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.

Le phénomène de résistance des bactéries à Gram négatif aux antibiotiques est aujourd’hui un problème sanitaire mondial majeur. La production de β-lactamases, et plus particulièrement de carbapénémases, enzymes capables d’hydrolyser la plupart des agents antibactériens de la classe des β-lactames, est le mécanisme de résistance le plus répandu. Dans ce mémoire, nous décrivons la conception et la synthèse de nouveaux inhibiteurs fluorés de carbapénémases. Notre objectif a été la synthèse de monobactames trifluorométhylés en position C4.Nous avons développé une nouvelle voie de synthèse diastéréosélective par expansion de cycle d’aziridines pour l’accès à des 3-bromo-4-CF3-azétidin-2-ones. Ces composés ont été fonctionnalisés par substitution nucléophile, réactions radicalaires et organométalliques en position C3. Dans une seconde partie, des essais de cyclisation de β-hydroxyaminoesters et acides ainsi que de β-hydroxy-hydroxamates ont été entrepris. Pour obtenir ces intermédiaires β-hydroxyaminoesters et acides, nous avons étudié la réactivité des hydroxylamines sur des accepteurs de Michael trifluorométhylés.Enfin, l’évaluation biologique des composés a été réalisée par des tests enzymatiques en suivi par spectroscopie UV. L’évaluation par RMN 19F a également été entreprise, et a permis le développement d’outil de diagnostique et de screening, toujours en cours d’optimisation. / Multidrug resistant gram-negative pathogens are emerging worldwide. β-lactamases production, especially carbapenemases, enzymes with broad hydrolytic capabilities towards β-lactams, is a global spread mechanism of resistance among gram-negative bacteria. We report here the design and the synthesis of new fluorinated inhibitors of carbapenemases. Our aim was to synthesize trifluoromethylated monobactams in C4 position. We have developed a new diastereoselective pathway by ring expansion of aziridines to access to 3-bromo-4-CF3-azetidin-2-ones. These compounds have been successfully functionalized in C3 position via nucleophilic substitution, radical and organometallic reactions.In a second part, cyclisation attempts of β-hydroxyaminoesters and acids, as well as β-hydroxy-hydroxamates have been conducted. A study of Michael addition of hydroxylamines on trifluoromethylated Michael acceptors have been achieved in order to obtain the β-hydroxyaminoesters and acids derivatives.Finally, biological evaluations of synthesized compounds have been realized through enzymatic tests. 19F NMR evaluation have been accomplished and led to development of diagnostic and screening tools, and it is still in under optimisation.

Made available in DSpace on 2015-03-26T13:51:59Z (GMT). No. of bitstreams: 1 01 - capa_abstract.pdf: 112846 bytes, checksum: f260bcc7fb2e9aa227782fd9e02a5c86 (MD5) Previous issue date: 2006-10-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The diversity of resistance mechanisms to antimicrobial compounds was investigated in E. coli strains isolated from poultry. All 30 isolates were resistant to ampicillin and 26 of them were resistant to at least two antibiotics. Multi-resistance profiles were confirmed, including intermediate levels. Forty percent of the strains presented four resistance markers and 93% of the isolates were resistant to tetracycline, an antibiotic commonly used in poultry farms. The β-lactamase diversity among the strains was probed against seven compounds. Among strains bearing the same resistance profile for other groups of antimicrobial drugs, it was possible to observe different β-lactamases activity against the tested substrates. This fact suggests either a pool of resistance genes or