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12 pages, 1968 KiB

Open AccessArticle

Development of TaqMan Real-Time PCR Protocols for Simultaneous Detection and Quantification of the Bacterial Pathogen Ralstonia solanacearum and Their Specific Lytic Bacteriophages

by Edson Bertolini, Àngela Figàs-Segura, Belén Álvarez and Elena G. Biosca

Viruses 2023, 15(4), 841; https://doi.org/10.3390/v15040841 - 25 Mar 2023

Cited by 8 | Viewed by 3094

Abstract

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three [...] Read more.

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three specific lytic R. solanacearum bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants. To optimize their applications, the phages and the bacterium need to be accurately monitored and quantified, which is laborious and time-consuming with biological methods. In this work, primers and TaqMan probes were designed, and duplex and multiplex real-time quantitative PCR (qPCR) protocols were developed and optimized for the simultaneous quantification of R. solanacearum and their phages. The quantification range was established from 10⁸ to 10 PFU/mL for the phages and from 10⁸ to 10² CFU/mL for R. solanacearum. Additionally, the multiplex qPCR protocol was validated for the detection and quantification of the phages with a limit ranging from 10² targets/mL in water and plant extracts to 10³ targets/g in soil, and the target bacterium with a limit ranging from 10³ targets/mL in water and plant extracts to 10⁴ targets/g in soil, using direct methods of sample preparation. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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15 pages, 3186 KiB

Open AccessArticle

The Broad-Spectrum Endolysin LySP2 Improves Chick Survival after Salmonella Pullorum Infection

by Hewen Deng, Mengjiao Li, Qiuyang Zhang, Chencheng Gao, Zhanyun Song, Chunhua Chen, Zhuo Wang and Xin Feng

Viruses 2023, 15(4), 836; https://doi.org/10.3390/v15040836 - 24 Mar 2023

Cited by 4 | Viewed by 2408

Abstract

Salmonella pullorum causes typical “Bacillary White Diarrhea” and loss of appetite in chicks, which leads to the death of chicks in severe cases; thus, it is still a critical issue in China. Antibiotics are conventional medicines used for Salmonella infections; however, due to [...] Read more.

Salmonella pullorum causes typical “Bacillary White Diarrhea” and loss of appetite in chicks, which leads to the death of chicks in severe cases; thus, it is still a critical issue in China. Antibiotics are conventional medicines used for Salmonella infections; however, due to the extensive long-term use and even abuse of antibiotics, drug resistance becomes increasingly severe, making treating pullorum disease more difficult. Most of the endolysins are hydrolytic enzymes produced by bacteriophages to cleave the host’s cell wall during the final stage of the lytic cycle. A virulent bacteriophage, YSP2, of Salmonella was isolated in a previous study. A Pichia pastoris expression strain that can express the Salmonella bacteriophage endolysin was constructed efficiently, and the Gram-negative bacteriophage endolysin, LySP2, was obtained in this study. Compared with the parental phage YSP2, which can only lyse Salmonella, LySP2 can lyse Salmonella and Escherichia. The survival rate of Salmonella-infected chicks treated with LySP2 can reach up to 70% and reduce Salmonella abundance in the liver and intestine. The treatment group showed that LySP2 significantly improved the health of infected chicks and alleviated organ damage caused by Salmonella infection. In this study, the Salmonella bacteriophage endolysin was expressed efficiently by Pichia pastoris, and the endolysin LySP2 showed good potential for the treatment of pullorum disease caused by Salmonella pullorum. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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28 pages, 682 KiB

Open AccessArticle

Gender-Responsive Design of Bacteriophage Products to Enhance Adoption by Chicken Keepers in Kenya

by Zoë A. Campbell, Nelly Njiru, Amos Lucky Mhone, Angela Makumi, Sylvain Moineau and Nicholas Svitek

Viruses 2023, 15(3), 746; https://doi.org/10.3390/v15030746 - 14 Mar 2023

Viewed by 3631

Abstract

Women and men keeping chickens in Kenya aspire to have a source of income, feed their families healthy food, and grow their businesses. Managing animal diseases and minimizing input costs enable their success. This study uses qualitative methods to recommend design opportunities for [...] Read more.

