Rapid means of biofilm disruption induce the newly released (NRel) phenotype of enhanced antibiotic sensitivity

Introduction: Biofilms are communities of microorganisms encased in a self-produced matrix, a structure that makes resident bacteria up to 1,000 times more resistant to antibiotics than their free-living, or planktonic, counterparts. Intriguingly, new methods that use reagents to release the biofilm-resident bacteria result in a transitory yet highly antibiotic sensitive phenotype. These newly released (NRel) bacteria are at least, if not more, sensitive to antibiotics than those in their planktonic form. Here, we sought to determine if the production of the NRel phenotype is reagent- dependent or can be accomplished by alternative means. Methods: specifically, we further characterized the phenotype by measuring the kinetics of antibiotic sensitivity via comparative plate counts over time. We also analyzed the relative expression levels of known NRel-associated genes using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: We show that using either intense mechanical disruption or a cationic depletion method not only facilitate rapid biofilm disruption but produce the NRel phenotype. In each case, both NRel signature gene expression and the transient antibiotic sensitivity phenotypes were observed compared to planktonic cells. Discussion: Similar to methods using reagents, we found that mechanical or cationic depletion disruption of pathogenic bacterial biofilms were sufficient to trigger the NRel phenotype. These results are consistent with the NRel state potentially being a rate-dependent physiological response rather than being induced by specific chemical or biological agents. We discuss the changes in gene expression permissive to NRel and the possibility that rapid and premature release of bacteria from a biofilm fails to allow the resident bacteria to be sufficiently prepared for their biofilm free state. This new insight both expands our understanding of the NRel phenotype and provides further validation for our rapid-release therapeutic strategy.