Ökande antibiotikaresistens utgör en allvarlig samhällsutmaning. För resistensbestämning av bakterier används standardmetoden buljongspädning. Då bestäms minsta inhiberande koncentration (MIC) som den lägsta koncentration av antibiotika som kan förhindra tillväxt. Examensarbetets syfte var att undersöka om val av odlingsbuljong påverkar MIC-värdet för Gramnegativa bakterier. En litteraturundersökning genomfördes av studier som jämfört resistensbestämning i standardbuljong med alternativ buljong. Därutöver gjordes en experimentell jämförelse. Litteraturstudien visade att buljongens sammansättning kan ha betydelse för MIC-värdet, men resultatet varierade med både bakteriearter och typer av antibiotika. Viktigt att beakta är det begränsade materialet i studien, möjliga förklaringar till MIC-varianser är exempelvis bikarbonat och antibiotikums olika känslighet för ämnet. Den experimentella studien visade att buljongvalet inte hade någon inverkan på MIC-värdet när det undersöktes för Klebsiella pneumoniae och antibiotikan meropenem. Det kan möjligen förklaras av att effekten av meropenem inte påverkas av buljongvalet. Slutsatsen är att många faktorer påverkar MIC-värdet och att standardbuljongen är en tillgång med stor mängd historiska data som kan nyttjas för att följa resistensutvecklingen, även om alternativa buljonger i vissa fall kan vara bättre lämpade för att förutsäga behanlingsbarheten. Buljongval bör väljas med noggrannhet efter rådande omständigheter. / Increasing antibiotic resistance is a concerning societal challenge. The standard test for measuring antibiotic susceptibility of bacteria is the method of broth microdilution. This method is used to determine the minimum inbibitory concentration (MIC), defined as the lowest concentration of an antibiotic that can inhibit the growth of a specific strain of a bacterium. This study aimed to investigate whether the choice of broth affects the MIC-value for Gram-negative bacteria. A literature review was conducted to examine studies comparing standard broth against alternative broth, alongside an experimental comparison of the same. The result of the literature review indicated that the composition of the broth does have an effect. However, the effect varies depending on the bacterial species and type of the antibiotic that was analyzed. Its important to note the limited amount of data from both prior work and this study, but one possible explanation to the variance in MIC-values could be e.g., bicarbonate and the specific antibiotic´s sensitivity to this substance. The experimental part of this study concluded that the choice of broth did not affect the resulting MIC-value when the antibiotic susceptibility of Klebsiella pneumoniae to meropenem was analyzed. This could be attributed to the efficacy of meropenem being unaffected by the broths used in this study. The conclusing of this study is that multiple factors contribute to the resulting MIC-value, and that the standard broth with its large volume of historical data, is a valuable asset to use when tracking the evolution of antibiotic resistance, even though an alternative broth may be better suited to predict treatability. The choice of broth should be made with great care and be adapted to actual circumstances and conditions.

Biocides and antibiotics are commonly used in Irish agriculture. This could lead to accumulation at sublethal levels in water and resistance development. The risk of this has earlier been assessed as non-existent. However, resistant strains have been found in Irish farm waste water. Due to possible horizontal gene transfer between bacterial populations the risk may be higher. Antibiotic resistance mechanisms have worked against certain biocides and antibiotic resistant strains have in certain cases showed reduced biocidal susceptibility. The aim of this project was to characterise bacterial populations from Irish aquatic samples and investigate their susceptibility to agriculturally common biocides and relevant antibiotics. Isolates from Dublin Bay water samples (n=15) were characterised using basic techniques. Minimum inhibitory concentration (MIC) tests with a broth microdilution method were performed with eight biocides and complimentary minimum bactericidal concentration (MBC) tests. Antibiotic disc diffusions were performed with eight antibiotics. The samples contained gram-negative isolates (n=3), Staphylococcus aureus (n=1) and Bacillus spp. (n=8) isolates. All isolates were on average resistant towards methylated spirits and iodine at the 2% v/v starting concentration. MIC values for Tri Scrub and the generic biocide were high. All MBC values were on average higher than the corresponding MIC values. A significant amount of the Bacillus spp. isolates were resistant towards β-lactams. As there is statistical uncertainty around the results, further investigations are needed. In conclusion, a trend of both high MIC and MBC values while showing resistance towards the largest number of antibiotics could be seen in Bacillus spp. isolates.

