ABSTRACT
Introduction
Salmonella is a significant foodborne pathogen responsible for severe gastroenteritis and systemic infections in humans and animals. The increasing prevalence of antimicrobial‐resistant Salmonella strains poses a major public health challenge. This study aimed to investigate the antimicrobial resistance patterns of Salmonella isolates obtained from various sources, including food and human clinical samples, and to study the effectiveness of bacteriophages against isolated strains.
Materials and Methods
A total of 80 S. Typhimurium isolates were obtained from two clinics between 2015 and 2022, and 16 ground meat samples were collected from agricultural markets. Standard microbiological methods were used for Salmonella isolation, including culture‐based techniques and biochemical confirmation. The antimicrobial susceptibility of the isolates was determined using the disk diffusion method using 8 common antibiotics. New bacteriophages were isolated against multidrug‐resistant Salmonella isolates. Electron microscopy for bacteriophage morphology investigation was employed.
Results
Antimicrobial susceptibility testing revealed high resistance rates to tetracycline, streptomycin, ampicillin, chloramphenicol and ciprofloxacin, with multidrug resistance (MDR) observed in 16% of the isolates. The phage cocktail composed of six phages showed efficacy against 88.3% S. Typhimurium strains of different origins.
Conclusion
The study highlights the widespread occurrence of antimicrobial‐resistant Salmonella in food and human samples. The presence of MDR strains poses a serious threat to public health, necessitating biosecurity interventions to limit the spread of resistant Salmonella strains. The bacteriophages assessed in this study demonstrated significant potential for the biocontrol of Salmonella strains. The phage cocktail may be used for both food safety and therapeutic purposes, particularly against antibiotic‐resistant bacteria causing salmonellosis.
1. Introduction
Salmonella is a significant zoonotic foodborne pathogen, responsible for 93.8 million cases of gastroenteritis and 155,000 deaths annually [1]. It is one of the major causes of enteric infections worldwide, often transmitted through contaminated animal products such as eggs, meat, and poultry. Salmonella Typhimurium is a major cause of foodborne illness and poses economic challenges to both humans and animals [1, 2, 3].
Antimicrobial resistance is an escalating global health concern, leading to an increased frequency of bacteremia, urinary tract infections, pneumonia, and endocarditis as a result of S. Typhimurium infection. Multidrug resistance to common antibiotics complicates the treatment options of Salmonella infections. In the U.S., Salmonella remains a leading cause of foodborne infections, with an increasing number of cases showing resistance to antibiotics like ampicillin, streptomycin, and tetracycline [4].
In Europe, there has been a rise in MDR Salmonella, especially strains resistant to fluoroquinolones and third‐generation cephalosporins. A significant outbreak of MDR S. Typhimurium across 10 countries affected 150 people, predominantly young children, with the strain showing resistance to six antibiotic classes and in veterinary practices [5].
The increasing prevalence of MDR Salmonella highlights the urgent need for both new antibiotics and alternative therapies, such as bacteriophages. Bacteriophages are one such tool, widely used in various fields, including medicine and agriculture. The main advantages of phage therapy over traditional antibiotic treatment are as follows: Phages are highly effective against multidrug‐resistant pathogenic bacteria and are generally safer, as they do not disrupt the normal microbial flora—phages specifically target and eliminate only the harmful bacteria. Moreover, phages can quickly respond to the emergence of resistant bacterial strains due to their relatively high mutation rates compared to those of bacteria. Side effects associated with phage therapy are extremely rare, and the vast diversity of naturally occurring phages offers an almost unlimited pool for therapeutic applications. Phage application is reported to be highly efficient, highly specific, and cost‐effective when applied for pathogen eradication in food products and water, and in veterinary. Bacteriophages have demonstrated high effectiveness against foodborne pathogenic microorganisms [6, 7, 8, 9, 10, 11, 12, 13]. Salmonella phages may be used in the following ways to biocontrol foodborne pathogens: pre‐harvest pathogen control in livestock and poultry reared for food production; decontamination of inanimate surfaces in food processing facilities; and post‐harvest foodborne pathogen control by directly applying phages to harvested or processed foods [14, 15, 16, 17]. As a result, reducing Salmonella in the food chain will contribute to a consistent decline in related human infections.
