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Journal of Bacteriology, December 2001, p. 6746-6751, Vol. 183, No. 23
Department of Biology, Northeastern
University, Boston, Massachusetts
Received 26 June 2001/Accepted 10 September 2001
Biofilms are considered to be highly resistant to antimicrobial
agents. Strictly speaking, this is not the case Biofilm infections are difficult to
eradicate with antimicrobial treatment, and in vitro susceptibility
tests show considerable resistance of biofilm cells to killing (for
reviews, see references 8, 16, and 22). Up to 60% of all
human infections are caused by biofilms. It is commonly accepted that
biofilms are more resistant to antibiotics than are planktonic cells.
This, however, is a misconception Much progress has been made in the understanding of biofilm development
(9, 13, 28-30, 38; see reference 27 for a recent comprehensive review), but the molecular basis of biofilm tolerance remains elusive. Our previous work suggested a general mechanism of biofilm resistance to antimicrobials. In a study of
P. aeruginosa biofilms, we found that the vast majority of cells were eliminated by fairly low, clinically achievable
concentrations of fluoroquinolones, but a small fraction of persisters
remained essentially invulnerable to killing (6). Biphasic
killing kinetics revealing similar persister populations can be found
in papers describing treatment of biofilms of various bacterial species with a range of bactericidal antibiotics (for reviews, see references 20 and 21). We suggested that persisters are largely
responsible for the resistance of biofilms to killing and explain, in
principle, the nature of biofilm resistance (6, 20, 21).
Persisters are not mutants. Reculturing of persisters produces a
wild-type population with a new population of persisters (3,
6). Are persisters produced primarily by biofilms? In this
paper, we report the results of a detailed comparison of tolerance
between biofilms and stationary-state populations. We found that,
contrary to popular belief, planktonic stationary-phase cells are
somewhat more tolerant than biofilms. This increased resistance to
killing is due to slow growth and high levels of persisters produced in
stationary-phase planktonic populations.
Bacterial strain and culture conditions.
The bacterial
strain used in this study was P. aeruginosa wild-type strain
PAO1 (6). Mueller-Hinton broth (MHB; Difco, Detroit,
Mich.) was used to culture PAO1 in all experiments.
Antibiotics.
Tobramycin and carbenicillin were obtained from
Sigma. Ofloxacin and peracetic acid were obtained from the R. W. Johnson Research Institute and Aldrich, respectively.
Susceptibility testing.
The MIC of each antibiotic was
determined by the standard NCCLS broth microdilution method
(25).
Testing of planktonic and biofilm cell resistance to
killing.
Planktonic stationary-phase cultures were prepared by
inoculating 105 cells/ml and incubating them for
18 h at 37°C with aeration. Cells were then sedimented, washed
twice, and resuspended in fresh MHB at the original concentration
(~109 cells/ml). Planktonic logarithmic-phase
cultures were prepared by inoculating 105
cells/ml and incubating them for 3 h at 37°C with aeration. For dose-dependent determination of killing, 200 µl of either stationary- or logarithmic-phase cells was dispensed into microtiter plates and
incubated with an antibiotic for 6 h at 37°C. Following the challenge, the number of live cells was determined by colony counting.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6746-6751.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Biofilms and Planktonic Cells of Pseudomonas
aeruginosa Have Similar Resistance to Killing by
Antimicrobials
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
biofilms do not grow
in the presence of antimicrobials any better than do planktonic cells.
Biofilms are indeed highly resistant to killing by bactericidal
antimicrobials, compared to logarithmic-phase planktonic cells, and
therefore exhibit tolerance. It is assumed that biofilms are also
significantly more tolerant than stationary-phase planktonic cells. A
detailed comparative examination of tolerance of biofilms versus
stationary- and logarithmic-phase planktonic cells with four different
antimicrobial agents was performed in this study. Carbenicillin
appeared to be completely ineffective against both stationary-phase
cells and biofilms. Killing by this 
-lactam antibiotic depends on
rapid growth, and this result confirms the notion of slow-growing
biofilms resembling the stationary state. Ofloxacin is a
fluoroquinolone antibiotic that kills nongrowing cells, and biofilms
and stationary-phase cells were comparably tolerant to this antibiotic.
