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.

Mycobacterium abscessus est une mycobactérie à croissance rapide qui a récemment émergé en tant que pathogène opportuniste, en particulier chez les patients atteints de mucoviscidose. Elle est naturellement résistante aux antibiotiques les plus couramment disponibles, limitant sérieusement les options thérapeutiques chez les patients infectés. Il apparaît donc urgent d’identifier et de caractériser de nouvelles cibles d’intérêt thérapeutique dans la lutte contre ce pathogène. Contrairement au variant lisse (S) de M. abscessus, caractérisé par une forte production de glycopeptidolipides (GPL), le variant rugueux (R) associé à une faible production de GPL est responsable des manifestations cliniques les plus sévères. Chez l’embryon de poisson zèbre, le variant R s’accompagne d’une virulence accrue avec formation de cordes mycobactériennes extracellulaires et d’abcès, engendrant une mortalité rapide des larves infectées. Les mécanismes moléculaires de la pathogénicité de M. abscessus demeurent toutefois très peu connus.Dans cette étude, nous avons identifié le gène MAB_4780, codant une déshydratase distincte du complexe HadABC impliqué dans la biosynthèse des acides mycoliques. Le gène MAB_4780, tout comme son homologue chez M. smegmatis, MSMEG_6754, ont été identifiés comme responsables de la résistance innée de ces deux espèces au thiacetazone (TAC), un agent antituberculeux de seconde intention. Nous avons inactivé le gène MAB_4780 dans le variant R de M. abscessus, ce qui a entraîné une modification de la composition en acides mycoliques, un défaut de formation des cordes mycobactériennes ainsi qu'un phénotype extrêmement atténué chez les embryons de poisson zèbre. L'atténuation in vivo du mutant MAB_4780 qui résulte très vraisemblablement de l’incapacité i) à former des cordes et ii) à inhiber la fusion phagolysosomale, s’accompagne d’une croissance intracellulaire diminuée. En outre, le mutant MAB_4780, tout comme le mutant de M. smegmatis dépourvu de MSMEG_6754, présente une croissance altérée dans l’amibe, qui représente un réservoir pour les mycobactéries environnementales. Ces résultats reflètent le rôle crucial de cette nouvelle déshydratase dans la survie de M. abscessus dans son environnement et dans l'établissement d'infections aiguës et létales. Par conséquent, cibler MAB_4780 pourrait représenter une stratégie particulièrement prometteuse pour contrôler les infections à M. abscessus. De futures études seront consacrées à l'identification d’inhibiteurs spécifiques de MAB_4780, grâce au développement d’un test d’activité et d’une structure cristalline de la protéine obtenue à très haute résolution.Nous avons également criblé contre M. abscessus une chimiothèque d’analogues structuraux du TAC, préalablement validés pour leur activité inhibitrice de la déshydratase HadABC de Mycobacterium tuberculosis. Trois d’entre eux ont montré une efficacité accrue d’un facteur 50 par rapport à la molécule parentale. La surexpression d’EthA, l’activateur du TAC, augmente la susceptibilité de M. abscessus à ces trois analogues. Ces résultats suggèrent que leur mode d'activation est similaire à celle de la TAC et indiquent que l'optimisation d’analogues du TAC pourrait conduire à une nouvelle génération de composés plus efficaces pour le traitement de M. abscessus. Des études de relations structure/activité sont envisagées afin d’améliorer l’efficacité et les propriétés pharmacologiques des analogues du TAC. / Mycobacterium abscessus, a rapidly-growing mycobacterium (RGM), has emerged in recent years as an important opportunistic pathogen especially in cystic fibrosis (CF) patients. M. abscessus is naturally resistant to most commonly available antibiotics, which seriously limits the treatment options, emphasizing the urgent need for more efficient drugs and innovative therapeutic strategies to combat M. abscessus infections. The M. abscessus rough (R) low-glycopeptidolipids (GPL) producer is responsible for more severe clinical infections than the smooth (S) high-GPL producer, and is associated with increased virulence in zebrafish, including the formation of massive serpentine cords, abscesses, and rapid larval death. However, the molecular mechanisms responsible for the pathogenicity of the R strains remain elusive.Herein, we identified a novel gene, MAB_4780, encoding a dehydratase distinct from the HadABC complex, known to participate in mycolic acid biosynthesis. Both MAB_4780 and its homologue in Mycobacterium smegmatis, MSMEG_6754, are responsible for the innate resistance in these species to thiacetazone (TAC), a second-line antitubercular drug. The successful deletion of MABS_4780 in the R variant of M. abscessus resulted in an altered mycolic acid composition, a pronounced defect in cording, and an extremely attenuated phenotype in zebrafish embryos. The in vivo attenuation of the MAB_4780 mutant results from both the deficiency in cord formation and the impaired intracellular growth, presumably due to limited inhibition of the phagolysosomal fusion events. In addition, similarly to the MSMEG_6754 deletion mutant, the MAB_4780 mutant showed impaired growth in amoeba, which represents a possible reservoir for environmental mycobacteria. These results reflect the critical role of this new dehydratase in the survival of M. abscessus in its environmental hosts and its importance in establishing acute and lethal infections. Therefore, targeting MAB_4780 may represent a promising strategy to control M. abscessus infections. Future work will focus on identifying specific inhibitors targeting MAB_4780, which could greatly benefit from the combination of our dehydratase assay and our high-resolution crystal structure of the protein.We have also screened against M. abscessus a library of TAC analogues, previously validated for their inhibitory effects against the Mycobacterium tuberculosis HadABC dehydratase. Among these compounds, three exhibited a 50-fold increased potency as compared to TAC. Overexpression of EthA, known as the activator of TAC, increased the susceptibility of M. abscesuss to the three analogues, suggesting that that their mode of activation is similar to that of TAC. Overall, these data indicate that optimizing the TAC scaffold may lead to more efficient compounds against M. abscessus. Additional structure/activity relationship studies are required to further improve the efficacy and pharmacological properties of TAC analogues.

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.