Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) in a pH-dependent manner. At high pH (>8) Spm is a potent nucleophile, and able to form Spm-β-lactam adduct. At physiological pH (or lower), Spm carries positive charges, and can bind to DNA through charge interactions. The potential of Spm interfering with cell wall was first investigated. A spontaneous mutant of MRSA Mu50 selected for Spm resistance conferred resistance to Spm/β-lactam synergy. This phenotype was due to the presence of a 7-bp deletion in pbpB as identified by genome resequencing and confirmed by complementation. Analysis of cell wall composition by HPLC revealed the combination of Spm and β-lactam can reduce the cross-linkage of peptidoglycan. These two lines of evidence suggest Spm may perturb cell wall integrity in favor of β-lactam efficacy with PBPs as a promising target. However, from the results of microarray analysis and fluorescent Bocillin labeling, Spm did not appear to suppress the PBPs expression or alter their interactions with β-lactams. Next, transcriptome analyses reveal the genes responsive to the synergy effect overlap extensively with those to high Spm challenge, implying the enhanced detrimental effect of Spm facilitated by β-lactams in inhibition on cell growth. In particular the induction of iron transport and reduction of energy production under synergy were depicted in this study, and high dose Spm was found to turn off the SigB regulon. Of interest, the tetM gene encoding a ribosomal protection protein for tetracycline (Tc) resistance exhibited the most significant fold change and high signals by both high and low dose Spm. Further analysis by qRT-PCR demonstrated the tetM expression was specifically induced by Tc and Spm to a comparable level but not by other polyamines, suggesting a similar mode of action by Spm and Tc in interactions with the ribosome to initiate tetM induction. Collectively, these data indicated the role of Spm could be multifarious with more than one target, and a combination of Spm and β-lactams may inhibit growth of MRSA in a more complicated manner than just potentiating β-lactam inhibition on PBPs.

While antibiotics have saved the lives of millions of people since the discovery of the first β-lactam, penicillin, their continued effectiveness is being increasingly threatened by resistant bacteria. Bacterial resistance to β-lactams is mainly achieved through the production of serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs). Although both types of β-lactamases are commonly isolated in clinical settings, MBLs represent the greatest threat to public health since they are resistant to SBL inhibitors and most β-lactams. However, aspergillomarasmine A (AMA), a fungal natural product synthesized by Aspergillus versicolor, was shown to be a rapid and potent inhibitor against two clinically relevant MBLs: NDM-1 and VIM-2. In bacteria possessing these enzymes, AMA could rescue the activity of meropenem, a broad-spectrum β-lactam that is usually reserved for the treatment of the most severe bacterial infections. However, many questions remain revolving around AMA's inhibitory potency and spectrum. Therefore, the activity of AMA in combination with six β-lactams from three subclasses (carbapenem, penam, cephem) was explored against 19 MBLs from three subclasses (B1, B2, B3). After determining that AMA activity was linked to MBL zinc affinity and that AMA was more potent when paired with a carbapenem, the efficacy of an AMA/meropenem combination was evaluated with and without avibactam, a potent SBL inhibitor. This study used ten Escherichia coli and ten Klebsiella pneumoniae laboratory strains as well as 30 clinical strains producing at least one MBL and one SBL. Once establishing that the AMA/avibactam/meropenem combination was effective against carbapenemase-producing Enterobacterales, new Acinetobacter and Pseudomonas shuttle vectors were created. With these shuttle vectors, it was determined that the AMA/avibactam/meropenem combination was effective against some of the bacteria topping the World Health Organization’s priority pathogen list. / Thesis / Doctor of Philosophy (PhD) / Bacteria are all around us. While some bacteria can promote human health, others can cause serious infections. These infections are typically treated with antibiotics. β-Lactam antibiotics, such as penicillins and cephalosporins, are especially important to medicine. Unfortunately, an increasing number of bacteria employ enzymes, known as β-lactamases, which negate the effects of β-lactam antibiotics. Previous studies demonstrated that a natural product, known as aspergillomarasmine A (AMA), could inhibit some β-lactamase enzymes. Consequently, the inhibitory power of AMA was further explored against a larger number of β-lactamase enzymes and in combination with different β-lactam antibiotics. After discovering that AMA had more inhibitory power when combined with a β-lactam antibiotic known as meropenem, the efficacy of the AMA/meropenem pairing was evaluated against resistant bacteria in the presence and absence of avibactam, another β-lactamase inhibitor. The AMA/avibactam/meropenem combination was shown to be effective against some of the world’s most antibiotic-resistant bacteria.

