a single phage or phage cocktail significantly reduced bacteria loads in the lung, liver, and spleen, and effectively rescued mice from the infection of the SCNJ1 strain, with a survival rate of 70-80%. These findings suggested the three phages have great potential as an alternative therapy with favorable stability and strong antibacterial activity both in vivo and in vitro for the treatment of CR-hvKP infection.

We describe the genome of a lytic phage EKq1 isolated on Klebsiella quasipneumoniae, with activity against Klebsiella pneumoniae. EKq1 is an unclassified representative of the class Caudoviricetes, similar to Klebsiella phages VLCpiS8c, phiKp_7-2, and vB_KleS-HSE3. The 48,244-bp genome has a GC content of 56.43% and 63 predicted protein-coding genes.

Salmonella enterica serotype Typhi is one of the major pathogens causing typhoid fever and a public health burden worldwide. Recently, the increasing number of multidrug-resistant strains of Salmonella spp. has made this utmost necessary to consider bacteriophages as a potential alternative to antibiotics for S. Typhi infection treatment. Salmonella phage STWB21, isolated from environmental water, has earlier been reported to be effective as a safe biocontrol agent by our group. In this study, we evaluated the efficacy of phage STWB21 in reducing the burden of salmonellosis in a mammalian host by inhibiting Salmonella Typhi invasion into the liver and spleen tissue.
Phage treatment significantly improved the survival percentage of infected mice. This study also demonstrated that oral administration of phage treatment could be beneficial in both preventive and therapeutic treatment of salmonellosis caused by S. Typhi. Altogether the result showed that the phage treatment could control tissue inflammation in mice before and after Salmonella infection.
To the best of our knowledge, this is the first report of phage therapy in a mouse model against a clinically isolated Salmonella Typhi strain that includes direct visualization of histopathology and ultrathin section microscopy images from the liver and spleen sections.

Erwinia amylovora is a devastating phytobacterium causing fire blight in the Rosaceae family. In this study, ΦFifi106, isolated from pear orchard soil, was further purified and characterized, and its efficacy for the control of fire blight in apple plants was evaluated. Its genomic analysis revealed that it consisted of 84,405 bp and forty-six functional ORFs, without any genes encoding antibiotic resistance, virulence, and lysogenicity. The phage was classified into the genus Kolesnikvirus of the subfamily Ounavirinae. ΦFifi106 specifically infected indigenous E. amylovora and E. pyrifoliae. The lytic activity of ΦFifi106 was stable under temperature and pH ranges of 4-50 °C and 4-10, as well as the exposure to ultraviolet irradiation for 6 h. ΦFifi106 had a latent period of 20 min and a burst size of 310 ± 30 PFU/infected cell. ΦFifi106 efficiently inhibited E. amylovora YKB 14808 at a multiplicity of infection (MOI) of 0.1 for 16 h. Finally, the pretreatment of ΦFifi106 at an MOI of 1000 efficiently reduced disease incidence to 37.0% and disease severity to 0.4 in M9 apple plants. This study addressed the use of ΦFifi106 as a novel, safe, efficient, and effective alternative to control fire blight in apple plants.