Dissertations / Theses on the topic 'Minimum inhibitory concentrations (MICs)'

This study was undertaken to compare the MIC (minimum inhibitory concentration) and MPC (mutant prevention concentration) values for oxytetracycline and florfenicol against strains of Pasteurella multocida isolated from cattle and pigs, and for enrofloxacin against strains of Salmonella Typhimurium isolated from horses. Isolates of P. multocida from cattle and pigs, and S. Typhimurium from horses were obtained from specimens or isolates from contributing laboratories. All the equine isolates and 50% of the cattle and pig isolates were from clinically sick animals. All isolates were tested in duplicate with both the MIC and the MPC methods. The MIC method used was the standardized microdilution method performed in microtitre plates. The MPC method used was according to the method described by Blondeau. This method was modified, to make use of smaller plates and lower volumes of antimicrobials, but retaining a final bacterial concentration of 109 colony-forming units per ml. The antimicrobials were dissolved as described in the certificates of analyses. Enrofloxacin and oxytetracycline were dissolved in water, and florfenicol was dissolved in alcohol. For the MPC method, an additional control was added to one quadrant of a four-quadrant 90mm plate/petri dish. The antimicrobials were tested as individual antimicrobials and not as combinations. Both the MIC and MPC methods included ATCC (American Type Culture Collection) strains as control organisms and were evaluated according to the guidelines of the CLSI (Clinical and Laboratory Standards Institute). The MIC50 values for enrofloxacin against Salmonella Typhimurium isolates from horses was 0.25 ìg/ml and the MPC50 values 0.5 ìg/ml. A comparative reference range was not available as enrofloxacin is not registered in South Africa for use in horses, and is used extra-labelly. The results for florfenicol against P. multocida yielded an MIC50 value of 0.5 ìg/ml and an MPC50 value of <2 ìg/ml. The close relationship of these two concentrations is an indication of the effectiveness of florfenicol when used against P. multocida. The PD/PK data with a value of 141.78 for AUC/MIC provided additional support for the efficacy of florfenicol against P. multocida. The PD/PK value of >125, is an effective parameter for treatment of Gram-negative bacteria. The corresponding results for oxytetracycline were above the MIC value but fell within the mutant selection window. The results point to the fact that the use of oxytetracycline against P. multocida may not be effective in preventing the appearance of first step mutant strains when used at current recommended dosages. The PK/PD data, using AUC/MIC, yielded a value of 56. Some of the isolates (55.17%) had an MPC value of 16 ìg/ml. Whereas the MIC method is used routinely in diagnostic laboratories, the MPC method can be employed to generate data that can be applied where antimicrobial treatment of certain bacteria is problematic and standard treatment may lead to the development of resistance. Data obtained from such studies will enable manufacturers of antimicrobial drugs to adapt antimicrobial therapy where practical and feasible to prevent the development of first step mutants.
Dissertation (MSc)--University of Pretoria, 2012.
Veterinary Tropical Diseases
unrestricted

The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi.

Background Broth microdilution (BMD) is a gold-standard reference method to determine minimum inhibitory concentration (MIC) of antibiotics. For this, a standardized concentration of bacterial inoculum (2e5–8e5 colony-forming units, CFU/ml) is added to progressively higher concentrations of antibiotics. Bacteria stop growing at a particular antibiotic concentration termed MIC. Like other assays, various biological and/or technical factors can affect BMD results.   Aims To investigate the effects of inoculum concentration (5e4–5e6 CFU/ml), growth-medium concentration (cation-adjusted Mueller-Hinton Broth (CAMHB)), ranging 0.5x to 2x (1x as standard)) and age (<6-months or >1-year old) of fastidious medium on MIC results. And to compare BMD results using 5 different brands of CAMHBs and 1 cation-non-adjusted MH-broth (non-CAMHB).   Methods 12 isolates of bacteria (gram-positive (n=3), gram-negative(n=5), fastidious isolates (n=7)) and custom-made antibiotics-containing plates for gram-positive (11 antibiotics) or gram-negative bacteria (10 antibiotics) were used. Overnight-grown colonies were used to prepare BMD solutions (MH-broth + inoculum +/- fastidious) which were plated on antibiotic-plates as well as diluted prior to plating on agar-plates. Antibiotic- and agar-plates were incubated (18–20hr, 35°C) and used to determine MICs (following European Committee on Antimicrobial Susceptibility Testing instructions) and actual number of viable bacteria in BMD solutions, respectively.   Results Increasing inoculum concentration increased MICs of all antibiotics except cefoxitin. Piperacillin–tazobactam, levofloxacin, benzylpenicillin and ampicillin were especially sensitive to increase in inoculum and showed a 4-fold increase in >50% isolates. MICs for tobramycin, tigecycline and gentamicin increased by 2-fold in >50% isolates every time MH-broth concentration increased. Age of fastidious medium had no decipherable pattern of effects on MIC. All MH-broths gave similar results except when testing daptomycin which gave higher MICs with non-CAMHB compared to CAMHB.    Conclusion This research reveals some technical factors affecting MIC results. These results could help define parameters for automated BMD-performing-systems. However, this research shows only trends as more replicates are needed to determine statistically significant results.