Women and men keeping chickens in Kenya aspire to have a source of income, feed their families healthy food, and grow their businesses. Managing animal diseases and minimizing input costs enable their success. This study uses qualitative methods to recommend design opportunities for a veterinary product under development in Kenya that contains bacteriophages (phages) that target pathogenic Salmonella strains responsible for fowl typhoid, salmonellosis, and pullorum in chickens and foodborne illness in people. Our findings revealed the interplay between gender and two production systems: free-range and semi-intensive. Chicken keepers in both systems could benefit from phages combined with the orally administered Newcastle disease vaccine, one of the most commonly used preventive veterinary interventions, or phages as a treatment for fowl typhoid. Oral administration is less labor intensive, with greater benefits for women who have less control over family labor and reported doing more care tasks themselves. Men in free-range systems usually pay for veterinary inputs. In semi-intensive production systems, a phage-based product used prophylactically could be an alternative to expensive, intramuscular fowl typhoid vaccines. Keeping layers was common for women in semi-intensive systems, as they are more economically impacted by reduced laying caused by bacterial diseases. Awareness of zoonoses was low, but men and women were concerned about the negative health effects of drug residues in meat and eggs. Therefore, highlighting the lack of a withdrawal period for a phage product may appeal to customers. Antibiotics are used to both treat and prevent diseases, and phage products will need to do both to compete in the Kenyan market. These findings guide the ongoing design of a phage-based product with the goal of introducing a new veterinary product that meets the diverse needs of chicken keepers in Africa and serves as an alternative or complement to antibiotics. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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11 pages, 620 KiB

Open AccessArticle

A Glimpse at the Anti-Phage Defenses Landscape in the Foodborne Pathogen Salmonella enterica subsp. enterica serovar Typhimurium

by Cedric Woudstra and Sophie A. Granier

Viruses 2023, 15(2), 333; https://doi.org/10.3390/v15020333 - 24 Jan 2023

Cited by 1 | Viewed by 3026

Abstract

Bacteriophages, which specifically infect and kill bacteria, are currently used as additives to control pathogens such as Salmonella in human food (PhageGuard S^®) or animal feed (SalmoFREE^®, Bafasal^®). Indeed, salmonellosis is among the most important zoonotic foodborne [...] Read more.

Bacteriophages, which specifically infect and kill bacteria, are currently used as additives to control pathogens such as Salmonella in human food (PhageGuard S^®) or animal feed (SalmoFREE^®, Bafasal^®). Indeed, salmonellosis is among the most important zoonotic foodborne illnesses. The presence of anti-phage defenses protecting bacteria against phage infection could impair phage applications aiming at reducing the burden of foodborne pathogens such as Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) to the food industry. In this study, the landscape of S. Typhimurium anti-phage defenses was bioinformatically investigated in publicly available genomes using the webserver PADLOC. The primary anti-phage systems identified in S. Typhimurium use nucleic acid degradation and abortive infection mechanisms. Reference systems were identified on an integrative and conjugative element, a transposon, a putative integrative and mobilizable element, and prophages. Additionally, the mobile genetic elements (MGEs) containing a subset of anti-phage systems were found in the Salmonella enterica species. Lastly, the MGEs alone were also identified in the Enterobacteriaceae family. The presented diversity assessment of the anti-phage defenses and investigation of their dissemination through MGEs in S. Typhimurium constitute a first step towards the design of preventive measures against the spread of phage resistance that may hinder phage applications. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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20 pages, 2196 KiB

Open AccessArticle

Tailoring the Host Range of Ackermannviridae Bacteriophages through Chimeric Tailspike Proteins

by Jose Gil, John Paulson, Matthew Brown, Henriett Zahn, Minh M. Nguyen, Marcia Eisenberg and Stephen Erickson

Viruses 2023, 15(2), 286; https://doi.org/10.3390/v15020286 - 19 Jan 2023

Cited by 8 | Viewed by 2580

Abstract

Host range is a major determinant in the industrial utility of a bacteriophage. A model host range permits broad recognition across serovars of a target bacterium while avoiding cross-reactivity with commensal microbiota. Searching for a naturally occurring bacteriophage with ideal host ranges is [...] Read more.

