Exploring the Activity of Bacteriocins Against Bacterial Biofilms Using a Simple and Inexpensive Assay

There is an urgent need for novel antimicrobials that can combat antibiotic resistant bacteria. Biofilms constitute a particular niche where bacteria display an intrinsic higher resistance to antibiotics and that favors resistance appearance and spread among species. However, the susceptibility tests for sessile bacteria are not well standardized. This work provides a high-throughput, affordable and simple methodology to grow biofilms on a three-dimensional surface represented by glass beads placed in 96-well microtiter plates. Two Gram-positive and two Gram-negative bacterial species were tested. Biofilms showed the highest cell number after 48-h or 72-h incubation, depending on the bacterial species, using brain heart infusion, and typical biofilms were observed by electron microscopy. It has been benchmarked with the commonly used Calgary Biofilm device proving similar adhesion properties and susceptibility to ampicillin, erythromycin, vancomycin, ciprofloxacin, kanamycin, and gentamicin. Bacteriocins are potent antimicrobial peptides produced by bacteria with promising properties for clinical use but poorly studied against biofilms. Thus, upon biofilm formation optimization, the bacteriocins nisin and the enterocin AS-48 have been purified and used against Gram-positive species biofilms showing a mild increase, below tenfold, in the bactericidal concentration compared to planktonic cells. Remarkably, due to the ability of pore formation in the cell membrane, these bacteriocins were able to provide an eradication concentration of 500 mg/L of nisin and 25 mg/L of AS-48 for Enterococcus faecalis and 83.3 mg/L of nisin and 18.5 mg/L of AS-48 for Staphylococcus aureus. These data highlight the value of bacteriocins as novel antimicrobial agents for further therapeutic development.