The present study aims to investigate the antibiotic resistance profiles of Salmonella strains isolated from both human samples and food products, and to develop a phage cocktail effective against these strains, including multidrug‐resistant (MDR) isolates from both sources.
2. Materials and Methods
2.1. Bacterial Strains
A total of 80 clinical isolates of Salmonella Typhimurium were obtained between 2015 and 2022 from two hospitals in Tbilisi, Georgia. These bacterial strains were isolated from patient stool samples and studied in the hospital's laboratories. All strains were identified as Salmonella Typhimurium by the respective hospital laboratories. To protect patient identities, microbiologists at these hospitals assigned codes to the strains. All strains were confirmed as Salmonella Typhimurium by us using culture‐based microbiological, biochemical, and serological methods, which we employed for the identification of Salmonella strains from food samples.
Additionally, 14 Salmonella Typhimurium strains were isolated from 16 ground meats purchased from agricultural markets near Tbilisi.
2.2. Isolation of Salmonella Typhimurium From Food Samples (Ground Meat)
Sample Collection and Preparation: A total of 16 ground meat samples were collected from various agricultural markets. The samples were homogenized, and 25 g of each sample was transferred into 225 mL of buffered peptone water (BPW) (Oxoid, UK) and mixed thoroughly.
Enrichment: The meat sample suspension was incubated at 37°C for 24 h to allow potential Salmonella cells to proliferate.
Subculture onto Selective Agar: After incubation, 100 µL of the enriched suspension was streaked onto SS (Salmonella, Shigella) agar and XLD agar and incubated at 37°C for 24–48 h.
Identification of Salmonella Colonies: Colonies presumptive for Salmonella were identified by colony morphology on SS and XLD agars. Colonies that appeared typical of Salmonella were selected for further analysis.
Biochemical and Serological Confirmation: Presumptive Salmonella colonies were biochemically identified through API 20E. Salmonella species were confirmed by serological methods using specific antisera for Salmonella Typhimurium (Bio‐Rad) (in accordance with established protocols. Salmonella serogroups were identified serologically through a slide agglutination test, employing both standard polyvalent and monovalent Salmonella antisera (Bio‐Rad). This method allowed for the precise classification of the strains based on their antigenic properties [18].
2.3. Antibiotics Susceptibility Test for S. Typhimurium Strains
Three to five bacterial colonies were transferred into 10 ml Brain Heart Infusion Broth (Oxoid), incubated at 37°C for 24 h, diluted 1:10 in 9 ml of 0.1% Peptone water (10 g of peptone and 5 g of Sodium chloride/L), and spread over the surface of a dried Mueller‐Hinton Agar (MHA) plate using a cotton swab. The MHA plates were kept at room temperature for 10 min to allow absorption of excess liquid. Antibiotic discs were placed over the surface of each inoculated plate (four discs per plate). After incubation for 24 h at 37°C, the diameter of the growth inhibition zone around each disc was measured, and in accordance with the resultant diameter, each strain was identified as susceptible (S), intermediate (I), or resistant (R) in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [19]. The following antibiotics were used in S. Typhimurium tests: Ampicillin −10 µg; Chloramphenicol − 30 µg; Gentamicin −10 µg; Streptomycin − 10 µg; Ciprofloxacin − 5 µg; Tetracycline − 30 µg; Trimethoprim − 5 µg; Kanamycin − 30 µg.
2.4. Isolation of Salmonella Bacteriophages From Sewage
From the investigated strains, 3 human and 3 food multidrug‐resistant strains were used as host strains for the isolation and investigation of bacteriophages.
Sewage samples were filtered through a 0.45‐µm Millipore filters, and the filtrates were examined for the presence of phages. 90 ml of each filtrate was enriched with 10 ml of concentrated broth, and added to 1 ml of 18‐h culture of the target bactria. Then, the whole mixture was incubated at 37°C. After 18–24 h, the incubated material was centrifuged at 9000 g for 15 min to remove debris and then filtered through 0.22 µm Millipore filters. The presence of phage was determined by spotting methods.
