Optimization of wall material for phage encapsulation via freeze-drying and antimicrobial efficacy of microencapsulated phage against Salmonella.

Microencapsulated phage as dry powder provides a protection to the phage particles from the harsh conditions while improving efficacy for controlling Salmonella. In this study, wall materials for phage encapsulation were optimized by altering the ratios of whey protein isolate (WPI) and trehalose prior to freeze-drying. Combination of WPI/trehalose at ratio of 3:1 (w/w) represented the optimal formulation with the highest encapsulation efficiency (91.9%). Fourier transform infrared spectroscopy analysis showed H-bonding in the mixture system and glass transition temperature presented at 63.43 °C. Encapsulated form showed the phage survivability of > 90% after 5 h of exposure to pH 1.5, 3.5, 5.5, 7.5 and 9.5. Phages in the non-encapsulated form could not survive at pH 1.5. In addition, microencapsulated phage showed high effectiveness in decreasing the numbers of S. Enteritidis and S. Typhimurium by approximately 1 log CFU/ml at 10 °C and 30 °C for both serovars. Phage powder newly developed in this study provides a convenient form for Salmonella control application and this form exhibits high stability over a wide range of temperatures and pH. This encapsulated phage thus can be used in various food applications without being interfered by physiological acidic or alkaline pH of foods or environments where phages are applied.

Evaluation of storage conditions and efficiency of a novel microencapsulated Salmonella phage cocktail for controlling S. enteritidis and S. typhimurium in-vitro and in fresh foods.

S. Enteritidis and S. Typhimurium are typically linked to foodborne outbreaks. Phages have continued to expand in various food applications. In this study, microencapsulation is applied for enhancing the stability and efficacy of phages as bio-control agent. Microencapsulated phage cocktail kept in aluminium laminated foil bag (LF) at 4 °C showed the highest survivability with a titer loss of 0.5 log PFU/g after 12 weeks of storage. Titer loss of phage cocktail lysate >4 log PFU/mL was observed after 12 weeks, at 4 °C. Color change of microencapsulated phage cocktail kept in LF at 4 °C did not show any significant difference during storage, and water activity (free water content) at 0.13 was found in these conditions. In-vitro study, S. Enteritidis and S. Typhimurium were decreased 1.79 and 3.63 log CFU/mL, respectively at 37 °C. Whereas, 0.43 and 0.76 log CFU/mL, respectively were observed at 10 °C. In foods, S. Enteritidis and S. Typhimurium were decreased 0.57 and 1.78 log CFU/cm2, respectively in meat. Whereas, 0.86 and 1.2 log CFU/g, respectively were observed in sprout. Foods with/without microencapsulated phage cocktail showed non-significant differences in liking scores after 2 days of storage. Overall, microencapsulated phage cocktail suggests another alternative for phage-based biocontrol with improved stability and efficacy for food application.

Antioxidant Properties and Antibacterial Effects of Eucalyptus camaldulensis Ethanolic Leaf Extract on Biofilm Formation, Motility, Hemolysin Production, and Cell Membrane of the Foodborne Pathogen Listeria monocytogenes.

Consumer concerns toward chemical preservatives have resulted in increased search for healthy green alternative. In this study, the antioxidant activity and antibacterial effects of Eucalyptus camaldulensis ethanolic leaf extract against Listeria monocytogenes, a serious foodborne pathogen, was evaluated. Total phenolic and flavonoid contents of the extract were 11.10 mg garlic acid equivalent/mg extract and 15.05 mg quercetin equivalent/mg extract, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration of the extract was 64-128 µg/mL and 256-512 µg/mL, respectively. Time-kill assay revealed growth inhibitory effects after 4-h treatment of the bacteria with the extract. A reduction of ≈2-3 log colony-forming units per milliliter was observed against the tested food and environmental isolates after challenging the pathogens with the extract at MIC for 6 h. Sub-MICs of the extract significantly inhibited motility and listeriolysin O production up to 80%, with 60% inhibition of biofilm formation (p < 0.05). Antioxidant assay revealed free radical scavenging activity with 50% inhibitory concentration (IC50) of 57.07 µg/mL for 2,2-diphenyl-1-picrylhydrazyl and 29.01 µg/mL for ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] assay. Ferric reducing antioxidant power assay further showed a total antioxidant power equivalent to 92.93 µM ascorbic acid equivalent/mg extract. As the extract exhibited profound antilisterial activity and good radical scavenging ability, it might serve as a potential alternative source of biopreservative agent against L. monocytogenes.

