Biofilms are commonly viewed as being resistant to killing by a broad range of antimicrobial agents. Indeed, biofilms show more tolerance to antimicrobials than do planktonic logarithmic-phase cells. But this is not surprising—it is well established that rapidly growing cells are more susceptible to both growth inhibition and killing. Some antibiotics, like older β-lactams, absolutely require rapid growth in order to kill cells. However, it is also generally believed that biofilms are more resistant to killing than are stationary-phase planktonic cells. This has led to suggestions that specific resistance mechanisms might be expressed in biofilms (8
). But growth of biofilm cells has not been found to be more resistant to antibiotics than that of logarithmic-phase planktonic cells, suggesting that there is no basis for proposing the existence of broadly specific biofilm resistance mechanisms (20
). Are biofilms really more resistant to killing than are stationary-phase planktonic cells? A systematic comparison of susceptibility to killing between biofilm and logarithmic- and stationary-phase planktonic cells was undertaken in this study.
Carbenicillin kills rapidly growing cells. In our experiments, carbenicillin had no effect on biofilm or stationary-phase planktonic cultures. This indicates that biofilm cells are essentially in the stationary state. Slow growth may satisfactorily explain resistance to killing by antibiotics like carbenicillin. We then tested whether biofilms are more resistant to killing by antibiotics that have activity against nongrowing cells.
Ofloxacin at low concentrations produced significant killing of logarithmic-phase planktonic cells. A small percentage (0.001%) of persister cells resistant to killing was evident. The killing of biofilm cells similarly followed biphasic kinetics, but the proportion of persisters was significantly higher than in logarithmic-phase planktonic cells, comprising 0.1% of the population. These results are in agreement with our previous findings (6
) and clearly show that the majority of biofilm cells are very sensitive to killing by ofloxacin and that the overall biofilm resistance to killing is due to the presence of persisters. Note that in these experiments, the biofilm was transferred into a fresh, antibiotic-containing growth medium. In order to make a meaningful comparison with the biofilm, stationary-phase cells were also transferred, essentially without dilution, into fresh, antibiotic-containing growth medium. Unexpectedly and contrary to the general assumption, a stationary-state culture produced relatively more persisters than did a biofilm and was more resistant to killing by ofloxacin. In our previous study, we found that a stationary-phase planktonic population was very sensitive to killing by ofloxacin. Cells in that experiment were intentionally diluted (about 100-fold) in order to arrive at a population of a size comparable to that of the biofilm growing on a single peg. That seemingly reasonable procedure produced a misleading result. We now find that the formation and maintenance of persisters depend strongly on the density of the population—dilution of stationary-phase cells leads to a collapse in the number of persisters. This indicates that the dense population of either stationary-phase cells or biofilms favors persister formation. It is possible that a quorum-sensing factor(s) controls persister formation (Kaldalu et al., submitted). To our knowledge, undiluted stationary-phase cell populations have not been used in comparative studies with biofilms in tests with antibiotics prior to this study. The common assumption that biofilms are more resistant to killing than are planktonic cells derives from experiments with either logarithmic-phase or diluted stationary-phase cultures.
Tobramycin was probably the first antibiotic capable of killing nongrowing cells that was reported to be very ineffective in killing biofilms (2
). It was subsequently found that the biofilm exopolysaccharide binds and restricts the penetration of cationic aminoglycosides like tobramycin (17
). This seemed to be a group of antibiotics for which a biofilm-specific mechanism of resistance was justified. However, our results show that a stationary-phase cell population is even more resistant to killing by tobramycin than is a biofilm. These stationary-phase cells were washed twice and then resuspended in fresh medium, suggesting that exopolysaccharide was probably removed from the culture. Slow growth rather than sequestration of the antibiotic might be the critical contributing factor. Indeed, tobramycin activity was found to be growth rate dependent (15
The nature of persistence and the mechanism of cell death are interrelated but virtually unexplored. Mutations dramatically increasing the production of persisters in a logarithmic-phase population of Escherichia coli
) have been described (4
). This observation suggests that in bacteria, death might be a regulated event. We have argued that, similarly to metazoan tissues, populations of kin bacterial cells would benefit from elimination of defective members through a programmed cell death (PCD) mechanism (20
). According to this hypothesis, bactericidal antibiotics do not kill cells but inflict damage that activates PCD. This logic is identical to what we know of metazoan cells damaged by toxins that induce apoptosis. The problem with this scenario is that an antibiotic diffusing uniformly through a bacterial population will lead to total suicide, which is counterproductive. We proposed that persisters are cells with a disabled PCD mechanism whose function is survival (20
Unlike conventional antibiotics, biocides are likely to inflict sufficient damage to kill cells directly. Biofilms have been reported to be resistant to killing by biocides. We find that peracetic acid, a strong oxidant and a widely used biocide, is indeed far less effective in killing biofilm cells than in killing logarithmic-phase planktonic cells. However, stationary-phase cells were even more resistant to killing than were biofilms. In this respect, the action of peracetic acid appeared to be similar to that of the bactericidal antibiotics ofloxacin and tobramycin. The apparent lack of persisters in this dose-dependent experiment was a distinct feature of peracetic acid killing. Note that persisters are virtually invulnerable to killing by antibiotics and survive in the presence of antibiotic levels 100- to 1,000-fold higher than the MIC. By contrast, the killing concentration range was very narrow in the case of the biocide and cells in all of the cultures were eradicated at four times the MIC. This comparison further supports the idea that damage from antibiotics is relatively limited and death requires active participation on the part of cells (PCD) while biocides kill cells directly.
The main unexpected conclusion of this study is that biofilms of P. aeruginosa
are not different from stationary-phase planktonic cells in their resistance to killing by antibiotics and a biocide. Our experiments were limited to a single bacterial species, but P. aeruginosa
has served as a main model organism for biofilm studies. The presence of persisters in other species (20
) and the increase in resistance to killing with an increase in cell density in Burkholderia cepacia
), E. coli
, and Staphylococcus aureus
(Kaldalu et al., submitted) suggest that this is a general phenomenon. The critical role of persisters in the survival of both biofilm and planktonic populations suggests a new paradigm in our understanding of biofilm infections. A biofilm sheds planktonic cells that are primarily responsible for the manifestation of a disease. Antibiotics like ofloxacin eliminate most planktonic and biofilm cells but leave the persisters intact. The immune system is likely to eliminate the remaining planktonic persisters. However, biofilm cells are physically protected from the components of the immune system by the exopolysaccharide matrix (18
) and biofilm persisters will persevere. Once the antibiotic level drops, persisters will recreate the biofilm (20
). This model explains the relapsing nature of biofilm infections.
The persister hypothesis provides a satisfactory explanation for the puzzling observation that bacterial biofilms are resistant to killing by all known antibiotics. This hypothesis (6
) is gaining acceptance as a general explanation for the phenomenon of biofilm tolerance (14
). The challenge is to understand the nature of persistence. Development of drugs that disable the persister phenotype might lead to compounds that can enable conventional antibiotics to eradicate a biofilm. An important practical conclusion from our results is that there might not be a need to study biofilm resistance per se—genes and proteins responsible for persistence can be identified in planktonic populations that are much easier to manipulate. Similarly, planktonic populations can be used for the discovery of antipersistence drugs.