2.5. Spotting Assay
The host bacteria were grown overnight, and then 100 μl of cell suspension was added to 5 ml of soft LB agar (0.6% agar), which had been pre‐heated to 42°C in a water bath. The resultant mixture was gently vortexed, poured over LB agar plates (1.5% agar), and allowed to solidify at room temperature during 30 min to produce bacterial lawns. Then, 10 μl of phage stock dilutions (10‐fold serial dilutions in SM buffer) were spotted onto the upper agar layer, and the plates were dried at room temperature for 30 min. Then, the plates were incubated overnight at 37°C, and inspected for single plaques or bacterial growth inhibition zones after 24 h [20].
2.6. Preparation of Concentrated Phage Stocks (Bilayer Agar Method)
A total of 100 μl of the host bacterium culture grown overnight and 1 ml of phage (103PFU/ml) were mixed; then 3 ml of molten soft‐agar (0.7%) was added to each tube, maintained at 45°C, and the mixture was gently vortexed and poured over LB agar plates (1.5% agar). The Petri dishes were incubated at 37°C. After 18–20 h incubation, 3 ml of broth was spread over the agar and left for 15–20 min. Using a spreading rod or spatula, the soft agar with broth was scraped and transferred to a centrifuge tube, and centrifuged at 6000 g for 45 min. The supernatant was filtered through a 0.22 nm filter, transferred into the sterile vial and titrated. Plaques were counted, and titers were expressed as plaque‐forming units per milliliter (PFU/mL). All assays were performed in triplicate. Plaque morphology (size, clarity, halo formation) was recorded after incubation.
2.6.1. Phage Purification
Individual well‐isolated plaques were selected using sterile pipette tips and suspended in 500 µL of SM buffer. The suspension was vortexed and incubated at 4°C for 2–4 h to allow phage diffusion. The eluate was centrifuged briefly and filtered (0.22 µm).
To ensure purity, at least three successive rounds of plaque purification were performed using the double‐layer agar technique. High‐titer lysates were obtained by propagating purified phages on their respective host strains and harvesting lysates after complete lysis.
2.7. Study of the Host Range of Bacteriophage and Selection of the Most Efficient Phage
The phages were investigated for host range specificity and lysis efficiency against Salmonella strains. Each strain was inoculated on Tryptic Soy Agar (TSA), and 10 μl of phages (1 × 107 PFU ml−1) was dropped over the plate with the inoculated culture. The plates were then incubated during 18 h at 37°C, and the presence of plaques was observed.
2.8. Characterization of Selected Salmonella Bacteriophages By Electron Microscopy
Pure phage stocks with titers of ~109 PFU/ml were prepared. A sample of each stock was stained negatively with 1% uranyl acetate, and electron microphotographs were taken at various magnifications. Based on the morphology of the phage, all phages were classified into their respective family according to the International Committee on Taxonomy of Viruses [3].
2.9. Statistical Analysis
All experiments were conducted in triplicate. To evaluate whether the observed difference in lytic efficacy was statistically significant, a two‐sample z‐test for proportions was performed. A p‐value of less than 0.05 (p < 0.05) was considered statistically significant.
3. Results
3.1. Antimicrobial Susceptibility Testing of Clinical and Food Strains of Salmonella Typhimurium
A total of 80 clinical strains and 14 food‐derived strains of Salmonella Typhimurium were tested for susceptibility to eight commonly used antibiotics in clinical and veterinary practice. The results revealed that the majority of Salmonella strains exhibited resistance to the antibiotics tested (Table 1).
Table 1.