O uso imprudente de antibióticos para o tratamento da mastite bovina pode levar ao aumento da resistência bacteriana e ao comprometimento da eficácia dos tratamentos atuais. Desta maneira, novas alternativas para o tratamento da mastite devem ser procuradas. As actinobactérias são capazes de produzir extratos brutos que contêm metabólitos secundários ativos, que inibem o crescimento de outras bactérias. Portanto, o presente estudo teve como objetivo avaliar a concentração inibitória mínima (CIM) de extratos brutos de actinobactérias para isolados de Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae e Streptococcus uberis, provenientes de 23 rebanhos leiteiros. Estes isolados foram identificados pela cultura microbiológica e por espectrometria de massas. Assim, foi realizada uma triagem, pelo bioensaio colorimétrico, de 15 extratos brutos, previamente testados e identificados, provenientes de actinobatérias isoladas de diferentes ambientes e plantas (caatinga, áreas de reflorestamento e do eucalipto) para 10 isolados de cada espécie causadora de mastite. Em seguida, os extratos Caat 1-54 e Caat P5-8 foram selecionados para determinação da CIM, a 50% (CIM50) e a 90% (CIM90). Para determinar a CIM dos extratos brutos, foram utilizados 20 isolados de cada espécie, totalizando 80 isolados. Foi utilizada a análise de sobrevivência para avaliação dos resultados. O extrato Caat 1-54 apresentou os menores valores de CIM50 e CIM90 para S. aureus (0,39 µg/mL e 6,25 µg/mL, respectivamente) e S. chromogenes (CIM50 = CIM90 = 0,78 µg/mL). O ceftiofur apresentou os menores valores de CIM tanto para Strep. dysgalactiae (CIM50 ≤ 0,048 µg/mL; CIM90 = 100 µg/mL), como para Strep. uberis (CIM50 ≤ 0,19 µg/mL; CIM90 = 0,39 µg/mL). Seguido do ceftiofur, o extrato Caat 1-54 foi o que apresentou menores valores CIM, com apenas uma diluição seriada de diferença entre as CIM50 das duas espécies (1,56 e 0,78 µg/mL para Strep. dysgalactiae e Strep. uberis, respectivamente). O extrato Caat P5-8 apresentou os maiores valores de CIM50 e CIM90 para todas as espécies estudadas (CIM ≥ 25 µg/mL). Os resultados obtidos indicam o potencial dos extratos brutos provenientes de actinobactérias para o tratamento da mastite bovina causada por S. aureus, S. chromogenes, Strep. dysgalactiae e Strep. uberis / The reckless use of antibiotics for the treatment of bovine mastitis can increase bacterial resistance and harm the effectiveness of current treatments. Thus, new alternatives for mastitis treatment must be searched. Actinomycetes are capable to produce crude extracts with secondary active metabolites that inhibit the growth of other bacteria. Therefore, the aim of this study was to evaluate the minimum inhibitory concentration (MIC) of crude extracts obtained from actinomycetes for Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae and Streptococcus uberis isolated from 23 dairy herds. These bacteria were identified by microbiology culture and mass spectrometry. Thus, the colorimetric bioassay was performed. In this screening 15 crude extracts previously tested and identified, obtained from actinomycetes from different Brazilian environments and plants (caatinga, reforestation areas and eucalyptus) were used for 10 isolates of each mastitis pathogens species. Then the Caat 1-54 and Caat P5-8 extracts were selected to the MIC 50% (MIC50) and 90% (MIC90) determination. Twenty mastitis pathogens isolates of each bacteria species were used. Survival analysis was used to evaluate the results. Caat 1-54 extract presented the lowest values of MIC50 e MIC90 for S. aureus (0.39 µg/mL and 6.25 µg/mL, respectively) and S. chromogenes (MIC50 = MIC90 = 0.78 µg/mL). On the other hand, ceftiofur presented the lowest MIC values for both Strep. dysgalactiae (MIC50 ≤ 0.048 µg/mL; MIC90 = 100 µg/mL) and Strep. uberis (MIC50 ≤ 0.19 µg/mL; MIC90 = 0.39 µg/mL). After ceftiofur, Caat 1-54 presented the highest efficiency against Strep. dysgalactiae and Strep. uberis and MIC50 for both species differed by only one serial dilution (1.56 e 0.78 µg/mL for Strep. dysgalactiae and Strep. uberis, respectively). Caat P5-8 extract presented the highest MIC50 and MIC90 values for all pathogens (MIC ≥ 25 µg/mL). Results indicated the potential of crude extracts from actinomycetes to treat bovine mastitis caused by S. aureus, S. schromogenes, Strep. dysgalactiae and Strep. uberis

O uso imprudente de antibióticos para o tratamento da mastite bovina pode levar ao aumento da resistência bacteriana e ao comprometimento da eficácia dos tratamentos atuais. Desta maneira, novas alternativas para o tratamento da mastite devem ser procuradas. As actinobactérias são capazes de produzir extratos brutos que contêm metabólitos secundários ativos, que inibem o crescimento de outras bactérias. Portanto, o presente estudo teve como objetivo avaliar a concentração inibitória mínima (CIM) de extratos brutos de actinobactérias para isolados de Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae e Streptococcus uberis, provenientes de 23 rebanhos leiteiros. Estes isolados foram identificados pela cultura microbiológica e por espectrometria de massas. Assim, foi realizada uma triagem, pelo bioensaio colorimétrico, de 15 extratos brutos, previamente testados e identificados, provenientes de actinobatérias isoladas de diferentes ambientes e plantas (caatinga, áreas de reflorestamento e do eucalipto) para 10 isolados de cada espécie causadora de mastite. Em seguida, os extratos Caat 1-54 e Caat P5-8 foram selecionados para determinação da CIM, a 50% (CIM50) e a 90% (CIM90). Para determinar a CIM dos extratos brutos, foram utilizados 20 isolados de cada espécie, totalizando 80 isolados. Foi utilizada a análise de sobrevivência para avaliação dos