Abstract

Purpose: Biofilms are complex and highly resistant microbial communities that have been found in different chronic infections (1,2). For in vitro studies, biofilms can be grown in each well of microtiter plates which allow the high-throughput screening of antimicrobial agents (3). The traditional method to evaluate bacterial viability is by plating serially diluted planktonic bacterial suspensions followed by colony (CFU) counting. Similarly, when evaluating biofilm bacterial viability, the same procedure is used after dispersion of the biofilm cells. Another traditional method is the crystal violet (CV) staining to quantify the biofilm biomass. The metabolic activity inside the biofilms is generally determined by using tetrazolium salt (XTT) (4), or fluorescein diacetate (FDA)-based assays. However, these methods present disadvantages, are laborious, and individually each assay only quantifies one aspect of the biofilms susceptibility (5). In this study, we propose a new method to study biofilms that combines the traditional microtiter method (well-biofilms) with the MBECTM biofilm inoculator method (peg-biofilms) (6). This modification allows the determination of biofilm susceptibility to a treatment by using different complementary techniques at the same time in the same well. After exposure to a treatment, well-biofilms can be assayed by XTT or FDA and CV, while in parallel peg-biofilms can be dispersed to determine the number of CFU. Methods: Pseudomonas aeruginosa PAO1 strain (ATCC® 15692TM) was grown planktonically from an overnight agar subculture. The bacterial suspension was adjusted to a concentration of 3 x 107 CFU/mL. For biofilm formation, 150 µL of this standard suspension were added into each well of a round bottom 96-well plate (Costar®, Corning Inc.) and then the 96-peg lid (MBECTM, Innovotech®) was placed. The plate was incubated in anaerobic conditions at 37°C and 75 rpm for 24 h. For validation, we compared the results of XTT and CV assays of untreated biofilms between well- and peg-biofilms. Then, different concentrations of tobramycin (from 2 to 1024 µg/mL) were used to treat the 24 h old biofilms. The susceptibility of the biofilms to tobramycin was evaluated using the same assays and by counting the number of CFU after biofilm dispersion by sonication. Each condition was tested 6 times. Statistical analysis was done using Wilcoxon test (α: 0.05). Results: The XTT and CV absorbance values measured for the well-biofilms were significantly higher (p<0.0001) than the values measured for the untreated peg-biofilms. This can be related to the larger surface available for biofilm growth on the wells than on the pegs. However, in tobramycin susceptibility tests, no significant differences were observed between well- and peg-biofilms metabolic activities and biomasses, expressed as a percentage of the control without treatment (p>0.05) at all tested concentrations. To compare the susceptibilities of well- and peg-biofilms, we defined as MIC80 the minimum concentration of tobramycin that caused 80% of reduction in the metabolic activity. The MIC80 values were 64 µg/mL for both biofilms. Under these experimental conditions, the XTT response of well-biofilms was in average 4 times higher than peg-biofilms (4.1 ± 0.7), while the amount of CV (µg) recovered was 3 times higher (2.9 ± 0.6) than peg-biofilms. Finally, the %survival of cells dispersed from peg-biofilm showed an 80% reduction with respect to control with 64 µg/mL of tobramycin which was consistent with the results obtained by previous methods. Conclusions: Here we report a modified method that can be useful to determine PAO1 biofilms susceptibility to antimicrobials by complementary techniques used within a single well in parallel. We demonstrated there was no significant difference between well- and peg-biofilms when metabolic activities and biomasses are compared as percentage with respect to the control without treatment. When raw data is compared (XTT absorbance and µg of CV) there is a significant difference related with the difference in size of the biofilms. Using complementary techniques, we are able to interpret the biofilm susceptibility to tobramycin as biomass, metabolic activity and number of CFU per one microtiter device. Additional studies may be necessary to adapt this method for different bacterial species and strains.