The role of the Gerontologist in the fight against Ageism

O envelhecimento progressivo das sociedades é um fenómeno que tende a acentuar-se devido aos avanços científicos e à melhoria das condições de vida que permitem às pessoas viver mais. Mas se as conquistas em termos de anos de vida são, em si mesmas, positivas, a elas surge associado um conjunto de preconceitos relativos à idade. Assim, o idadismo tornou-se uma realidade presente, tanto nos discursos, como nas práticas. Os gerontólogos, como profissionais com conhecimentos teóricos e práticos sobre o envelhecimento, têm o dever ético e moral de lutar contra o idadismo. Disso se pretende dar conta neste artigo.The progressive aging of societies is an unavoidable phenomenon that tends to be accentuated due to the scientific advances and the improvement of living conditions that allow people to live longer. But if the achievements in terms of years of life are, themselves, positive, they are accompanied by a set of prejudices associated with age. Thus, ageism has become a present reality, both in speeches and in practices. Gerontologists, as professionals with theoretical and practical knowledge on aging have an ethical and moral duty to fight ageism. This is intended to be accounted for in this article

OMP proteomic analysis of benzalkonium chloride and ciprofloxacin adapted biofilm cells

Adaptive resistance to antimicrobials has been widely reported in planktonic studied trough phenotypic characterization and proteomic analysis. Concerning biofilm adaptation, the response of biofilm-entrapped cells to chemical stress conditions is not yet well studied. There is evidence that proteins involved in oxidative stress response, cell envelope synthesis, as well as in synthesis of EPS become up- or down-regulated in biofilms, indicating that these altered phenotypes might contribute to antimicrobial tolerance. This work aimed to examine whether exposure of Pseudomonas aeruginosa biofilms to benzalkonium chloride (BC) and ciprofloxacin (CIP) could induce an adaptive response in bacteria. This was attained by inspection of proteome alterations of the outer membrane (OMP) in biofilm cells. Biofilms were formed in 6-well plates for 24h being after submitted to the presence of 0.9 mM BC and 6.0 ug/ml CIP, during 13 days. The obtained biofilm-cells were separated and the OMP extracted. Protein patterns were analysed by 2-DE and gels by the SameSpot software. Biofilm-proteome showed that P. aeruginosa adaptation to BC promoted the down-regulation of 36 OMP and the up-regulation of only one. OMP 2DE of P. aeruginosa adapted to CIP revealed the down-regulation of 29 OMP. Six OMPs were changed in common by both antimicrobials, revealing a possible similar stress response. Proteins identification is in progress. This study highlighted that there might be an OMP regulation when bacteria within biofilms are submitted to chemical adaptation. This particular response to the environment can be one of the causes of the well-known biofilm resistance phenotype

Motility and biofilm formation ability of isolated vs collection P. aeruginosa : effect of single and combined antimicrobial application

IBB-CEB and FC

Membranome analysis of benzalkonium chloride adapted planktonic and biofilm cells

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/1096

Ciprofloxacin susceptibility patterns of planktonic and sessile S. aureus, E. coli, and P. aeruginosa – effect of the exposure time