Yes / There is a need to develop asymmetric routes to functionalised β-lactams, which remain the most important group of antibacterials. Here we describe biocatalytic and protein engineering studies concerning carbapenem biosynthesis enzymes, aiming to enable stereoselective production of functionalised carbapenams with three contiguous chiral centres. Structurallyguided substitutions of wildtype carboxymethylproline synthases enable tuning of their C-N and C-C bond forming capacity to produce 5-carboxymethylproline derivatives substituted at C-4 and C-6, from amino acid aldehyde and malonyl-CoA derivatives. Use of tandem enzyme incubations comprising an engineered carboxymethylproline synthase and an alkylmalonylCoA forming enzyme (i.e. malonyl-CoA synthetase or crotonyl-CoA carboxylase reductase) can improve stereocontrol and expand the product range. Some of the prepared 4,6-disubstituted-5-carboxymethylproline derivatives are converted to bicyclic β-lactams by carbapenam synthetase catalysis. The results illustrate the utility of tandem enzyme systems involving engineered crotonases for asymmetric bicyclic β-lactam synthesis.

Yes / Stereoselective biocatalysis by crotonase superfamily enzymes is exemplified by use of engineered 5-carboxymethylproline synthases (CMPSs) for preparation of functionalized 5-carboxymethylproline (5-CMP) derivatives methylated at two positions (i.e. C2/C6, C3/C6 and C5/C6), including products with a quaternary centre, from appropriately-substituted-amino acid aldehydes and C-2 epimeric methylmalonyl-CoA. The enzymatically-produced disubstituted 5-CMPs were converted by carbapenam synthetase into methylated bicyclic Β-lactams, which manifest improved hydrolytic stability compared to the unsubstituted carbapenams. The results highlight the use of modi-fied carbapenem biosynthesis enzymes for production of new carbapenams with improved properties. / Medical Research Council, Biotechnology and Biological Sciences Research Council (BB/L000121/1)

N-sec -butylthioloated β-lactam (NsβL) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxoacyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that NsβL inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of NsβL on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant NsβL-resistant mutants. Phenotypic analysis of the mutants was performed, as well as sequence analysis of fabH; along with comparative proteomic analysis of intracellular proteomes. Our results indicate that NsβL resistance is mediated by drastic changes in the cell wall, oxidative stress response, virulence regulation, and those pathways associated with CoA. It is our conclusion that Nsβ L has activity towards CoA, resulting in wide-spread effects on metabolism, virulence factor production, stress response, and antimicrobial resistance.

Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-08-08T13:21:02Z No. of bitstreams: 2 Dissertação - Andressa Liberal Santos - 2018.pdf: 2603632 bytes, checksum: 0a1312a46fb6a806189f5479682576a2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Olhe a citação : Dissertação (Mestrado em Biologia da Relação Parasito-Hospedeiro (IPTSP)) - Universidade Federal de Goiás, Goiânia, 2018. on 2018-08-08T13:28:59Z (GMT) / Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-08-09T11:46:17Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Andressa Liberal Santos - 2018.pdf: 2603632 bytes, checksum: 0a1312a46fb6a806189f5479682576a2 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-08-09T12:21:53Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Andressa Liberal Santos - 2018.pdf: 2603632 bytes, checksum: 0a1312a46fb6a806189f5479682576a2 (MD5) / Made available in DSpace on 2018-08-09T12:21:53Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação - Andressa Liberal Santos - 2018.pdf: 2603632 bytes, checksum: 0a1312a46fb6a806189f5479682576a2 (MD5) Previous issue date: 2018-06-28 / Enterobacteria are microorganisms involved with bacteria and the health of women with care. Treatment of bacterial infections is most often done with the use of antibiotics and one of the major classes of antimicrobials is one of the β-lactams. Among the main mechanisms of resistance to the observational β-lactam antibiotics: alteration of the antimicrobial target; alteration of β-lactam permeability; Flow pumps and the entry of enzymatic signals that destroy the β-lactate totally or with the development of an