Ana Margarida Sousa

Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review.

Pseudomonas aeruginosa is the most prevalent pathogen of cystic fibrosis (CF) lung disease. Its long persistence in CF airways is associated with sophisticated mechanisms of adaptation, including biofilm formation, resistance to antibiotics, hypermutability and customized pathogenicity in which virulence factors are expressed according the infection stage. CF adaptation is triggered by high selective pressure of inflamed CF lungs and by antibiotic treatments. Bacteria undergo genetic, phenotypic, and physiological variations that are fastened by the repeating interplay of mutation and selection. During CF infection development, P. aeruginosa gradually shifts from an acute virulent pathogen of early infection to a host-adapted pathogen of chronic infection. This paper reviews the most common changes undergone by P. aeruginosa at each stage of infection development in CF lungs. The comprehensive understanding of the adaptation process of P. aeruginosa may help to design more effective antimicrobial treatments and to identify new targets for future drugs to prevent the progression of infection to chronic stages.

Adaptive response of single and binary Pseudomonas aeruginosa and Escherichia coli biofilms to benzalkonium chloride

The main goal of this work was to examine whether the continuous exposure of single and binary P. aeruginosa and E. coli biofilms to sub‐lethal benzalkonium chloride (BC) doses can induce adaptive response of bacteria. Biofilms were formed during 24 h and then put continuously in contact with BC for more 5 days. The six‐day‐old adapted biofilms were then submitted to BC challenge, characterized and inspected by SEM. Both single and binary adapted biofilms have clearly more biomass, polysaccharides and proteins and less activity even though the number of cells was identical. After BC treatment, adapted biofilms maintained their mass and activity. SEM examination revealed that those adapted biofilms had a slimier and denser matrix that became thicker after BC treatment. Continuous exposure of bacteria to antimicrobials can lead to development of biofilms encompassing more virulent and tolerant bacteria. This adaptive resistance can be the result of a phenotypic adaptation, a genetic acquired resistance or both. Instead of eradicating biofilms and kill microorganisms, the use of a disinfectant can, favour biofilm formation and tolerance. This must be a genuine concern as it can happen in clinical environments, where the use of antimicrobials is unavoidable. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Effect of antimicrobial residues on early adhesion and biofilm formation by wild-type and benzalkonium chloride-adapted Pseudomonas aeruginosa

Antimicrobial residue deposition can change the physico-chemical properties of bacteria and surfaces and thus promote or impair bacterial adhesion. This study focuses on benzalkonium chloride (BC) deposition on polystyrene (PS) surfaces and the influence of this conditioning film on the physico-chemical properties of PS and on early adhesion and biofilm formation by Pseudomonas aeruginosa wild-type and its laboratory BC-adapted strain. The latter readily acquired the ability to grow in BC, and also exhibited physico-chemical surface changes. The existence of residues on PS surfaces altered their hydrophobicity and favoured adhesion as determined by the free energy and early adhesion characterization. Adapted bacteria revealed a higher ability to adhere to surfaces and to develop biofilms, especially on BC-conditioned surfaces, which thereby could enhance resistance to sanitation attempts. These findings highlight the importance of investigations concerning the antimicrobial deposition effect after cleaning procedures, which may encourage bacterial adhesion, especially of bacteria that have been previously exposed to chemical stresses.

Minimum information about a biofilm experiment ( MIABiE): standards for reporting experiments and data on sessile microbial communities living at interfaces

The minimum information about a biofilm experiment (MIABiE) initiative has arisen from the need to find an adequate and scientifically sound way to control the quality of the documentation accompanying the public deposition of biofilm-related data, particularly those obtained using high-throughput devices and techniques. Thereby, the MIABiE consortium has initiated the identification and organization of a set of modules containing the minimum information that needs to be reported to guarantee the interpretability and independent verification of experimental results and their integration with knowledge coming from other fields. MIABiE does not intend to propose specific standards on how biofilms experiments should be performed, because it is acknowledged that specific research questions require specific conditions which may deviate from any standardization. Instead, MIABiE presents guidelines about the data to be recorded and published in order for the procedure and results to be easily and unequivocally interpreted and reproduced. Overall, MIABiE opens up the discussion about a number of particular areas of interest and attempts to achieve a broad consensus about which biofilm data and metadata should be reported in scientific journals in a systematic, rigorous and understandable manner.

Improvements on colony morphology identification towards bacterial profiling

Colony morphology may be an indicator of phenotypic variation, this being an important adaptive process adopted by bacteria to overcome environmental stressors. Furthermore, alterations in colony traits may reflect increased virulence and antimicrobial resistance. Despite the potential relevance of using colony morphological traits, the influence of experimental conditions on colony morphogenesis has been scarcely studied in detail. This study aims to clearly and systematically demonstrate the impact of some variables, such as colony growth time, plate colony density, culture medium, planktonic or biofilm mode of growth and strain genetic background, on bacterial colony morphology features using two Pseudomonas aeruginosa strains. Results, based on 5-replicate experiments, demonstrated that all variables influenced colony morphogenesis and 18 different morphotypes were identified, showing different sizes, forms, colours, textures and margins. Colony growth time and composition of the medium were the variables that caused the highest impact on colony differentiation both derived from planktonic and biofilm cultures. Colony morphology characterization before 45 h of incubation was considered inadequate and TSA, a non-selective medium, provided more colony diversity in contrast to P. aeruginosa selective media. In conclusion, data obtained emphasized the need to perform comparisons between colony morphologies in equivalent experimental conditions to avoid misinterpretation of microbial diagnostics and biomedical studies. Since colony morphotyping showed to be a reliable method to evaluate phenotypic switching and also to infer about bacterial diversity in biofilms, these unambiguous comparisons between morphotypes may offer a quite valuable input to clinical diagnosis, aiding the decision-making towards the selection of the most suitable antibiotic and supportive treatments.

