Meropenem-ANT3310, a unique ß-lactam-ß-lactamase inhibitor combination with expanded antibacterial spectrum against Gram-negative pathogens including carbapenem-resistant Acinetobacter baumannii.

ANT3310 is a novel broad-spectrum diazabicyclooctane serine ß-lactamase inhibitor being developed in combination with meropenem (MEM) for the treatment of serious infections in hospitalized patients where carbapenem-resistant Gram-negative pathogens are expected. In this study, we evaluated the in vitro antibacterial activity of MEM in the presence of ANT3310 at 8 µg/mL against global clinical isolates that included Acinetobacter baumannii (n = 905), carbapenem-resistant Enterobacterales (CRE), carrying either oxacillinase (OXA) (n = 252) or Klebsiella pneumoniae carbapenemase (KPC) (n = 180) carbapenemases, and Pseudomonas aeruginosa (n = 502). MEM was poorly active against A. baumannii, as were MEM-vaborbactam, ceftazidime-avibactam, aztreonam-avibactam, cefepime-taniborbactam, cefepime-zidebactam, and imipenem-relebactam (MIC90 values of ≥32 µg/mL). On the other hand, MEM-ANT3310 displayed an MIC90 value of 4 µg/mL, similar to that observed with sulbactam-durlobactam, a drug developed to specifically treat A. baumannii infections. ANT3310 (8 µg/mL) additionally restored the activity of MEM against OXA- and KPC-producing CREs decreasing MEM MIC90 values from >32 µg/mL to 0.25 and 0.5 µg/mL, respectively. The combination of 8 µg/mL of both MEM and ANT3310 prevented growth of 97.5% of A. baumannii and 100% of OXA- and KPC-positive CREs, with ~90% of P. aeruginosa isolates also displaying MEM MICs ≤8 µg/mL. Furthermore, MEM-ANT3310 was efficacious in both thigh and lung murine infection models with OXA-23 A. baumannii. This study demonstrates the potent in vitro activity of the MEM-ANT3310 combination against both carbapenem-resistant A. baumannii and Enterobacterales clinical isolates, a key differentiator to other ß-lactam/ß-lactamase combinations.

Novel Specific Metallo-ß-Lactamase Inhibitor ANT2681 Restores Meropenem Activity to Clinically Effective Levels against NDM-Positive Enterobacterales.

The global dissemination of metallo-ß-lactamase (MBL)-producing carbapenem-resistant Enterobacterales (CRE) is a serious public health concern. Specifically, NDM (New Delhi MBL) has been a major cause of carbapenem therapy failures in recent years, particularly as effective treatments for serine-ß-lactamase (SBL)-producing Enterobacterales are now commercially available. Since the NDM gene is carried on promiscuous plasmids encoding multiple additional resistance determinants, a large proportion of NDM-CREs are also resistant to many commonly used antibiotics, resulting in limited and suboptimal treatment options. ANT2681 is a specific, competitive inhibitor of MBLs with potent activity against NDM enzymes, progressing to clinical development in combination with meropenem (MEM). Susceptibility studies have been performed with MEM-ANT2681 against 1,687 MBL-positive Enterobacterales, including 1,108 NDM-CRE. The addition of ANT2681 at 8 µg/ml reduced the MEM MIC50/MIC90 from >32/>32 µg/ml to 0.25/8 µg/ml. Moreover, the combination of 8 µg/ml of both MEM and ANT2681 inhibited 74.9% of the Verona integron-encoded MBL (VIM)-positive and 85.7% of the imipenem hydrolyzing ß-lactamase (IMP)-positive Enterobacterales tested. The antibacterial activity of MEM-ANT2681 against NDM-CRE compared very favorably to that of cefiderocol (FDC) and cefepime (FEP)-taniborbactam, which displayed MIC90 values of 8 µg/ml and 32 µg/ml, respectively, whereas aztreonam-avibactam (ATM-AVI) had a MIC90 of 0.5 µg/ml. Particularly striking was the activity of MEM-ANT2681 against NDM-positive Escherichia coli (MIC90 1 µg/ml), in contrast to ATM-AVI (MIC90 4 µg/ml), FDC (MIC90 >32 µg/ml), and FEP-taniborbactam (MIC90 >32 µg/ml), which were less effective due to the high incidence of resistant PBP3-insertion mutants. MEM-ANT2681 offers a potential new therapeutic option to treat serious infections caused by NDM-CRE.