alternative metabolic pathway. These enzymes are known as beta-lactamases and are encoded by specific genes. Thus, the objective of this study was to correlate the resistance profile of enterobacteria, using phenotype methodologies and identifying 14 genes that encode as beta-lactamase enzymes: blaOXA genes; blaIMP; blaNDM; blaSME; blaDHA; blaCMY, blaTEM, blaKPC, blaSPM, blaCTX-M, blaVIM, blaSIM, blaGim and blaSHV. The phenotypic methodologies used were the Antimicrobial Sensitivity Test for Disk-Diffusion (antibiogram), and complementary tests for the detection of resistance mechanisms of beta-lactamases (ESBL, MBL, AmpC and Carbapenemase). The molecular methodology used was Real Time PCR using the Sybr Green system. Among the results found in the tests it was observed that 74.28% were resistant to ampicillin, 34.28% were resistant to aztreonam, 62.85% were resistant to amoxicillin associated with clavalunate, 51.42% were resistant to ceftazidime, 41 , 42% were resistant to cefoxitin, 54.28% were resistant to cefazolin, 44.28% were resistant to cefepime, 41.42% were resistant to cefuroxime, 8.57% were resistant to cefuroxime, 35.71% were resistant an imipenem and 41.42% were resistant to piperacillin associated with tazobactam. Among the total samples, the mechanism of resistance that presented the highest expression was ESBL (17.14%). The genes studied that were detected in a greater number of genera were blaGIM and blaSIM (66.66% of the samples). The gene that was amplified in a smaller number of samples was blaVIM (16.66%). It is concluded that although there is a low correlation between the methodologies analyzed, the levels of antimicrobial resistance in enterobacteria are high and worrying, and a way to minimize the accelerated emergence of resistance includes the development or improvement of techniques that generate diagnoses with high efficiency and speed. / As enterobactérias são microrganismos envolvidos com infecções bacterianas adquiridas na comunidade e nos ambientes dos cuidados com a saúde. O tratamento das infecções bacterianas na maioria das vezes é realizado com a utilização de antibióticos e uma das maiores classes de antimicrobianos é a dos β-lactâmicos. Entre os principais mecanismos de resistência aos antimicrobianos β-lactâmicos observa-se: alteração do alvo antimicrobiano; alteração da permeabilidade ao β-lactâmico; bombas de e-fluxo e a presença de mecanismos enzimáticos que destroem o β-lactâmico totalmente ou parcialmente com desenvolvimento de uma via metabólica alternativa. Essas enzimas são conhecidas como beta-lactamases e são codificadas por genes específicos. Dessa forma, o objetivo deste estudo foi o de correlacionar o perfil de resistência das enterobactérias, utilizando metodologias fenotípicas e identificar 14 genes que codificam as enzimas beta-lactamases: genes blaOXA; blaIMP; blaNDM; blaSME; blaDHA; blaCMY, blaTEM, blaKPC, blaSPM, blaCTX-M, blaVIM, blaSIM, blaGIM e blaSHV. As metodologias fenotípicas utilizadas foram o Teste de Sensibilidade aos Antimicrobianos por disco-difusão (antibiograma), e testes complementares para detecção dos mecanismos de resistência das beta-lactamases (ESBL, MBL, AmpC e Carbapenemase). A metodologia molecular utilizada foi a PCR em Tempo Real, utilizando o sistema Sybr Green. Entre os resultados fenotípicos encontrados nas bactérias observou-se que 74,28% foram resistentes a ampicilina, 34,28% foram resistentes a aztreonam, 62,85% foram resistentes a amoxicilina associado ao clavalunato, 51,42% foram resistentes a ceftazidima, 41,42% foram resistentes cefoxitina, 54,28% foram resistentes a cefazolina, 44,28% foram resistentes a cefepime, 41,42% foram rsistentes a ceftriaxona, 8,57% foram resistentes a cefuroxima, 35,71% foram resistentes a imipenem e 41,42% foram resistentes a piperacilina associada tazobactam. Entre o total de amostras, o mecanismo de resistência que apresentou maior expressão foi o ESBL (17,14%). Os genes estudados que foram detectados em um maior número de gêneros foram o blaGIM e o blaSIM (66,66% das amostras). O gene amplificado em menor número de amostras foi o blaVIM (16,66%). Conclui-se que apesar de haver baixa correlação entre as metodologias analisadas, os níveis de resistência a antimicrobianos em enterobactérias são altos e preocupantes e uma maneira de minimizar a acelerada emergência de resistência é desnvolver e aprimorar técnicas de diagnósticos com alta eficiência e rapidez.

