In Vitro Mechanisms of Resistance Development to Imipenem-Relebactam in KPC-Producing Klebsiella pneumoniae.

Carbapenem-resistant Enterobacterales, such as KPC-producing Klebsiella pneumoniae, represent a major threat to public health. Novel drug combinations including imipenem-relebactam (IPM-REL) have recently been introduced and have been shown to exhibit excellent activity toward such strains. However, there has recently been reports of the in vivo emergence of IPM-REL resistance in KPC-producing K. pneumoniae. Here, we evaluated, in vitro, the nature of the mutations that lead to IPM-REL resistance in 5 KPC-producing K. pneumoniae strains, including 2 that produce KPC enzymes conferring ceftazidime-avibactam resistance. An in vitro multi-step selection assay was performed and corresponding mutants obtained. Mutations were identified in OmpK36 as well as 2 different mutant derivatives of KPC. Mutant strains exhibited decreased susceptibility to ß-lactams, including the carbapenems, and meropenem-vaborbactam (MEM-VAB). Expression of blaKPC gene variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to carbapenems and decreased susceptibility to CAZ-AVI, and enzymatic assays showed that the inhibitory activity of both AVI and REL was significantly lowered for both KPC mutants compared to parental enzymes. Complementation assays showed that OmpK36 plays a major role in IPM-REL resistance as well resistance to other ß-lactams and ß-lactam/ß-lactamase inhibitor combinations. Overall, this study showed that (i) IPM-REL resistant strains can be obtained from CAZ-AVI-susceptible or -resistant KPC producers, (ii) selection of IPM-REL resistance has a collateral effect on MEM-VAB susceptibility - indicative of shared resistance mechanisms, (iii) and mutations in the KPC sequence may be obtained using IPM-REL selection leading to the possibility of vertical and horizontal transfer of this resistance trait.

Roles for phthiocerol dimycocerosate lipids in Mycobacterium tuberculosis pathogenesis.

The success of Mycobacterium tuberculosis as a pathogen is well established: tuberculosis is the leading cause of death by a single infectious agent worldwide. The threat of multi- and extensively drug-resistant bacteria has renewed global concerns about this pathogen and understanding its virulence strategies will be essential in the fight against tuberculosis. The current review will focus on phthiocerol dimycocerosates (PDIMs), a long-known and well-studied group of complex lipids found in the M. tuberculosis cell envelope. Numerous studies show a role for PDIMs in several key steps of M. tuberculosis pathogenesis, with recent studies highlighting its involvement in bacterial virulence, in association with the ESX-1 secretion system. Yet, the mechanisms by which PDIMs help M. tuberculosis to control macrophage phagocytosis, inhibit phagosome acidification and modulate host innate immunity, remain to be fully elucidated.

Aloe Emodin Reduces Phthiodiolone Dimycocerosate Potentiating Vancomycin Susceptibility on Mycobacteria.

Treatment of tuberculosis still represent a major public health issue. The emergence of multi-and extensively-drug resistant (MDR and XDR) Mycobacterium tuberculosis clinical strains further pinpoint the urgent need for new anti-tuberculous drugs. We previously showed that vancomycin can target mycobacteria lacking cell wall integrity, especially those lacking related phthiocerol and phthiodolone dimycocerosates, PDIM A and PDIM B, respectively. As aloe emodin was previously hypothesized to be able to target the synthesis of mycobacterial cell wall lipids, we tested its ability to potentiate glycopeptides antimycobacterial activity. The aloe emodin with the vancomycin induced a combination effect beyond simple addition, close to synergism, at a concentration lower to reported IC50 cytotoxic value, on M. bovis BCG and on H37Rv M. tuberculosis. Interestingly, out of six MDR and pre-XDR clinical strains, one showed a strong synergic susceptibility to the drug combination. Mycobacterial cell wall lipid analyses highlighted a selective reduction of PDIM B by aloe emodin.

Effects of Lipid-Lowering Drugs on Vancomycin Susceptibility of Mycobacteria.

