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.

The Novel Concept of Synergically Combining: High Hydrostatic Pressure and Lytic Bacteriophages to Eliminate Vegetative and Spore-Forming Bacteria in Food Products.

The article focuses on the ongoing challenge of eliminating vegetative and spore-forming bacteria from food products that exhibit resistance to the traditional preservation methods. In response to this need, the authors highlight an innovative approach based on the synergistic utilization of high-hydrostatic-pressure (HHP) and lytic bacteriophages. The article reviews the current research on the use of HHP and lytic bacteriophages to combat bacteria in food products. The scope includes a comprehensive review of the existing literature on bacterial cell damage following HHP application, aiming to elucidate the synergistic effects of these technologies. Through this in-depth analysis, the article aims to contribute to a deeper understanding of how these innovative techniques can improve food safety and quality. There is no available research on the use of HHP and bacteriophages in the elimination of spore-forming bacteria; however, an important role of the synergistic effect of HHP and lytic bacteriophages with the appropriate adjustment of the parameters has been demonstrated in the more effective elimination of non-spore-forming bacteria from food products. This suggests that, when using this approach in the case of spore-forming bacteria, there is a high chance of the effective inactivation of this biological threat.

Characterization and Genome Study of a Newly Isolated Temperate Phage Belonging to a New Genus Targeting Alicyclobacillus acidoterrestris.

The spoilage of juices by Alicyclobacillus spp. remains a serious problem in industry and leads to economic losses. Compounds such as guaiacol and halophenols, which are produced by Alicyclobacillus, create undesirable flavors and odors and, thus, decrease the quality of juices. The inactivation of Alicyclobacillus spp. constitutes a challenge because it is resistant to environmental factors, such as high temperatures, and active acidity. However, the use of bacteriophages seems to be a promising approach. In this study, we aimed to isolate and comprehensively characterize a novel bacteriophage targeting Alicyclobacillus spp. The Alicyclobacillus phage strain KKP 3916 was isolated from orchard soil against the Alicyclobacillus acidoterrestris strain KKP 3133. The bacterial host's range and the effect of phage addition at different rates of multiplicity of infections (MOIs) on the host's growth kinetics were determined using a Bioscreen C Pro growth analyzer. The Alicyclobacillus phage strain KKP 3916, retained its activity in a wide range of temperatures (from 4 °C to 30 °C) and active acidity values (pH from 3 to 11). At 70 °C, the activity of the phage decreased by 99.9%. In turn, at 80 °C, no activity against the bacterial host was observed. Thirty minutes of exposure to UV reduced the activity of the phages by almost 99.99%. Based on transmission-electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the Alicyclobacillus phage strain KKP 3916 was classified as a tailed bacteriophage. The genomic sequencing revealed that the newly isolated phage had linear double-stranded DNA (dsDNA) with sizes of 120 bp and 131 bp and 40.3% G+C content. Of the 204 predicted proteins, 134 were of unknown function, while the remainder were annotated as structural, replication, and lysis proteins. No genes associated with antibiotic resistance were found in the genome of the newly isolated phage. However, several regions, including four associated with integration into the bacterial host genome and excisionase, were identified, which indicates the temperate (lysogenic) life cycle of the bacteriophage. Due to the risk of its potential involvement in horizontal gene transfer, this phage is not an appropriate candidate for further research on its use in food biocontrol. To the best of our knowledge, this is the first article on the isolation and whole-genome analysis of the Alicyclobacillus-specific phage.

Effectiveness of a Phage Cocktail as a Potential Biocontrol Agent against Saprophytic Bacteria in Ready-To-Eat Plant-Based Food.

