Conceptual Model of Biofilm Antibiotic Tolerance that Integrates Phenomena of Diffusion, Metabolism, Gene Expression, and Physiology.

Abstract

Transcriptomic, metabolomic, physiological, and computational modeling approaches were integrated to gain insight into the mechanisms of antibiotic tolerance in an in vitro biofilm system. Pseudomonas aeruginosa biofilms were grown in drip-flow reactors on a medium composed to mimic the exudate from a chronic wound. After four days, the biofilm was 114 μm thick with 9.45 log10 cfu cm-2 These biofilms exhibited tolerance, relative to exponential-phase planktonic cells, to subsequent treatment with ciprofloxacin. The biofilm specific growth rate was estimated via elemental balances to be approximately 0.37 h-1 and with a reaction-diffusion model to be 0.32 h-1 or one-third of the planktonic maximum specific growth rate. Global analysis of gene expression indicated decreased transcription of ribosomal genes and other anabolic functions in biofilms compared to exponential-phase planktonic cells and revealed the induction of multiple stress responses in biofilm cells including those associated with growth arrest, zinc limitation, hypoxia, and acyl-homoserine lactone quorum sensing. Metabolic pathways for phenazine biosynthesis and denitrification were transcriptionally activated in biofilms. A customized reaction-diffusion model predicted that steep oxygen concentration gradients form when these biofilms are thicker than about 40 μm. Mutant strains that were deficient in Psl polysaccharide synthesis, stringent response, stationary phase response, and membrane stress response exhibited increased ciprofloxacin susceptibility when cultured in biofilms. These results support a sequence of phenomena leading to biofilm antibiotic tolerance involving oxygen limitation, electron acceptor starvation and growth arrest, induction of associated stress responses, and differentiation into protected cell states.IMPORTANCE Bacteria in biofilms are protected from killing by antibiotics and this reduced susceptibility contributes to the persistence of infections such as those in the cystic fibrosis lung and chronic wounds. A generalized conceptual model of biofilm antimicrobial tolerance with these mechanistic steps is proposed: 1) establishment of concentration gradients in metabolic substrates and products; 2) active biological responses to these changes in the local chemical microenvironment; 3) entry of biofilm cells into a spectrum of states involving alternative metabolisms, stress responses, slow growth, cessation of growth, or dormancy (all prior to antibiotic treatment); 4) adaptive responses to antibiotic exposure; and 5) reduced susceptibility of microbial cells to antimicrobial challenges in some of the physiological states accessed through these changes.