resultados. O extrato Caat 1-54 apresentou os menores valores de CIM50 e CIM90 para S. aureus (0,39 µg/mL e 6,25 µg/mL, respectivamente) e S. chromogenes (CIM50 = CIM90 = 0,78 µg/mL). O ceftiofur apresentou os menores valores de CIM tanto para Strep. dysgalactiae (CIM50 ≤ 0,048 µg/mL; CIM90 = 100 µg/mL), como para Strep. uberis (CIM50 ≤ 0,19 µg/mL; CIM90 = 0,39 µg/mL). Seguido do ceftiofur, o extrato Caat 1-54 foi o que apresentou menores valores CIM, com apenas uma diluição seriada de diferença entre as CIM50 das duas espécies (1,56 e 0,78 µg/mL para Strep. dysgalactiae e Strep. uberis, respectivamente). O extrato Caat P5-8 apresentou os maiores valores de CIM50 e CIM90 para todas as espécies estudadas (CIM ≥ 25 µg/mL). Os resultados obtidos indicam o potencial dos extratos brutos provenientes de actinobactérias para o tratamento da mastite bovina causada por S. aureus, S. chromogenes, Strep. dysgalactiae e Strep. uberis / The reckless use of antibiotics for the treatment of bovine mastitis can increase bacterial resistance and harm the effectiveness of current treatments. Thus, new alternatives for mastitis treatment must be searched. Actinomycetes are capable to produce crude extracts with secondary active metabolites that inhibit the growth of other bacteria. Therefore, the aim of this study was to evaluate the minimum inhibitory concentration (MIC) of crude extracts obtained from actinomycetes for Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae and Streptococcus uberis isolated from 23 dairy herds. These bacteria were identified by microbiology culture and mass spectrometry. Thus, the colorimetric bioassay was performed. In this screening 15 crude extracts previously tested and identified, obtained from actinomycetes from different Brazilian environments and plants (caatinga, reforestation areas and eucalyptus) were used for 10 isolates of each mastitis pathogens species. Then the Caat 1-54 and Caat P5-8 extracts were selected to the MIC 50% (MIC50) and 90% (MIC90) determination. Twenty mastitis pathogens isolates of each bacteria species were used. Survival analysis was used to evaluate the results. Caat 1-54 extract presented the lowest values of MIC50 e MIC90 for S. aureus (0.39 µg/mL and 6.25 µg/mL, respectively) and S. chromogenes (MIC50 = MIC90 = 0.78 µg/mL). On the other hand, ceftiofur presented the lowest MIC values for both Strep. dysgalactiae (MIC50 ≤ 0.048 µg/mL; MIC90 = 100 µg/mL) and Strep. uberis (MIC50 ≤ 0.19 µg/mL; MIC90 = 0.39 µg/mL). After ceftiofur, Caat 1-54 presented the highest efficiency against Strep. dysgalactiae and Strep. uberis and MIC50 for both species differed by only one serial dilution (1.56 e 0.78 µg/mL for Strep. dysgalactiae and Strep. uberis, respectively). Caat P5-8 extract presented the highest MIC50 and MIC90 values for all pathogens (MIC ≥ 25 µg/mL). Results indicated the potential of crude extracts from actinomycetes to treat bovine mastitis caused by S. aureus, S. schromogenes, Strep. dysgalactiae and Strep. uberis

A mastite bovina apresenta alta prevalência nos rebanhos leiteiros e a antibioticoterapia é o procedimento mais utilizado no tratamento da mesma. O Staphylococcus aureus, agente etiológico mais frequentemente isolado em casos de mastite, tem demonstrado em diversos estudos aumento crescente no padrão de resistência aos antimicrobianos. O desenvolvimento de bactérias resistentes e a presença de resíduos de antimicrobianos no leite estimulam o desenvolvimento de novos tratamentos que solucionem estes problemas. O muco dos moluscos Achatina fulica é composto por uma glicoproteína, denominada achacin, com atividade antimicrobiana sobre bactérias Gram positivas. Sendo assim, os objetivos deste estudo foram avaliar a atividade antimicrobiana in vitro do muco de A. fulica sobre S. aureus, bem como determinar e avaliar a concentração inibitória mínima (CIM) do muco de A. fulica purificado sobre S. aureus ATCC 25923 e isolados de S. aureus provenientes de infecções intramamárias bovina. O muco foi purificado mediante cromatografia líquida de alta eficiência e a fração com atividade antimicrobiana foi submetida à quantificação protéica e microdiluição em caldo. A CIM do muco purificado determinada para a cepa ATCC 25923 correspondeu a 50 µg/mL e para os isolados de S. aureus, variou entre 12,5 e 100 µg/mL. Sendo que destes, dois isolados (6,7 %) apresentaram CIM de 12,5 µg/mL, três (10 %) CIM de 25µg/mL, vinte e três (76,6 %) CIM de 50 µg/mL e dois (6,7 %) apresentaram CIM de 100 µg/mL. A CIM50 para os isolados correspondeu a 50 µg/mL e a CIM90 a 100 µg/mL. No presente estudo observou-se que o muco purificado apresentou atividade antimicrobiana com ação bactericida. Estes resultados proporcionam perspectivas para a compreensão e otimização de protocolos microbiológicos deste potencial biofármaco com a finalidade de empregá-lo futuramente na terapêutica veterinária para o controle de infecções intramamárias causadas por S. aureus. / The bovine mastitis is highly prevalent in dairy herds and antibiotic therapy is the procedure most commonly used to treat it. Staphylococcus aureus is the most frequently isolated etiologic agent in cases of mastitis and it has been shown in several studies the pattern of increasing antimicrobial resistance. Due to the development of resistant bacterias and the presence of antimicrobial residues in the milk the development of new treatments to solve those problems are required. The mucus of the molluscs Achatina fulica contains a glycoprotein called achacin that presents antimicrobial activity against Gram positive bacteria. The aim of this study was the evaluation of the in vitro antimicrobial activity of the A. fulica mucus of on S. aureus, as well the determination of the minimum