Methicillin-resistant Staphylococcus Aureus (MRSA) is now the most challenging bacterial pathogen affecting patients in hospitals and in care centers, and has brought on the need to develop new drugs for MRSA. This thesis centers on studies of N-thiolated β-lactams, a new family of potent antibacterial compounds that selectively inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA). Chapter 1 describes MRSA in more detail. Chapter 2 outlines experiments on the effect of a fatty ester group (CO2R) on the C4-phenyl ring of N-methylthio β-lactams, expecting that attachment of long chain ester moieties might increase the hydrophobicity, and thus enhance the drugs ability to penetrate through the cell membrane. However, the results indicate that antibacterial activity drops off rapidly when more than seven carbon atoms are in the chain. These results led to the idea about examining a β-lactam conjugated polymer as a possible pro-drug delivery method, which is the focus of Chapter 3. To synthesize the initial drug-polymer candidate, microemulsion polymerization of an acrylate-substituted lactam was done in aqueous solution to form hydrophilic polymeric nanoparticles containing the highly water-insoluble solid antibiotic, N-methylthio fO-lactam. This method has advantages over the conventional emulsion polymerization methods because a solid co-monomer (β-lactam drug) can be utilized. SEM studies show that these polymeric nanoparticles have a microspherical morphology with nano-sizes of 40-150 nm. The N-thiolated fO-lactam containing nanoparticles display potent anti-MRSA activity at much lower drug amounts compared with free lactam drug, penicillin G or vancomycin. Although at this time the relationship between particle size and activity is not clear and the mode of action is unknown, the Nthiolated β-lactam containing nanoparticles dramatically enhance bioactivity, possibly due to increased bioavailability of the antibiotic via endocytosis. In chapter 4, Fluorescence-active emulsified nanoparticles containing naphthyl or anthracenyl side chains were also successfully prepared by microemulsion polymerization for possible use in fluorescence studies to determine if the drug enters the cell of MRSA through endocytosis, and where possible bioaccumulation site are located.

Mycobacterium abscessus est une mycobactérie responsable principalement d’infections pulmonaires, en particulier chez les patients atteints de mucoviscidose ou de dilatation des bronches. M. abscessus est naturellement résistante aux antituberculeux, laissant peu d’options thérapeutiques. Le traitement de référence associait classiquement un aminoside, un macrolide (clarithromycine) et une β-lactamine (céfoxitine ou imipénème), avec un taux de succès d’environ 50 %. Cependant, des souches résistantes à la clarithromycine sont fréquemment isolées, remettant en cause l’utilisation de cet antibiotique. M. abscessus produit naturellement une β-lactamase à large spectre (BlaMab) mais les mécanismes d’action des β-lactamines n’ont pas été étudiés chez cette espèce, ce qui constitue une entrave à l’optimisation des traitements par cette classe d’antibiotiques. Le premier objectif était d’identifier et de caractériser les cibles des β-lactamines chez cette espèce. Inhibant la dernière étape de polymérisation du peptidoglycane, les cibles potentielles des β-lactamines sont trois familles d’enzymes : les D,D-transpeptidases et les D,D­carboxypeptidases appartenant à la famille des protéines de liaison à la pénicilline (PLP), ainsi que les L,D-transpeptidases qui sont majoritairement responsables de cette dernière étape chez cette espèce. Pour identifier les cibles, des mutants résistants aux β-lactamines ont été sélectionnés à partir de la souche de référence M. abscessus CIP104536 et d’un dérivé portant une délétion du gène blaMab (∆blaMab). Pour les