Prevention of Staphylococcus aureus biofilm formation by antibiotics in 96-Microtiter Well Plates and Drip Flow Reactors : critical factors influencing outcomes

Biofilm formation leads to the failure of antimicrobial therapy. Thus, biofilm prevention is a desirable goal of antimicrobial research. In this study, the efficacy of antibiotics (doxycycline, oxacillin and rifampicin) in preventing Staphylococcus aureus biofilms was investigated using Microtiter Well Plates (MWP) and Drip Flow Reactors (DFR), two models characterized by the absence and the presence of a continuous flow of nutrients, respectively. Planktonic culture of S. aureus was exposed to antibiotics for one hour followed by 24 hours incubation with fresh nutrients in MWP or continuous flow of nutrients in DFR. The DFR grown biofilms were significantly more tolerant to the antibiotics than those grown in MWP without the continuous flow. The differences in log reductions (LR) between the two models could not be attributed to differences in the cell density, the planktonic inoculum concentration or the surface-area-to-volume ratios. However, eliminating the flow in the DFR significantly restored the antibiotic susceptibility. These findings demonstrate the importance of considering differences between experimental conditions in different model systems, particularly the flow of nutrients, when performing anti-biofilm efficacy evaluations. Biofilm antibiotic efficacy studies should be assessed using various models and more importantly, in a model mimicking conditions of its clinical application.Peer reviewe

α,α-disubstituted β-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal

Bacterial biofilms account for up to 80% of all infections and complicate successful therapies due to their intrinsic tolerance to antibiotics. Biofilms also cause serious problems in the industrial sectors, for instance due to the deterioration of metals or microbial contamination of products. Efforts are put in finding novel strategies in both avoiding and fighting biofilms. Biofilm control is achieved by killing and/or removing biofilm or preventing transition to the biofilm lifestyle. Previous research reported on the anti-biofilm potency of α,α-disubstituted β-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Dalton. In the current study it was investigated if these derivatives cause a fast disintegration of biofilm bacteria and removal of Staphylococcus aureus biofilms. One hour incubation of biofilms with all three derivatives resulted in reduced metabolic activity and membrane permeabilization in S. aureus (ATCC 25923) biofilms. Bactericidal properties of these derivatives were attributed to a direct effect on membranes of biofilm bacteria. The green fluorescence protein expressing Staphylococcus aureus strain AH2547 was cultivated in a CDC biofilm reactor and utilized for disinfectant efficacy testing of A3, following the single-tube method (American Society for Testing and Materials designation number E2871). A3 at a concentration of 90 μM acted as fast as 100 μM chlorhexidine and was equally effective. Confocal laser scanning microscopy studies showed that chlorhexidine treatment lead to fluorescence fading indicating membrane permeabilization but did not cause biomass removal. In contrast, A3 treatment caused a simultaneous biofilm fluorescence loss and biomass removal. These dual anti-biofilm properties make α,α-disubstituted β-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms

Minimum information guideline for spectrophotometric and fluorometric methods to assess biofilm formation in microplates

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioflm.2019.100010.The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the necessary information that a manuscript should include for the experiments in a study to be clearly interpreted and independently reproduced. As a result of several discussions between international groups working in the area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm formation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set of recommendations. The intention of the minimum information guideline is to improve the quality of scientific communication that will augment interlaboratory reproducibility in biofilm microplate assays.This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska – Curie grant agreement No 722467, as part of the Print-Aid consortium. The information and views set out in this article are those of the authors and do not necessarily reflect the official opinion of the European Union. Neither the European Union institutions and bodies nor any person acting on their behalf may be held responsible for the use which may be made of the information contained therein. This work received additional financial support by: project UID/EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE funded by national funds through FCT/MCTES (PIDDAC); Project “LEPABE-2-ECO-INNOVATION” – NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log(10)-scale, the reproducibility SD (S-R) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/S-R=1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.Peer reviewe

Infrared Space Observatory Spectra of R Coronae Borealis Stars. I. Emission Features in the Interval 3 - 25 microns

Infrared Space Observatory 3 - 25 $\mu$m spectra of the R Coronae Borealis stars V854 Cen, R CrB, and RY Sgr are presented and discussed. Sharp emission features coincident in wavelengths with the well known Unidentified Emission Features are present in the spectrum of V854 Cen but not of R CrB or RY Sgr. Since V854 Cen is not particularly H-poor and has a 1000 times more H than the other stars, the emission features are probably from a carrier containing hydrogen. There is a correspondence between the features and emission from laboratory samples of hydrogenated amorphous carbon. A search for C$_{60}$ in emission or absorption proved negative. Amorphous carbon particles account for the broad emission features seen between 6 - 14 $\mu$m in the spectrum of each star.Comment: 11 pages with 2 tables and 4 figures in ApJ print page form. Accepted for publication in Ap

The influence of binarity on dust obscuration events in the planetary nebula M 2-29 and its analogues

