Biology subjects

Ramon-Garcia, S.

Publications and source records attributed to Ramon-Garcia, S..

11 recordsLinked to original sources

bioRxiv2026

OPTIKA, a new high content kill-kinetic assay to longitudinally assess in vitro drug combinations against Mycobacterium tuberculosis

In vitro methods to characterize drug combinations typically involve phenotypic screenings using checkerboard assays (CBA) or, more recently, DiaMOND. Such approaches rely on the Fractional Inhibitory Concentration Index (FICI), a fixed-time measurement of growth inhibition that, nonetheless, necessitates secondary validation by time-kill assays (TKA). Longitudinal time-kinetics of bacterial killing are considered the gold standard in vitro proxy for antimicrobial activity, but they required increased assay complexity, particularly against the slow growing Mycobacterium tuberculosis. Here, we developed a new methodology named OPTIKA (Optimized Time Kill Assays) that enhances the capacity of traditional TKA by over 1000-fold. This allows for easy and dynamic examination of n-way drug interactions by simultaneously monitoring bactericidal and sterilizing capacities in a longitudinal manner. We then replicated previous DiaMOND studies and performed comparisons using CBA and OPTIKA methodologies. We demonstrate that selection of the efficacy parameters (either routed on bacteriostatic, bactericidal or sterilizing properties) affects the interpretation of in vitro drug interactions and, consequently, its potential translational value. The increased assay throughput provided by OPTIKA offers a novel framework for developing tuberculosis treatment regimens. TeaserOPTIKA is a new methodology that increases time-kill assay performance against Mycobacterium tuberculosis by over 1,000-fold

microbiology↗

bioRxiv2025

In vitro pharmacokinetics and pharmacodynamics of the diarylquinoline TBAJ-587 and its metabolites against Mycobacterium tuberculosis

The first-in-class diarylquinoline (DARQ) bedaquiline (BDQ) is in the medicines list for drug-resistant tuberculosis. TBAJ-587 is a next-generation DARQ with improved anti-Mycobacterium tuberculosis (Mtb) activity and reduced cardiac repolarization abnormalities. Methods. The in-vitro efficacy of TBAJ-587 and its main metabolites (M2, M3 and M12) was analyzed under standard (ST) growth conditions, with cholesterol (CHO), or fatty acids (FA) as alternative carbon sources. Minimal inhibitory concentration (MIC) assays and time-kill assays (TKA) linked to drug measurements in bacterial samples were performed to allow correlation of pharmacodynamics (PD) with actual pharmacokinetics (PK). The most active compounds, TBAJ-587 and its M3 metabolite, exhibited broth media and concentration dependent efficacy showing a bactericidal effect at [≥]5x MIC. Bacterial cultures treated with 1x MIC and 2x MIC of TBAJ-587 resumed growth after 28 days and displayed moderate increased MIC values compared to untreated conditions, which were linked to new variants of BDQ resistance mutations in the atpE, atpB, and Rv0678 genes. This study revealed TBAJ-587 and metabolites bind to polystyrene plastic-ware, the most commonly used material in antimicrobial research, being the effective unbound drug concentration dependent on the media composition. PKPD analyses determined that Mtb was killed with lower exposures of TBAJ-587 and M3 than expected in ST and FA broth, suggesting previously underestimated potency in these media. This is the first in-vitro study to precisely link compound activities (PD) to their effective concentration (PK) over time in Mtb cultures, providing improved longitudinal data to feed models for translational research.

pharmacology and toxicology↗

bioRxiv2025

Novel antimicrobial activity of photoactive compounds originally synthesized for industrial applications

The emergence of antimicrobial resistance threatens advances achieved by medicine in the last century. This situation has been aggravated by the suboptimal outcome of screening campaigns to provide novel antibiotics. In this study, we took an alternative approach to discover new chemical scaffolds with antimicrobial activity. We screened a collection of photoactive compounds originally synthesized for industrial purposes and found that 4H-pyran-4-ylidenes are active against Gram-positive bacteria. Compounds belonging to this family displayed dose-dependent bactericidal activity against wild-type Staphylococcus aureus and Methicillin Resistant S. aureus (MRSA). Importantly, they were not cytotoxic to hepatic cell lines at the concentrations at which antimicrobial activity was observed. Resistance to 4H-pyran-4-ylidenes in S. aureus was achieved via mutations in the rny locus, likely via changes in mRNA levels of genes critical for their activity. Overall, we demonstrate that chemical libraries not originally intended for drug discovery can be a fruitful source of chemical diversity for the development of novel antimicrobials.

