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 4 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 specific growth rate of the biofilm 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 maximum specific growth rate for planktonic cells. Global analysis of gene expression indicated lower transcription of ribosomal genes and genes for other anabolic functions in biofilms than in 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 will form when these biofilms are thicker than about 40 μm. Mutant strains that were deficient in Psl polysaccharide synthesis, the stringent response, the stationary-phase response, and the 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 the following mechanistic steps is proposed: (i) establishment of concentration gradients in metabolic substrates and products; (ii) active biological responses to these changes in the local chemical microenvironment; (iii) 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); (iv) adaptive responses to antibiotic exposure; and (v) reduced susceptibility of microbial cells to antimicrobial challenges in some of the physiological states accessed through these changes.

Modelling experimentally measured of ciprofloxacin antibiotic diffusion in Pseudomonas aeruginosa biofilm formed in artificial sputum medium

“…In the above equations, we took into account that the biofilm coefficients may be different for the cases described by Eqs (8) and (9). When κ = 0, the coefficients are denoted asD M ,q A , andq B .…”

“…A biofilm has a complex structure that changes over time. Special biofilm defence mechanisms, not fully understood, that affect the diffusion of antibiotic molecules in the biofilm can be activated [6][7][8]. Modelling the diffusion of antibiotics in a biofilm, we therefore propose to use a different strategy.…”

Pulse Dosing of Antibiotic Enhances Killing of a Staphylococcus aureus Biofilm

“…The tolerance of biofilms to antibiotics is linked to many physiological factors. Low nutrient and oxygen availability, upregulation of innate resistance factors, cell-cell signaling, heterogeneity in population gene expression profiles, the extracellular matrix, stress responses, and cell dormancy have all been implicated (Mah et al, 2003;Walters et al, 2003;Rani et al, 2007;Mulcahy et al, 2008;Macia et al, 2011;Nguyen et al, 2011;Kirby et al, 2012;Koch et al, 2014;Van Acker et al, 2014;Stewart, 2015;Ciofu et al, 2017;Hall and Mah, 2017;Stewart et al, 2019). Moreover, the extracellular matrix protects bacteria in biofilms from clearance by the immune system (Otto, 2018;Yan and Bassler, 2019).…”

Subpopulations of Stressed Yersinia pseudotuberculosis Preferentially Survive Doxycycline Treatment within Host Tissues

“…Biofilm structures also limit antibiotic diffusion into the center of bacterial communities. Both granulomas and biofilms contain stressed, slow-growing bacterial subpopulations at the center of the bacterial community, and it is thought that inhibitory concentrations of antibiotic cannot reach these cells, so they remain following treatment (4)(5)(6)(7)(8). Mass spectrometry could be used to better understand antibiotic diffusion within tissues but requires specialized technology for tissue processing, data collection, and analysis.…”