Colistin surface conditioning impairs Pseudomonas aeruginosa biofilm formationand enhances ciprofloxacin antimicrobial activity

Bacterial biofilms on medical devices (MD) are commonly associated with persistent infections. Biofilm formation is central to pathogenesis due to the ability of the biofilm-entrapped bacteria to evade the host immune responses and the increased antimicrobial resistance phenotype often shown by sessile microorganisms. In order to control the infections related with MD, antimicrobial peptide conditioning of MD surfaces can be an attractive answer. Colistin (COL), an important cationic antimicrobial peptide (AMP) produced by Bacillus polymyxa strains, was used to coat polystyrene (PS) surfaces. This work aims at characterizing the antimicrobial effect of COL surface coating to impair Pseudomonas aeruginosa adhesion and subsequent biofilm formation. The response of P. aeruginosa 24 h biofilms treatment with Ciprofloxacin (CIP) in non-conditoned and COL conditioned surfaces was also assessed P. aeruginosa from collection (ATCC 10145) (PAC) and P. aeruginosa isolated (PAI) from a medical device (endoscope) were used as biofilm producers. PS surfaces were pre-conditioned with 64 mg/L of COL during 30 min. Biofilms were then developed in unconditioned and COL-conditioned surfaces, being after treated with Ciprofloxacin (CIP) at 0.75 mg/L. Biofilms were phenotypically characterized in terms of biomass, respiratory activity and cell number. Results showed that, in general, the MD isolate produces biofilms with more mass and activity but less number of cells than the reference strain, being the action of COL conditioning or CIP treatment similar for both strains. The surface conditioning with COL was very efficient, as it impaired significantly biofilm formation in terms of mass and activity, allowing the adhesion of just 3-log of cells. The CIP treatment of biofilms developed in unconditioned and COL-conditioned surfaces, promoted reduction of biofilm mass, activity and 2-log of number of biofilm cells. Concerning the combined application of COL surface conditioning and biofilm treatment with CIP it was observed an increase in CIP efficacy in biofilm sanitation, especially regarding biofilm-entrapped cell reduction. In fact, the combination of conditioning/treatment promoted an accentuated reduction of the biofilm mass and activity and caused a reduction of 4-log of biofilm-entrapped cells. This study demonstrates the potential use of COL surface conditioning since this surface treatment impairs biofilm formation, probably interfering in the transition from irreversible attached cells to mature biofilms.Moreover, and as consequence of the reduced amount of biofilms attached to COL-conditioned surfaces, adhered cells or thin biofilms become more exposed to the subsequent action of CIP. This study highlights a promising use of COL as MD coating and a synergistic effect between COL surface conditioning and CIP antimicrobial activity. The financial support from IBB-CEB and Fundação para a Ciência e Tecnologia (FCT) and European Community fund FEDER, trough Program COMPETE, in the ambit of the Project PTDC/SAU-SAP/113196/2009 /FCOMP-01-0124-FEDER-016012 and Idalina Machado PhD Grant (SFRH/BD/31065/2006) are gratefully acknowledged

Adaptive response of single and binary biofilms formed in the presence of benzalkonium chloride

In actual situations bacteria can live nicely in hostile environments in part due to its ability to live in biofilms and to regulate gene expression as an adaptive response to a variety of stresses. This altered profile, compared to planktonic counterparts, as well as the interactions amongst the several strains existing within the biofilm seems to be responsible for the gradual loss of susceptibility to antimicrobials. The presence of biofilms on the surfaces of various types of medical surfaces and equipment can be the source of pathogens and contribute to biofilm-associated infections. The aim of this work was to examine whether exposure of P.aeruginosa and E.coli to an antimicrobial agent – benzalkoniun chloride (BC) – during single and binary biofilm formation could induce an adaptive response in bacteria and further evaluate the biofilm behaviour after aggression with the same agent. Biofilms were formed in microtiter plates for 5 days in presence of 0.9 mM of BC, being afterwards submitted to BC attack (1 mM). Biofilms were characterized by total mass, through crystal violet, metabolic activity, through XTT method, and number of cells. The data showed that both single and binary biofilms formed in the presence of BC have clearly more mass, less activity and slight less number of cells. These events could be sign of a different metabolic state of the cells entrapped in the biofilms previously exposed to the surfactant. The simultaneous growth of both species seems not prejudice or favour binary biofilm development. After BC attack, biofilms developed in the absence of the surfactant suffered mass and activity reduction and had fewer cells, probably due to the toxic effect of the product. Conversely, when the biofilms were developed in the presence of BC, the attack leads to biofilms with more mass and activity, being however the number of cells reduced. This discrepancy may be due to the loss of culturability of bacteria in solid media. These facts emphasise that BC is not effective against biofilms developed in its presence. It can be concluded that the presence of BC during single and binary sessile growth seems to induce an adaptive resistance of P. aeruginosa and E. coli biofilms. This biofilm features, resulting from an adverse situation, can play a significant role in the selection of resistant pathogenic bacteria and be on the basis of recalcitrant biofilms, contaminating medical surfaces and causing nosocomial infections.IBB-CEB and FCT, for the financial support (Project PTDC/SAU-ESA/64609/2006; PhD Grant SFRH/BD/31065/2006)

