Next generation sequencing reveals the antibiotic resistant variants in the genome of Pseudomonas aeruginosa.

Babu Ramanathan,Ranganath Gudimella,Rozaimi Razali,Hassan Mahmood Jindal,Johan Poole-Johnson,Rishya Manikam,Shamala Devi Sekaran,Arif Anwar,Cheng Foh Le,Chiyu Zhang

doi:10.1371/journal.pone.0182524

Abstract

Rapid progress in next generation sequencing and allied computational tools have aided in identification of single nucleotide variants in genomes of several organisms. In the present study, we have investigated single nucleotide polymorphism (SNP) in ten multi-antibiotic resistant Pseudomonas aeruginosa clinical isolates. All the draft genomes were submitted to Rapid Annotations using Subsystems Technology (RAST) web server and the predicted protein sequences were used for comparison. Non-synonymous single nucleotide polymorphism (nsSNP) found in the clinical isolates compared to the reference genome (PAO1), and the comparison of nsSNPs between antibiotic resistant and susceptible clinical isolates revealed insights into the genome variation. These nsSNPs identified in the multi-drug resistant clinical isolates were found to be altering a single amino acid in several antibiotic resistant genes. We found mutations in genes encoding efflux pump systems, cell wall, DNA replication and genes involved in repair mechanism. In addition, nucleotide deletions in the genome and mutations leading to generation of stop codons were also observed in the antibiotic resistant clinical isolates. Next generation sequencing is a powerful tool to compare the whole genomes and analyse the single base pair variations found within the antibiotic resistant genes. We identified specific mutations within antibiotic resistant genes compared to the susceptible strain of the same bacterial species and these findings may provide insights to understand the role of single nucleotide variants in antibiotic resistance.

Highlights

Pseudomonas aeruginosa is a major opportunistic pathogen causing acute and chronic infections in human community
This approach can be used to identify the single nucleotide polymorphism that either alters a single amino acid or leads to a stop codon, and frame-shift mutations that alters a gene sequence itself resulting in truncated proteins
We have investigated the genetic variation in P. aeruginosa hospital isolates by high-throughput next-generation sequencing (NGS) technology and comparative genomics

Summary

Background

Pseudomonas aeruginosa is a major opportunistic pathogen causing acute and chronic infections in human community. NGS is quick and high throughput technique that follows a single unified workflow Using this technique, it is possible to identify the single-base-pair mutations within same bacterial species that can replace the traditional molecular typing methods for bacterial pathogens [13]. In the present study, we examined single nucleotide variations within the antibiotic resistant and susceptible P. aeruginosa clinical isolates collected from patients admitted at the University Malaya Medical Centre, Kuala Lumpur, Malaysia. These nsSNPs found within the antibiotic resistant isolates could potentially alter the amino acid sequence and may affect the stability and/or the function of the resulting protein expression

Materials and methods

Results

Discussion