Biological Characterization and Genomic Analysis of Three Novel Serratia- and Enterobacter-Specific Virulent Phages.

Due to the high microbiological contamination of raw food materials and the increase in the incidence of multidrug-resistant bacteria, new methods of ensuring microbiological food safety are being sought. One solution may be to use bacteriophages (so-called phages) as natural bacterial enemies. Therefore, the aim of this study was the biological and genomic characterization of three newly isolated Serratia- and Enterobacter-specific virulent bacteriophages as potential candidates for food biocontrol. Serratia phage KKP_3708 (vB_Sli-IAFB_3708), Serratia phage KKP_3709 (vB_Sma-IAFB_3709), and Enterobacter phage KKP_3711 (vB_Ecl-IAFB_3711) were isolated from municipal sewage against Serratia liquefaciens strain KKP 3654, Serratia marcescens strain KKP 3687, and Enterobacter cloacae strain KKP 3684, respectively. The effect of phage addition at different multiplicity of infection (MOI) rates on the growth kinetics of the bacterial hosts was determined using a Bioscreen C Pro growth analyzer. The phages retained high activity in a wide temperature range (from -20 °C to 60 °C) and active acidity values (pH from 3 to 12). Based on transmission electron microscopy (TEM) imaging and whole-genome sequencing (WGS), the isolated bacteriophages belong to the tailed bacteriophages from the Caudoviricetes class. Genomic analysis revealed that the phages have linear double-stranded DNA of size 40,461 bp (Serratia phage KKP_3708), 67,890 bp (Serratia phage KKP_3709), and 113,711 bp (Enterobacter phage KKP_3711). No virulence, toxins, or antibiotic resistance genes were detected in the phage genomes. The lack of lysogenic markers indicates that all three bacteriophages may be potential candidates for food biocontrol.

Newly Isolated Virulent Salmophages for Biocontrol of Multidrug-Resistant Salmonella in Ready-to-Eat Plant-Based Food.

Due to irrational antibiotic stewardship, an increase in the incidence of multidrug resistance of bacteria has been observed recently. Therefore, the search for new therapeutic methods for pathogen infection treatment seems to be necessary. One of the possibilities is the utilization of bacteriophages (phages)-the natural enemies of bacteria. Thus, this study is aimed at the genomic and functional characterization of two newly isolated phages targeting MDR Salmonella enterica strains and their efficacy in salmonellosis biocontrol in raw carrot-apple juice. The Salmonella phage vB_Sen-IAFB3829 (Salmonella phage strain KKP 3829) and Salmonella phage vB_Sen-IAFB3830 (Salmonella phage strain KKP 3830) were isolated against S. I (6,8:l,-:1,7) strain KKP 1762 and S. Typhimurium strain KKP 3080 host strains, respectively. Based on the transmission electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the viruses were identified as members of tailed bacteriophages from the Caudoviricetes class. Genome sequencing revealed that these phages have linear double-stranded DNA and sizes of 58,992 bp (vB_Sen-IAFB3829) and 50,514 bp (vB_Sen-IAFB3830). Phages retained their activity in a wide range of temperatures (from -20 °C to 60 °C) and active acidity values (pH from 3 to 11). The exposure of phages to UV radiation significantly decreased their activity in proportion to the exposure time. The application of phages to the food matrices significantly reduced the level of Salmonella contamination compared to the control. Genome analysis showed that both phages do not encode virulence or toxin genes and can be classified as virulent bacteriophages. Virulent characteristics and no possible pathogen factors make examined phages feasible to be potential candidates for food biocontrol.

Isolation and Characterization of Pseudomonas aeruginosa and its Virulent Bacteriophages.

BACKGROUND AND OBJECTIVE: Pseudomonas aeruginosa is a free living bacterium in widely different areas such as plants, soil, water and other moist locations. It is pathogenic to plants and humans. P. aeruginosa causes several disease symptoms to plants such as wet rot and curved leaves. The virulent bacterial viruses of P. aeruginosa were found to be of widespread occurrence in nature and isolated from widely different sources. Bacterial viruses were applied to control pathogenic bacteria in different fields and successfully. Therefore, this work aimed to study the different characteristics of P. aeruginosa lytic phage isolates. Moreover, the bio-control of P. aeruginosa by lytic phage isolates was also studied. MATERIAL AND METHODS: Different physical and molecular characteristics were assayed and determined of P. aeruginosa lytic bacteriophages. Also, the effect of phage isolates on P. aeruginosa as a bio-control under lab condition was studied. RESULTS: Pseudomonas aeruginosa pathogenic bacterium was isolated from a sewage water sample. Two lytic bacteriophages specific to P. aeruginosa were isolated from same sewage water sample and designated Pa1 and Pa2. Both phage isolates (Pa1 and Pa2) found to be stable in 90°C and different pH low and high levels. The total count of P. aeruginosa decreased after 48 h in broth treated with lytic phages. RAPD-PCR amplification was indicated that the two phage isolates (Pa1 and Pa2) are belonging to two different phage types. CONCLUSION: The results of this study indicated that both lytic phage isolates could be used as a biological control agents against the plant pathogen P. aeuroginosa.

Guidelines to Compose an Ideal Bacteriophage Cocktail.

Correctly designed bacteriophage therapeutics are the cornerstone for a successful outcome of bacteriophage therapy. Here we overview strategies on how to choose bacteriophages and their bacterial hosts at different steps of a bacteriophage cocktail development in order to comply with all quality and safety requirements based on the already existing essentially empirical experience in bacteriophage therapy and current accomplishments in modern biomedical sciences. A modification of the classic Appelmans' method (1922) to assess stability of bacteriophage activity in liquid media is presented in order to improve the overall performance of therapeutic bacteriophages individually and collectively in the cocktail.

Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9.

Phages are biological entities found in every ecosystem. Although much has been learned about them in past decades, significant knowledge gaps remain. Manipulating virulent phage genomes is challenging. To date, no efficient gene-editing tools exist for engineering virulent lactococcal phages. Lactococcus lactis is a bacterium extensively used as a starter culture in various milk fermentation processes, and its phage sensitivity poses a constant risk to the cheese industry. The lactococcal phage p2 is one of the best-studied models for these virulent phages. Despite its importance, almost half of its genes have no functional assignment. CRISPR-Cas9 genome editing technology, which is derived from a natural prokaryotic defense mechanism, offers new strategies for phage research. Here, the well-known Streptococcus pyogenes CRISPR-Cas9 was used in a heterologous host to modify the genome of a strictly lytic phage. Implementation of our adapted CRISPR-Cas9 tool in the prototype phage-sensitive host L. lactis MG1363 allowed us to modify the genome of phage p2. A simple, reproducible technique to generate precise mutations that allow the study of lytic phage genes and their encoded proteins in vivo is described.