Antimicrobial Susceptibility Pattern of Salmonella Typhimurium.
| Antibiotic group | Antibiotic | Antibiotic susceptibility | |||||
|---|---|---|---|---|---|---|---|
| Susceptible | Intermediate | Resistant | |||||
| N | % | N | % | N | % | ||
| Aminoglycosides | Streptomycin | 56 | 59.6 | 16 | 17 | 22 | 23.4 |
| Gentamicin | 64 | 68.1 | 10 | 10.6 | 20 | 21.3 | |
| Kanamycin | 57 | 60.1 | 20 | 21.3 | 17 | 18.1 | |
| Phenicol | Phenicol | 53 | 56.4 | 25 | 26.6 | 16 | 17 |
| Tetracyclines | Tetracycline | 53 | 56.4 | 13 | 13.8 | 28 | 29.8 |
| β‐Lactams | Ampicillin | 60 | 63.8 | 17 | 18.1 | 17 | 18.1 |
| Diaminopyrimidine | Trimethoprim | 67 | 71.3 | 16 | 17 | 11 | 11.7 |
| Quinolones | Ciprofloxacin | 47 | 50 | 32 | 34 | 15 | 15.9 |
Resistance in clinical strains was most frequent to tetracycline (26.25%), streptomycin (22.5%), gentamycin (21.25%), ampicillin (18.75%), kanamycin (17.5%), phenicol and ciprofloxacin (15%), followed by lower levels of resistance to trimethoprim (11.25%). Food isolates were resistant most frequently to tetracycline (46.6%), phenicol and streptomycin (26.6%), ciprofloxacin, gentamycin and kanamycin (20%), followed by lower levels of resistance to ampicillin and trimethoprim (13.3%). From investigates 94 strains 9 clinical and 6 food strains were characterized with resistance to 3 or more different group of antibiotics (Table 2).
Table 2.
Antibiotic Resistance Pattern of the MDR Salmonella Strains.
| N | Strains | Source | Antibiotic Resistance Profiles | ||
|---|---|---|---|---|---|
| 3 | 4 | 5 | |||
| 1 | S. Typhimurium #1 | Clinical isolate | Amp, Chl, Str, Cip, Tmp | ||
| 2 | S. Typhimurium #5 | Clinical isolate | Amp, Chl, Tet, Cip | ||
| 3 | S. Typhimurium #8 | Clinical isolate | Chl, Str, Tet | ||
| 4 | S. Typhimurium #12 | Clinical isolate | Chl, Tet, Gen | ||
| 5 | S. Typhimurium #13 | Clinical isolate | Amp, Str, Tet, Cip, Tmp | ||
| 6 | S. Typhimurium #15 | Clinical isolate | Str, Tet, Cip | ||
| 7 | S. Typhimurium #24 | Clinical isolate | Chl, Str, Gen | ||
| 8 | S. Typhimurium #38 | Clinical isolate | Tet, Cip, Gen | ||
| 9 | S. Typhimurium #40 | Clinical isolate | Amp, Chl, Tet, Cip | ||
| 10 | S. Typhimurium #21 | Ground meat | Amp, Chl, Str, Tet | ||
| 11 | S. Typhimurium #22 | Ground meat | Chl, Str, Tmp | ||
| 12 | S. Typhimurium #25 | Ground meat | Str, Tet, Cip, Tmp | ||
| 13 | S. Typhimurium #26 | Ground meat | Amp, Chl, Tet | ||
| 14 | S. Typhimurium #27 | Ground meat | Str, Tet, Cip | ||
| 15 | S. Typhimurium #31 | Ground meat | Tet, Cip, Kan, Tmp | ||
Abbreviations: Amp, Ampicillin; Chl, Chloramphenicol; Cip, Ciprofloxacin; Gen, Gentamicin; Kan, Kanamycin; Str, Streptomycin; Tet, Tetracycline; Tmp: Trimethoprim.
3.2. Isolation of Bacteriophages
To isolate bacteriophages effective against newly isolated pathogenic Salmonella Typhimurium strains, we analyzed 40 samples collected from sewage, river water, and other environmental sources. Sixteen of these samples contained phages capable of lysing pathogenic S. Typhimurium strains. From these, six phages with the ability to infect and lyse a broad range of Salmonella strains—particularly multidrug‐resistant (MDR) isolates—were selected for further study: vB_Stm 16, vB_Stm 17, vB_Stm 18, Sal phi 21, vB_Stm 29, and Sal phi 13 (Table 3).
Table 3.
