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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, March 2000, p. 640-646, Vol. 44, No. 3
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Dose-Response Study of Antibiotic Resistance in
Pseudomonas aeruginosa Biofilms
Alexei
Brooun,
Songhua
Liu, and
Kim
Lewis*
Biotechnology Center, Tufts University,
Medford, Massachusetts 02155
Received 12 August 1999/Returned for modification 10 November
1999/Accepted 16 December 1999
 |
ABSTRACT |
Bacterial biofilms show enormous levels of antibiotic resistance,
but little is known about the underlying molecular mechanisms. Multidrug resistance pumps (MDRs) are responsible for the extrusion of
chemically unrelated antimicrobials from the bacterial cell. Contribution of the MDR-mediated efflux to antibiotic resistance of
Pseudomonas aeruginosa biofilms was examined by using
strains overexpressing and lacking the MexAB-OprM pump. Resistance of P. aeruginosa biofilms to ofloxacin was dependent on the
expression of MexAB-OprM but only in the low concentration range.
Unexpectedly, biofilm resistance to ciprofloxacin, another substrate of
MexAB-OprM, did not depend on the presence of this pump. Dose-dependent
killing indicated the presence of a small "superresistant" cell
fraction. This fraction was primarily responsible for very high
resistance of P. aeruginosa biofilms to quinolones.
Bacterial cells recovered from a biofilm and tested under nongrowing
conditions with tobramycin exhibited higher resistance levels than
planktonic cells but lower levels than cells of an intact biofilm.
| |
INTRODUCTION |
Biofilms, communities of cells
adhering to a substratum, likely represent the prevalent form of
microorganisms in nature (7). Resistance to antimicrobials
is a general feature of all biofilms that are the major cause of
recalcitrant infections (8, 13, 26). Biofilms might be
responsible for 65% of all bacterial infections (39).
Life-threatening infection caused by Pseudomonas aeruginosa
biofilms in cystic fibrosis patients is a well-known example
(15). One obvious difference between planktonic cells and
biofilm is the presence of a polysaccharide matrix enveloping the
community that retards diffusion of antimicrobials into the biofilm.
However, direct measurements of diffusion rates show that at least some
antibiotics equilibrate throughout the biofilm within minutes or hours
(9, 29, 41). P. aeruginosa expresses a
-lactamase, and a combination of retarded diffusion and an enzyme
that destroys the antibiotic at the rate at which it arrives at the
cell surface could explain resistance to ampicillin (41). However, this would not explain resistance to quinolones, for example.
Retarded diffusion alone can postpone but not prevent the death of
biofilm cells from quinolones. This realization has left the mechanism
of biofilm resistance largely unexplained (6).
Do biofilm cells express mechanisms that contribute to biofilm
resistance? Not much is known apart from an example of increased -lactamase production in P. aeruginosa biofilms
(14). The genetic analysis of biofilms has just recently
begun, and its focus has been on genes required for biofilm formation.
It has been found that pili and flagella are important for the early
stages of biofilm formation in Pseudomonas (32,
33) and Escherichia coli (40). It was also
reported that a quorum-sensing factor of P. aeruginosa is
required for the formation of the biofilm architecture; in the absence
of the factor, biofilms were thin and dense (10). This
altered biofilm was solubilized by sodium dodecyl sulfate (SDS), unlike
a wild-type biofilm. It remained unclear whether the quorum-sensing
factor induced an increase in the intrinsic antimicrobial resistance of
biofilm cells.
All known organisms have multidrug resistance pumps (MDRs) that can
extrude chemically unrelated antimicrobials from the cell (20, 30,
34). In P. aeruginosa, the MexAB-OprM MDR is expressed in wild-type planktonic cells and is largely responsible for high levels of "intrinsic resistance" of this microorganism to
antibiotics (21, 30, 37). Mutations leading to
overexpression of the pump have been identified in clinical isolates of
multidrug-resistant strains (6). The substrates of
MexAB-OprM include quinolones, tetracycline, -lactams, trimethoprim,
and many other compounds. The pump extrudes antimicrobials across the
outer membrane, which explains its ability to confer resistance to
-lactams that target the cell wall synthesis. The pump is composed
of three different peptides: a MexB translocase belonging to the
resistance-nodulation-division (RND) family of solute/proton
antiporters, an outer membrane porin, OprM; and a "membrane fusion
protein," MexA, that apparently docks MexB to OprM. The broad
specificity of MDRs seems to qualitatively match the broad resistance
of biofilms to antimicrobials. However, biofilms can show very high
levels of resistance (for example, a minimal bactericidal concentration
of 1 mg/ml in the case of P. aeruginosa and tobramycin), and
it is unclear whether mechanisms operating in planktonic cells that
confer significantly lower levels of resistance play a role in
biofilms. Nothing is currently known with regard to the role of MDRs in
biofilm resistance to antibiotics.
The contribution of MDR-mediated efflux to biofilm antibiotic
resistance was examined in this study. A detailed examination of
dose-response killing of biofilm cells expressing or lacking MexAB-OprM
led to a number of unexpected findings.
| |
MATERIALS AND METHODS |
Bacterial strains and culture conditions.
Bacterial strains
used in this study were K767 PAO1 prototroph (25), K1119
PAO1 
mexAB-oprM (22), K766 PAO1 nalB
mexAB-oprM++, PAO-JP1 PAO1 lasI
Tcr (35), and PA14 pilB
(31). Mueller-Hinton broth (MHB) (Difco, Detroit, Mich.) was
used to culture P. aeruginosa unless otherwise noted.
Antibiotics.
Ciprofloxacin and ofloxacin were obtained from
Bayer and the R.W. Johnson Research Institute, respectively.
