Molecular study of Salmonella typhimurium integrons gene isolated from food sources and the effect of probiotic Bifidobacterium bifidum on its expression by Real time PCR

Salmonella have general characteristics of the Enterobacteriaceae family, gram-negative bacilli, voluntary anaerobes, non-acid fast and without spores, a large group of bacteria. Salmonella typhimurium is one of the most important causes of food poisoning in humans in the world. This organism is one of the most common pathogens that can be transmitted from animals to humans. Gastroenteritis is the most common Salmonella infection caused by these serotypes. Salmonella is transmitted through the consumption of contaminated food, which has led to increased public health concerns. Antibiotic resistance genes in Salmonella cause them to become resistant to antibiotics. Salmonella species have the ability to acquire antibiotic resistance in a variety of ways. The emergence of antibiotic resistance has now become a growing problem among Salmonella species and has created increasing health and medical problems in the control and treatment of infections caused by this bacterium. Therefore, the pattern of antibiotic resistance in samples Salmonella is clinically very important. Rapid and reliable tests in medical, veterinary and food laboratories for the diagnosis of Salmonella are important and necessary. Salmonella affects a wide range of vertebrates and Salmonella infections in humans occur mainly through the consumption of substances contaminated with this bacterium. The disease must be diagnosed very quickly. Invasion of the intestinal mucosa is an important and fundamental step in the pathogenesis of Salmonella. Salmonella typhimurium is mainly transmitted through direct and indirect human contact with an infected source. Salmonella carries chromosomal and plasmid genes that play a major role in the virulence and invasion of this bacterium. One of the most important virulence factors is the Salmonella Vir, Inv, and Int genes. Bifidobacterium bacteria can be named as the most important probiotics used in food products. Bifidobacterium (Langum, Catnulatum, Bruch, Bifidum) are gram-positive bacteria that are the dominant and normal intestinal microflora in 80% of children and 25% of adults is considered. Due to the probiotic effects of Bifidobacteria, these bacteria as living microorganisms that have a positive effect on the treatment of pathogenic conditions, our findings showed that the integron I gene has a high prevalence among Salmonella species. Bifidobacterium is one of the most important species of probiotics known and today extensive efforts are being made to use them in food products. Probiotics are in fact food supplements containing living microorganisms that affect the health of their host by balancing the microbial flora of the gastrointestinal tract. Yogurt is the most common dairy product containing probiotics, and other ingredients such as cheese, fermented and non-fermented milk, and fruit juices can also be used as probiotics. Dietary supplements can generally also contain probiotics. The aim of this study was to molecularly investigate Salmonella typhimurium integron gene isolated from food sources and the effect of Bifidobacterium bifidum probiotic on its expression by Real time PCR.

In the present study, 138 food samples (milk, meat, chicken, fish, etc.) were collected to advance the project and transferred to a microbiology laboratory in a sterile container with a special lid at 4° C. In the laboratory for preparation, ‌ samples were precipitated with a uniform solution of phosphatase phosphate and then solid particles and woody substances in food, using a centrifuge at 3000 rpm for 5 minutes and the upper liquid in another sterile container. Stored at 4° C for subsequent experiments. Culture and Isolation: After culturing and growing the samples, Salmonella-Shigla agar (MC) agar, hectone, bismuth sulfite agar were transferred to differentiated mediums of McConkey Agar (MC) and placed at 37° C for 24 hours. Suspected Salmonella colonies (green colonies with black halo or without black halo) to confirm the diagnosis by biochemical tests and differential media TSI, SIM, Simon citrate, urease, MRVP, lysine iron agar, Broth malonate, IMViC was performed. After culturing in solid medium, BHI was refrigerated at 4° C. Isolated bacteria that had a + - + - reaction in the IMViC test and an alkaline / acid reaction in TSI medium were selected and isolated with suspicion of oxidase and ONPG colonies suspecting Salmonella and by comparing the results with tables Biochemical, biochemical confirmation of bacteria was performed. After extracting DNA from isolated Salmonella typhimurium isolate, the polymerase chain reaction was performed using the necessary materials and a suitable program, the presence of II, Int, Int III IntI genes was investigated with a specific primer. Determination of intI gene expression under the influence of Bifidobacterium bifidum probiotic. Before performing the polymerase chain reaction, 0.1 mg / ml gram of Bifidobacterium bifidum probiotic was infused in 20 ml of BHI and half of McFarland was added to Bifidobacterium bifidum intI positive. After 15 hours of incubation (Late log phase) is the best step for RNA extraction. After 15 hours, RNA extraction was performed. Bacterial suspension (bacteria with intI gene and determined by their MIC in the presence of probiotic Bifidobacterium bifidum, which are in the logarithmic phase of growth) (OD = 0.4-0.6 = 600-4) was used. The Qiagen DNase kit was used to remove genomic DNA. cDNA synthesis was performed using Reverse AMV enzyme at a concentration of 25 l / unit (Roche) then to calculate the expression of integron gene and draw diagrams Corresponding software was determined and the amount of target gene expression was calculated. Expression rate analysis was performed by relative measurement of mRNA expression compared to standard strains.

In this study, Salmonella typhimurium integron virulence gene isolated from clinical cases under the influence of bifidum probiotic was identified by Real time PCR technique. From 138 food samples, 12 Salmonella strains were isolated, all of which had integron I gene. And the effect of antimicrobial agent on gene expression and according to the research done in this study, the Fold Change rate for intI gene is -1.13, which indicates that this gene in the treated group compared to the untreated group /13. Decreased 1 times. Which indicates a decrease in the expression and relative inhibitory effect of Bifidobacterium bifidum and a 1.13% decrease in the expression of the gene under the influence of probiotic treatment.

In fact, combination treatment of Bifidobacterium bifidum probiotics with common antimicrobial drugs showed good antibacterial effects against Salmonella species and the rate of increased bacterial growth was dependent on the probiotic concentration. As a result, low-dose probiotics can be a viable alternative to single-drug treatment for Salmonella infections, with benefits such as preventing adverse effects during treatment, saving on medication, and reducing costs.