Blocking of Pseudomonas aeruginosa biofilm formation by a colistin coating

Bacterial colonisation of indwelling devices followed by biofilm formation remains a serious threat in clinical field as it is commonly associated to persistent infections. Once adhered to a surface, bacteria embed themselves in a self-produced matrix mainly composed of extracellular polymeric substances which confers them protection against antimicrobial agents and the host immune system. Early bacterial adhesion is a crucial step in biomaterial associated infections pathogenesis, representing therefore a promising target for the development of biofilm preventive measures. Several strategies have been developed to prevent bacterial adhesion and biofilm formation on the surfaces of medical devices, based mainly on the use of anti-adhesive, antiseptic and antibiotic coatings. Although some of these coatings have been shown efficient in the prevention of biofilm formation, an important drawback associated to them is the development of microbial resistance that limits the usefulness of classical antimicrobials. A promising solution to overcome this problem may rely on the use of new alternatives, as antimicrobial peptides (AMPs) that are unlikely to induce any resistance because of their evolutionary path. The aim of this work was to evaluate the potential role of colistin, a traditional AMP, as an antimicrobial coating for biomaterials. Based on the observation that the presence of colistin as a biofilm growth media complement was able to significantly impair Pseudomonas aeruginosa biofilm formation at concentrations below its MBC, polystyrene (PS) surfaces were coated with this AMP and its ability to prevent biofilm formation was assessed. A P. aeruginosa reference strain (ATCC 10145) and a P. aeruginosa clinical isolate (U147016) were used as biofilm producers. PS surfaces were pre-coated with several concentrations of colistin and the biofilms formed on conditioned and clean surfaces were then characterized in terms of biomass (CV), respiratory activity (XTT) and number of viable cells (CFU). The susceptibility of biofilms formed on colistin-conditioned surfaces to ciprofloxacin (CIP) treatment was further investigated. Results showed that the clinical isolate produces biofilms with more activity, less biomass and similar number of cells than the reference strain. Random deposition of colistin residues on the adhesion surfaces significantly reduced biofilm activity and mass accumulated in a dose-dependent manner for both strains. Regarding biofilm entrapped cells, the conditioning film proved to be less efficient, causing significant reductions for the highest concentrations tested. Concerning the combined application of colistin surface conditioning and biofilm treatment with CIP, it was observed that biofilms formed on colistin-conditioned surfaces were more susceptible to CIP treatment in terms of biofilm-entrapped cells. The presence of colistin during biofilm formation may have interfered in the transition from reversible to irreversible interactions during the early steps of bacterial adhesion to PS, disturbing and delaying the mature biofilm development. Biomaterial associated infections remains a major drawback to the long-term use of medical devices. This study demonstrates the potential of the AMP colistin as an excellent candidate for biomaterials coating limiting biofilm formation on their surfaces

Susceptibility patterns and cross-resistance evaluation of several biofilm-producing P. aeruginosachallenged by antibiotics

