Orientador: Carlos Magno Castelo Branco Fortaleza / Resumo: A resistência microbiana causa grande número de mortes todos os anos. Sua emergência e disseminação são fenômenos complexos, que devem ser compreendidos no contexto de redes de assistência. Entre os microrganismos multidroga-resistentes (MDR), causam especial preocupação os bacilos Gram-negativos, especialmente Acinetobacter baumannii e Enterobactérias (Escherichia coli, Klebsiella spp e Enterobacter spp) resistentes aos carbapenêmicos (CRAB e CRE, respectivamente). Nós realizamos dois estudos com o objetivo de descrever o comportamento espaço-temporal de CRAB e CRE. O primeiro deles teve delineamento ecológico e se baseou em notificações de agentes de infecção da corrente sanguínea em Unidades de Terapia Intensiva do Estado de São Paulo. O segundo foi uma revisão sistemática de literatura científica e “literatura cinzenta” para identificar relatos de ocorrência de infecções por CRAB e CRE no Brasil. Nossos resultados no Estado de São Paulo demonstraram tendências temporais opostas para CRAB (redução) e CRE (crescimento). Em ambos os casos a incidência é maior nas Regiões Metropolitanas de São Paulo e Campinas, a partir de onde os microrganismos parecem espalhar-se para outras áreas. Infecções foram mais frequentes em hospitais públicos e naqueles com menor proporção de leitos de UTI. Fatores socio-econômicos e demográficos apresentaram associações variáveis, porém plausíveis, com a incidência dos microrganismos de interesse. Os resultados da revisão sistemática apontam para ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Antimicrobial resistance causes great numbers of deaths every year. Its emergence and dissemination are complex phenomena, which must be understood in the context of healthcare networks. Among multidrug-resistant microorganisms (MDR), carbapenem-resistant Gram-negative bacilli, especially Acinetobacter baumannii (CRAB) and Enterobacteriaceae (Escherichia coli, Klebsiella spp and Enterobacter spp; CRE) are of special concern. We performed two studies with the objective of describing the space-time behavior of CRAB and CRE. The first study had an ecological design and was based on reports of microorganisms causing bloodstream infections in Intensive Care Units in the São Paulo State, Brazil. The second was a systematic review of scientific literature and "gray literature" to identify reports of CRAB and CRE infections in Brazil. Our results in the State of São Paulo showed opposite temporal trends for CRAB (reduction) and CRE (growth). In both cases the incidence is higher in the Metropolitan Regions of São Paulo and Campinas, from where the microorganisms seem to spread to other areas. Infections were more frequent in public hospitals and in those with a lower proportion of ICU beds. Socio-economic and demographic factors presented variable but plausible associations with the incidence of microorganisms of interest. Results of the systematic review point to an initial concentration of CRAB and CRE reports in the Brazilian Southeast, followed by a dispersion in the country. The... (Complete abstract click electronic access below) / Doutor

Orientador: Pasqual Barretti / Resumo: Peritonite por bacilos Gram-negativos não fermentadores (BGNNF) é complicação importante da diálise peritoneal (DP), com curso clínico grave e elevada taxa de falência do método. Fatores associados à virulência, resistência antimicrobiana, formação de biofilme, entre outros, têm sido relatados, mas o limitado conjunto de evidências não permite concluir sobre os fatores responsáveis pelo pior curso clínico dessas infecções. O objetivo deste trabalho foi avaliar a influência das características microbiológicas, das condições clínicas do paciente e do tratamento na evolução de peritonites por BGNNF, ocorridas num único centro, em período de 18 anos. A sensibilidade in vitro aos antimicrobianos, produção de biofilme, além da análise do perfil clonal das bactérias pela técnica de eletroforese em gel de campo pulsado foram realizadas em todos os isolados bacterianos. Foram pesquisados genotipicamente, em isolados de Pseudomonas aeruginosa, a presença de marcadores de virulência (alginato, exoenzima S, fosfolipases C, exotoxina A, protesase alcalina, elastase e ramnolipídeos). Associações entre as características microbiológicas do paciente e tratamento com a taxa de resolução da peritonite foram estudadas. A espécie mais frequente foi Pseudomonas aeruginosa (45,59%), seguida por isolados do complexo Acinetobacter baumannii (17,65%). O estudo dos fatores de virulência da Pseudomonas aeruginosa revelou a presença de fatores de virulência em 100% dos casos, exceto exoenzima S (58,33%)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Peritonitis due to non-fermentative Gram-negative bacilli (NFGNB) is a serious complication of peritoneal dialysis (PD), with a severe clinical course and high technique failure rate. Factors as bacterial virulence, antimicrobial resistance, biofilm formation, among others, have been reported, but the limited amount of evidence does not allow to conclude on the factors responsible for the worst clinical course of these infections. The objective of this study was to evaluate the influence of the microbiological characteristics, patients conditions, and treatment on evolution of peritonitis episodes at a single center in an 18 - year period. In vitro susceptibility, biofilm production, and clonal profile analysis of bacteria by pulsed-field gel electrophoresis (PFGE) were performed in all isolates. The presence of virulence markers (alginate, exoenzyme S, phospholipases C, exotoxin A, alkaline protease, elastase, and ramnolipids) was genotyped in bacterial isolates of Pseudomonas aeruginosa. From the data referring to the patient and causal agent, associations between the microbiological, patient characteristics, and treatment on the resolution rate of peritonitis were analyzed. The most frequent species was Pseudomonas aeruginosa (45.59%), followed by Acinetobacter baumannii complex (17.65%). The study of the virulence factors of Pseudomonas aeruginosa revealed the presence of virulence factors in 100% of the cases, except for exonzyme S (58.33%) and hemolytic phospholipase C ... (Complete abstract click electronic access below) / Mestre

This study focuses on the question of the epidemiology of resistance to antibiotics in Acinetobacter baumannii, which is nowadays one of the most problematic bacterial patho- gens associated with failing antimicrobial therapy. Its aim was to define population-genetic properties, epidemiology and the nature of multidrug resistance for a sample of the current population of A. baumannii from Czechia. A total of 55 isolates were collected in eight medi- cal facilities in central Bohemia from October 2016 to May 2018. The isolates were assessed for their identity at the species, clonal and strain levels as well as resistance phenotype and genotype; they were classified into five clonal groups, each of which encompassed isolates that were likely to be epidemiologically related. The 55 isolates studied belonged, nearly exclusively, to global clone ECII, with 53 % of them forming a genetically relatively homoge- neous group characterized by extensive resistance to antibiotics (susceptible only to col- istin), the presence of genes encoding ArmA a OXA-23 (resistance to all aminoglycosides and carbapenems) and spread in all locations. The in-depth epidemiological analysis of isolates from the city of Příbram and its vicinity indicated the regional spread of two strains, one of which belonged to the...

