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)

Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs

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

The impact of carbon source in Candida albicans virulence: participation of RLM1 in pathogen host interaction

Dissertação de mestrado em Genética Molecular

Prevalência e susceptibilidade de isolados clínicos de Pseudomonas aeruginosa numa unidade hospitalar de Portugal

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina)

MMP-9/RECK imbalance: a mechanism associated with high-grade cervical lesions and genital infection by Human Papillomavirus (HPV) and Chlamydia trachomatis

"Manuscript"

Biosurfactants produced by marine microorganisms with therapeutic applications

Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of the existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to discover novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments.

Escherichia coli and Enterococcus faecalis are able to incorporate and enhance a pre-formed Gardnerella vaginalis biofilm

Gardnerella vaginalis is the most frequent microorganism found in bacterial vaginosis (BV), while Escherichia coli and Enterococcus faecalis are amongst the most frequent pathogens found in urinary tract infections (UTIs). This study aimed to evaluate possible interactions between UTIs pathogens and G. vaginalis using an in vitro dual-species biofilm model. Our results showed that dual-species biofilms reached significantly higher bacterial concentration than mono-species biofilms. Moreover, visualization of dual-populations species in the biofilms, using the epifluorescence microscopy, revealed that all of the urogenital pathogens co-existed with G. vaginalis. In conclusion, our work demonstrates that uropathogens can incorporate into mature BV biofilms.

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.

Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity

The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0-10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 hours. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (> 5 log) and more than 4 logs P. aeruginosa and Salmonella Typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine.

Bacterial cellulose-lactoferrin as an antimicrobial edible packaging

Bacterial cellulose (BC) films from two distinct sources (obtained by static culture with Gluconacetobacter xylinus ATCC 53582 (BC1) and from a commercial source (BC2)) were modified by bovine lactoferrin (bLF) adsorption. The functionalized films (BC+bLF) were assessed as edible antimicrobial packaging, for use in direct contact with highly perishable foods, specifically fresh sausage as a model of meat products. BC+bLF films and sausage casings were characterized regarding their water vapour permeability (WVP), mechanical properties, and bactericidal efficiency against two food pathogens, Escherichia coli and Staphylococcus aureus. Considering their edibility, an in vitro gastrointestinal tract model was used to study the changes occurring in the BC films during passage through the gastrointestinal tract. Moreover, the cytotoxicity of the BC films against 3T3 mouse embryo fibroblasts was evaluated. BC1 and BC2 showed equivalent density, WVP and maximum tensile strength. The percentage of bactericidal efficiency of BC1 and BC2 with adsorbed bLF (BC1+bLF and BC2+bLF, respectively) in the standalone films and in inoculated fresh sausages, was similar against E. coli (mean reduction 69 % in the films per se versus 94 % in the sausages) and S. aureus (mean reduction 97 % in the films per se versus 36 % in the case sausages). Moreover, the BC1+bLF and BC2+bLF films significantly hindered the specific growth rate of both bacteria. Finally, no relevant cytotoxicity against 3T3 fibroblasts was found for the films before and after the simulated digestion. BC films with adsorbed bLF may constitute an approach in the development of bio-based edible antimicrobial packaging systems.

Construction of an innocuous Salmonella phage

Dissertação de mestrado em Bioengenharia

Activity of carvacrol against Coagulase-negative Staphylococci biofilm related infections

Dissertação de mestrado em Bioengenharia

Influence of oxygen conditions on bacterial interactions within biofilms related with cystic fibrosis

Dissertação de mestrado em Bioengineering

Antibacterial properties of multifunctional coatings Ag-ZrCN for biomedical devices: a novel approach

PhD in Sciences Specialty in Physics

The effects of sodium hypochlorite against selected drinking water-isolated bacteria in planktonic and sessile states

Chlorine is the most commonly used agent for general disinfection, particularly for microbial growth control in drinking water distribution systems. The goals of this study were to understand the effects of chlorine, as sodium hypochlorite (NaOCl), on bacterial membrane physicochemical properties (surface charge, surface tension and hydrophobicity) and on motility of two emerging pathogens isolated from drinking water, Acinetobacter calcoaceticus and Stenotrophomonas maltophilia. The effects of NaOCl on the control of single and dual-species monolayer adhered bacteria (2 h incubation) and biofilms (24 h incubation) was also assessed. NaOCl caused significant changes on the surface hydrophobicity and motility of A. calcoaceticus, but not of S. maltophilia. Planktonic and sessile S. maltophilia were significantly more resistant to NaOCl than A. calcoaceticus. Monolayer adhered co-cultures of A. calcoaceticus-S. maltophilia were more resilient than the single species. Oppositely, dual species biofilms were more susceptible to NaOCl than their single species counterparts. In general, biofilm removal and killing demonstrated to be distinct phenomena: total bacterial viability reduction was achieved even if NaOCl at the higher concentrations had a reduced removal efficacy, allowing biofilm reseed. In conclusion, understanding the antimicrobial susceptibility of microorganisms to NaOCl can contribute to the design of effective biofilm control strategies targeting key microorganisms, such as S. maltophilia, and guarantying safe and high-quality drinking water. Moreover, the results reinforce that biofilms should be regarded as chronic contaminants of drinking water distribution systems and accurate methods are needed to quantify their presence as well as strategies complementary/alternative to NaOCl are required to effectively control the microbiological quality of drinking water.