Silencing and reactivation of antimicrobial resistance genes in Enterobacteriaceae

The rising number of antimicrobial resistant bacterial infections threaten global public health. Bacteria can obtain antimicrobial resistant genes (ARG) on mobile genetic elements, such as plasmids, which enables bacteria to become resistant to multiple antimicrobials in a single acquisition event. However, ARGs present on plasmids often negatively affect bacterial growth when antibiotics are not present. One reason is that ARGs can contain more A+T bases than G+C relative to the bacterial chromosome and A+T nucleotide availability can be limited in bacteria. To circumvent this, in the absence of antimicrobials, bacteria can silence plasmid-encoded ARGs using a range of mechanisms that target A+T-rich DNA, including the histone-like protein H-NS. ARG silencing can cause mismatch between the phenotype and genotype, where the bacterial cell contains an ARGs (genotype) but does not express them, appearing phenotypically susceptible (phenotype). This mismatch can cause problems during the treatment of infections, as antimicrobial resistance (AMR) is determined using phenotypic antimicrobial susceptibility assays, which help clinicians choose the most appropriate antimicrobial to use to treat an infection. This is an important part of the control of antimicrobial usage and to limit the development of AMR during therapeutic use, termed antimicrobial stewardship. Reactivation of silenced ARGs can result in the antimicrobial treatment no longer working, which will negatively impact the outcomes of treatment and antimicrobial stewardship. ARG silencing also complicates AMR prediction and monitoring of the number of AMR infections, which are essential for the development of tests to diagnose antimicrobial resistant infections and antimicrobial stewardship.
The aim of this project is to understand the silencing and reactivation of plasmid encoded ARGs in Enterobacteriaceae and will comprise of three main components:
1. ARG silencing in clinical isolates of Enterobacteriaceae. ARGs will be synthesised with varying A+T-richness while maintaining an identical amino acid sequence and introduced into clinical isolates of Enterobacteriaceae on a plasmid. Frequency of ARG silencing will be determined using an experimental evolution approach in nutrient-rich and nutrient-poor environments and confirmed using qPCR and RT-PCR. This will provide important information on factors which effect ARG silencing, as well as producing a library of clinical isolates containing silenced ARGs.
2. ARG reactivation in clinical isolates of Enterobacteriaceae. Using an experimental evolution approach, reactivated ARGs will be selected for in increasing concentrations of antimicrobials which are both targeted by the ARG and other unrelated antimicrobials. This will tell us the effect of antimicrobial concentration on ARG reactivation and whether the use of unrelated antimicrobials can also reactivate these ARGs.
3. Molecular mechanisms of ARG reactivation. Whole genome and RNA sequencing of isolates displaying ARG reactivation will be used to identify the underlining mechanisms of reactivation. This will determine any mutations and/or changes in gene expression which are involved in ARG reactivation.
This project will fill a significant knowledge gap about the role of ARG silencing in mismatch between phenotype and genotype, enhancing the monitoring and prediction of AMR. Importantly, this data will improve treatment options and treatment outcomes by informing the selection of the best antimicrobial for therapeutic use.
References:
Enne, V. I., et al. 2006. Evidence of Antibiotic Resistance Gene Silencing in Escherichia coli. Antimicrobial Agents and Chemotherapy. DOI: 10.1128/AAC.00137-06
Fang, F.C. and Rimsky, S. 2008. New insights into transcriptional regulation by H-NS. Current Opinion in Microbiology. DOI: 10.1016/j.mib.2008.02.011.