regulatory differences. The isolate that displayed confirmed activity against six of the β-lactamic antibiotics bears a large molecular mass plasmid that confers the AmpR phenotype. A fragment of this plasmid with approximately 5 kb was subcloned in the pCCR9 vector which is commonly used to detect β-lactamase genes. The amino acid deduced from nucleotide sequencing displayed 100% of identity to TEM-1, a class A serine β-lactamase. Next to the gene encoding TEM-1 was found putative transposon related to the Tn3 family, albeit with some differences in the order and direction of transcription of the genes. / A diversidade dos mecanismos de resistência a antimicrobianos foi investigada em Escherichia coli, originadas de frangos de corte. Dos 30 isolados, todos resistentes à ampicilina, 26 apresentaram mais de uma marca de resistência. Foram confirmados modelos de multirresistência, incluindo níveis intermediários de resistência. Em 40% dos casos, ocorreram quatro marcas e 93% dos isolados resistiram à ação de tetraciclina, um promotor de crescimento usual na agropecuária. A diversidade de β-lactamases entre os isolados foi demonstrada pela ação direta contra sete β-lactâmicos. Os isolados com mesmo modelo de resistência possuíam, sob mesmas condições, espectro de ação e de atividade diferentes, mostrando que há amplo pool de genes de resistência, ou diferenças regulatórias nessas bactérias. O isolado com maior espectro de ação, confirmado sobre seis β-lactâmicos, contém um plasmídeo de alta massa molecular, que confere resistência a ampicilina. Um fragmento deste plasmídeo, com aproximadamente 5 kb, foi clonado em pCCR9, vetor usado para detecção de genes de resistência a β-lactâmicos. As análises das seqüências obtidas revelaram 100 % de identidade com TEM-1, uma serina β-lactamase da classe A. Próximo ao gene codificador da TEM-1, foi encontrado um transposon putativo relacionado com os da família Tn3, porém com particularidades na ordem e direção de transcrição dos genes componentes.

Mycobacterium tuberculosis et Mycobacterium abscessus produisent les β-lactamases BlaC et BlaMab qui contribuent à la résistance intrinsèque de ces bactéries aux β-lactamines. Notre objectif est de caractériser l’inhibition de ces β-lactamases par l’avibactam et le clavulanate pour contribuer au développement de nouveaux traitements. Nous avons déterminé le profil de substrat et d’inhibition de BlaMab ainsi que sa structure cristalline, révélant trois différences majeures avec BlaC. BlaMab a une activité supérieure à celle de BlaC pour toutes les β-lactamines sauf la céfoxitine qui est utilisée pour les infections dues à M. abscessus. BlaC est inhibée irréversiblement par le clavulanate et inefficacement par l’avibactam alors que BlaMab présente le comportement inverse impliquant une hydrolyse du clavulanate et une inhibition très rapide par l’avibactam. La structure de BlaMab diffère de celle de BlaC principalement par le remplacement du motif conservé SDN par SDG. L’introduction de SDG dans BlaMab et de SDN dans BlaC a montré que cette différence détermine le profil d’inhibition des β-lactamases. Une seule mutation peut donc entraîner l’émergence d’une résistance aux combinaisons d’une β-lactamine avec le clavulanate ou l’avibactam mais pas avec les deux inhibiteurs. L’avibactam et le clavulanate offrent donc des alternatives thérapeutiques en cas de résistance à l’un des inhibiteurs. Nous nous sommes également intéressés aux β-lactamines partenaires du clavulanate, pour le traitement de la tuberculose et montrer que la structure des carbapénèmes pouvait être optimisée pour améliorer l’inactivation des cibles et diminuer l’hydrolyse par BlaC. / Mycobacterium