To better understand the role of cellular state and gene-environment interactions in antibiotic tolerance, we examined the relative importance of each gene in P. aeruginosa
to fitness in the presence of tobramycin as a function of whether the bacterium is living in a biofilm or growing planktonically. Several previous studies identified a small number of P. aeruginosa
genes whose contribution to antibiotic tolerance depends on whether the cells are in a biofilm or planktonic state 
, but this work represents a substantially more comprehensive and systematic examination of the question. Here, we competed the mutants in a transposon library en masse
in each of four conditions: biofilms with and without tobramycin, and planktonic growth with and without tobramycin. We then characterized the changes in the population by genetic-footprinting and microarray hybridization.
All the biofilms were formed on plastic slides under static conditions with limited oxygen availability, likely creating micro-aerobic conditions. Cellular physiology in oxygen-limited biofilms is clinically relevant as during chronic, late-stage cystic fibrosis, P. aeruginosa
grows under reduced oxygen tension and is capable of respiring anaerobically within the thickened airway mucus 
Although it is well-known that the biofilm state increases drug tolerance 
, we find that the strains that are most fit in biofilm environments do not necessarily have higher antibiotic tolerance. In fact, our population-level data shows that the set of mutants with high fitness in biofilms (not exposed to tobramycin) has minimal overlap with the set of mutants with high fitness in biofilms in the presence of tobramycin. This implies that the general resistance provided by a biofilm against antibiotics does not protect all members equally and that genetic factors contribute to fitness in the biofilm context.
Moreover, while there is a considerable overlap between the loci that modulate antibiotic tolerance in biofilm and planktonic cells, the relative importance of most genes is state-specific. For example, the vast majority of mutants with the strongest fitness advantages in the biofilm state also have only a weak to moderate advantage in the planktonic state. Such strains, however, perform poorly in planktonic tobramycin challenges when in complex mutant populations due to the presence of a myriad of other strains, such as NADH dehydrogenase mutants, with more pronounced planktonic drug tolerance capacities. Similarly, while NADH dehydrogenase mutants are among the fittest strains in planktonic tobramycin challenges, these mutants exhibit only a comparatively moderate advantage in biofilms exposed to tobramycin. We expect that such differential fitness reflects not only the physiological state of the cells but also environmental differences, such as lower oxygen availability.
To better understand the pathways contributing to tobramycin tolerance in biofilms, we undertook a broad characterization of a subset of the strains that demonstrated a fitness advantage in biofilms in the presence of tobramycin and whose role in antibiotic tolerance had not been previously identified. We found mutants with changes in membrane permeability, quorum sensing, efflux pump abundance, and oxidative respiration activity—changes previously associated with planktonic antibiotic tolerance 
. Some mutants, however, did not show changes in any of the above pathways, suggesting that additional mechanisms are at play (). One such mechanism that we did not explore is conversion to the RSCV (Rough Small-Colony Variant) state, which is associated with hyper-adherence to solid surfaces and higher antibiotic tolerance 
Mechanisms for altering biofilm-mediated antibiotic tolerance.
As P. aeruginosa
cells in microcolonies have elevated mutation rates 
and many clinical isolates of P. aeruginosa
are hypermutable 
, each population explores a large part of the fitness landscape. In the course of this real-time evolution, each new mutant starts as a minority and competes against the pre-existing population. In this study, we attempted to capture some elements of natural conditions by analyzing our library of mutants as a heterogeneous pool rather than as homogeneous cultures of individual mutants as has been done more commonly 
. Each individual mutant was present at low abundance and was tested for a fitness advantage or disadvantage within a diverse population that was expected to function collectively as a wild-type proxy.
Our choice of experimental paradigm leads to some important discrepancies with previous works. For example, while Bjarnsholt et al.
showed that quorum sensing enhances tobramycin tolerance in P. aeruginosa 
, our results indicate that some quorum sensing-defective mutants have a fitness advantage in the presence of tobramycin in competition with a quorum sensing-capable strain. The cheating behavior of quorum-sensing mutants in a mixed population 
can explain the incongruity, and, indeed, quorum sensing-deficient mutants, specifically lasR
mutants, have been frequently isolated from Pseudomonas–
associated infections 
. Hence, the mixed population approach utilized here, in both the initial selections and the follow-up competitions, appears to capture some real-world, biological phenomena not observed in homogeneous cultures.
Efforts to develop effective therapeutic strategies against P. aeruginosa infections can benefit from a thorough understanding of how each gene contributes to the organism's antibiotic tolerance in the range of microenvironments present within an infection. We hope that this work and future studies using similar tools in other natural and clinical isolates will contribute to that effort.