Development of diagnostic tools to identify MDR bacteria
Jean-Michel Bolla
Axis 1: Clinical prevalence of impermeability and efflux
Detecting an overexpressed efflux mechanism in bacteria remains a major challenge to this day. While the world is focusing its efforts on combating drug efflux by developing an appropriate inhibitor, relatively less attention is being paid to developing a diagnostic tool that determines whether resistance is due to efflux. Knowing the type of resistance is a crucial factor in treatment selection, otherwise it may result in toxicity due to overdose or delayed patient recovery. The 2019 OECD (Organization for Economic Co-operation and Development) has announced a global strategy to address the problem of drug resistance, which includes effective and rapid diagnosis of AB resistance beyond controlling AB use. While methods for identifying resistance by enzyme or target mutation exist, although researchers have discovered ways to determine efflux-mediated drug resistance in bacteria, these procedures need specific equipment and are time consuming.
Apart from gene amplification or complex spectrometric techniques, the most common method for identifying drug resistance is to use susceptibility testing techniques to determine the minimum inhibitory concentration (MIC). However, the MIC does not indicate whether the resistance is caused by an efflux pump or some other mechanism. Consequently, further analyses are required to determine efflux-mediated resistance, so there is currently a strong need for a diagnostic tool/compound that is fast, cost-effective, safe and easy to use. This tool must be able to differentiate between a WT strain with a low level of efflux to a MDR strain that overexpress the efflux. In collaboration with the CINaM (CNRS-Marseille) we have developed a new class of compounds called phenazinium, that serve as substrates for several efflux pump systems. Our results demonstrate that our compounds possess all these characteristics and allow easy detection of efflux-mediated resistance.
Our phenazinium compound (PCT Application no. PCT/EP2023/051578) is unique in that it detects bacterial efflux activity through quantitative differential staining of cell pellets. This provides a new and improved method that enables the quick identification of efflux-mediated MDR phenotypes. At present, the scope of our study and its findings are restricted to Gram-positive bacteria. The optimized phenazinium compound was licensed and pre-marketed under the name “ColorFlux” by Idylle Labs in France.
We intend now to set up the same assay on Gram-negatives. Thanks to our strain libraries, we have numerous strains (E. coli, Pseudomonas aeruginosa, Enterobacter, and Klebsiella spp., …) with different levels of efflux activity to test our compound. Preliminary results showed that the selected compound can very efficiently stain an acrB deleted mutant of E. coli but no coloration is obtained in a wild-type strain. We hypothesized that either the entry of the compound is slowed down by the presence of the double membrane of E. coli or that the efflux through AcrAB-TolC is more efficient than in Gram positives. Experiments are currently ongoing to test these hypotheses. We will take advantage of the natural fluorescence of 053 (ex: 530, em: 670 nm) and of the other compounds of the same family to follow the kinetics of entry of the molecules by flow-cytometry, in collaboration with the platform of the “Institut Microbiologie, Bioénergies et Biotechnologie” (IM2B). In addition, we are currently determining the condition of growth of the bacteria that could modify efflux activity (growth phase, culture conditions like temperature, and media). In parallel, we will test numerous BG+ from our strain library and from our hospital partners (HIA Laveran, HIA St Anne, Aubagne’s Hospital) to validate our assay. We also initiate a collaboration between our two labs (CINaM and MCT) and the University of Cagliari to perform molecular docking of compounds from the same family to better understand their interaction with the NorA and AcrB pumps.
Because bacteria considered as putative biological weapons (Burkholderia pseudomallei, Yersinia pestis, Francisella tularensis, Bacillus anthracis) are also putatively MDR either by intentional or non-intentional manipulations, it is of great importance to determine their efflux level. In that purpose we recently obtained a financial support to develop the same assay for this kind of bacteria. At a first level we will setup the process on level 2 strains from the same species (attenuated strains) and then apply it to the actual pathogens thank to the NSB3 IRBA facilities.
New regulators
Muriel Masi
Bacteria have evolved mechanisms to preserve the structural integrity of their cell envelope. In Escherichia coli, the Cpx system detects alterations to the envelope and regulates gene expression to facilitate adaptation. This system comprises the transmembrane kinase CpxA and the response regulator CpxR. Activation of CpxA results in autophosphorylation and the transfer of phosphate to CpxR, which subsequently functions as a transcription factor. The CpxRA operon also encompasses CpxP, a protein that serves to downregulate Cpx activity. Cpx is linked to other physiological signals and adaptations, including the detection and repair of disruptions in peptidoglycan, which is essential for bacterial survival.
A mutation in the cpxA gene was identified in a clinical strain of Klebsiella aerogenes isolated from a patient who had been treated with imipenem. This mutation resulted in increased resistance to β-lactams, which was associated with a loss of outer membrane porins (OmpF/C) and higher β-lactamase activity. Our findings demonstrated that this mutation disrupts the interaction between the periplasmic sensor domain of CpxA and CpxP, leading to the constant activation of the Cpx system. Of interest, we found that the β-lactam-induced damage of peptidoglycan can trigger β-lactamase AmpC expression via the Cpx pathway, indicating an intimate connection between the Cpx and AmpG pathways and validating the Cpx system as a novel antibacterial target.