Host Strains and Sources of Selected Bacteriophages.
| # | Bacteriophage | Source of bacteriophages | Host strains | Source of host strains |
|---|---|---|---|---|
| 1 | vB_Stm 16 | Sewage water “Rioni” | S. Typhimurium #8 | Stool samples |
| 2 | vB_Stm 17 | Sewage water “Mtkvari” | S. Typhimurium #15 | Stool samples |
| 3 | vB_Stm 18 | Sewage water “Rioni” | S. Typhimurium #24 | Stool samples |
| 4 | vB_Stm 21 | Sewage water “Mtkvari” | S. Typhimurium #26 | Ground meat |
| 5 | vB_Stm 29 | Sewage water “Mtkvari” | S. Typhimurium #22 | Ground meat |
| 6 | Sal. phi 13 | Sewage water “Mtkvari” | S. Typhimurium #31 | Ground meat |
The lytic activity of six individual bacteriophages and a phage cocktail was evaluated against Salmonella enterica serovar Typhimurium strains isolated from both clinical (human) and food sources (Table 4, Figure 1). Individual phages demonstrated variable lysis profiles, with efficacy ranging from 47.9% to 73.4%, depending on the phage and the strain tested. Among these, phage vB_Stm 18 showed the highest individual activity, lysing 73.4% of the tested strains. In contrast, the phage cocktail—composed of six partially complementary individual phages—demonstrated broader lytic activity, lysing 88.3% of Salmonella Typhimurium strains overall. To assess whether this difference in efficacy was statistically significant, a two‐sample z‐test for proportions was performed. The result (z = 0.33, p = 0.744) indicated no statistically significant difference between the two groups, suggesting that the phage cocktail shows strong lytic activity against Salmonella strains from different sources.
Table 4.
A Host Range of Selected Salmonella Phages and Phage Cocktail.
| Strains | Antibiotics resistance profiles | Lysis by phage* | |||||||
|---|---|---|---|---|---|---|---|---|---|
| vB_Stm 16 | vB_Stm 17 | vB_Stm 18 | vB_Stm 21 | vB_Stm 29 | Sal phi 13 | Cocktail | |||
| 1 | S. Typhimurium #1 | Amp, Chl, Str, Cip, Trm | |||||||
| 2 | S. Typhimurium #5 | Amp, Chl, Tet, Cip | |||||||
| 3 | S. Typhimurium #8 | Chl, Str, Tet | |||||||
| 5 | S. Typhimurium #13 | Amp, Str, Tet, Cip, Tmp | |||||||
| 6 | S. Typhimurium #15 | Str, Tet, Cip, Gen, Kan | |||||||
| 7 | S. Typhimurium #20 | Str, Tet | |||||||
| 8 | S. Typhimurium #23 | Chl, Str | |||||||
| 9 | S. Typhimurium #24 | Chl, Str, Gen | |||||||
| 10 | S. Typhimurium #28 | Str | |||||||
| 11 | S. Typhimurium #29 | Amp, Chl | |||||||
| 12 | S. Typhimurium #30 | Tet, Cip | |||||||
| 13 | S. Typhimurium #32 | Gen, Tmp | |||||||
| 14 | S. Typhimurium #33 | Tmp | |||||||
| 15 | S. Typhimurium #38 | Tet, Cip, Gen | |||||||
| 16 | S. Typhimurium #40 | Amp, Chl, Tet, Cip | |||||||
| 17 | S. Typhimurium #46 | Chl, Gen | |||||||
| 18 | S. Typhimurium #48 | Amp, Chl | |||||||
| 19 | S. Typhimurium #50 | Tet, Kan | |||||||
| 20 | S. Typhimurium #51 | Gen, Kan | |||||||
| 21 | S. Typhimurium #52 | Gen, Tmp | |||||||
| 22 | S. Typhimurium #53 | Str, Tmp | |||||||
| 23 | S. Typhimurium #54 | Str, Tet | |||||||
| 24 | S. Typhimurium #55 | Str, Tet | |||||||
| 25 | S. Typhimurium #56 | Tet, Cip | |||||||
| 26 | S. Typhimurium #57 | Tet | |||||||
