Qualitative and quantitative agar invasion test based on bacterial colony/biofilm

Invasion of the culture medium is a feature frequently studied in yeasts, in which it has been related to a greater virulence, but it is practically unknown in bacteria. Recently, it has been demonstrated that several clinically relevant bacterial species were also able of invading agar media, so it was necessary to design a microbiological assay to study the expression of this character in bacteria. Accordingly, a bacterial agar invasion test based on colony/biofilm development was designed, which allows qualitative and quantitative characterization of bacterial growth into the agar culture medium. Once the culture conditions were optimized, the test was applied to 90 strains from nine bacterial species, validating its usefulness for differentiating invasive strains (positive) from those non invasive (negative). The test also allows sorting invasive strains according to agar invasion intensity (low, moderate, high) and topographic invasion pattern (peripheral, homogeneous, mixed). Moreover, an image analysis routine to quantify the invasion was developed. Implemented method enables direct measuring of two invasion parameters (invasion area and number of invasion dots), automated calculation of three relative variables (invasion relative area, invasion dots relative density, and invasion dot average area), and the establishment of strain specific frequency histograms.This new methodology is simple, fast, reproducible, objective, inexpensive and can be used to study a great number of specimens simultaneously, all of which make it suitable for incorporation to the routine of any microbiology laboratory. It could also be a useful tool for additional studies related to clinical aspects of bacterial isolates such as virulence and antimicrobial response.