tuberculosis and Mycobacterium abscessus produce the β-lactamases BlaC and BlaMab that contribute to the intrinsic resistance of those bacteria to β-lactams. Our objective was to characterize the inhibition of these β-lactamases by avibactam and clavulanate in order to contribute to the development of new treatments. We have determined the inhibition and substrate profiles of BlaMab, as well as its crystal structure, revealing three major differences with BlaC. BlaMab is more active than BlaC with respect to hydrolysis of all β-lactams except cefoxitin, which is used for the treatment of infections due to M. abscessus. BlaC is inhibited irreversibly by clavulanate and inefficiently by avibactam. In contrast, BlaMab shows the opposite behavior involving hydrolysis of clavulanate and a rapid inhibition by avibactam. Structurally BlaC differs from BlaMab mainly by the replacement of the conserved motif SDN by SDG. The introduction of SDG in BlaMab and of SDN in BlaC revealed that this difference determines the inhibition profile of the β-lactamases. A single mutation can therefore lead to the emergence of resistance to the association of β-lactam with clavulanate or avibactam, but not to both associations. Thus, avibactam and clavulanate offer therapeutic alternatives in case of resistance to one of the two inhibitors. We have also investigated the β-lactam partners of clavulanate for the treatment of tuberculosis and showed that the structure of carbapenems could be optimized to enhance the inactivation of the targets and to reduce hydrolysis by BlaC.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2018-06-28T20:38:21Z No. of bitstreams: 2 Tese- Cláudia Castelo Branco Artiaga Kobayashi - 2013.pdf: 3424505 bytes, checksum: 8f6814112115a98fa01748f80463bf8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-06-29T11:46:54Z (GMT) No. of bitstreams: 2 Tese- Cláudia Castelo Branco Artiaga Kobayashi - 2013.pdf: 3424505 bytes, checksum: 8f6814112115a98fa01748f80463bf8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-29T11:46:54Z (GMT). No. of bitstreams: 2 Tese- Cláudia Castelo Branco Artiaga Kobayashi - 2013.pdf: 3424505 bytes, checksum: 8f6814112115a98fa01748f80463bf8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-04-12 / Carbapenemase-producing Klebsiella pneumoniae is an enterobacteria, commonly associated with resistance to multiple antibiotics. The presence of multidrug resistance phenotype is an important challenge in the epidemiological point of view, regarding the therapeutic management and infection control. A retrospective observational study was conducted in a tertiary-care hospital in Goiânia, Brazil, in the period of 2006 to 2011 to investigate the carbapenemases phenotypes, the in vitro activity of tigecycline and polymyxin B and determine the risk factors associated with infection or colonization by carbapenemase-producing K. pneumoniae. The serine-carbapenemase production was detected by modified Hodge test and combined disk test with boronic acid; metallo-β-lactamases - MβL by double-disk synergy test with imipenem and EDTA; extended spectrum β-lactamases - ESBL by combined disk with cefotaxime and ceftazidime with and without clavulanate, and plasmid AmpC type by combined disk with boronic acid and cefoxitin. The in vitro activity of tigecycline and polymyxin B against the enzyme-producing isolates was assessed using the broth microdilution method and for not β-lactam antimicrobials by disk diffusion and semi-automated system. The association between independent risk factors and colonization or infection by carbapenemase-producing K. pneumoniae was determined by logistic regression analysis. The carbapenem resistance was found in 