Host range is a major determinant in the industrial utility of a bacteriophage. A model host range permits broad recognition across serovars of a target bacterium while avoiding cross-reactivity with commensal microbiota. Searching for a naturally occurring bacteriophage with ideal host ranges is challenging, time-consuming, and restrictive. To address this, SPTD1.NL, a previously published luciferase reporter bacteriophage for Salmonella, was used to investigate manipulation of host range through receptor-binding protein engineering. Similar to related members of the Ackermannviridae bacteriophage family, SPTD1.NL possessed a receptor-binding protein gene cluster encoding four tailspike proteins, TSP1-4. Investigation of the native gene cluster through chimeric proteins identified TSP3 as the tailspike protein responsible for Salmonella detection. Further analysis of chimeric phages revealed that TSP2 contributed off-target Citrobacter recognition, whereas TSP1 and TSP4 were not essential for activity against any known host. To improve the host range of SPTD1.NL, TSP1 and TSP2 were sequentially replaced with chimeric receptor-binding proteins targeting Salmonella. This engineered construct, called RBP-SPTD1-3, was a superior diagnostic reporter, sensitively detecting additional Salmonella serovars while also demonstrating improved specificity. For industrial applications, bacteriophages of the Ackermannviridae family are thus uniquely versatile and may be engineered with multiple chimeric receptor-binding proteins to achieve a custom-tailored host range. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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24 pages, 398 KiB

Open AccessArticle

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

by Michał Wójcicki, Olga Świder, Iwona Gientka, Stanisław Błażejak, Paulina Średnicka, Dziyana Shymialevich, Hanna Cieślak, Artur Wardaszka, Paulina Emanowicz, Barbara Sokołowska and Edyta Juszczuk-Kubiak

Viruses 2023, 15(1), 172; https://doi.org/10.3390/v15010172 - 6 Jan 2023

Cited by 13 | Viewed by 3505

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

19 pages, 4005 KiB

Open AccessArticle

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

by Dziyana Shymialevich, Michał Wójcicki, Artur Wardaszka, Olga Świder, Barbara Sokołowska and Stanisław Błażejak

Viruses 2023, 15(1), 9; https://doi.org/10.3390/v15010009 - 20 Dec 2022

Cited by 4 | Viewed by 3358

Abstract

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. [...] Read more.

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. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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19 pages, 2187 KiB

Open AccessArticle

Phage Encounters Recorded in CRISPR Arrays in the Genus Oenococcus

by Yasma Barchi, Cécile Philippe, Amel Chaïb, Florencia Oviedo-Hernandez, Olivier Claisse and Claire Le Marrec

Viruses 2023, 15(1), 15; https://doi.org/10.3390/v15010015 - 20 Dec 2022

Cited by 5 | Viewed by 2237

Abstract

The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected [...] Read more.

The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected through their bacteriophages. First, we investigated the diversity of CRISPR-Cas systems using a genome-mining approach, and CRISPR-endowed strains were identified in three species. A census of the spacers from the four identified CRISPR-Cas loci showed that each spacer space was mostly dominated by species-specific sequences. Yet, we characterized a limited records of potentially recent and also ancient infections between O. kitaharae and O. sicerae and phages of O. oeni, suggesting that some related phages have interacted in diverse ways with their Oenococcus hosts over evolutionary time. Second, phage-host interaction analyses were performed experimentally with a diversified panel of phages and strains. None of the tested phages could infect strains across the species barrier. Yet, some infections occurred between phages and hosts from distinct beverages in the O. oeni species. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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13 pages, 3030 KiB

Open AccessArticle

Influence of Physicochemical Factors on Adsorption of Ten Shigella flexneri Phages

by David Tomat, Virginia Aquili, Cecilia Casabonne and Andrea Quiberoni

Viruses 2022, 14(12), 2815; https://doi.org/10.3390/v14122815 - 16 Dec 2022

Cited by 2 | Viewed by 1886

Abstract

Bacterial viruses known as bacteriophages have been demonstrated to be effective in killing foodborne pathogens such as Shigella flexneri. Adsorption is the first step in the phage–host interaction. In the present work, 10 Shigella phages were used to characterize the adsorption process [...] Read more.