microbiology↗

bioRxiv2024

Sanfetrinem, an oral beta-lactam antibiotic repurposed for the treatment of tuberculosis

Tuberculosis (TB) is historically the worlds deadliest infectious disease. New TB drugs that can avoid pre-existing resistance are desperately needed. The {beta}-lactams are the oldest and most widely used class of antibiotics to treat bacterial infections but, for a variety of reasons, they were largely ignored until recently as a potential treatment option for TB. Recently, a growing body of evidence indicates that later-generation carbapenems in the presence of {beta}-lactamase inhibitors could play a role in TB treatment. However, most of these drugs can only be administered intravenously in the clinic. We performed a screening of {beta}-lactams against intracellular Mycobacterium tuberculosis (Mtb) and identified sanfetrinem cilexetil as a promising oral {beta}-lactam candidate. Preclinical in vitro and in vivo studies demonstrated that: (i) media composition impacts the activity of sanfetrinem against Mtb, being more potent in the presence of physiologically relevant cholesterol as the only carbon source, compared to the standard broth media; (ii) sanfetrinem shows broad spectrum activity against Mtb clinical isolates, including MDR/XDR strains; (iii) sanfetrinem is rapidly bactericidal in vitro against Mtb despite being poorly stable in the assay media; (iv) there are strong in vitro synergistic interactions with amoxicillin, ethambutol, rifampicin and rifapentine and, (v) sanfetrinem cilexetil is active in an in vivo model of infection. These data, together with robust pre-clinical and clinical studies of broad-spectrum carbapenem antibiotics carried out in the 1990s by GSK, identified sanfetrinem as having potential for treating TB and catalyzed a repurposing proof-of-concept Phase 2a clinical study (NCT05388448) currently underway in South Africa.

microbiology↗

bioRxiv2024

Emergence of resistance to the antiparasitic selamectin in Mycobacterium smegmatis is improbable and contingent on cell wall integrity

Tuberculosis remains the deadliest infectious disease of the 21st century. New antimicrobials are needed to improve treatment outcomes and enable therapy shortening. Drug repurposing is an alternative to the traditional drug discovery process. The avermectins are a family of macrocyclic lactones with anthelmintic activity active against Mycobacterium tuberculosis. However, their mode of action in mycobacteria remains unknown. In this study, we employed traditional mutant isolation approaches using Mycobacterium smegmatis, a non-pathogenic Mycobacterium tuberculosis surrogate. We were only able to isolate mutants with decreased susceptibility to selamectin using the {Delta}nucS mutator M. smegmatis strain. This phenotype was caused by mutations in mps1 and mmpL11. Two of these mutants were used for a second experiment in which high-level selamectin-resistant mutants were isolated; however, specific mutations driving the phenotypic change to high-level resistance could not be identified. The susceptibility to selamectin in these mutants was restored to the basal level by subinhibitory concentrations of ethambutol. The selection of ethambutol resistance in a high-level selamectin-resistant mutant also resulted in multiple colonies becoming susceptible to selamectin again. These colonies carried mutations in embB, suggesting that integrity of the cell envelope is a prerequisite for selamectin resistance. The absence of increased susceptibility to selamectin in an embB deletion strain demonstrated that the target of selamectin is not cytosolic. Our data show that concurrence of specific multiple mutations and complete integrity of the mycobacterial envelope are necessary for selamectin resistance. Our studies provide first-time insights into the antimycobacterial mode of action of the antiparasitic avermectins.

microbiology↗

bioRxiv2023

Amoxicillin/clavulanate in combination with rifampicin/clarithromycin is bactericidal against Mycobacterium ulcerans