8.06% of all isolates K. pneumoniae and 6.90% were carbapenemase producers. Among carbapenemases producers isolates, 77.78% were KPC type β-lactamase, 37.04% demonstrated the simultaneous production of plasmid AmpC β-lactamase and KPC, 29.63% associated with MβL or ESBL and KPC, 11.11% MβL/ESBL/KPC and 7.41% MβL/ESBL/KPC/plasmid AmpC. High percentages of not β-lactams antimicrobial resistance were observed, ranging from 61.10% - 74.70% to fluoroquinolones and 52.10% - 54.30% to aminoglycosides. Polymyxin B showed limited activity against the carbapenemase-producing K. pneumoniae (35.70%) and tigecycline was the only antimicrobial that inhibited 75.0% of these multidrug-resistant strains. The independent risk factors for K. pneumoniae carbapenemase producers were prolonged hospitalization (p = 0.031), previous use of carbapenems (p = 0.041) and exposure to more than three antimicrobials (p = 0.034). The detection of multidrug- resistant carbapenemase-producing isolates and the knowledge of different resistance mechanisms can result in significant impact on the treatment outcomes and strategies for infection control and prevention. / Klebsiella pneumoniae produtora de carbapenemase é uma enterobactéria, comumente associada à resistência aos múltiplos antimicrobianos. A presença deste fenótipo de multirresistência consiste em um importante desafio do ponto de vista epidemiológico, quanto ao manejo terapêutico e ao controle de infecção. Um estudo observacional retrospectivo foi conduzido em um hospital público terciário em Goiânia, Go, Brasil, no período entre 2006 e 2011, para investigar os fenótipos de carbapenemases, a atividade in vitro da tigeciclina e polimixina B e determinar os fatores de risco associados à infecção ou colonização por K. pneumoniae produtora de carbapenemase. A presença de serina carbapenemase foi detectada por meio do teste de Hodge modificado e disco combinado com ácido borônico; metalo-β-lactamases – MβL por sinergismo de disco duplo com imipenem e EDTA; β-lactamases de espectro estendido – ESBL, através do disco combinado com cefotaxima e ceftazidima com e sem ácido clavulânico e AmpC plasmidial por disco combinado com ácido borônico e cefoxitina. A atividade in vitro da tigeciclina e polimixina B foi avaliada através do método de microdiluição em caldo e dos antimicrobianos não β-lactâmicos por disco-difusão e sistema semiautomatizado. A associação entre fatores de risco independentes e colonização ou infecção foi determinada através de regressão logística. A resistência aos carbapenêmicos foi verificada em 8,06% do total de K. pneumoniae e a atividade de carbapenemase em 6,90%. Dentre os isolados produtores de carbapenemases, 77,78% foram considerados β-lactamase tipo KPC, 37,04% demonstraram a produção simultânea de AmpC plasmidial e KPC, 29,63% associação com MβL ou ESBL e KPC, 11,11% MβL/ESBL/KPC e 7,41% MβL/ESBL/KPC/AmpC plasmidial. Altas taxas de resistência aos antimicrobianos não β-lactâmicos foram observadas, variando de 61,10% - 74,70% para fluoroquinolonas e 52,10% - 54,30% para aminoglicosídeos. A polimixina B apresentou baixa atividade in vitro contra estes isolados (35,70%) e a tigeciclina foi o único antimicrobiano que inibiu 75,0% dos mesmos. Os fatores de risco independentes para K. pneumoniae produtora de carbapenemase foram a internação prévia prolongada (p = 0,031), uso prévio de carbapenêmicos (p = 0,041) e exposição a mais de três antimicrobianos (p = 0,034). A detecção de isolados produtores de carbapenemase multirresistentes e o conhecimento dos diferentes mecanismos de resistência podem resultar em significante impacto nos resultados terapêuticos e estratégias de controle de infecção e prevenção.

