Development of encapsulated salmonella bacteriophages to reduce salmonella spp. in food matrices

Abstract

Salmonella Enteritidis and Salmonella Typhimurium are the most important serovars that have often been linked to food contamination and foodborne outbreaks. Bacteriophage (phage) has the outstanding properties over antibiotics and other chemical agents with regards to its specificity to kill bacterial pathogens. Phage applications have gained interest as alternative strategy for controlling bacterial pathogens in the food industry. This study aimed to develop microencapsulated phage as dry powder to improve phage applications as an effective strategy to control Salmonella serovars. Abundance and diversity of Salmonella phages in various animal farms (chicken, swine, goat and bovine) in Songkhla province, Thailand were evaluated upon phage isolation. A total of 36 Salmonella phages were obtained, suggesting that animal farms in our study are common sources of abundant Salmonella phages. Phenotypic characterization of all isolated phages was determined by phage lysis profiles. Phages were evaluated for their lysis profiles on 47 Salmonella strains (28 serovars) isolated from various sources (animal farms, seafood processing plant and humans) in Thailand and the US. Most of isolated phages showed the broader host range on Salmonella strains isolated from animal farms in Thailand as compared to other sources. However, a few phages showed a broad spectrum against Salmonella strains from different communities and continents. Genotypic characterization by pulsed-field gel electrophoresis (PFGE) analysis revealed the estimated genome sizes ranging from 50 ± 2 kb to 200 ± 2 kb among isolated phages. Phenotypic and genotypic characterizations indicated that our isolated phages could effectively control important Salmonella serovars. To improve the antibacterial efficiency of our phages against important Salmonella serovars (S. Enteritidis and S. Typhimurium), we thus selected three phages which showed the highest efficiency and further develop as a phage cocktail. Based on efficiency of plating (EOP) and lytic ability assays, KP4, KP5 and KP50 were selected. Electron microscopy analysis classified these phages in the order Caudovirales and family Siphoviridae. One-step growth curve assay revealed the burst size of three phages of approximately 25-98 PFU/cell and latent period of 5-40 min on S. Enteritidis. On S. Typhimurium the burst size revealed approximately 70-112 PFU/cell and latent period of 10-15 min. Genome sequencing analysis and annotation revealed that phages KP4, KP5 and KP50 presented as virulent phages due to the absence of the lysogeny module, genes associated virulence/toxins and genes associated antibiotic resistance. Antibacterial efficiency of our developed phage cocktail was investigated in in-vitro, foods and also feed model. In-vitro study, S. Enteritidis and S. Typhimurium were decreased by more than 4 log CFU/mL after 4 h of phage cocktail treatment. In addition, phage-resistant development was not found in Salmonella after phage cocktail treatment. In chicken meat, S. Enteritidis and S. Typhimurium were decreased by 0.66 and 1.73 log CFU/cm2, respectively. In sunflower sprout, S. Enteritidis and S. Typhimurium were decreased by 1.27 and 1.17 log CFU/g, respectively. In animal feed, S. Enteritidis and S. Typhimurium were decreased by 1.87 and 2.38 log CFU/g, respectively. Antibacterial efficiency studies indicated that our developed phage cocktail provided high efficiency to control Salmonella contamination in food matrices. A new form of microencapsulated phage as dry powder was developed to improve the limitations and extend the application of phage as traditional lysate form. To protect phage particles from drying method (freeze-drying), the formulations presented the combination of coating materials (whey protein isolate; WPI and trehalose) were optimized. The ratio of WPI/trehalose at 3:1 presented the optimal formulation with the highest encapsulation efficiency (EE) of 91.9%. SEM images indicated the complex physical structure of coating materials. Fourier transform infrared spectroscopy (FTIR) analysis revealed the chemical interaction of coating materials as H-bonding. Differential scanning calorimetry (DSC) analysis indicated Tg of our develop dry phage powder with optimal formulation at 63.43°C. Our dry phage powder with optimal formulation showed the specific functional properties to protect phage particles as high stability in wide range of pH (1.5 to 9.5) and various temperatures (4°C, 25°C and 50°C). A developed phage cocktail was transformed as dry powder by the optimized formulation using freeze-drying. Stability and storage conditions studies revealed that dry phage cocktail powder kept in aluminium laminated foil bag at 4°C showed the suitable conditions of dry phage cocktail powder. The phage titer was decreased by only 0.5 log PFU/mL and the physio-chemical properties (color and aw) of dry phage cocktail powder remained unchanged over 12 weeks of storage. In addition, the study revealed that dry phage cocktail powder showed higher phage survivability as compared to the lysate form. Dry phage cocktail powder showed the desirable antibacterial efficiency to decrease the number of S. Enteritidis and S. Typhimurium in in-vitro, foods and also feed model. In-vitro study showed that S. Enteritidis and S. Typhimurium were decreased by 1.79 and 3.63 log CFU/mL, respectively at 37°C, and 0.43 and 2.36 log CFU/mL, respectively at 10°C. In chicken meat, S. Enteritidis and S. Typhimurium were decreased by 0.57 and 1.78 log CFU/cm2, respectively. In sunflower sprout, S. Enteritidis and S. Typhimurium were decreased by 0.86 and 1.2 log CFU/g, respectively. In animal feed, S. Enteritidis and S. Typhimurium were decreased by 1.92 and 1.74 log CFU/g, respectively. Sensory evaluation (30 panelists) indicated that the quality of foods (chicken meat and sunflower sprout) and feed applied with dry phage cocktail powder were accepted for 3 days and 2 days of storage, respectively. Results indicated that the developed dry phage cocktail powder provided high antibacterial efficiency to control Salmonella serovars in different food matrices without negative effect on the acceptability of consumers. In addition, this study also investigated the effect of a combination of antibiotic and bacteriophage in controlling Salmonella. The results showed that antibiotic and bacteriophage provided the synergistic effect against Salmonella compared to using antibiotic alone. Moreover, this effective strategy could lower the mechanism of antibiotic resistance at the molecular level, indicating that this combination can be used as the effective strategy to control Salmonella. Overall, this study indicates that animal farm environments in Thailand provide abundance and diversity of Salmonella phages which could effectively eliminate important Salmonella serovars isolated from different sources and continents. Our developed phage cocktail in both lysate and dry powder form showed high efficiency to control Salmonella serovars in different food matrices. Dry phage cocktail powder developed here showed high efficiency to protect phage particles in the harsh conditions. This study suggests that phage as dry powder is a novel convenient form for applications, storage and transportation. In addition, this developed form can be used in various food matrices and also in high-risk sources of Salmonella contamination to improve safety along the food production chain.