Identification of antibiotic tolerance determinants that are unique to biofilms has proven to be an arduous task, due in part to the physiological heterogeneity of these bacterial communities 
. Here we screened for E. coli
mutants forming biofilms with increased antibiotic tolerance in order to reveal transient physiological states and genetic modifications as tolerance mechanisms potentially occurring within biofilm subpopulations.
This approach showed that most identified mutants were amino acid auxotrophs displaying strong tolerance to either ticarcillin or ofloxacin upon starvation. Biofilm heterogeneity through generation of amino acid auxotrophs had been previously observed. Pseudomonas aeruginosa
amino acid auxotrophs are commonly isolated from sputa of cystic fibrosis patients and are generally more resistant to antibiotics than their prototrophic parental strains 
. In contrast to the planktonic cells and biofilm ticarcillin tolerance displayed by all tested starved amino acid auxotrophs, leucine, lysine, and cysteine auxotrophs exhibited increased tolerance to ofloxacin, which occurred only in starved biofilms. Since ofloxacin is active even against non-growing bacteria 
, general growth arrest did not explain the observed increase in ofloxacin tolerance. Moreover, while bacteria starved for glucose, leucine, cysteine, and lysine were highly tolerant to ofloxacin in biofilms, tryptophan starvation had no significant effect and starvation for other amino acids led to intermediate levels of tolerance. While these results confirm that the observed tolerance was not due to growth arrest, but rather to an active mechanism, it also suggests that distinct starvation types may have different physiological consequences in biofilms, consistent with a recent study demonstrating that starvation for different amino acids resulted in variations in growth rates and ß-galactosidase activity 
We demonstrated that both a functional RecA and a cleavable LexA were essential for the ofloxacin tolerance phenotype exhibited by biofilms starved either for glucose, leucine or lysine using prototrophic and auxotrophic mutant strains. A RecA-mediated SOS response was not necessary for increased ticarcillin tolerance in biofilms suggesting growth arrest as tolerance determinant unlike ofloxacin. We previously showed that recA
and other SOS response genes were significantly induced in mature biofilms compared to exponentially grown planktonic cells 
. Consistently, our findings demonstrated that induction of the SOS response was significantly greater in biofilm bacteria than in their planktonic counterparts. This could account for the biofilm specificity of ofloxacin tolerance, in which RecA or SOS-regulated proteins reached a critical level that is not achieved in planktonic cells, which may explain the lack of ofloxacin tolerance induction in starved planktonic populations.
Interestingly, despite a lower level of SOS-response, non-starved planktonic bacteria were more tolerant than their biofilm counterparts to both ticarcillin and ofloxacin, which is in agreement with previous observations 
. The ticarcillin tolerance could be explained by the fact that planktonic stationary cells are under growth arrest and therefore highly tolerant to ticarcillin while biofilms are heterogeneous populations containing a mix of growing and non-growing bacterial cells. For ofloxacin, the hypothesis of slow or absence of growth cannot satisfactorily explain the higher planktonic cells tolerance since it kills independently of growth. Stationary phase cultures are known to generate more persister cells than exponentially growing cultures, even more than biofilm cells, therefore suggesting some level of planktonic and biofilm specificity involved in generation of persisters 
. In absence of starvation, it is therefore possible to imagine that a higher level of persisters occurring in planktonic cells would explain their higher tolerance. Moreover, the ofloxacin tolerance of planktonic bacteria is likely due to a mechanism other than the SOS response since a ΔrecA
did not significantly impair the overall tolerance of both non-starving and starving populations. The latter results strengthen the notion that the induction of ofloxacin tolerance in starving biofilms is likely to involve mechanisms different than those currently described in planktonic cells 
Although our study evaluates ofloxacin only, fluoroquinolones are known to induce the SOS response in E. coli
. Here we showed that induction of the SOS response upon exposure to bactericidal concentrations of ofloxacin (80× MIC) was unlikely to occur in absence of carbon source, i.e. in full deprivation state, due to the absence of protein synthesis. Hence, both induction of the SOS response during biofilm formation and nutrient depletion (carbon source or amino acids) are necessary but not sufficient to lead to the observed biofilm-specific high tolerance to ofloxacin.