New Fluorescent Heterocyclic Materials: Synthesis, Solvatochromic and Fluorescence Properties

Thienyl- and bithienyl-1,3-benzothiazoles 1 and 2 were synthesised by reacting various formyl thienyl and bithienyl derivatives with o-aminobenzenethiol in moderate to excellent yields. Evaluation of the solvatochromic and fluorescence properties of these compounds was carried out. Due to their strong fluorescence and also the strong push-pull character, benzothiazole derivatives 1 and 2 can be used as potential NLO materials or as fluorescent markers.

Donor-acceptor substituted thienylpyrrole azo dyes : Synthesis, solvatochromic and electrochemical properties

The synthesis of thienyl- substituted pyrrole azo dyes and their UV-visible, solvatochromic and electrochemical properties are described. In agreement with the solvatochromic data and also with the electrochemical study the new donor-acceptor systems synthesized could have applications in NLO.

Heteroresistance to colistin in Klebsiella pneumoniae is triggered by small colony variants sub-populations within biofilms

The emergence of Klebsiella pneumoniae multidrug-resistant strains paves the way to the re-introduction of colistin as a salvage therapy. However, recent planktonic studies have reported several cases of heteroresistance to this antimicrobial agent. The aim of this present work was to gain better understanding about the response of K. pneumoniae biofilms to colistin antibiotherapy and inspect the occurrence of heteroresistance in biofilm-derived cells. Biofilm formation and its susceptibility to colistin were evaluated through the determination of biofilm-cells viability. The profiling of planktonic and biofilm cell populations was conducted to assess the occurrence of heteroresistance. Colony morphology was further characterized in order to inspect the potential role of colistin in K. pneumoniae phenotypic differentiation. Results show that K. pneumoniae was susceptible to colistin in its planktonic form, but biofilms presented enhanced resistance. Population analysis profiles pointed out that K. pneumoniae manifest heteroresistance to colistin only when grown in biofilm arrangements, and it was possible to identify a resistant sub-population presenting a small colony morphology (diameter around 5 mm). To the best of our knowledge, this is the first report linking heteroresistance to biofilm formation and a morphological distinctive sub-population. Moreover, this is the first evidence that biofilm formation can trigger the emergence of heteroresistance in an apparently susceptible strain.

Computational approaches to standard-compliant biofilm data for reliable analysis and integration

The study of microorganism consortia, also known as biofilms, is associated to a number of applications in biotechnology, ecotechnology and clinical domains. Nowadays, biofilm studies are heterogeneous and data-intensive, encompassing different levels of analysis. Computational modelling of biofilm studies has become thus a requirement to make sense of these vast and ever-expanding biofilm data volumes. The rationale of the present work is a machine-readable format for representing biofilm studies and supporting biofilm data interchange and data integration. This format is supported by the Biofilm Science Ontology (BSO), the first ontology on biofilms information. The ontology is decomposed into a number of areas of interest, namely: the Experimental Procedure Ontology (EPO) which describes biofilm experimental procedures; the Colony Morphology Ontology (CMO) which characterises morphologically microorganism colonies; and other modules concerning biofilm phenotype, antimicrobial susceptibility and virulence traits. The overall objective behind BSO is to develop semantic resources to capture, represent and share data on biofilms and related experiments in a regularized fashion manner. Furthermore, the present work also introduces a framework in assistance of biofilm data interchange and analysis - BiofOmics (http://biofomics.org) - and a public repository on colony morphology signatures - MorphoCol (http://stardust.deb.uminho.pt/morphocol).

Immobilization of Mo(IV) complex in hybrid matrix obtained via sol–gel technique

A molybdenum(IV) complex, trans-bis-[1,2-bis(diphenylphosphino)ethane]-fluoro-(diazopropano)-molybdenum tetraphenylborate, [MoF(DIAZO)(dppe)2][BPh4], was prepared and immobilized in a hybrid matrix synthesized by the sol–gel process. The host matrix, designated as U(500), is an organic–inorganic network material, classed as ureasil, that combines a reticulated siliceous backbone linked by short polyether-based segments. Urea bridges make the link between these two components, and the polymerization of silicate substituted terminal groups generates the inorganic network. The free Mo(IV) complex and all new materials were characterized by spectroscopic techniques (FT-IR and UV–Vis) and thermal analysis (DSC). The ionic conductivity of the resulting material was also studied. The results indicate that immobilized Mo(IV) complex has kept its solid-state structure, although there is evidence of inter-molecular interactions between the Mo(IV) complex and some groups/atoms of the hybrid host matrix.