<p>Mycobacterium abscessus (Mab) is a non-tuberculous mycobacterium that is highly resistant to many antibiotics. Mab causes pulmonary infections in immunocompromised individuals. The presence of efflux pumps to pump out antibiotics and its ability to form biofilms makes Mab a virulent pathogen. Studies have been done on Mab antibiotic tolerance but there are still a lot of gaps in knowledge about the effects of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis in this bacterium during biofilm formation. In this study, we investigated the effects of the EPIs chlorpromazine (CPZ), 1-(1-naphthylmethyl)-piperazine (NMP), thioridazine (TRZ), Phenylalanine-arginine β-naphthylamide (PBN) and plumbagin (PLU) on antibiotic resistance, efflux, biofilm formation and lipid biosynthesis associated with log-phase growth and biofilm formation in Mab. We used the resazurin assay to determine the minimum inhibitory concentration (MIC) of the EPIs. We investigated the effects of the EPIs during biofilm-forming growth conditions on the MICs of antibiotics such as clarithromycin, amikacin, cefoxitin, ciprofloxacin which are the frontline antibiotics used to treat non-tuberculous mycobacterial infections. We also assessed the effects of the EPIs on the accumulation and efflux activities of the Mab cells through ethidium bromide (EtBr) assay. Furthermore, we evaluated the effects of the EPIs at sub-MIC concentrations on Mab biofilm formation under normoxic and hypoxic conditions. We utilized metabolic radiolabeling methods using 14C-palmitic acid and 14C- acetic acid which are precursors of lipid biosynthesis and analyzed lipids by silica-thin layer chromatography and autoradiography. We observed that Mab cells developed higher tolerance to the EPIs in a biofilm-stimulating medium. Furthermore, a decrease in the MICs of antibiotics was observed in the presence of the EPIs. Also, in the presence of the EPIs, there was less efflux activity within the Mab cells. In addition, EPIs inhibited biofilm formation significantly. We also noticed that NMP and PBN inhibited 14C-palmitic acid and 14C- acetic acid incorporation into polar lipids such as glycopeptidolipids, trehalose monomycolate, phosphatidylethanolamine, phosphatidylglycerol/cardiolipin, phosphatidylinositol mannosides at specific tested conditions. Our findings suggest that the EPIs inhibited the activities of the efflux pumps associated with the efflux of the antibiotics and lipid biosynthesis involved in biofilm formation. In conclusion, the results from this study gives insights on possible therapeutic opportunities.</p>

No description available.

<p dir="ltr">Mycobacterium abscessus (Mab) is a type of mycobacterium that is known for its remarkable resistance to a variety of antibiotics. This pathogen poses a significant risk for individuals with weakened immune systems as it can cause skin and soft tissue infections, pulmonary disease and disseminated infections. Mab's ability to expel antibiotics through efflux pumps and form strong biofilms makes it even more challenging to treat infections. Lipids form a major part of the extracellular matrix of Mab biofilms. Efflux pumps have been shown to export lipids to the cell surface. Despite ongoing research into Mab's antibiotic tolerance, there is still much to learn about the impact of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis during biofilm development in Mab. In this study, we investigated the impact of the EPIs; CCCP (carbonyl cyanide m-chlorophenyl hydrazone), piperine (PIP), reserpine (RES), berberine (BER), and verapamil (VER) on efflux activity, biofilm formation, antibiotic resistance, and lipid biosynthesis in Mab during planktonic and biofilm growth conditions. We found that Mab cells had a higher tolerance to EPIs in biofilm-stimulating medium and that the presence of EPIs led to a decrease in minimum inhibitory concentrations of frontline antibiotics, reduced efflux activity within Mab cells, and significantly inhibited biofilm formation. We examined the effects of EPIs that inhibited biofilm