This study aimed to evaluate the effectiveness of the phage cocktail to improve the microbiological quality of five different mixed-leaf salads: rucola, mixed-leaf salad with carrot, mixed-leaf salad with beetroot, washed and unwashed spinach, during storage in refrigerated conditions. Enterobacterales rods constituted a significant group of bacteria in the tested products. Selected bacteria were tested for antibiotic resistance profiles and then used to search for specific bacteriophages. Forty-three phages targeting bacteria dominant in mixed-leaf salads were isolated from sewage. Their titer was determined, and lytic activity was assessed using the Bioscreen C Pro automated growth analyzer. Two methods of phage cocktail application including spraying, and an absorption pad were effective for rucola, mixed leaf salad with carrot, and mixed leaf salad with beetroot. The maximum reduction level after 48 h of incubation reached 99.9% compared to the control sample. In washed and unwashed spinach, attempts to reduce the number of microorganisms did not bring the desired effect. The decrease in bacteria count in the lettuce mixes depended on the composition of the autochthonous saprophytic bacteria species. Both phage cocktail application methods effectively improved the microbiological quality of minimally processed products. Whole-spectral phage cocktail application may constitute an alternative food microbiological quality improvement method without affecting food properties.

Application of Lytic Bacteriophages and Their Enzymes to Reduce Saprophytic Bacteria Isolated from Minimally Processed Plant-Based Food Products-In Vitro Studies.

The aim of this study was to isolate phage enzymes and apply them in vitro for eradication of the dominant saprophytic bacteria isolated from minimally processed food. Four bacteriophages-two Enterobacter-specific and two Serratia-specific, which produce lytic enzymes-were used in this research. Two methods of phage enzyme isolation were tested, namely precipitation with acetone and ultracentrifugation. It was found that the number of virions could be increased almost 100 times due to the extension of the cultivation time (72 h). The amplification of phage particles and lytic proteins was dependent on the time of cultivation. Considering the influence of isolated enzymes on the growth kinetics of bacterial hosts, proteins isolated with acetone after 72-hour phage propagation exhibited the highest inhibitory effect. The reduction of bacteria count was dependent on the concentration of enzymes in the lysates. The obtained results indicate that phages and their lytic enzymes could be used in further research aiming at the improvement of microbiological quality and safety of minimally processed food products.

Bacterial Pathogens in the Food Industry: Antibiotic Resistance and Virulence Factors of Salmonella enterica Strains Isolated from Food Chain Links.

Salmonella is one of the most important foodborne pathogens. Fifty-three strains of Salmonella deposited in the Culture Collection of Industrial Microorganisms-Microbiological Resources Center (IAFB) were identified using molecular and proteomic analyses. Moreover, the genetic similarity of the tested strains was determined using the PFGE method. Main virulence genes were identified, and phenotypical antibiotic susceptibility profiles and prevalence of resistance genes were analyzed. Subsequently, the occurrence of the main mechanisms of ß-lactam resistance was determined. Virulence genes, invA, fimA, and stn were identified in all tested strains. Phenotypic tests, including 28 antibiotics, showed that 50.9% of the strains were MDR. The tet genes associated with tetracyclines resistance were the most frequently identified genes. Concerning the genes associated with ESBL-producing Salmonella, no resistance to the TEM and CTX-M type was identified, and only two strains (KKP 1597 and KKP 1610) showed resistance to SHV. No strains exhibited AmpC-type resistance but for six Salmonella strains, the efflux-related resistance of PSE-1 was presented. The high number of resistant strains in combination with multiple ARGs in Salmonella indicates the possible overuse of antibiotics. Our results showed that it is necessary to monitor antimicrobial resistance profiles in all food chain links constantly and to implement a policy of proper antibiotic stewardship to contain or at least significantly limit the further acquisition of antibiotic resistance among Salmonella strains.

The use of bacteriophages against saprophytic mesophilic bacteria in minimally processed food.