Submitted by MIRIAN MARCOLAN DE MELLO null (marcolanmirian@yahoo.com.br) on 2016-02-16T08:13:42Z No. of bitstreams: 1 TESE Doutorado Mirian14_02_2016REPOSITÓRIO.pdf: 1649527 bytes, checksum: c26e5ed45e88f8a3889873caa917015a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-02-17T13:45:10Z (GMT) No. of bitstreams: 1 mello_mm_dr_sjc.pdf: 1649527 bytes, checksum: c26e5ed45e88f8a3889873caa917015a (MD5) / Made available in DSpace on 2016-02-17T13:45:10Z (GMT). No. of bitstreams: 1 mello_mm_dr_sjc.pdf: 1649527 bytes, checksum: c26e5ed45e88f8a3889873caa917015a (MD5) Previous issue date: 2015-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Devido ao rápido aumento dos micro-organismos resistentes aos antibióticos e ao desenvolvimento limitado de novos agentes antimicrobianos, as infecções por bactérias Gram-negativas estão se tornando um desafio para os profissionais da saúde e uma ameaça para a saúde pública internacional. O objetivo desse estudo foi avaliar o efeito sinérgico dos antibióticos convencionais associados a terapia fotodinâmica antimicrobiana (PDT) no controle de Acinetobacter baumannii. Para realização desse trabalho, foram obtidos isolados clínicos de A. baumannii do Laboratório de Análises Clínicas Valeclin da cidade de São José dos Campos/SP, identificados pelo método de bioquimismo e submetidos ao teste de difusão em disco para verificar a sensibilidade antimicrobiana. Os isolados selecionados foram transferidos para o ICT/UNESP, onde foi realizado testes para determinação da Concentração Inibitória Mínima aos antibióticos Imipenem e Meropenem seguindo as normas da CLSI. Cepas sensíveis e resistentes aos antibióticos foram avaliadas quanto a sensibilidade in vitro à terapia fotodinâmica antimicrobiana. Além disso, foram testados os efeitos dos antibióticos convencionais, da PDT e da terapia combinada de antibióticos e PDT nas infecções experimentais induzidas em G. mellonella por isolados clínicos de A. baumannii resistentes aos antibióticos. Os resultados das terapias na infecção experimental foram avaliados por meio da curva de sobrevivência das lagartas de G. mellonella. Os dados dos testes in vitro foram submetidos à Análise de Variância e teste de Tukey. Os dados obtidos na curva de sobrevivência de G. mellonella foram analisados pelo método de Log-rank. Em todos os testes, foi considerado nível de significância de 5%. Nos resultados desse estudo, observou-se que o Laboratório Valeclin identificou 1,54% de amostras positivas para A. baumannii entre as 13.715 amostras clínicas analisadas em um período de 8 meses. Entre os isolados de A. baumannii, 58% demonstraram resistência aos antibióticos imipenem e meropenem por meio de teste de difusão em disco. A seguir 3 isolados clínicos sensíveis e 18 isolados resistentes a esses antibióticos foram selecionados para o presente estudo. O valor de CIM para os isolados sensíveis variou de ˂ 0,5 a 1µg/mL e para os isolados resistentes de 64 a >128µg/mL. A PDT in vitro reduziu o número de células de A. baumannii em todos os isolados testados, mas o percentual de redução foi dependente dos isolados analisados. Além disso, verificou-se nos testes in vivo, que o tratamento com PDT, antibióticos (Imipenem e Meropenem) e associação de PDT+Antibióticos resultaram na sobrevivência das lagartas de G. mellonella, porém sem efeito sinérgico. Conclui-se que a PDT teve ação antimicrobiana contra isolados clínicos de A. baumannii sensíveis e resistentes aos carbapenêmicos, mas não apresentou efeito sinérgico quando associada com antibióticos. / Due to the rapid growth of microorganisms resistant to antibiotics and the limited development of new antimicrobial agents, infections by Gram-negative bacteria are becoming a challenge for health professionals and a threat to international public health. The aim of this study was to evaluate the synergistic effect of conventional antibiotics associated with antimicrobial photodynamic therapy (PDT) in control of Acinetobacter baumannii. In order to conduct this project were obtained clinical isolates of A. baumannii at the Clinical Laboratory Valeclin situated in the city of São José dos Campos / SP, identified by bioquimismo method and submitted to disk diffusion test to verify the antimicrobial sensitivity. The selected isolates were transferred to the ICT / UNESP, which were conducted tests to determine the Minimum Inhibitory Concentration to Imipenem and Meropenem antibiotics following the rules of the CLSI. Sensitive and resistant strains to antibiotics were evaluated in vitro sensitivity to antimicrobial photodynamic therapy. Besides, the effects of conventional antibiotics, and combined PDT, and PDT of antibiotics in experimental infections induced in G. mellonella by clinical isolates of A. baumannii resistant to antibiotic therapy were tested. The results of therapies in experimental infection were evaluated by survival curve of worms G. mellonella. Data from in vitro tests were submitted to ANOVA and Tukey test. The data obtained in G. mellonella survival curve were analyzed by log-rank method. In all tests it was considered 5% significance level. The results of this study, it was observed that the Valeclin Laboratory identified 1.54% of positive samples for A. baumannii between the 13,715 clinical specimens analyzed in a period of 8 months. Among the isolates of A. baumannii, 58% were resistant to antibiotic imipenem and meropenem by disk diffusion test. Next, 3 isolates clinical sensitive and 18 isolates resistant to those antibiotics were selected for this study. The MIC value for sensitive isolates ranged from 0.5 to ˂ 1μg / mL and resistant isolates from 64 to> 128μg / mL. The PDT in vitro reduced the number of A. baumannii cells in all isolates tested, but the percentage of reduction was dependent on the analyzed isolates. Furthermore, it was found in in vivo tests, treatment with PDT, antibiotic (Imipenem and Meropenem) and PDT + Antibiotics association resulted in the survival of G. mellonella caterpillars, but no synergistic effect. It was concluded that PDT has antimicrobial activity against clinical isolates of A. baumannii sensitive and resistant to carbapenems, but it had no synergistic effect when combined with antibiotics.

