Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) är en viktig metod för artidentifiering av mikroorganismer på kliniska mikrobiologiska laboratorier. Enligt rekommendationer från Bruker Daltonics skall matrixapplicering ske relativt omedelbart efter att mikroorganismer har applicerats. Syftet med studien var att utvärdera effekter på artidentifiering vid förlängda tidsintervall mellan applicering av mikroorganismer och matrix med MALDI-TOF MS. Målet var att optimera arbetsflödet på mikrobiologilaboratoriet, laboratoriemedicin, Region Jönköping län (RJL). I studien undersöktes hur identifieringen av mikroorganismer påverkades av applicering avbakterie- och jästsvampstammar (n = 267) och α-Cyano-4-hydroxycinnaminsyra (HCCA)-matrix på en MALDI-provplatta vid varierande tidsintervall upp till tre timmar. Analys med MALDI-TOF MS genomfördes med masspektrometrarna MALDI Biotyper Sirius IVD System och Bruker MicroFlex LT. Resultaten visade att förlängda tidsintervall mellan applicering av mikroorganismer och matrix upp till tre timmar inte hade någon signifikant effekt på scorevärden vid artidentifiering, vilket innebar att tidsintervallen är acceptabla för artidentifiering med MALDI-TOF MS. Detta ger värdefull information för att optimera arbetsflödet och öka effektiviteten på mikrobiologilaboratoriet. / Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an important method for species level identification in clinical microbiology laboratories. According to recommendations from Bruker Daltonics matrix application should occur relatively immediately after microorganisms have been applied. The aim of the study was to evaluate the effects on microbial identification at extended time intervals between microbial and matrix application using MALDI-TOF MS. The objective was to optimize the workflow at the microbiology laboratory, laboratory medicine, Region Jönköping län (RJL). The study investigated the impact on identification of microorganisms between the application of bacterial and yeast strains (n = 267) and α-Cyano-4-hydroxycinnamic acid (HCCA) matrix on a MALDI target plate of various time intervals up to three hours. MALDI-TOF MS was performed using MALDI Biotyper Sirius IVD System and Bruker MicroFlex LT mass spectrometers.The results showed that a delay of matrix application up to three hours had no significant effect on score values, indicating that time did not affect the accuracy of microbial identification using MALDI-TOF MS. Consequently, the study provided valuable information for optimizing the workflow and increasing the efficiency of the microbiology laboratory.

Consumption of cocoa and dark chocolate products has been associated with positive health outcomes including reduced onset of cardiovascular disease, inflammation, diabetes, obesity, and platelet disorders. Cocoa polyphenols, putatively responsible for these beneficial activities, are highly impacted by cocoa variety, agronomic effects and processing history. However, the difference in polyphenol concentration and composition between cocoa products originating from different hybrid clones (selected for high yield) or from different fermentation conditions is not fully understood. Detailed polyphenol characterization including determination of total polyphenol and total procyanidin concentrations, and qualitative and quantitative analysis of (mean) degree of polymerization was conducted. Significant differences in total polyphenol and procyanidin concentrations were observed between five genetic clones grown by the USDA-ARS Cocoa Germplasm Repository located in Mayagüez, Puerto Rico. To facilitate cocoa fermentation research in laboratories distant from cocoa harvesting sites, a laboratory-scale cocoa fermentation model system was developed in this study. This model system used dried, unfermented, cocoa beans and simulated pulp medium as the starting material. The model system supported growth of the essential succession of cocoa fermenting microorganisms and generated similar chemical changes to those observed in on-farm cocoa fermentation. Using this model system, the impact of inoculation with proprietary yeast strains Saccharomyces cerevisiae Lev F and Saccharomyces cerevisiae Lev B on cocoa polyphenol concentration and composition was evaluated. Inoculation with both yeast strains resulted in increased fermentation rate and Lev B inoculation resulted in higher total polyphenol and procyandin contents at the end of fermentation. Overall, the present work addressed the influence of cocoa variety selection and fermentation process conditions on the composition and concentration of polyphenols. These findings will contribute to continued efforts to develop cocoa products with optimized bioactivity and maximum disease preventative effects. / PHD / Annual worldwide cocoa production exceeds four million tons and the cocoa market impacts not only cocoa growing regions, but also countries producing cocoa products. Consumption of cocoa and dark chocolate products has been associated with positive health outcomes including reduced onset of cardiovascular disease, inflammation, diabetes, obesity, and platelet disorders. Cocoa polyphenols, compounds increasingly associated with health benefits, are highly impacted by the variety and processing history of cocoa. However, the extent to which cocoa genetic variety and processing conditions impact polyphenol concentration and composition is not fully understood. Polyphenol characterization was conducted for five genetic varieties sourced from the USDA cocoa germplasm repository located in Mayagüez, Puerto Rico and it was found that genetic variance could contribute to differences in polyphenol concentration. The main focus in this thesis was to develop laboratory cocoa fermentation model system to facilitate cocoa research in laboratories distant from the cocoa growing region. This model system used dried, unfermented cocoa beans and simulated pulp medium as the starting material. The model system was effective in that it supported growth of the essential succession of coca fermenting microorganisms and generated similar chemical changes to those observed in on-farm cocoa fermentation. This model system could be further applied to test the impact of fermentation conditions on cocoa bean quality. The potential for using yeast starter cultures to improve control and consistency of cocoa fermentation was evaluated using the model fermentation system. Yeast inoculation improved fermentation rate, and polyphenol concentration in fermented beans was higher when cocoa fermentation was inoculated with the yeast strain Saccharomyces cerevisiae Lev B. Overall, the present work addressed the influence of cocoa variety selection and fermentation process conditions on the composition and concentration of polyphenols. These findings will contribute to continued efforts to develop cocoa products optimized for maximum health benefits.