Longitudinal monitoring of Listeria monocytogenes and Listeria phages in seafood processing environments in Thailand.

Listeria monocytogenes is a foodborne pathogen commonly found in environments of seafood processing, thus presenting a challenge for eradication from seafood processing facilities. Monitoring the prevalence and subtype diversity of L. monocytogenes together with phages that are specific to Listeria spp. ("Listeria phages") will provide knowledge on the bacteria-phage ecology in food processing plants. In this work, a total of 595 samples were collected from raw material, finished seafood products and environmental samples from different sites of a seafood processing plant during 17 sampling visits in 1.5 years of study. L. monocytogenes and Listeria spp. (non-monocytogenes) were found in 22 (3.7%) and 43 (7.2%) samples, respectively, whereas 29 Listeria phages were isolated from 9 (1.5%) phage-positive samples. DNA fingerprint analysis of L. monocytogenes isolates revealed 11 Random Amplified Polymorphic DNA (RAPD) profiles, with two subtypes were frequently observed over time. Our data reveal a presence of Listeria phages within the same seafood processing environments where a diverse set of L. monocytogenes subtypes was also found. Although serotype 4b was observed at lower frequency, data indicate that isolates from this seafood processing plant belonged to both epidemiologically important serotypes 1/2a and 4b, which may suggest a potential public health risk. Phages (all showed a unique genome size of 65 ± 2 kb) were classified into 9 host range groups, representing both broad- and narrow-host range. While most L. monocytogenes isolates from this facility were susceptible to phages, five isolates showed resistance to 12-20 phages. Variations in phage host range among Listeria phages isolated from food processing plant may affect a presence of a diverse set of L. monocytogenes isolates derived from the same processing environment in Thailand.

Microbiological and chemical changes of shrimp Acetes vulgaris during Kapi production.

Microbiological and chemical changes in shrimp Acetes vulgaris during production of Kapi (salted shrimp paste of Thailand) including salting, drying and fermentation were monitored. Moisture content of samples decreased rapidly after salting and drying steps. The lower water activity was found in the final product (0.694). The pH decreased within the first 10 days of fermentation and continuously increased as fermentation progressed. Protein underwent degradation throughout Kapi production as indicated by increasing TCA-soluble peptides and degree of hydrolysis. The increases in peroxide value as well as thiobarbituric acid reactive substances value revealed that lipid oxidation occurred throughout all processes. Total viable count, halophilic, proteolytic and lipolytic bacteria counts increased continuously during Kapi production, while lactic acid bacteria count slightly decreased at the final stage of fermentation. Thus, proteolysis and lipolysis took place throughout Kapi production, and contributed to the characteristics of finished product. These changes were governed by both endogenous and microbial enzymes.

Use of Rhodomyrtus tomentosa ethanolic leaf extract for the bio-control of Listeria monocytogenes post-cooking contamination in cooked chicken meat.

Controlling foodborne pathogen in ready-to-eat food is important in food safety. The present study accessed the potential use of Rhodomyrtus tomentosa ethanolic leaf extract as a bio-control agent against Listeria monocytogenes in cooked chicken meat model system. The antilisterial activity of the plant extract was better under microwave condition and enhanced as storage temperature increased from 4 to 37 °C. The extract could reduce L. monocytogenes numbers at low (104 CFU/g) and high (106 CFU/g) inoculum levels in cooked chicken by both rinse and injection application methods. A 5 min rinse in 8% w/v R. tomentosa extract reduced the bacterial number by ≥2-log before storage and ≥3-log after storage at 4 °C for 5 days. Injection with 0.4% w/w R. tomentosa extract resulted in approximately 2-log reduction in the cell numbers both before and after storage at 4 °C for 5 days. Five minutes rinse in the extract bath demonstrated better sensory preferences which were not significantly different from the control. Addition of black pepper powder to the extract rinsed samples improved odour but not appearance, colour, and texture preferences. Rhodomyrtus tomentosa extract was significantly effective for the bio-control of L. monocytogenes contaminations in cooked chicken meat model. The extract was observed as a potent bio-additive agent to control contaminations from L. monocytogenes and ensure safety in ready-to-eat meat.