Submitted by Ana Cláudia Moro Lima dos Santos (anna.moro@hotmail.com) on 2018-05-07T20:15:18Z No. of bitstreams: 1 Dissertaçãofinal.pdf: 1989003 bytes, checksum: c02493762a88023038e4fc08eea12f1c (MD5) / Approved for entry into archive by Sulamita Selma C Colnago null (sulamita@btu.unesp.br) on 2018-05-09T16:37:26Z (GMT) No. of bitstreams: 1 santos_acml_me_bot.pdf: 1989003 bytes, checksum: c02493762a88023038e4fc08eea12f1c (MD5) / Made available in DSpace on 2018-05-09T16:37:26Z (GMT). No. of bitstreams: 1 santos_acml_me_bot.pdf: 1989003 bytes, checksum: c02493762a88023038e4fc08eea12f1c (MD5) Previous issue date: 2018-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Peritonite por bacilos Gram-negativos não fermentadores (BGNNF) é complicação importante da diálise peritoneal (DP), com curso clínico grave e elevada taxa de falência do método. Fatores associados à virulência, resistência antimicrobiana, formação de biofilme, entre outros, têm sido relatados, mas o limitado conjunto de evidências não permite concluir sobre os fatores responsáveis pelo pior curso clínico dessas infecções. O objetivo deste trabalho foi avaliar a influência das características microbiológicas, das condições clínicas do paciente e do tratamento na evolução de peritonites por BGNNF, ocorridas num único centro, em período de 18 anos. A sensibilidade in vitro aos antimicrobianos, produção de biofilme, além da análise do perfil clonal das bactérias pela técnica de eletroforese em gel de campo pulsado foram realizadas em todos os isolados bacterianos. Foram pesquisados genotipicamente, em isolados de Pseudomonas aeruginosa, a presença de marcadores de virulência (alginato, exoenzima S, fosfolipases C, exotoxina A, protesase alcalina, elastase e ramnolipídeos). Associações entre as características microbiológicas do paciente e tratamento com a taxa de resolução da peritonite foram estudadas. A espécie mais frequente foi Pseudomonas aeruginosa (45,59%), seguida por isolados do complexo Acinetobacter baumannii (17,65%). O estudo dos fatores de virulência da Pseudomonas aeruginosa revelou a presença de fatores de virulência em 100% dos casos, exceto exoenzima S (58,33%) e fosfolipase C não hemolítica (87,5%). Houve elevada proporção de BGNNF resistentes aos antimicrobianos testados, em particular à amicacina (36,73%) e à ciprofloxacina (44,9%), sendo que a sensibilidade aos betalactâmicos esteve acima de 70%. Observou-se elevada proporção de isolados produtores de biofilme (73,08%). Os resultados da tipagem por PFGE revelaram um perfil policlonal para a maioria dos isolados, entretanto para isolados do complexo Acinetobacter baumannii a análise revelou um cluster, entre 2000-2008, com perfil de multiresistência aos antimicrobianos, sugerindo fonte hospitalar. A evolução dos episódios mostrou reduzida taxa de cura (35,29%). A sensibilidade à amicacina e cefepime, se associaram de modo independente à maior chance de cura, enquanto a presença concomitante de infecção do óstio de saída do cateter de DP foi preditor independente de não resolução do episódio. Não se observaram associações entre fatores de virulência, produção de biofilme e características do paciente e tratamento com o desfecho dos episódios. Em conclusão, peritonites em DP, por BGNNF, são infecções com reduzida taxa de cura; a resistência bacteriana é fator associado à menor chance de resolução e peritonite por bactérias do gênero Acinetobacter spp. podem representar infecção grave, potencialmente de origem hospitalar, o que deve fazer redobrar os cuidados quanto ao seu manejo clínico. / Peritonitis due to non-fermentative Gram-negative bacilli (NFGNB) is a serious complication of peritoneal dialysis (PD), with a severe clinical course and high technique failure rate. Factors as bacterial virulence, antimicrobial resistance, biofilm formation, among others, have been reported, but the limited amount of evidence does not allow to conclude on the factors responsible for the worst clinical course of these infections. The objective of this study was to evaluate the influence of the microbiological characteristics, patients conditions, and treatment on evolution of peritonitis episodes at a single center in an 18 - year period. In vitro susceptibility, biofilm production, and clonal profile analysis of bacteria by pulsed-field gel electrophoresis (PFGE) were performed in all isolates. The presence of virulence markers (alginate, exoenzyme S, phospholipases C, exotoxin A, alkaline protease, elastase, and ramnolipids) was genotyped in bacterial isolates of Pseudomonas aeruginosa. From the data referring to the patient and causal agent, associations between the microbiological, patient characteristics, and treatment on the resolution rate of peritonitis were analyzed. The most frequent species was Pseudomonas aeruginosa (45.59%), followed by Acinetobacter baumannii complex (17.65%). The study of the virulence factors of Pseudomonas aeruginosa revealed the presence of virulence factors in 100% of the cases, except for exonzyme S (58.33%) and hemolytic phospholipase C (87.5%). There was a high proportion of antimicrobial resistant, in particular to amikacin (36.73%) and ciprofloxacin (44.9%), with sensitivity to betalactam above 70%. A high (73.08%) proportion of biofilm producing isolates was observed. The results of the PFGE typing revealed a polyclonal profile for most of the isolates; however, for the Acinetobacter baumannii complex species the analysis revealed a cluster at interval from 2000 to 2008, with antimicrobial multi resistance profile, suggest that peritonitis by this agent had a hospital source. The evolution of the episodes showed a reduced resolution rate (35.29%). The susceptibility to amikacin and to cefepime were independently associated with a higher odds of resolution, while the concomitant presence of PD catheter exit site infection was an independent predictor of non-resolution. In conclusion, peritonitis due to NFGNB in PD are infections with reduced resolution rate; bacterial resistance is an independent predictor of lower odds of resolution. Peritonitis by Acinetobacter spp. can represent serious / 64736017.2.0000.5411