A qualidade microbiológica de medicamentos é fundamental para garantir sua eficácia e segurança. Os métodos convencionais para identificação microbiana em produtos não estéreis são amplamente utilizados, entretanto são demorados e trabalhosos. O objetivo deste trabalho é desenvolver método microbiológico rápido (MMR) para a identificação de contaminantes em produtos farmacêuticos utilizando a espectrofotometria de infravermelho com transformada de Fourier com reflectância total atenuada (FTIR-ATR). Análise de componentes principais (PCA) e análise de discriminantes (LDA) foram utilizadas para obter um modelo de predição com a capacidade de diferenciar o crescimento de oriundo de contaminação por Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), Enterococcus faecium (ATCC 8459), Escherichia coli (ATCC 8739), Micrococcus luteus (ATCC 10240), Pseudomonas aeruginosa (ATCC 9027), Salmonella Typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538) e Staphylococcus epidermidis (ATCC 12228). Os espectros de FTIR-ATR forneceram informações quanto à composição de proteínas, DNA/RNA, lipídeos e carboidratos provenientes do crescimento microbiano. As identificações microbianas fornecidas pelo modelo PCA/LDA baseado no método FTIR-ATR foram compatíveis com aquelas obtidas pelos métodos microbiológicos convencionais. O método de identificação microbiana rápida por FTIR-ATR foi validado quanto à sensibilidade (93,5%), especificidade (83,3%) e limite de detecção (17-23 UFC/mL de amostra). Portanto, o MMR proposto neste trabalho pode ser usado para fornecer uma identificação rápida de contaminantes microbianos em produtos farmacêuticos. / Microbiological quality of pharmaceuticals is fundamental in ensuring efficacy and safety of medicines. Conventional methods for microbial identification in non-sterile drugs are widely used, however are time-consuming and laborious. The aim of this paper was to develop a rapid microbiological method (RMM) for identification of contaminants in pharmaceutical products using Fourier transform infrared with attenuated total reflectance spectrometry (FTIR-ATR). Principal components analysis (PCA) and linear discriminant analysis (LDA) were used to obtain a predictive model with capable to distinguish Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), Enterococcus faecium (ATCC 8459), Escherichia coli (ATCC 8739), Micrococcus luteus (ATCC 10240), Pseudomonas aeruginosa (ATCC 9027), Salmonella Typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538), and Staphylococcus epidermidis (ATCC 12228) microbial growth. FTIR-ATR spectra provide information of protein, DNA/RNA, lipids, and carbohydrates constitution of microbial growth. Microbial identification provided by PCA/LDA based on FTIR-ATR method were compatible to those obtained using conventional microbiological methods. FTIR-ATR method for rapid identification of microbial contaminants in pharmaceutical products was validated by assessing the sensitivity (93.5%), specificity (83.3%), and limit of detection (17-23 CFU/mL of sample). Therefore, the RMM proposed in this work may be used to provide a rapid identification of microbial contaminants in pharmaceutical products.

