Thesis project whole-genome analysis of colistin-exposed pseudomonas aeruginosa ATCC 9027

Abstract

Colistin (polymyxin E), is an effective antimicrobial agent against a variety of Gram-negative bacterial infections. However, it is often used as a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains. With the emergence of colistin resistant P. aeruginosa worldwide, study on colistin resistance development of P.aeruginosa may be the key in controlling the spread of resistant strains. This project would provide a deeper look about the characteristics of colistin-exposed Pseudomonas aeruginosa ATCC 9027 which has not been studied before. Whole-genome sequencing (WGS) data of PA ATCC 9027 (wild type bacterial strain), PA-3, PA-4 (separate colistin-exposed strains after 14 days), PA-5, PA-6 (PA-3 and PA-4 cultured in antibiotic free medium for 10 days, respectively) were analyzed. FASTQC (v0.11.9) (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for quality checking of the sequence reads. De novo assembly was performed using a program called Unicycler (v.0.5.0) (Wick et al., 2017). Bowtie2 (v2.4.5) (Langmead and Salzberg et al., 2012), bcftools (v.1.15) and Picard Tools (v.2.27.4) (https://github.com/broadinstitute/picard) were used for alignment. Bcftools (v.1.15) were then continued being used for variant calling. SnpEff (v5.1) (https://github.com/pcingola/SnpEff ) was used for variant annotation. Lastly, Gene ontology (GO annotation) (https://geneontology.org/) would be used for further gene’s analysis. As a result, Pseudomonas aeruginosa ATCC 9027 with 6.03 Mb genome of 79 scaffolds with a scaffold N50 size of 277 Kb was obtained. The number of INDELs and SNPs from PA-3 to PA-6 were as followed: 204, 234, 206, 144 INDELs; 135, 129, 135, 130 SNPs , respectively. At the end, only 10 genes that were indentified share the possibility of participating in the colistin resistance mechanism, which mainly involved the biofilm formation (pstS, phzM, phzD2 and puuA), and the efflux transmembrane transporter activity (czcC_1, dppA_2). 146 of the newly annotated genes (using locus as their gene names), was found in all four samples. 19 genes that have yet to be identified in P. aeruginosa so their specific roles still remain unknown until further researches are conducted for confirmation.