Objetivos: Avaliar o perfil de suscetibilidade, a prevalência da produção de AmpC, β-lactamase de espectro estendido (ESBL) e Metalo-β-lactamase (M-βla) em Pseudomonas aeruginosa obtidas de dois hospitais universitários distintos (ISCMPA e HCPA) em Porto Alegre. Em adição, tipagem molecular por PFGE foi realizada entre os isolados produtores de M-βla para avaliar a relação clonal. Métodos: Foi determinada a suscetibilidade de 238 isolados de P. aeruginosa para 8 agentes antimicrobianos, através do teste de disco-difusão, usando agar Müller-Hinton (MH) de acordo com “National Committee for Clinical Laboratory Standards” (NCCLS) . Todos isolados foram avaliados para produção de AmpC com o disco de imipenem (indutor) próximo ao disco de cefepima/ceftazdima (substrato). Um achatamento no halo de cefepime/ceftazidima pela indução da enzima pelo imipenem, indicava resultado positivo para AmpC. Todos isolados forma avaliados para a presença de ESBL através do teste de aproximação de disco com ceftazidima, cefepima, cefotaxima, ceftriaxona e ticarcilina-clavulanato como inibidor de β-lactamase. A produção de M-βla foi determinada através do teste de aproximação de discos de CAZ a discos impregnados com ácido2-mercatopropiônico (2-MPA). As taxas de resistência foram comparadas através do Teste Exato de Fisher. Valor de P<0,05 foi considerado estatisticamente significativo. Análise de macrorestrição com a enzima speI foi realizada em isolados produtores de M-βla. Resultados: As taxas de resistência para todos os agentes foram superiores entre os isolados obtidos na ISCMPA em relação aos do HCPA. A ceftazidima mostrou ser o antibiótico mais efetivo contra os isolados de ambos Hospitais (ISCMPA e HCPA) com taxa de resistência de 25,7% (ISCMPA) e 6,1% (HCPA). A expressão de AmpC foi observada em 190 isolados (83,7% HCPA e 77,1% ISCMPA). Não foi possível detectar a presença de ESBL entre todos as P. aeruginosa avaliadas em ambos hospitais. Foi observada a presença de M-βla em 28 isolados (20,0%) da ISCMPA. Mas não foi detectada M-βla em nenhuma P. aeruginosa do HCPA. A análise de macrorestrição mostrou que 14 de 16 P. aeruginosa Mβla positivas pertenciam a um clone (denominado clone A), e seus subclones. Apenas dois outros clones (B e C) foram identificados em um isolado cada. / Objectives: To evaluate susceptibility profile, the prevalence of extendedspectrum β-lactamases (ESBL) production, AmpC and Metallo-β-lactamases (M-βla) in Pseudomonas aeruginosa obtained from two distinct hospitals (ISCMPA and HCPA) in Porto Alegre, Brazil. In addiction, molecular typing by PFGE was perfomed among isolates producing M-βla in order to evaluate probably clonal relatedness. Methods: The susceptibility of 238 P. aeruginosa to 8 antimicrobial agents was determined by the disk diffusion method, using Müller-Hinton agar (MH) in accordance with “National Committee for Clinical Laboratory Standards” guidelines. All isolates were evaluated for AmpC production with the imipenem disk (strong inducer) near of the cefepime/ceftazidime disk (substrate). A blunting of the cefepime/ceftazidime zone by imipenem-induced enzyme, indicated positive result for AmpC. All isolates were evaluated for ESBL production by disk approximation test with ceftazdime, cefepime, cefotaxime, ceftriaxone plus ticarcillin-clavulanate as inhibitor. M-βla production was determined by disk approximation test with disks containing CAZ and 2-mercatopropionic acid (2-MPA). The results were compared by the Fisher’s Exact Test. Macrorestriction analysis by SpeI, followed by PFGE, was perfomed in isolates M-βla positive. The resistance rates were compared by The Fisher’s Exact Test. P values < 0.05 were considered to be statistically significant. Results: The resistance rates to all antimicrobial agents were higher among isolates obtained from ISCMPA than those obtained from HCPA. The ceftazidime was the more active antibiotic against the isolates in both hospitals with resistance rates of 25,7% (ISCMPA) and 6,1% (HCPA). The derepression of AmpC was observed in 190 isolates (83,7% HCPA and 77,1% ISCMPA). It was not possible to detect the presence of ESBL among all P. aeruginosa evaluated in both hospitals. Positive results for M-βla production were observed in 28 isolates (20,0%) from ISCMPA. But none M-βla production was identified in P. aeruginosa from HCPA. The macrorestriction analysis by PFGE, showed that 14 of 16 M-βla positive P. aeruginosa beloneed to one clone (named clone A) and its subclones.Only two others clones (B and C) were identified in one isolate each.