Extended-spectrum β-lactamases (ESBLs) are plasmid-mediated bacterial enzymes that confer resistance for most β-lactams antibiotics. These enzymes are widespread in microorganisms in hospital settings worldwide. This study estimated the distribution and prevalence of the main ESBLs families among samples of Escherichia coli and Klebsiella spp. in the university hospital of Santa Maria (HUSM), Rio Grande do Sul. During a period of 14 months 90 microorganisms were selected as probable ESBL producers according to the recommendations of Clinical and Laboratory Standards Institute (CLSI). The isolated microorganisms were submitted to phenotypic confirmatory tests for the presence of ESBL. Samples that showed negative results were tested against their susceptibility to cefoxitin. The ESBLs types found in each organism were determined by the research of the genes bla TEM, bla SHV e bla CTX-M by polymerase chain reaction (PCR). Fifty-five (61.1%) samples were confirmed as ESBL positive by the combined disc method and fifty-seven (63.3%) by the double disc method. In the cefoxitin susceptibility test 16 of the 39 samples presented resistance to this agent. Based on PCR, 74 (82,2%) samples harbored TEM-type ESBL gens, 61 (67,8%) SHV-type and 19 (21,1%) CTX-M-type. Only one Escherichia coli isolate appeared harboring genes for the CTX-M family of ESBLs. The distribution of TEM, SHV and CTX-M ESBL families from HUSM presented some similarities and differences compared with ESBLs of other hospital settings. / As β-lactamases de Espectro Estendido (ESBLs) são enzimas bacterianas mediadas por plasmídeos que conferem resistência à maioria dos antibióticos β-lactâmicos. Estas enzimas estão amplamente disseminadas em microrganismos nos ambientes hospitalares do mundo. Este estudo estimou a distribuição e prevalência das principais famílias de ESBLs entre amostras de Escherichia coli e Klebsiella spp. no Hospital Universitário de Santa Maria (HUSM), Rio Grande do Sul. Durante um período de 14 meses, 90 microrganismos foram selecionados como prováveis produtores de ESBLs de acordo com os critérios estabelecidos pelo Clinical and Laboratory Standards Institute (CLSI). Os microrganismos isolados foram submetidos a testes fenotípicos confirmatórios para a presença de ESBL. As amostras que apresentaram resultado negativo nestes testes tiveram sua susceptibilidade testada frente à cefoxitina. Os tipos de ESBLs presentes em cada microrganismo foram determinados pela pesquisa dos genes bla TEM, bla SHV e bla CTX-M através da reação em cadeia da polimerase (PCR). Empregando-se o método do disco combinado, a presença de ESBLs foi confirmada em 55 (61,1%) amostras; quando o método do duplo disco foi utilizado, 57 (63,3%) amostras foram confirmadas. No teste de susceptibilidade à cefoxitina, 16 das 39 amostras testadas apresentaram resistência a este substrato. Com base na PCR, 74 (82,2%) amostras possuíam genes para a família TEM de ESBLs, 61 (67,8%) para a família SHV e 19 (21,1%) para a família CTX-M. Apenas um isolado de Escherichia coli demonstrou possuir genes para a família CTX-M de ESBLs. A distribuição de ESBLs das famílias TEM, SHV e CTX-M no HUSM apresentou semelhanças e diferenças em comparação com ESBLs de outros ambientes hospitalares.

Submitted by Luciana de Cenço Correa Lacerda (lulacerda2@yahoo.com.br) on 2018-06-21T19:31:05Z No. of bitstreams: 1 TESE Luciana 21.06.18 PRONTA.pdf: 1525467 bytes, checksum: a6d6df3286d60500baf769d3035234e1 (MD5) / Approved for entry into archive by Neli Silvia Pereira null (nelisps@fcav.unesp.br) on 2018-06-25T18:40:29Z (GMT) No. of bitstreams: 1 lacerda_lcc_dr_jabo.pdf: 1525467 bytes, checksum: a6d6df3286d60500baf769d3035234e1 (MD5) / Made available in DSpace on 2018-06-25T18:40:29Z (GMT). No. of bitstreams: 1 lacerda_lcc_dr_jabo.pdf: 1525467 bytes, checksum: a6d6df3286d60500baf769d3035234e1 (MD5) Previous issue date: 2018-05-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Lesões na pele podem resultar em feridas que, dependendo do tempo de reparação tissular podem ser classificadas como agudas ou crônicas, sendo crônicas aquelas que não apresentaram cicatrização dentro do período de quatro semanas. A ferida é contaminada por diferentes espécies