Shelf-life extension of refrigerated sea bass slices wrapped with fish protein isolate/fish skin gelatin-ZnO nanocomposite film incorporated with basil leaf essential oil.

Microbiological, chemical and sensory changes of sea bass slices wrapped with fish protein isolate (FPI)/fish skin gelatin (FSG) films incorporated with 3 % ZnO nanoparticles (ZnONP) (w/w, based on protein content) and 100 % basil leaf essential oil (BEO) (w/w, based on protein content) during storage of 12 days at 4 °C were investigated. Sea bass slices wrapped with FPI/FSG-ZnONP-BEO film had the lowest growth of psychrophilic bacteria, lactic acid bacteria and spoilage microorganisms including Pseudomonas , H2S-producing bacteria and Enterobacteriaceae throughout storage of 12 days in comparison with those wrapped with FPI/FSG-BEO, FPI/FSG-ZnONP, FPI/FSG film, polypropylene film (PP film) and the control (without wrapping), respectively (P < 0.05). Lowered increases in pH, total volatile base, peroxide value and TBARS value were found in FPI/FSG-ZnO-BEO film wrapped samples, compared with others (P < 0.05). Sensory evaluation revealed that shelf-life of sea bass slices was longest for samples wrapped with FPI/FSG-ZnONP-BEO film (12 days), as compared to the control (6 days) (P < 0.05).

Comparative genomic and morphological analyses of Listeria phages isolated from farm environments.

The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large genome (~135-kb) myoviruses belonging to the genus "Twort-like viruses," three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (~66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (~56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.

Phage cocktail administration to reduce Salmonella load in broilers.

Salmonella is a serious foodborne pathogen that can cause gastrointestinal disease through the consumption of contaminated foods; including poultry meat. Salmonella is commonly present in the intestinal tract of poultry and farm environments, posing a potential risk of contamination during the processing of poultry meat. This study was a continuation in evaluating the effects of our previously developed phage cocktail targeting Salmonella at large-scale trials in commercial broiler farms, in which this cocktail considerably lowered Salmonella colonization in the gut of broilers. The phage cocktail given to broilers showed resistance to temperatures of up to 65 °C (> 60% survivability), pH ranging from 2 to 12 (> 96% survivability), 0.5 to 15% (w/v) NaCl (> 98% survivability), chlorine up to 0.5% (v/v) (53% survivability), and chlorine neutralizer (100% survivability). In the animal challenge study, phage treatments, designed as "prevention" and "exclusion" programs, could control Salmonella on day 20 and 32 of the experiment, respectively; as indicated by the absence of Salmonella detection in cloacal swabs from broilers (0% prevalence). In the commercial-scale trial I, Salmonella was not detected in the phage-treated group from cloacal swabs, boot cover swabs, and bedding material samples after 16 days (0% prevalence) of phage administration. In the commercial-scale trial II, phage treatment extended the Salmonella control period in broilers during a 40-day growout period. In summary, a phage cocktail demonstrated high efficiency in controlling various serovars of Salmonella historically linked to contamination on these broiler farms. Phage cocktail application offers an effective, alternative to enhance food safety within the poultry value chain, protecting consumers and as well as the economic sustainability of the poultry sector.

Genomic characterization provides new insight into Salmonella phage diversity.

