RespiriNTM – Specificity of the innate immune responses to different classes of non-tuberculous mycobacteria

Abstract: Mycobacterium avium is the most common non-tuberculous mycobacterium (NTM) species causing infectious disease. Here, we characterized a M. avium infection model in zebrafish larvae, and compared it to M. marinum infection, a model of tuberculosis. M. avium bacteria are efficiently phagocytosed and frequently induce granuloma-like structures in zebrafish larvae. Although macrophages can respond to both mycobacterial infections, their migration speed is faster in infections caused by M. marinum. Tlr2 is conservatively involved in most aspects of the defense against both mycobacterial infections. However, Tlr2 has a function in the migration speed of macrophages and neutrophils to infection sites with M. marinum that is not observed with M. avium. Using RNAseq analysis, we found a distinct transcriptome response in cytokine-cytokine receptor interaction for M. avium and M. marinum infection. In addition, we found differences in gene expression in metabolic pathways, phagosome formation, matrix remodeling, and apoptosis in response to these mycobacterial infections. In conclusion, we characterized a new M. avium infection model in zebrafish that can be further used in studying pathological mechanisms for NTM-caused diseases.

Authors: Wanbin, H., Koch, B. E. V., Lamers, G. E. M., Forn-Cuní, G., Spaink, H. P.

Source: Frontiers in Immunology, Section Microbial Immunology

DOI: https://doi.org/10.3389/fimmu.2022.1075473

UNITE4TB – Implementing molecular tuberculosis diagnostic methods in limited-resource and high-burden countries

Abstract:Tuberculosis (TB) is one of the deadliest infectious diseases in the world with more than a million people dying of TB each year. Accurate and timely TB diagnosis has the potential to alleviate the global TB burden; therefore, one of the pillars of the End TB Strategy developed by the World Health Organization (WHO) is the early diagnosis of TB, including universal drug-susceptibility testing (DST). The WHO emphasises the importance of DST before treatment initiation, using molecular WHO-recommended rapid diagnostic tests (mWRDs). Currently available mWRDs are nucleic acid amplification tests, line probe assays, whole genome sequencing, and targeted next-generation sequencing. However, implementing the sequencing mWRDs in routine laboratories in low-income countries is constrained by the existing infrastructure, high cost, the specialised skills needed, data storage, and the current delay in results compared with other routine methods. These limitations are pronounced in resource-limited settings, which often have a high TB burden and need for innovative TB diagnostic technologies. In this article we propose several possible solutions, like adapting infrastructure capacity to needs, advocating for lowering costs, building bioinformatics and laboratory capacity, and increasing the use of open-access resources for software and publications.

Authors: Vasiliu, A., Iswari Saktiawati, A. M., Duarte, R., Lange, C., Cirillo, D. M.

Source: European Respiratory Society, Breathe

DOI: https://doi.org/10.1183/20734735.0226-2022

ERA4TB – Microfluidic dose–response platform to track the dynamics of drug response in single mycobacterial cells

Abstract: Preclinical analysis of drug efficacy is critical for drug development. However, conventional bulk-cell assays statically assess the mean population behavior, lacking resolution on drug-escaping cells. Inaccurate estimation of efficacy can lead to overestimation of compounds, whose efficacy will not be confirmed in the clinic, or lead to rejection of valuable candidates. Time-lapse microfluidic microscopy is a powerful approach to characterize drugs at high spatiotemporal resolution, but hard to apply on a large scale. Here we report the development of a microfluidic platform based on a pneumatic operating principle, which is scalable and compatible with long-term live-cell imaging and with simultaneous analysis of different drug concentrations. We tested the platform with mycobacterial cells, including the tubercular pathogen, providing the first proof of concept of a single-cell dose–response assay. This dynamic in-vitro model will prove useful to probe the fate of drug-stressed cells, providing improved predictions of drug efficacy in the clinic.

Authors: Mistretta, M., Gangneux, N., Manina, G.

Source: Nature, Scientific Reports

DOI: https://doi.org/10.1038/s41598-022-24175-9

PrIMAVeRa – Estimating antimicrobial resistance burden in Europe: what are the next steps?

Abstract: No abstract

Authors: Hassoun-Kheir, N., Harbarth, S.

Source: The Lancet, Public Health

DOI:https://doi.org/10.1016/S2468-2667(22)00250-X

GNA NOW – Exploring Cluster-Dependent Antibacterial Activities and Resistance Pathways of NOSO-502 and Colistin against Enterobacter cloacae Complex Species