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Infections caused by Acinetobacter baumannii and Pseudomonas aeruginosa multiresistant are responsible for high morbidity and mortality, failure of drug therapy, increased hospital stay and consequently the financial impact on the health system. However, while the occurrence of these bacteria to configure a public health problem, numerous studies reveal that it is scarce information about the resistance genes present in multi-drug resistant bacteria. This reality associated with the negative impact of these on society, justifies the importance of detecting the resistance genes of A. baumannii and P. aeruginosa multiresistant and clinically characterize patients from a public hospital in Sergipe. It is an analytical prospective cohort study and a quantitative approach. The collection of clinical data of patients was carried out through a specifically designed form. Strains of A. baumannii were subjected to PCR for identification of resistance genes (blaIMP, blaVIM, blaSIM, bla OXA-51, blaOXA-58, blaOXA-23 and blaOXA-24) and P. aeruginosa the blaSPM genes, blaVIM, blaIMP, blaKPC. Descriptive analyzes were performed, the Chi-square and Fisher exact tests, with 5% significance level. The software used was R version 3.1.2. The sample consisted of 119 patients. Of the 43 patients with P. aeruginosa isolates, 33 were male (76.7%) with mean age of 46.2 years. Twenty-eight were admitted to the ICU (65.1%) and 13 (30.2%) diagnosed with head trauma (TBI). Of the 76 patients with isolates of A. baumannii, 59 (77.6%) were male, mean age of 44.4 years. Fifty patients (65.8%) were from the ICU and 18 (23.7%) diagnosed with TBI. The median number of days of hospitalization was statistically significant between bacteria. Among the isolation sites, there is urine to P. aeruginosa, with 16 samples (37.2%) and tracheal aspirate for A. baumannii with 32 (42.1%) strains. A urinary catheter was the most used device in patients with isolates of A. baumannii (93.4% - 71) and the central venous catheter in patients with P. aeruginosa (93% - 40). All patients with P. aeruginosa isolates made use of carbapenems and 98.6% (75) of A. baumannii. It found statistically significant differences between bacteria in the use of aminoglycosides, 3rd generation cephalosporins and tigecycline. In P. aeruginosa was no significant difference in the use of oxacillin and cephalosporins of 1st and 3rd generations and polymyxin in the various sectors of the hospital. All the samples of A. baumannii and P. aeruginosa were susceptible to colistin, ranging between MIC <= 0.5 and 2. The majority (55.8% - 24) patients with P. aeruginosa and A. baumannii (52.6% - 40) died. Among the 76 strains of A. baumannii, 56 (73.6%) had concomitant both blaOXA-51 and blaOXA-23 genes. Among the 43 strains of P. aeruginosa, 28 (65.1%) had the blaSPM gene. It was concluded that the A.baumannii was more frequent than P. aeruginosa. There was a significant predominance of Pseudomonas and Acinetobacter in the urine in tracheal aspirates. Carbapenems was widely used throughout the hospital stay of patients with Acinetobacter and Pseudomonas isolates in different hospital departments. Most Pseudomonas strains showed blaSPM resistance gene and Acinetobacter blaOXA-23 genes and blaOXA-51 concurrently. The mortality of patients with Acinetobacter and Pseudomonas was greater than 50%. / As infecções causadas por Acinetobacter baumannii e Pseudomonas aeruginosa multirresistentes são responsáveis pela alta morbidade e mortalidade, falência da terapia medicamentosa, aumento do período de internação e consequentemente impacto financeiro no sistema de saúde. Todavia, embora a ocorrência destas bactérias se configure um problema de saúde pública, inúmeros estudos revelam que é escasso o conhecimento acerca dos genes de resistência presentes nas bactérias multirresistentes. Essa realidade associada ao impacto negativo destas na sociedade, justifica a importância de detectar os genes de resistência de A. baumannii e P. aeruginosa multirresistentes e caracterizar clinicamente os pacientes de um hospital público de Sergipe. Trata-se de um estudo analítico de coorte prospectiva e abordagem quantitativa. A coleta dos dados clínicos dos pacientes foi realizada através de um formulário especificamente elaborado. As cepas de A. baumannii foram submetidas à técnica PCR para identificação dos genes de resistência (blaIMP, blaVIM, blaSIM, bla OXA-51, blaOXA-58, blaOXA-23 e blaOXA-24) e em P. aeruginosa os genes blaSPM, blaVIM, blaIMP, blaKPC. Foram realizadas análises descritivas, os testes de Qui-Quadrado e Exato de Fisher, com nível de significância de 5%. O software utilizado foi o R versão 3.1.2. A amostra foi constituída de 119 pacientes. Dos 43 pacientes com isolados de P.aeruginosa, 33 eram do sexo masculino (76,7%), com idade média de 46,2 anos. Vinte e oito estavam internados na UTI (65,1%) e 13 (30,2%) com diagnóstico de trauma crânio encefálico (TCE). Dos 76 pacientes com isolados de A. baumannii, 59 (77,6%) era do sexo masculino, média de idade de 44,4 anos. Cinquenta pacientes (65,8%) eram procedentes da UTI e 18 (23,7%) com diagnóstico de TCE. A mediana de dias de internamento foi estatisticamente significante entre as bactérias. Dentre os sítios de isolamento, destaca-se a urina para P. aeruginosa, com 16 amostras (37,2%) e o aspirado traqueal para A. baumannii com 32 (42,1%) cepas. A sonda vesical foi o dispositivo mais usado nos pacientes com isolados de A. baumannii (93,4% - 71) e o cateter venoso central nos pacientes com P. aeruginosa (93% - 40). Todos os pacientes com isolados de P.aeruginosa fizeram uso dos carbapenêmicos e 98,6% (75) dos A. baumannii. Foi encontrado diferença estatisticamente significante entre as bactérias quanto ao uso dos aminoglicosídeos, cefalosporinas de 3ª geração e tigeciclina. Em P. aeruginosa houve diferença significativa no uso da oxacilina e cefalosporinas de 1ª e 3ª gerações e polimixina nos diversos setores do hospital. Todos as amostras de A. baumannii e P. aeruginosa apresentaram sensibilidade à colistina, com variação da MIC entre < = 0,5 e 2. A maioria (55,8% - 24) dos pacientes com P. aeruginosa e A. baumannii (52,6% - 40) foram a óbito. Dentre as 76 cepas de A. baumannii, 56 (73,6%) apresentaram concomitantemente os dois genes blaOXA-51 e blaOXA-23. Dentre as 43 cepas de P. aeruginosa, 28 (65,1%) apresentaram o gene blaSPM. Concluiu-se que o A.baumannii foi mais frequente do que a P.aeruginosa. Houve predomínio significante da Pseudomonas na urina e do Acinetobacter na secreção traqueal. Os carbapenêmicos foi amplamente utilizado ao longo da internação dos pacientes com isolados de Acinetobacter e Pseudomonas nos diversos setores do hospital. A maioria das cepas de Pseudomonas apresentaram gene de resistência blaSPM e Acinetobacter os genes blaOXA-23 e blaOXA-51 concomitantemente. A mortalidade dos pacientes com Acinetobacter e Pseudomonas foi superior a 50%.