Bacterial viruses known as bacteriophages have been demonstrated to be effective in killing foodborne pathogens such as Shigella flexneri. Adsorption is the first step in the phage–host interaction. In the present work, 10 Shigella phages were used to characterize the adsorption process on Shigella flexneri ATCC12022 in several physicochemical conditions related to food and in a food matrix. One-step growth curves were drawn for all the Shigella-phages evaluated. Furthermore, the adsorption rate for each of the 10 phages was determined. In addition, the influence of temperature, Na⁺, Mg²⁺, pH, sucrose and glycerol on phage adsorption was investigated. Two phages (Shi22 and Shi30) showed higher burst sizes values (67 and 64 PFU cell⁻¹, respectively) and burst times of 25 min to 30 min, while the other eight phages exhibited burst sizes ranging from 14 to 17 PFU cell⁻¹ with slower burst times. Furthermore, most phages achieved a high adsorption rate, and the adsorption constants (k) ranged from ~10⁻⁹ to 10⁻¹⁰ mL min⁻¹. Regarding the influence of temperature, cations and pH, a high or moderate percentage of adsorption was observed for most of the phages evaluated. The adsorption decreased at increasing concentrations of Na⁺, sucrose and glycerol, although at different levels, since adsorption was more affected by sucrose than by glycerol and Na⁺ for most phages. The adsorption obtained in Triptein soy broth (TSB) for most of the phages/strain systems evaluated was moderate or high, as well as those observed in a food matrix. Thus, our phages could potentially be used to improve food safety under a wide range of environmental conditions against foodborne pathogens. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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16 pages, 1649 KiB

Open AccessArticle

Phages Shape Microbial Dynamics and Metabolism of a Model Community Mimicking Cider, a Fermented Beverage

by Pierre Ledormand, Nathalie Desmasures, Margot Schlusselhuber, André Sesboüé, Jérôme Ledauphin and Marion Dalmasso

Viruses 2022, 14(10), 2283; https://doi.org/10.3390/v14102283 - 17 Oct 2022

Cited by 5 | Viewed by 2101

Abstract

Model microbial communities are often studied to better understand interactions and fluxes during fermentation processes. However, models that take into account the potential impact of bacteriophages (phages), which are recognized as drivers of microbial communities, are scarce, especially in fermented foods. This study [...] Read more.

Model microbial communities are often studied to better understand interactions and fluxes during fermentation processes. However, models that take into account the potential impact of bacteriophages (phages), which are recognized as drivers of microbial communities, are scarce, especially in fermented foods. This study aimed at investigating the behavior of a cider model microbial community, which was subjected to disturbance in the presence or absence of phages and at two different temperatures (25 °C and 15 °C). The model microbial community was composed of three lactic acid bacteria (LAB) strains belonging to the species Liquorilactobacillus mali, Leuconostoc mesenteroides and Oenococcus oeni, and of a Saccharomyces uvarum yeast strain. Two phages were selected, targeting L. mali and Ln. mesenteroides strains. In order to follow the behavior of the microbial community model, the phages and microbial strains were enumerated at several time points, and the metabolic signatures (sugar consumption, production of organic acids and volatile organic compounds) of the model microbial community were monitored. At 25 °C, the community with phages (P) was significantly closer to the control condition (C) than to the condition without phages (D). Microbial levels were similar between conditions C and P, which were characterized by high concentrations of compounds such as 2-phenylethanol, ethyl octanoate and isoamyl alcohol, and more globally by a more complex metabolic signature than that of condition D. In condition D, L. mali and Ln. mesenteroides were dominant while S. uvarum and O. oeni were less present, and this condition was characterized by a high concentration of ethyl lactate. At 15 °C, condition P differed from conditions C and D, as Ln. mesenteroides was not detected while the other strains all reached approximately the same levels. The metabolic range of condition P was less important than for conditions C and D. The current study showed that the influence of phages on the model microbial community dynamics and metabolisms after a disturbance phenomenon was temperature-dependent. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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16 pages, 3050 KiB

Open AccessArticle

Biological and Molecular Characterization of the Lytic Bacteriophage SoKa against Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast and Black Pit in Tunisia

by Maroua Oueslati, Dominique Holtappels, Kiandro Fortuna, Mohamed Rabeh Hajlaoui, Rob Lavigne, Najla Sadfi-Zouaoui and Jeroen Wagemans

Viruses 2022, 14(9), 1949; https://doi.org/10.3390/v14091949 - 2 Sep 2022

Cited by 6 | Viewed by 2736

Abstract

Pseudomonas syringae pv. syringae (Pss), the causal agent of citrus blast and black pit lesion of lemon fruit, continues to cause serious damage in citrus production in Tunisia. Faced with the rapid emergence of the disease and the inefficiency of conventional control methods, [...] Read more.