The SOS response is involved in bacterial adaptive responses and horizontal gene transfer potentially leading to the onset of antibiotic resistance in a broad range of bacterial species 
. The SOS response was also previously shown to induce persistent cells in planktonic populations during treatment with the fluoroquinolone ciprofloxacin 
. “Persisters” are phenotypic variants that can revert to wild-type antibiotic sensitivity 
. They are believed to be the end result of stochastic endogenous stress leading to growth arrest, resulting in the shutdown of bactericidal antibiotic targets and therefore the creation of multidrug-tolerant cells 
. Among possible targets, the SOS response was shown to induce expression of the TA module TisAB 
. However, TisAB-dependent ciprofloxacin persisters were only observed in exponentially growing bacteria 
, consistent with the fact that none of the TA modules tested in our study was required for the biofilm-associated increased tolerance to ofloxacin, including known SOS-induced TA modules TisAB, SymER, DinJ-YafQ and YafNO. Our results therefore indicate that starvation in biofilm bacteria induces a SOS-dependent ofloxacin tolerance, but independently of TA loci induced by the SOS response. As raised above, the ability in generating persisters differs greatly between planktonic and biofilms cells 
, therefore it can be speculated that ofloxacin-tolerant persisters in biofilms could be physiologically distinct from their planktonic persister counterparts. Future work should concentrate precisely on identifying which SOS-gene(s), among the over 40 known SOS-regulated genes 
, is required to support this biofilm increased tolerance towards ofloxacin.
Since we showed that starvation, but not auxotrophy per se
, promotes the high ofloxacin tolerance observed within biofilms, our results demonstrate a general link between nutrient limitation and formation of highly antibiotic-tolerant populations. Starvation to amino acids or carbon sources induces the stringent response through the synthesis of (p)ppGpp via both RelA and SpoT 
. Indeed, while we could not assess the role of SpoT since a relA spoT
double mutant, i.e. a ppGpp0 strain, is auxotroph to multiple amino acids 
, we demonstrated that relA
plays a role in observed ofloxacin biofilm-specific hypertolerance. However, the role of this response seemed less important than that of the SOS response, since the stringent response was only partially involved following leucine starvation. Nguyen et al.
recently demonstrated that antibiotic tolerance exhibited in nutrient-limited biofilms of P. aeruginosa
depended on the stringent response 
. It can therefore be imagined that multiple pathways might play a significant role in the biofilm-associated ofloxacin tolerance of starved biofilms, with their role depending on conditions prevailing within biofilms. The manner in which these two distinct responses become integrated at the molecular level to promote high biofilm-specific ofloxacin tolerance remains unclear at the moment.
Nutrient deprivation might occur in biofilm populations located in micro-niches of heterogeneous biofilms, leading to higher tolerance to antibiotics 
. Alternatively, adverse physicochemical parameters such as oxygen rarefaction prevailing within deep biofilm layers could slow down bacterial metabolism, thereby causing bacteria to enter a quasi-auxotrophic state even in the presence of sufficient available carbon sources. In support of this hypothesis, we showed that ofloxacin tolerance increased with biofilm age, suggesting that nutrient-depleted pockets or starved layers of cells may increase in number or size with biofilm maturation, or else be accompanied by a reduction in nutrient flow. Interestingly, Allison et al.
recently showed that reactivation of the metabolism of E. coli
persister cells restored antibiotic sensitivity both in planktonic and biofilms cells 
. In parallel with our study, the addition of exogenous leucine during antibiotic treatment of leucine auxotroph biofilms restored sensitivity to levels greater than those of the parent wild-type prototroph treated without leucine, suggesting that the addition of amino acids may also impact overall antibiotic tolerance. While the study by Allison et al.
did not specifically involve ofloxacin, it clearly suggests that nutrient depletion might play a central role in the ability of biofilm-associated bacteria to tolerate otherwise lethal concentrations of antibiotics. Although lethal concentrations of ofloxacin induce the SOS response, we show here that increased tolerance of aging biofilms is SOS-dependent and is correlated with increased SOS induction in biofilm bacteria. While this result is consistent with SOS induction in aging biofilm-like E. coli
colonies on agar plates 
, it also suggests that biofilm areas in which local nutrient depletion and/or various stresses (pH drop, reduction of oxygen, etc) occur may cause SOS induction and favour high antibiotic tolerance and thus persistence.
In conclusion, we show that starvation potentially occurring locally in heterogeneous and diffusion-limited biofilm microenvironments leads to biofilm-specific SOS-dependent tolerance to the fluoroquinolone ofloxacin. Identification of the SOS response as a key molecular determinant in antibiotic tolerance in nutrient-limited biofilms reveals a possible general mechanism leading to high, but transient tolerance to a medically relevant class of antibiotics. It underlines the importance of the SOS response in the different bacterial mechanisms counteracting the effects of antibiotics, and it raises the possibility of using the SOS response as a target for reducing the emergence of biofilm tolerance to antibiotics in clinical settings