Over time, “superbugs,” or bacteria that have become resistant to antibiotics, have become a great concern in modern medicine. Viable alternates are currently being looked into as effective and safe ways to prevent or treat infections caused by these superbugs. One such method is through the utilization of the second messenger molecule cyclic-di-GMP (c-di-GMP) that has been shown to regulate phenotypes within other bacteria that may control surface colonization in Acinetobacter baumannii. Through a series of experiments, the active enzymes that create c-di-GMP - diguanylate cyclases - and break down c-di- GMP - phosphodiesterases - have been inactivated in mutants to test phenotypes including biofilm formation, motility, antibiotic resistance, and desiccation survival. The research’s objective is to show that manipulation of c-di-GMP within the multi-drug resistant strain of Acinetobacter baumannii may serve as a means to control this bacteria.

Bibliography / Thesis (MMed (Pathology. Medical Microbiology))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Acinetobacter baumannii has emerged as one of the most troublesome nosocomial pathogens globally. This organism causes infections that are often extremely difficult to treat because of the widespread resistance to the major antibiotic groups. Colonization or infection with multidrugresistant A. baumannii is associated with the following risk factors: prolonged hospital stay, admission to an intensive care unit (ICU), mechanical ventilation, and exposure to broad spectrum antibiotics, recent surgery, invasive procedures, and severe underlying disease. A. baumannii has been isolated as part of the skin flora, mostly in moist regions such as axillae, groin and toe webs. It has also been isolated from the oral cavity and respiratory tract of healthy adults. Debilitated hospitalized patients have a high rate of colonization, especially during nosocomial Acinetobacter outbreaks. This organism is an opportunistic pathogen as it contains few virulence factors. Clinical manifestations of A. baumannii include nosocomial pneumonia, nosocomial bloodstream infections, traumatic battlefield and other wound infections, urinary tract infections, and post-neurological surgery meningitis. Fulminant community-acquired pneumonia has recently been reported, indicating that this organism can be highly pathogenic. The number of multidrug-resistant A. baumannii strains has been increasing worldwide in the past few years. Therefore the selection of empirical antibiotic treatment is very challenging. Antibiotic combinations are used mostly as empirical therapy in critically ill patients. One rationale for the use of combination therapy is to achieve synergy between agents. The checkerboard and time-kill methods are two traditional methods that have been used for synergy testing. These methods are labor intensive, cumbersome, costly, and time consuming. The E-test overlay method is a modification of the E-test method to determine synergy between the different antibiotics. This method is easy to perform, flexible and time efficient. The aim of this study was to assess the in vitro activity of different combinations of colistin, rifampicin, imipenem, and tobramycin against selected clinical strains of A. baumannii using the checkerboard and the E-test synergy methods. The MICs obtained with the E-test and broth microdilution method were compared. The results of the disk diffusion for imipenem and tobramycin as tested in the routine microbiology laboratory were presented for comparison. Overall good reproducibility was obtained with all three methods of sensitivity testing. The agreement of MICs between the broth dilution and E-test methods was good with not more than two dilution differences in MIC values for all isolates, except one in which the rifampicin E-test MIC differed with three dilutions from the MIC obtained with the microdilution method. However, the categorical agreement between the methods for rifampicin was poor. Although MICs did not differ with more than two dilutions in most cases, many major errors occurred because the MICs clustered around the breakpoints. The combinations of colistin + rifampicin, colistin + imipenem, colistin + tobramycin, rifampicin + tobramycin, and imipenem + tobramycin all showed indifferent or additive results by the E-test method. No results indicating synergy were obtained for all the above-mentioned combinations. There was one result indicating antagonistic effect for the combination of colistin + tobramycin. The results of the checkerboard method showed results indicating synergy in four of the six isolates for which the combination of colistin and rifampicin was tested. The other two isolates showed indifferent/additive results. All the other combinations showed indifferent/additive results for all isolates except isolate 30 (col + tob) and isolate 25 (rif + tob) which showed synergism. No antagonistic results were observed by the checkerboard method. When the results obtained with the E-test and checkerboard methods were compared, it was noted that for most antibiotic combinations an indifferent/additive result was obtained. However, for the colistin + rifampicin combination, the checkerboard method showed synergism for 4 of 6 isolates, whereas the E-test method showed indifference and an additive result in one. For the rifampicin + tobramycin, and colistin + tobramycin combinations, synergism was also shown with the checkerboard method in one isolate for each combination. The E-test method however showed an indifferent and additive result respectively. . The E-test method was found to be a rapid, reproducible, easy-to-perform, and flexible method to determine synergistic antibiotic activity. This study was however limited by low numbers of isolates. This might explain why no synergistic results were obtained with the E-test method and few synergistic results with the checkerboard method. Genotypic analysis using pulse-field gel electrophoresis (PFGE) may be considered in future studies to determine relatedness of the isolates which will facilitate the selection of different strains for synergy testing. Furthermore, clinical studies are needed to establish whether in vitro synergy testing is useful in the clinical setting and whether the results of synergy testing will have any bearing on the clinical outcome of patients