| 27 | S. Typhimurium #58 | Tet, Cip | |||||||
| 28 | S. Typhimurium #59 | Amp, Chl | |||||||
| 29 | S. Typhimurium #99 | Cip, Gen | |||||||
| 30 | S. Typhimurium #100 | Tet, Cip | |||||||
| 31 | S. Typhimurium #101 | Str, Gen | |||||||
| 32 | S. Typhimurium #102 | Tmp, Kan | |||||||
| 33 | S. Typhimurium #103 | Str, Gen | |||||||
| 34 | S. Typhimurium #104 | Kan | |||||||
| 35 | S. Typhimurium #105 | Amp | |||||||
| 36 | S. Typhimurium #106 | Tet, Kan | |||||||
| 37 | S. Typhimurium #107 | Amp, Tet | |||||||
| 38 | S. Typhimurium #108 | Str | |||||||
| 39 | S. Typhimurium #109 | — | |||||||
| 40 | S. Typhimurium #110 | — | |||||||
| 41 | S. Typhimurium #111 | — | |||||||
| 42 | S. Typhimurium #112 | Kan | |||||||
| 43 | S. Typhimurium #113 | Amp | |||||||
| 44 | S. Typhimurium #114 | — | |||||||
| 45 | S. Typhimurium #115 | Amp, Gen | |||||||
| 46 | S. Typhimurium #116 | Str | |||||||
| 47 | S. Typhimurium #117 | — | |||||||
| 48 | S. Typhimurium #118 | Str | |||||||
| 49 | S. Typhimurium #119 | Kan | |||||||
| 50 | S. Typhimurium #120 | Amp | |||||||
| 51 | S. Typhimurium #121 | Kan | |||||||
| 52 | S. Typhimurium #122 | Amp, Kan | |||||||
| 53 | S. Typhimurium #123 | Str, Gen | |||||||
| 54 | S. Typhimurium #124 | — | |||||||
| 55 | S. Typhimurium #125 | Gen, Tmp | |||||||
| 56 | S. Typhimurium #126 | — | |||||||
| 57 | S. Typhimurium #127 | — | |||||||
| 58 | S. Typhimurium #128 | — | |||||||
| 59 | S. Typhimurium #129 | — | |||||||
| 60 | S. Typhimurium #130 | — | |||||||
| 61 | S. Typhimurium #131 | Amp | |||||||
| 62 | S. Typhimurium #132 | Tet | |||||||
| 63 | S. Typhimurium #133 | Gen | |||||||
| 64 | S. Typhimurium #134 | — | |||||||
| 65 | S. Typhimurium #135 | — | |||||||
| 66 | S. Typhimurium #136 | Str | |||||||
| 67 | S. Typhimurium #137 | — | |||||||
| 68 | S. Typhimurium #138 | Amp | |||||||
| 69 | S. Typhimurium #139 | — | |||||||
| 70 | S. Typhimurium #140 | Tmp, Kan | |||||||
| 71 | S. Typhimurium #141 | — | |||||||
| 72 | S. Typhimurium #142 | Cip, Gen | |||||||
| 73 | S. Typhimurium #143 | Kan | |||||||
| 74 | S. Typhimurium #144 | Gen | |||||||
| 75 | S. Typhimurium #145 | Tet | |||||||
| 76 | S. Typhimurium #146 | — | |||||||
| 77 | S. Typhimurium #147 | Chl | |||||||
| 78 | S. Typhimurium #148 | Tet | |||||||
| 79 | S. Typhimurium #149 | — | |||||||
| 80 | S. Typhimurium #150 | — | |||||||
| 81 | S. Typhimurium #21 | Amp, Chl, Str, Tet, Cip | |||||||
| 82 | S. Typhimurium #22 | Chl, Str, Gen, Tmp | |||||||
| 83 | S. Typhimurium #25 | Str, Tet, Cip, Tmp, Kan | |||||||
| 84 | S. Typhimurium #26 | Amp, Chl, Tet | |||||||
| 85 | S. Typhimurium #27 | Str, Tet, Cip | |||||||
| 86 | S. Typhimurium #31 | Tet, Cip, Gen, Tmp, Kan | |||||||
| 87 | S. Typhimurium #77 | Tet | |||||||
| 88 | S. Typhimurium #79 | — | |||||||
| 89 | S. Typhimurium #82 | Chl | |||||||
| 90 | S. Typhimurium #85 | Tet | |||||||
| 91 | S. Typhimurium #87 | — | |||||||
| 92 | S. Typhimurium #91 | — | |||||||
| 93 | S. Typhimurium #93 | — | |||||||
| 94 | S. Typhimurium #95 | — | |||||||
Dark zones correspond to complete lysis of the bacterial lawn; transparent zones correspond to no lysis.