Oral Biology
M.S.
Objectives: Systemic antibiotics are generally recognized as providing a beneficial impact in treatment of both aggressive and chronic periodontitis. Since strains of periodontal pathogens among periodontitis patients may vary in their antibiotic drug resistance, the American Academy of Periodontology recommends antimicrobial susceptibility testing of suspected periodontal pathogens prior to administration of systemic periodontal antibiotic therapy, to reduce the risk of a treatment failure due to pathogen antibiotic resistance. E-test and MIC Test Strip assays are two in vitro antimicrobial susceptibility testing systems employing plastic- and paper-based, respectively, carriers loaded with predefined antibiotic gradients covering 15 two-fold dilutions. To date, no performance evaluations have been carried out comparing the Etest and MIC Test Strip assays in their ability to assess the in vitro antimicrobial susceptibility of periodontal bacterial pathogens. As a result, the purpose of this study was to compare the in vitro performance of E-test and MIC Test Strip assays in assessing minimal inhibitory concentration (MIC) values of four antibiotics frequently utilized in systemic periodontal antibiotic therapy against 11 fresh clinical subgingival isolates of the putative periodontal pathogen, Prevotella intermedia/ nigrescens, and to compare the distribution of P. intermedia/ nigrescens strains identified with interpretative criteria as "susceptible" and "resistant" to each of the four antibiotics using MIC values determined by the two antimicrobial susceptibility testing methods. Methods: Standardized cell suspensions, equivalent to a 2.0 McFarland turbidity standard, were prepared with 11 fresh clinical isolates of P. intermedia/nigrescens, each recovered from the subgingival microbiota of United States chronic periodontitis subjects, and plated onto to the surfaces of culture plates containing enriched Brucella blood agar. After drying, pairs of antibiotic-impregnated, quantitative, gradient diffusion strips from two manufacturers (E-test, bioMérieux, Durham, NC, USA, and MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, clindamycin, metronidazole, and doxycycline were each placed apart from each other onto the inoculated enriched Brucella blood agar surfaces, so that an antibiotic test strip from each manufacturer was employed per plate against each P. intermedia/ nigrescens clinical isolate for antibiotic susceptibility testing. After 48-72 hours anaerobic jar incubation, individual MIC values for each antibiotic test strip against P. intermedia/nigrescens were read in μg/ml at the point where the edge of the bacterial inhibition ellipse intersected with the antibiotic test strip. MIC50, MIC90, and MIC range were calculated and compared for each of the test antibiotics, with essential agreement (EA) values determined per test antibiotic for the level of outcome agreement between two antimicrobial susceptibility testing methods. In addition, the identification of antibiotic "susceptible" and "resistant" strains among the P. intermedia/nigrescens clinical isolates was determined for each test antibiotic using MIC interpretative criteria from the MIC interpretative standards developed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for gram-negative anaerobic bacteria for amoxicillin, clindamycin, and metronidazole findings, and from the French Society of Microbiology breakpoint values for anaerobic disk diffusion testing for doxycycline data. Results: For amoxicillin, higher MIC50 and MIC90 values against the P. intermedia/ nigrescens strains were found with the MIC Test Strip assay than with E-test strips, resulting in a relatively low EA value of 45.5% between the two susceptibility testing methods. A higher percentage of amoxicillin "resistant" P. intermedia/nigrescens strains (72.7%) were identified by MIC Test Strips as compared to E-test strips (54.5%), although both methods found the same proportion of amoxicillin "susceptible" strains (27.3%). For clindamycin, both susceptibility testing methods provided identical MIC values (EA value = 100%), and exactly the same distributions of "susceptible" and "resistant" strains of P. intermedia/nigrescens. For metronidazole, only very poor agreement (EA value = 9.1%) was found between the two susceptibility testing methods, with MIC Test Strips exhibiting markedly higher MIC50 and MIC90 values against P. intermedia/nigrescens as compared to E-test strips. However, the distribution of "susceptible" and "resistant" P. intermedia/ nigrescens were identical between