The majority of cells in both populations were eradicated at low levels
of ofloxacin, leaving a fraction of essentially invulnerable
persisters. The bulk of the population in both biofilm and
stationary-phase cultures was tolerant to tobramycin. At very high
tobramycin concentrations, a fraction of persister cells became
apparent in stationary-phase culture. Stationary-phase cells were more
tolerant to the biocide peracetic acid than were biofilms. In general,
stationary-phase cells were somewhat more tolerant than biofilms in all
of the cases examined. We concluded that, at least for
Pseudomonas aeruginosa, one of the model organisms for
biofilm studies, the notion that biofilms have greater resistance than
do planktonic cells is unwarranted. We further suggest that tolerance
to antibiotics in stationary-phase or biofilm cultures is largely
dependent on the presence of persister cells.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
the biofilm literature does not
point to the ability of biofilms to grow at higher concentrations of
antimicrobials compared to planktonic cells (20, 21).
Rather, biofilms are highly resistant to killing by bactericidal
antibiotics. This should properly be referred to as phenotypic
tolerance or tolerance, for short. Several factors have been suggested
to account for biofilm toleranceslow growth (15), the
presence of an exopolysaccharide matrix that can slow the diffusion of
antibiotics, and the presence of unknown resistance mechanisms
(8). Slow growth undoubtedly contributes to resistance to
killing by antimicrobials, but this is not a unique feature of biofilm
cells. With the exception of that of aminoglycosides (17, 19, 26,
33), the exopolysaccharide matrix has not been found to notably
retard the diffusion of antibiotics. For example, fluoroquinolones
readily penetrate the biofilm (1, 19, 33, 37). Multidrug
resistance pumps represent a generalized resistance mechanism and have
been considered as candidates for a biofilm resistance mechanism.
However, we and two other groups reported that, at least in
Pseudomonas aeruginosa, multidrug resistance pumps do
not noticeably contribute to biofilm survival (6, 10,
22). In retrospect, this is not surprisingbiofilms are not
usually more resistant to growth inhibition than are planktonic cells,
so there does not seem to be a need to invoke special drug resistance mechanisms.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Several different bactericidal antimicrobials were chosen to test
the relative resistance to killing of planktonic and biofilm cellsofloxacin, a fluoroquinolone; tobramycin, an aminoglycoside; carbenicillin, a -lactam; and peracetic acid, an oxidant.
Carbenicillin is a bactericidal antibiotic that, similarly to
other -lactams, kills only rapidly growing cells (35).
If biofilm cells were primarily slow growing, they would be expected to
be resistant to killing by carbenicillin. Logarithmic-phase, stationary-phase, and biofilm cultures were challenged with
carbenicillin over a wide range of concentrations, from 1.67 times the
MIC (50 µg/ml) to 20 times the MIC (600 µg/ml). After a 6-h
incubation with the antibiotic, viability was determined by colony
counting. As expected, carbenicillin produced little killing in
stationary-phase cells while the majority of logarithmic-phase cells
were killed at 1.67 times the MIC (Fig.
1). The amount of killing of
logarithmic-phase cells dropped off significantly at concentrations
above 1.67 times the MIC, indicating the presence of a persister
subpopulation. This 0.1% of the cells in the rapidly growing
logarithmic-phase culture were invulnerable to killing by carbenicillin
at 600 µg/ml. Biofilm cells were resistant to killing by
carbenicillin. This indicates that the biofilms used in this study are
made of slow-growing, essentially stationary-phase cells, in agreement
with previous reports (15).
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Unlike carbenicillin, ofloxacin can kill nongrowing cells
(6), providing a useful tool with which to examine the
relative tolerance of stationary-phase and biofilm cultures.
Logarithmic-phase, stationary-phase, and biofilm cultures were
challenged with ofloxacin over a wide range of concentrations, from the
MIC (0.5 µg/ml) to 30 times the MIC (15 µg/ml). After a 6-h
incubation with the antibiotic, viability was determined by colony
counting. The majority of cells in the three populations examined were
killed by low concentrations of ofloxacin (Fig.
2A). The killing in all three cultures
was distinctly biphasic, indicating the presence of persister cells.