infected with multidrug resistant A. baumannii. / AFRIKAANSE OPSOMMING: Acinetobacter baumannii het wêreldwyd as een van die mees problematiese nosokomiale patogene verskyn. Hierdie organisme veroorsaak infeksies wat dikwels baie moeilik is om te behandel weens wydverspreide weerstandigheid teen major antibiotikagroepe. Kolonisasie of infeksie met multi-weerstandige A. baumannii word geassosieer met die volgende riskofaktore: verlengde hospitaalverblyf, toelating tot ‘n intensiewe sorgeenheid (ICU), meganiese ventilasie, blootstelling aan breëspektrum antibiotika, onlangse chirurgie, indringende prosedures en ernstige onderliggende siekte. A. baumannii kan deel vorm van die normale velflora, veral in die axillae, inguinale area en tussen die tone. Dit is ook al vanuit die mondholte en die respiratoriese traktus van gesonde volwassenes geïsoleer. Verswakte gehospitaliseerde pasiënte word veral gekoloniseer gedurende nosokomiale Acinetobacter uitbrake. Hierdie organisme is ‘n opportunistiese patogeen en bevat min virulensie faktore. Kliniese manifestasies van A. baumannii sluit nosokomiale pneumonie, nosokomiale bloedstroom infeksies, troumatiese slagveld- en ander wondinfeksies, urienweginfeksies en meningitis wat volg op neurologiese chirurgie in. Fulminerende gemeenskapsverworwe pneumonie is onlangs beskryf en dui aan dat hierdie organisme hoogs patogenies kan wees. Die aantal multi-weerstandige A. baumannii stamme het wêreldwyd toegeneem oor die laaste paar jare. Daarom is die seleksie van empiriese antibiotiese behandeling ‘n uitdaging. Antibiotika kombinasies word meestal as empiriese behandeling in ernstige siek pasiënte gebruik. Die beginsel hiervan is om sinergistiese werking tussen agente te verkry. Die “checkerboard” en “time-kill” metodes is twee tradisionele metodes van sinergisme toetsing. Hierdie metodes is werksintensief, duur en tydrowend. Die E-toets sinergisme metode is gebaseer op die E-toets metode. Hierdie metode is maklik, buigbaar en tydseffektief. Die doel van hierdie studie was om die in vitro aktiwiteit tussen verskillende antibiotika kombinasies van colistin, rifampisien, imipenem, en tobramisien teen geselekteerde kliniese A. baumannii isolate te toets met die “checkerboard” en E-toets sinergisme toetsing metodes. Die minimum inhibitoriese konsentrasies (MIKs) verkry met die E-toets en “broth microdilution” metode is ook vergelyk. Die resultate van die skyfie diffusie metode (die metode wat in die roetiene mikrobiologie laboratorium gebruik word) vir imipenem en tobramisien word ook verskaf vir vergelyking van die resultate van verskillende sensitiwiteitsmetodes. In oorsig is goeie herhaalbaarheid van resultate verkry met al drie metodes van sensitiwiteitstoetsing. Die ooreenstemming van MIKs tussen die “broth dilution” en E-toets metodes was goed en resultate het met nie meer as twee verdunnings in MIK waardes verskil nie. Daar is een uitsondering waar die rifampisien E-toets MIK waarde met drie verdunnings van die MIK waarde verkry met die “microdilution” metode verskil. Die ooreenstemming tussen die sensitiwiteitskategorie resultate tussen die twee metodes was egter swak vir rifampisien. Alhoewel die MIKs in die meeste gevalle met nie meer as twee verdunnings in waarde verskil het nie, was daar baie major foute aangetoon omdat die MIKs rondom die breekpunte geval het. Die kombinasies van colistin + rifampisien, colistin + imipenem, colistin + tobramisien, rifampisien + tobramisien, en imipenem + tobramisien het oorwegend slegs matige interaksie met die E-toets metode getoon. Geen sinergisme is verkry met enige van die antibiotika kombinasies met hierdie metode nie. Daar was egter een resultaat wat antagonisme getoon het vir die kombinasie van colistin + tobramycin. Die resultate van die “checkerboard” metode het sinergisme getoon in vier van die ses isolate wat vir die kombinasie van colistin en rifampisien getoets was. Die ander twee isolate het slegs matige interaksie getoon. Al die ander kombinasies het ook slegs matige interaksie getoon, behalwe in isolaat 30 (col + tob) en isolaat 25 (rif + tob) waar die spesifieke kombinasies sinergisme getoon het. Geen antagonisme is waargeneem met die “checkerboard” metode nie. Met vergelyking van die E-toets en “checkerboard” metodes, is dit opmerklik dat vir die meeste van die antibiotika kombinasies slegs matige interaksie verkry is. Vir die colistin + rifampisien kombinasie toon die “checkerboard” metode egter sinergisme vir 4 uit 6 isolate, terwyl die E-toets metode slegs matige interaksie toon. Vir rifampisien + tobramisien, en colistin + tobramisien kombinasies is sinergisme getoon met die “checkerboard” metode in een isolaat vir elke kombinasie. Die E-toets metode het slegs matige interaksie getoon. Die E-toets sinergisme metode was vinnig, herhaalbaar en maklik om uit te voer. Hierdie studie word egter beperk deur lae getalle van isolate. Dit mag verklaar waarom geen sinergistiese resultate met die E-toets metode verkry is nie en die min sinergistiese resultate met die “checkerboard” metode. Genotipiese analiese met “pulse-field gel electrophoresis” mag in aanmerking geneem word in toekomstige studies om die verwantskap tussen isolate te bepaal wat die seleksie van verskillende stamme vir sinergisme toetsing sal vergemaklik. Verder, kliniese studies is nodig om te bepaal of in vitro sinergisme toetsing van waarde is en of die resultate van sinergisme toetsing ‘n rol speel in die kliniese uitkoms van pasënte geïnfekteer met multiweerstandige A. baumannii. / The National Health Laboratory Serivice