the two susceptibility testing methods. For doxycycline, relatively good agreement (EA value = 72.7%) was found in MIC concentrations between the two susceptibility testing methods, although generally lower MIC values were associated with MIC Test Strips. In addition, identical distributions of "susceptible" and "resistant" P. intermedia/nigrescens were provided by both susceptibility testing methods. Conclusions: Relative to MIC values measured against periodontal strains of P. intermedia/nigrescens, MIC Test Strips gave higher MIC values with amoxicillin and metronidazole, equal MIC values with clindamycin, and lower MIC values with doxycycline, as compared to MIC values measured with the E-test assay. Relative to the identification of antibiotic "susceptible" periodontal P. intermedia/ nigrescens strains, both susceptibility testing methods provided identical findings, suggesting that both methods appear to be interchangeable for clinical decision making in regard to identification of antibiotic-sensitive strains of periodontal P. intermedia/nigrescens. However, for epidemiologic surveillance of drug susceptibility trends, where exact MIC values are important to track over time, the relatively higher proportion of non-exact MIC differences between the two susceptibility testing methods argues against using them interchangeably. Instead, one or the other method should be used consistently for such studies. Further comparative studies of the E-test and MIC Test Strip assays are indicated using other periodontopathic bacterial species besides P. intermedia/ nigrescens, and to assess the reproducibility of MIC values provided by both in vitro susceptibility testing methods over time.
Temple University--Theses

Raw and processed foods are vulnerable to contamination during their production, distribution and sale. Thus, a wide variety of chemical preservatives are used in the food industry to prevent the growth of food spoilage and pathogenic bacteria. However, health and economic concerns have led to an intensive search for natural alternatives, such as plant extracts, that can safely be used as substitutes for synthetic antimicrobials and preservatives to partially or completely inhibit the growth of bacteria. This study evaluated the antimicrobial effects of natural phenolic compounds extracted from vegetables, fruits, herbs and spices. The main objective was to determine the lowest concentration of phenolics to inhibit the visible growth of the pathogenic bacteria which is defined as the minimum inhibitory concentration (MIC). Some of the most common Gram-positive and Gram-negative foodborne pathogens were treated with several natural phenolic compounds. Concentrations of 5, 10, 15, and 20 ppm (pH 5-6) of each compound were evaluated by broth micro-dilution method and the MICs were determined by using official density (OD) assay. The results demonstrated that the phenolic compounds have varying antimicrobial activities against foodborne pathogens. Natural sources of phenolic compounds contain major antibacterial components and have great potential to be used as natural antimicrobials and food preservatives.

Feeding sub-therapeutic levels of antibiotics to livestock has been associated withdevelopment and spread of antibiotic resistant bacteria. The present experiment was conductedto investigate the effect of antibiotic alternatives (caffeic acid, chlorogenic acid, and carbadox)on the microbial ecology of swine feces in vitro.Minimum inhibitory concentrations of caffeic and chlorogenic acids were determined forseveral pathogens using macrobroth and agar dilution techniques. Gram-negative bacteria werenot inhibited. Caffeic acid inhibited four Staphylococcus aureus strains at 200 ppm or less, andtwo Clostridium perfringens strains at 300 ppm. Chlorogenic acid inhibited all S. aureus strainsat 500 ppm, and one C. perfringens strain at 400 ppm.Effects of antibiotic alternatives on fecal microbial ecology were determined using an invitro incubation. Caffeic acid lowered total anaerobes, Bifidobacteria, Escherichia coli, andpercent E. coli (pandlt;0.01). Chlorogenic acid lowered total anaerobes, Bifidobacteria, andlactobacilli (pandlt;0.01), and increased acetate concentration (pandlt;0.0001). Carbadox lowered totalanaerobes, Bifidobacteria, E. coli, and coliforms (pandlt;0.01), and lowered acetate, propionate,butyrate, valerate, and total volatile fatty acid concentrations (pandlt;0.01). It can be concluded thataddition of caffeic acid, chlorogenic acid, or carbadox effected bacterial and chemicalcomponents of the microbial ecology of swine feces.