BACKGROUND: Salmonella is a widely distributed foodborne pathogen that causes tens of millions of salmonellosis cases globally every year. While the genomic diversity of Salmonella is increasingly well studied, our knowledge of Salmonella phage genomic diversity is still rather limited, despite the contributions of both lysogenic and lytic phages to Salmonella virulence, diversity and ecology (e.g., through horizontal gene transfer and Salmonella lysis). To gain a better understanding of phage diversity in a specific ecological niche, we sequenced 22 Salmonella phages isolated from a number of dairy farms from New York State (United States) and analyzed them using a comparative genomics approach. RESULTS: Classification of the 22 phages according to the presence/absence of orthologous genes allowed for classification into 8 well supported clusters. In addition to two phage clusters that represent novel virulent Salmonella phages, we also identified four phage clusters that each contained previously characterized phages from multiple continents. Our analyses also identified two clusters of phages that carry putative virulence (e.g., adhesins) and antimicrobial resistance (tellurite and bicyclomycin) genes as well as virulent and temperate transducing phages. Insights into phage evolution from our analyses include (i) identification of DNA metabolism genes that may facilitate nucleotide synthesis in phages with a G+C % distinct from Salmonella, and (ii) evidence of Salmonella phage tailspike and fiber diversity due to both single nucleotide polymorphisms and major re-arrangements, which may affect the host specificity of Salmonella phages. CONCLUSIONS: Genomics-based characterization of 22 Salmonella phages isolated from dairy farms allowed for identification of a number of novel Salmonella phages. While the comparative genomics analyses of these phages provide a number of new insights in the evolution and diversity of Salmonella phages, they only represent a first glimpse into the diversity of Salmonella phages that is likely to be discovered when phages from different environments are characterized.

Persistent Listeria monocytogenes subtypes isolated from a smoked fish processing facility included both phage susceptible and resistant isolates.

Contamination of Ready-To-Eat foods with Listeria monocytogenes can typically be traced back to post-processing contamination from environmental sources; contamination is often linked to subtypes that persist in food associated environments. Although phage-based biocontrol strategies have been proposed for controlling this pathogen, information on the efficacy of phage treatment against diverse L. monocytogenes subtypes from food associated environments is still limited. We identified subtypes that were repeatedly found ("persistent") in a smoked fish processing facility by using EcoRI ribotyping data for isolates obtained in 1998-2009. PFGE analysis of 141 isolates (9 ribotypes) supported persistence for up to 11 years. Characterization of selected isolates, representing persistent subtypes, against a panel of 28 listeriaphages showed a wide range of likelihood of phage susceptibility, ranging from 4.6% (for 7 ribotype DUP-1043A isolates) to 95.4% (for 7 ribotype DUP-1044A isolates). In challenge studies with 10(5) and 10(6) CFU/ml L. monocytogenes, using phage cocktails and a commercial phage product at different phage-host ratios, one isolate (ribotype DUP-1043A) was not affected by any treatment. A reduction in L. monocytogenes counts of up to 4 log units was observed, after 8 h of treatment, in isolates of two ribotypes, but subsequent re-growth occurred. Survivor isolates obtained after 24 h of treatment showed decreased susceptibility to individual phages included in the phage cocktail, suggesting rapid emergence of resistant subtypes.

Salmonella bacteriophage diversity reflects host diversity on dairy farms.

Salmonella is an animal and human pathogen of worldwide concern. Surveillance programs indicate that the incidence of Salmonella serovars fluctuates over time. While bacteriophages are likely to play a role in driving microbial diversity, our understanding of the ecology and diversity of Salmonella phages is limited. Here we report the isolation of Salmonella phages from manure samples from 13 dairy farms with a history of Salmonella presence. Salmonella phages were isolated from 10 of the 13 farms; overall 108 phage isolates were obtained on serovar Newport, Typhimurium, Dublin, Kentucky, Anatum, Mbandaka, and Cerro hosts. Host range characterization found that 51% of phage isolates had a narrow host range, while 49% showed a broad host range. The phage isolates represented 65 lysis profiles; genome size profiling of 94 phage isolates allowed for classification of phage isolates into 11 groups with subsequent restriction fragment length polymorphism analysis showing considerable variation within a given group. Our data not only show an abundance of diverse Salmonella phage isolates in dairy farms, but also show that phage isolates that lyse the most common serovars causing salmonellosis in cattle are frequently obtained, suggesting that phages may play an important role in the ecology of Salmonella on dairy farms.

Control of Salmonella in Chicken Meat by a Phage Cocktail in Combination with Propionic Acid and Modified Atmosphere Packaging.

