Indiana University-Purdue University Indianapolis (IUPUI) / The primary goal of root canal treatment is to eliminate microbes from the root canal system, which is the cause of pulpal and periapical infections. Research shows that after a single visit of chemomechanical debridement microbes continue to remain within the canal system. An interappointment medication step has been advocated to maximize potential elimination of microbes within the root canal system. Previous studies have shown propolis to be antibacterial against common endodontic microbes. Studies have shown trends in different microbes being present in primary verus secondary endodontic infections. The majority of literature has focused on the efficacy of propolis against Enterococcus faecalis, a microbe commonly implicated in secondary endodontic 95 infections. The aim of this study was to demonstrate the efficacy of propolis against Fusobacterium nucleatum, a microbe commonly found in primary endodontic infections. This study aims to demonstrate the efficacy of propolis against a bacterium of primary endodontic infections (F. nucleatum) as well as against microbial biofilm to further support its potential use as a novel intracanal medicament. Dilutions of propolis were added to cultures of F. nucleatum in microtiter plates in a range from 390 μg/ml to 50,000 μg/ml. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and the minimum biofilm inhibitory concentration (MBIC) were determined. The MIC was determined of the total solution (biofilm+planktonic), planktonic, and biofilm (MBIC) after a 48-hour incubation period. The MBIC was determined by fixing biofilm to the wells and using crystal violet staining with spectrophotometry. The MBC was examined by plating solution from each concentration test well and reading the plates after 48 hours of incubation. The results show that the MIC of the total (biofilm+planktonic) appears to occur at a concentration of 6250 μg/ml. The MBIC appears to occur at the concentration of 1562.5 μg/ml. The planktonic results exhibit no significant difference in test and control wells. There was no MBC at any of the test concentrations. The propolis appears to inhibit bacterial growth and biofilm formation but does not appear to be bactericidal at any of the tested concentrations. The results of this study indicate that propolis has an MIC and MBIC when tested in vitro against F. nucleatum, although it does not show an MBC. There appears to be potentially significant interaction of propolis with biofilm as displayed by the lower concentration needed to exibit inhibitory effects on biofilm formation. This information 96 may contribute to the ability to develop a proper concentration of propolis to use in vivo when treating endodontic infections.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: One of the main objectives of endodontic therapy is to reduce microbes and remove inflamed pulpal tissue within the root canal system (RCS). This is accomplished through chemomechanical debridement of the RCS using hand and rotary instrumentation along with an antimicrobial irrigant. Today, the most commonly used irrigant is sodium hypochlorite (NaOCl), often at concentrations toxic to human cells. The use of ozone as an endodontic irrigant is a novel technique that has been proven to be antimicrobial against several microorganisms. However, independent research is lacking on ozone’s efficacy against an established endodontic biofilm. If ozone’s efficacy against biofilms is confirmed, the use of toxic and potentially dangerous sodium hypochlorite could be replaced in some clinical situations (i.e., regeneration, immature teeth, resorption) with a safer and effective alternative. Objective: The aim of the current study was to evaluate the anti-biofilm activity of different concentrations of ozonated water compared to various concentrations of NaOCl against an established endodontic biofilm of Enterococcus faecalis in root canal treated teeth in vitro. Materials and Methods: The crowns of similarly sized, maxillary anterior teeth were removed, and the roots cut to a standard length (12 mm). All root canals were instrumented to a standard size. Specimens were sterilized and then inoculated with E. faecalis, which were allowed to grow for two weeks to form an established biofilm. There were six treatment groups: 1) 6% NaOCl; 2) 1.5% NaOCl; 3) 16µg/mL ozonated water; 4) 25µg/mL ozonated water; 5) 50µg/mL ozonated water, and 6) saline. Following treatment, samples were collected, plated, and incubated for two days. The number of CFU/mL were determined, and samples visualized using confocal imaging. The effect of treatment group on bacterial counts was made using one-way ANOVA followed by pair-wise comparisons. Null Hypothesis: Endodontically treated teeth irrigated with ozonated water will not demonstrate a statistically significant decrease in the E. faecalis biofilm compared to those treated with sodium hypochlorite Results: CFUs were converted to log10 and compared using Fisher’s Exact tests or one-way ANOVA followed by pair-wise tests. In all observations utilizing NaOCl irrigation, no colonies formed following treatment. The two NaOCl groups, with 0 CFU/mL, were significantly different than the other four groups (p=0.009). Saline showed a trend towards higher CFU/mL than 50 µg/ml O3 (p=0.068). None of the other comparisons approached statistical significance (p=0.453 25 µg/ml O3, p=0.606 16 µg/ml O3, p=0.999 25 µg/ml O3 vs 50 µg/ml O3, p=0.990 16 µg/ml O3 vs 50 µg/ml O3, p=1.000 16 µg/ml O3 vs 25 µg/ml O3). Confocal imaging helped illustrate effects of irrigation and confirm CFU findings. Conclusion: The results of this study failed to reject the null hypothesis. There was a statistically significant difference in the E. faecalis biofilm remaining in the groups treated with ozonated water compared to those treated with NaOCl. However, there was a trend towards higher CFU/mL in the saline group compared to the 50µg/mL ozonated water group. According to this finding, future studies should evaluate the effects of higher concentrations of ozonated water against an established E. faecalis biofilm. In addition, other follow-up studies might include ozonated water’s effect on human cells, such as the stem cells of the apical papilla that are so critical to the success of regenerative endodontic procedures. Due to university and laboratory closures caused by the COVID-19 pandemic, this project was stopped short and an insufficient sample size did not allow for proper statistical power. Additional occasions should be run upon the university’s re-opening to allow for proper statistical power.

