Yeast resistance and its reversion

Actualmente, a resistência a drogas é um dos grandes problemas na terapia de diferentes patologias e também na biotecnologia e agricultura. O objectivo do presente estudo foi elucidar a resistência a drogas em leveduras patogénicas ou alimentares, que mais frequentemente são encontradas. Assim, diferentes antifungicos clínicos e novos compostos recentemente sintetizados foram testados por um método para avaliar a resistência e sua reversão que foi previamente desenvolvido. Foi testado um painel de diferentes drogas para reverter a resistência. Mostramos que alguns destes moduladores foram capazes de reverter eficazmente a resistência.

Mercury, cadmium, lead and arsenic levels in three pelagic fish species from the Atlantic Ocean: intra- and inter-specific variability and human health risks for consumption

Three commonly consumed and commercially valuable fish species (sardine, chub and horse mackerel) were collected from the Northeast and Eastern Central Atlantic Ocean in Portuguese waters during one year. Mercury, cadmium, lead and arsenic amounts were determined in muscles using graphite furnace and cold vapour atomic absorption spectrometry. Maximum mean levels of mercury (0.1715 ± 0.0857 mg/kg, ww) and arsenic (1.139 ± 0.350 mg/kg, ww) were detected in horse mackerel. The higher mean amounts of cadmium (0.0084 ± 0.0036 mg/kg, ww) and lead (0.0379 ± 0.0303 mg/kg, ww) were determined in chub mackerel and in sardine, respectively. Intra- and inter-specific variability of metals bioaccumulation was statistically assessed and species and length revealed to be the major influencing biometric factors, in particular for mercury and arsenic. Muscles present metal concentrations below the tolerable limits considered by European Commission Regulation and Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO). However, estimation of non-carcinogenic and carcinogenic health risks by the target hazard quotient and target carcinogenic risk, established by the US Environmental Protection Agency, suggests that these species must be eaten in moderation due to possible hazard and carcinogenic risks derived from arsenic (in all analyzed species) and mercury ingestion (in horse and chub mackerel species).

Diversity and characterisation of coagulasenegative staphylococci isolated from healthy individuals in Portugal

In the past few years the interest in coagulase-negative staphylococci (CoNS) has significantly increased in human medicine. CoNS are common commensal colonisers of the human skin, although now also recognised as major nosocomial pathogens. Over the last decades, several studies have been carried out in order to understand the pathogenicity mechanisms of CoNS. The well known determinants in the pathogenesis of CoNS infections are their ability to form biofilms and an exceptional resistance to several antibiotics. Nevertheless, there is a lack of studies regarding the commensal lifestyle of these microorganisms. Additionally, it is now hypothesised that commensal bacteria might be a reservoir of pathogenic determinants. Therefore, the work described throughout this thesis was aimed to perform a phenotypic and genotypic characterisation of different CoNS species isolated from healthy Portuguese individuals. A total of 61 CoNS isolates, comprising 7 different species, were obtained and characterised at the level of biofilm formation and antibiotic susceptibility profiles. According to the results, biofilm formation ability and presence of biofilm-associated genes were commonly found features, highlighting their pivotal role in the colonising lifestyle of CoNS. This study also addressed the correlation between phenotypic and genotypic characteristics of biofilm formation, corroborating and raising questions about the importance of some genes in this process. Moreover, it was observed a great proportion of isolates with decreased susceptibility and multiple resistances to some important antibiotics. A significant association between antibiotic resistance and biofilm formation was also demonstrated, and some hypotheses about the nature of such association were provided. Lastly, the expression patterns of two biofilm-associated genes at two distinct biofilm developmental stages were determined, confirming their importance in the accumulative stage of biofilm formation. Overall, the results presented in this thesis indicate that staphylococcal skin flora might be an important reservoir of potentially pathogenic bacteria and, simultaneously, bring to light new perceptions about the molecular basis of staphylococcal biofilm formation, and the nature of the association between antibiotic resistance and biofilm formation.

Candida glabrata susceptibility to antifungals and phagocytosis is modulated by acetate

Candida glabrata is considered a major opportunistic fungal pathogen of humans. The capacity of this yeast species to cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon sources available. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids, contributes to the virulence of this fungus. Transcriptional studies on C. glabrata cells identified a similar response, upon nutrient deprivation. In this work, we aimed at analyzing biofilm formation, antifungal drug resistance, and phagocytosis of C. glabrata cells grown in the presence of acetic acid as an alternative carbon source. C. glabrata planktonic cells grown in media containing acetic acid were more susceptible to fluconazole and were better phagocytosed and killed by macrophages than when compared to media lacking acetic acid. Growth in acetic acid also affected the ability of C. glabrata to form biofilms. The genes ADY2a, ADY2b, FPS1, FPS2, and ATO3, encoding putative carboxylate transporters, were upregulated in C. glabrata planktonic and biofilm cells in the presence of acetic acid. Phagocytosis assays with fps1 and ady2a mutant strains suggested a potential role of FPS1 and ADY2a in the phagocytosis process. These results highlight how acidic pH niches, associated with the presence of acetic acid, can impact in the treatment of C. glabrata infections, in particular in vaginal candidiasis.