Salmonella contamination in poultry meat is an important food safety issue as this pathogen can lead to serious illness and economic losses worldwide. In poultry meat processing, a variety of strong bacteriostatic agents has been introduced for controlling Salmonella including bacteriophages (phages), organic acids, and modified atmosphere packaging (MAP). In our study, two selected phages including vB_SenM_P7 and vB_SenP_P32 were used in combination with propionic acid (PA) and MAP for controlling Salmonella of multiple serovars on chicken meat under storage at 4 °C. The two phages showed strong lytic activity against over 72 serovars of Salmonella tested (25.0 to 80.6%). Phages, vB_SenM_P7 and vB_SenP_P32 showed 40% and 60% survival rates, respectively, after the exposure to temperatures up to 70 °C. Both phages remained active, with nearly 100% survival at a wide range of pH (2 to 12) and 15% NaCl (w/v). The available chlorine up to 0.3% (v/v) led to a phage survival rate of 80-100%. A combination of Salmonella phage cocktail and 0.5% PA could reduce Salmonella counts in vitro by 4 log CFU/mL on day 3 whereas a phage cocktail and 0.25% PA showed a 4-log reduction on day 5 during storage at 4 °C. For the phage treatment alone, a 0.3-log reduction of Salmonella was observed on day 1 of storage at 4 °C. In the chicken meat model, treatment by a phage cocktail and PA at both concentrations in MAP conditions resulted in a complete reduction of Salmonella cells (4-5 log unit/g) on day 2 of storage whereas each single treatment under MAP conditions showed a complete cell reduction on day 4. For the meat sensory evaluation, chicken meat treated with a phage cocktail-PA (0.5%) in MAP condition showed the highest preference scores, suggesting highly acceptability and satisfactory. These findings suggest that a combined treatment using a phage cocktail and PA in MAP conditions effectively control Salmonella in poultry meat during storage at low temperature to improve the quality and safety of food.

Silage collected from dairy farms harbors an abundance of listeriaphages with considerable host range and genome size diversity.

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium ("listeriaphages") are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10(4) PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.

Effectiveness of the Organic Acid-Based Antimicrobial Agent to Prevent Bacterial Contamination in Fish Meal.

Animal feed production is an important step of the food animal production chain in a farm-to-table model. The contamination of raw ingredients with foodborne pathogens in feed production remains as an important safety issue where pathogens may spread into food animals to cause illnesses in humans when affected food animals are consumed. In the present study, we aimed to examine the quality and microbial contamination of fish meal and to investigate the effectiveness of the organic acid-based antimicrobial agent SALTEC 514TM against Salmonella to prevent bacterial contamination in fish meal. Fish meal samples (n = 4) collected from feed mills at different locations were analyzed for protein and total volatile basic nitrogen (TVBN) content to assess their nutritional value and freshness, and its microbiological quality. The protein and TVBN content ranged from 53.2 ± 3.1 to 67.5 ± 2.3 g/100 g and 73.8 ± 4.5 to 100.4 ± 11.2 mg/100 g meal, respectively. Total plate count of the fish meal samples ranged from 2.0 ± 0.3 to 4.5 ± 0.5 log units, whereas suspected foodborne bacteria, Escherichia coli and Salmonella, were not detected in all samples. Fish meal samples were artificially contaminated (day 0) and re-challenged (day 30 and 90) with Salmonella Enteritidis (3 log CFU/g) to test for the effectiveness of SALTEC 514TM, an organic acid-based antimicrobial formulation, in preventing Salmonella contamination and recontamination during storage. SALTEC 514TM, when applied at three different doses, was found to reduce the number of Salmonella in monitored samples after one day of storage. A low dose of 0.5 kg/ton SALTEC 514TM prevented Salmonella recontamination from occurring in fish meal samples stored for 37 days. In medium (1.0 kg/ton) and high doses (3.0 kg/ton), applications of SALTEC 514TM prevented the Salmonella recontamination for a maximum storage duration of 97 days. The application of SALTEC 514TM in fish meal and/or other feed ingredients may prove to be a safe alternative to reduce the microbial load, especially of foodborne-related microorganisms, to contribute to feed and food safety.

Broad lytic spectrum of novel Salmonella phages on ciprofloxacin-resistant Salmonella contaminated in the broiler production chain.