Contexto: O Acinetobacter spp. é um cocobacilo gram-negativo, considerado patógeno oportunista e de grande importância nas infecções hospitalares. Estão envolvidos em amplo espectro de infecções nosocomiais, incluindo bacteremia, meningite secundária e infecção do trato urinário, mas sua maior prevalência é como agente de pneumonia associada à ventilação mecânica em pacientes internados em unidades de terapia intensiva (UTIs); podendo ocasionar um agravamento do quadro clínico e o óbito desses pacientes. Considera-se como um patógeno de baixa virulência, podendo permanecer sobre a pele ou dentro do corpo humano sem causar doença. A disseminação pelas mãos dos profissionais de saúde geralmente não é detectada e quando as infecções pelo Acinetobacter tornam-se aparentes o número de pacientes colonizados é, provavelmente, muito elevado. Assim sendo, as precauções para prevenir um surto tornam-se tardias. Estudos prévios indicaram como fatores de risco para aquisição de infecção por Acinetobacter a gravidade da doença dos pacientes, uso prévio de antimicrobiano, número de dias com procedimento invasivo, tempo de permanência no hospital, contaminação ambiental. Os fatores de risco associados à mortalidade de pacientes com A. baumannii ainda não foram totalmente elucidados pela literatura, mas a idade, colonização prévia por esta bactéria, neutropenia, escore de gravidade APACHE II (Acute Physiology and Chronic Health Evaluation) elevado, procedimentos como ventilação mecânica, terapia antimicrobiana inapropriada são apontados como alguns dos fatores relacionados. Objetivos: Caracterizar a mortalidade atribuível a infecções causadas por Acinetobacter baumannii resistente à carbapenêmicos (CRAB) em um surto no 13 Centro de Terapia Intensiva adulto de um hospital universitário. Métodos: Foi realizado um estudo de coorte retrospectivo pareado como parte da investigação do surto de pacientes no Centro de Tratamento Intensivo (CTI) Adulto infectados com a bactéria Acinetobacter baumannii apresentando resistência à carbapenêmicos. Os pacientes foram selecionados entre 01/01/2007 a 31/07/2008 e foram considerados como casos os pacientes com cultura positiva para CRAB. Os controles foram pacientes internados no CTI no mesmo período que os casos, mas que não apresentaram infecção na qual foi isolada a presença da bactéria em questão. Os fatores avaliados como possível associação com o risco de mortalidade foram avaliados. Determinou-se a mortalidade atribuível a infecções causadas por CRAB e através da curva de sobrevivência avaliou-se essa distribuição entre casos e controles. Resultados: Foram selecionados 90 pacientes como casos e 179 pacientes pareados como controles. A média de idade, as proporções de pacientes com Escore de Chalson ³ 2, de pacientes internados não eletivamente, as reinternações e a freqüência de realização de cirurgias foram muito semelhantes entre os grupos estudados. Entre os casos, houve maior proporção de pacientes transferidos de outro hospital (P<0,001), internados em área contígua à presença de casos de colonização ou infecção por CRAB (P<0,001), de pacientes submetidos a alimentação parenteral (P<0,001); ventilação mecânica (P<0,001), cateteres urinários (P=0,031), cateteres para acesso vascular central (P=0,006) e cateteres para hemodiálise (P<0,001) comparativamente aos controles. Da mesma maneira, casos apresentaram maior freqüência de exposição prévia a antimicrobianos, comparativamente aos controles: penicilinas (P<0,001), cefalosporinas de 1ª e/ou 2ª gerações (P<0,001), carbapenêmicos (P<0,001), aminoglicosídeos (P=0,046), quinolonas (P=0,004) e 14 glicopeptídeos (P=0,001). Os casos apresentaram tempo médio de internação superior aos controles, incluindo duração total da internação (P=0,002), permanência na CTI (P<0,001) e permanência na CTI antes da infecção por CRAB (P=0,03). O escore de APACHE II por ocasião da admissão no CTI também teve média significativamente maior entre os casos comparativamente aos controles (P<0,001). Houve diferença na taxa de mortalidade bruta intra-hospitalar entre casos e controles, respectivamente, 58,9% (53/90) e 36,9% (66/179) (P=0,001). A mortalidade atribuível foi 22% (IC 95%; 8,8%-35,2%) e as curvas de sobrevivência cumulativa para casos e controles não apresentaram diferença significativa entre os grupos (P=0,207; log rank test) A análise multivariável indica que pacientes com escore de APACHE II maiores e que mais freqüentemente foram submetidos a procedimentos invasivos como ventilação mecânica, suporte nutricional (dieta parenteral) e que permaneceram um período maior no hospital estiveram mais propensos a risco de mortalidade associada à infecção por CRAB. Conclusões: Nesse estudo os fatores associados com a mortalidade e a taxa de mortalidade atribuível identificados vão ao encontro da literatura e indica que pacientes mais graves estão mais propensos a risco de morte associada à infecção por CRAB. A literatura enfatiza também a necessidade de consistentes estratégias de controle de infecção para prevenir infecções por Acinetobacter multirresistente. A investigação da mortalidade atribuível ao A. baumannii apresenta muitas limitações e ainda não é conclusiva. / Context: Acinetobacter spp. is a bacilli gram-negative considered an opportunistic pathogen and of great importance in nosocomial infections. They are involved in a wide spectrum of nosocomial infections, including bacteremia, secondary meningitis and urinary tract infection, but is prevalent as an agent of mechanical ventilatorassociated pneumonia in patients admitted to intensive care units (ICUs); this factor can lead to an increase morbidity and mortality of these patients. It is considered as a pathogen of low virulence and may remain on or within the human body without causing disease. The spread by the hands of clinical staff is often not detected and when Acinetobacter infections become apparent, the number of colonized patients is probably very high, therefore, precautions to prevent an outbreak are late. Previous studies have observed as risk factors for acquisition of Acinetobacter infection by the disease severity of patients, prior use of antimicrobials, number of days with invasive procedures, length of stay in hospital environmental contamination. Risk factors associated with mortality of patients with A. baumannii have not been fully elucidated in the literature, but showed that age, previous colonization by this bacterium, neutropenia, high severity score APACHE II (Acute Physiology and Chronic Health Evaluation), procedures such as mechanical ventilation, inappropriate antimicrobial therapy as some of the factors related to mortality. Objectives: To characterize attributable mortality to infections caused by Acinetobacter baumannii resistant to carbapenem (CRAB) in an outbreak in the adult intensive care unit of a university hospital. 