bacterianas, sendo o sistema imunológico da pele o responsável por impedir que tais contaminações evoluam para infecções. No entanto, muitas vezes o quadro infeccioso é instalado, havendo necessidade de tratamento com antimicrobianos. Tendo em vista que o mau uso de antimicrobianos provoca resistência a multidrogas em estirpes bacterianas potencialmente patogênicas, este trabalho teve como objetivo identificar as bactérias prevalentes em feridas agudas e crônicas de cães por meio de sequenciamento da região 16S rRNA e testar a sensibilidade dos isolados a diferentes antimicrobianos. Para tanto, foram amostradas 20 feridas, sendo cada uma de um cão atendido no Hospital Veterinário da UNESP, Câmpus de Jaboticabal. De cada ferida foram obtidos dez isolados, os quais foram selecionados para o sequenciamento de DNA por meio de comparação entre os perfis genéticos obtidos pelo emprego de marcador molecular randômico. Foram sequenciados 74 isolados identificados como pertencentes a oito gêneros de bactérias gram-negativas, Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Enterobacter spp., Acinetobacter baumannii, Klebsiella spp., Kluyvera georgiana e Providencia stuartii, e três de gram-positivas, Enterococcus sp., Staphylococcus spp. e Bacillus spp. Casos de cães com feridas agudas ou crônicas, associadas a mais de um gênero bacteriano, foram de 85,7% e 30,7%, respectivamente. Isolados da espécie S. aureus apresentaram amplificação para quatro genes codificadores das enterotoxinas sea, seh, see e hlg, enquanto um isolado de B. cereus foi positivo para a presença dos genes hblA, hblC, hblD, nheA, nheB, nheC e entFM. Dois dos isolados de E. coli (6%) apresentaram o gene blaCTX-M-2 e provaram ser resistentes à cefotaxima, um antibiótico do grupo dos β-lactâmicos. Todos os isolados avaliados apresentaram resistência a pelo menos um dos antimicrobianos testados, sendo metronidazol, cefalexina e cefazolina, aqueles pelos quais os isolados mostraram maior resistência. Cinco pacientes que estavam sob antibioticoterapia no momento da coleta possuíam estirpes resistentes aos antimicrobianos pelos quais estavam sendo tratados. Tendo em vista que há uma grande diversidade bacteriana resistente à multidrogas colonizando feridas cutâneas agudas e crônicas de cães, a implantação de antibiogramas previamente à recomendação de antimicrobianos é prática imprescindível e deve ser implantada para preservação da saúde animal e, consequentemente, pública. / Skin lesions can result in cutaneous wounds that may be classified as acute or chronic depending on the period of time spent in tissue repairment. Wounds that have not healed within four weeks are generally classified as chronic. The wound is contaminated by different bacterial species and the immune system of the skin is responsible for preventing infections. Nonetheless, the infectious process is often developed and antimicrobial treatment become necessary. Considering that the misuse of antimicrobials provokes multidrug resistance in potentially pathogenic bacterial strains, this work aimed to identify prevalent bacteria in acute and chronic wounds of dogs by 16S rRNA sequencing and to test the sensitivity of the isolates to different antimicrobials. For that, 20 wounds were sampled, each one from a dog admitted at the Veterinary Hospital of UNESP, Jaboticabal, São Paulo, Brazil. From each wound ten isolates were obtained, which were selected for DNA sequencing through genetic profiles comparison by applying a random molecular marker. Seventy-five isolates were identified as belonging to eight Gram-negative bacteria genera, Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Enterobacter spp., Acinetobacter baumannii, Klebsiella spp., Kluyvera georgiana and Providencia stuartii, and to three gram-positive bacteria genera, Enterococcus sp., Staphylococcus spp. and Bacillus spp. Cases of dogs with acute or chronic wounds associated to more than one bacterial genus were 85.7% and 30.7%, respectively. Staphylococcus aureus isolates presented amplification to four enterotoxin encoding the genes sea, seh, see and hlg, whereas a B. cereus isolate was positive for hblA, hblC, hblD, nheA, nheB, nheC and entFM genes. Two of the E. coli isolates (6%) presented