formation on lipid metabolism in Mab using radiolabeling with 14C?palmitic acid and 14C-acetic acid which are precursors of lipid biosynthesis. We observed that the EPI berberine inhibited the incorporation of 14C-palmitic acid into glycopeptidolipids in the surface lipids of planktonic cells and increased cellular glycopeptidolipid (GPL) in biofilm cells. Verapamil-treated cells showed a 55 % increase in cellular trehalose monomycolate (TMM) compared to controls. Piperine-treated cells exhibited a 50 % increase in cardiolipin. The incorporation of 14C-acetate into biofilm cells showed that piperine-treated biofilm cells showed a 146 % increase in surface glycopeptidolipids. Overall, our study enhances our understanding of lipid biosynthesis in Mab, the effects of EPIs on Mab biofilms, efflux mechanisms, and antibiotic resistance and offers insights for combating Mab-related infections.</p>

Práticas inadequadas de descontaminação, desinfecção e esterilização de materiais médico-hospitalares têm propiciado o surgimento de inúmeros surtos de infecções por \'Micobactérias de Crescimento Rápido\' (MCR), em todo o Brasil. Entre os anos de 2000 a 2008 foram relatados mais de 2000 casos confirmados de infecções por MCR, sendo que os procedimentos por vídeo se constituíram como os maiores veiculadores destes microrganismos. O aumento do emprego de dispositivos de natureza polimérica em procedimentos médico-cirúrgicos e ausência/não cumprimento de protocolos de processamento destes materiais podem estar envolvidos na disseminação, principalmente, pela capacidade de MCR produzirem e sobreviverem em sistemas de biofilmes. Desta forma, este trabalho teve como objetivo a avaliação da susceptibilidade de cepas de Mycobacterium abscessus subsp. bolettii (suspensão e biofilmes), causadoras ou não de surto, frente a desinfetantes químicos constituídos de: Glutaraldeído 2%, Ácido Peracético 0,2%, Peróxido de Hidrogênio 35%, Solução Alcoólica de Digluconato de Clorexidina 0,5%, Solução Aquosa de Clorexidina 0,2%, Compostos de Amônio Quaternário 1,2%, Iodo 1% e Fenol 5% e Sistemas de Esterilização por Plasma (RIE e ICP) empregando mistura gasosa O2-H2O2. Paralelamente, suportes poliméricos (PVC, PEAD, PP, PUR e PC) empregados como carreadores de MCR foram analisados por Espectroscopia Fotoacústica no Infravermelho (PAS-FTIR), Microscopia Eletrônica de Varredura (MEV), microanálise em Sistema Energy Dispersive Spectroscopy (EDS) e Perfilometria. Resultados destas investigações demonstraram a resistência das cepas de M. abscessus subsp. bolettii, isolada do surto ocorrido em Belém, frente a Glutaraldeído 2%, Solução Alcoólica de Digluconato de Clorexidina 0,5%, Solução Aquosa de Clorexidina 0,2%, Compostos de Amônio Quaternário 1,2% e Iodo 1%. Entretanto, estas cepas foram altamente sensíveis à Ácido Peracético 0,2%, Peróxido de Hidrogênio 35% e Fenol 5% e Sistema de Esterilização por Plasma (RIE). Em sistemas de biofilmes, as cepas padrão e M. abscessus subsp. bolletii se apresentaram resistentes a todos os desinfetantes estudados. Nos estudos envolvendo a integridade de polímeros frente a processos por plasma foram demonstradas modificações em nível superficial (oxidação e aumento da rugosidade) em todos os materiais processados, sendo que o sistema ICP apresentou-se mais agressivo em relação àquele empregando RIE. / Numerous outbreaks of Rapid Growth of Mycobacteria (RGM) have been associated with decontamination, disinfection and sterilization malpractices, in Brazil. Between 2000-2008 were reported more than 2,000 confirmed cases due to RGM infections, and the video procedures were considered to carry these microorganisms. The increased use of medical devices in surgical procedures may be involved in the RGM spreading by its ability to grow and survive in biofilm systems. The aim of this study was evaluate the susceptibility of Mycobacterium abscessus subsp. bolletii (outbreak and nonoutbreak strains) to chemical disinfectants: Glutaraldehyde 2%, Peracetic Acid 0.2%, Hydrogen Peroxide 35%, Chlorhexidine Digluconate 0.5%, Chlorhexidine 