BackgroundBuruli ulcer (BU) is a skin neglected tropical disease (NTD) caused by Mycobacterium ulcerans. WHO-recommended treatment requires 8-weeks of daily rifampicin (RIF) and clarithromycin (CLA) with wound care. Treatment compliance may be challenging due to socioeconomic determinants. Minimum Inhibitory Concentration and checkerboard assays showed that amoxicillin/clavulanate (AMX/CLV) combined with RIF+CLA were synergistic against M. ulcerans. However, in vitro time kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug. Colony forming units (CFU) enumeration is the in vitro reference method to measure bacterial load, although this is a cumbersome method due to the slow growth of M. ulcerans. The aim of this study was to assess the in vitro activity of RIF, CLA and AMX/CLV combinations against clinical M. ulcerans isolates by TKA, while comparing four methodologies: CFU/mL enumeration, luminescence by relative light unit (RLU) and optical density (at 600 nm) measurements, and 16S rRNA/IS2404 genes quantification. Methodology/Principal findingsTKA of RIF, CLA and AMX/CLV alone and in combination were performed against different M. ulcerans clinical isolates. Bacterial loads were quantified after 1, 3, 7, 10, 14, 21 and 28 days of treatment with different methodologies. RIF+AMX/CLV and the triple RIF+CLA+AMX/CLV combination were bactericidal and more effective in vitro than the currently used RIF+CLA combination to treat BU. All methodologies provided similar results with a general good correlation between CFU counting and the other methodologies. Measuring luminescence (RLU) was the most cost-effective methodology to quantify M. ulcerans bacterial loads in in vitro TKA. Conclusions/SignificanceOur study suggests that alternative and faster TKA methodologies can be used in BU research instead of the cumbersome CFU quantification method. These results provide an in vitro microbiological support to the design of the BLMs4BU clinical trial (NCT05169554, PACTR202209521256638) to shorten BU treatment. Author summarySince 2004, when only surgery was available, Buruli ulcer (BU) treatment has improved reaching to the efficient 8-weeks all oral antibiotic course of rifampicin and clarithromycin together with wound care and, sometimes, tissue grafting and surgery. This skin neglected tropical disease caused by Mycobacterium ulcerans mainly affects people living in rural areas in under-resourced countries with limited access to health services and medicines, thus compromising patients treatment adherence. The inclusion of amoxicillin/clavulanate in BU therapy was previously described with the potential to shorten BU treatment. In this study, we confirmed the high bactericidal activity over time of rifampicin and amoxicillin/clavulanate-containing combinations against M. ulcerans, being even more effective in vitro than the antibiotics currently used to treat BU. A comparison of different methodologies that are applied in the laboratory and in the clinical environment showed good correlation between them, leading to a wide variety of biomarkers for BU research and giving the opportunity for clinical translation; the choice of the most suitable one being driven by the purpose and the context of the study. These in vitro results provide further support to the ongoing clinical trial in West Africa to evaluate if BU treatment can be shortened from 8 to 4 weeks (BLMs4BU trial: NCT05169554, PACTR202209521256638).

microbiology↗

bioRxiv2022

Repurposing azithromycin in combination with last-line fosfomycin, colistin and tigecycline against Multi-Drug Resistant Klebsiella pneumoniae

BackgroundNew therapeutical strategies are urgently needed against multidrug-resistant (MDR) Enterobacterales. Azithromycin is a widely prescribed antibiotic with additional immunomodulatory properties, but traditionally underused for the treatment of enterobacterial infections. We previously identified azithromycin as a potent enhancer of colistin, fosfomycin and tigecycline against Klebsiella pneumoniae ATCC 13883. ObjectivesThe aim of this work was to evaluate the antibacterial in vitro activity of azithromycin-based combinations with last-line antibiotics against an expanded panel of MDR/XDR K. pneumoniae isolates. MethodsTime-kill assays of azithromycin alone and in pair-wise combinations with fosfomycin, colistin and tigecycline were performed against a collection of 12 MDR/XDR K. pneumoniae isolates. Synergistic and bactericidal activities of azithromycin-based combinations were analyzed after 8, 24 and 48 hours of treatment, and compared with antimicrobial combinations frequently used in the clinic for the treatment of MDR Enterobacterales. ResultsSynergistic interactions were detected in 100% (12/12) for azithromycin/fosfomycin, 58.3% (7/12) for azithromycin/colistin and 75% (9/12) for azithromycin/tigecycline of the strains, showing potent killing activities. Clinical combinations currently used in the clinic showed synergy in 41.6% (5/12) for meropenem/ertapenem, 33.33% (4/12) for meropenem/colistin, 75% (9/12) for fosfomycin/colistin and 66.6% (8/12) for fosfomycin/tigecycline of the strains, with lower bactericidal efficacy. ConclusionsNovel azithromycin-based combinations with last-line MDR/XDR K. pneumoniae antibiotics were identified showing in vitro capacity to eradicate MDR/XDR K. pneumoniae. Our results provide an in vitro basis supporting azithromycin used in combinatorial treatment for MDR-related infections.

microbiology↗

bioRxiv2022

Zidovudine multi-combos with last-line fosfomycin, ceftazidime-avibactam, colistin and tigecycline against Multi-Drug Resistant Klebsiella pneumoniae