inhibitory concentration (MIC) of A. fulica mucus purified on S. aureus ATCC 25923 and isolates of S. aureus from bovine intramammary infections. The mucus was purified by high performance liquid chromatography. The protein content in portion presenting the was quantified and microdiluted. The MIC determined for the purified mucus strain ATCC 25923 was 50 µg/mL and for the isolates of S. aureus ranged between 12,5 and 100 µg/mL. Among these samples, two isolates (6,7 %) showed MIC of 12,5 µg/mL, three (10 %) of MIC 25 µg/mL, twenty-three (76,6 %) MIC of 50 µg/mL and two (6,7 %) had an MIC of 100 µg/mL. The MIC50 for isolates was 50 µg/mL and MIC90 to100 µg/mL. It was found that the purified mucus showed bactericidal activity. The results showed in the present work improved the understanding and optimization of the biopharmaceutical microbiological potential that could be applied in new protocols in future veterinary therapy for the control of intramammary infections caused by S. aureus.

<i>Staphylococcus aureus</i> is a ubiquitous organism causing world-wide morbidity and mortality. This species readily develops resistance to antimicrobial agents. Current dosing strategies are based, in part, on minimum inhibitory concentrations (MICs). This susceptibility test fails to detect the presence of first-step resistant mutants often present in large heterogeneous populations of infecting bacteria. Dosing strategies based on MIC results may, in fact, allow for the selective proliferation of resistant subpopulations. The mutant-prevention concentration (MPC) is the drug concentration at which all first-step resistant mutants will be eradicated along with the susceptible cells. Determination of the mutant-selection window (MSW) is possible using MIC and MPC data. When considered together with achievable drug concentrations in human bodily sites, the MSW helps determine which antimicrobials are likely to select for resistance. MIC and MPC testing on clinical isolates of methicillin-susceptible (MSSA) and -resistant (MRSA) S. aureus was performed. Characterization via the polymerase chain reaction, sequencing, and electron microscopy (EM) was done on selected organisms recovered from MPC studies (MPC-recovered). MIC and MPC testing was performed on organisms isolated sequentially from patients with recurring S. aureus infections. Pulsed field gel electrophoresis was performed on these sequential isolates. Based on the MIC and the MPC values, the most potent agents for systemic MSSA and MRSA infections are gemifloxacin and vancomycin, respectively. Re-testing MPC-recovered populations by the MIC showed increased MIC results compared to the parent populations. Macrolide-resistance genes were discovered in S. aureus MPC-recovered populations; in contrast, parental isolates lacked these resistance determinants. EM revealed an increase in cell wall thickness of a vancomycin MPC-recovered population compared to its parental population. Moxifloxacin and vancomycin had the lowest and narrowest MSWs for systemic MSSA and MRSA infections, respectively, compared to the other agents tested. Sequential isolates showed no change in MIC and MPC values. The data presented provides evidence for the application of the MPC test to S. aureus organisms. The MPC data is significant when determining appropriate dosing strategies aimed at preventing resistance.

<i>Staphylococcus aureus</i> is a ubiquitous organism causing world-wide morbidity and mortality. This species readily develops resistance to antimicrobial agents. Current dosing strategies are based, in part, on minimum inhibitory concentrations (MICs). This susceptibility test fails to detect the presence of first-step resistant mutants often present in large heterogeneous populations of infecting bacteria. Dosing strategies based on MIC results may, in fact, allow for the selective proliferation of resistant subpopulations. The mutant-prevention concentration (MPC) is the drug concentration at which all first-step resistant mutants will be eradicated along with the susceptible cells. Determination of the mutant-selection window (MSW) is possible using MIC and MPC data. When considered together with achievable drug concentrations in human bodily sites, the MSW helps determine which antimicrobials are likely to select for resistance. MIC and MPC testing on clinical isolates of methicillin-susceptible (MSSA) and -resistant (MRSA) S. aureus was performed. Characterization via the polymerase chain reaction, sequencing, and electron microscopy (EM) was done on selected organisms recovered from MPC studies (MPC-recovered). MIC and MPC testing was performed on organisms isolated sequentially from patients with recurring S. aureus infections. Pulsed field gel electrophoresis was performed on these sequential isolates. Based on the MIC and the MPC values, the most potent agents for systemic MSSA and MRSA infections are gemifloxacin and vancomycin, respectively. Re-testing MPC-recovered populations by the MIC showed increased MIC results compared to the parent populations. Macrolide-resistance genes were discovered in S. aureus MPC-recovered populations; in contrast, parental isolates lacked these resistance determinants. EM revealed an increase in cell wall thickness of a vancomycin MPC-recovered population compared to its parental population. Moxifloxacin and vancomycin had the lowest and narrowest MSWs for systemic MSSA and MRSA infections, respectively, compared to the other agents tested. Sequential isolates showed no change in MIC and MPC values. The data presented provides evidence for the application of the MPC test to S. aureus organisms. The MPC data is significant when determining appropriate dosing strategies aimed at preventing resistance.

