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.

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.

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.

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.

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.

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.

Organic acids and 2,4-Di-tert-butylphenol: major compounds of Weissella confusa WM36 cell-free supernatant against growth, survival and virulence of Salmonella Typhi.

BACKGROUND: Salmonella Typhi (S. Typhi), the causative agent of typhoid fever, causes serious systemic disease in humans. Antibiotic treatment is required for the S. Typhi infection, while the inappropriate use of antibiotics causes increased drug-resistant S. Typhi. Hence, alternative therapies through non-antibiotic approaches are urgently needed. The use of beneficial lactic acid bacterium and/or its metabolites to control typhoid fever represent a promising approach, as it may exert protective actions through various mechanisms. METHOD: In this study, the cell-free culture supernatant (CFCS) of Weissella confusa WM36 was evaluated via the antibacterial activity, and its metabolites were identified. In addition, the effects of CFCS on Salmonella virulence behaviors were also investigated. RESULT: Based on strong inhibition the growth of S. Typhi DMST 22842, organic acids (lactic acid and acetic acid) and 2,4-Di-tert-butylphenol (2,4 DTBP), were the main antibacterial metabolites presented in CFCS of strain WM36. Minimum inhibitory concentration (MIC) at 40% WM36-CFCS dramatically reduced the S. Typhi population to more than 99.99% at 4 h and completely inhibited biofilm formation, while sub-MIC at 20% (v/v) and MIC could reduce 100% of motility. Additionally, sub-MIC at only 10% (v/v) WM36-CFCS did down-regulate the expression of virulence genes which are responsible for the type-III secretion system, effector proteins, and quorum sensing system in this pathogen. CONCLUSION: W. confusa WM36 and its metabolites are shown to be a promising candidates, and an effective approach against typhoid Salmonella burden.

Cell-free supernatants from cultures of lactic acid bacteria isolated from fermented grape as biocontrol against Salmonella Typhi and Salmonella Typhimurium virulence via autoinducer-2 and biofilm interference.

BACKGROUND: Salmonella Typhi and Salmonella Typhimurium are the causative pathogens of salmonellosis, and they are mostly found in animal source foods (ASF). The inappropriate use of antibiotics enhances the possibility for the emergence of antibiotic resistance in pathogens and antibiotic residue in ASF. One promising alternative to antibiotics in animal farming is the use of lactic acid bacteria (LAB). METHODS: The present study was carried out the cells and/or the cell-free culture supernatants (CFCS) from beneficial LAB against S. Typhi and S. Typhimurium. The antibacterial mechanisms of LAB-CFCS as biocontrol agents against both Salmonella serovars were investigated through the analysis of anti-salmonella growth activity, biofilm inhibition and quorum quenching activity. RESULTS: Among 146 LAB strains isolated from 110 fermented food samples, the 2 strong inhibitory effect strains (WM33 and WM36) from fermented grapes against both Salmonella serovars were selected. Out of the selected strains, WM36 was the most effective inhibitor, which indicated S. Typhi by showing 95.68% biofilm inhibition at 20% biofilm inhibition concentration (BIC) and reduced 99.84% of AI-2 signaling interference. The WM33 was the best to control S. Typhimurium by producing 66.46% biofilm inhibition at only 15% BIC and 99.99% AI-2 signaling a reduction. The 16S rDNA was amplified by a polymerase chain reaction (PCR). The selected isolates were identified as Weissella viridescens WM33 and Weissella confusa WM36 based on nucleotide homology and phylogenetic analysis. CONCLUSION: The metabolic extracts from Weissella spp. inhibit Salmonella serovars with the potential to be used as biocontrol agents to improve microbiological safety in the production of ASF.