Thesis (M.Sc.(Medical Sciences)) -- University of Limpopo, 2022 / Background: Wound infections continue to be problematic in clinical practice where empiric treatment of infections is a routine, with non-healing wounds being a burden to the health care system. A gap has been noted between antimicrobial resistance and demographic factors as an existing relationship. This necessitates an investigation of patterns of isolates and susceptibility profiles of microorganisms in wounds to modify the preventative and therapeutic strategies against the resistant strains leading to the stall of wound healing, which could aid in empiric treatment. Objective: The aim of this study was to determine the antimicrobial patterns and their associated demographic determinants in bacteria isolated from patients with non healing wounds at Pietersburg and Mankweng Hospitals, Limpopo Province. Methods: The study was conducted using antimicrobial susceptibility data collected from National Health Laboratory Service through Academic Affairs and Research Management System for the period 2016-2020. A total of 797 Antimicrobial Susceptibility Test results were analysed using Statistical Package for Social Sciences version 27.0. The susceptibility rates for the bacterial isolates by age and gender were calculated. The mean percentages for sensitivity and resistance were also calculated. Pearson’s Chi-square test was used to compare age and gender with drug susceptibility. A p-value of ≤ 0.05 was considered significant. Results: Of the 797 patient Antimicrobial Susceptibility Test results, 372 (46.7%) were males and 425 (53.3%) females, with mean age of 31.42 ± 21.75 years. The most common isolates were, Klebsiella pneumoniae (23%), Pseudomonas aeruginosa (21.7%), Escherichia coli (16%) and Proteus mirabilis (13.5%). Highest percentage of resistance to any antibiotic was amoxicillin, ampicillin (85.15%) then trimethoprim sulfamethoxazole (60.85%), amoxicillin ampicillin (49.1%), tigecycline (46.35%), cefepime (32.7%), gentamycin (25.4%), ciprofloxacin (22.5%), colistin (17.6%), and meropenem (12.3%). Furthermore, the general view of the study is no statistically clinical significance on the effect of age and gender on bacterial resistance although statistical significance was noted on age the resistance Acinetobacter baumannii vi (p=0.018), and gender on K. pneumoniae (p=0.015), P. mirabilis (p=0.024). Major resistance to A. baumannii, K. pneumoniae and P. mirabilis were from female patients. Conclusions: The most effective antibiotics were meropenem, colistin, and ciprofloxacin. The highest number of isolates were K. pneumoniae, E. coli, P. aeruginosa, P. mirabilis and A. baumannii with the most effective antibiotics gentamycin, meropenem, ciprofloxacin, and cefepime. Although the general view of the study is that no statistically clinical significance was noted on the effect of age and gender on bacterial resistance, it is important to note the significant observation that there was an observed relation of age to amoxicillin-clavulanic acid and Ciprofloxacin and gender to amoxicillin ampicillin. As such, there is insufficient evidence that supports the effect of age and gender on antimicrobial susceptibility. The study suggests caution against the use of amoxicillin ampicillin in the treatment of wound infections as it confers low levels of efficacy and high resistance and ultimately the call to revise minimum inhibitory concentrations and critical concentrations of all less effective drugs to increase their efficacy. / National Research Foundation (NRF)

INTRODUÇÃO: A combinação de sepse e insuficiência renal com necessidade de diálise é bastante comum nas Unidades de Terapia Intensiva e esta situação tem elevada mortalidade. Um desafio neste cenário é prescrever a dose correta dos antimicrobianos para o tratamento destas infecções. Em pacientes críticos e hemodinamicamente instáveis que necessitam de terapia renal substitutiva, um dos métodos mais utilizados é a diálise contínua, entretanto, recentemente, tem-se utilizado a diálise de baixa eficiência - conhecida como SLED, da abreviação do inglês \"sustained low-efficiency dialysis\". Esta modalidade de terapia renal substitutiva combina características da hemodiálise contínua com a intermitente, utilizando o equipamento da diálise intermitente, com menores fluxos sanguíneos e de dialisato, e com vantagem de menor custo. Apesar do fluxo mais baixo, por ser utilizado tempo