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Un grupo de científicos de China ha encontrado un nuevo gen en las bacterias gram negativas, el MCR-1. Este gen ha sido responsable de otorgarle resistencia a estas bacterias patógenas ante la colistina, el antibacteriano que los médicos emplean como última arma para combatir ciertas infecciones multi-resistentes. Con este gen, las superbacterias serán potencialmente epidémicas y las enfermedades que se creían controladas, como la neumonía, la tuberculosis y las infecciones del tracto urinario, podrían ser nuevamente letales. / A group of Chinese scientists have found a new gene in gram negative bacteria, the MCR-1. This gene has given pathogenic bacteria resistance to colistin, which is the antibacterial that medical doctors use as last line of defense against multi-drug resistant infections. Superbacteria with MCR-1 will be potentially epidemic, and diseases that were believed to be controlled such as pneumonia, tuberculosis and urinary tract infections could be lethal again.

The bacterium Rhodococcus is a potential source for novel antimicrobial metabolites. Recently, the Rhodococcus strain MTM3W5.2 was isolated from a soil sample collected from Morristown, East Tennessee and was found to produce an inhibitor molecule that is active against similar Rhodococcus species. The aim of this research is to extract, purify, and characterize the active compound. The compound was obtained from both agar and broth cultures of strain MTM3W5.2 and purified by primary fractionation of crude extract on a Sephadex LH-20 column, followed by semi-preparative reversed phase column chromatography. Final purification was achieved using multiple rounds of an analytical C18 HPLC column. Based on the results obtained from UV-Vis, FT-IR, and HR-MS, the molecule is a polyketide with a molecular formula of C52H78O13 and an exact mass of 911.5490 amu. The partial structure of this compound has been determined using 1D and 2D NMR spectroscopy.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-10-02T14:48:12Z No. of bitstreams: 1 marianasilvalopesneves.pdf: 523366 bytes, checksum: 2c33e978d12652cf1b17217fe3509198 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-10-09T19:44:11Z (GMT) No. of bitstreams: 1 marianasilvalopesneves.pdf: 523366 bytes, checksum: 2c33e978d12652cf1b17217fe3509198 (MD5) / Made available in DSpace on 2017-10-09T19:44:11Z (GMT). No. of bitstreams: 1 marianasilvalopesneves.pdf: 523366 bytes, checksum: 2c33e978d12652cf1b17217fe3509198 (MD5) Previous issue date: 2013-04-25 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A prata é conhecida por sua atividade antibacteriana e tem se mostrado eficiente como alternativa no contexto da resistência bacteriana a drogas, sobretudo na forma de nanopartículas (AgNPs). Nossos objetivos foram a avaliação da susceptibilidade de bactérias representativas contra AgNPs e pesquisa de efeito sinérgico ou antagônico quando associadas a quitosana (QIT) e antimicrobianos de uso terapêutico humano. Foram utilizadas oito linhagens bacterianas de referência, representativas de: Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, S. epidermides e Enterococcus faecalis. O perfil de susceptibilidade foi determinado através do método de microdiluição em caldo, de acordo com as recomendações do CLSI. A concentração inibitória mínima (CIM90) das AgNPs para as amostras testadas foi de 64μg/mL, enquanto que para a combinação AgNPs-QIT foi de 16μg/mL. Considerando-se atividade antimicrobiana somente da QIT, a CIM90 foi de 16 μg/mL. Entre os antimicrobianos testados (meropenem, amicacina, gentamicina, levofloxacina, rifampicina, sulfametoxazol-trimetropim, tetraciclina, e oxacilina e vancomicina, estes apenas para Gram positivos), apesar de a CIM para cada linhagem bacteriana estar de acordo com os valores determinados pelo CLSI observou-se diminuição significativa para todas as drogas testadas quando combinadas com AgNPs-QIT. De modo geral, para todas as espécies bacterianas avaliadas, a associação de AgNPs com QIT ou drogas antimicrobianas apresentaram grande potencial inibitório que pode estar relacionado a estabilidade das AgNPs associadas ao polímero ou a interação positiva das nanopartículas com os antimicrobianos. O ensaio de Checkerboard foi realizado para estabelecer os efeitos sinérgicos ou antagônicos em cada combinação. A possibilidade da interação das AgNPs e da QIT com antimicrobianos já existentes é altamente relevante e reforça as recomendações da literatura acerca da reformulação de uso de antimicrobianos tradicionais, além da pesquisa de novas drogas e estratégias para sobrepujar o crescente fenômeno da resistência bacteriana aos antimicrobianos. / Silver is known for its antibacterial activity and has been shown to be effective as a potential alternative in the context of bacterial resistance to drugs, particularly in the form of nanoparticles (AgNPs). Our objectives were to evaluate the susceptibility of bacteria against AgNPs and representative survey of synergistic or antagonistic when combined with chitosan (QIT) and antimicrobials for human therapeutic use. A total of eight reference strains were evaluated of Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, S. epidermidis and Enterococcus faecalis. The susceptibility patterns were determined by broth microdilution method, according to the CLSI guidelines. The minimum inhibitory concentration (MIC90) of AgNPS to the samples tested was 64μg/mL, while for the combination AgNPs-QIT was 16μg/mL. Considering only antimicrobial activity of QIT, the MIC90 was 16 mg / mL. Among the antimicrobials (meropenem, amikacin, gentamicin, levofloxacin, rifampicin, sulfamethoxazole- trimethoprim, tetracycline, and vancomycin and oxacillin, these only to Gram positive), while the MIC for each bacterial strain is in accordance to the reference CLSI, significant decrease was observed for all tested drugs when combined with AgNPs-QIT. In general, association of AgNPs with QIT or with antimicrobial drugs showed higher inhibitory potential, which may be related to the stability of the polymer associated AgNPs or positive interaction of nanoparticles with antibiotics. The Checkerboard assay was then performed to establish antagonistic or synergistic effects in each combination. The possibility of interaction of AgNPs and QIT with existing antimicrobials is highly relevant and reinforces the recommendations of the literature on the reformulation of traditional antimicrobial use, as well as research into new drugs and strategies to overcome the growing phenomenon of bacterial resistance to antimicrobials.

