تعیین ژنوتیپ ایزوله‌های بالینی Serratia marcescens با استفاده از تکنیک‌های مولکولی

نوع مقاله : مقاله پژوهشی

نویسندگان

گروه زیست‌شناسی، دانشکده آموزش، دانشگاه القادسیه، عراق

چکیده

هدف: Serratia marcescens یک باسیل گرم‌منفی، بی‌هوازی اختیاری و متعلق به خانواده Enterobacteriaceae است. این باکتری یک پاتوژن فرصت‌طلب محسوب می‌شود که به‌دلیل نقش آن در طیف گسترده‌ای از عفونت‌های بیمارستانی و اکتسابی از جامعه، از جمله عفونت‌های دستگاه ادراری، عفونت‌های دستگاه تنفسی، عفونت‌های خونی و عفونت‌های زخم، مورد توجه فزاینده‌ای قرار گرفته است. این باکتری همچنین به‌دلیل توانایی زنده‌ماندن در شرایط محیطی متنوع و مقاومت ذاتی به چندین نوع آنتی‌بیوتیک شناخته می‌شود که درمان عفونت‌های ناشی از آن را در محیط‌های بالینی چالش‌برانگیز می‌کند. ژن 16S rRNA، که در میان باکتری‌ها بسیار محافظت‌شده است، ولی دارای نواحی متغیر نیز می‌باشد، به‌طور گسترده برای مطالعات فیلوژنتیک و شناسایی باکتریایی استفاده می‌شود. تعیین ژنوتیپ با استفاده از آنالیز پلی‌مورفیسم طول قطعات برشی (PCR-RFLP) ژن 16S rRNA امکان تمایز سویه‌های باکتریایی را بر اساس حضور جایگاه‌های برش آنزیمی خاص فراهم می‌سازد. این تکنیک دیدگاه‌هایی در مورد تنوع ژنتیکی و روابط تکاملی بین ایزوله‌ها ارائه می‌دهد. هدف این مطالعه تعیین ژنوتیپ ایزوله‌های بالینی S. marcescens به‌دست‌آمده از منابع مختلف بهداشتی و درمانی در استان دیوانیه، عراق با استفاده از تکنیک‌هایی نظیر PCR، تعیین توالی و PCR-RFLP با آنزیم‌های برشی AluI و MspI و بررسی تنوع ژنتیکی ایزوله‌های محلی بود.
مواد و روش‌ها: در مجموع ۲۰۰ نمونه بالینی از بیماران مراجعه‌کننده به بیمارستان آموزشی دیوانیه و چندین درمانگاه خصوصی در سطح استان دیوانیه عراق جمع‌آوری شد. نمونه‌ها بر روی محیط‌های کشت انتخابی و افتراقی استاندارد کشت داده شدند. DNA ژنومی از ایزوله‌های خالص‌شده S. marcescens با استفاده از کیت تجاری استخراج DNA باکتری استخراج شد. شناسایی مولکولی با تکثیر ژن 16S rRNA با استفاده از پرایمرهای جهانی باکتریایی انجام شد. محصولات PCR ایزوله‌های تأییدشده S. marcescens خالص‌سازی شده و به یک مرکز تعیین توالی تجاری در کره جنوبی ارسال شدند. درخت‌های فیلوژنتیکی با روش Neighbor-Joining ساخته شدند. برای ارزیابی تنوع ژنتیکی در میان ایزوله‌های S. marcescens، آنالیز PCR-RFLP ژن 16S rRNA انجام شد. الگوهای RFLP متمایز تحلیل شدند و تعداد ژنوتیپ‌های مختلف بر اساس الگوی باندی حاصل از هر آنزیم تعیین گردید.
نتایج: در مجموع ۲۰ ایزوله از منابع مختلف به‌دست آمد که شامل ۱۵ ایزوله (۷۵٪) از عفونت‌های دستگاه ادراری، ۳ ایزوله (۱۵٪) از سوختگی و زخم‌ها، و ۲ ایزوله (۱۰٪) از آسیب‌های چشمی بودند.
نتیجه‌گیری: این مطالعه نشان داد که تکنیک‌های مولکولی داده‌های دقیقی در مورد ساختار ژنتیکی Serratia marcescens فراهم می‌کنند که موجب بهبود دقت تشخیص داده شده و مسیر توسعه ابزارهای تشخیصی حساس‌تر و قابل‌اعتمادتر را در آینده هموار می‌سازد.

کلیدواژه‌ها

عنوان مقاله [English]

Genotyping of clinical Serratia marcescens isolates using molecular techniques

نویسندگان [English]

  • Aintizar Kazar
  • Azhar Hussein
Department of Biology, College of Education, University of Al-Qadisiyah, Iraq.
چکیده [English]

Objective
Serratia marcescens is a Gram-negative, facultatively anaerobic bacillus belonging to the Enterobacteriaceae family. It is an opportunistic pathogen that has gained increasing attention due to its involvement in a wide range of nosocomial and community-acquired infections, including urinary tract infections, respiratory tract infections, bloodstream infections, and wound infections. The bacterium is also known for its ability to survive in diverse environmental conditions and its intrinsic resistance to several antibiotics, making its infections particularly challenging to treat in clinical settings. The 16S ribosomal RNA (16S rRNA) gene, which is highly conserved among bacteria but also contains hypervariable regions, is widely used for phylogenetic studies and bacterial identification. Genotyping using PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene allows differentiation of bacterial strains based on the presence of specific restriction sites. This technique provides insights into genetic diversity and evolutionary relationships among isolates. The aim of this study was to genotype clinical isolates of S. marcescens obtained from various healthcare sources in Al-Diwaniyah Governorate, Iraq using techniques such as PCR, sequencing, and PCR-RFLP with the restriction enzymes AluI and MspI, and explore the genetic diversity of local isolates.
Materials and methods
A total of 200 clinical samples were collected from patients attending Diwaniyah Teaching Hospital as well as from several private medical clinics across Al-Diwaniyah Governorate, Iraq. Samples were inoculated onto standard selective and differential media. Genomic DNA was extracted from purified S. marcescens isolates using a commercial bacterial DNA extraction kit. Molecular identification was performed by amplifying the 16S rRNA gene using universal bacterial primers. PCR products of confirmed S. marcescens isolates were purified and sent to a commercial sequencing facility in South Korea. Phylogenetic trees were constructed using the neighbor-joining method. To assess genetic variability among S. marcescens isolates, PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene was performed. Distinct RFLP patterns were analyzed visually, and the number of different genotypes was determined based on banding profiles for each enzyme.
Results
A total of 20 isolates were obtained from different sources as follows: 15 (75%) isolates from urinary tract infections, 3 (15%) isolates from burn and wound injuries, and 2 (10%) isolates from eye injuries.
Conclusions
The study showed that molecular techniques provided accurate data on the genetic makeup of Serratia marcescens, improving diagnostic accuracy, paving the way for the development of future diagnostic tools that are more sensitive and reliable.

کلیدواژه‌ها [English]

  • 16srRNA
  • genotyping
  • PCR-RFLP
  • Serratia marcescens

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مجله بیوتکنولوژی کشاورزی
دوره 17، شماره 2
اردیبهشت 1404
صفحه 263-284

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