The thermophilic Campylobacters, Campylobacter jejuni and Campylobacter coli are found as commensals in the intestinal tract of healthy mammals and birds. Campylobacter jejuni is one of the leading causes of sporadic food-borne bacterial disease in humans which is predominantly contracted from poultry products. Although the vast majority of these infections are mild, life-threatening complications should be treated with antimicrobials. Patients are usually treated with either macrolides of fluoroquinolones. However, globally there is an increased trend in the development of resistance to these antibiotics. This trend has also been observed in infection of poultry and pigs. The aim of this investigation was to determine antimicrobial sensitivity of thermophilic Campylobacters isolated from pigs and poultry by broth microdilution minimum inhibitory concentration testing. A total of 482 samples of the small intestinal content from poultry and pigs from the Western Cape and Gauteng Provinces were collected and analysed. Thirty-eight Campylobacter isolates were obtained. Statistical analyses included percentage resistance, minimum inhibitory concentrations (MIC50 and MIC90) as well as the distribution percentages of the MICs. The non-parametric Mann-Whitney U test was used to establish any significant differences at an interspecies, interhost and interprovincial level. Analyses of the data obtained revealed indications of decreasing susceptibility to several antibiotic groups including the tetracyclines, macrolides, erythromycin and tylosin, as well as the lincoasamides, and fluoroquinolones. It was found that isolates from the Western Cape were more likely to be resistant to the fluoroquinolones (p = 0.0392), macrolides (p = 0.0262), and lincoasmides (p = 0.0001) and, as well as to a certain extent the pleuromutulins (p = 0.0985), whereas isolates from Gauteng were more resistant to the tetracycyclines (p = <.0001). Poultry Campylobacter spp. were more prone to be resistant to enrofloxacin (p = 0.0021). Campylobacter jejuni, mainly isolated from poultry, was more liable to be resistant to the tetracyclines (chlrotetracycline p= 0.0307), whereas C. coli, predominatly isolated from pigs was more likely to be resistant to the macrolides (tylosin p= 0.063). Four of the bacteria isolated from the Western Cape were resistant to three or more antibiotic classes, namely; tetracyclines, macrolides, lincosamides, pleuromutulins and fluoroquinolones. No multi-resistant Campylobacter spp. were isolated from the flocks in Gauteng. With the exception of tiamulin, the bacterial populations could clearly be divided into resistant and susceptible populations. As consequence of the increased resistance to the antimicrobial classes used for human therapy and the geographical differences in antimicrobial susceptibility, it is recommended that an antimicrobial resistance monitoring system for the thermophilic Campylobacter spp. be initiated in the South Africa National Veterinary Surveillance and Monitoring Programme for Resistance to Antimicorbial Drugs (SANVAD) Copyright
Dissertation (MSc)--University of Pretoria, 2009.
Veterinary Tropical Diseases
unrestricted

Os Mollicutes causam doenças em várias espécies animais de importância econômica, inclusive em bovinos. Neste estudo, foi avaliada por concentração inibitória mínima (CIM) e citometria de fluxo, a atividade de oito agentes antibacterianos (enrofloxacina, ciprofloxacina, gentamicina, claritromicina, cloranfenicol, oxitetraclina, tiamulina e tilosina) contra Ureaplasma diversum. Foram analisadas 24 amostras de isolados de campo oriundas da mucosa genital de fêmeas bovinas. As amostras foram confirmadas por crescimento em caldo, placa e por PCR. Os inóculos foram submetidos à analise de suscetibilidade aos antibióticos pelo método da microdiluição em microplaca e posteriormente analisados pelo citômetro de fluxo a fim de avaliar a atividade antimicrobiana nas células. A claritromicina apresentou os maiores índices de inibição in vitro, sendo a gentamicina considerada o antibiótico de menor espectro de ação nesse estudo. De acordo com as análises do citômetro, a gentamicina apresentou o menor número de células viáveis enquanto a tiamulina apresentou o maior número. Embora haja resultados destoantes entre as técnicas utilizadas, o citômetro de fluxo pode ser utilizado como uma boa ferramenta para auxiliar a avaliação da suscetibilidade desses microrganismos a antibióticos.
The Mollicutes cause disease in several economically important species, including cattle. In this study, was evaluated by minimum inhibitory concentration (MIC) and flow cytometry, the activity of eight antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, clarithromycin, chloramphenicol, oxitetraclina, tiamulin and tylosin) against Ureaplasma diversum. We analyzed 24 samples of field isolates originating from the genital mucosa of cows. The samples were confirmed by growth in broth, plate, and PCR. The inoculations were subjected to analysis of susceptibility to antibiotics by the method of micro-dilution plate and then analyzed by flow cytometry to assess the antimicrobial activity in cells. Clarithromycin showed the highest levels of inhibition in vitro, the antibiotic gentamicin considered lower spectrum of action in this study. According to the analysis of the flow cytometer, gentamicin showed the lowest number of viable cells as tiamulin showed the greatest number. Although there are divergent results between the techniques used, flow cytometry can be used as a good tool even help assess the susceptibility of microorganisms to antibiotics.

