Browsing by Author "Fallarero, Adyary"

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Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods
(2021-07) Allkja, Jontana; van Charante, Frits; Aizawa, Juliana; Reigada, Ines; Guarch-Perez, Clara; Vazquez-Rodriguez, Jesus Augusto; Cos, Paul; Coenye, Tom; Fallarero, Adyary; Zaat, Sebastian A. J.; Felici, Antonio; Ferrari, Livia; Azevado, Nuno F.; Parker, Albert E.; Goeres, Darla M.
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 log10-scale, the reproducibility SD (SR) 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/SR = 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.
Minimum information guideline for spectrophotometric and fluorometric methods to assess biofilm formation in microplates
(2020-12) Allkja, Jontana; Bjarnsholt, Thomas; Coenye, Tom; Cos, Paul; Fallarero, Adyary; Harrison, Joe J.; Lopes, Susana P.; Oliver, Antonio; Pereira, Maria Olivia; Ramage, Gordon; Shirtliff, Mark E.; Stoodley, Paul; Webb, Jeremy S.; Zaat, Sebastian A. J.; Goeres, Darla M.; Azevedo, Nuno Filipe
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.
Minimum information guideline for spectrophotometric and ﬂuorometric methods to assess bioﬁlm formation in microplates
(Elsevier BV, 2020) Allkja, Jontana; Bjarnsholt, Thomas; Coenye, Tom; Cos, Paul; Fallarero, Adyary; Harrison, Joe J.; Lopes, Susana P.; Oliver, Antonio; Pereira, Maria Olivia; Ramage, Gordon; Shirtliff, Mark E.; Stoodley, Paul; Webb, Jeremy S.; Zaat, Sebastian A.J.; Goeres, Darla M.; Azevedo, Nuno Filipe
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.
Prevention of Staphylococcus aureus biofilm formation by antibiotics in 96-Microtiter Well Plates and Drip Flow Reactors: critical factors influencing outcomes
(2017-03) Manner, Suvi; Goeres, Darla M.; Skogman, Malena; Vuorela, Pia; Fallarero, Adyary
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.
Revisiting an agar-based plate method: What the static biofilm method can offer for biofilm research
(2014-12) Oja, Terhi; Blomqvist, Brianna; Buckingham-Meyer, Kelli; Goeres, Darla M.; Vuorela, Pia; Fallarero, Adyary
The development of biofilms in static plates was monitored. Glass coupons were placed on agar covered with filter paper, which was inoculated with suspended bacteria. The viable cell density, biofilms matrix and biomass were quantified. The method is excellent for adhesion and material studies, due to its simplicity and flexibility.
Systematic exploration of natural and synthetic flavonoids for the inhibition of Staphylococcus aureus biofilms
(2013-09) Manner, Suvi; Skogman, Malena; Goeres, Darla M.; Vuorela, Pia; Fallarero, Adyary
When single-cell (or suspended) bacteria switch into the biofilm lifestyle, they become less susceptible to antimicrobials, imposing the need for anti-biofilms research. Flavonoids are among the most extensively studied natural compounds with an unprecedented amount of bioactivity claims. Most studies focus on the antibacterial effects against suspended cells; fewer reports have researched their anti-biofilm properties. Here, a high throughput phenotypic platform was utilized to screen for the inhibitory activity of 500 flavonoids, including natural and synthetic derivatives, against Staphylococcus aureus biofilms. Since discrepancies among results from earlier antibacterial studies on flavonoids had been noted, the current study aimed to minimize sources of variations. After the first screen, flavonoids were classified as inactive (443), moderately active (47) or highly active (10). Further, exclusion criteria combining bioactivity and selectivity identified two synthetic flavans as the most promising. The body of data reported here serves three main purposes. First, it offers an improved methodological workflow for anti-biofilm screens of chemical libraries taking into account the (many times ignored) connections between anti-biofilm and antibacterial properties. This is particularly relevant for the study of flavonoids and other natural products. Second, it provides a large and freely available anti-biofilm bioactivity dataset that expands the knowledge on flavonoids and paves the way for future structure-activity relationship studies and structural optimizations. Finally, it identifies two new flavans that can successfully act on biofilms, as well as on suspendedbacteria and represent more feasible antibacterial candidates.
Α,α-disubstituted β-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal
(Elsevier BV, 2023-12) Ausbacher, Dominik; Miller, Lindsey A.; Goeres, Darla M.; Stewart, Philip S.; Strøm, Morten B.; Fallarero, Adyary
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 Da. 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.