Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

Phage therapy: a step forward in the treatment of Pseudomonas aeruginosa infections

Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics and nowadays, a common infection can be fatal. To revert this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since P. aeruginosa is one of the most common causes of healthcare-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review will further address the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.

A Gß protein and the TupA Co-Regulator bind to protein kinase A Tpk2 to act as antagonistic molecular switches of fungal morphological changes

A Gß protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes

Polymicrobial interactions in infections: the case Pseudomonas aeruginosa and Candida albicans in ventilator-associated pneumonia

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clinica)

Strategies to combat infections of Acinetobacter baumannii biofilms

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Microscopy- or DNA-based analyses: Which methodology gives a truer picture of stream-dwelling decomposer fungal diversity?

We assessed aquatic hyphomycete diversity in autumn and spring on oak leaves decomposing in five streams along a gradient of eutrophication in the Northwest of Portugal. Diversity was assessed through microscopy-based (identification by spore morphology) and DNA-based techniques (Denaturing Gradient Gel Electrophoresis and 454 pyrosequencing). Pyrosequencing revealed five times greater diversity than DGGE. About 21% of all aquatic hyphomycete species were exclusively detected by pyrosequencing and 26% exclusively by spore identification. In some streams, more than half of the recorded species would have remained undetected if we had relied only on spore identification. Nevertheless, in spring aquatic hyphomycete diversity was higher based on spore identification, probably because many species occurring in this season are not yet connected to ITS barcodes in genetic databases. Pyrosequencing was a powerful tool for revealing aquatic hyphomycete diversity on decomposing plant litter in streams and we strongly encourage researchers to continue the effort in barcoding fungal species.

Introducing resistance genes against ToMV, TYLCV and TSWV infections in two tomato varieties (Muchamiel and De la Pera)

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Saprotrophic fungi Hypholoma fasciculare effect on the fungal community associated to Castanea sativa

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.

Global validation of the WSES Sepsis Severity Score for patients with complicated intra-abdominal infections: a prospective multicentre study (WISS Study).

BACKGROUND To validate a new practical Sepsis Severity Score for patients with complicated intra-abdominal infections (cIAIs) including the clinical conditions at the admission (severe sepsis/septic shock), the origin of the cIAIs, the delay in source control, the setting of acquisition and any risk factors such as age and immunosuppression. METHODS The WISS study (WSES cIAIs Score Study) is a multicenter observational study underwent in 132 medical institutions worldwide during a four-month study period (October 2014-February 2015). Four thousand five hundred thirty-three patients with a mean age of 51.2 years (range 18-99) were enrolled in the WISS study. RESULTS Univariate analysis has shown that all factors that were previously included in the WSES Sepsis Severity Score were highly statistically significant between those who died and those who survived (p < 0.0001). The multivariate logistic regression model was highly significant (p < 0.0001, R2 = 0.54) and showed that all these factors were independent in predicting mortality of sepsis. Receiver Operator Curve has shown that the WSES Severity Sepsis Score had an excellent prediction for mortality. A score above 5.5 was the best predictor of mortality having a sensitivity of 89.2 %, a specificity of 83.5 % and a positive likelihood ratio of 5.4. CONCLUSIONS WSES Sepsis Severity Score for patients with complicated Intra-abdominal infections can be used on global level. It has shown high sensitivity, specificity, and likelihood ratio that may help us in making clinical decisions.

Exploring silk based biomaterials functionalized with antimicrobial peptides to prevent surgical site infections

Surgical site infections (SSI) often occur after invasive surgery, which is as a serious health problem, making it important to develop new biomaterials to prevent infections. Spider silk is a natural biomaterial with excellent biocompatibility, low immunogenicity and controllable biodegradability. Through recombinant DNA technology, spider silk-based materials can be bioengineered and functionalized with antimicrobial (AM) peptides 1. The aim of this study is to develop new materials by combining spider silk chimeric proteins with AM properties and silk fibroin extracted from Bombyx mori cocoons to prevent microbial infection. Here, spider silk domains derived from the dragline sequence of the spider Nephila clavipes (6 mer and 15 mer) were fused with the AM peptides Hepcidin and Human Neutrophil peptide 1 (HNP1). The spider silk domain maintained its self-assembly features allowing the formation of beta-sheets to lock in structures without any chemical cross-linking. The AM properties of the developed chimeric proteins showed that 6 mer + HNP1 protein had a broad microbicidal activity against pathogens. The 6 mer + HNP-1 protein was then assembled with different percentages of silk fibroin into multifunctional films. In vitro cell studies with a human fibroblasts cell line (MRC5) showed nontoxic and cytocompatible behavior of the films. The positive cellular response, together with structural properties, suggests that this new fusion protein plus silk fibroin may be good candidates as multifunctional materials to prevent SSI.

Activity of carvacrol against Coagulase-negative Staphylococci biofilm related infections

Dissertação de mestrado em Bioengenharia

Effect of pH on Candida tropicalis virulence factors

Dissertação de mestrado em Bioengenharia