Application of Next-Generation Sequencing for 16S rRNA Gene Analysis of P. aeruginosa and S. maltophilia in Keratitis Patients from Baghdad

Background: Next-generation sequencing (NGS) is a more recent method for evaluating infections. It can sequence DNA fragments from a biopsy of infected ocular tissue or from intraocular fluid. Objective: This study aims to view the data gained from NGS of Pseudomonas aeruginosa and Stenotrophomonas maltophilia isolated from keratitis patients by giving an overview of the genomes, exporting their 16s rRNA gene for identification of the hypervariable regions. Methodology: Nine samples of P. aeruginosa and 1 sample of S. maltophilia were obtained from keratitis patients directed to Ibn Al-Haitham Hospital in Baghdad, their DNA was extracted, quantified, went through NGS procedures, and analyzed via various bioinformatic tools. Variable regions for 16S rRNA were estimated by using MAFFT alignment tool powered by Geneious Prime® and comparing it to Escherichia coli 16S rRNA gene and a primer was designed and tested in silico. Results: Genome assembly of P. aeruginosa and S. maltophilia samples showed genome lengths that are within the range of the corresponding species. 16S rRNA of P. aeruginosa only showed one polymorphism in only one sample, For the S. maltophilia, 10 polymorphisms were found, however, the GC content remained the same but the molecular weight differed with the clinical sample being heavier than the reference. Conclusion:16s rRNA gene from P. aeruginosa isolated from keratitis patients showed no significant difference with the same gene in Pseudomonas aeruginosa PAO1, while S. maltophilia clinical sample’s 16s rRNA gene differs in 10 polymorphisms from the same gene in Stenotrophomonas maltophilia strain NCTC10257 which gives novelty to this eye isolate.