Biology subjects

Dumitrescu, O.

Publications and source records attributed to Dumitrescu, O..

8 recordsLinked to original sources

bioRxiv2025

NCY-I beta-lactamase activity correlates with antimicrobial susceptibility of a clinical strain of Nocardia cyriacigeorgica

Nocardiosis is an infectious disease caused by several Nocardia species, among which Nocardia cyriacigeorgica is one of the most frequently isolated species in the clinic. Albeit most isolates of this species are susceptible to standard treatment combining trimethoprim and sulfamethoxazole, resistance has been reported, necessitating alternative or combination therapies. {beta}-lactam antibiotics are of particular interest in this context. In this study, we aimed to address the {beta}-lactam susceptibility profile of a clinical strain of N. cyriacigeorgica and assessed whether it correlated with the enzymatic activity of purified {beta}-lactamase of the strain. We herein established that the strain is highly susceptible to imipenem and ceftriaxone, moderately sensitive to meropenem and resistant to amoxicillin. The resistance could be counteracted by {beta}-lactamase inhibitors from two distinct chemical classes: vaborbactam, and avibactam while clavulanate was less potent. We demonstrated that the {beta}-lactam susceptibility of the strain is in direct line with the enzymatic activity of purified NCY-I, a class A {beta}-lactamase. NCY-I was indeed only active with amoxicillin but displayed poor activity towards other classes of {beta}-lactams. The NCY-I activity could be inhibited in vitro by vaborbactam, clavulanate and avibactam. We consolidated these data by determining the high-resolution structure of NCY-I bound to avibactam. The structural analysis supported a conserved inhibitor binding site among other Nocardia class A {beta}-lactamases strongly suggesting a broad inhibition spectrum of avibactam across Nocardia species.

microbiology↗

bioRxiv2024

Impact of autologous serum on an in vitro granuloma model to study Mycobacterium tuberculosis infection dynamics

Tuberculosis (TB) remains a global health challenge, with Mycobacterium tuberculosis (Mtb) persisting in granulomas, complex immune structures that balance containment and survival of the pathogen. In vitro granuloma models have advanced TB research but often rely on commercial human serum, which may not fully replicate the native immune environment. Here, we investigated the role of autologous serum, either heat-inactivated or native, in granuloma-like structure (GLS) formation, bacterial dynamics, and immune responses. Using a human PBMC-based model to induce spontaneous in vitro granuloma formation after infection with Mtb, we found that autologous serum significantly enhanced GLS formation and altered cytokine profiles compared to commercial serum. Native serum also reduced bacterial growth, likely due to active complement system-mediated immune responses. These findings highlight the importance of autologous serum in creating physiologically relevant models, crucial for preclinical evaluation of TB therapies. Incorporating patient-specific serum can improve model accuracy, enabling better exploration of host-pathogen interactions and targeted therapeutic strategies.

microbiology↗

bioRxiv2024

Autologous host-pathogen pairing enhances early immune responses and bacterial growth control in tuberculosis in vitro granuloma model

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a global health challenge, characterized by significant heterogeneity in its clinical presentation and physiopathology, underscoring the need for optimized models to better understand host-pathogen interactions. This study evaluated the impact of both host and Mtb clinical isolate variability on bacterial growth and dormancy and immune responses using an in vitro granuloma model. Peripheral blood mononuclear cells from active TB patients were infected with autologous and heterologous Mtb clinical isolates, as well as the H37Rv Mtb reference strain. Bacterial growth and dormancy dynamics and cytokines release were assessed at 0-, 7-, 14- and 21-days post-infection. Results revealed that bacterial growth and dormancy dynamics were mostly isolate-dependent, with almost no impact of PBMC donor. However, at 7-days post-infection, autologous infections exhibited lower bacterial loads and elevated Th1-type cytokines, such as TNF- and IFN-{gamma}. Yet, this early advantage in bacterial control did not persist at later time points. These findings highlight the importance of host-pathogen pairing in shaping immune responses and validate the in vitro granuloma model as a relevant tool for studying TB pathogenesis and evaluating therapeutic interventions. Furthermore, further research is needed to decipher the specificities of autologous immune response.

microbiology↗

bioRxiv2023

Crystal structure of a Class A beta-lactamase from Nocardia cyriacigeorgica

Nocardia are gram-positive bacteria from the Actinobacteria phylum. Some Nocardia species can infect humans and are usually considered opportunist pathogens as they often infect immunocompromised patients. Albeit, their clinical incidence is low, many Nocardia species are nowadays considered emerging pathogens. Primary sites of infection of Nocardia are the skin or the lungs but dissemination to other body parts is very frequent. These disseminated infections are very difficult to treat and thus, tackled with multiple classes of antibiotics, on top of the traditional treatment targeting the folate pathway. {beta}-lactams are often included in the regimen but many Nocardia species present moderate or strong resistance to some of this drug class. Genomic, microbiological, and biochemical studies have reported the presence of class A {beta}-lactamases (ABL) in a handful of Nocardia species but no structural investigation of Nocardia {beta}-lactamases has been performed yet. In this study, we report the expression, purification, and preliminary biochemical characterization of the ABL from a Nocardia cyriacigeorgica (NCY-1) clinical strain. We describe, as well, the crystallization and the very high-resolution crystal structure of NCY-1. The protein sequence and structural analysis demonstrate that NCY-1 belongs to {beta}-lactamase of class A1 and attest to its very high conservation with ABL from other human pathogenic Nocardia. In addition, the presence of one molecule of citrate tightly bound in the catalytic site of the enzyme is described. This structure may provide a solid basis for future drug development to specifically target Nocardia spp. {beta}-lactamases. SynopsisThe crystal structure at high resolution of a class A {beta}-lactamase from a clinical strain of Nocardia cyriacigeorgica is reported.