The central star of the planetary nebula (CSPN) M 2-29 shows an extraordinary R Coronae Borealis-like fading event in its optical lightcurve. The only other CSPN to show these events are CPD-568032 (Hen 3-1333) and V651 Mon (NGC 2346). Dust cloud formation in the line of sight appears responsible but the exact triggering mechanism is not well understood. Understanding how planetary nebulae (PNe) trigger dust obscuration events may help understand the same process in a wide range of objects including Population-I WC9 stars, symbiotic stars and perhaps Asymptotic Giant Branch (AGB) stars with long secondary periods (LSPs). A binary scenario involving an eccentric, wide companion that triggers dust formation via interaction at periastron is a potential explanation that has been suggested for LSP variables. Model fits to the lightcurves of CPD-568032 and M 2-29 show the dust forms in excess of 70 AU at the inner edge of a dust disk. In the case of CPD-568032 this radius is far too large to coincide with a binary companion trigger, although a binary may have been responsible for the formation of the dust disk. We find no direct evidence to support previous claims of binarity in M 2-29 either from the OGLE lightcurve or deep medium-resolution VLT FLAMES spectroscopy of the CSPN. We classify the CSPN as Of(H) with T_eff=50+-10 kK and log g=4.0+-0.3. We find a mean distance of 7.4+-1.8 kpc to M 2-29 at which the M_V=-0.9 mag CSPN could potentially hide a subgiant luminosity or fainter companion. A companion would help explain the multiple similarities with D'-type symbiotic stars whose outer nebulae are thought to be bona-fide PNe. The 7.4 kpc distance, oxygen abundance of 8.3 dex and Galactic coordinates (l=4.0, b=-3.0) prove that M 2-29 is a Galactic Bulge PN and not a Halo PN as commonly misconceived.Comment: 15 pages, 14 figures, 7 tables. Accepted for publication in A\&

Detection of an asymmetry in the envelope of the carbon Mira R Fornacis using VLTI/MIDI

Aims. We present a study of the envelope morphology of the carbon Mira R For with VLTI/MIDI. This object is one of the few asymptotic giant branch (AGB) stars that underwent a dust-obscuration event. The cause of such events is still a matter of discussion. Several symmetric and asymmetric scenarios have been suggested in the literature. Methods. Mid-infrared interferometric observations were obtained separated by two years. The observations probe different depths of the atmosphere and cover different pulsation phases. The visibilities and the differential phases were interpreted using GEM-FIND, a tool for fitting spectrally dispersed interferometric observations with the help of wavelength-dependent geometric models. Results. We report the detection of an asymmetric structure revealed through the MIDI differential phase. This asymmetry is observed at the same baseline and position angle two years later. The observations are best simulated with a model that includes a uniform-disc plus a Gaussian envelope plus a point-source. The geometric model can reproduce both the visibilities and the differential phase signatures. Conclusions. Our MIDI data favour explanations of the R For obscuration event that are based on an asymmetric geometry. We clearly detect a photocentre shift between the star and the strongly resolved dust component. This might be caused by a dust clump or a substellar companion. However, the available observations do not allow us to distinguish between the two options. The finding has strong implications for future studies of the geometry of the envelope of AGB stars: if this is a binary, are all AGB stars that show an obscuration event binaries as well? Or are we looking at asymmetric mass-loss processes (i.e. dusty clumps) in the inner part of a carbon-rich Mira?Comment: accepted for publication as A&A lette

An overview on the reactors to study drinking water biofilms

The development of biofilms in drinking water distribution systems (DWDS) can cause pipe degradation, changes in the water organoleptic properties but the main problem is related to the public health. Biofilms are the main responsible for the microbial presence in drinking water (DW) and can be reservoirs for pathogens. Therefore, the understanding of the mechanisms underlying biofilm formation and behavior is of utmost importance in order to create effective control strategies. As the study of biofilms in real DWDS is difficult, several devices have been developed. These devices allow biofilm formation under controlled conditions of physical (flow velocity, shear stress, temperature, type of pipe material, etc), chemical (type and amount of nutrients, type of disinfectant and residuals, organic and inorganic particles, ions, etc) and biological (composition of microbial community e type of microorganism and characteristics) parameters, ensuring that the operational conditions are similar as possible to the DWDS conditions in order to achieve results that can be applied to the real scenarios. The devices used in DW biofilm studies can be divided essentially in two groups, those usually applied in situ and the bench top laboratorial reactors. The selection of a device should be obviously in accordance with the aim of the study and its advantages and limitations should be evaluated to obtain reproducible results that can be transposed into the reality of the DWDS. The aim of this review is to provide an overview on the main reactors used in DW biofilm studies, describing their characteristics and applications, taking into account their main advantages and limitations.This work was supported by the Operational Programme for Competitiveness Factors COMPETE and by Portuguese Foundation for Science and Technology through Project Phyto disinfectants - PTDC/DTPSAP/1078/2012 (COMPETE: FCOMP-01-0124-FEDER-028765), the Post-Doc grant awarded to Lucia Simoes (SFRH/BPD/81982/2011). Also, this work was undertaken as part of the European Research Project SUS-CLEAN (Contract n_FP7-KBBE-2011-5, project number: 287514) and the COST Action FA1202. The authors are solely responsible for this work. It does not represent the opinion of the Community, and the Community is not responsible for any use that might be made of data appearing herein