Carbon sources and XlnR-dependent transcriptional landscape of CAZymes in the industrial fungus Talaromyces versatilis: when exception seems to be the rule.

BACKGROUND: Research on filamentous fungi emphasized the remarkable redundancy in genes encoding hydrolytic enzymes, the similarities but also the large differences in their expression, especially through the role of the XlnR/XYR1 transcriptional activator. The purpose of this study was to evaluate the specificities of the industrial fungus Talaromyces versatilis, getting clues into the role of XlnR and the importance of glucose repression at the transcriptional level, to provide further levers for cocktail production. RESULTS: By studying a set of 62 redundant genes representative of several categories of enzymes, our results underlined the huge plasticity of transcriptional responses when changing nutritional status. As a general trend, the more heterogeneous the substrate, the more efficient to trigger activation. Genetic modifications of xlnR led to significant reorganisation of transcriptional patterns. Just a minimal set of genes actually fitted in a simplistic model of regulation by a transcriptional activator, and this under specific substrates. On the contrary, the diversity of xlnR+ versus ΔxlnR responses illustrated the existence of complex and unpredicted patterns of co-regulated genes that were highly dependent on the culture condition, even between genes that encode members of a functional category of enzymes. They notably revealed a dual, substrate-dependant repressor-activator role of XlnR, with counter-intuitive transcripts regulations that targeted specific genes. About glucose, it appeared as a formal repressive sugar as we observed a massive repression of most genes upon glucose addition to the mycelium grown on wheat straw. However, we also noticed a positive role of this sugar on the basal expression of a few genes, (notably those encoding cellulases), showing again the strong dependence of these regulatory mechanisms upon promoter and nutritional contexts. CONCLUSIONS: The diversity of transcriptional patterns appeared to be the rule, while common and stable behaviour, both within gene families and with fungal literature, the exception. The setup of a new biotechnological process to reach optimized, if not customized expression patterns of enzymes, hence appeared tricky just relying on published data that can lead, in the best scenario, to approximate trends. We instead encourage preliminary experimental assays, carried out in the context of interest to reassess gene responses, as a mandatory step before thinking in (genetic) strategies for the improvement of enzyme production in fungi.

Tracking the best reference genes for RT-qPCR data normalization in filamentous fungi.

BACKGROUND: A critical step in the RT-qPCR workflow for studying gene expression is data normalization, one of the strategies being the use of reference genes. This study aimed to identify and validate a selection of reference genes for relative quantification in Talaromyces versatilis, a relevant industrial filamentous fungus. Beyond T. versatilis, this study also aimed to propose reference genes that are applicable more widely for RT-qPCR data normalization in filamentous fungi. RESULTS: A selection of stable, potential reference genes was carried out in silico from RNA-seq based transcriptomic data obtained from T. versatilis. A dozen functionally unrelated candidate genes were analysed by RT-qPCR assays over more than 30 relevant culture conditions. By using geNorm, we showed that most of these candidate genes had stable transcript levels in most of the conditions, from growth environments to conidial germination. The overall robustness of these genes was explored further by showing that any combination of 3 of them led to minimal normalization bias. To extend the relevance of the study beyond T. versatilis, we challenged their stability together with sixteen other classically used genes such as ß-tubulin or actin, in a representative sample of about 100 RNA-seq datasets. These datasets were obtained from 18 phylogenetically distant filamentous fungi exposed to prevalent experimental conditions. Although this wide analysis demonstrated that each of the chosen genes exhibited sporadic up- or down-regulation, their hierarchical clustering allowed the identification of a promising group of 6 genes, which presented weak expression changes and no tendency to up- or down-regulation over the whole set of conditions. This group included ubcB, sac7, fis1 and sarA genes, as well as TFC1 and UBC6 that were previously validated for their use in S. cerevisiae. CONCLUSIONS: We propose a set of 6 genes that can be used as reference genes in RT-qPCR data normalization in any field of fungal biology. However, we recommend that the uniform transcription of these genes is tested by systematic experimental validation and to use the geometric averaging of at least 3 of the best ones. This will minimize the bias in normalization and will support trustworthy biological conclusions.