Several aspects of human infections within a clinical arena are related to biofilm development. Various surfaces, like indwelling devices and medical equipments, are prone to biofilm formation, causing subsequent pathogenesis. The application of antimicrobial agents is one of the main strategies to eradicate biofilms. However, the action of antibiotics could be inefficient duo to the high tolerance of biofilmes comparatively to the bacteria in planktonic state. Thus, it is of upmost importance the use of suitable antimicrobials with high efficacies to eradicate biofilms and to control nosocomial infections. Ciprofloxacin (CIP) is a broad spectrum fluoroquinolone antibiotic, often use against both Gram-positive and Gram-negative bacteria, that causes inhibition of bacterial cell division by the inhibition of DNA gyrase and topoisomerase IV. With this study it was aimed to analyze the antimicrobial efficacy of CIP against S. aureus, E. coli K-12, and P. aeruginosa PAO1, in planktonic cultures and in biofilm state, and also to characterize the time–kill kinetics of ciprofloxacin in pre-established 1-day-old biofilms. In planktonic cultures, the CIP susceptibility patterns were achieved by the broth microdilution method and by the determination of the number of viable cells. In sessile bacteria, the anti-biofilm activity of CIP was assessed using a standardized biofilm assay, quantifying the biofilm mass, through crystal violet, the respiratory activity, using the XTT reduction assay, and the number of viable cells. The time-kill kinetics of CIP were attained exposing 24-hour-old biofilms of each bacteria to ciprofloxacin (6 ug/ml) over time (until 24 h), and determining biofilm metabolic activity, biomass and cellular viability at regular time intervals. Ciprofloxacin displayed dose-dependent activity against both bacteria in planktonic and biofilm states. The MIC values ranged between 0.185 ug/ml for S. aureus, 0.5 ug/ml for E. coli and 0.75 ug/ml for P. aeruginosa. These values revealed that it was needed a 4-fold increase in CIP dose to cause growth inhibition of Gram- bacteria comparatively to the Gram+ strain. The presence of CIP during biofilm formation did not kill totally the biofilm-associated cells neither eradicated the biomass adhered. CIP exhibited only a bacteriostatic effect for all strains studied emphasizing that this antibiotic is more effective on suspended than on biofilm cells. The application of a constant dose of 6 ug/ml of CIP against pre-formed biofilms revealed an evident time-dependent effect in the antibiotic action, since a gradual reduction of biofilm activity, biomass and cell number occurred over time. However, the time of CIP exposure needed to reduce the biofilm characteristics varied from each bacterial species. In fact, the antibacterial effect was evident after 4 h of exposure of the antibiotic with the S. aureus, around 8 h of exposure with the E. coli and 6 h of exposure with the P. aeruginosa pre-formed biofilms. Once more, ciprofloxacin only presented bacteriostatical activity against all the bacterial biofilms, even when the exposure time reached 24 h. In the range of experimental concentrations and exposure times tested, CIP didn´t reveal to be effective against bacteria cells neither in the pre-established biofilm nor during the process of biofilm development, in spite of the improved reduction of the metabolic activity, biofilm biomass and in the number of viable cells of S. aureus, E. coli and P. aeruginosa biofilms. Alternative treatments or combination of antibiotic therapies should be studied for the implementation of more effective ways of eradication biofilm-associated infections.IBB-CEB, and the Portuguese Foundation for Science and Technology (Project PTDC/SAU-ESA/64609/2006; PhD Grant SFRH/BD/31065/2006

Phenotypic switching : an opportunity to bacteria thrive

Bacteria in nature are “plagued“ by various unpredictable environmental stresses, being population diversity one of the strategies adopted to survive. Phenotypic switching is one of the evolution processes that causes commutation between phenotypic states. This phenomenon shows up as variation in colony morphology. Alterations in colony morphotype traits may reveal altered cellular basis phenotype which can confer ensured virulence, antimicrobial resistance and persistence. However, the specific correlation between those traits and the biological impact is unknown. Phenotypic switching also occurs during biofilm formation, in that, bacteria have to adapt to this mode of growth expressing different phenotype traits often distinct from those expressed during planktonic growth. Often, after severe stresses, it is observed the survival of a small non-growing population, the persisters cells. The persister-state is fully reversible under growth stimulating conditions and therefore does not depend on genetic alterations. Bacterial persistence has been pointed out as switching between growing and dormant cells. Nevertheless, not only the responsive switching to environmental changes is important for survival of bacteria. Inherent heterogeneity may also be of major importance in environmental adaptation and persistence. Control of stress-response gene expression determines whether bacteria can survive to changing conditions and compete for the resources it needs to proliferate. Reversible phenotypic switching offers considerable advantages over conventional irreversible mutations. This chapter discusses the impact of generating population-level diversity on important clinical issues as resistance, virulence and persistence. It highlights that even though the growing interest and relevance of this phenomenon and its impact on bacterial ecology, the evolutionary origins and adaptive significance remain poorly understood

Effect of benzalkonium chloride residues on the initial adhesion of adapted P. aeruginosa

