Emerging antibiotic resistance in Gram-negative bacilli; deciphering acquired resistance mechanisms to ß-lactam/ ß-lactamase inhibitor combinations and to fosfomycin

Antibiotic resistance in bacteria is a key topic of interest due to the escalation of multi- and pandrug resistance currently observed mostly in Gram-negatives such as in Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii This is particularly worrisome in view of the current dearth of compounds active against multidrug-resistant (MDR) bacteria. Curtailing their spread requires at least a perfect understanding of the biochemical and genetic mechanisms leading to the development of acquired resistance. In this context, novel molecules corresponding to ß-lactam / ß-lactamase inhibitor combinations, but also old but still active molecules such as fosfomycin, are of particular interest. A first part of the study is aimed to analyze the genetic bases of acquired resistance to ceftazidime/avibactam, ceftolozane/tazobactam, aztreonam/avibactam and meropenem/varbobactam taken as clinically-relevant representatives of ß-lactam/ß-lactamase inhibitor combinations. Isogenic Escherichia coli and Klebsiella pneumoniae reference strains possessing broad-spectrum ß-lactamases CTX-M, KPC, OXA-48, NDM-type encoding genes will be constructed. A focus is made on those two enterobacterial species since they are the most common ones in which acquired MDR is observed. In-vitro selection of mutant strains being resistant to those drug combinations will be performed by using plates containing increasing concentrations of those antibiotics. The genetics basis of the resistance will be deciphered by entire genome sequencing of the mutant strains. Similarly, in-vitro obtained resistant mutants to those antibiotics will be obtained using P. aeruginosa reference strains. Prediction of occurrence of mutants under selection pression with ß-lactam/ß-lactam inhibitors with and without additional antibiotics given in combination (colistin, fosfomycin) will be obtained. The structure/function relationship of those obtained mutated enzymes will be evaluated. In addition, by screening clinical strains obtained from our emerging antibiotic reference center for Switzerland as well as collections of clinical isolates of worldwide origin, novel resistant determinants might also be identified. As an example, a novel KPC mutant (KPC-41) from a clinical isolate from Switzerland conferring resistance to ceftazidime-avibactam is currently under investigation.A second part of the project is focused on acquired resistance to fosfomycin. Our preliminary results show that the naturally-occurring and chromosomally-located fosfomycin (fos) gene in K. pneumoniae does usually not confer resistance in that species. Preliminary genetic investigations showed that this gene is associated to a gene encoding a LysR-type regulatory protein. We have observed that both genes are overexpressed in resistant strains. By performing a series of genetic constructions, our aim will be to analyze the functional relationships between those genes and the regulatory effect of the LysR-type protein. We have recently identified a novel plasmid-borne fos gene, namely fos249, in an E. coli clinical isolate recovered in Switzerland. This gene was located into a putative remnant of a Tn7 transposon structure. We will attempt to experimentally mobilize the fos249 gene through the action of Tn7, and to analyze the biochemical activity of this Fos249 protein. By screening a large collection of MDR Gram-negatives, we will seek for novel and transferable fosfomycin resistance genes. This might contribute to gain knowledge on the epidemiology and functionality of those resistance determinants. This study will contribute to predict to what extent bacterial isolates may be ?prompt to develop resistance to those novel ß-lactamase/ß-lactamase inhibitor combinations, and to fosfomycin. Furthermore, it may help to better characterize which are the conditions (drug concentration, drug combination) that may enhance or conversely antagonize the development of resistance. Additionally, a significant outcome of this project might be the detection of novel resistant determinants to those molecules, that may emerge in clinically-significant strains.