BACKGROUND: ood producers strive to meet the changing needs of consumers while maintaining the highest nutritional value of the products they supply. Physicochemical methods, which include modified atmosphere packaging, membrane techniques or ultrasounds, are the most frequently used to preserve food. Alternatively, biological methods can be applied, one of which is the use of bacteriophages (phages) to limit bacterial growth in the food environment. The purpose of our research was to verify the possibility of the use of bacteriophages as an antibacterial agent in minimally processed food environments of vegetable origin. The first stage of the research involved the isolation of phages against the dominant bacterial microflora in the analyzed products: broccoli sprouts, spinach leaves and freshly squeezed carrot-celery juice. Bacteriophages were isolated from municipal waste collected from sewage-treatment plants. Specific bacteriophages were isolated for twenty-nine out of thirty identified bacterial strains. The lytic activity of the phages was tested using a Bioscreen C automatic growth analyzer. Three methods for applying the phage cocktail were tested: direct addition of the cocktail, spraying it on, and placing the food product on a pad soaked with the phage mixture. The food products were packaged in a protective atmosphere and stored at 20°C. The total number of bacteria after adding the phage cocktail to the products was determined during the subsequent hours of incubation using classical microbial culturing. A significant decrease in the total number of bacteria was observed in the products containing phage suspensions. The obtained results suggest that application of the phage cocktail offers the possibility to extend the shelf life of the analyzed minimally processed food products by reducing the total number of saprophytic. METHODS: , food producers strive to meet the changing needs of consumers while maintaining the highest nutritional value of the products they supply. Physicochemical methods, which include modified atmosphere packaging, membrane techniques or ultrasounds, are the most frequently used to preserve food. Alternatively, biological methods can be applied, one of which is the use of bacteriophages (phages) to limit bacterial growth in the food environment. The purpose of our research was to verify the possibility of the use of bacteriophages as an antibacterial agent in minimally processed food environments of vegetable origin. The first stage of the research involved the isolation of phages against the dominant bacterial microflora in the analyzed products: broccoli sprouts, spinach leaves and freshly squeezed carrot-celery juice. Bacteriophages were isolated from municipal waste collected from sewage-treatment plants. Specific bacteriophages were isolated for twenty-nine out of thirty identified bacterial strains. The lytic activity of the phages was tested using a Bioscreen C automatic growth analyzer. Three methods for applying the phage cocktail were tested: direct addition of the cocktail, spraying it on, and placing the food product on a pad soaked with the phage mixture. The food products were packaged in a protective atmosphere and stored at 20°C. The total number of bacteria after adding the phage cocktail to the products was determined during the subsequent hours of incubation using classical microbial culturing. A significant decrease in the total number of bacteria was observed in the products containing phage suspensions. The obtained results suggest that application of the phage cocktail offers the possibility to extend the shelf life of the analyzed minimally processed food products by reducing the total number of saprophytic bac. RESULTS: , food producers strive to meet the changing needs of consumers while maintaining the highest nutritional value of the products they supply. Physicochemical methods, which include modified atmosphere packaging, membrane techniques or ultrasounds, are the most frequently used to preserve food. Alternatively, biological methods can be applied, one of which is the use of bacteriophages (phages) to limit bacterial growth in the food environment. The purpose of our research was to verify the possibility of the use of bacteriophages as an antibacterial agent in minimally processed food environments of vegetable origin. The first stage of the research involved the isolation of phages against the dominant bacterial microflora in the analyzed products: broccoli sprouts, spinach leaves and freshly squeezed carrot-celery juice. Bacteriophages were isolated from municipal waste collected from sewage-treatment plants. Specific bacteriophages were isolated for twenty-nine out of thirty identified bacterial strains. The lytic activity of the phages was tested using a Bioscreen C automatic growth analyzer. Three methods for applying the phage cocktail were tested: direct addition of the cocktail, spraying it on, and placing the food product on a pad soaked with the phage mixture. The food products were packaged in a protective atmosphere and stored at 20°C. The total number of bacteria after adding the phage cocktail to the products was determined during the subsequent hours of incubation using classical microbial culturing. A significant decrease in the total number of bacteria was observed in the products containing phage suspensions. The obtained results suggest that application of the phage cocktail offers the possibility to extend the shelf life of the analyzed minimally processed food products by reducing the total number of saprophytic bacteria.