the blaCTX-M-2 gene and proved to be resistant to cefotaxime, a β-lactam antibiotic. All isolates evaluated were resistant to at least one of the antimicrobials tested, being metronidazole, cephalexin and cefazolin the ones for those the isolates showed to be more resistant. Five patients undergoing antibiotic therapy at the same period of sampling presented resistants bacterial strains to the antibiotics by which the patients were being treated. Considering that there is great multidrug resistant bacterial diversity colonizing acute and chronic cutaneous wounds of dogs, the implantation of antibiograms prior to the antimicrobials recommendation is a practice to be implemented in order to preserve animal and, consequently, public health.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In recent years, the isolation of bacteria producing β-lactamases has caused concern around the world, due to the fact these enzymes hydrolysis the ring β-lactam antimicrobials used in the main clinic. This aim of this study was asses the prevalence metallo-β-lactamases (MbL) in isolates of Pseudomonas aeruginosa and Acinetobacter baumannii obtained from patients admitted at the University Hospital of Santa Maria (HUSM). The profile of susceptibility for all isolates was evaluated by the disk diffusion method standardized by CLSI. The antimicrobial disks were distributed in a way that allows the identification of strains producers of AmpC and ESBL. For the identification of the producers of MbL the test of disk approximation with EDTA 0.1 M, EDTA 0,5M and acid 2-mercaptopropionic were performed. Isolates that did not have any of the mechanisms of resistance search were classified as multiresistant (MDR). The minimum inhibitory concentration (MIC) for ceftazidima, imipenem and polymyxin B was assessed by broth method microdilution for all isolated, according to CLSI. From January to June 2006, were obtained 32 isolates the P.aeruginosa and 41 the A. baumannii, the those 17 (23.29%) were β-lactamase AmpC-type producers, 11 (15.07%) were MbL producers, and 45 (61,64%) were classified as MDR. All strains producing MbL were Pseudomonas aeruginosa. The sensitivity of the isolates according to the CIM for antimicrobial evaluated were: 90,28% for polymyxin B, 36,11% for imipenem and 18% for ceftazidima. There was a high prevalence of MDR isolates and producers of β-lactamase-type AmpC and MbL in HUSM, this is extremely worrying once there is limiting therapy available. This situation becomes even more worrying with the find of isolates resistant the polymyxin B, witch is one of the last options of treatment for MDR isolates and producers of MbL. The detection of microorganisms is extremely important for the committees of infection hospital with the goal of preventing outbreaks, as well as guide the medical team on the conduct therapy, since there are few effective antimicrobial clinically for these pathogens and no prospects for development the new antimicrobial in the near future. / Nos últimos anos, o isolamento de bactérias produtoras de β-lactamases tem causado preocupação em todo o mundo, devido ao fato dessas enzimas hidrolisarem o anel β- lactâmico dos principais antimicrobianos utilizados na clínica. Este trabalho teve por objetivo avaliar a prevalência de metalo-β-lactamases (MbL) em isolados de Pseudomonas aeruginosa e Acinetobacter baumannii obtidos de pacientes atendidos no Hospital Universitário de Santa Maria (HUSM). O perfil de sensibilidade para todos os isolados foi avaliado pelo método de disco difusão padronizado pelo CLSI. Os discos de antimicrobianos utilizados foram distribuídos de forma que permitisse a identificação dos isolados produtores de AmpC e ESBL. Para a identificação dos produtores de MbL utilizou-se o teste de disco aproximação com os seguintes agentes quelantes: EDTA 0,1M, EDTA 0,5 M e ácido 2-mercaptopropiônico. Os isolados que não possuíam nenhum dos mecanismos de resistência pesquisados foram classificados como multirresistentes (MDR). A concentração inibitória mínima (CIM) para ceftazidima, imipenem e polimixina B foi avaliada pelo método de microdiluição em caldo para todos os isolados, de acordo com o CLSI. Durante o período de janeiro a junho de 2006 foram obtidos 