Aquaculture is the fastest growing food industry in the world and it accounts for roughly half of the world's fish supply. The majority of global aquaculture production occurs in freshwater systems that are increasingly subject to multiple uses by different stakeholders. Given the overall scarcity of freshwater on a global scale, freshwater aquaculture will face increasing environmental constraints that will demand an ever better understanding of its potential impacts on the aquatic environment and human health. This thesis consists of a series of studies that, collectively, contribute to further our understanding on the effects of freshwater aquaculture effluents on aquatic ecosystems, on the effects and environmental safety of antibiotics used in freshwater aquaculture on aquatic bacterial communities and on the link between antibiotic pollution and antibiotic resistance. Chapter 2 reviews the effects of freshwater aquaculture effluents on stream ecosystems using land-based salmonid farms as a case study. In this chapter I discuss relevant considerations related to the temporal and spatial scales of effluent discharge and ecological effects that highlight the need to characterize the patterns of stressor discharge when assessing environmental impacts and designing ecological effects studies. I also discuss the potential role of multiple stressors - with an emphasis on veterinary medicines - in disrupting ecosystem structure and function. Overall, the critical analysis presented in this chapter indicates that further research on the effects of veterinary medicines using relevant exposure scenarios would significantly contribute to our understanding of their impact in relation to other effluent stressors. Chapter 3 is a general methods chapter that describes the stream microcosm system used to assess the effects of erythromycin thiocyanate (ERT) and florfenicol (FFC) on bacterial communities of stream biofilms. This chapter presents the results of preliminary experiments whose results provided relevant information on the overall operation of the microcosms and on the variability of major physical and biological variables. This information guided the experimental designs used to assess the effects of FFC and ERT on the bacterial community structure of stream biofilms. Chapter 4 presents the results of the experiment conducted to assess the effects of FFC on the bacterial community structure of developing biofilms. The objective was to assess changes in bacterial community structure along a gradient of FFC concentrations that could provide insight into the type and magnitude of effects that could be expected from episodic exposure of stream biofilms to FFC in headwater streams. At 10 and 20 days of biofilm development, bacterial community structure differentiated in a pattern consistent with the FFC concentration gradient and there was a positive relationship between bacterial richness and bacterial diversity with FFC concentration. At 15 days of biofilm development there was also a positive relationship between FFC concentration and the surface coverage of bacteria and extracellular polymeric substances. These trends declined as the biofilm developed a more complex architecture, in terms of thickness and in the surface coverage of algae. The results are consistent with an initial stimulatory effect of FFC on biofilm formation that triggered changes in bacterial community structure that were gradually compressed as the development of a complex biofilm architecture increased the relative importance of autogenic ecological processes. The results suggest that the co-occurrence of FFC with bacterial pathogens in effluents and wastewaters may favour their persistence in the environment by enhancing biofilm formation. Chapter 5 presents the results of the experiment conducted to assess the effects of ERT on the bacterial community structure of developing biofilms. Currently, Aquamycin® 100 - a Type A medicated article (i.e., Premix) containing 100 g ERT lb-1 and used to produce a Type C medicated feed - is a candidate drug for approval by the US FDA to control mortality associated with bacterial kidney disease in freshwater salmonids. The objective of this experiment was to assess the effects of ERT on the bacterial community structure of stream biofilms using an exposure period consistent with the 28-day treatment regime suggested for Aquamycin® 100. The results provide no evidence to suggest that a 30-day exposure to ERT concentrations in the range of 10 μg L-1 (i.e., 7.3 ± 3.9 μg L-1) would lead to changes in the bacterial community structure or overall bacterial abundance of stream biofilms, while they suggest that these effects may occur at concentrations in the range of 100 μg L-1 (i.e., 87.2 ± 31.1 μg L-1). Chapter 6 attempts to determine whether environmental concentrations of antibiotics and concentrations representing action limits used in environmental risk assessment may exert a selective pressure on clinically relevant bacteria in the environment. In this chapter I use bacterial inhibition as an assessment endpoint to link antibiotic selective pressures to the prevalence of resistance in bacterial populations. Species sensitivity distributions were derived for three antibiotics by fitting log-logistic models to endpoints calculated from minimum inhibitory concentration (MIC) distributions based on worldwide data collated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Bacteria represented in these distributions were placed in a broader context by performing a brief phylogenetic analysis. The potentially affected fraction of bacterial genera at measured environmental concentrations of antibiotics and environmental risk assessment action limits was used as a proxy for antibiotic selective pressure. Measured environmental concentrations and environmental risk assessment action limits were also directly compared to wild-type cut-off values. Results suggest that measured environmental concentrations of antibiotics and concentrations representing environmental risk assessment action limits are high enough to exert a selective pressure on clinically relevant bacteria that may lead to an increase in the prevalence of resistance. Chapter 7 presents the results of an exploratory analysis conducted to assess the abundance of class 1 integrons in stream biofilms exposed to FFC and ERT. There was no pattern in the abundance of intI1 genes consistent with the treatment of FFC and ERT, suggesting either the absence of gene cassettes involved in dealing with selective pressures caused by these antibiotics or that the concentrations tested were below those required to give them a selective advantage. Chapter 8 is a brief general discussion that brings together the findings of the thesis and makes suggestions for future research. Key areas identified for future research include assessing in further detail the stimulatory effect of FFC on biofilm formation in complex bacterial communities, the interactive effects of multiple aquaculture effluent stressors on aquatic bacterial communities and their potential effects on the development of antibiotic resistance, the fate of FFC and ERT in stream ecosystems, and further developing the analysis based on MIC distributions presented in chapter 6 to assess the potential effects of antibiotic pollution on the selection of multi-drug resistance in the environment.