An alpha-amino ester hydrolase (AEH) applicable to synthesis of semi-synthetic antibiotics was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913. AEHs catalyze the synthesis and hydrolysis of alpha-amino beta-lactam antibiotics. The enzyme was characterized for thermodynamic and kinetic parameters. The enzyme shows optimal ampicillin hydrolytic activity at 25C and pH 6.8. The AEH enzymes have been shown to have excellent synthetic capability. Additionally, we demonstrated the first fully aqueous enzymatic one-pot synthesis of ampicillin direct from the natural product penicillin G eliminating the isolation of the intermediate 6-APA. Lastly, to improve the thermostability of the AEH a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-factors from the available crystal structures of the known AEH from Xanthomonas citri. Our best variant, which is a quadruple mutant, E143H/A275P/N186D/V622I, which has a T_50_30, the temperature at which the half-life is 30 minutes, of 34C and 1.3-fold activity compared to wild-type. Overall, we have successfully improved the understanding of the AEH class of enzymes and applied a novel cascade application, demonstrating AEHs unique applicability in the synthesis of beta-lactam antibiotics. The improved thermostability will further improve the industrial relevance of AEHs.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The use of antibiotics and antimicrobial drugs has played a large role in the elimination of many infectious diseases, however the wide spread use of such drugs has given rise to the phenomenon of antimicrobial resistance and has rendered antibiotics ineffective to a broad range of bacteria. The aim of the study was to ascertain the differences if any in the phenotypic and genotypic resistance profiles of K. pneumoniae isolated from a single tertiary hospital in two surveillance studies undertaken at different times, viz., 2001 and 2007 with special emphasis on ESBLs. A correlation with antibiotic use was also undertaken. ESBL positives were identified and phenotypic resistance profiles were generated based on the resistance profiles of individual isolates by means of their MIC data. The molecular detection of ESBLs was carried out using representative isolates and sequencing was based on the phenotypic expression of the most common ESBL genes. The data was summarized using median values and interquartile ranges. Antibiotic use and susceptibility in 2000 was compared to that in 2007 using a Wilcoxon signed rank test for paired data since the same drugs were tested in both years. Of the isolates that were tested, sequencing revealed that TEM – 1 was identified in all isolates and SHV-1 and SHV-2 were identified in 60 % in the isolates collected in 2000 and 77 % and 11 % respectively in the isolates collected in 2007. SHV – 11 was present in 67% of isolates from 2007 and 55% of those were in combination with SHV – 1. Sequencing also revealed CTXM-15 present in one of the isolates collected in 2007. There was 100% susceptibility to cefoxitin and only one isolate in 2007 showing an intermediate result to imipenem. No novel β-lactamases were identified in this study; however the decrease in susceptibility over time is proof of bacterial evolution. The variety of β-lactamases and diversity of plasmid profiles in these two small populations provides proof to the claim that dissemination of resistance in Klebsiella pneumonia is effortless. Statistical analysis showed an increase in resistance from the year 2000 to 2007 however the correlation between overall antibiotic use and the increase in resistance did not reach statistical significance. It was observed that resistance increased despite only a slight increase in the use of a few antibiotics to which we attributed co-carriage of resistance genes. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2008. / Title from second page of PDF document. Includes bibliographical references.

Thesis (MSc.(Medical Microbiology)--Faculty of Health Sciences)-University of Pretoria, 2008. / Summary in English and Afrikaans. Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2007. / Title from second page of PDF document. Includes bibliographical references.