32 isolados de P.aeruginosa e 41 de A. baumannii, destes 17 (23,29%) foram produtores de β-lactamase do tipo AmpC, 11 (15,07%) foram produtores de MbL e 45 (61,64%) foram classificados como MDR. Todas as cepas produtoras de MbL foram de Pseudomonas aeruginosa. A sensibilidade dos isolados de acordo com a CIM para os antimicrobianos avaliados foram as seguintes: 90,28% para polimixina B, 36,11% imipenem e 18% ceftazidima. Observou-se uma alta prevalência de isolados MDR no HUSM, além de isolados produtores de β-lactamase do tipo AmpC e MbL, o que é extremamente preocupante devido limitar a terapia a poucos antimicrobianos. Esta situação torna-se ainda mais preocupante com a detecção de isolados resistentes a polimixina B, a qual é uma das últimas opções de tratamento para infecções causadas por isolados de P. aeruginosa e Acinetobacter baumannii MDR e produtores de MbL. A detecção desses microrganismos é de grande importância para as comissões de controle de infecção hospitalar com o objetivo de prevenir surtos, bem como orientar a equipe médica sobre a conduta terapêutica, uma vez que há poucos antimicrobianos efetivos clinicamente para esses patógenos e as perspectivas para o desenvolvimento de novos antimicrobianos em um futuro próximo são mínimas.

Genetic typing of Klebsiella pneumoniae is used for epidemiological referencing. In the clinical setting it can be useful in outbreak investigations, understanding transmission and managing hospital infections. Multi-drug resistant bacteria exist and proliferate either due to natural selection of clonal lineages or the transfer of mobile genetic elements, sometimes in response to antibiotic-use selective pressure. Pulsed-field gel electrophoresis (PFGE) is highly discriminatory and the gold standard typing method for the characterisation of K. pneumoniae isolates. The aim of the study was to genetically characterise K. pneumoniae isolates by PFGE and multilocus sequence typing (MLST). One hundred unrepeated ESBL-producing K. pneumoniae isolates were collected from the National Health Laboratory Service (NHLS). The PFGE was performed on a Rotaphor VI system (Biometra, Germany). Clonal representatives were further characterised by MLST. All the strains were typeable by PFGE using XbaI, which discerned multiple pulsotypes and MLST identified 10 different STs including a novel sequence type, ST1632. The diverse pulsotypes of K. pneumoniae isolates are not suggestive of clonal spread of particular strains. The MLST results further confirmed the variability among isolates tested and elucidated several STs, some of which have been identified internationally and often associated with carbapenem-resistance. Data on K. pneumoniae STs is still limited in the South African clinical setting, although the close monitoring of resistance profiles and characterisation of isolates is imperative for outbreak analysis, identification of prominent STs in clinical settings as compared to international counterparts and surveillance of expanding resistance. / Dissertation (MSc (Medical Microbiology))--University of Pretoria, 2015. / Medical Microbiology / MSc (Medical Microbiology) / Unrestricted

β-lactam antibiotics have been the most widely used drug of choice to combat infectious disease caused by bacteria. Unfortunately, their effectiveness is drastically threatened by bacterial β-lactamases. β-lactamases is responsible for the resistance to most antibiotic drugs. For decades, β-lactam β-lactamases inhibitors have been used to reduce bacterial resistance; however, in this study 1,2,4-oxadiazolidinone derivatives as a non-β-lactam β-lactamases inhibitor against TEM-1 and P99 β-lactamases. The significance of oxadiazolidinone is the prominent five-membered ring scaffold in its structure, which is configurationally stable and present in other biologically active compounds such as linezolid and avibactam. Oxadiazolidinones were synthesized by treating nitrones with isocyanates. The synthesized compounds were characterized using 1H and 13C NMR, GC-MS, and FTIR. Afterward, they were tested using Nitrocefin as substrate to determine their effectiveness against TEM-1 and P99 serine β-lactamase. Compound 2a-2c, and 3 showed inhibition ranging from 12-38%.