mais prolongado, a SLED frequentemente resulta em maior clearance e especula-se que a remoção dos fármacos seria maior. Há escassez de estudos que avaliaram a farmacocinética e farmacodinâmica de antimicrobianos em pacientes submetidos à SLED.OBJETIVOS: Avaliar adequação farmacodinâmica de meropenem e vancomicina em pacientes submetidos a diálise estendida de baixa eficiência. Avaliar a depuração paramêtros farmacocinéticos durante a sessão de SLED. MÉTODOS: Foi realizado estudo prospectivo descritivo observacional com coleta de material biológico julho de 2012 a julho de 2014 HC-FMUSP. Foram incluídos pacientes submetidos à SLED em uso de vancomicina e/ou meropenem. Foram coletadas amostras de sangue seriadas (tempos: imediatamente antes do início da sessão de diálise, 0,5h, 1h, 2h, 4h após o início do tratamento e ao final da sessão). A quantificação dos antimicrobianos foi realizada através dos métodos analíticos de quantificação em Cromatografia Líquida de Alta Eficiência (CLAE). Os parâmetros farmacocinéticos foram calculados apenas durante a sessão de diálise utilizando-se o software WinNonlin. A área sob a curva foi determinada para a vancomicina. Para o meropenem, calculou-se o tempo acima da MIC. Resultados: Foram incluídos 24 pacientes tratados com vancomicina e 21 com meropenem eforam obtidas 170 amostras de plasma. As concentrações médias de vancomicina sérica e meropenem: antes da sessão de SLED foram 24,5 e 28,0 ?g / ml, respectivamente; e após SLED 14 e 6 ?g / ml, respectivamente. A depuração média foi de 41% para a vancomicina e 78% para o meropenem. Para vancomicina, 22 (96%), 19 (83%) e 16 (70%) pacientes teriam atingido o alvo (AUC0-24 > 400) considerando-se MIC 0,5; <= 1mg/l e <= 2 mg/l respectivamente. Para meropenem, 19 (95%), 18 (90%) e 11 (55%) pacientes teriam atingido a meta (70% de tempo acima da CIM) se infectados com isolados com MIC <= 1, <= 4 e <= 8 mg/l, respectivamente. Conclusões: Em pacientes críticos, meropenem evancomicina foram removidas durante o SLED. Entretando, a maioria dos pacientes alcançaria alvo PK-PD, excepto para CIMs mais altas. Sugerimos doses de manutenção de 1g a cada 12 ou 8 horaspara meropenem. Para a vancomicina, deve-se utilizar abordagem mais individualizada com monitorização sérica, uma vez que ensaios comerciais são disponíveis / Background: Antibiotic dosing is a challenge in critically ill patients undergoing renal replacement therapy. Our aim was to evaluate pharmacokinetics and pharmacodynamics of meropenem and vancomycin in patients undergoing SLED.Methods: ICU patients undergoing SLED, receiving meropenem and/or vancomycin, were prospectively evaluated. Blood samples were collected at the start of SLED and 0.5; 1; 2; 4 and 6 hours later. Antimicrobial levels were determined by HPLC. Noncompartimental pharmacokinetic analysis was performed. Area under the curve was determined for vancomycin. For meropenem, time above MIC was calculated. Results: 24 patients receiving vancomycin and 21 receiving meropenem were included; 170 plasma samples were obtained. Median serum vancomycin and meropenem concentrations: before SLED were 24.5 and 28.0 ?g/ml, respectively; and after SLED 14 and 6 ?g/ml, respectively. Mean removal was 41% for vancomycin and 78% for meropenem. For vancomycin, 22 (96%), 19(83%) and 16(70%) patients would have achieved the target (AUC0-24>400) considering MIC 0.5; <= 1mg/l and <= 2 mg/l, respectively. For meropenem, 19 (95%), 18 (90%) and 11(55%) patients would have achieved the target (70% of time above MIC) if infected with isolates with MIC <= 1, <= 4 and <= 8mg/l, respectively. Conclusions: In critically ill patients, meropenem and vancomycin were removed during SLED. Despite this, overall high PK/PD target attainment was obtained, except for higher MICs. We suggest maintenance doses of 1g tid or bid for meropenem. For vancomycin, more individualized approach using therapeutic drug monitoring should be used, as commercial assays are available