Tuberculosis is still a cause of major concern, partly due to the emergence of multidrug-resistant strains. New drugs are therefore needed. Vancomycin can target mycobacteria with cell envelope deficiency. In this study, we used a vancomycin susceptibility assay to detect drugs hampering lipid synthesis in Mycobacterium bovis BCG and in Mycobacterium tuberculosis We tested three drugs already used to treat human obesity: tetrahydrolipstatin (THL), simvastatin, and fenofibrate. Only vancomycin and THL were able to synergize on M. bovis BCG and on M. tuberculosis, although mycobacteria could also be inhibited by simvastatin alone. Lipid analysis allowed us to identify several lipid modifications in M. tuberculosis H37Rv treated with those drugs. THL treatment mainly reduced the phthiocerol dimycocerosate (PDIM) content in the mycobacterial cell wall, providing an explanation for the synergy, since PDIM deficiency has been related to vancomycin susceptibility. Proteomic analysis suggested that bacteria treated with THL, in contrast to bacteria treated with simvastatin, tried to recover, inducing, among other reactions, lipid synthesis. The combination of THL and vancomycin should be considered a promising solution in developing new strategies to treat multidrug-resistant tuberculosis.

Increased Vancomycin Susceptibility in Mycobacteria: a New Approach To Identify Synergistic Activity against Multidrug-Resistant Mycobacteria.

Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing Mycobacterium bovis BCG and M. tuberculosis mutants that lack phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids with wild-type strains, we observed that glycopeptides strongly inhibited PDIM-deprived mycobacteria. Vancomycin together with a drug targeting lipid synthesis inhibited multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential antituberculosis (TB) agents and might provide a new antimycobacterial drug-screening strategy.

The Mycobacterium bovis BCG GroEL1 Contributes to Isoniazid Tolerance in a Dormant-Like State Model.

Due to the Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis, tuberculosis still causes 1.6 million deaths per year. Therefore, efforts to improve tuberculosis treatment are necessary. We previously showed that the GroEL1 protein is involved in antibiotic intrinsic resistance. Indeed, the M. bovis BCG cpn60.1 gene (encoding GroEL1)-disrupted strain (Δcpn60.1) exhibits higher rifampicin and vancomycin susceptibility due to defective cell wall integrity. Here, we show that during hypoxia-triggered growth stasis, in the Wayne dormancy model, the mutant exhibited comparable rifampicin and ethionamide susceptibility but higher isoniazid susceptibility compared to the wild-type strain. Although the Δcpn60.1 strain showed compromised induction of the DosR regulon, growth stasis was achieved, but an ATP burst and a higher reactive oxygen species (ROS) production were observed in the isoniazid-treated Δcpn60.1 strain. GroEL1 could contribute to INH tolerance by reducing ROS.

Mycobacterium tuberculosis Infection of THP-1 Cells: A Model for High Content Analysis of Intracellular Growth and Drug Susceptibility.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the leading cause of death from any infectious agent worldwide, with an estimated 10 million new cases in 2019. Drug development efforts for TB have classically relied on in vitro screening campaigns without consideration for Mtb's established intracellular lifestyle, which may not reflect true drug susceptibility in vivo. Here, we introduce two intracellular screening techniques based on the detection of different fluorescent markers to enumerate bacterial burden in THP-1 monocyte derived macrophages. These techniques are able to distinguish actively growing bacteria from killed bacteria by two distinct methodologies, with the use of cell wall intercalating dye DMN-Tre or an RFP expressing Mtb. This method may also be utilised in the screening of mutant Mtb libraries to evaluate the mutations' effect on drug susceptibility and vice versa. As current high content platform technologies are able to perform a variety of functions, these techniques are broadly applicable to a multiplicity of intracellular screens. We further provide a comparison of infection techniques that may be used for drug screening (batch infection) and high content host-pathogen interaction analysis (2-day differentiation). The aim of this text is to provide the user with a solid and reproducible starting point to high content screening of intracellular Mtb, and to highlight adaptations to the protocol that may aid in future assay development.

Inhibition of glycogen synthase kinase-3-beta (GSK3ß) blocks nucleocapsid phosphorylation and SARS-CoV-2 replication.