Orientador: Glaucia Maria Pastore / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-13T13:08:43Z (GMT). No. of bitstreams: 1 Quadros_CedenirPereirade_D.pdf: 2098145 bytes, checksum: bb56ba34ac3fa84f3dc7493e47485a6d (MD5) Previous issue date: 2009 / Resumo: Na perspectiva de se tornar uma alternativa aos produtos já existentes para a indústria, foi proposto neste trabalho uma mistura inédita e original composta por uma lipase alcalina (LA) produzida por Fusarium oxysporum e biossurfatante (BS) produzido por Bacillus subtilis, a qual foi denominada de mistura LA/BS. O objetivo desta mistura foi remover materiais gordurosos de tecidos de algodão e de superfícies de aço inoxidável, assim como remover biofilmes de micro-organismos indesejáveis para a indústria de alimentos. Primeiramente, a mistura LA/BS foi submetida a uma caracterização e verificação da manutenção das propriedades individuais de cada composto quando submetida aos diferentes tratamentos físico-químicos. A lipase alcalina manteve sua atividade enzimática na presença de 0,0001 a 0,01% de biossurfatante. O tensoativo, na mistura LA/BS, manteve sua característica de reduzir a tensão superficial da água para valores em torno de 30 mN.m-1. Quando submetida a variações de temperatura (-4 a 30?C) por 24 h e de pH (6 a 10) por 48 h, a mistura LA/BS se mostrou bastante estável em suas propriedades, demonstrando um comportamento bastante semelhante ao encontrado na literatura. A concentração micelar crítica (CMC) do BS não foi alterada na presença da LA. A mistura não perdeu suas características frente a diversos sais inorgânicos (exceto pelo CuSO4 para LA e Fe3+ para o BS), agentes oxidantes, redutores, quelantes e preservantes de alimentos. Além disso, funcionou como um ótimo agente emulsificante na presença de diversos hidrocarbonetos e ?eos vegetais, apresentando um ?dice de emulsificação após 24 h (IE24) em torno de 60%. Subsequentemente, a lipase alcalina se apresentou mais estável frente a 0,1% de BS (98% de atividade residual) do que aos surfatantes sintéticos testados. Em seguida, avaliou-se a sua capacidade de remover azeite de oliva impregnado em tecidos de algodão em uma solução. Uma alíquota de 0,5 mg.ml-1 de LA foi capaz de remover 48% de gordura dos tecidos e 3 mg.ml-1 de BS cerca de 50%. A presença de 3 mg.ml-1 de BS no sistema contendo 0,5 mg.ml-1 de LA aumentou a remoção do azeite de oliva em 10%, demonstrando um efeito de sinergismo na mistura. Além disso, LA/BS teve um comportamento similar aos surfatantes alquil benzeno linear sulfonado (LAS) e o dodecil sulfato de sódio (SDS). Superfícies rígidas de aço inoxidável foram recobertas com diferentes tipos de gorduras como gordura suína, gordura de frango, manteiga e creme de leite como forma de simular situações vistas em frigoríficos e laticínios. A presença do BS e da mistura LA/BS, de uma forma geral, melhorou a remoção das gorduras presentes nas placas conforme aumenta a concentração dos compostos. A mistura LA/BS melhorou a remoção de gordura suína, de frango e de creme de leite em 30; 35,1 e 29%, respectivamente. A remo?o da manteiga foi a menos influenciada pelos detergentes. Como padrão de detergente foi utilizado o surfatante SDS na concentração de 1,5%. A atividade antimicrobiana da mistura LA/BS foi realizada por meio de ensaio quantitativo pela concentração inibitóia mínima (CIM). Entre todos os micro-organismos testados, somente Bacillus subtilis, Bacillus cereus e a Listeria innocua tiveram seus crescimentos afetados pela mistura. A bactéria B. cereus foi a mais sensível (CIM 0,013 mg.ml-1). Em adição, o efeito de sanitizantes contra L. innocua aderidas ao aço inoxidável foi determinado por contagem em placas. Este método demonstrou que a presença de biossurfatante ou da mistura melhorou a remoção das células aderidas em relação ao tratamento sem sanitizantes, reduzindo a contagem, em média, 0,97 log.cm-2. Entretanto, não houve diferença significativa entre os sanitizantes propostos e o detergente padrão SDS (p<0,05) / Abstract: Within the perspective of becoming an alternative to products already used in the industry, this study proposed an unprecedented and original mixture consisting of na alkaline lipase (AL) from Fusarium oxysporum and a biosurfactant (BS) from Bacillus subtilis, which will be called AL/BS mix. The aim of this mixture was to remove fatty residues from cotton fabrics and stainless steel surfaces, as well as to remove biofilms of undesirable microorganisms on rigid surface to food industry. Firstly, the AL/BS mix was submitted to a characterization to verify the preservation of each compound individual properties when submitted to different physical and chemical treatments. The AL kept its enzymatic activity in the presence of 0.0001 to 0.01% of biosurfactant. The