Through a statistical meta-analysis of published data on antimicrobial efficacy against biofilms formed by two common bacterial species it was concluded that the particular experimental method used is the most important factor determining the outcome of the test. An expected dose-response behavior (greater killing with higher dose concentrations or longer treatment times) was observed for data sets derived from a single method, but was not observed when data from multiple studies using diverse methods were pooled. Method specific properties such as the surface area to volume ratio, areal biofilm cell density, and microbial species were shown to influence quantitative measurements of biofilm killing. A better appreciation of the method characteristics that affect anti-biofilm efficacy tests could aid decision-making related to investment in research and development and regulatory approvals for biofilm control strategies. These recommendations are offered to those working in research and development related to biofilm control: 1) report the log reduction, surface area to volume ratio, and biofilm areal cell density; 2) include data for a benchmark agent, making sure that this agent performs competitively at the dose tested; 3) measure a dose response, i.e., make measurements at multiple treatment concentrations or dose durations; and 4) use a standardized method in addition to research methods.
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