Pseudomonas syringae pv. syringae (Pss), the causal agent of citrus blast and black pit lesion of lemon fruit, continues to cause serious damage in citrus production in Tunisia. Faced with the rapid emergence of the disease and the inefficiency of conventional control methods, an alternative strategy based on the use of bacteriophages was pursued in this study. The lytic Pss bacteriophage SoKa was isolated from soil collected from Tunisian citrus orchards. Analysis of the host range showed that SoKa was able to lyse seven other Pss strains. Interestingly, Pseudomonas syringae pv. porri, pathogenic to leek, could also be infected by SoKa. The activity of SoKa was maintained at pH values between 2 and 10, at temperatures between −80 and 37 °C; the phage could resist UV radiation at an intensity of 320 nm up to 40 min. Whole genome sequencing revealed that the Pseudomonas phage SoKa is a novel phage that belongs to the Bifseptvirus genus of the Autographiviridae family. The absence of virulence proteins and lysogeny-associated proteins encoded on the phage genome, its anti-biofilm activity, and the significant reduction of tissue necrosis in different fruit bioassays make SoKa potentially suitable for use in phage biocontrol. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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21 pages, 4723 KiB

Open AccessArticle

Salmonella Enteritidis Bacteriophages Isolated from Kenyan Poultry Farms Demonstrate Time-Dependent Stability in Environments Mimicking the Chicken Gastrointestinal Tract

by Amos Lucky Mhone, Angela Makumi, Josiah Odaba, Linda Guantai, K. M. Damitha Gunathilake, Stéphanie Loignon, Caroline Wangari Ngugi, Juliah Khayeli Akhwale, Sylvain Moineau and Nicholas Svitek

Viruses 2022, 14(8), 1788; https://doi.org/10.3390/v14081788 - 16 Aug 2022

Cited by 9 | Viewed by 4172

Abstract

Multi-drug resistant (MDR) Salmonella enterica Enteritidis is one of the major causes of foodborne illnesses worldwide. This non-typhoidal Salmonella (NTS) serovar is mainly transmitted to humans through poultry products. Bacteriophages (phages) offer an alternative to antibiotics for reducing the incidence of MDR NTS [...] Read more.

Multi-drug resistant (MDR) Salmonella enterica Enteritidis is one of the major causes of foodborne illnesses worldwide. This non-typhoidal Salmonella (NTS) serovar is mainly transmitted to humans through poultry products. Bacteriophages (phages) offer an alternative to antibiotics for reducing the incidence of MDR NTS in poultry farms. Phages that survive the harsh environment of the chicken gastrointestinal tract (cGIT), which have low pH, high temperatures, and several enzymes, may have a higher therapeutic or prophylactic potential. In this study, we analysed the stability of 10 different S. Enteritidis phages isolated from Kenyan poultry farms in different pH-adjusted media, incubation temperatures, as well as simulated gastric and intestinal fluids (SGF and SIF, respectively). Furthermore, their ability to persist in water sources available in Kenya, including river, borehole, rain and tap water, was assessed. All phages were relatively stable for 12 h at pHs ranging from 5 to 9 and at temperatures ranging from 25 °C to 42 °C. At pH 3, a loss in viral titre of up to three logs was observed after 3 h of incubation. In SGF, phages were stable for 20 min, after which they started losing infectivity. Phages were relatively stable in SIF for up to 2 h. The efficacy of phages to control Salmonella growth was highly reduced in pH 2- and pH 3-adjusted media and in SGF at pH 2.5, but less affected in SIF at pH 8. River water had the most significant detrimental effect on phages, while the other tested waters had a limited impact on the phages. Our data suggest that these phages may be administered to chickens through drinking water and may survive cGIT to prevent salmonellosis in poultry. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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24 pages, 3436 KiB

Open AccessArticle

Virulent Phages Isolated from a Smear-Ripened Cheese Are Also Detected in Reservoirs of the Cheese Factory

by Thomas Paillet, Julien Lossouarn, Clarisse Figueroa, Cédric Midoux, Olivier Rué, Marie-Agnès Petit and Eric Dugat-Bony

Viruses 2022, 14(8), 1620; https://doi.org/10.3390/v14081620 - 25 Jul 2022

Cited by 7 | Viewed by 3030

Abstract

Smear-ripened cheeses host complex microbial communities that play a crucial role in the ripening process. Although bacteriophages have been frequently isolated from dairy products, their diversity and ecological role in such this type of cheese remain underexplored. In order to fill this gap, [...] Read more.

