Bacterial preparation and susceptibility testing

Laboratory studies of this study included bacteriophage sampling and isolation in Food Microbiology Laboratory of School of Public Health, Tehran University of Medical Sciences. Salmonella infantis (ATCC 51741) serotypes were obtained from Faculty of Veterinary Medicine, University of Tehran. Antibiotic susceptibility was tested using E-test (AB.BioDisk, Solna, Sweden) in accordance with the guidelines from the Clinical and Laboratory Standards Institute (CLSI.2018).

E.test method for determination of susceptibility of Salmonella infantis to ciprofloxacin antibiotic

The E-test (AB.Biodisk, Solna, Sweden) was used .In this method, after preparation of a bacterial suspension by a semi-McFarland method, it was transferred onto a Mueller Hinton Agar plate by a sterile swab using a cotton seed dressing method, and then the E.test strips, which represented the ciprofloxacin antibiotic, were applied to the agar plate And after 24 hours of incubation at 37 °C, the area of the inhibition zone was triangular, and then by referring to the table provided by the company E.test tape (AB.BioDisk, Solna Sweden) sensitivity Serotype Infec-tion of Salmonella was determined by the antibiotic.

Bacteriophage isolation and purification

The bird stool sample was first mixed in a certain amount of distilled water. Using centrifuge (Sigma, Germany), the mixed solution was centrifuged at 15 00 gr for 20 minutes at 4 ° C. The centrifuge solution was then filtered with a 0.22 μm filter (JetBiofil, China). In order to search the plaque, a double-layer cultures were used on an agar plate as follows. A portion of the centrifugation solution and a few milliliters of the 12-hour culture of Salmonella bacterial enterotoxin were added to the Brain Heart Infusion (BHI) medium (Merck, Germany) with 0.7% agar and placed on a plate containing a BHI fluid with a base of 5 / 1% agar was added superficially (12). After closing the agar in the medium and incubating at 37 ° C for 24 hours plaques were observed (transparent points caused by bacterial lysis of bacteria by bacteri-ophage). The plaques were removed by sterile Pasteur pipette and added to the BHI medium for 24 hours for enrichment, then the enriched phage was used to the research of plaques. In order to purify the phage, all phases were repeated five times (13).

Morphological assessment of the phage

The isolated bacteriophage was examined for size and morphology using a transmission electron microscope (TEM). For this purpose, a drop of phage was poured over a carbon-coated copper grade and was then painted with 2% uranyl acetate.

Bacteriophage titration

Bacteriophage titration was carried out in three steps. First, Salmonella infantis was inoculated on LB agar and plated at 37 ֯C for 24h. Then, a single colony of the bacterium was inoculated in 25ccs of tryptone broth at 37 ֯C. Finally, 1cc of SM buffer was pipetted in a tube and a dilution of 10-10 was prepared using serial dilution. Besides, 300μL of bacterial culture was poured in 9 tubes, and 100μL of each dilution was serially added to each tube. Tubes were then vortexed and place at room temperature for 20min.
Nine tubes were labeled and 4ccs of molten agar (40 ֯C-50 ֯C) were poured in tube No. 1 and the mixture in the tube was added to agar using a sterile pipette. After vortex, this suspension was transferred to Luria–Bertani agar (LB). This procedure was carried out for each dilution and the plates were incubated at 37 ֯C. Finally, a plaque assay was carried out to determine plaque formation.

Phage susceptibility to heat

A certain concentration (aliquot) of phage suspension (1×108 CFU/ml) was inoculated at 4, 25, 37, 50, 60, and 70 ֯C at pH=7 for 60 min. Bacteriophage titration was carried out using the Double-Layer Agar method.

Phage susceptibility to pH

A certain concentration (aliquot) of phage suspension (1×10⁸ CFU/ml) was inoculated at 4 ֯C at a pH range of 3 to 12 for 60 min. Bacteriophage titration was carried out using Double-Layer Agar.

Determining the Host and Specificity of Bacteriophage

Spot test was used to determine the bacteriophage specificity of Salmonella infantis. During this process, two groups were selected for the specificity of the bacteriophage:
Group 1: Non-Salmonella bacteria (Staphylococcus aureus (ATCC 43300), Shigella dysenteriae (ATCC 13313) and Entero-pathogenic Escherichia coli (ATCC 43887)
Group 2: Salmonella infantis serotypes
All the bacteria were placed in BHI Broth for 24 hours. 100 μl of the culture suspension was mixed with ml of agar-BHI medium and repeated with BHI agar for making two-layer Pure Plate. When medium became solid 10 μl of the bacteriophage suspension was added and incubated at 37 ° for 24 hours. Bacteriophage sensitivity was determined by translucent area of non-growth.