Drug repurposing is a novel strategy for the development of new therapies against antibiotic-resistant bacteria. Zidovudine, an antiviral largely used in the HIV-therapy, exerts antibacterial activity against Gram-negative bacteria. Zidovudine was identified in a previous drug repurposing synergy screening as fosfomycin enhancer against Klebsiella pneumoniae ATCC 13883. Our aim was to evaluate the antibacterial in vitro activity of zidovudine-based combinations with last-line antibiotics against MDR/XDR K. pneumoniae isolates. We validated the zidovudine/fosfomycin combination against a collection of 12 MDR K. pneumoniae isolates by the checkerboard assay (CBA). In addition, we performed time-kill assays (TKA) to analyze synergistic and bactericidal activities of zidovudine paired combinations with fosfomycin, ceftazidime-avibactam, colistin and tigecycline. These were compared with frequent clinical combinations in the treatment of MDR Enterobacteriaceae. The potential of the triple zidovudine/fosfomycin/colistin was also assessed by TKA. CBA synergy confirmation rate between zidovudine/fosfomycin was 83.33%. TKA yielded synergy confirmation rates of 83.3% for zidovudine/ceftazidime-avibactam, 75% for zidovudine/fosfomycin, 75% for zidovudine/colistin and 66.6% for zidovudine/tigecycline with potent killing activities. Frequent clinical combinations displayed synergy rates of 41.6% for meropenem/ertapenem, 33.33% for meropenem/colistin, 75% for fosfomycin/colistin and 66.6% for fosfomycin/tigecycline with lower bactericidal efficacy than zidovudine-based combinations. The triple zidovudine/fosfomycin/colistin combination exhibited activities similar to fosfomycin/colistin and fosfomycin/zidovudine. As conclusion, zidovudine is an effective partner in in vitro combinations with existing antibiotics against MDR K. pneumoniae, especially with ceftazidime-avibactam, fosfomycin or colistin. Further studies are needed to elucidate the clinical potential of zidovudine as a repurposed drug in the antibacterial therapy.

microbiology↗

bioRxiv2022

Novel synergistic combinations of last-line antibiotics and FDA-approved drugs against Klebsiella pneumoniae revealed by in vitro synergy screenings

Treatment of infections caused by multi-drug resistant (MDR) enterobacteria remains challenging due to the limited therapeutic options. Drug repurposing could accelerate the development of urgently needed successful interventions. This work aimed to identify and characterize novel drug combinations against Klebsiella pneumoniae based on the concepts of synergy and drug repurposing. We performed a semi-qualitative high-throughput synergy screening (sHTSS) with tigecycline, colistin and fosfomycin (last-line antibiotics against MDR Enterobacteriaceae) combined with an FDA-library containing 1,430 clinically approved drugs. Selected hits were further validated by secondary checkerboard (CBA) and time-kill (TKA) assays. Our sHTSS results yielded 37, 31 and 41 hits showing synergy with tigecycline, colistin and fosfomycin, respectively. Most hits (75%) were known antibiotics. Non-antibiotic compounds included other anti-infective agents (7%), antineoplastics (7%) or antipsychotics (3%). Overall, 15.09% and 65.85% of hits were further confirmed by CBA and TKA, respectively, indicating that TKA is more useful than CBA for the validation of synergistic combinations. Accordingly, TKA were used for synergy classification based on determination of the bactericidal activities at 8, 24 and 48 hours. Twenty-seven combinations were validated with effective synergistic activity against K. pneumoniae by TKA, six of them novel non-antibiotic combinations. Based on our observations we conclude that repurposing approaches allowed to enhance the activity of last-line antibiotics in the treatment of MDR K. pneumoniae. sHTSS paired to TKA was a powerful tool for the identification of novel synergistic drug combinations against K. pneumoniae. Further pre-clinical studies might support the translational potential of these novel combinations.

microbiology↗

bioRxiv2021

The veterinary anti-parasitic selamectin is a novel inhibitor of the mycobacterial DprE1 enzyme

Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-{beta}-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

microbiology↗

bioRxiv2018

Triple oral beta-lactam containing therapy for Buruli ulcer treatment shortening

The potential use of clinically approved beta-lactams for Buruli ulcer (BU) treatment was investigated with representative classes analyzed in vitro for activity against Mycobacterium ulcerans. Beta-lactams tested were effective alone and displayed a strong synergistic profile in combination with antibiotics currently used to treat BU, i.e. rifampicin and clarithromycin; this activity was further potentiated in the presence of the beta-lactamase inhibitor clavulanate. In addition, quadruple combinations of rifampicin, clarithromycin, clavulanate and beta-lactams resulted in multiplicative reductions in their minimal inhibitory concentration (MIC) values. The MIC of amoxicillin against a panel of clinical isolates decreased more than 200-fold within this quadruple combination. Amoxicillin/clavulanate formulations are readily available with clinical pedigree, low toxicity, and orally and pediatric available; thus, supporting its potential inclusion as a new anti-BU drug in current combination therapies.

microbiology↗