Objetivos: Avaliar o perfil de suscetibilidade, a prevalência da produção de AmpC, β-lactamase de espectro estendido (ESBL) e Metalo-β-lactamase (M-βla) em Pseudomonas aeruginosa obtidas de dois hospitais universitários distintos (ISCMPA e HCPA) em Porto Alegre. Em adição, tipagem molecular por PFGE foi realizada entre os isolados produtores de M-βla para avaliar a relação clonal. Métodos: Foi determinada a suscetibilidade de 238 isolados de P. aeruginosa para 8 agentes antimicrobianos, através do teste de disco-difusão, usando agar Müller-Hinton (MH) de acordo com “National Committee for Clinical Laboratory Standards” (NCCLS) . Todos isolados foram avaliados para produção de AmpC com o disco de imipenem (indutor) próximo ao disco de cefepima/ceftazdima (substrato). Um achatamento no halo de cefepime/ceftazidima pela indução da enzima pelo imipenem, indicava resultado positivo para AmpC. Todos isolados forma avaliados para a presença de ESBL através do teste de aproximação de disco com ceftazidima, cefepima, cefotaxima, ceftriaxona e ticarcilina-clavulanato como inibidor de β-lactamase. A produção de M-βla foi determinada através do teste de aproximação de discos de CAZ a discos impregnados com ácido2-mercatopropiônico (2-MPA). As taxas de resistência foram comparadas através do Teste Exato de Fisher. Valor de P<0,05 foi considerado estatisticamente significativo. Análise de macrorestrição com a enzima speI foi realizada em isolados produtores de M-βla. Resultados: As taxas de resistência para todos os agentes foram superiores entre os isolados obtidos na ISCMPA em relação aos do HCPA. A ceftazidima mostrou ser o antibiótico mais efetivo contra os isolados de ambos Hospitais (ISCMPA e HCPA) com taxa de resistência de 25,7% (ISCMPA) e 6,1% (HCPA). A expressão de AmpC foi observada em 190 isolados (83,7% HCPA e 77,1% ISCMPA). Não foi possível detectar a presença de ESBL entre todos as P. aeruginosa avaliadas em ambos hospitais. Foi observada a presença de M-βla em 28 isolados (20,0%) da ISCMPA. Mas não foi detectada M-βla em nenhuma P. aeruginosa do HCPA. A análise de macrorestrição mostrou que 14 de 16 P. aeruginosa Mβla positivas pertenciam a um clone (denominado clone A), e seus subclones. Apenas dois outros clones (B e C) foram identificados em um isolado cada. / Objectives: To evaluate susceptibility profile, the prevalence of extendedspectrum β-lactamases (ESBL) production, AmpC and Metallo-β-lactamases (M-βla) in Pseudomonas aeruginosa obtained from two distinct hospitals (ISCMPA and HCPA) in Porto Alegre, Brazil. In addiction, molecular typing by PFGE was perfomed among isolates producing M-βla in order to evaluate probably clonal relatedness. Methods: The susceptibility of 238 P. aeruginosa to 8 antimicrobial agents was determined by the disk diffusion method, using Müller-Hinton agar (MH) in accordance with “National Committee for Clinical Laboratory Standards” guidelines. All isolates were evaluated for AmpC production with the imipenem disk (strong inducer) near of the cefepime/ceftazidime disk (substrate). A blunting of the cefepime/ceftazidime zone by imipenem-induced enzyme, indicated positive result for AmpC. All isolates were evaluated for ESBL production by disk approximation test with ceftazdime, cefepime, cefotaxime, ceftriaxone plus ticarcillin-clavulanate as inhibitor. M-βla production was determined by disk approximation test with disks containing CAZ and 2-mercatopropionic acid (2-MPA). The results were compared by the Fisher’s Exact Test. Macrorestriction analysis by SpeI, followed by PFGE, was perfomed in isolates M-βla positive. The resistance rates were compared by The Fisher’s Exact Test. P values < 0.05 were considered to be statistically significant. Results: The resistance rates to all antimicrobial agents were higher among isolates obtained from ISCMPA than those obtained from HCPA. The ceftazidime was the more active antibiotic against the isolates in both hospitals with resistance rates of 25,7% (ISCMPA) and 6,1% (HCPA). The derepression of AmpC was observed in 190 isolates (83,7% HCPA and 77,1% ISCMPA). It was not possible to detect the presence of ESBL among all P. aeruginosa evaluated in both hospitals. Positive results for M-βla production were observed in 28 isolates (20,0%) from ISCMPA. But none M-βla production was identified in P. aeruginosa from HCPA. The macrorestriction analysis by PFGE, showed that 14 of 16 M-βla positive P. aeruginosa beloneed to one clone (named clone A) and its subclones.Only two others clones (B and C) were identified in one isolate each.