Figure 1.
The efficacy of individual bacteriophages and phage cocktails against S. Typhimurium strains isolated from clinical and food samples.
Morphological characteristics of the six phages were examined, and the TEM images showed that two phages vB_Stm 18, and Sal phi 13, were members of the Siphoviridae family. Siphoviruses have an icosahedral head that measures approximately 45–60 nm in diameter. Both phages contained a long, non‐contractile tail with a length of 190 nm for vB_Stm 18 and 100 nm for Sal phi 13. On the other hand, four phages vB_Stm 16, vB_Stm 17, vB_Stm 21, and vB_Stm 29, were classified as myoviruses, where they belong to the Myoviridae family, per the criteria established by the International Committee on Taxonomy of Viruses. The TEM image of vB_Stm 16, vB_Stm 17 phages showed that they contained a round head in shape with isometric symmetry (50–60 nm in diameter) and a contractile sheathed tail (95 nm in length for vB_Stm 17 and 75 nm for vB_Stm 16), and vB_Stm 21 and vB_Stm 29 phages had icosahedral head with 54–60 nm in diameter and a contractile sheathed tail 120 nm in length for vB_Stm 21 and 95 nm in length for vB_Stm 29 (Table 5, Figure 2).
Table 5.
Morphological Characteristics of Selected Salmonella Phages.
| Bacteriophage | Head diameter, nm | Head length, nm | Tail diameter, nm | Tail length, nm | Family |
|---|---|---|---|---|---|
| vB_Stm 16 | 60 | 60 | 10 | 75 | Myoviridae |
| vB_Stm 17 | 50 | 60 | 20 | 95 | Myoviridae |
| vB_Stm 18 | 50 | 50 | 15 | 190 | Siphoviridae |
| vB_Stm 21 | 54 | 54 | 18 | 120 | Myoviridae |
| Sal.phi13 | 45 | 45 | 15 | 100 | Siphoviridae |
| vB_Stm 29 | 60 | 50 | 15 | 95 | Myoviridae |
Figure 2.
TEM images of bacteriophages: 1‐vB_Stm 16, 2‐vB_Stm 17, 3‐vB_Stm 18, 4‐vB_Stm 21, 5‐Sal.phi13, 6‐vB_Stm 29.
4. Discussion
This study demonstrates the high prevalence of antimicrobial resistance among Salmonella Typhimurium isolates from both clinical and food sources in Georgia. Resistance was most pronounced against tetracycline, streptomycin, ampicillin, chloramphenicol, and ciprofloxacin, consistent with global reports of widespread resistance to these antibiotics in Salmonella due to their extensive use in human and veterinary medicine. The detection of multidrug‐resistant (MDR) strains in 16% of isolates highlights the growing public health threat, particularly given the presence of MDR strains in food samples, which suggests a direct transmission route from contaminated food products to humans.
Our findings align with previous reports from Europe and North America, where MDR Salmonella strains resistant to fluoroquinolones and β‐lactams have been increasingly documented. The resistance patterns observed here—especially against tetracycline and streptomycin—likely reflect the continued reliance on these antibiotics in agriculture, which contributes to persistence of resistant strains in the food chain. This underscores the urgent need for stricter antimicrobial stewardship and biosecurity measures [21].
The bacteriophage cocktail developed in this study showed strong lytic activity, effectively targeting 88.3% of S. Typhimurium strains, including MDR isolates. This broad host range is significant, as phage therapy has been proposed as a viable alternative or complement to antibiotics in both therapeutic and food safety applications. The inclusion of phages isolated from diverse environmental sources likely contributed to the cocktail's efficacy, reflecting the adaptability and diversity of naturally occurring phages. Similar studies have demonstrated that phage cocktails can control MDR Salmonella infections in poultry and reduce bacterial loads in food matrices [22, 23].