GSK3ß has been proposed to have an essential role in Coronaviridae infections. Screening of a targeted library of GSK3ß inhibitors against both SARS-CoV-2 and HCoV-229E to identify broad-spectrum anti-Coronaviridae inhibitors resulted in the identification of a high proportion of active compounds with low toxicity to host cells. A selected lead compound, T-1686568, showed low micromolar, dose-dependent activity against SARS-CoV-2 and HCoV-229E. T-1686568 showed efficacy in viral-infected cultured cells and primary 2D organoids. T-1686568 also inhibited SARS-CoV-2 variants of concern Delta and Omicron. Importantly, while inhibition by T-1686568 resulted in the overall reduction of viral load and protein translation, GSK3ß inhibition resulted in cellular accumulation of the nucleocapsid protein relative to the spike protein. Following identification of potential phosphorylation sites of Coronaviridae nucleocapsid, protein kinase substrate profiling assays combined with Western blotting analysis of nine host kinases showed that the SARS-CoV-2 nucleocapsid could be phosphorylated by GSK3ß and PKCa. GSK3ß phosphorylated SARS-CoV-2 nucleocapsid on the S180/S184, S190/S194 and T198 phospho-sites, following previous priming in the adjacent S188, T198 and S206, respectively. Such inhibition presents a compelling target for broad-spectrum anti-Coronaviridae compound development, and underlies the mechanism of action of GSK3ß host-directed therapy against this class of obligate intracellular pathogens.

Apoptosis assessment in high-content and high-throughput screening assays.

Here the authors describe the development of AUTOptosis, an economical and rapid apoptosis monitoring method suitable for high-content and high-throughput screening assays. AUTOptosis is based on the quantification of nuclei intensity via staining with Hoechst 33342. First, the authors calibrated the method using standard apoptosis inducers in multiple cell lines. Next, the authors validated the applicability of this approach to high-content screening using a small library of compounds and compared it with the terminal deoxynucleotidyl transferase dUTP nick end labeling gold standard. Finally, the authors demonstrated the specificity of the method by using AUTOposis to detect apoptosis triggered by Mycobacterium tuberculosis intracellular infections.

DMN-Tre Labeling for Detection and High-Content Screening of Compounds against Intracellular Mycobacteria.

4-N,N-Dimethylamino-1,8-naphthalimide conjugate of trehalose (DMN-Tre) is a fluorogenic dye recently developed as a diagnostic tool for tuberculosis. DMN-Tre selectively labels the mycobacterial cell wall through the Ag85 enzymes. In this work, we disclose a protocol describing the total synthesis of DMN-Tre with more than 99% purity. We further developed a protocol for in vitro and intercellular labeling of various mycobacterial strains. DMN-Tre labeling was found to be a useful tool to study in vitro and intracellular Mycobacterium tuberculosis (Mtb) physiology and as an end-point readout system in high-content image-based screening (HCS) of drug molecules. Such uses of DMN-Tre labeling provide a simple, fast, and cheap alternative to the existing, time-consuming approach that requires Mtb strains to be genetically transformed with fluorescent reporter genes.

High-Content Screening of Eukaryotic Kinase Inhibitors Identify CHK2 Inhibitor Activity Against Mycobacterium tuberculosis.

A screen of a eukaryotic kinase inhibitor library in an established intracellular infection model identified a set of drug candidates enabling intracellular killing of Mycobacterium tuberculosis (M.tb). Screen validity was confirmed internally by a Z' = 0.5 and externally by detecting previously reported host-targeting anti-M.tb compounds. Inhibitors of the CHK kinase family, specifically checkpoint kinase 2 (CHK2), showed the highest inhibition and lowest toxicity of all kinase families. The screen identified and validated DDUG, a CHK2 inhibitor, as a novel bactericidal anti-M.tb compound. CHK2 inhibition by RNAi phenocopied the intracellular inhibitory effect of DDUG. DDUG was active intracellularly against M.tb, but not other mycobacteria. DDUG also had extracellular activity against 4 of 12 bacteria tested, including M.tb. Combined, these observations suggest DDUG acts in tandem against both host and pathogen. Importantly, DDUG's validation highlights the screening and analysis methodology developed for this screen, which identified novel host-directed anti-M.tb compounds.

I3-Ag85 effect on phthiodiolone dimycocerosate synthesis.

The multiplicity of drug resistant Mycobacterium tuberculosis (Mtb) strains is a growing health issue. New therapies are needed, acting on new targets. The I3-Ag85 was already reported to reduce the amount of trehalose dimycolate lipid of the mycobacterial cell wall. This inhibitor of Ag85C increased the mycobacterial wall permeability. We previously showed that M. tuberculosis strains, even multi-drug resistant and extensively-drug resistant strains, can be susceptible to vancomycin when concomitantly treated with a drug altering the cell envelope integrity. We investigated the effect of the I3-Ag85 on vancomycin susceptibility of M. tuberculosis. Although no synergy was observed, a new target of this drug was discovered: the production of phthiodiolone dimycocerosate (PDIM B).