Quaternary ammonium compounds (QACs) have long served as lead disinfectants in residential, industrial, and hospital settings. Their simple yet effective amphiphilic nature makes them an ideal class of compounds through which to explore antibacterial activity. We have developed novel multiQAC scaffolds through simple and cost-efficient syntheses, yielding hundreds of diverse compounds strategically designed to examine various aspects of antibacterial and anti-biofilm activity, as well as toxicity. Many of these bis-, tris-, and tetraQACs display antibacterial activity 10 to 100 times greater than conventional monoQACs, and are among the most potent biofilm eradicators to date. Through analyzing their activity against several strains, we have uncovered and provided further evidence for key tenets of amphiphilic QAC bioactivity: a balance of hydrophobic side chains with cationic head groups generates optimal antibacterial activity, though toxicity to eukaryotic cells needs to be mitigated. Given their ubiquitous nature and chemical robustness, the overuse of QACs has led to the development of QAC resistance genes that are spreading throughout the microbial world at an alarming rate. These resistant strains, when found in bacterial biofilms, are able to persist in the presence of lead commercial QAC disinfectants, warranting the development of next-generation biocides. Several of our scaffolds were designed with QAC resistance machinery in mind; thus, we utilized these compounds not only as antibacterial agents but also as chemical probes to better understand and characterize QAC-resistance in methicillin-resistant Staphylococcus aureus (MRSA). Our findings support previous postulations that triscationic QACs would retain potency against QAC-resistant strains. Furthermore, we have identified monocationic and aromatic moieties, as well as conformational rigidity, as being more prone to recognition by the resistance machinery. Using our chemical toolbox comprised of QACs of various charge state and scaffold, we explored both the mechanism and scope of QAC-resistance by examining their structure-resistance relationship. Our holistic findings have allowed us to better understand the dynamics of this system towards the design and development of next-generation QACs that will: (1) allow us to better probe the resistance machinery, and (2) remain efficacious against a variety of microbial pathogens. / Chemistry

The Pseudomonas aeruginosa VIM-4 metallo-β-lactamase is a member of the subclass B1. It belongs to the VIM-type β-lactamases encoded by genetic mobile element such as class 1 integrons. This particularity enhances the relevance of VIM type enzymes since these enzymes are now disseminated among important nosocomial strains such as Klebsiella pneumoniae and Pseudomonas aeruginosa. In consequence, the study of VIM-4 was performed to determine its kinetic parameters, its 3D structure and the dependence of its activity on Zn2+ concentration. VIM-4 has been described as an efficient enzyme able to hydrolyze all the β-lactam compounds. We observed that VIM-4 activity is dependent on the Zn2+ concentration in the buffer, with a maximal activity obtained at 50 µM. Two different forms of VIM-4 were observed by X-ray crystallography in the presence or absence of Zn2+ in the crystallogenesis buffer. The first form contains two Zn2+ and the second possesses only one Zn2+ in the histidine site. The apoenzyme form was obtained. Its study revealed that this form was poorly stable with a less structured shape. The active form is not restored even in presence of a large Zn2+ excess. In order to determine the Zn2+ stoechiometry in VIM-4, ICPMS (Inductively coupled plasma mass spectrometry) experiments were performed. The results of this study indicated that the mono-zinc form of VIM-4 is favored in absence of Zn2+ ions in the buffer and the di-zinc form appears in the presence of added Zn2+ ions. The dissociation constants of the Zn2+ ions with VIM-4 enzyme were determined using benzylpenicillin. The dissociation constants of the first, the second and the third Zn2+ are respectively KD1 which is clearly below 1 µM, KD2 equal to 8.5 µM and KD3 within a range of 200 to 500 µM. A complementary titration of the free Zn2+ ions from denatured VIM-4 with a chromophore chelating agent seems to confirm this feature as only one Zn2+ could be titrated. Then with no added Zn2+, VIM-4 would be in a mono-zinc form and with a Zn2+ concentration higher than 10 µM, the di-zinc form of VIM-4 would be present. In the last decade, numerous studies have reported an increasing mortality among patients in intensive care due to multiresistant pathogens. These strains are producing at least two different classes of β-lactamases. In the eighties, a strategy of combination of β-lactamase inactivator and β-lactam antibiotics was developed against serine β-lactamases. Some of the members of this family have already developed variants resistant to these inhibitors; however this combination remains efficient. In the case of the MBLs, extensive studies have been performed to find a generic inhibitor. Unfortunately, due to the heterogeneity of this enzyme group, no solution has been found. The development of a metallo-β-lactamase inhibitor, particularly active against acquired MBLs, which are the most relevant enzymes, is thus needed. Our second purpose was the identification of new molecules that could be developed as broad spectrum MBLs inhibitors. We found a new class of compounds, mercaptophosphonates derivatives, which can be used as leads for finding generic MBL inhibitors.