Phage therapy offers several advantages over conventional antibiotics. Unlike broad‐spectrum antimicrobials, bacteriophages specifically target pathogenic bacteria without disturbing commensal microbiota, thereby reducing collateral damage. Their ability to co‐evolve with bacterial hosts provides a dynamic mechanism to counteract resistance development. Importantly, phage‐based interventions have been shown to be safe in both animal and human applications, with rare adverse effects. The demonstrated effectiveness of phages against foodborne Salmonella suggests potential applications in pre‐harvest pathogen control, food processing environments, and direct food decontamination [24, 25].
In conclusion, this study highlights the dual challenge posed by MDR Salmonella strains in clinical and food settings, while also demonstrating the potential of bacteriophage cocktails as effective biocontrol agents. Integrating phage therapy into food safety protocols and clinical practice could significantly reduce the burden of antibiotic‐resistant Salmonella infections [26]. Future research should focus on optimising phage formulations, evaluating their stability under real‐world conditions, and exploring synergistic applications with existing antimicrobial strategies.
5. Conclusion
This study reveals the significant antimicrobial resistance of Salmonella Typhimurium in both clinical and food sources. All S. Typhimurium strains showed multidrug resistance to three or more antibiotics of different groups: ciprofloxacin, tetracycline, gentamycin, trimethoprim and chloramphenicol. These resistance patterns highlight serious challenges in current treatment strategies for Salmonella infections and emphasize concerns about food safety and the risk of transmission to humans. In response to this growing threat, a bacteriophage cocktail formulated using six of the most effective phages was developed. This cocktail showed high efficacy, successfully targeting 88.75% of clinical isolates and 87.5% of food‐derived strains, indicating strong potential as an alternative therapeutic approach against multidrug‐resistant Salmonella infections. Given the high lytic activity and broad spectrum of this phage cocktail, it represents a promising alternative for the prevention and treatment of salmonellosis caused by multidrug‐resistant Salmonella pathogens. This approach could enhance therapeutic options and address the challenges posed by antibiotic resistance in Salmonella infections.
The therapeutic use of phage cocktails—combinations of two or more phage types to create pharmacologically diverse formulations—is gaining increasing attention. The main advantage of using phage cocktails lies in their broader spectra of activity in comparison to individual phage isolates: they can target a wider range of bacterial types and remain effective under more varied conditions. Overall, this approach offers greater potential for presumptive or empirical treatment compared to single phage applications. Additionally, phage cocktails are believed to have a lower likelihood of inducing microbial resistance [7, 8].
This research was conducted at P. Shotadze Tbilisi Medical Academy, the G. Eliava Institute of Bacteriophage, Microbiology and Virology, Laboratory of Applied Microbiology and New Vision University.
Author Contributions
Manana Loladze: conceptualization, investigation, writing – original draft, methodology, validation, resources. Maia Zarnadze: methodology, validation, visualization, data curation, investigation. Natia Tamarashvili: methodology, visualization, data curation, investigation, supervision. Natela Chakhunashvili: methodology, visualization, data curation, investigation. Manana Nadiradze: investigation, methodology, visualization. Maia Alibegashvili: investigation, methodology, visualization. Tatiana Eliava: investigation, methodology, visualization. Tamar Kalandarishvili: investigation, methodology, visualization. Teimuraz Katamadze: investigation, methodology, visualization. Taras Gabisonia: conceptualization, research, overall management and supervision, writing the manuscript, reviewing the manuscript.
Ethics Statement
The activities and their related tasks described in this paper were approved (IRB approval #04‐11052023) by the Bioethics International Committee of the Petre Shotadze Tbilisi Medical Academy.
Consent
All affiliated institutions and authors express consent for publication.
Conflicts of Interest
The authors declare no conflicts of interest.
Transparency Statement
The lead author, Gabisonia T, affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.
Acknowledgments
The research was conducted with funding from the Shota Rustaveli National Science Foundation, Georgia, Grant #RF 17‐293.
Data Availability Statement
The data that support the findings of this study are available in the Supporting Information of this article.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available in the Supporting Information of this article.