Bacterial invasion and the effect of a specific bacteriophage using HEp-2 cell line

MEM medium containing 10% FBS is used to culture HEp-2 cells. Thus, for every 90 ml of MEM, 10 ml of deactivated FCS is used. Then add 2 ml of 10% sodium bicarbonate and 1ml of penicillin-streptomycin solution and 2ml of L_glutamine and thus the environment is ready for consumption. But we have to make sure it is sterile before use. If necessary, 5% and 2% FBS media can be prepared in the same way, which is mostly used to maintain or replace the cell culture medium. At this point, the 1.5 ml cell suspension was added to sterile Leighton tubes to cover the entire lamellar surface of the tube. Then tubes were placed horizontally in an incubator at 37 ° C. After 24 hours, the medium was replaced with 1.5 ml of warm minimum essential medium (MEM). The previous culture was discarded and the warm antibiotic-free MEM medium was added one day before the experiment respectively. The tubes were placed again horizontally in an incubator at 37 ° C for testing the next day. To infect the cell line, at first, the culture medium of HEp-2 cells in the previously prepared Leighton tubes was discarded and cells wash thoroughly with 1.5 ml of Earle's Salt. Then tubs were placed horizontally in an incubator at 37 °C for 3 hours. During 3 hours, the invading bacteria use their invasive power to infect the cells, and after 3 hours, the cell culture medium, which has turned yellow due to the growth of bacteria, is discarded and the cells were treated several times with warm Salt and paint finally.

Addition of bacteriophage to infectious HEp-2 cell line

A definite dilution of an overnight culture of Salmonella infantis was added to LB broth and vortexed at 150 rpm at 37 ֯C until reaching the optic density of 0.6 at 650 nm. Next, a certain concentration of bacteriophage (1×108 CFU/ml) was prepared. Isolated bacteriophage was inoculated on the HEp-2 cell line and was then incubated at 37 ֯C for 24 h. Cells were then assessed under microscopy after 24 h.

Antibiotic susceptibility testing

The Standard strain of Salmonella infantis was confirmed by E-test according to CLSI(2018) guideline (Fig. 1).

 
 
Figure 1. Bacterial susceptibility to Ciprofloxacin

Determination of bacteriophage morphology

Using microscopy, bacteriophages with an icosahedral head of approximately 90 nm were observed along with a very short tail 135 nm. Transmission electron microscopy showed isolated bacteriophages belonging to the Myoviridae family (Fig. 2).

 
 
Figure 2. Determination of morphology of Myovirida family

Bacteriophage titration

To count the bacteriophage in the sample, the serial dilution method was used and double-layer agar was performed for plaque assay. The results of the double-layer agar assay showed that the titer of bacteriophages was 1.8×10⁷ PFU/mL. To be able to report the number of plaques, 30-300 plaques should be formed on the media (Table1). Bacterial dilution of 10⁴ was used to report the number of phages according to the following formula (14):

 Number of phage in 1ml suspention=1.8×10⁷ PFU/mL..

Susceptibility of bacteriophage to heat

As indicated in Table 2, this bacteriophage was stable at 4 ֯C, indicating the shelf life of 3 months in the refrigerator (Table 2).

Susceptibility of bacteriophage to pH

Bacteriophages were quantified at different pH values. Each quantification was carried out in triplicates (Table3).

Bacteriophage specificity

The specificity of bacteriophage against Salmonella infantis isolate was confirmed using the Spot test (Table4). Also, Salmonella infantis Susceptibility to bacteriophage was observed based on the formation of inhibition zone.

The effect of isolated bacteriophage on Salmonella infantis using HEP-2 cell-line

Isolated bacteriophages under sterile conditions were applied to pre-prepared infectious cell lines and a tube with healthy cells was applied for control. Then they were incubated for 24 hours at 37 °C. The next day, we removed, stained, and examined them under a microscope (Figure.3). According to the images related to the study of the effect of isolated bacteriophage on the HEp-2 cell line infected with Salmonella infantis, the isolated bacteriophages showed a good ability to reduce the number and eliminate the studied bacteria. The bactericidal effects of isolated bacteriophage against Salmonella infantis were evaluated as completely positive. As shown in the figure, infected HEp-2 cell lines have lost their original state (deformation) and intercellular connections have been largely lost. After adding the specific bacteriophage of this infectious bacterium, after the incubation period as expected, it was observed that the bacteriophages were able to lysis the cells of their specific bacterium and largely removed this infectious bacterium, which As a result, they maintain the original shape and condition of the cells and the stability of intracellular com-ponents, which is the result of the absence of bacterial contamination.