Smear-ripened cheeses host complex microbial communities that play a crucial role in the ripening process. Although bacteriophages have been frequently isolated from dairy products, their diversity and ecological role in such this type of cheese remain underexplored. In order to fill this gap, the main objective of this study was to isolate and characterize bacteriophages from the rind of a smear-ripened cheese. Thus, viral particles extracted from the cheese rind were tested through a spot assay against a collection of bacteria isolated from the same cheese and identified by sequencing the full-length small subunit ribosomal RNA gene. In total, five virulent bacteriophages infecting Brevibacterium aurantiacum, Glutamicibacter arilaitensis, Leuconostoc falkenbergense and Psychrobacter aquimaris species were obtained. All exhibit a narrow host range, being only able to infect a few cheese-rind isolates within the same species. The complete genome of each phage was sequenced using both Nanopore and Illumina technologies, assembled and annotated. A sequence comparison with known phages revealed that four of them may represent at least new genera. The distribution of the five virulent phages into the dairy-plant environment was also investigated by PCR, and three potential reservoirs were identified. This work provides new knowledge on the cheese rind viral community and an overview of the distribution of phages within a cheese factory. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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16 pages, 2800 KiB

Open AccessArticle

Streptococcus thermophilus Phages in Whey Derivatives: From Problem to Application in the Dairy Industry

by Mariángeles Briggiler Marcó, Nicolás Machado, Andrea Quiberoni and Viviana Suárez

Viruses 2022, 14(4), 810; https://doi.org/10.3390/v14040810 - 14 Apr 2022

Cited by 4 | Viewed by 2635

Abstract

Fifteen samples of whey protein concentrate (WPC) were tested against 37 commercial Streptococcus thermophilus strains to detect infective bacteriophages. Seventy-three diverse phages were isolated from 12 samples, characterized by using DNA restriction patterns and host range analyses. Sixty-two of them were classified as [...] Read more.

Fifteen samples of whey protein concentrate (WPC) were tested against 37 commercial Streptococcus thermophilus strains to detect infective bacteriophages. Seventy-three diverse phages were isolated from 12 samples, characterized by using DNA restriction patterns and host range analyses. Sixty-two of them were classified as cos, two as pac, and nine as 5093, according to PCR multiplex assays. Phage concentration was greater than 10⁴ PFU/g for 25.3% of isolated phages. Seven phages showed an unusual wide host range, being able to infect a high number of the tested strains. Regarding thermal resistance, pac phages were the most sensitive, followed by cos phages, those classified as 5093 being the most resistant. Treatments at 85 °C for 5 min in TMG buffer were necessary to completely inactivate all phages. Results demonstrated that the use, without control, of these whey derivatives as additives in dairy fermentations could be a threat because of the potential phage infection of starter strains. In this sense, these phages constitute a pool of new isolates used to improve the phage resistance of starter cultures applied today in the fermentative industry. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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13 pages, 2018 KiB

Open AccessArticle

A Bacteriophage Cocktail Significantly Reduces Listeria monocytogenes without Deleterious Impact on the Commensal Gut Microbiota under Simulated Gastrointestinal Conditions

by Rasmus Riemer Jakobsen, Jimmy T. Trinh, Louise Bomholtz, Signe Kristine Brok-Lauridsen, Alexander Sulakvelidze and Dennis Sandris Nielsen

Viruses 2022, 14(2), 190; https://doi.org/10.3390/v14020190 - 19 Jan 2022

Cited by 14 | Viewed by 2860

Abstract

In this study, we examined the effect of a bacteriophage cocktail (tentatively designated as the Foodborne Outbreak Pill (FOP)) on the levels of Listeria monocytogenes in simulated small intestine, large intestine, and Caco-2 model systems. We found that FOP survival during simulated passage [...] Read more.