Bacteria have developed different strategies to sense, respond and adapt to chemical antimicrobial compounds. One of them involves bacterial adhesion to solid surfaces and subsequent biofilm formation. The biofilm lifestyle, that is usually resistant to antimicrobial treatment, is a phenomenon well noticed in almost all industrial and medical arenas. In the latter, the existence of pathogenic bacteria, that easily adhere to a surface and form biofilms, can give rise to equipment contamination and persistent infections. For instance, Pseudomonas aeruginosa has become increasingly recognized as an emerging opportunistic pathogen of clinical relevance. This bacterium, often found in a biofilm, attached to some surface, can frequently cause life-threatening infections under conditions where the host is injured and/or has the immune system compromised. Benzalkonium chloride (BZK) is a quaternary ammonium compound (QAC) used as a general clinical disinfectant and antiseptic in health care facilities and domestic households. QACs are bacteriostatic at low concentrations and bactericidal at high concentrations, thus their indiscriminate and improper use may favor the development of adaptive resistance. Therefore, they have been pointed out as the possible cause for the selection and persistence of bacterial strains with antibiotic and biocidal resistance. With this study it was aimed to characterize the adhesion ability and biofilm formation of P. aeruginosa non-adapted and adapted to BZK on polyethylene (PET) surfaces non-conditioned and pre-conditioned with BZK solution. For that purpose, bacterial hydrophobicity and changes in surface properties after conditioning were evaluated by means of contact angles determination. The initial bacterial adhesion was followed up along time (2, 4 and 6 h) by the quantification of the adhered biomass, through crystal violet (CV) and respiratory activity, using XTT. The adaptation of P. aeruginosa to BZK was attained after 6 cycles of re-inoculation every 48 h selected in TSA also supplemented with increasing doses of BZK. The free energy of adhesion of the bacteria with the PET surfaces, calculated trough the hydrophobicity components, revealed that the precontioning of the substrata increases the potential initial adhesion of P.aeruginosa, being this more significant for the adapted bacteria. Thus, it can be stated that, in the thermodynamic point of view, the initial adhesion of the P. aeruginosa adapted and non-adapted to the PET is favoured by surface preconditioning. Moreover, BZK preconditioning did not prevent or impair P.aeruginosa adhesion. In fact, the initial adhesion data showed that the accumulation of biomass increased with time, being the non-adapted bacterial cells more able to attach and establish a biofilm. The presence of BZK residues in the adhesion surfaces (preconditioning) promotes the attachment of the bacterial cells, since more biomass was determined. Concerning the activity of the adhered cells, preconditioning seem not cause any disturbance of cells, since equal or higher cellular activity was determined. The overall results suggest that preconditioning of PET surfaces with BZK promotes the attachment and activity of the bacterial cells, especially of the adapted bacteria. These evidences suggest that when the bacteria were previously exposed to a sub-lethal concentration of a chemical antimicrobial compound, they acquired some insusceptibility to that compound allowing their growth when facing adverse conditions. This study allowed gaining insights about the impact of unsuitable disinfection and rinsing procedures that might instigate the residues deposition, leading to bacteria adaptation, bacterial adhesion and biofilm development

The antimicrobial action of Pseudomonas aeruginosa byproducts in the control of single and mixed biofilms

Since bacteria are continuously acquiring resistance to conventional chemical agents, it is urgently needed the development of new strategies for biofilm control. It is well recognised that certain microorganisms represent an important source of novel biologically active compounds, with pronounced antibacterial activity, as secondary metabolites. Such substances are accepted to be essential for their producers, inhibiting other bacteria that compete for common resources. The main goal of this work was to investigate the antimicrobial effect of secondary metabolites secreted by P. aeruginosa on planktonic and sessile growth of several pathogens, in order to later use those molecules as bio-regulation agents. P. aeruginosa supernatants had potential as anti-biofilm agents but only against staphylococcal biofilms since they failed in disturb other biofilm consortia that encompassed Grambacteria. This trait makes them quite ineffective chemical countermeasures against real biofilms

Relevance of porin OprE on adaptive resistance of Pseudomonas aeruginosa biofilms to benzalkonium chloride

Biofilm formation is a phenomenon that happens naturally in the environment, representing one of the strategies adopt by microorganisms in attempt to protect themselves from external pressures. In clinical area, the most common practice to eliminate unwanted biofilms is the application of antimicrobial agents. However, current disinfection practices show often inefficacy in the eradication of biofilms, contributing for decreased susceptibility of microorganisms. The high tolerance of Pseudomonas aeruginosa to several antibiotics has been reported and is caused, partially, by reduced permeability through membrane. Concerning biocide resistance, the membrane alterations adopted by microorganisms is still unclear. The main goal of this work was the characterization of the membrane protein profile of sessile P. aeruginosa after induction of adaptive phenotype by exposed to antimicrobial stress with the biocide benzalkonium chloride (BC). Previous to biofilm exposure to BC, biofilms were formed on glass wool for 5 days. Biofilm adaptation was made by serially exposure of P. aeruginosa in increasing concentrations for each 48 hours. After 6 days of adaptation, alterations in outer membrane proteins expressed by sessile bacteria and matrix proteins were examined by SDS-PAGE. From adaptation resulted biofilms able to survive in media with 324 mg/L of BC. Analyzing the protein profiles of adapted and non-adapted biofilms, it was verified the decrease expression of a serie of proteins with 45 kDa. Certainly, the outer membrane porin OprE must be present on this serie. Indeed, this OprE is often associated with phenomena of resistance [1]. The results suggest that, as similar as chromate resistance, OprE can have a role in efflux of BC from periplasm to cell exterior [2]. Regarding protein profiles of the polymeric matrix of biofilms were found differences between adapted and non-adapted biofilms. However, there was no evidence that these changes could be associated with resistance phenomenon. This work revealed that a simple in vitro process of induction of adaptive resistance generated P. aeruginosa more resistant to BC action. This finding may, partially, explain the rapid development of resistance by microorganisms in a real environment, notably in clinical area, where antimicrobials are often used and sometimes at inadequate levels. Knowledge about mechanisms underlying the biocide resistance can be useful to design new antimicrobial agents with specific target as OprE