Razi Journal of Medical Sciences
مجله علوم پزشکی رازی
RJMS
Medical Sciences
http://rjms.iums.ac.ir
39
journal39
2228-7043
2228-7051
en
jalali
1399
7
1
gregorian
2020
10
1
27
7
online
1
fulltext
fa
مطالعه مولکولی ژن اینتگرون سالمونلا تیفی موریوم جدا شده از منابع غذایی و تاثیر پروبیوتیک بیفیدوباکتریوم بیفیدوم بر بیان آن با روش Real time PCR
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
میکروبیولوژی
Microbiology
پژوهشي
Research
<strong><span style="color:#0070c0;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">زمینه و هدف: </span></span></span></strong><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">باکتری<em> سالمونلا </em>از خانواده</span></span></span> <em><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">انتروباکتریاسه</span></span></span></em><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;"> باکتری رودهای، گرم منفی، بی هوازی اختیاری است.<em> سالمونلا</em></span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">طیف</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">وسیعی</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">از</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">مهرهداران</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">را</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">تحت</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">تأثیر</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">قرار داده</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">عمدتاً از طریق تماس مستقیم و غیرمستقیم با منشأ آلوده منتقل می­شود. سالمونلوزیس بیماری خطرناک عفونی و بیماری مشترک انسان و حیوانات است.</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">ژن اینتگرون (</span></span></span><em><span dir="LTR"><span style="color:black;"><span style="font-family:Times New Roman,serif;"><span style="font-size:10.0pt;">int</span></span></span></span></em><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">) در مقاومت دارویی و انتشار</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">باکتری در بدن میزبان موثر و عامل مهم در مقاومت دارویی باکتریها است. هدف </span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">بررسی مولکولی ژن <em>اینتگرون سالمونلا تیفی موریوم</em> جدا شده از منابع غذایی و تاثیر پروبیوتیک <em>بیفیدوباکتریوم بیفیدوم</em> بر بیان آن با روش </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:Times New Roman,serif;"><span style="font-size:10.0pt;">Real time PCR</span></span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;"> می­باشد.</span></span></span><br>
<strong><span style="color:#0070c0;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">روش کار:</span></span></span></strong> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">در این پروژه 138 نمونه مواد غذایی (شیر، گوشت، مرغ، ماهی و غیره) از شهر اراک جداسازی گردید. </span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">در ظرف استریل و درپوش دار مخصوص با رعایت شرایط دمایی به آزمایشگاه میکروب شناسی منتقل شد. در آزمایشگاه جهت آماده سازی نمونه ها پس از سانتریفوژ از مایع فوقانی در ظرف استریل جهت آزمایشات نگهداری شد. </span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">محیطهای انتخابی افتراقی مک کانکی آگار </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:Times New Roman,serif;"><span style="font-size:10.0pt;">(MC)</span></span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;"> <em>سالمونلا-شیگلا</em></span> (</span></span><span dir="LTR"><span style="color:black;"><span style="font-family:Times New Roman,serif;"><span style="font-size:10.0pt;">SS</span></span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">) آگار، بیسموت سولفیت آگار برای 24 ساعت در دمای 37 درجه سانتی گراد قرار گرفت. </span></span></span><br>
<strong><span style="color:#0070c0;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">یافتهها:</span></span></span></strong><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;"> از بین 138 نمونه موادغذایی با روش بیوشیمیایی تعداد 12 جدایه <em>سالمونلا</em> تیفی موریوم جدا گردید، روش واکنش زنجیرهای پلیمراز جهت افتراق و بررسی حضور ژن اینتگرون انجام گرفت. <em>سالمونلا تیفی موریوم </em>دارای بیشترین اهمیت بیماریزایی در انسان و دام و طیور است که عامل رایج منتقله از طریق مدفوع و مواد غذایی جهانی میباشد.</span></span></span><br>
<strong><span style="color:#0070c0;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">نتیجهگیری:</span></span></span></strong> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">در این مطالعه </span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">میزان </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:Times New Roman,serif;"><span style="font-size:10.0pt;">Fold Change</span></span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;"> برای ژن </span></span></span><em><span dir="LTR"><span style="color:black;"><span style="font-family:Times New Roman,serif;"><span style="font-size:10.0pt;">intI</span></span></span></span></em><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;"> برابر 13/1- که بیانگر انست که این ژن در گروه تیمار شده نسبت به گروه غیر تیمار 13/1 برابر</span></span></span> <span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">کاهش دارد و این </span></span></span><span style="color:black;"><span style="font-family:B Mitra;"><span style="font-size:10.0pt;">عمل اثر ضد میکروبی پروبیوتیک <em>بیفیدو باکتریوم بیفیدوم</em> بر بیان ژن اینتگرون نشان داد. در واقع درمان ترکیبی پروبیوتیک <em>بیفیدوباکتریوم بیفیدوم</em> با داروهای ضد میکروبی رایج، اثرات ضد باکتری خوبی را علیه گونههای<em> سالمونلا </em>نشان میدهند.</span></span></span>
<strong>Background:</strong> 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.<br>
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.<br>
<strong>Methods:</strong> 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.<br>
<strong>Results: </strong>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.<br>
<strong>Conclusion:</strong> 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.
سالمونلا تیفی موریوم, حساسیت آنتی بیوتیکی,Real time PCR
Salmonella typhimurium, integron I, II, III, Probiotic, Real time PCR
65
77
http://rjms.iums.ac.ir/browse.php?a_code=A-10-5075-3&slc_lang=fa&sid=1
Ehsan
Estabergi
احسان
استبرقی
stabraghi@yahoo.com
3900319475328460054292
3900319475328460054292
No
Department of Microbiology, Faculty of Basic Sciences, Islamic Azad University, shahrebabak Branch, kerman, Iran
دانشکده دامپزشکی، دانشگاه آزاد اسلامی واحد شهربابک، ، شهربابک، ایران
Kumarss
Amini
کیومرث
امینی
dr_kumarss_amini@yahoo.com
3900319475328460054293
3900319475328460054293
Yes
Department of Microbiology, Faculty of Basic Sciences, Saveh Branch, Islamic Azad University, Saveh, Iran
دانشکده علوم پایه، واحد ساوه، دانشگاه آزاد اسلامی، ساوه، ایران
Behrooz
Shojaee Saadi
بهروز
شجاعی سعدی
bhshojaii@yahoo.com
3900319475328460054294
3900319475328460054294
No
Department of Microbiology, Faculty of Basic Sciences, Islamic Azad University, Arak Branch, Arak, Iran
دانشکده علوم پایه، دانشگاه آزاد اسلامی ،واحد اراک ، اراک، ایران
Saeed
Eghdamian
سعید
اقدامیان
saeedarak@yahoo.com
3900319475328460054295
3900319475328460054295
No
Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Arak Branch, Arak, Iran
گروه کشاورزی، دانشکده کشاورزی، دانشگاه آزاد اسلامی، واحد ابهر، زنجان، ایران