0.2%, Iodine 1%, Quaternary Ammonium Compounds 1.2%, and Phenol 5%. Plasma Sterilization Technologies (Reactive Ion Etching and Inductively Coupled Plasma) were also evaluated. Polymers employed in medical devices (Polyvinyl chloride, High-Density Polyethylene, Polycarbonate, Polypropylene, and Polyurethane) were analyzed by Photoacoustic Infrared Spectroscopy (PAS-FTIR), Scanning Electron Microscopy (SEM), System Energy Dispersive Spectroscopy (EDS) and Profilometry. The results have demonstrated the resistance of Mycobacterium abscessus subsp. bolletii isolated from the Belém (PA) outbreak considering chemical exposition to Glutaraldehyde 2%, Chlorhexidine Digluconate 0.5%, Chlorhexidine 0.2%, Quaternary Ammonium Compounds 1.2%, and Iodine 2%. However, these strains were highly sensitive to Peracetic Acid 0.2%, Hydrogen Peroxide 35%, and Phenol 5%. The M. abscessus subsp. bolletii strains have been presented resistant to all disinfectants studied, in biofilm systems. Studies involving polymer integrity demonstrated changes in surface (oxidation and roughness) on all processed materials, and the ICP system was more aggressive in contrast to Reactive Ion Etching.

Práticas inadequadas de descontaminação, desinfecção e esterilização de materiais médico-hospitalares têm propiciado o surgimento de inúmeros surtos de infecções por \'Micobactérias de Crescimento Rápido\' (MCR), em todo o Brasil. Entre os anos de 2000 a 2008 foram relatados mais de 2000 casos confirmados de infecções por MCR, sendo que os procedimentos por vídeo se constituíram como os maiores veiculadores destes microrganismos. O aumento do emprego de dispositivos de natureza polimérica em procedimentos médico-cirúrgicos e ausência/não cumprimento de protocolos de processamento destes materiais podem estar envolvidos na disseminação, principalmente, pela capacidade de MCR produzirem e sobreviverem em sistemas de biofilmes. Desta forma, este trabalho teve como objetivo a avaliação da susceptibilidade de cepas de Mycobacterium abscessus subsp. bolettii (suspensão e biofilmes), causadoras ou não de surto, frente a desinfetantes químicos constituídos de: Glutaraldeído 2%, Ácido Peracético 0,2%, Peróxido de Hidrogênio 35%, Solução Alcoólica de Digluconato de Clorexidina 0,5%, Solução Aquosa de Clorexidina 0,2%, Compostos de Amônio Quaternário 1,2%, Iodo 1% e Fenol 5% e Sistemas de Esterilização por Plasma (RIE e ICP) empregando mistura gasosa O2-H2O2. Paralelamente, suportes poliméricos (PVC, PEAD, PP, PUR e PC) empregados como carreadores de MCR foram analisados por Espectroscopia Fotoacústica no Infravermelho (PAS-FTIR), Microscopia Eletrônica de Varredura (MEV), microanálise em Sistema Energy Dispersive Spectroscopy (EDS) e Perfilometria. Resultados destas investigações demonstraram a resistência das cepas de M. abscessus subsp. bolettii, isolada do surto ocorrido em Belém, frente a Glutaraldeído 2%, Solução Alcoólica de Digluconato de Clorexidina 0,5%, Solução Aquosa de Clorexidina 0,2%, Compostos de Amônio Quaternário 1,2% e Iodo 1%. Entretanto, estas cepas foram altamente sensíveis à Ácido Peracético 0,2%, Peróxido de Hidrogênio 35% e Fenol 5% e Sistema de Esterilização por Plasma (RIE). Em sistemas de biofilmes, as cepas padrão e M. abscessus subsp. bolletii se apresentaram resistentes a todos os desinfetantes estudados. Nos estudos envolvendo a integridade de polímeros frente a processos por plasma foram demonstradas modificações em nível superficial (oxidação e aumento da rugosidade) em todos os materiais processados, sendo que o sistema ICP apresentou-se mais agressivo em relação àquele empregando RIE. / Numerous outbreaks of Rapid Growth of Mycobacteria (RGM) have been associated with decontamination, disinfection and sterilization malpractices, in Brazil. Between 2000-2008 were reported more than 2,000 confirmed cases due to RGM infections, and the video procedures were considered to carry these microorganisms. The increased use of medical devices in surgical procedures may be involved in the RGM spreading by its ability to grow and survive