Inhibitory efficacy of various antibiotics on matrix and viable mass of Staphylococcus aureaus and Pseudomonas aeruginosa biofilms

Both bacteria and the matrix are essential for the development of biofilms. Antimicrobials should therefore be tested against both components. The aim of this study was to determine the structure-activity relationships of different antibiotics against biofilm-forming Staphylococcus aureus and Pseudomonas aeruginosa strains using in vitro biofilm discriminatory assays. Only four of twelve antibiotics showed efficacy against S. aureus biofilms. Rifampicin had a 50% inhibitory activity both against the matrix and bacteria at 16 x the minimum bactericidal concentration (MBC). Polymyxin B killed nearly all bacteria at 8 x MBC, but left the matrix undisturbed. Both P. aeruginosa biofilms responded differently to antibiotic treatment. Rifampicin showed the greatest activity, with 100% killing of microorganisms combined with 91% destruction of the matrix at the MBC. In conclusion, rifampicin showed the highest activity on biofilm matrix and bacteria in S. aureus and P. aeruginosa biofilms. Our results also indicated that biofilm viable mass was more susceptible to treatment than the biofilm matrix, which is mainly responsible for biofilm persistence. Future research should specifically focus on compounds destroying the matrix that can be used as an adjunct to antibiotic therapy

Validation of a Simple Resazurin-Based Promastigote Assay for the Routine Monitoring of Miltefosine Susceptibility in Clinical Isolates of Leishmania Donovani

Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases (n017) against standard amastigote assay, in two different labs in India. The interstage MIL susceptibility correlated strongly (r00.70, p0 0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation (r00.72, p00.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions (n07, r00.78, p00.046) and MIL-induced parasites (r00.92, p00.0001; n03) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patient

A flow cytometric approach to quantify biofilms

Abstract: Since biofilms are important in many clinical, industrial, and environmental settings, reliable methods to quantify these sessile microbial populations are crucial. Most of the currently available techniques do not allow the enumeration of the viable cell fraction within the biofilm and are often time consuming. This paper proposes flow cytometry (FCM) using the single-stain viability dye TO-PRO\uae-3 iodide as a fast and precise alternative. Mature biofilms of Candida albicans and Escherichia coli were used to optimize biofilm removal and dissociation, as a single-cell suspension is needed for accurate FCM enumeration. To assess the feasibility of FCM quantification of biofilms, E. coli and C. albicans biofilms were analyzed using FCM and crystal violet staining at different time points. A combination of scraping and rinsing proved to be the most efficient technique for biofilm removal. Sonicating for 10 min eliminated the remaining aggregates, resulting in a single-cell suspension. Repeated FCM measurements of biofilm samples revealed a good intraday precision of approximately 5 %. FCM quantification and the crystal violet assay yielded similar biofilm growth curves for both microorganisms, confirming the applicability of our technique. These results show that FCM using TO-PRO\uae-3 iodide as a single-stain viability dye is a valid fast alternative for the quantification of viable cells in a biofilm

An Overview of European Efforts in Generating Climate Data Records

The Coordinating Earth Observation Data Validation for Reanalysis for Climate Services project (CORE-CLIMAX) aimed to substantiate how Copernicus observations and products can contribute to climate change analyses. CORE-CLIMAX assessed the European capability to provide climate data records (CDRs) of essential climate variables (ECVs), prepared a structured process to derive CDRs, developed a harmonized approach for validating essential climate variable CDRs, identified the integration of CDRs into the reanalysis chain, and formulated a process to compare the results of different reanalysis techniques. With respect to the Copernicus Climate Change Service (C3S), the systematic application and further development of the CORE-CLIMAX system maturity matrix (SMM) and the spinoff application performance metric (APM) were strongly endorsed to be involved in future implementations of C3S. We concluded that many of the current CDRs are not yet sufficiently mature to be used in reanalysis or applied in climate studies. Thus, the production of consistent high-resolution data records remains a challenge that needs more research urgently. Extending ECVs to close climate cycle budgets (e.g., essential water variables) is a next step linking CDRs to sectoral applications