deux souches, l’émergence de la résistance aux β-lactamines a requis de multiples étapes, ce qui constitue un atout pour leur utilisation thérapeutique. Pour les mutants obtenus à partir de la souche CIP104536, les analyses phénotypiques ont montré que la résistance aux β-lactamines n'est pas due à une augmentation de l’efficacité catalytique de BlaMab, à une surproduction de cette enzyme, ou à une diminution de la perméabilité. Le séquençage des génomes de mutants résistants n’a pas révélé de mutations dans les gènes codant pour les L,D-transpeptidases, mais des mutations ont été trouvées dans des gènes codant pour deux PLP. D’autres mutations se situent dans des gènes codant en particulier pour des protéines non caractérisées. L’acquisition de la résistance pourrait donc dépendre de mutations affectant des facteurs essentiels à l’activité des cibles des β­lactamines. Le deuxième objectif était d’étudier et de comparer l’activité in vitro des β-lactamines sur M. abscessus. Des expériences de bactéricidie et d’activité intracellulaire chez le macrophage infecté ont été effectuées pour les souches CIP104536 et ∆blaMab. Parmi les antibiotiques étudiés (amikacine, céfoxitine, imipénème, ceftaroline, et amoxicilline), l’imipénème est le plus efficace sur les deux souches. Sur la souche ∆blaMab, l’association d’imipénème et d’amikacine est bactéricide. En l’absence de BlaMab, l’amoxicilline est aussi efficace que l’imipénème. L’avibactam augmente l’activité de la ceftaroline mais l’inhibition de BlaMab est seulement partielle en intracellulaire. Les résultats obtenus in vitro montrent que l’imipénème est supérieur à la céfoxitine pour des concentrations atteignables dans le sérum. L’inhibition de BlaMab pourrait augmenter l’efficacité de l’imipénème et d’autres composés utilisés pour traiter les infections pulmonaires à M. abscessus. / Mycobacterium abscessus is an important pathogen responsible for pulmonary infections in cystic fibrosis patients or in patients suffering from bronchiectasis. The treatment of infections due to M. abscessus is complicated since this bacterium is naturally resistant to the anti­tuberculous agents. The recommended treatment includes an aminoglycoside, a macrolide (clarithromycin) and a β-lactam (cefoxitin or imipenem), with a success rate of about 50 %. However, strains resistant to clarithromycin are frequently isolated, questioning the use of this antibiotic. M. abscessus naturally produces a broad spectrum β-lactamase (BlaMab) but the mechanisms of action of the β-lactams have not been studied in this species, impairing the optimization of the treatment by these antibiotics. The first objective was to identify and characterize the targets of β-lactams antibiotics in this species. Inhibiting the final stage of the peptidoglycan polymerization, the potential targets of β-lactams are three families of enzymes: the D,D-transpeptidases and D,D­carboxypeptidases belonging to the family of penicillin-binding proteins (PBP), and the L,D-transpeptidases which are mainly responsible for this final stage in this species. To identify the targets, mutants resistant to β-lactams have been selected from the reference strain M. abscessus CIP104536 and from its β-lactamase-deficient derivative ΔblaMab. For both strains, the emergence of resistance to β­lactams has required multiple steps, which is an advantage for the therapeutic use of these antibiotics. For the mutants derived from the strain CIP104536, phenotypic analyzes showed that the resistance to β-lactams is not due to an increase in the catalytic efficiency of BlaMab, to an overproduction of this enzyme, or to a decrease in permeability. Genomes sequencing of the resistant mutants did not reveal mutations in the genes encoding the L,D-transpeptidases, but mutations have been found in genes encoding two PBPs. Other mutations have been