Abstract: The Enterobacter cloacae complex (ECC) is a group of diverse environmental and clinically relevant bacterial species associated with a variety of infections in humans. ECC have emerged as one of the leading causes of nosocomial infections worldwide. The purpose of this paper is to evaluate the activity of NOSO-502 and colistin (CST) against a panel of ECC clinical isolates, including different Hoffmann’s clusters strains, and to investigate the associated resistance mechanisms. NOSO-502 is the first preclinical candidate of a novel antibiotic class, the odilorhabdins (ODLs). MIC50 and MIC90 of NOSO-502 against ECC are 1 μg/mL and 2 μg/mL, respectively, with a MIC range from 0.5 μg/mL to 32 μg/mL. Only strains belonging to clusters XI and XII showed decreased susceptibility to both NOSO-502 and CST while isolates from clusters I, II, IV, and IX were only resistant to CST. To understand this phenomenon, E. cloacae ATCC 13047 from cluster XI was chosen for further study. Results revealed that the two-component system ECL_01761-ECL_01762 (ortholog of CrrAB from Klebsiella pneumoniae) induces NOSO-502 hetero-resistance by expression regulation of the ECL_01758 efflux pump component (ortholog of KexD from K. pneumoniae) which could compete with AcrB to work with the multidrug efflux pump proteins AcrA and TolC. In E. cloacae ATCC 13047, CST-hetero-resistance is conferred via modification of the lipid A by addition of 4-amino-4-deoxy-l-arabinose controlled by PhoPQ. We identified that the response regulator ECL_01761 is also involved in this resistance pathway by regulating the expression of the ECL_01760 membrane transporter.

Authors: Pantel, L., Guérin, F., Serri, M., Gravey, F., Houard, J., Maurent, K., Attwood, M., Noel, A., MacGowan, A., Racine, E., Cattoir, V., Gualtieri, M.

Source: American Society for Microbiology, Antimicrobial Agents and Chemotherapy

DOI: https://doi.org/10.1128/aac.00776-22

COMBINE – Expert workshop summary: Advancing toward a standardized murine model to evaluate treatments for antimicrobial resistance lung infections

Abstract: The rise in antimicrobial resistance (AMR), and increase in treatment-refractory AMR infections, generates an urgent need to accelerate the discovery and development of novel anti-infectives. Preclinical animal models play a crucial role in assessing the efficacy of novel drugs, informing human dosing regimens and progressing drug candidates into the clinic. The Innovative Medicines Initiative-funded “Collaboration for prevention and treatment of MDR bacterial infections” (COMBINE) consortium is establishing a validated and globally harmonized preclinical model to increase reproducibility and more reliably translate results from animals to humans. Toward this goal, in April 2021, COMBINE organized the expert workshop “Advancing toward a standardized murine model to evaluate treatments for AMR lung infections”. This workshop explored the conduct and interpretation of mouse infection models, with presentations on PK/PD and efficacy studies of small molecule antibiotics, combination treatments (β-lactam/β-lactamase inhibitor), bacteriophage therapy, monoclonal antibodies and iron sequestering molecules, with a focus on the major Gram-negative AMR respiratory pathogens Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Here we summarize the factors of variability that we identified in murine lung infection models used for antimicrobial efficacy testing, as well as the workshop presentations, panel discussions and the survey results for the harmonization of key experimental parameters. The resulting recommendations for standard design parameters are presented in this document and will provide the basis for the development of a harmonized and bench-marked efficacy studies in preclinical murine pneumonia model.

Authors: Arrazuria, R., Kerscher, B., Huber, K. E., Hoover, J. L., Vingsbo Lundberg, C., Hansen, J. U., Sordello, S., Renard, S., Aranzana-Climent, V., Hughes, D., Gribbon, P., Friberg, L. E., Bekeredjian-Ding, I.

Source: Frontiers Microbiology, Section Antimicrobials, Resistance and Chemotherapy

DOI: https://doi.org/10.3389/fmicb.2022.988725

COMBINE – Variability of murine bacterial pneumonia models used to evaluate antimicrobial agents

Abstract: Antimicrobial resistance has become one of the greatest threats to human health, and new antibacterial treatments are urgently needed. As a tool to develop novel therapies, animal models are essential to bridge the gap between preclinical and clinical research. However, despite common usage of in vivo models that mimic clinical infection, translational challenges remain high. Standardization of in vivo models is deemed necessary to improve the robustness and reproducibility of preclinical studies and thus translational research. The European Innovative Medicines Initiative (IMI)-funded “Collaboration for prevention and treatment of MDR bacterial infections” (COMBINE) consortium, aims to develop a standardized, quality-controlled murine pneumonia model for preclinical efficacy testing of novel anti-infective candidates and to improve tools for the translation of preclinical data to the clinic. In this review of murine pneumonia model data published in the last 10 years, we present our findings of considerable variability in the protocols employed for testing the efficacy of antimicrobial compounds using this in vivo model. Based on specific inclusion criteria, fifty-three studies focusing on antimicrobial assessment against Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii were reviewed in detail. The data revealed marked differences in the experimental design of the murine pneumonia models employed in the literature. Notably, several differences were observed in variables that are expected to impact the obtained results, such as the immune status of the animals, the age, infection route and sample processing, highlighting the necessity of a standardized model.

Authors: Arrazuria, R., Kerscher, B., Huber, K. E., Hoover, J. L., Vingsbo Lundberg, C., Hansen, J. U., Sordello, S., Renard, S., Aranzana-Climent, V., Hughes, D., Gribbon, P., Friberg, L. E., Bekeredjian-Ding, I.