Background and Aim: Ciprofloxacin (CIP) is recommended for salmonellosis treatment as the drug of choice; however, overuse of this drug can cause drug resistance issues and failure to treat diseases. Phage therapy is an alternative approach for combatting CIP-resistant infection. This study aimed to estimate the prevalence of CIP-resistant Salmonella isolated from the broiler production chain and evaluated the lytic ability of novel Salmonella phages isolated from water samples. Materials and Methods: Samples were obtained from the broiler production chain and used for Salmonella isolation. serovar and CIP resistance of each isolate were characterized through latex agglutination and agar disk diffusion test, respectively. Water samples from different sources were acquired for phage isolation. The lytic activity of novel-isolated phages was also examined. Results: In this study, 51 Salmonella isolates were recovered from the broiler production chain (two commercial farms, one free-range farm, two slaughterhouses, and three stalls from the wet market). Kentucky was the major serovar characterized (16), followed by Typhimurium (9), Agona (5), Corvalis (5), Schwarzengrund (5), Singapore (3), Weltevreden (3), Mbandaka (2), Give (2), and Albany (1). The serovars that exhibited CIP resistance were 14/16 isolates of serovar Kentucky (87.5%) and one isolate of serovar Give (50%), whereas eight other serovars were susceptible to this drug. Overall, the prevalence of CIP-resistant Salmonella recovered from the sources included in this study was 29.4%. This study identified 11 Salmonella phages isolated from wastewater samples derived from broiler farms, wastewater treatment stations, and natural reservoirs. Our phages showed the total percentage of lysis ability ranging from 33.3% to 93.3% against CIP-resistant isolates. However, only one bacterial isolate, namely 210SL, recovered from the food contact surface of a wet market stall and was resistant to all phages. Conclusion: Diverse serovars of Salmonella were recovered in the broiler production chain in this study, while the isolates presenting CIP-resistant Salmonella were as high as 29.4%. Overall, Salmonella phages showed high lysis ability against these CIP-resistant Salmonella isolates, suggesting the potential application of phage-based treatments or biocontrol in the broiler production chain.

Oral Administration of a Phage Cocktail to Reduce Salmonella Colonization in Broiler Gastrointestinal Tract-A Pilot Study.

Salmonella contamination in poultry meat products can lead to serious foodborne illness and economic loss from product recalls. It is crucial to control Salmonella contamination in poultry from farm to fork. Bacteriophages (phages) are viruses of bacteria that offer several advantages, especially their specificity to target bacteria. In our study, three Salmonella phages (vB_SenS_KP001, vB_SenS_KP005, and vB_SenS_WP110) recovered from a broiler farm and wastewater treatment stations showed high lysis ability ranging from 85.7 to 96.4% on over 56 serovars of Salmonella derived from several sources, including livestock and a broiler farm environment. A three-phage cocktail reduced S. Enteritidis and S. Typhimurium, in vitro by 3.9 ± 0.0 and 3.9 ± 0.2 log units at a multiplicity of infection (MOI) of 103 and 3.8 ± 0.4 and 4.1 ± 0.2 log units at MOI of 104 after 6 h post-phage treatment. A developed phage cocktail did not cause phage resistance in Salmonella during phage treatments for three passages. Phages could survive under simulated chicken gastrointestinal conditions in the presence of gastric acid for 2 h (100.0 ± 0.0% survivability), bile salt for 1 h (98.1 ± 1.0% survivability), and intestinal fluid for 4 h (100 ± 0.0% survivability). Each phage was in the phage cocktail at a concentration of up to 9.0 log PFU/mL. These did not cause any cytotoxicity to human fibroblast cells or Caco-2 cells as indicated by the percent of cell viability, which remained nearly 100% as compared with the control during 72 h of co-culture. The phage cocktail was given to broilers raised in commercial conditions at a 9 log PFU/dose for five doses, while naturally occurring Salmonella cells colonized in the gastrointestinal tract of broilers were significantly reduced as suggested by a considerably lower Salmonella prevalence from over 70 to 0% prevalence after four days of phage treatment. Our findings suggest that a phage cocktail is an effective biocontrol agent to reduce Salmonella present in the guts of broilers, which can be applied to improve food safety in broiler production.