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Pig farming has become increasingly important in recent years in Brazil, because of this, studies for the treatment of diseases that cause the loss of mass of meat animals has increased significantly, such as the Glasser's disease caused by Haemophilus parasuis. Some initial studies have shown human resistance to antibiotics due to the consumption of meat produced with high levels of these substances, and alternatively treatments have been developed from natural products. Lippia origanoides Kunth is presented as a natural source of antimicrobial substances due to the composition of the essential oil obtained, mainly, from the leaves of this plant. In this study the antimicrobial activity of the essential oil from Lippia origanoides Kunth, by determining the minimum inhibitory concentration (MIC) and Minimum Bactericidal Concentration (MBC) against Haemophilus parasuis serotypes 1,2,4,5,9,10,12,13,14 and one untypable was studied. Essential oils were obtained by hydrodistillation of the dried leaves and the chemical composition analysis revealed the presence of carvacrol as the predominant component, which characterizes the chemotype B. The results of the antimicrobial activity demonstrated the inhibitory effect of essential oil samples for all tested bacteria. The best result was 0.005% against the sample MV12315 (serotype 10) while the least satisfactory was 0.078% against the sample MV12196 (serotype 12). Results demonstrate the bactericidal action of the oil against the different serotypes of Haemophilus parasuis.
A suinocultura vem se sobressaindo nos ?ltimos anos no Brasil, por isso aumentam os estudos para tratamento das doen?as que causam perdas de carca?a dos animais, como a doen?a de Gl?sser, provocada pelo Haemophilus parasuis. Alguns trabalhos incipientes demonstram a resist?ncia humana a antibi?ticos devido ao consumo de carnes produzidas com altos ?ndices destas subst?ncias, e tratamento alternativos com produtos naturais vem sendo desenvolvidos. Lippia origanoides Kunth se apresenta como uma fonte natural de subst?ncias antimicrobianas devido ? composi??o do seu ?leo essencial obtido principalmente das folhas desta planta. Este trabalho avaliou a atividade antimicrobiana do ?leo essencial de Lippia origanoides atrav?s da determina??o da Concentra??o Inibit?ria M?nima (CIM) e Concentra??o Bactericida M?nima (CBM) frente a amostras de campo do Haemophilus parasuis com sorotipos 1,2,4,5,9,10,12,13,14 e um n?o sorotip?vel. Os ?leos essenciais foram obtidos por hidrodestila??o das folhas secas ap?s tr?s horas, e na an?lise da composi??o qu?mica, o carvacrol foi identificado como componente predominante, caracterizando-o como quimiotipo B. Os resultados de atividade antimicrobiana demonstram o efeito inibit?rio do ?leo essencial para todas as amostras de bact?rias testadas. O melhor resultado encontrado foi de 0,005% frente a amostra MV12315 (sorotipo 10) enquanto o menos satisfat?rio foi de 0,078% contra a amostra MV12196 (sorotipo 12). Os resultados obtidos demonstram a a??o bactericida do ?leo para os diferentes sorotipos do Haemophilus parasuis.