in biofilm systems. The aim of this study was evaluate the susceptibility of Mycobacterium abscessus subsp. bolletii (outbreak and nonoutbreak strains) to chemical disinfectants: Glutaraldehyde 2%, Peracetic Acid 0.2%, Hydrogen Peroxide 35%, Chlorhexidine Digluconate 0.5%, Chlorhexidine 0.2%, Iodine 1%, Quaternary Ammonium Compounds 1.2%, and Phenol 5%. Plasma Sterilization Technologies (Reactive Ion Etching and Inductively Coupled Plasma) were also evaluated. Polymers employed in medical devices (Polyvinyl chloride, High-Density Polyethylene, Polycarbonate, Polypropylene, and Polyurethane) were analyzed by Photoacoustic Infrared Spectroscopy (PAS-FTIR), Scanning Electron Microscopy (SEM), System Energy Dispersive Spectroscopy (EDS) and Profilometry. The results have demonstrated the resistance of Mycobacterium abscessus subsp. bolletii isolated from the Belém (PA) outbreak considering chemical exposition to Glutaraldehyde 2%, Chlorhexidine Digluconate 0.5%, Chlorhexidine 0.2%, Quaternary Ammonium Compounds 1.2%, and Iodine 2%. However, these strains were highly sensitive to Peracetic Acid 0.2%, Hydrogen Peroxide 35%, and Phenol 5%. The M. abscessus subsp. bolletii strains have been presented resistant to all disinfectants studied, in biofilm systems. Studies involving polymer integrity demonstrated changes in surface (oxidation and roughness) on all processed materials, and the ICP system was more aggressive in contrast to Reactive Ion Etching.

Recent years have seen the emergence of Mycobacterium abscessus, a highly drug-resistant non-tuberculous mycobacterium, which causes life-threatening infections in people with chronic lung conditions like cystic fibrosis. This opportunistic pathogen is refractory to treatment with standard anti-tuberculosis drugs and most currently available antibiotics, often resulting in accelerated lung function decline. This project aims to use a structure-guided fragment-based drug discovery approach to develop effective drugs to treat M. abscessus infections. During the early stage of the project, three bacterial targets were identified, based on analysis of the structural proteome of M. abscessus and prior knowledge of M. tuberculosis drug targets, followed by gene knockout studies to determine target essentiality for bacterial survival. The three targets from M. abscessus were then cloned, expressed and purified and suitable crystallization conditions were identified leading to the determination of high resolution structures. Further, a large number of starting fragments that hit the three target proteins were determined, using a combination of biophysical screening methods and by defining crystal structures of the complexes. For target 3, PPAT (Phosphopantethiene adenylyl transferase), a chemical linking of two fragments followed by iterative fragment elaboration was carried out to obtain two compounds with low micromolar affinities in vitro. However, these compounds afforded only low inhibitory activity on M. abscessus whole cell. All starting fragments of target 2, PurC (SAICAR synthase), occupied the ATP indole pocket. Efforts were then made to identify further fragment hits by screening diverse libraries. Sub-structure searches of these initial fragment hits and virtual screening helped to identify potential analogues amenable to further medicinal chemistry intervention. While fragment hits of target 1, TrmD (tRNA-(N1G37) methyl transferase), were prioritized, whereby two chemical series were developed using fragment growing and merging approaches. Iterative fragment elaboration cycle, aided by crystallography, biophysical and biochemical assays led to the development of several potential lead candidates having low nano-molar range of in vitro affinities. Two such compounds afforded moderate inhibition of M. abscessus and stronger inhibition of M. tuberculosis and S. aureus cultures. Further chemical modifications of these compounds as well as others are now being done, to optimize cellular and in vivo activities, to be ultimately presented as early stage clinical candidates.