A presente investigação teve como objetivo avaliar a prática de cirurgiões dentistas em uma unidade de terapia intensiva (UTI) de um hospital militar, o estabelecimento de um protocolo de higiene oral e os seus efeitos sobre a redução de pneumonias associadas à ventilação mecânica (PAVM). As percepções da equipe da UTI sobre as atividades dos cirurgiões dentistas também foram avaliadas por meio de um questionário. O perfil de colonização microbiana da mucosa oral antes e depois do estabelecimento das medidas de higiene oral também foi avaliado tanto por diluição e plaqueamento em meios de cultura microbiológicos seletivos e enriquecidos e através da amplificação pelo método de PCR e eletroforese em gel desnaturante em gradiente (DGGE), subsequente ao sequenciamento dos amplicons. A carga microbiana foi avaliada após a contagem de placas de agar e através da amplificação por PCR em tempo real (qPCR) do gene rrs nas amostras. O protocolo de higiene oral, realizado pelos cirurgiões dentistas, foi capaz de reduzir a incidência de PAVM (p <0,05). O questionário revelou que a modificação da halitose foi percebida por 93,33% dos participantes. A redução da ocorrência das úlceras orais e dos lábios durante a internação dos pacientes foi observada por 80% da equipe da UTI. Foi observada a redução da produção das secreções nasais e bucais por 70% da equipe dos profissionais da UTI. Para 86,66% dos participantes a assistência aos pacientes tornou-se mais agradável após a instituição dos cuidados bucais. O protocolo, realizado com a utilização de solução 0,12% de clorexidina, não foi capaz de evitar a colonização da mucosa oral por patógenos microbianos usualmente encontrados no ambiente hospitalar tais como os bastonetes Gram-negativos entéricos e não fermentadores, nem foi capaz de eliminá-los quando tais micro-organismos já se encontravam presentes antes dos procedimentos de higiene bucal. Alguns Bastonetes Gram-positivos (Lactobacillus sp e corinebactérias) e Staphylococcus epidermidis permaneceram após a realização dos procedimentos. O protocolo de higiene oral permitiu a redução da carga microbiana na mucosa oral de 50% dos pacientes considerando-se o método de contagem microbiana e para 35% dos pacientes pela avaliação dos números de cópias de genes rrs através de qPCR. Em conclusão, o protocolo de higiene oral desenvolvido pelos cirurgiões dentistas foi capaz de reduzir a incidência de PAV na UTI, embora não tenha sido capaz de prevenir a colonização da mucosa oral por supostos patógenos microbianos. O protocolo de higiene oral com a participação ativa dos cirurgiões dentistas foi bem aceito pelos profissionais da UTI e foi capaz de melhorar a qualidade da assistência aos pacientes críticos. / This investigation aimed to evaluate the practice of dentists in an intensive care unit (ICU) of a military hospital, the establishment of a protocol for oral hygiene, and the effect of the protocol on the reduction of ventilator associated pneumonia (VAP) after the introduction of the oral hygiene protocol. The opinion of the ICU staff about the activity of the dentists was also evaluated by means of a questionnaire. In addition, the microbial colonization profile of the oral mucosa before and after the establishment of the oral hygiene measures was evaluated by means of both dilution and plating the samples in microbiological culture mediums (both selective and rich agar media) and DGGE technique, with sequencing of amplicons. The microbial load was evaluated after counting agar plates and by real time rrs gene PCR amplification (qPCR) in the samples. The oral hygiene protocol performed by dentists was capable to reduce the incidence of VAP (p< 0.05). The questionnaire revealed that the change of the oral odor was noticed by 93.33% of the participants. The reduction of oral and lip ulcers during the hospitalization of the patients was observed by 80% of the staff. The patients were observed to reduce the production of oral and nasal secretions after the establishment of the oral hygiene procedures by 70% of the ICU professionals. The approach to the patients developed by the staff became more pleasant after the establishment of the oral protocol for 86.66%. The protocol, with the use of 0,12% chlorhexidine solution, was not capable to avoid the colonization of the oral mucosa by the microbial pathogens usually found in nosocomial environment, in especial Gram-negative enteric and non-fermentative rods, nor eliminated these organisms previously found before the oral care procedures. Gram-positive rods (Lactobacillus sp, and corynebacteria) and Staphylococcus epidermidis remained after the procedures. The protocol reduced the microbial load in the oral mucosa of 50% of the patients considering the microbial counting and in 35% of the patients evaluated by the numbers of copies of rrs genes by qPCR. In conclusion, the oral hygiene protocol developed by dentists was capable to reduce the incidence of VAP in the studied ICU, though was not capable to prevent the colonization of oral mucosa by putative microbial pathogens. The oral hygiene protocol with active participation of dentists was well accepted by the ICU professionals and was capable to improve the quality of assistance to critically ill patients.