Genomic Analysis of Prophages Recovered from Listeria monocytogenes Lysogens Found in Seafood and Seafood-Related Environment.

A prophage is a phage-related sequence that is integrated into a bacterial chromosome. Prophages play an important role in bacterial evolution, survival, and persistence. To understand the impact of Listeria prophages on their host genome organizations, this work sequenced two L. monocytogenes strains (134LM and 036LM), previously identified as lysogens by mitomycin C induction. Draft genomes were generated with assembly sizes of 2,953,877 bp and 3,000,399 bp. One intact prophage (39,532 bp) was inserted into the comK gene of the 134LM genome. Two intact prophages (48,684 bp and 39,488 bp) were inserted in tRNA-Lys and elongation-factor genes of the 036LM genome. The findings confirmed the presence of three corresponding induced phages previously obtained by mitomycin C induction. Comparative genomic analysis of three prophages obtained in the newly sequenced lysogens with 61 prophages found in L. monocytogenes genomes, available in public databases, identified six major clusters using whole genome-based phylogenetic analysis. The results of the comparative genomic analysis of the prophage sequences provides knowledge about the diversity of Listeria prophages and their distribution among Listeria genomes in diverse environments, including different sources or geographical regions. In addition, the prophage sequences and their insertion sites contribute to the genomic diversity of L. monocytogenes genomes. These data of prophage sequences, prophage insertion sites, and prophage sequence comparisons, together with ANIb confirmation, could be useful for L. monocytogenes classification by prophages. One potential development could be refinement of prophage typing tools for monitoring or surveillance of L. monocytogenes contamination and transmission.

Isolation and Characterization of Potential Salmonella Phages Targeting Multidrug-Resistant and Major Serovars of Salmonella Derived From Broiler Production Chain in Thailand.

Salmonella is a major foodborne pathogen that causes foodborne disease in humans through consumption of contaminated foods, especially those of animal origin. Multiple Salmonella strains are antibiotic-resistant due to the common use of antibiotics in farm animals, including broiler farms. In this study, an alternative strategy using phage-based treatment was evaluated against Salmonella isolated from the broiler production. The prevalence of Salmonella spp. showed up to 46.2 and 44.4% in bedding samples from the broiler farms located in eastern and southern Thailand, respectively. Overall, 21 samples (36.2%) were positive for Salmonella and eight serovars were recovered from cloacal swabs, bedding materials (rice husk), and boot swabs collected from five farms. Up to 20 Salmonella phages were isolated from seven water samples from wastewater treatment ponds, a river, and a natural reservoir in Songkhla province. Isolated phages were investigated, as well as their lysis ability on eight target Salmonella serovars derived from broiler farms, five foodborne outbreak-related serovars, and 10 multidrug-resistant (MDR) serovars. All phages showed a strong lytic ability against five serovars of Salmonella derived from broiler farms including Kentucky, Saintpaul, Schwarzengrund, Corvalis, and Typhimurium; three foodborne outbreak serovars including Enteritidis, Typhimurium, and Virchow; and eight MDR serovars including Agona, Albany, Give, Kentucky, Typhimurium, Schwarzengrund, Singapore, and Weltevreden. Three phages with the highest lysis potential including vB_SenS_WP109, vB_SenS_WP110, and vB_SenP_WP128 were selected for a phage cocktail preparation. Overall, a phage cocktail could reduce Salmonella counts by 2.2-2.8 log units at 6 h of treatment. Moreover, Salmonella did not develop a resistant pattern after being treated with a phage cocktail. Findings here suggest that a phage cocktail is an effective biocontrol to combat Salmonella derived from broiler production chain, other serovars linked to foodborne outbreaks, and MDR serovars.

Whole-Genome Sequencing Analysis of Salmonella Isolates from Poultry Farms, a Slaughterhouse, and Retail Stalls in Thailand.

The draft genome sequences of 21 Salmonella isolates obtained from poultry production chains in Hat Yai City, Songkhla Province, Thailand, are reported in this study. Our study revealed that there was Salmonella environmental contamination along poultry production chains and cross-contamination among poultry through inanimate surfaces in the environment.