Isolation and Characterization of Two Lytic Bacteriophages Infecting a Multi-Drug Resistant Salmonella Typhimurium and Their Efficacy to Combat Salmonellosis in Ready-to-Use Foods

Abstract

Foodborne salmonellosis is a global threat to public health. In the current study, we describe the isolation and characterization of two broad-spectrum, lytic Salmonella phages: SPHG1 and SPHG3 infecting a multidrug-resistant Salmonella Typhimurium EG.SmT3. Electron microscopy and whole genome analysis identified SPHG1 as a Myovirus, while SPHG3 as a new member of the genus "Kuttervirus" within the family Ackermannviridae. SPHG1 and SPHG3 had a lysis time of 60 min. with burst sizes of 104 and 138 PFU/cell, respectively. The two phages were robust at variable temperatures and pH ranges that match the corresponding values of most of the food storage and processing conditions. A phage cocktail containing the two phages was stable in the tested food articles for up to 48 h. The application of the phage cocktail at MOIs of 1000 or 100 resulted in a significant reduction in the viable count of S. Typhimurium by 4.2 log₁₀/sample in milk, water, and on chicken breast. Additionally, the phage cocktail showed a prospective ability to eradicate and reduce the biofilm that formed by S. Typhimurium EG.SmT3. A phage cocktail of SPHG1 and SPHG3 is considered as a promising candidate as a biocontrol agent against foodborne salmonellosis due to its broad host ranges, highly lytic activities, and the absence of any virulence or lysogeny-related genes in their genomes.

Keywords: Bacteriophage; Biocontrol; Foodborne salmonellosis; Salmonella Typhimurium.

Figure 1

(A) Lytic ability of the five isolated phages on S. Typhimurium, EG.SmT3 as a host at multiplicity of infection (MOI) of 1 in TSB broth, (B,C) Lytic ability of phages SPHG1 and SPHG3, respectively to lyse S. Typhimurium EG.SmT3 in TSB medium at different MOIs of 10, 5, 1, 0.1, and 0.01 at 37 °C, and (D) Lytic activity of a phage cocktail developed from phages SPHG1 and SPHGThe bacteria were challenged with the isolated phages at the designated MOI in 96-well microtiter plates and incubated at 37 °C. The bacterial growth was estimated by measuring optical densities (OD₆₀₀ nm) up to 6 h post-infection. The data shown are the mean of three replicates and error bars show the deviation in the values.

Figure 2

Morphology and Growth kinetics of phages SPHG1 and SPHG3: (A,B) Transmission Electron Microscope analysis of phages SPHG1 and SPHG3, respectively, head and tail measurements of each phage are represented below each micrograph, scale bar = 100 nm and (C,D) one-step growth curves of SPHG1 and SPHG3 phages on S. Typhimurium EG.SmT3. The data shown are the mean of three replicates and error bars show the deviation in the values.

Figure 3

The SPHG1 genome organization represented as a circle. The 62 CDSs are represented as blue arrows showing the predicted genes transcribed clockwise (outer side) and counterclockwise (inner side) along the genome. The genome map is circle, but there is a break at the 12 o’clock position (at the dotted straight red line) because the phage genome is a linear molecule. CDSs with known functions are labelled along with their position; however, other non-labelled CDSs represent hypothetical proteins. The following cycle is the GC content of the genome. The inner two circles represent the BLASTn alignment of SPHG1 against Salmonella phage vB_SenM-1 and Salmonella phage SE13, respectively.

Figure 4

The SPHG3 genome organization represented as a circle. The 154 CDSs are represented as blue arrows showing the predicted genes transcribed clockwise (outer side) and counterclockwise (inner side) along the genome. Genes encoding tRNAs are represented as red arrows at about 3 o’clock in the genome. The genome map is circle, but there is a break at the 12 o’clock position (at the dotted straight red line) because the phage genome is a linear molecule. CDSs with known functions are labelled along with their position; however, other non-labelled CDSs represent hypothetical proteins. The following cycle is the GC content of the genome. The inner two circles represent the BLASTn alignment of SPHG3 against Salmonella phage ST-W77 and Salmonella phage vB_SalM_SJ3, respectively.

Figure 5

Thermal and pH stability test of phages SPHG1 and SPHG3. (A,B) Thermal tolerance of SPHG1 and SPHG3 phages respectively, and (C,D) pH stability of SPHG1 and SPHG3 phages, respectively. Temperature experiments were performed for 30 min., and 60 min. at pH 7. pH experiments were performed for 24 h at 37 °C. The data showed the percentages of the remaining phages after each treatment, as normalized from the control. The data reported are means of three independent trials and error bars show the deviation in the values.

Figure 6

Stability of Salmonella phage cocktail in different food articles. (A) Stability in milk, (B) stability in water, and (C) stability on chicken breast. Phage cocktail titer of 8.3 log₁₀ PFU/mL was mixed with each food sample and incubated at either 4 °C or 25 °C for 48 h. The values represent mean with a standard deviation of three replicates of each time point.

Figure 7

Biocontrol of S. Typhimurium EG.SmT3 using Salmonella phage cocktail at different food matrices. (A,D) Application in milk, (B,E) application in water, and (C,F) application on chicken breasts. Phage cocktail at to different MOIs (100 and 1000) were added separately to each food object and incubated either at 4 °C or 25 °C for 48 h, the un-infected control consisted of phage-free bacteria with SM buffer added. Values represent mean CFU/mL with a standard deviation of three replicates of each time point.

Figure 8

Effect of a phage cocktail on 72-h-old biofilm of S. Typhimurium EG.SmT3 in 96-well microplate. The values represent mean biofilm reduction with standard deviation of three replicates.