Source: Frontiers Microbiology, Section Antimicrobials, Resistance and Chemotherapy

DOI: https://doi.org/10.3389/fmicb.2022.988728

UNITE4TB – Machine Learning and Pharmacometrics for Prediction of Pharmacokinetic Data: Differences, Similarities and Challenges Illustrated with Rifampicin

Abstract: Pharmacometrics (PM) and machine learning (ML) are both valuable for drug development to characterize pharmacokinetics (PK) and pharmacodynamics (PD). Pharmacokinetic/pharmacodynamic (PKPD) analysis using PM provides mechanistic insight into biological processes but is time- and labor-intensive. In contrast, ML models are much quicker trained, but offer less mechanistic insights. The opportunity of using ML predictions of drug PK as input for a PKPD model could strongly accelerate analysis efforts. Here exemplified by rifampicin, a widely used antibiotic, we explore the ability of different ML algorithms to predict drug PK. Based on simulated data, we trained linear regressions (LASSO), Gradient Boosting Machines, XGBoost and Random Forest to predict the plasma concentration-time series and rifampicin area under the concentration-versus-time curve from 0–24 h (AUC0–24h) after repeated dosing. XGBoost performed best for prediction of the entire PK series (R2: 0.84, root mean square error (RMSE): 6.9 mg/L, mean absolute error (MAE): 4.0 mg/L) for the scenario with the largest data size. For AUC0–24h prediction, LASSO showed the highest performance (R2: 0.97, RMSE: 29.1 h·mg/L, MAE: 18.8 h·mg/L). Increasing the number of plasma concentrations per patient (0, 2 or 6 concentrations per occasion) improved model performance. For example, for AUC0–24h prediction using LASSO, the R2 was 0.41, 0.69 and 0.97 when using predictors only (no plasma concentrations), 2 or 6 plasma concentrations per occasion as input, respectively. Run times for the ML models ranged from 1.0 s to 8 min, while the run time for the PM model was more than 3 h. Furthermore, building a PM model is more time- and labor-intensive compared with ML. ML predictions of drug PK could thus be used as input into a PKPD model, enabling time-efficient analysis.

Authors: Keutzer, L., You, H., Farnoud, A., Nyberg, J., Wicha, S. G., Maher-Edwards, G., Vlasakakis, G., Moghaddam, G. K., Svensson, E. M., Menden, M. P., Simonsson, U.

Source: Pharmaceutics, Artificial Intelligence Enabled Pharmacometrics

DOI: https://doi.org/10.3390/pharmaceutics14081530

UNITE4TB – Tuberculosis Treatment Monitoring and Outcome Measures: New Interest and New Strategies

Abstract: Despite the advent of new diagnostics, drugs and regimens, tuberculosis (TB) remains a global public health threat. A significant challenge for TB control efforts has been the monitoring of TB therapy and determination of TB treatment success. Current recommendations for TB treatment monitoring rely on sputum and culture conversion, which have low sensitivity and long turnaround times, present biohazard risk, and are prone to contamination, undermining their usefulness as clinical treatment monitoring tools and for drug development. We review the pipeline of molecular technologies and assays that serve as suitable substitutes for current culture-based readouts for treatment response and outcome with the potential to change TB therapy monitoring and accelerate drug development.

Authors: Heyckendorf, J., Georghiou, S., Frahm, N., Heinrich, N., Kontsevaya, I., Reimann, M., Holtzman, D., Imperial, M., Cirillo, D. M., Gillespie, S. H., Ruhwald, M.

Source: American Society for Microbiology, Clinical Microbiology Reviews

DOI: https://doi.org/10.1128/cmr.00227-21

RespiriNTM & RespiriTB – Host-directed therapy to combat mycobacterial infections

Abstract: Upon infection, mycobacteria, such as Mycobacterium tuberculosis (Mtb) and nontuberculous mycobacteria (NTM), are recognized by host innate immune cells, triggering a series of intracellular processes that promote mycobacterial killing. Mycobacteria, however, have developed multiple counter-strategies to persist and survive inside host cells. By manipulating host effector mechanisms, including phagosome maturation, vacuolar escape, autophagy, antigen presentation, and metabolic pathways, pathogenic mycobacteria are able to establish long-lasting infection. Counteracting these mycobacteria-induced host modifying mechanisms can be accomplished by host-directed therapeutic (HDT) strategies. HDTs offer several major advantages compared to conventional antibiotics: (a) HDTs can be effective against both drug-resistant and drug-susceptible bacteria, as well as potentially dormant mycobacteria; (b) HDTs are less likely to induce bacterial drug resistance; and (c) HDTs could synergize with, or shorten antibiotic treatment by targeting different pathways. In this review, we will explore host-pathogen interactions that have been identified for Mtb for which potential HDTs impacting both innate and adaptive immunity are available, and outline those worthy of future research. We will also discuss possibilities to target NTM infection by HDT, although current knowledge regarding host-pathogen interactions for NTM is limited compared to Mtb. Finally, we speculate that combinatorial HDT strategies can potentially synergize to achieve optimal mycobacterial host immune control.

Authors: Kilinç, G., Saris, A., Ottenhoff, T., Haks, M. C.

Source: Wiley Online Library, Immunological Reviews

DOI: https://doi.org/10.1111/imr.12951