As espécies do Complexo Acinetobacter calcoaceticus-A. baumannii (ACB) são importantes causadoras de Infecções Relacionadas à Assistência à Saúde em todo o mundo. Detêm maior relevância os isolados com resistência aos antimicrobianos, os quais impactam negativamente no prognóstico, na mortalidade e custos associados ao cuidado com o paciente. O objetivo deste estudo foi avaliar a diversidade genética e o perfil de susceptibilidade antimicrobiana de 134 isolados multirresistentes de A. baumannii presentes no Hospital das Clínicas da Faculdade de Medicina de Botucatu, entre 2007 e 2014. A identificação de A. baumannii deu-se pela pesquisa dos genes blaOXA-51-like e gltA, detectados em 85% (n=114) dos isolados Os isolados de Acinetobacter não-baumannii foram identificados por sequenciamento gênico como A. nosocomialis (n=4; 3,1%), A. pittii, A. bereziniae (n=2; 1,7%, cada), A. ursingii, A. variabilis, A. gyllenbergii (n=1; 0,9% cada) e Acinetobacter spp (n=2; 1,7%). Os isolados de A. baumannii foram submetidos às técnicas de PCR multiplex para detecção de outras oxacilinases, pesquisa de ISAba1, teste de susceptibilidade antimicrobiana, tipagem molecular por eletroforese em campo pulsado (PFGE), por sequência trilocus (3LST) e por sequência multilocus (MLST). Detectou-se o gene blaOXA-23-like em 105 isolados (92,1%), estando 100% associados a ISAba1; blaOXA-72 em um isolado (0,9%) e blaOXA-231 em dois isolados (1,7%). A maior parte (n=66; 57,9%) dos isolados foi classificada como extensivamente resistentes (XDR). O PFGE agrupou os isolados em 11 clusters (A-K) e o MLST identificou os isolados pertencentes majoritariamente aos clones CC79 (42,4%), CC1 (16,6%), CC15 (12,1%) e ao ST317 (18,2%). Os resultados do MLST e 3LST concordaram em 95,6%. Foi verificada a ocorrência de diferentes perfis de PFGE em A. baumannii MDR e XDR, predominando cepas carreadoras de ISAba1/OXA-23-like e pertencentes aos CC1, CC15, CC79, ST317. Predominaram o ST317 nos anos iniciais e o CC79 (ST730) de 2011 a 2014. Estes resultados fornecem subsídios que ressaltam a necessidade de monitoramento e controle de patógenos multirresistentes / Species of Acinetobacter calcoaceticus-A. baumannii Complex (ACB) are important causes of Healthcare Associated Infections worldwide. More relevant are isolates with antimicrobial resistance, which have a negative impact on the outcome, mortality and costs associated with patient care. The aim of this study was to evaluate the genetic diversity and the antimicrobial susceptibility profile of 134 multiresistant ACB spcies strains present at Botucatu Medical School Teaching Hospital between 2007 and 2014. Identification of A. baumannii species was by detection of blaOXA-51-like and gltA genes, detected in 85% (n=114) of the isolates. Non-baumannii Acinetobacter species were identified by gene sequencing as A. nosocomialis (n=4, 3.1%), A. ursingii, A. variabilis, A. gyllenbergii (n=1, 0.9% each) and Acinetobacter spp (n=2; 1.7%). A. baumannii isolates were submitted to multiplex PCR for other oxacillinases and ISAba1 detection, antimicrobial susceptibility testing, molecular typing by pulsed field gel electrophoresis (PFGE), trilocus sequence typing (3LST) and multilocus sequence typing (MLST). blaOXA-23-like gene was detected in 105 isolates (92.1%), of which 100% were associated with ISAba1; blaOXA-72 was present in one isolate (0.9%) and blaOXA-231, in two isolates (1.7%). The majority (n=66; 57.9%) of isolates were classified as extensively resistant (XDR). The PFGE grouped the isolates into 11 clusters (A-K) and MLST identified the isolates belonging mainly to CC79 (42.4%), CC1 (16.6%), CC15 (12.1%) and ST317 (18.2%). MLST and 3LST results were 95.6% concordant. We verified the occurrence of different PFGE profiles in multidrug-resistant (MDR) and extensively drug-resistant (XDR) A. baumannii, predominantly presenting ISAba1/OXA-23-like genes and belonging to CC1, CC15, CC79 and ST317. There was a prevalence of ST317 in the early years and CC79 (ST730) from 2011 to 2014. Our results highlight the importance of surveillance and control of multiresistant pathogens

Made available in DSpace on 2017-07-24T19:22:34Z (GMT). No. of bitstreams: 1 Adelaida S Aliaga.pdf: 5670394 bytes, checksum: 9cdda52bc2a6b833409747ce8d7cf507 (MD5) Previous issue date: 2014-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Purpose: The purpose of this study was to evaluate the surface morphology and roughness and the in vitro leachability of temporary soft liners modified by the incorporation of antifungals, generally used for the denture stomatitis treatment, in their minimum inhibitory concentrations (MIC) for the biofilm of Candida albicans. Material and methods: The surface analyses of the tissue conditioner Softone (S) and the resilient liner Trusoft (T) modified or not by the addition of nystatin (Ny), miconazole (Mc), ketoconazole (Ke), chlorhexidine diacetate (Chx), and itraconazole (It) were made by using scanning electron microscopy and confocal laser microscopy. In vitro leachability of Ny and Chx was measured using Ultraviolet visible spectroscopy. Additional analyses of the modified materials containing Ny and Chx were made using differential scanning calorimetry (DSC). The antifungals were incorporated at their previously determined MIC for the biofilm of C. albicans (Ny = 0.032 g; Mc = 0.256 g; Ke = 0.128 g; Chx = 0.064 g; and It = 0.256 g/g of material). The specimens were stored in distilled water at 37ºC for up to 14 days previously to the analyses. Results: Softone had more irregular surface morphology than Trusoft did. Morphological changes were noted in both materials with increasing immersion time, particularly in those containing drugs. Ny and Ke showed the smallest particle sizes, while Chx and It showed the largest ones. Groups containing Chx and It presented extremely porous and irregular surface. Modified specimens had superior roughness (Ra) values in comparison with the control specimens. There was a trend towards an increase in Ra parameter after 7 days, followed by a decrease to values lower than the initial ones after 14 days, in the control and specimens with Ny, Mc, and Ke. Both materials had biexponential kinetics of release: a rapid initial release followed by a slower leaching. Softone leached more concentration of the antifungals than Trusoft and chlorhexidine was released at higher concentration than nystatin. DSC analysis revealed low Tg for Softone and that the fusion temperature of the drugs changed little after they had been added to the materials. Conclusion: The addition of Chx or It changed more significantly the surface of the materials. Softone was able to release more drug concentration and it was noted a weak chemical bond between the drugs and the evaluated materials. / Objetivo: A proposta deste estudo foi avaliar a morfologia e a rugosidade de superfície e a liberação in vitro de materiais macios temporários com incorporação de fármacos antifúngicos, comumente utilizados para o tratamento da estomatite protética, em suas concentrações mínimas inibitórias (CMI) ao biofilme de Candida albicans. Material e métodos: As análises de superfície do condicionador de tecido Softone (S) e do reembasador resiliente Trusoft (T) tanto controles como modificados pela incorporação de nistatina (Ni), miconazol (Mc), cetoconazol (Ce), diacetato de clorexidina (Clx) e itraconazol (It) foram feitas por meio de microscopia eletrônica de varredura e microscopia confocal laser. A liberação in vitro dos fármacos Ni e Clx foi quantificada utilizando espectrofotometria na região do Ultravioleta visível. Análises adicionais dos materiais contendo Ni e Clx foram feitas utilizando calorimetria exploratória diferencial (DSC). Os antifúngicos foram incorporados em suas CMI ao biofilme de C. albicans determinadas em estudo prévio (Ni = 0,032 g; Mc = 0,256 g; Ce = 0,128 g; Clx = 0,064 g e It = 0,256 g/g do material). Os corpos de prova foram armazenados em água destilada a 37ºC por até 14 dias previamente às análises. Resultados: O Softone apresentou morfologia mais irregular que o Trusoft. Foi notada alteração de superfície em ambos os materiais, principalmente naqueles contendo fármacos, com o aumento do tempo de imersão. Os maiores e os menores tamanhos de partículas foram dos fármacos Clx e It e Ni e Ce, respectivamente. Os grupos contendo Clx e It demonstraram superfícies extremamente porosas e irregulares. Os espécimes modificados apresentaram valores superiores de rugosidade média (Ra) em relação aos controles. Houve uma tendência de aumento de Ra após 7 dias, seguida por uma diminuição a valores inferiores aos iniciais após 14 dias para o grupo controle e aqueles contendo Ni, Mc e Ce. Ambos os materiais apresentaram cinética de liberação biexponencial: rápida liberação inicial seguida por uma liberação mais lenta. O Softone liberou maior concentração dos fármacos que o Trusoft e a clorexidina foi liberada em maior quantidade que a nistatina. As análises em DSC revelaram Tg mais baixa para o Softone e que a temperatura de fusão dos fármacos pouco alterou após terem sido incorporados aos materiais. Conclusão: A incorporação de Clx ou It alterou mais significativamente a superfície dos materiais. O Softone foi capaz de liberar maior concentração dos fármacos Clx e Ni e foi detectada uma fraca ligação química entre estes fármacos e os materiais avaliados.