detected in genes encoding uncharacterized proteins. Acquisition of resistance could therefore depend on mutations affecting key factors essential for the activity of β-lactams targets. The second objective was to study and compare the in vitro activities of β-lactams against M. abscessus. Bactericidal experiments and intracellular activity in the infected macrophage were performed for the strains CIP104536 and ΔblaMab. Among the antibiotics tested (amikacin, cefoxitin, imipenem, ceftaroline, and amoxicillin), imipenem is the most effective agent against the two strains. Combination of imipenem and amikacin was bactericidal against the ΔblaMab mutant. In the absence of BlaMab, amoxicillin was as active as imipenem. Avibactam increased the intracellular activity of ceftaroline but inhibition of BlaMab was only partial intracellularly. Evaluation of the killing and intracellular activities of β-lactams indicates that imipenem is superior to cefoxitin at clinically achievable drug concentrations. Inhibition of BlaMab could improve the efficacy of imipenem and extend the spectrum of drug potentially useful to treat pulmonary infections.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-07-16T11:25:09Z No. of bitstreams: 2 Dissertação - Adailton Pereira dos Santos - 2018.pdf: 2470326 bytes, checksum: b19858377cb05cc2abd767e4560cd12a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2018-07-17T13:12:23Z (GMT) No. of bitstreams: 2 Dissertação - Adailton Pereira dos Santos - 2018.pdf: 2470326 bytes, checksum: b19858377cb05cc2abd767e4560cd12a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-07-17T13:12:23Z (GMT). No. of bitstreams: 2 Dissertação - Adailton Pereira dos Santos - 2018.pdf: 2470326 bytes, checksum: b19858377cb05cc2abd767e4560cd12a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-06-29 / β-lactamases are enzymes that hydrolyze the β-lactam ring, inactivating the action of β-lactam antibiotics. The objective of this work was to diagnose phenotypically and molecularly 14 β- lactamase resistance genes expressed in Pseudomonas aeruginosa and to correlate the results found. A total of 99 samples of Pseudomonas aeruginosa were selected and the antibiogram was performed. Real-time PCR is being performed using the Sybr Green system to amplify the genes corresponding to the resistances found in phenotyping. Three/14 (21.4%) genes blaSME, blaOXA, blaGIM were found simultaneously in three samples. According to the statistical test, when evaluating the amplification results obtained for PPT, the molecular method was more sensitive for the detection of the gene coding for multidrug resistance, presenting values of (p <0.05). This information suggests that gene research is more sensitive and specific compared to the antibiogram. / As β-lactamases são enzimas que hidrolisam o anel β-lactâmico, inativando a ação de antibióticos β-lactâmicos. O objetivo deste trabalho foi diagnosticar fenotipicamente e molecularmente 14 genes de resistência à β-lactamases expressos em Pseudomonas aeruginosa e correlacionar os resultados encontrados. Um total de 99 amostras de Pseudomonas aeruginosa foi selecionado e o antibiograma foi realizado. A PCR em tempo real foi realizada usando o sistema Sybr Green para amplificar os genes correspondentes às resistências encontradas na fenotipagem. Das 99 amostras, 14 foram identificadas como fenotipicamente resistentes ao ATM antimicrobiano. Três/14 (21,4%) genes blaSME, blaOXA, blaGIM foram encontrados simultaneamente em três amostras. De acordo com o teste estatístico, ao avaliar os resultados de amplificação obtidos para o PPT, o método molecular foi ma s sensível para a detecção do gene que codifica a resistência a múltiplos fármacos, apresentando valores de (p <0,05). Esta informação sugere que a pesquisa genética é mais sensível e específica em comparação com o antibiograma.