Development of an unmarked gene deletion system for the filamentous fungi Aspergillus niger and Talaromyces versatilis.

In this article, we present a method to delete genes in filamentous fungi that allows recycling of the selection marker and is efficient in a nonhomologous end-joining (NHEJ)-proficient strain. We exemplify the approach by deletion of the gene encoding the transcriptional regulator XlnR in the fungus Aspergillus niger. To show the efficiency and advantages of the method, we deleted 8 other genes and constructed a double mutant in this species. Moreover, we showed that the same principle also functions in a different genus of filamentous fungus (Talaromyces versatilis, basionym Penicillium funiculosum). This technique will increase the versatility of the toolboxes for genome manipulation of model and industrially relevant fungi.

Higher levels of Pseudomonas aeruginosa LasB elastase expression are associated with early-stage infection in cystic fibrosis patients.

Pseudomonas aeruginosa is a common pathogen in cystic fibrosis (CF) patients and a major contributor to progressive lung damage. P. aeruginosa elastase (LasB), a key virulence factor, has been identified as a potential target for anti-virulence therapy. Here, we sought to differentiate the P. aeruginosa isolates from early versus established stages of infection in CF patients and to determine if LasB was associated with either stage. The lasB gene was amplified from 255 P. aeruginosa clinical isolates from 70 CF patients from the Toulouse region (France). Nine LasB variants were identified and 69% of the isolates produced detectable levels of LasB activity. Hierarchical clustering using experimental and clinical data distinguished two classes of isolates, designated as 'Early' and 'Established' infection. Multivariate analysis revealed that the isolates from the Early infection class show higher LasB activity, fast growth, tobramycin susceptibility, non-mucoid, pigmented colonies and wild-type lasR genotype. These traits were associated with younger patients with polymicrobial infections and high pFEV1. Our findings show a correlation between elevated LasB activity in P. aeruginosa isolates and early-stage infection in CF patients. Hence, it is this patient group, prior to the onset of chronic disease, that may benefit most from novel therapies targeting LasB.

Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1ß in Cellular and Animal Infection Models.

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1ß. Furthermore, inhibition of LasB-mediated IL-1ß activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen.

Discovery of ANT3310, a Novel Broad-Spectrum Serine ß-Lactamase Inhibitor of the Diazabicyclooctane Class, Which Strongly Potentiates Meropenem Activity against Carbapenem-Resistant Enterobacterales and Acinetobacter baumannii.

The diazabicyclooctanes (DBOs) are a class of serine ß-lactamase (SBL) inhibitors that use a strained urea moiety as the warhead to react with the active serine residue in the active site of SBLs. The first in-class drug, avibactam, as well as several other recently approved DBOs (e.g., relebactam) or those in clinical development (e.g., nacubactam and zidebactam) potentiate activity of ß-lactam antibiotics, to various extents, against carbapenem-resistant Enterobacterales (CRE) carrying class A, C, and D SBLs; however, none of these are able to rescue the activity of ß-lactam antibiotics against carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO "critical priority pathogen" producing class D OXA-type SBLs. Herein, we describe the chemical optimization and resulting structure-activity relationship, leading to the discovery of a novel DBO, ANT3310, which uniquely has a fluorine atom replacing the carboxamide and stands apart from the current DBOs in restoring carbapenem activity against OXA-CRAB as well as SBL-carrying CRE pathogens.