As espécies do Complexo Acinetobacter calcoaceticus-A. baumannii (ACB) são importantes causadoras de Infecções Relacionadas à Assistência à Saúde em todo o mundo. Detêm maior relevância os isolados com resistência aos antimicrobianos, os quais impactam negativamente no prognóstico, na mortalidade e custos associados ao cuidado com o paciente. O objetivo deste estudo foi avaliar a diversidade genética e o perfil de susceptibilidade antimicrobiana de 134 isolados multirresistentes de A. baumannii presentes no Hospital das Clínicas da Faculdade de Medicina de Botucatu, entre 2007 e 2014. A identificação de A. baumannii deu-se pela pesquisa dos genes blaOXA-51-like e gltA, detectados em 85% (n=114) dos isolados Os isolados de Acinetobacter não-baumannii foram identificados por sequenciamento gênico como A. nosocomialis (n=4; 3,1%), A. pittii, A. bereziniae (n=2; 1,7%, cada), A. ursingii, A. variabilis, A. gyllenbergii (n=1; 0,9% cada) e Acinetobacter spp (n=2; 1,7%). Os isolados de A. baumannii foram submetidos às técnicas de PCR multiplex para detecção de outras oxacilinases, pesquisa de ISAba1, teste de susceptibilidade antimicrobiana, tipagem molecular por eletroforese em campo pulsado (PFGE), por sequência trilocus (3LST) e por sequência multilocus (MLST). Detectou-se o gene blaOXA-23-like em 105 isolados (92,1%), estando 100% associados a ISAba1; blaOXA-72 em um isolado (0,9%) e blaOXA-231 em dois isolados (1,7%). A maior parte (n=66; 57,9%) dos isolados foi classificada como extensivamente resistentes (XDR). O PFGE agrupou os isolados em 11 clusters (A-K) e o MLST identificou os isolados pertencentes majoritariamente aos clones CC79 (42,4%), CC1 (16,6%), CC15 (12,1%) e ao ST317 (18,2%). Os resultados do MLST e 3LST concordaram em 95,6%. Foi verificada a ocorrência de diferentes perfis de PFGE em A. baumannii MDR e XDR, predominando cepas carreadoras de ISAba1/OXA-23-like e pertencentes aos CC1, CC15, CC79, ST317. Predominaram o ST317 nos anos iniciais e o CC79 (ST730) de 2011 a 2014. Estes resultados fornecem subsídios que ressaltam a necessidade de monitoramento e controle de patógenos multirresistentes / Species of Acinetobacter calcoaceticus-A. baumannii Complex (ACB) are important causes of Healthcare Associated Infections worldwide. More relevant are isolates with antimicrobial resistance, which have a negative impact on the outcome, mortality and costs associated with patient care. The aim of this study was to evaluate the genetic diversity and the antimicrobial susceptibility profile of 134 multiresistant ACB spcies strains present at Botucatu Medical School Teaching Hospital between 2007 and 2014. Identification of A. baumannii species was by detection of blaOXA-51-like and gltA genes, detected in 85% (n=114) of the isolates. Non-baumannii Acinetobacter species were identified by gene sequencing as A. nosocomialis (n=4, 3.1%), A. ursingii, A. variabilis, A. gyllenbergii (n=1, 0.9% each) and Acinetobacter spp (n=2; 1.7%). A. baumannii isolates were submitted to multiplex PCR for other oxacillinases and ISAba1 detection, antimicrobial susceptibility testing, molecular typing by pulsed field gel electrophoresis (PFGE), trilocus sequence typing (3LST) and multilocus sequence typing (MLST). blaOXA-23-like gene was detected in 105 isolates (92.1%), of which 100% were associated with ISAba1; blaOXA-72 was present in one isolate (0.9%) and blaOXA-231, in two isolates (1.7%). The majority (n=66; 57.9%) of isolates were classified as extensively resistant (XDR). The PFGE grouped the isolates into 11 clusters (A-K) and MLST identified the isolates belonging mainly to CC79 (42.4%), CC1 (16.6%), CC15 (12.1%) and ST317 (18.2%). MLST and 3LST results were 95.6% concordant. We verified the occurrence of different PFGE profiles in multidrug-resistant (MDR) and extensively drug-resistant (XDR) A. baumannii, predominantly presenting ISAba1/OXA-23-like genes and belonging to CC1, CC15, CC79 and ST317. There was a prevalence of ST317 in the early years and CC79 (ST730) from 2011 to 2014. Our results highlight the importance of surveillance and control of multiresistant pathogens

[Truncated abstract] Antimicrobial growth promoters are added to feed to prevent lactic acidosis in ruminant animals by selectively inhibiting rumen bacteria that produce lactic acid. However, recently imposed or impending bans on the use of antimicrobial growth promoters in animal production have lead to a critical need to find practical alternatives that are safe for the animal and consumer and that obtain similar production benefits. I investigated bioactive plants of Australia for their potential to prevent lactic acidosis in ruminants. The unifying hypothesis tested was that plants would be identified that selectively inhibit lactic acid-producing bacteria and consequently protect against lactic acidosis. This hypothesis was tested in a three phase process: phase 1, plant selection and collection; phase 2, a three stage protocol for screening plants and essential oils; phase 3, in vivo experiments and chemical fractionation of the most