Mycobacterium tuberculosis est responsable de 8,7 millions de nouveaux cas de tuberculose et de 1,4 millions de décès en 2011. L’émergence de souches résistantes aux deux antituberculeux majeurs, isoniazide et rifampicine, (MDR) et aux antibiotiques de seconde ligne (XDR), ainsi que la difficulté d’éradiquer les formes « dormantes » du bacille nécessitent la recherche de nouveaux antibiotiques. Les β-lactamines n’ont jamais été utilisées en thérapeutique car M. tuberculosis produit une β-lactamase à large spectre, BlaC. Cependant, l’association d’une β-lactamine appartenant à la classe des carbapénèmes, le méropénème, et d’un inhibiteur de β-lactamase, l’acide clavulanique, est active sur M. tuberculosis incluant des souches XDR. Notre objectif a été de caractériser les cibles des carbapénèmes qui sont atypiques chez M. tuberculosis, parce que le peptidoglycane de cette bactérie contient majoritairement (80%) des ponts interpeptidiques formés par une classe particulière de transpeptidases, les L,D-transpeptidases. Nous avons comparé les cinq L,D-transpeptidases de M. tuberculosis au niveau de leur activité in vitro dans la formation des ponts interpeptidiques du peptidoglycane et dans la réaction d’inactivation par les carbapénèmes. Nous avons ainsi pu montrer que les cinq L,D-transpeptidases sont fonctionnelles in vitro. LdtMt1, LdtMt2, LdtMt4 et LdtMt5 sont capables de former des ponts interpeptidiques du peptidoglycane reliant l’acide aminé en position 3 d’un substrat tétrapeptidique donneur à l’acide aminé en position 3 d’un substrat tétrapeptidique accepteur. Ces mêmes enzymes peuvent également utiliser la D-méthionine comme accepteur dans une réaction d’échange de la D-Ala4 du substrat tétrapeptidique. LdtMt1, LdtMt2, LdtMt3 et LdtMt4 forment un complexe covalent avec les carbapénèmes. La réaction d’inactivation des L,D-transpeptidases par les carbapénèmes se déroulent en deux étapes. Dans un premier temps, un intermédiaire covalent réversible est formé (constante catalytique k1) puis la deuxième étape aboutit à la formation de l’acylenzyme (constante catalytique k2). La détermination des constantes catalytiques d’inactivation k1 et k2 a révélé d’importantes différences entre les carbapénèmes. Excepté pour LdtMt1, l’imipénème inactive plus rapidement les L,D-transpeptidases que les autres carbapénèmes suggérant que des modifications de la chaine latérale pourraient être envisagées pour optimiser l’activité « anti-mycobactérienne » de cette classe de β-lactamines. Nous avons en parallèle initié l’étude de la régulation des L,D-transpeptidases dans différentes conditions de culture ce qui permettra à terme d’identifier les L,D-transpeptidases essentielles pour la croissance et la persistance de M. tuberculosis. Ce travail pourrait déboucher sur l’identification de cibles essentielles permettant l’éradication des formes dormantes de M. tuberculosis qui sont très difficile à traiter. / Mycobacterium tuberculosis is responsible for 8.7 million of new cases of tuberculosis (TB) and 1.4 million of deaths in 2011. The emergence of strains resistant to the two first-line anti-TB drugs, isoniazid and rifampicin, (MDR), to second line-drugs (XDR) and the difficult to kill dormant forms of the bacilli require the discovery of new anti-TB antibiotics. β-lactams are usually not considered for tuberculosis treatment since M. tuberculosis produces a broad-spectrum β-lactamase, BlaC. However, the combination of β-lactam belonging to the carbapenem class, meropenem, with β-lactamase inhibitor, clavulanate, is notably active on XDR strains. Our aim was to characterize the carbapenem targets, atypical in M. tuberculosis, since peptidoglycan of this bacteria contains a majority (80%) of cross-links formed by a special transpeptidase family, the L,D-transpeptidases. We have compared the five L,D-transpeptidases of M. tuberculosis for their in vitro activities with respect to peptidoglycan dimers formation and for inactivation reaction by carbapenems. Thus, we have showed that the five L,D-transpeptidases were functional in vitro. LdtMt1, LdtMt2, LdtMt4 et LdtMt5 were able to form peptidoglycan cross-links binding the third amino acid of a donor tetrapeptide substrate with the third amino acid of an acceptor tetrapeptide substrate. These enzymes were also able to use D-methionine as an acceptor in exchange reaction of D-Ala4 of the donor tetrapeptide substrate. LdtMt1, LdtMt2, LdtMt3 et LdtMt4 formed a covalent adduct with carbapenems. The inactivation reaction of L,D-transpeptidases by carbapenems proceed through two steps. In first, a reversible covalent adduct is formed (catalytic constant k1), followed by a second step leading to acylenzyme formation (catalytic constant k2). The determination of kinetic constants of inactivation k1 et k2 revealed important differences between carbapenems. Except for LdtMt1, Imipenem inactivates L,D-transpeptidases more rapidly than other carbapenems indicating that modification of the carbapenem side chain could be used to optimize their anti-mycobacterial activity. In parallel, we have started the study of the L,D-transpeptidases regulation in various culture conditions will allow identifying the L,D-transpeptidases essential for growth and persistence of M. tuberculosis. This work might lead to identification of essential targets allowing eradication of M. tuberculosis dormant forms, which are difficult to treat with conventional anti-TB drugs.