P.aeruginosa (PA) is an opportunistic pathogen responsible for high percentage of nosocomial infections. Its virulence and persistence rises when bacteria switch from planktonic to biofilm state and when facing antimicrobial pressures. This study aimed to evaluate the antimicrobial tolerance and regrowth of several biofilmproducing PA after antibiotic treatment, and the occurrence of cross-resistance to other antimicrobials. PAO, ATCC, CGCT and an isolated strain were used to form 1-day old biofilms in the presence of ciprofloxacin (CIP). Biofilm regrowth was evaluated after 48h, 72h and 96h after addition of, respectively, fresh medium, medium with antibiotic and antibiotic-free medium again. The final 4-days old biofilms were then attacked with CIP, gentamicin, rifampicin and benzalkonium chloride. After 24h and 72h of biofilm growth, it was observed a good reduction of the biofilm mass, respiratory activity and CFU/cm2. However, PA biofilm regrowth with similar or even higher cell numbers, comparatively to control, were noticed always the antibiotic-free medium was added. Data allowed speculating that the adhered bacteria that survived to antibiotic therapy may become more resistant to CIP or to other antimicrobials. Luckily, data did not show evidence of cross-resistance to the antimicrobials tested. Biofilms formed in the presence of CIP appeared to be more susceptible to the anti-biofilm action of antimicrobials. Although it has been observed marked biofilm regrowth after good levels of biofilm eradication due to antibiotic treatment, the resulting biofilms challenged by CIP seemed to be less tolerant to the action of other antimicrobials.IBB-CEB and FCT (PTDC/SAU-ESA/64609/2006; SFRH/BD/31065/2006; SFRH/ BD/47613/2008

Longe dos partidos e perto da federação: uma avaliação dos gastos municipais na saúde pública

The institutionalization of SUS made the municipalities important players in the public health policy. Thus, it was expected that the amount of resources allocated to public health would be determinate in some measure by the political party preferences. Given these political and institutional contexts, we analyzed the importance of the political party policies at the municipality level as a determinant of the expenditure on public health, as well was other conventional determinants of the expenditures on public health (region, per capita income, GDP, etc). Applying some econometric techniques (quantile regressions and econometric decomposition), we found that the parties’ political preferences have minor influence on the allocation of resources in public health. We conclude that the constitutional regulation (Constitutional Emend 29 or EC29) seems to be a more important determinant to explain the allocation of resources in the period 2002-2006 than the supposed political preferences announced by parties.SUS, health economics, Brazil

Enzyme-assisted release of antioxidant peptides from porphyra dioica conchocelis

peer-reviewedThe conchocelis life cycle stage of P. dioica represents an unexplored source of bioactive compounds. The aim of this study was to generate and characterise, for the first time, hydrolysates of conchocelis using a specific combination of proteases (Prolyve® and Flavourzyme®). Hydrolysate molecular mass distribution and free amino acid contents were assessed, and the antioxidant activity was determined using a range of in vitro assays. The protein content and the total amino acid profiles of conchocelis were also studied. Conchocelis contained ~25% of protein (dry weight basis) and had a complete profile of essential amino acids. Direct sequential enzymatic treatment modified the profile of the generated compounds, increasing the amount of low molecular weight peptides (<1 kDa). There was a significant improvement in the antioxidant activity of the hydrolysates compared with the control (up to 2.5-fold), indicating their potential as a novel source of antioxidant ingredients

Towards the construction of health workforce metrics for Latin America and the Caribbean

<p>Abstract</p> <p>Introduction</p> <p>One of the components of the Health Observatory for Latin American and the Caribbean (HO-LAC) is the design and implementation of metrics for human resources for health. Under the HO-LAC initiative, researchers from nine countries in the region formed the Collaborative Community on Human Resources for Health in Latin America and the Caribbean to identify common metrics applicable to the field of human resources for health (HRH).</p> <p>Case description</p> <p>The case description comprises three stages: a) the origins of an initiative in which a non-governmental organization brings together researchers involved in HRH policy in LAC, b) a literature search to identify initiatives to develop methods and metrics to assess the HRH field in the region, and c) subsequent discussions held by the group of researchers regarding the possibilities of identifying an appropriate set of metrics and indicators to assess HRH throughout the region.</p> <p>Discussion and evaluation</p> <p>A total of 101 documents produced between 1985 and 2008 in the LAC region were identified. Thirty-three of the papers included a variety of measurements comprising counts, percentages, proportions, indicators, averages and metrics, but only 13 were able to fully describe the methods used to identify these metrics and indicators. Of the 33 articles with measurements, 47% addressed labor market issues, 25% were about working conditions, 23% were on HRH training and 5% addressed regulations. Based on these results, through iterative discussions, metrics were defined into three broad categories (training, labor market and working conditions) and available sources of information for their estimation were proposed. While only three of the countries have data on working conditions, all countries have sufficient data to measure at least one aspect of HRH training and the HRH labor market.</p> <p>Conclusions</p> <p>Information gleaned from HRH metrics makes it possible to carry out comparisons on a determined experience in space and time, in a given country and/or region. The results should then constitute evidence for policy formulation and HRH planning and programs, with improved health system performance ultimately contributing to improved population health. The results of this study are expected to guide decision making by incentivizing the construction of metrics that provide information about HRH problems in LAC countries.</p