Shahid Bahonar University of Kerman and Iranian Biotechnology Society Agricultural Biotechnology Journal 2228-6705 17 2 2025 04 21 Genotyping of clinical Serratia marcescens isolates using molecular techniques Genotyping of clinical Serratia marcescens isolates using molecular techniques 263 284 4827 10.22103/jab.2025.24830.1665 FA Aintizar Kazar Department of Biology, College of Education, University of Al-Qadisiyah, Iraq. Azhar Hussein Department of Biology, College of Education, University of Al-Qadisiyah, Iraq Journal Article 2025 02 14 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. 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.

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