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2011 / Made available in DSpace on 2012-10-25T23:32:21Z (GMT). No. of bitstreams: 1 294997.pdf: 3221362 bytes, checksum: 05c3e867a109c64c20174ece942ee493 (MD5) / Nas últimas duas décadas, índices crescentes de resistência têm minado a eficácia dos agentes antimicrobianos. Pseudomonas aeruginosa são importantes patógenos hospitalares e esta espécie é considerada um reservatório de genes determinantes de resistência, inclusive genes que codificam para as metalo-ß-lactamases (MßL). MßL são betalactamases capazes de hidrolisar os antimicrobianos betalactâmicos carbapenêmicos, os quais constituem o tratamento empírico de infecções causadas por bactérias Gram-negativas multirresistentes. Neste trabalho foram analisados 42 isolados clínicos de P. aeruginosa quanto à susceptibilidade aos principais antimicrobianos utilizados na clínica. Através da determinação das concentrações inibitórias mínimas, 41 desses não eram sensíveis a imipenem e meropenem e 31 se apresentaram simultaneamente resistentes a estes dois carbapenêmicos e às cefalosporinas ceftazidima e cefepima. Vinte e quatro isolados foram considerados produtores de MßL através do teste de sinergismo de disco duplo (DDST) utilizando ácido 2-mercaptopropiônico como inibidor seletivo de MßL. Trinta e cinco isolados (35/42) foram avaliados quanto à presença dos genes de MßL. Em dezessete dos isolados em que os testes fenotípicos se apresentaram positivos, quinze (15/17) corresponderam às subclasses SPM-1 (14/15) e IMP-1 (1/15). Nos demais isolados onde os testes fenotípicos se apresentaram negativos, também em quatro (4/18) deles pôde ser verificada a presença da subclasse SPM-1. A ocorrência de isolados produtores desta enzima naqueles isolados não sensíveis aos carbapenêmicos foi de 58,54% (24/41; . = 0,4322), no entanto, naqueles simultaneamente resistentes aos carbapenêmicos e cefalosporinas, aliados à observação de CIM elevadas, foi de 85,71% (24/28; . < 0,001), sugerindo que o isolamento de P. aeruginosa apresentando este último fenótipo é um indicativo mais apurado da presença de MßL. Os isolados desta amostragem também demonstraram uma associação positiva entre a presença de MßL e perfis de multirresistência. Foi observado que 81,5% (22/27) dos isolados sensíveis a, no máximo, colistina e aztreonam, produziam MßL. Assim, a correlação entre a presença dessa enzima e a observação de multirresistência foi de 91,67% (. < 0,001) e a uma razão 3,3 vezes superior à correlação obtida para os isolados não produtores de MßL. / In the last two decades, increasing rates of resistance have weakened the effectiveness of antimicrobial agents. Pseudomonas aeruginosa is an important nosocomial pathogen and the species is considered as a reservoir of resistant genes, including those coding for metallo-â-lactamases (MâL). MâL are enzymes able to hydrolyze the carbapenem beta-lactam antibiotics, which are the empirical treatment of infections caused by multiresistant Gram-negative bacteria. In the present work, 42 clinical isolates of P. aeruginosa with different susceptibility to the main antimicrobials used in the clinic were analyzed. By determining the minimum inhibitory concentrations, 41 of them were not sensitive to imipenem and meropenem, and 31 were simultaneously resistant to these carbapenems and to the cephalosporins ceftazidime and cefepime. Twenty-four isolates were considered as producers of MâL through double-disk synergy test (DDST) using the acid 2-mercaptopropionic as an MâL selective inhibitor. Thirty-five isolates (35/42) were evaluated for the presence of MâL genes. In seventeen of them, in which the phenotypic tests were positive, fifteen (15/17) corresponded to subclasses SPM-1 (14/15) and IMP-1 (1/15). In the other eighteen isolates where phenotypic tests were negative, four of them (4/18) also presented the SPM-1 gene. In the group of isolates resistant to carbapenems, the occurrence of producers of this enzyme was 58.54% (24/41; ñ = 0.4322). However, when the resistant isolates to both carbapenem and cephalosporin were combined with the observation of high MIC values, this value increased to 85.71% (24/28; ñ <0.001). This suggests that the occurrence of isolates of P. aeruginosa showing the latter phenotype is a reliable indicative of the presence of MâL. Isolates of this sample also showed a positive association between the presence of MâL and profiles of multidrug resistance. The results showed that 81.5% (22/27) of the isolates sensitive at least to colistin and aztreonam, produced MâL. The correlation between the presence of MâL and the observation of multidrug resistance was of 91.67% (ñ <0.001), in a rate 3.3 times greater than the risk obtained for the isolates that did not produce MâL.