This work was aimed at developing novel tools that utilize HINT, an empirical forcefield capable of quantitating both hydrophobic and hydrophilic (hydropathic) interactions, for implementation in theoretical biology and drug discovery/design. The role of hydrophobicity in determination of macromolecular structure and formation of complexes in biological molecules is undeniable and has been the subject of research across several decades. Hydrophobicity is introduced, with a review of its history and contemporary theories. This is followed by a description of various methods that quantify this all-pervading phenomenon and their use in protein folding and contemporary drug design projects – including a detailed overview of the HINT forcefield. The specific aim of this dissertation is to introduce our attempts at developing new methods for use in the study of antibacterial drug resistance and antiviral drug discovery. Multidrug efflux is commonly regarded as a fast growing problem in the field of medicine. Several species of microbes are known to have developed resistance against almost all classes of antibiotics by various modes-of-action, which include multidrug transporters (a.k.a. efflux pumps). These proteins are present in both gram-positive and gram-negative bacteria and extrude molecules of various classes. They protect the efflux pump-expressing bacterium from harmful effects of exogenous agents by simply evacuating the latter. Perhaps the best characterized mechanism amongst these is that of the AcrA-AcrB-TolC efflux pump. Data is available in literature and perhaps also in proprietary databases available with pharmaceutical companies, characterizing this pump in terms of the minimum inhibitory concentration ratios (MIC ratios) for various antibiotics. We procured a curated dataset of 32 β-lactam and 12 antibiotics of other classes from this literature. Initial attempts at studying the MIC ratios of β-lactam antibiotics as a function of their three dimensional topology via 3D-quantitative structure activity relationship (3D-QSAR) technology yielded seemingly good models. However, this methodology is essentially designed to address single receptor-ligand interactions. Molecules being transported by the efflux pump must undoubtedly be involved in multiple interactions with the same. Notably, such methods require a pharmacophoric overlap of ligands prior to the generation of models, thereby limiting their applicability to a set of structurally-related compounds. Thus, we designed a novel method that takes various interactions between antibiotic agents and the AcrA-AcrB-TolC pump into account in conjunction with certain properties of the drugs. This method yielded mathematical models that are capable of predicting high/low efflux with significant efficiency (>93% correct). The development of this method, along with the results from its validation, is presented herein. A parallel aim being pursued by us is to discover inhibitors for hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 3 (HPIV3) by in silico screening. The basis for targeting HN is explored, along with commentary on the methodology adopted during this effort. This project yielded a moderate success rate of 34%, perhaps due to problems in the computational methodology utilized. We highlight one particular problem – that of emulating target flexibility – and explore new avenues for overcoming this obstacle in the long run. As a starting point towards enhancing the tools available to us for virtual screening in general (and for discovering antiviral compounds in specific), we explored the compatibility between sidechain rotamer libraries and the HINT scoring function. A new algorithm was designed to optimize amino acid residue sidechains, if provided with the backbone coordinates, by generating sidechain positions using the Dunbrack and Cohen backbone-dependent rotamer library and scoring them with the HINT scoring function. This rotamer library was previously used by its developers previously to design a very successful sidechain optimization algorithm called SCWRL. Output structures from our algorithm were compared with those from SCWRL and showed extraordinary similarities as well as significant differences, which are discussed herein. This successful implementation of HINT in our sidechain optimization algorithm establishes the compatibility between this forcefield and sidechain rotamer libraries. Future aims in this project include enhancement of our current algorithm and the design of a new algorithm to explore partial induced-fit in targets aimed at improving current docking methodology. This work shows significant progress towards the implementation of our hydropathic force field in theoretical modeling of biological systems in order to enhance our ability to understand atomistic details of inter- and intramolecular interactions which must form the basis for a wide variety of biological phenomena. Such efforts are key to not only to understanding the said phenomena, but also towards a solid basis for efficient drug design in the future.

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This study describes the therapeutic potential of extracts and standardized fractions of Arrabidaea chica leaves. A. chica is a Bignoniaceae popularly known as “crajiru”. The genus Arrabidaea occurs in Tropical America, from the Southern Mexico to the Southern Brazil. The red color of its dried leaves is attributed to two flavonoidal pigments: carajurina (main pigment) and carajurona. Chemical studies described the isolation of saponins and flavonoids from the plant leaves; purified 3- desoxyanthocyanidins were reported as anti-inflammatory. The infusion or decoction of the plant leaves is used in the folk medicine to treat anemia, inflammation and in skin wound-healing. In this work, the antimicrobial activity of the standardized extracts and fractions of A. chica cultivated at Embrapa Amazônia Ocidental, Manaus, were evaluated against fungal and bacterial microorganisms grown either from local domestic dogs and cats, or from human samples supplied by the Microorganism Collection of FIOCRUZ, in Manaus, Brazil. The plant dried leaves were extracted with increasing polarity solvents and progressively purified in preparative thin layer chromatography (TLC) and silica-gel column; the semi-purified extracts were standardized in TLC. The agar diffusion method; bioautography; minimum inhibitory concentrations tested against Staphylococcus epidermidis (CBAM 293), Staphylococcus aureus (CBAM 324), Pseudomonas aeruginosa (CBAM 232), Escherichia coli (CBAM 002), Trichophyton mentagrophytes (CFAM 1288), Microsporum canis (CFAM 1289), Malassezia pachydermatis (CFAM 1290) e Candida albicans (CFAM 1285). The standardized fractions were effective against all these microorganisms, but more intensively against Microsporum canis and Staphylococcus epidermidis. The results might favour the use of the standardized sub-fractions of A. chica as topic phytotherapic agent to treat canine external otitis. So far oleanolic and ursolic acids were identified as the main compounds in the active semi-purified fraction but other compounds of the leaves extract were not discarded. Later studies will consider the veterinarian use of the standardized extract, of the active pure entities and the convenience of the natural active antibiotic mix.