16 Methods: We performed a matched retrospective cohort as part of outbreak investigation of patients in the ICU adult infected with the bacteria Acinetobacter baumannii exhibiting resistance to carbapenems. Patients were selected from 01/01/2007 to 31/07/2008 and the cases were considered patients with positive culture for CRAB. Controls were patients admitted to the ICU during the same period as cases, but showed no infection in which was isolated the presence of the bacterium in question. Factors evaluated as possible association with the risk of mortality were evaluated. Determined the attributable mortality to infections caused by CRAB and through the survival curve was evaluated this distribution between cases and controls. Results: 90 patients were selected as cases and 179 patients matched as controls. The average age, the proportions of patients with a Chalson score ³ 2 from inpatients not elective, the frequency of hospitalizations and surgeries were similar among studied groups. Among the cases, a greater proportion of patients transferred from another hospital (P <0.001), admitted in an area contiguous to the presence of cases of colonization or infection by CRAB (P <0.001) in patients undergoing parenteral nutrition (P <0.001) ; mechanical ventilation (P <0.001), urinary catheters (P = 0.031), central catheters for vascular access (P = 0.006) and catheters for hemodialysis (P <0.001) compared to controls. Likewise, cases had higher frequency of prior exposure to antimicrobials, compared with controls: penicillin (P <0.001), cephalosporins of 1st and / or 2nd generation (P <0.001), carbapenems (P <0.001), aminoglycosides (P = 0.046), quinolones (P = 0.004) and glycopeptides (P = 0.001). The cases presented mean length of stay higher than controls, including total duration of hospitalization (P = 0.002), stay in ICU (P <0.001) and stay in the ICU before 17 infection by CRAB (P = 0.03). The APACHE II score on admission to the ICU was also significantly higher average among cases compared with controls (P <0.001). There was a difference in the rate of in-hospital crude mortality among cases and controls, respectively, 58,9% (53/90) e 36,9% (66/179) (P = 0.001). The attributable mortality was 22% (95% CI 8.8% -35.2%) and cumulative survival curves for cases and controls showed no significant difference between groups (P = 0.207, log rank test) Multivariate analysis indicates that patients with APACHE II score higher and more frequently underwent invasive procedures such as mechanical ventilation, nutritional support (parenteral nutrition) and remained a longer period in hospital were more likely to risk of mortality associated with infection by CRAB. Conclusions: In this study the factors associated with mortality and the attributable mortality rate identified are in line with the literature and indicates that more severe patients are more prone to risk of mortality associated with infection by CRAB. The literature also emphasizes the need for consistent infection control strategies to prevent infection by multidrug resistant Acinetobacter. The investigation of attributable mortality to A. baumannii has many limitations and is not conclusive yet.

Introdução: O gênero Acinetobacter representa um importante patógeno relacionado a infecções hospitalares, principalmente, à pneumonia associada à ventilação mecânica (PAV). O aumento de isolados de Acinetobacter baumannii resistentes aos carbapenêmicos (ABRC) representa um problema mundial, pois limita drasticamente as opções terapêuticas. A produção da carbapenamase OXA-23, uma β-lactamase da classe D de Ambler, representa o principal mecanismo responsável por esta resistência em nosso país. Objetivo: O presente estudo tem como objetivo padronizar a técnica de PCR em tempo real (qPCR) para a detecção do gene blaOXA-23 diretamente de aspirados traqueais (ATs) de pacientes com suspeita de pneumonia associada à ventilação mecânica (PAV) de unidades de tratamento intensivo (UTIs). Métodos e resultados: O DNA das amostras de ATs coletadas de pacientes em ventilação mecânica foi analisado pela técnica de qPCR, utilizando o SYBR green, para a detecção do gene blaOXA-23. Dentre os 20 ATs analisados, ABRC foi isolado em 10, A. baumannii sensível aos carbapemêmicos (ABSC) em 3 e 7 foram negativos na culura bacteriológica. O gene blaOXA-23 foi detectado tanto na colônia quanto no AT em 8 das 10 amostras de ABRC. Em uma amostra não houve detecção do gene por qPCR em nenhum dos materiais e em outra amostra houve detecção só no AT. Dos ABSC, em duas amostras não houve detecção do gene na colônia e no AT enquanto que em uma amostra o gene foi detectado somente no AT. Em nenhuma das amostras de ATs negativas na cultura foi detectado o gene. Conclusão: O estudo sugere que a técnica qPCR pode ser aplicada para a detecção do gene blaoxa-23 diretamente de amostras de AT, reduzindo assim, o tempo para o início de uma terpia antimicrobiana adequada e melhorando, consequentemente, o desfecho clínico deste pacientes. / Background: The genus Acinetobacter is an important pathogen associated with nosocomial infections mainly ventilator-associated pneumonia (VAP). The increasing of carbapenemresistant Acinetobacter baumannii (CRAB) isolates is a worldwide concern since it limits drastically the range of therapeutic alternatives. The OXA-23 producing, a carbapenemhydrolysing class D β-lactamase, is the major mechanism responsible for CRAB in our country. Objective: The study objective is to develop a real time PCR (qPCR) to detect the Acinetobacter baumannii blaOXA-23 gene directly in endotracheal aspirate (ETA) of patients under mechanical ventilation, with suspected ventilator-associated pneumonia (VAP). Methods and Results: DNA extracted from ETA samples from patients under mechanical ventilation was analyzed by qPCR, using SYBR green, for the presence of blaOXA-23 gene. Among the 20 ETAs examined, CRAB isolates were recovered in 10 quantitative cultures; carbapenem-susceptible A. baumannii (CSAB) in 3 and 7 cultures were negative to A. baumanni. Of the 10 CRAB, 8 were positive for blaOXA-23 on both the colony and ETA. In one blaOXA-23 qPCR was negative in colony and directly from ETA, while the other showed a qPCR negative result in the colony and positive in the ETA. In 3 CSAB, 2 samples showed negative results in colony and ETA and one showed a blaOXA-23 positive result only from the ETA. None of the 7 negative ETAs were positive for blaOXA-23 gene in the qPCR of the ETA. Conclusion: Our study suggests that qPCR can be applied to detect the presence of blaOXA-23 gene directly from ETAs reducing the time to an earlier initiation of appropriate therapy improving, consequently, the clinical outcomes for these patients.