tensoactive agent, in the AL/BS mix, kept its characteristic of reducing the superficial tension of aqueous solutions to values around 30 mN.m-1. When submitted to temperature (-4 to 30ºC for 24 h) and pH (6 to 10 for 48 h) variations, the AL/BS mix proved quite stable in its properties, showing a behavior very similar to the ones found in literature. The BS critical micelle concentration (CMC) did not change in AL presence. The mix did not lose its characteristics in the presence of several inorganic salts (except CuSO4 for AL and Fe3+ for BS), oxidizing, reducing, chelating and food preservative compounds. Apart from that, the AL/BS mix also proved an excellent emulsifying agent in the presence of several hydrocarbons and vegetable oils, showing emulsifying index around 60% after 24 h (EI24). Subsequently, the AL proved more stable in the presence of 0.1% BS (98% residual activity) than the tested synthetic surfactant, in the same concentrations. Next, the ability of the AL/BS mix to remove olive oil from cotton fabric in a solution was evaluated. The 0.5 mg.mL-1 AL managed to remove 48% of the oil from cotton fabrics and 3 mg.mL-1BS managed to remove approximately 50%. The presence of 3 mg.mL-1 BS in the system which contained 0.5 mg.mL-1 AL increased the olive oil removal by 10%, showing a synergy effect in the mixture. Apart from that, AL/BS mix had a similar performance to sodium linear alkyl benzene sulphonate (LAS) and sodium dodecyl sulphate (SDS) surfactants. Rigid stainless steel surfaces were covered with different types of fatty materials, such as pork fat, chicken fat, butter and whipped cream, to simulate situations existing in meat and dairy industries. The presence of BS and AL/BS mix, generally speaking, improved the fat removal from plates in pace with the increase of compounds concentration. The AL/BS mix improved the removal of pork and chicken fat, and whipped cream in 30, 35.1 and 29% respectively. Butter was the fat least influenced by detergents. Surfactant SDS concentration at 1.5% was used as detergent standard. This testing showed efficiency, sensitivity and it can be used to develop new formulations. The anti-microbial activity of AL/BS mix was carried out by minimum inhibitory concentration (MIC) quantitative assays. Among all tested microorganisms, only Bacillus subtilis, Bacillus cereus and Listeria innocua had their growth affected by the mixture. B. cereus bacteria was the most sensitive (MIC 0.013 mg.mL-1). Besides, the sanitizing effect against L. innocua that was attached to stainless steel was determined by plates counting. This assay proved that the presence of biosurfactant or the AL/BS mix improved the removal of adhered cells in relation to the treatment without sanitizers, reducing the count, in average, 0.97 log CFU.cm-2. However, there has not been a significant difference between the proposed sanitizers and the standard detergent SDS (p<0.05) / Doutorado / Doutor em Ciência de Alimentos

Made available in DSpace on 2014-07-29T16:11:53Z (GMT). No. of bitstreams: 1 dissertacao tiago oliveira.pdf: 60590 bytes, checksum: 2becac23a5f99abe922b4af561c0a7d1 (MD5) Previous issue date: 2009-08-27 / In the last years the treatment with medicines made from medicinal plants has increased. A concern about the useful of these natural products is the quality control pharmacobotanical, phytochemical and purity tests, which make up the technical specifications of a particular plant species. Moreover, pharmacological and toxicological studies must be conducted in order to effectiveness and safety. This study aimed to carry out the assessment pharmacognostic, acute toxicity and evaluation of antimicrobial and leishmanicidal of the ethanol extract of the leaves, fruit, stem bark and root bark of R. elaeocarpum Reissek. Samples of R. elaeocarpum were collected in the municipality of Campestre-GO, a description of the macroscopic aspects of the leaves, stem bark and root bark was carried to the naked eye by observing the macroscopic general of vegetal drugs; the microscopical analysis was performed by observing the histological stained. The qualitative analysis of the major classes of secondary metabolites present in the sprayed sample. The tests for water content, total ashes and acid insoluble ashes had been carried through according to the brazilian pharmacopoeia IV. The rude ethanol extract was obtained by maceration and the fractions were obtained by partition, liquid-liquid, successive with hexane, dichloromethane and ethyl acetate. Minimum inhibitory concentration (MIC) was determined by agar dilution. The procedure was performed according to NCCLS standards 2004 (M7-A7). The evaluation of acute toxicity of ethanol extracts had reference to the Guidelines of 423 Organization for