Flexible biofilm monitoring device.

Biofilms and their analysis are increasingly attracting the attention of the scientific community due to the immense importance and impact of biofilms in various natural, technical and medical fields. For these purposes, an optimized and extended antibiofilm assay system based on the Calgary Biofilm Device (MBEC Assay® system) consisting of microtiter plate and PCR tubes was established. Its implementation was used to study the growth characteristics of the sessile phenotype of Pseudomonas fluorescens exposed to antimicrobial peptides. Inhibitory effects of an antimicrobial peptide on P. fluorescens biofilm formation could be determined at a concentration of 250 µg/ml (biofilm prevention concentration (BPC)) using the modified biofilm assay. Similarly, the biofilm bactericidal concentration (BBC) at 125 µg/ml and the minimum biofilm elimination concentration to remove 90% of the total biofilm mass (MBEC90) were measured at a concentration range of 15.625-1.95 µg/ml. In conclusion, this optimized system provides a highly variable, simple, and cost-effective alternative to high-throughput screening based on the Calgary Biofilm Device (CBD).

Adaptation of the Start-Growth-Time Method for High-Throughput Biofilm Quantification.

Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.

MBEC Versus MBIC: the Lack of Differentiation between Biofilm Reducing and Inhibitory Effects as a Current Problem in Biofilm Methodology.

BACKGROUND: Biofilms are communities of aggregated, matrix-embedded microbial cells showing a high tolerance to an in principle adequate antibiotic therapy, often resulting in treatment failure. A major challenge in the management of biofilm-associated infections is the development of adequate, standardized biofilm susceptibility testing assays that are clinically meaningful, i.e. that their results correlate with treatment outcome. Different biofilm susceptibility endpoint parameters like the minimal biofilm eradication concentration (MBEC) or the minimal biofilm inhibitory concentration (MBIC) have been suggested as a guide for treatment of biofilm-associated infections, however with inconsistent perception and use among biofilm researchers, leading to confusion and contradictions among different anti-biofilm component studies and clinical trials. FINDINGS: Evaluation of anti-biofilm effects is mostly based on the untreated reference growth control biofilm measured at the same endpoint as the treated biofilm, neglecting the possible change of the untreated reference biofilm from the time point of pre-antimicrobial exposure to the measured endpoint. In this commentary, we point out the importance of individual quantification of mature, established biofilms before antimicrobial treatment for each biofilm model in order to draw conclusions on the measured biofilm effect size, i.e. biofilm reducing (MBEC) or biofilm inhibitory (MBIC) effects. CONCLUSION: The assessment of pre-treatment biofilms contributes to a standardized use of biofilm susceptibility endpoint parameters, which is urgently needed to improve the clinical validity of future anti-biofilm assays.