Este estudo analisou o potencial terapêutico de extratos e frações purificadas da planta amazônica Arrabidaea chica visando seu uso tópico como medicamento e eficácia comprovada em doenças cutâneas. A. chica Verl., é uma Bignoniaceae conhecida popularmente como crajiru. O gênero Arrabidaea ocorre na América tropical, do sul do México ao sul do Brasil. A cor avermelhada da folha seca e sua propriedade tintorial são devidas a dois pigmentos flavonoídicos: a carajurina, que é o pigmento principal e a carajurona. Dela foram isolados saponinas e flavonóides; As 3-desoxiantocianidinas, descritas na planta parecem possuir atividade antiinflamatória. A medicina popular utiliza o decocto ou a infusão das folhas para tratar anemia, inflamações e na cicatrização da pele. Neste trabalho, a atividade antimicrobiana de extratos e frações padronizadas da A. chica cultivada na Embrapa Amazônia Ocidental, em Manaus/AM, foi avaliada contra fungos e bactérias de amostras clínicas coletadas de animais domésticos e contra amostras humanas depositadas na coleção de Microrganimos da FIOCRUZ, Manaus/AM. Para isso, as folhas secas da planta foram extraídas com solventes de polaridade crescente, as frações foram progressivamente purificadas em cromatografia de placa ou coluna de sílica-gel, os extratos semi-purificados foram padronizados em cromatografia líquida de alta eficiência acoplada à espectrometria de massas. Os testes de difusão em ágar, bioautografia e concentração inibitória mínima foram usados para avaliar a atividade antimicrobiana das subfrações padronizadas frente aos microrganismos Staphylococcus epidermidis (CBAM 293), Staphylococcus aureus (CBAM 324), Pseudomonas aeruginosa (CBAM 232), Escherichia coli (CBAM 002), Trichophyton mentagrophytes (CFAM 1288), Microsporum canis (CFAM 1289), Malassezia pachydermatis (CFAM 1290) e Candida albicans (CFAM 1285). As frações padronizadas foram ativas contra todos esses microrganismos, com melhores resultados contra M. pachydermatis e S. epidermidis. Os resultados foram favoráveis à utilização das subfrações padronizadas na formulação de um produto fitoterápico para uso tópico em otite canina. Nas frações ativas foram identificados os ácidos oleanólico e ursólico. Estudos posteriores deverão avaliar a possibilidade de uso humano das frações purificadas ou dos compostos identificados.

Nowadays, fungal infections are a serious public health issue due to the increasing drug resistance and small number of antifungals available. Candida albicans is the most prevalent human fungal pathogen, causing invasive fungal infections that are associated with high mortality rates. This fungus has a particular hybrid tRNA (tRNACAGSer) that is recognized by both the leucyl - and the seryl-tRNA synthetases (LeuRS and SerRS), allowing for incorporation of leucine (3%) and serine (97%) at CUG positions. It has already been shown that in the presence of antifungals, the level of Leu misincorporation increases up to 20% and that hypermistranslating strains have higher tolerance to drugs, namely azoles, which includes fluconazole. In this study, we tested the hypothesis that mistranslation could be directly linked with the appearance of drug tolerant subpopulations of C. albicans cells from which, with prolonged drug treatment, resistance may emerge. In order to understand if antifungals can select subpopulations of tolerant cells, we carried out in vitro competition experiments with fluorescently tagged C. albicans strains that were experimentally evolved with or without fluconazole during 400 generations. A wild-type strain (T0) was tagged with mCherry while hypermistranslating strains were tagged with GFP which allowed strain differentiation within the competition. Results showed an increase of hypermistranslating cells during evolution in the presence of the antifungal, but no significative alteration of the minimal inhibitory concentration (MIC) value was detected. On the other hand, microcolonies (constituted exclusively by hypermistranslator cells) appeared within the inhibition ellipse of the E test, suggesting the emergence of tolerance instead of resistance. These results suggest that prolonged antifungal therapy may select hypermistranslating clones that drive the appearance and persistence of tolerant cells within the population. Tolerance phenotypes may not be detected through significative MIC alterations but it must be taken into consideration since it could be associated with recurrent candidiasis