Economic Co-Operation and Development (OECD). The leishmanicidal activity was tested for the promastigotes forms of Leishmania (L) chagasi. Most of the morphological and anatomical characteristics observed are consistent with the typical family. In phytochemical screening of leaf, root bark, stem bark and fruit of R. elaeocarpum verified the presence of flavonoids and alkaloids. It was also the presence of heterosides saponins and tannins in leaf, root bark, stem bark and is absent in the fruit. The extracts and fractions that had obtained the lowest value of minimum inhibitory concentration (0,390 mg.mL-1) against microorganisms tested showed moderate inhibitory activity. None of the extracts and fractions showed good inhibitory activity. As is said about the toxicological research, throughout the period of observation of animals during the evaluation of acute toxicity was not death. Through behavioral observations made through the hippocratic screening is assigned a zero score for all evaluated items. The necropsy of the animals did not evidence any macroscopic alterations to justify the histopathology of the organs (kidney, spleen and liver) selected for analysis. This study demonstrated that the use of rude extract and fractions of leaves of R. elaeocarpum induced death of promastigotes forms of L. (L) chagasi in vitro. The presented results open perspectives for the future isolation, purification and identification of bioactive substances of R. elaeocarpum / Nos últimos anos o tratamento com medicamentos à base de plantas medicinais tem aumentado. Uma preocupação em relação à utilização desses produtos naturais consiste no controle de qualidade farmacobotânico, fitoquímico e ensaios de pureza, que compõem as especificações técnicas de uma determinada espécie vegetal. Além disso, estudos farmacológicos e toxicológicos devem ser realizados, visando a sua eficácia e segurança. Este trabalho teve por objetivo realizar a avaliação farmacognóstica, toxicidade aguda e avaliação das atividades antimicrobiana e leishmanicida do extrato etanólico das folhas, frutos, cascas do caule e cascas da raiz de R. elaeocarpum Reissek. Amostras da R. elaeocarpum foram coletadas no município de Campestre-GO, a descrição dos aspectos macroscópicos das folhas, casca do caule e casca da raiz foi realizada à vista desarmada observando as características macroscópicas gerais de drogas vegetais; a análise microscópica foi realizada através da observação de cortes histológicos submetidos à coloração. A análise qualitativa das principais classes de metabólitos secundários presentes foi realizada na amostra pulverizada. Os ensaios para determinação do teor de água, cinzas totais e cinzas insolúveis em ácido foram realizados segundo a Farmacopéia Brasileira IV. O extrato etanólico bruto foi obtido por maceração e as frações foram obtidas por partições, líquido-líquido, sucessivas com hexano, diclorometano e acetato de etila. A Concentração Inibitória Mínima (CIM) foi determinada pelo método de diluição em ágar. O procedimento foi realizado segundo normas do NCCLS 2004 (M7-A7). A avaliação da toxicidade aguda dos extratos etanólico brutos teve como referência as Diretrizes 423 da Organisation for Economic Co-Operation and Development (OECD). A atividade leishmanicida foi testada para as formas promastigotas de Leishmania (L) chagasi. A maior parte das características morfoanatômicas observadas está de acordo com o padrão típico da família. Na prospecção fitoquímica da folha, cascas da raiz, cascas do caule e fruto de R. elaeocarpum verificou-se a presença de flavonóides e alcalóides. Verificou-se, também, a presença de heterosídeos saponínicos e taninos na folha, cascas da raiz, cascas do caule sendo ausente no fruto. Os extratos e as frações que obtiveram o menor valor de Concentração Inibitória Mínima (0,390 mg.mL-1) frente aos microrganismos testados apresentaram atividade inibitória moderada. Nenhum dos extratos e frações apresentou boa atividade inibitória. No que se diz a respeito à investigação toxicológica, durante todo o período de observação dos animais durante a avaliação de toxicidade aguda não ocorreu morte. Através das observações comportamentais feitas por meio do screening hipocrático atribuiu-se pontuação zero para todos os itens avaliados. A necropsia dos animais não evidenciou nenhuma alteração macroscópica que justificasse o estudo histopatológico dos órgãos (rins, baço e fígado) selecionados para análise. Neste estudo foi demonstrado ainda que a utilização do extrato bruto e as frações de folhas de R. elaeocarpum induziu a morte das formas promastigotas de L. (L) chagasi in vitro. Os resultados apresentados abrem perspectivas para o futuro isolamento, purificação e identificação das substâncias bioativas de R. elaeocarpum