The levels of persisters were dramatically higher in the dense
stationary-phase planktonic and biofilm cultures than in the
logarithmic-phase cells. Note that the cell concentration of the
biofilm is presented as the number of cells per peg and that the
density of the biofilm is considerably higher than the density of the
stationary-phase culture. Dilution of stationary-phase cells causes a
drastic drop in persisters, to the level found in logarithmic-phase
cells (N. Kaldalu et al., submitted for publication). (The small
persister fraction was not detected in a diluted stationary-phase culture in our previous study [6], apparently due to
minor differences in the experimental protocol.) The plateau at
increasing antibiotic concentrations shows that persisters are
essentially invulnerable to killing by a fluoroquinolone. Note that
this experiment was performed with planktonic cells and biofilms that
were transferred into fresh medium containing the antibiotic (see
Materials and Methods for details). With ofloxacin at 5 µg/ml, which
is a clinically achievable concentration (32), the
percentage of live cells was 0.001% in the logarithmic-phase
population, 0.1% in the biofilm, and 2.5% in the stationary-phase
culture (Fig. 2B). Unexpectedly, stationary-phase cells appeared to
produce more persisters and were relatively more tolerant than the
biofilm (Fig. 2B). Microscopic examination of the suspension of
stationary-phase cells prior to antibiotic addition showed that it
consisted primarily of individual planktonic cells and a small
number of clumps. The total percentage of cells in clumps was 
0.01%.
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Tobramycin is another bactericidal antibiotic that can kill nongrowing
cells. Unlike ofloxacin, tobramycin reportedly binds to the biofilm
exopolysaccharide, and we expected biofilms to be considerably more
resistant to killing by this antibiotic. Logarithmic-phase,
stationary-phase, and biofilm cultures were challenged with tobramycin
over a wide range of concentrations, from the MIC (1 µg/ml) to 1,500 times the MIC (1,500 µg/ml). After a 6-h incubation with the
antibiotic, viability was determined by colony counting. Tobramycin was
exceptionally effective in killing logarithmic-phase cells, and no
logarithmic-phase persisters were detected (Fig.
3A). Tobramycin at 50 µg/ml (the
maximal clinically achievable concentration is 10 µg/ml
[32]) eliminated 90% of the biofilm cells, but the
remaining population declined very gradually with increasing amounts of
the antibiotic. This relative resistance of the biofilm to killing by
tobramycin is in accordance with previous observations in the
literature (18). We found tobramycin to be ineffective in
killing stationary-phase planktonic cells. Unlike the biofilm, the
majority of the cells were not eliminated at low tobramycin
concentrations. At a tobramycin concentration of 800 µg/ml, a
persistent population of 1% surviving cells became apparent (Fig. 3B).
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Biofilms have been reported to be tolerant to both specific antibiotics
and biocides. It was of interest to learn whether biofilms are indeed
more resistant to biocide killing than are planktonic cells.
Logarithmic-phase, stationary-phase, and biofilm cultures were
challenged with peracetic acid over a range of concentrations, from the
MIC (100 µg/ml) to four times the MIC (400 µg/ml). After a 6-h
incubation with the antibiotic, viability was determined by colony
counting. Biphasic killing by this antimicrobial oxidant was not
observed (Fig. 4). Biofilms showed
considerable tolerance to this biocide compared to logarithmic-phase
planktonic cells. However, stationary-phase planktonic cells were even
more tolerant to peracetic acid than were biofilms. Complete killing of
all three populations by 400 µg/ml was observed.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 surprisingit is well established that rapidly growing
cells are more susceptible to both growth inhibition and killing. Some
antibiotics, like older -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, 21). 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 populationdilution 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, 19, 26, 33). 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 (hip) have been described (4, 5, 12, 23, 24, 31, 39). 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 (11), 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, 36) and biofilm persisters will persevere. Once the antibiotic level drops, persisters will recreate the biofilm (20, 21). 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, 20, 21) is
gaining acceptance as a general explanation for the phenomenon of
biofilm tolerance (14, 34). 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 segenes 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.
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ACKNOWLEDGMENTS |
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We thank Niilo Kaldalu for helpful discussions and Slava Epstein for help with microscopy.
This research was supported by NIH grant R01 GM61162.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biology, Northeastern University, 405 Mugar, 360 Huntington Ave., Boston, MA 02115. Phone: (617) 373-8238. Fax: (617) 373-3724. E-mail: k.lewis{at}neu.edu.
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Journal of Bacteriology, December 2001, p. 6746-6751, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6746-6751.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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