Nos dias de hoje, as infeções fúngicas representam um grave problema de saúde pública devido ao aumento da resistência a tratamentos e à pouca variedade de antifúngicos disponíveis. Candida albicans é o fungo patogénico que mais causa infeções superficiais bem como graves infeções sistémicas que estão associadas a elevadas taxas de mortalidade. Este fungo tem uma característica particular, pois possui um tRNA (tRNACAGSer) híbrido responsável pela ambiguidade do codão CUG, que pode ser decodificado tanto como serina ou leucina, com níveis de incorporação de 97% e 3%, respetivamente. Já foi demonstrado que na presença de antifúngicos, este nível de incorporação de leucina pode subir até 20% e que as estirpes com maior erro de tradução têm uma maior tolerância à ação de drogas, nomeadamente aos azóis, na qual se inclui o fluconazol. Neste estudo, testamos a hipótese de que os erros de tradução podem estar diretamente associados com o aparecimento de sub-populações de células de C. albicans que são tolerantes à droga e a partir das quais, ao longo de tratamentos prolongados, a resistência pode emergir. De modo a perceber de que forma os antifúngicos podem ou não selecionar sub-populações de células tolerantes, foram feitas competições in vitro entre estirpes controlo e estirpes com elevado erro de tradução. Estas competições foram evoluídas experimentalmente ao longo de 400 gerações na presença e ausência de fluconazol. De modo a distinguir as populações, a estirpe controlo (T0) foi marcada com mCherry enquanto que as estirpes com erros de tradução foram marcadas com GFP. Os resultados obtidos mostraram que ao longo da evolução, na presença de antifúngico, houve um aumento do número de células com erro de tradução, sem que isso se resultasse num aumento da concentração inibitória mínima (MIC). Por outro lado, surgiram microcolónias (constituídas exclusivamente por células com elevado erro de tradução) dentro da elipse de inibição do E-test, o que sugere o aparecimento de um fenótipo de tolerância em vez de resistência. Estes resultados sugerem que as terapias prolongadas com antifúngicos podem selecionar clones com elevado erro de tradução que sustentam o aparecimento e persistência de células tolerantes dentro da população. Este fenótipo de tolerância pode não ser detetado através de alterações significativas da MIC, no entanto, é um factor a ter em consideração, pois pode conduzir ao desenvolvimento de candidíases recorrentes
Mestrado em Biomedicina Molecular

Saskatchewan is the only province in Canada to have routinely tested the antimicrobial susceptibility of all provincially reported human cases of campylobacteriosis. From 1999 to 2006, 1378 human Campylobacter species infections were tested for susceptibility at the Saskatchewan Disease Control Laboratory using the Canadian Integrated Program for Antimicrobial Resistance Surveillance panel and minimum inhibitory concentration (MIC) breakpoints. Of these, 1200 were C. jejuni, 129 were C. coli, with the remaining made up of C. lari, C. laridis, C. upsaliensis and undifferentiated Campylobacter species. Campylobacter coli had significantly higher prevalences of ciprofloxacin resistance (CIPr), erythromycin resistance (ERYr), combined CIPr-ERYr resistance and multidrug resistance (to three or greater drug classes) than C. jejuni. Logistic regression models indicated that CIPr in C. jejuni decreased from 1999 to 2004 and subsequently increased in 2005 and 2006. The risk of CIPr was significantly increased in the winter months (January to March) compared to other seasons. A comparison of logistic regression and Cox proportional hazard survival models found that the latter were better able to detect significant temporal trends in CIPr and tetracycline resistance by directly modeling MICs, but that these trends were more difficult to interpret. Scan statistics detected significant spatial clusters of CIPr C. jejuni infections in urban centers (Saskatoon and Regina) and temporal clusters in the winter months; the space-time permutation model did not detect any space-time clusters. Bernoulli scan tests were computationally the fastest for cluster detection, compared to ordinal MIC and multinomial antibiogram models. eBURST analysis of antibiogram patterns showed a marked distinction between case and non-case isolates from the scan statistic clusters. Multilevel logistic regression models detected significant individual and regional contextual risk factors for infection with CIPr C. jejuni. Patients infected in the winter, that were between the ages of 40-45 years of age, that lived in urban regions and that lived in regions of moderately high poultry density had higher risks of a resistant infection. These results advance the epidemiologic knowledge of CIPr C. jejuni in Saskatchewan and provide novel analytical methods for antimicrobial resistance surveillance data in Canada.
Saskatchewan Disease Control Laboratory (Saskatchewan Ministry of Health); Laboratory for Foodborne Zoonoses (Public Health Agency of Canada); Centre for Foodborne, Environmental and Zoonotic Infectious Diseases (Public Health Agency of Canada); Ontario Veterinary College Blake Graham Fellowship