Effect of β-Blockers on Cardiac and Pulmonary Events and Death in Older Adults With Cardiovascular Disease and Chronic Obstructive Pulmonary Disease

CONTEXT: In older adults with multiple conditions, medications may not impart the same benefits seen in patients who are younger, or without multi-morbidity. Furthermore, medications given for one condition may adversely affect other outcomes. Beta-blocker (β-Blocker) use with coexisting cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD) is such a situation. OBJECTIVE: To determine the effect of β-Blocker use on cardiac and pulmonary outcomes and mortality in older adults with coexisting COPD and CVD. DESIGN, SETTING, PARTICIPANTS: The 1062 participants were members of the 2004-2007 Medicare Current Beneficiary Survey cohorts, a nationally representative sample of Medicare beneficiaries. Study criteria included age 65+ years plus coexisting CVD and COPD/asthma. Follow-up occurred through 2009. We determined the association between β-Blocker use and the outcomes with propensity score-adjusted and covariate-adjusted Cox proportional hazards. MAIN OUTCOME MEASURES: The three outcomes were major cardiac and pulmonary events, and all-cause mortality. RESULTS: Half of the participants used β-Blockers. During follow-up 179 participants experienced a major cardiac event; 389 participants experienced a major pulmonary event; and 255 participants died. Each participant could have experienced any one or more of these events. The hazard ratio for β-blocker use was 1.18 (95% CI, 0.85-1.62) for cardiac events; 0.91 (95% CI, 0.73-1.12) for pulmonary events; and, 0.87 (95% CI, 0.67-1.13) for death. CONCLUSION: In this population of older adults, β-Blockers did not seem to affect occurrence of cardiac or pulmonary events or death in those with CVD and COPD.Keywords: cardiovascular disease, multiple chronic conditions, coronary artery disease, COPD, chronic obstructive pulmonary disease, cardiac events, beta-blocker, pulmonary events, multimorbidity, CAD, CV

Statins and Physical Activity in Older Men: The Osteoporotic Fractures in Men Study

IMPORTANCE: Muscle pain, fatigue, and weakness are common adverse effects of statin medications and may decrease physical activity in older men. OBJECTIVE: Determine whether statin use is associated with physical activity, longitudinally and cross-sectionally. DESIGN, SETTING, AND PARTICIPANTS: Men participating in the Osteoporotic Fractures in Men Study, a multicenter prospective cohort study of community-living men age 65+, enrolled between March 2000-April 2002. EXPOSURE: Statin use as determined by an inventory of medications (taken within last 30 days). In cross-sectional analyses, statin use categories were: users and nonusers. In longitudinal analyses, categories were: prevalent users (baseline use and throughout study), new users (initiated use during the study) and nonusers (never used). MAIN OUTCOMES AND MEASURE: Self-reported physical activity at baseline and 2 follow-up visits using the Physical Activity Scale for the Elderly (PASE). At the third visit, an accelerometer measured metabolic equivalents (METs; kcal/kg/hr) and minutes of moderate activity (METs ≥3.0), vigorous activity (METs ≥6.0), and sedentary behavior (METs ≤1.5). RESULTS: At baseline, 989 men (24%) were users and 3,148 (76%) were nonusers. The adjusted difference in baseline PASE between users and nonusers was -5.8 points (95% CI, -10.9 to -0.7). A total of 3,039 men met the inclusion criteria for longitudinal analysis: 727 (24%) prevalent users, 845 (28%) new users, 1,467 (48%) nonusers. PASE declined by an average of 2.5 points/year (2.0-3.0) for nonusers and 2.8 points/year (2.1, 3.5) for prevalent users, a nonstatistical difference (0.3 point, -0.5-1). For new users, annual PASE score declined at a faster rate than nonusers (0.9 point difference; 0.1-1.7). 3,071 men had adequate accelerometry data, 1,542 (50%) were statin users. Statin users expended less METS (0.03 kcal/kg/hr less; 0.02-0.04); engaged in less moderate physical activity (5.4 fewer minutes/day; 1.9-8.8), less vigorous activity (0.6 fewer minutes/day; 0.1-1.1), and more sedentary behavior (7.6 greater minutes/day; 2.6-12.4). CONCLUSION AND RELEVANCE: Statin use was associated with modestly lower physical activity among community-living men, even after accounting for medical history and other potentially confounding factors. The clinical significance of these findings deserves further investigation