23943

BIO2 1032

كارشناسي ارشد

زيست شناسي -ميكروبيولوژي

علوم و فناوري‌‌‌هاي زيستي

1403/6/28

76 ص.

دكتر مجيد بوذري

دكتر عباس سليماني دلفان

كرونوباكتر ساكازاكي , پودر شير خشك نوزادان (PIF) , باكتريوفاژ حاد , فاژ درماني

كرونوباكتر ساكازاكي از اعضاي خانواده انتروباكترياسه است كه در سال 1980 به‌عنوان گونه جديد باكتريايي شناسايي شد. كرونوباكتر ساكازاكي نوعي پاتوژن انساني قابل‌انتقال از طريق غذا و فرصت‌طلب است كه در انواع مختلفي از منابع غذايي و محيطي جداسازي شده است. ايجاد عفونت در نوزادان به‌واسطه كرونوباكتر ساكازاكي ناشي از مصرف شير خشك‌هاي آلوده به اين باكتري است. نوزادان كم‌وزن و زودرس بيشتر در معرض خطر ابتلا به عفونت هستند. علائم باليني شامل عفونت جريان خون (37%)، انتروكوليت نكروزه كننده (41%)، سپتي سمي، مننژيت (63%)، تأخير در رشد، تشكيل كيست، تشنج، آبسه مغزي (21%)، هيدروسفالي مغز و مرگ در 30 تا 40 درصد از موارد است. در سال‌هاي اخير به علت افزايش استفاده از آنتي‌بيوتيك‌ها سبب پيدايش مقاومت نسبت به آنتي‌بيوتيك‌ها شده است. لذا استفاده از روش‌هاي درماني جايگزين ازجمله باكتريوفاژها براي درمان باكتري‌هاي مقاوم به آنتي‌بيوتيك ازجمله كرونوباكتر ساكازاكي به‌عنوان راهكاري جديد مطرح‌شده است. در اين مطالعه در فاصله زماني تيرماه 1402 تا مهرماه 1402، 4 نمونه از پساب فاضلاب تصفيه‌خانه جنوب اصفهان و همچنين چند نمونه از درياچه دانشگاه اصفهان، درياي خزر و خليج‌فارس جمع‌آوري و تحت شرايط استريل به آزمايشگاه ويروس‌شناسي دانشگاه اصفهان منتقل شدند. سپس اقدام به جداسازي فاژ حاد عليه كرونوباكتر ساكازاكي شد و خصوصيات اين فاژ از قبيل مورفولوژي، دامنه ميزباني، پايداري فاژ در شرايط مختلف از قبيل دما و شوري و pH هاي مختلف، زمان جذب، رشد تك‌مرحله‌اي فاژ و تأثير باكتريوفاژ بر بيوفيلم باكتري موردمطالعه قرار گرفت. فاژ حاد جداسازي شده عليه كرونوباكتر ساكازاكي در محدوده دمايي 20- تا 55 درجه سانتي‌گراد، محدوده 2 ـ 12=pH و غلطت نمك سديم كلريد 1 تا 30 درصد پايدار بود. دوره نهفتگي آن 20 دقيقه بود و به ازاي هر سلول باكتري 250 فاژ توليد شد. همچنين پس از گذشت 35 دقيقه حدود 60 درصد فاژ و در حضور يون كلسيم و منيزيم حدود 85 درصد فاژ به باكتري ميزبان جذب مي‌شوند و باكتريوفاژ مذكور فعاليت ضد باكتريايي مناسبي در MOI برابر با 1 از خود نشان داد. با توجه به اطلاعات به‌دست‌آمده در اين تحقيق احتمال مي‌رود كه بتوان از اين فاژ درزمينهٔ فاژ درماني كرونوباكتر ساكازاكي استفاده نمود كه درنتيجه باعث كاهش مصرف آنتي‌بيوتيك و به‌تبع آن مقاومت آنتي‌بيوتيكي خواهيم بود.

Cronobacter sakazakii , bacteriophages , antibiotic resistance , phage therapy

Isolation and identification of avirulant bacteriophage of Cronobacter sakazakii and assessment of its function in controlling growth and biofilm formation

زيست شناسي سلولي مولكولي و ميكروبيولوژي

Cronobacter sakazakii is a Gram-negative bacterium belonging to the Enterobacteriaceae family; it was identified as a separate species in 1980. This organism has been regarded as a human pathogen, and its main route of transmission is via foods. Infants, especially premature or low birth weight babies, are highly susceptible to this organism. Among the high-risk group, C. sakazakii infections are most often associated with the consumption of contaminated powdered infant formula. Neonatal infection due to this organism shows clinical presentations that include bacteremia and necrotizing enterocolitis, an infection of the intestine mainly occurring in premature infants, which results in inflammation of the tissue of the intestine followed by tissue death. All such infections have a potential to advance into meningitis, an infection of membranes casing the brain that, in turn causes massive neurological damages. This infection during the developmental stage not only arrested the growth and maturation of a child but also led to the cyst formation in many other vital organs of the human body. Infection with these anaerobic bacteria have been known to cause mortality rates as high as 30 to 40 percent. Conclusion The trend over the last ten years shows a significant increase in the use of antibiotics, which has resulted in the resistance of most types of bacteria. The usual efficiency and reliability associated with these drugs have been reduced, making it difficult to treat an infection. A significant example is C. sakazakii. The development of antibiotic resistance by C. sakazakii has enhanced the challenges in its treatment. In the light of this, bacteriophage therapy has been one of the ways current research is exploring alternative therapeutic methods of treatment. Bacteriophages are viruses that infect bacteria and hence can be a strong weapon in combating bacterial types resistant to antibiotics. Bacteriophage therapy against C. sakazakii infection is a new trend. Sampling was performed from the South Isfahan wastewater treatment facility, a lake at Isfahan University, the Caspian Sea, and the Persian Gulf between July and October of 2023. The samples were filtered and sterilized upon collection and were sent to the virology lab of Isfahan University for the isolation of bacteriophages infecting C. sakazakii . Other supplementary characteristics that were also analyzed included morphology, host range, stability at different temperatures, salinity and pH, adsorption rate, one-step growth curve, and efficacy of the bacteriophage in control of bacterial biofilm. The virulent phage screened against C. sakazakii had various distinguishing features. The phage was stable at a temperature from -20 to 55°C and in the pH range from 4 to 12, besides the sodium chloride concentration from 1% to 30%. Further, the latent period was 20 minutes, and this particular phage was able to produce 250 phages for one bacterial cell. Such characteristics of the phage may enable it to work more effectively under variant and harsh environmental conditions. Additionally, the rate of adsorption to host bacteria upon a time of 35 minutes reached about 60% that further reached up to about 85% with adding calcium and magnesium ions. This phage showed a very successful antibacterial activity with MOI 1 since it had attached well to C. sakazakii and was destroying it efficiently. The results obtained herein therefore point to a potential application of phage therapy in C. sakazakii infection, which is the reduction in antibiotics, and consequently hindering the emergence of antibiotic resistance-a phenomenon that many consider highly significant with regard to public health concerns.

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