A presente investigação teve como objetivo avaliar a prática de cirurgiões dentistas em uma unidade de terapia intensiva (UTI) de um hospital militar, o estabelecimento de um protocolo de higiene oral e os seus efeitos sobre a redução de pneumonias associadas à ventilação mecânica (PAVM). As percepções da equipe da UTI sobre as atividades dos cirurgiões dentistas também foram avaliadas por meio de um questionário. O perfil de colonização microbiana da mucosa oral antes e depois do estabelecimento das medidas de higiene oral também foi avaliado tanto por diluição e plaqueamento em meios de cultura microbiológicos seletivos e enriquecidos e através da amplificação pelo método de PCR e eletroforese em gel desnaturante em gradiente (DGGE), subsequente ao sequenciamento dos amplicons. A carga microbiana foi avaliada após a contagem de placas de agar e através da amplificação por PCR em tempo real (qPCR) do gene rrs nas amostras. O protocolo de higiene oral, realizado pelos cirurgiões dentistas, foi capaz de reduzir a incidência de PAVM (p <0,05). O questionário revelou que a modificação da halitose foi percebida por 93,33% dos participantes. A redução da ocorrência das úlceras orais e dos lábios durante a internação dos pacientes foi observada por 80% da equipe da UTI. Foi observada a redução da produção das secreções nasais e bucais por 70% da equipe dos profissionais da UTI. Para 86,66% dos participantes a assistência aos pacientes tornou-se mais agradável após a instituição dos cuidados bucais. O protocolo, realizado com a utilização de solução 0,12% de clorexidina, não foi capaz de evitar a colonização da mucosa oral por patógenos microbianos usualmente encontrados no ambiente hospitalar tais como os bastonetes Gram-negativos entéricos e não fermentadores, nem foi capaz de eliminá-los quando tais micro-organismos já se encontravam presentes antes dos procedimentos de higiene bucal. Alguns Bastonetes Gram-positivos (Lactobacillus sp e corinebactérias) e Staphylococcus epidermidis permaneceram após a realização dos procedimentos. O protocolo de higiene oral permitiu a redução da carga microbiana na mucosa oral de 50% dos pacientes considerando-se o método de contagem microbiana e para 35% dos pacientes pela avaliação dos números de cópias de genes rrs através de qPCR. Em conclusão, o protocolo de higiene oral desenvolvido pelos cirurgiões dentistas foi capaz de reduzir a incidência de PAV na UTI, embora não tenha sido capaz de prevenir a colonização da mucosa oral por supostos patógenos microbianos. O protocolo de higiene oral com a participação ativa dos cirurgiões dentistas foi bem aceito pelos profissionais da UTI e foi capaz de melhorar a qualidade da assistência aos pacientes críticos. / This investigation aimed to evaluate the practice of dentists in an intensive care unit (ICU) of a military hospital, the establishment of a protocol for oral hygiene, and the effect of the protocol on the reduction of ventilator associated pneumonia (VAP) after the introduction of the oral hygiene protocol. The opinion of the ICU staff about the activity of the dentists was also evaluated by means of a questionnaire. In addition, the microbial colonization profile of the oral mucosa before and after the establishment of the oral hygiene measures was evaluated by means of both dilution and plating the samples in microbiological culture mediums (both selective and rich agar media) and DGGE technique, with sequencing of amplicons. The microbial load was evaluated after counting agar plates and by real time rrs gene PCR amplification (qPCR) in the samples. The oral hygiene protocol performed by dentists was capable to reduce the incidence of VAP (p< 0.05). The questionnaire revealed that the change of the oral odor was noticed by 93.33% of the participants. The reduction of oral and lip ulcers during the hospitalization of the patients was observed by 80% of the staff. The patients were observed to reduce the production of oral and nasal secretions after the establishment of the oral hygiene procedures by 70% of the ICU professionals. The approach to the patients developed by the staff became more pleasant after the establishment of the oral protocol for 86.66%. The protocol, with the use of 0,12% chlorhexidine solution, was not capable to avoid the colonization of the oral mucosa by the microbial pathogens usually found in nosocomial environment, in especial Gram-negative enteric and non-fermentative rods, nor eliminated these organisms previously found before the oral care procedures. Gram-positive rods (Lactobacillus sp, and corynebacteria) and Staphylococcus epidermidis remained after the procedures. The protocol reduced the microbial load in the oral mucosa of 50% of the patients considering the microbial counting and in 35% of the patients evaluated by the numbers of copies of rrs genes by qPCR. In conclusion, the oral hygiene protocol developed by dentists was capable to reduce the incidence of VAP in the studied ICU, though was not capable to prevent the colonization of oral mucosa by putative microbial pathogens. The oral hygiene protocol with active participation of dentists was well accepted by the ICU professionals and was capable to improve the quality of assistance to critically ill patients.

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