biochemistry↗

bioRxiv2022

Mycobacterium tuberculosis genetic features associated with pulmonary tuberculosis severity

Mycobacterium tuberculosis (Mtb) infections result in a wide spectrum of clinical presentations but without proven Mtb genetic determinants. Herein, 234 pulmonary tuberculosis (TB) patients were stratified according to TB disease severity and Mtb genetic features were explored using whole genome sequencing, including heterologous single nucleotide polymorphism (SNP) calling to explore micro-diversity. Clinical isolates from patients with mild TB carried mutations in genes associated with host-pathogen interaction, while those from patients with moderate/severe TB carried mutations associated with regulatory mechanisms. Genome-wide association study identified a SNP in the promoter of the gene coding for the virulence regulator EspR associated with moderate/severe disease. Structural equation modelling and model comparisons indicated that TB severity was associated with the detection of Mtb micro-diversity within clinical isolates and to the espR SNP. Taken together, these results provide a new insight to better understand TB pathophysiology and could provide new prognosis tool for pulmonary TB severity.

microbiology↗

bioRxiv2021

In-depth immunophenotyping with mass cytometry during TB treatment reveals non-canonical T-cell subsets associated with sputum culture conversion.

Tuberculosis (TB) is a difficult-to-treat infection because of multidrug regimen requirements based on drug susceptibility profiles and treatment observance issues. TB cure is defined by mycobacterial sterilization, technically complex to systematically assess. We hypothesized that microbiological outcome was associated with stage-specific immune changes in peripheral whole blood during TB treatment. The T-cell phenotypes of treated TB patients were prospectively characterized in a blinded fashion using mass cytometry after Mycobacterium tuberculosis (Mtb) antigen stimulation, and then correlated to sputum culture status. At two months of treatment, cytotoxic and terminally differentiated CD8+ T-cells were under-represented and naive CD4+ T-cells were over-represented in positive-versus negative-sputum culture patients, regardless of Mtb drug susceptibility. At treatment completion, an antigen-driven T-cell immune shift towards differentiated subpopulations was associated with TB cure. Overall, we identified specific T-cell profiles associated with slow sputum converters, which brings new insights in TB prognostic biomarker research designed for clinical application. SummaryIn patients treated for pulmonary TB, high-dimensional immune profiling with mass cytometry revealed that Mycobacterium tuberculosis culture conversion is associated with newly characterized peripheral CD8+ T-cell phenotypes. This paves the way for new immune biomarkers associated with mycobacterial sterilization.

immunology↗

bioRxiv2021

Within-host genetic micro-diversity of Mycobacterium tuberculosis and the link with tuberculosis disease features

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex, is still the number one deadly contagious disease. Mtb infection results in a wide spectrum of clinical presentations and severity symptoms, but without proven Mtb genetic determinants. Thanks to a collection of 355 clinical isolates with associated patients clinical data, we showed that Mtb micro-diversity within patient isolates is strongly correlated with TB-associated severity scores. Interestingly, this diversity is driven by a selection pressure to adapt to different lifestyles related to the infection site. Taken together, these results provide a new insight to better understand TB pathophysiology. Furthermore, Mtb micro-diversity could be envisioned as a new prognostic tool to improve the management of TB patients.

microbiology↗

bioRxiv2020

Mycobacterium tuberculosis micro-diversity promotes intra-macrophagic persistence and antibiotic tolerance

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptive response during the course of tuberculosis (TB) and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in a cohort of patients with delayed Mtb culture conversion. We captured the genetic diversity of both the initial and the last Mtb isolate obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed the last Mtb isolate to rifampicin (RIF) prior to whole genome sequencing analysis. We were able to detect unfixed SNPs within the Mtb isolates for 9/12 patients, and non-synonymous (ns) SNPs in 6/12 patients. Furthermore, RIF exposure revealed 8 additional unfixed nsSNP unlinked to drug resistance. To better understand the dynamics of Mtb micro-diversity during the course of TB, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments chosen to mimic the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants, not necessarily linked to canonical drug-resistance and which may be able to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management. Author summaryTuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), bacteria that are able to persist inside the patient for many months or years, thus requiring long antibiotic treatments. Here we focused on TB patients with delayed response to treatment and we performed genetic characterization of Mtb isolates to search for sub-populations that may tolerate anti-TB drugs. We found that Mtb cultured from 9/12 patients contained different sub-populations, and in vitro drug exposure revealed 8 other sub-populations, none related to known drug-resistance mechanisms. Furthermore, we characterized a Mtb variant isolated from a sub-population growing in the presence of rifampicin (RIF), a major anti-TB drug. We found that this variant featured a modified lipidic envelope, and that it was able to develop in the presence of RIF and inside human macrophage cells. We performed pharmacological modelling and found that this kind of variant may be related to a poor response to treatment. In conclusion, searching for particular Mtb sub-populations may help to detect patients at risk of treatment failure and provide additional guidance for TB management.

microbiology↗