promising plant. I developed an in vitro bioassay that simulated acidosis by adding glucose to rumen fluid in Bellco tubes and incubating for 5 h (Chapter 4). The pH and gas production were used as indicators of acidosis and fermentation activity. I used this bioassay to screen ninety-five plants (dried and ground material from 79 species) and ten essential oils and included a negative control (oaten chaff) and a positive control (virginiamycin). One plant, Eremophila glabra, produced a similar pH (5.63) to the positive control (5.43) although it inhibited gas production to a moderate extent (P < 0.05). ... Seven serrulatane diterpenes were identified to be the major secondary metabolites in E. glabra. The metabolites were screened using a broth dilution and microtitre spectrophotometry method and were selective against S. bovis at between 320 and 1077 [mu]g/ mL. The serrulatanes from E. glabra were probably responsible for the activity against acidosis that I observed in vitro, because they selectively inhibited lactateproducing bacteria. It is also possible that a synergy between serrulatanes and possibly other metabolites are responsible for the activity observed in vitro. The results from my experiments support the role that bioactive plants may have to replace the antibiotics that are added to livestock feed. Australian plants were identified containing compounds that were active against the bacterial processes responsible for ruminant acidosis. To my knowledge this is the first work undertaken to identify bioactive plants of Australia for their potential to prevent acidosis. I developed in vitro screening bioassays that targeted key indicators of acidosis. These bioassays enabled me to identify 5 plants from the 104 screened that could potentially control acidosis. One of these plants in particular, E. glabra, showed a level of activity in vitro that was comparable to antibiotic protection against acidosis. The exciting in vitro results were not demonstrated in vivo but only one dose level of E. glabra was used, which was based on the in vitro work. In contrast to the in vitro system the rumen is a continuous flow system with greater complexity and it is possible that the concentration of E. glabra that I used in vivo was not optimum. This places importance on future dose response experiments to confirm the efficacy of E. glabra in vivo.

[Truncated abstract] Pseudomonas aeruginosa, an important opportunistic pathogen, is resistant to a wide array of functionally and structurally diverse antimicrobial agents including antibiotics, disinfectants and biocides. P. aeruginosa is more resistant than other Gram negative bacteria to tea tree oil (TTO), the essential oil steam distilled from the leaves of Melaleuca alternifolia and comprised of over 100 terpene hydrocarbon components and their oxygenated derivatives. TTO is an established topical antimicrobial agent, with antibacterial, antiviral and antifungal properties. Intrinsic antimicrobial resistance mechanisms in P. aeruginosa include the low permeability of the outer membrane and expression of multi-drug efflux pumps. A series of multi-drug efflux mutants from the resistance-nodulation-cell division family was obtained and their susceptibility to TTO and several components examined. This demonstrated that TTO and the components terpinen-4-ol, 1,8-cineole and a-terpineol were substrates of MexAB-OprM, using both pump deletion mutants and the pump inhibitor Phe-arg ß-naphthylamide dihydrochloride. In complementation studies, the addition of mexAB-oprM to deletion mutants restored susceptibility to these agents to that of the wild-type, confirming the role of MexAB-OprM in tolerance to TTO and these three components. ... An increase in susceptibility to ticarcillin and Timentin occurred in PAO1 following serial subculture in terpinen-4-ol. Susceptibility to ticarcillin has been associated with expression of the MexCD-OprJ system in P. aeruginosa. A library of transposon mutants was created to find additional mechanisms by which P. aeruginosa could tolerate TTO. The library yielded a total of 20 mutants that were more susceptible than parental strains to TTO and/or terpinen-4-ol. The insertion site of the transposon was identified in 14 mutants and, in four mutants, this was a gene related to flagellar biosynthesis. Flagella deficient mutants have previously demonstrated enhanced susceptibility to the membrane-disrupting surfactant sodium dodecyl sulfate and this echoes the increased susceptibility to TTO and terpinen-4-ol observed. Three non-sibling surA mutants were also identified. SurA is involved in the correct folding of outer membrane proteins, including porins, in Gram negative bacteria: surA mutants of Escherichia coli have phenotypes that are characteristic of a defective cell envelope, including an increased susceptibility to hydrophobic agents. The increase in susceptibility to hydrophobic TTO and terpinen-4-ol in the surA mutants is consistent with this and represents the first report linking SurA function to antimicrobial resistance in P. aeruginosa. In conclusion, several Mex efflux systems of P. aeruginosa including MexAB-OprM, MexCD-OprJ and MexEF-OprN, as well as the LPS core, outer membrane integrity and a functioning flagella biosynthetic pathway contribute to the tolerance of this organism to TTO and/or several components.