Molecular insights into the mechanisms of drug-induced resistance reversion in Staphylococcus aureus, Escherichia coli and Acinetobacter baumannii

Abstract

The development of multidrug resistant bacteria is currently a great concern, since the misuse of antibiotics have caused a strong selective pressure for these resistant bacteria and various treatment options are becoming ineffective. Reversion of antibiotic resistant bacteria into antibiotic sensitive phenotypes is becoming a promising approach to address this problem. This study set out to investigate the molecular mechanisms of drug induced resistance reversion in three multi-drug resistant bacteria, S. aureus, E. coli, and A. baumannii, after treatment with an iodine-containing drug, FS-1. Bacteria cultivated on medium containing FS-1, served as experimental bacteria (denoted as FS), and bacteria cultivated on normal medium, served as negative control cultures (denoted as NC). SMRT sequencing was used to generated long reads and high genome coverages for bacteria genomes. These sequences, together with tools for the SMRT-link software, enabled us to obtain complete bacterial genomes assemblies. The genomes were annotated using the RAST server, followed by manual corrections as needed. All genomes were submitted to the NCBI and genome announcements were published. Upon investigation of FS genomes compared to NC genomes, we found increased frameshift mutations occurring in FS-1 treated cultures. RNA sequences were also generated for these bacteria during different growth phases. This was used to investigate the effect of FS-1 on bacterial metabolism, as well as the direct effect of the drug. Bacteria were also cultivated over 10 passages to determine the effect of the drug on bacterial populations. For the immediate effect, treatment with FS-1 caused downregulation of various important pathway which consume the co-enzymes NADH and NADPH, while the metabolic processes associated with the production of the reduced species of these co-enzymes were generally up-regulated. It may be assumed that the pathways helping bacteria to withstand oxidative stress were up-regulated. Bacteria cultivated on a medium containing FS-1 regained their initial growth rate by adapting to the presence of FS-1, which required an alternative gene transcription regulation controlled either by accumulation of specific mutations in bacterial populations, or due to epigenetic phase variations. Lastly, modified bases were detected using the generated PacBio reads, together with tools available from the SMRT-link software. In both E. coli and S. aureus, bacterial cultures treated with FS-1 had an overall increase in modified bases, while the number of methylated nucleotides remained unchanged. This was specifically observed in G and A bases. It was hypothesized that the observed sporadically modified nucleotides might be due to oxidation, especially of G bases, by the iodine contained in FS-1. Therefore, these findings conclude that treatment with FS-1 possibly leads to DNA oxidation, especially of G bases, which caused frame shift mutations, as well as alternative gene transcription regulation. This is a possible explanation of how resistant bacteria which were treated with FS-1, had increased sensitivity to antibiotics.