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More than 65% of bacterial infections are caused by biofilms. However, standard biofilm susceptibility tests are not available for clinical use. All conventional biofilm models suffer from a long formation time and fail to mimic in vivo microbial biofilm conditions. Moreover, biofilms make it difficult to monitor the effectiveness of antibiotics. This work creates a powerful yet simple method to form a target biofilm and develops an innovative approach to monitoring the antibiotic's efficacy against a biofilm-associated infection. A paper-based culture platform can provide a new strategy for rapid microbial biofilm formation through capillary action. A combined electrical-electrochemical technique monitors bacterial metabolism rapidly and reliably by measuring microbial extracellular electron transfer (EET) and using electrochemical impedance spectroscopy (EIS) across a microbe-electrode interface. Three representative pathogens, Pseudomonas aeruginosa , Escherichia coli , and Staphylococcus aureus , form their biofilms controllably within an hour. Within another hour their susceptibilities to three frontline antibiotics with different action modes (gentamicin, ciprofloxacin, and ceftazidime) are examined. Our antibiotic susceptibility testing (AST) technique provides a quantifiable minimum inhibitory concentration (MIC) of those antibiotics against the in vitro biofilm models and characterizes their action mechanisms. The results will have an important positive effect because they provide immediately actionable healthcare information at a reduced cost, revolutionizing public healthcare.

There is a pressing need for evidence-based, non-surgical therapy guidance for biofilm-based infections. Conventional phenotypic or genotypic or emerging antibiotic susceptibility testing (AST) techniques cannot provide clinically relevant guidelines and widely adaptable stewardship for effective biofilm treatment because they are mainly limited to planktonic bacteria and suffer from many technical and operational challenges. Here, we created an all-electrical, reliable, rapid AST device to monitor antibiotic efficacy in bacterial biofilms that can be practically translatable to clinical settings and industrial antibiotic developments. The electrons metabolically produced by a Pseudomonas aeruginosa biofilm provided a strong signal for monitoring bacterial growth and treatment efficacy while a 3-D paper-based culturing platform provided a new strategy for rapid biofilm formation through capillary action. When antibiotics are effective against the pathogenic biofilm, their metabolic activities are inhibited, decreasing their electron transfer reactions. The changes in electrical outputs can be measured to assess the treatment effectiveness against pathogenic biofilms. Within 100 minutes, our six-well AST device successfully distinguished antibiotic-susceptible and -resistant P. aeruginosa biofilms, provided a quantifiable minimum inhibitory concentration (MIC) of antibiotics, and characterized the bacterial antibiotic action mechanisms.