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2013-04-10T13:59:40Z No. of bitstreams: 2 Patricia_M_Lima_Dois.doc: 131072 bytes, checksum: 98e1ab34d602b80d18a06d92c6bc168a (MD5) Patricia_M_Lima_um.pdf: 1534203 bytes, checksum: e84c34b0ca61e948c1a489cec5fb02f9 (MD5) / Approved for entry into archive by Priscila Nascimento(pnascimento@icict.fiocruz.br) on 2013-04-10T14:30:12Z (GMT) No. of bitstreams: 2 Patricia_M_Lima_Dois.doc: 131072 bytes, checksum: 98e1ab34d602b80d18a06d92c6bc168a (MD5) Patricia_M_Lima_um.pdf: 1534203 bytes, checksum: e84c34b0ca61e948c1a489cec5fb02f9 (MD5) / Made available in DSpace on 2013-04-10T14:30:13Z (GMT). No. of bitstreams: 2 Patricia_M_Lima_Dois.doc: 131072 bytes, checksum: 98e1ab34d602b80d18a06d92c6bc168a (MD5) Patricia_M_Lima_um.pdf: 1534203 bytes, checksum: e84c34b0ca61e948c1a489cec5fb02f9 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Klebsiella sp. é uma bactéria ubíqua, responsável por infecções nosocomiais oportunistas. Linhagens multirresistentes a antibióticos têm se tornado um problema cada vez mais freqüente no mundo todo. Estudos objetivando a determinação do potencial patogênico dos isolados ambientais são escassos. K. pneumoniae de origem ambiental poderia estar atuando como reservatório de genes de resistência que eventualmente poderiam ser transferidos e levar ao aparecimento linhagens multirresistentes. Nosso objetivo é determinar o perfil de resistência aos antimicrobianos de isolados clínicos e ambientais de K. pneumoniae, determinar os genótipos circulantes e sua clonalidade e caracterizar a genética da resistência observada. A susceptibilidade a 9 classes de antibióticos foi determinada para os 76 isolados clínicos e ambientais. De modo geral, os isolados clínicos mostraram-se resistentes a maioria das classes de antibióticos testadas, enquanto os isolados ambientais mostraram-se suscetíveis às diferentes classes. A pesquisa por elementos genéticos associados a resistência a antibióticos mostrou a presença de integron de classe 1 entre os isolados clínicos e integron de classe 2 em isolados ambientais. Os principais cassetes identificados no integron de classe 1 foram acetilt- e adenil-transferases ou dihidrofolatoredutase, os cassetes mais frequentemente encontrados nessa classe. No integron de classe 2, foi caracterizado o arranjo sat-aadA. Essa é a primeira identificação de integron de classe 2 em isolado ambiental de K. pneumoniae. A caracterização da relação genética entre os isolados, utilizando MLST, mostrou a existência de 45 sequencias-tipo(STs), das quais 24 novas. A análise por MLST mostrou que existe uma linhagem principal, distribuída pelo Brasil. Identificamos STs pandêmicos: ST11, ST23, ST37, ST423 e ST437 e sua distribuição mostra a existência de complexos clonais distribuídos em diferentes regiões geográficas do Brasil. / Klebsiella sp. Are ubiquitous bacteria associated with opportunistic nosocomial infections. Multiresistant strains to antibiotics have become an increasingly common problem worldwide. Studies focused on determining the pathogenic potential of environmental isolates are scarce. K. pneumoniae environmental strains could be acting as reservoirs of resistance genes that could be transferred and eventually lead to the emergence of multiply antibiotic-resistant strains. Our aim is to determine the antimicrobial resistance profiles of clinical and environmental K. pneumoniae strains, characterize the circulating genotypes and their clonality, and investigate the presence of genetic elements associated with the resistance observed, in Brazil. The susceptibility to nine classes of antibiotics was determined for 76 clinical and environmental isolates. There is a prevalence of the multidrug resistant phenotype (MDR) within the clinical isolates, whilst the environmental isolates are susceptible to different classes of antibiotics. The search for genetic elements associated with antibiotic resistance revealed the presence of class 1 and class 2 integrons among clinical and environmental isolates, respectively. The main gene cassettes present in class 1 integrons were aac and aad (acetylt- and adenyl-transferases) or dfr (dihydrofolateredutase), the most commonly found in class 1 integrons. The class 2 integron harbored the sat-aadA arrangement. This is the first observation of class 2 integrons in environmental K. pneumoniae isolates. Using the MLST approach, the genetic relationship among the strains showed 45 sequence-types (STs), of which 24 have not been described yet. The MLST analysis revealed a major K. pneumoniae lineage distributed throughout Brazil. Pandemic STs were identified among the clinical strains: ST11, ST23, ST37, ST423 and ST437. Their distribution shows the existence of clonal complexes throughout geographic regions of Brazil.