In this study, we examined the effect of a bacteriophage cocktail (tentatively designated as the Foodborne Outbreak Pill (FOP)) on the levels of Listeria monocytogenes in simulated small intestine, large intestine, and Caco-2 model systems. We found that FOP survival during simulated passage of the upper gastrointestinal was dependent on stomach pH, and that FOP robustly inhibited L. monocytogenes levels with effectiveness comparable to antibiotic treatment (ampicillin) under simulated ilium and colon conditions. The FOP did not inhibit the commensal bacteria, whereas ampicillin treatment led to dysbiosis-like conditions. The FOP was also more effective than an antibiotic in protecting Caco-2 cells from adhesion and invasion by L. monocytogenes (5-log reduction vs. 1-log reduction) while not triggering an inflammatory response. Our data suggested that the FOP may provide a robust protection against L. monocytogenes should the bacterium enter the human gastrointestinal tract (e.g., by consumption of contaminated food), without deleterious impact on the commensal bacteria. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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Bacteriophage Delivery Systems for Food Applications: Opportunities and Perspectives

by Maria J. Costa, Lorenzo M. Pastrana, José A. Teixeira, Sanna M. Sillankorva and Miguel A. Cerqueira

Viruses 2023, 15(6), 1271; https://doi.org/10.3390/v15061271 - 29 May 2023

Cited by 12 | Viewed by 4154

Abstract

Currently, one-third of all food produced worldwide is wasted or lost, and bacterial contamination is one of the main reasons. Moreover, foodborne diseases are a severe problem, causing more than 420,000 deaths and nearly 600 million illnesses yearly, demanding more attention to food [...] Read more.

Currently, one-third of all food produced worldwide is wasted or lost, and bacterial contamination is one of the main reasons. Moreover, foodborne diseases are a severe problem, causing more than 420,000 deaths and nearly 600 million illnesses yearly, demanding more attention to food safety. Thus, new solutions need to be explored to tackle these problems. A possible solution for bacterial contamination is using bacteriophages (phages), which are harmless to humans; these natural viruses can be used to prevent or reduce food contamination by foodborne pathogens. In this regard, several studies showed the effectiveness of phages against bacteria. However, when used in their free form, phages can lose infectivity, decreasing the application in foods. To overcome this problem, new delivery systems are being studied to incorporate phages and ensure prolonged activity and controlled release in food systems. This review focuses on the existent and new phage delivery systems applied in the food industry to promote food safety. Initially, an overview of phages, their main advantages, and challenges is presented, followed by the different delivery systems, focused in methodologies, and biomaterials that can be used. In the end, examples of phage applications in foods are disclosed and future perspectives are approached. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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27 pages, 3418 KiB

Open AccessReview

From Farm to Fork: Streptococcus suis as a Model for the Development of Novel Phage-Based Biocontrol Agents

by Emmanuel Kuffour Osei, Jennifer Mahony and John G. Kenny

Viruses 2022, 14(9), 1996; https://doi.org/10.3390/v14091996 - 9 Sep 2022

Cited by 5 | Viewed by 4063

Abstract

Bacterial infections of livestock threaten the sustainability of agriculture and public health through production losses and contamination of food products. While prophylactic and therapeutic application of antibiotics has been successful in managing such infections, the evolution and spread of antibiotic-resistant strains along the [...] Read more.

Bacterial infections of livestock threaten the sustainability of agriculture and public health through production losses and contamination of food products. While prophylactic and therapeutic application of antibiotics has been successful in managing such infections, the evolution and spread of antibiotic-resistant strains along the food chain and in the environment necessitates the development of alternative or adjunct preventive and/or therapeutic strategies. Additionally, the growing consumer preference for “greener” antibiotic-free food products has reinforced the need for novel and safer approaches to controlling bacterial infections. The use of bacteriophages (phages), which can target and kill bacteria, are increasingly considered as a suitable measure to reduce bacterial infections and contamination in the food industry. This review primarily elaborates on the recent veterinary applications of phages and discusses their merits and limitations. Furthermore, using Streptococcus suis as a model, we describe the prevalence of prophages and the anti-viral defence arsenal in the genome of the pathogen as a means to define the genetic building blocks that are available for the (synthetic) development of phage-based treatments. The data and approach described herein may provide a framework for the development of therapeutics against an array of bacterial pathogens. Full article

(This article belongs to the Special Issue Roles and Applications of Phages in the Food Industry and Agriculture)

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