Tetracycline and tobramycin were from Sigma, St. Louis, Mo.
Susceptibility testing of planktonic and biofilm cells.
Planktonic stationary-phase cells were used in dose-response killing
experiments, for a better comparison to biofilms. The minimal
bactericidal concentration for planktonic cells was defined as an
antimicrobial concentration required for complete eradication of the
cells during a 6-h incubation in MHB. Complete eradication was recorded
if one or no colonies were present on triplicate plates inoculated with
undiluted culture. This point was plotted as 100/ml on the
dose-response killing curves. Both modifications were implemented to
allow a better comparison with bacterial biofilms. Phosphate-buffered
saline (PBS), pH 7.4 (Sigma), was used to determine planktonic cell
resistance under nongrowing conditions.
The disposable MBEC device was used to form biofilms (MBEC Biofilm
Technologies Ltd., Calgary, Canada) (5). The device is a
platform carrying 96 polystyrene pegs that fit in a microtiter plate.
For biofilm formation, the device was placed in a tray filled with MHB
and cells (104/ml) and was incubated on a tilting shaker,
which provides a shearing force, for 16 h at 37°C. After
biofilms formed on the pegs, the pegs were washed in PBS, and the
device was placed in a microtiter plate with MHB for drug
susceptibility testing. Following 6 h of incubation in the
presence of an antimicrobial agent, the pegs were washed twice in PBS,
and the device was placed in a microtiter plate with PBS and sonicated
for 5 min in a water bath sonicator (Branson ultrasonic cleaner;
Branson Ultrasonics Corporation, Danbury, Conn.). For each
antimicrobial concentration tested, cells were collected from three
parallel pegs, plated with appropriate dilutions, and counted
separately, and the mean values are presented.
Dose-dependent killing of cells recovered from a biofilm.
Biofilm formation was carried out as described above, and cells were
dislodged from pegs by sonication into MHB containing a given amount of
antibiotic. Following a 6-h incubation with antibiotic, the number of
live cells was determined by plate counts.
| |
RESULTS |
In this study, we used the recently developed Calgary device for
biofilm susceptibility testing. The device allows us to test a fairly
large number of samples simultaneously, enabling us for the first time
to collect detailed data on the dose response of P. aeruginosa biofilms to several antibiotics. We have intentionally chosen two strains of P. aeruginosa for this study that have
a fixed status of MexAB-OprM expression, an overproducing mutant and a
deletion strain. This would limit the number of uncontrollable variants
and clearly show a potential role for an MDR in biofilm resistance. A
set of representative antibiotics was selected for this study. Of
these, ofloxacin, ciprofloxacin, and tetracycline are substrates of the
MexAB-OprM pump. MDRs primarily extrude amphipathic substances
(20), but recently RND-type MDRs extruding hydrophilic
aminoglycosides were found in Burkholderia pseudomallei (27) and P. aeruginosa (2).
MexAB-OprM and biofilm resistance.
P. aeruginosa
biofilms were formed overnight and treated with antimicrobials for
6 h. Cells were then dislodged from the plastic pegs by mild
sonication, and the number of live cells was determined by plate
counts. MexAB-OprM conferred a distinct protective effect on biofilms
exposed to ofloxacin (Fig. 1).
At low concentrations, ofloxacin was
50- to 100-fold more effective in killing biofilm cells of the deletion
mutant than the strain overexpressing the pump. Unexpectedly, as the
concentration of ofloxacin increased, the difference in resistance
between the two strains gradually diminished. After the number of live
cells dropped by 3 orders of magnitude, the remaining fraction,
consisting of about 105 cells, did not decline upon further
increase in antibiotic concentration. There was therefore no difference
in resistance to ofloxacin between these "superresistant" cells of
the two strains. By contrast, the rate of killing of planktonic cells
in growth medium by ofloxacin showed a rapid increase upon an increase
in the antibiotic concentration, and a large difference in resistance
between the MexAB-OprM expressing and deletion strains was observed
throughout the entire concentration range. Interestingly, the majority
of biofilm cells (~99.9%) were eradicated with a clinically
achievable antibiotic concentration (1 to 5 µg/ml), and the
MexAB-OprM pump contributed to resistance at this concentration range.
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FIG. 1.
Role of the MexAB-OprM pump in resistance of biofilms
and planktonic cells to killing by antibiotics. (A through C) Biofilms,
stationary-phase cells, and nongrowing cells, respectively, treated
with ofloxacin (Oflox). (D through F) Biofilms, stationary-phase cells,
and nongrowing cells, respectively, treated with ciprofloxacin (Cipro).
(G through I) Biofilms, stationary-phase cells, and nongrowing cells,
respectively, treated with tetracycline (Tet). (J through L) Biofilms,
stationary-phase cells, and nongrowing cells, respectively, treated
with tobramycin (Tobra).
|
|
In the case of ciprofloxacin, there was no difference in biofilm
resistance between cells expressing and lacking MexAB-OprM (Fig. 1). A
large difference in resistance between these strains was observed as
expected with planktonic cells (Fig. 1). The ability of MexAB-OprM to
confer resistance to ofloxacin shows that the pump is indeed active in
the biofilm. It appears that other drug resistance mechanisms operating
in the biofilm mask the contribution of the MexAB-OprM pump to
ciprofloxacin resistance. As in the case of ofloxacin, a distinct
plateau in the dose-response curve was observed with ciprofloxacin,
suggesting the presence of superresistant cells.
We found that tetracycline, a poorly lethal antibiotic, showed good
killing activity against planktonic cells of the MexAB-OprM deletion
strain (about a 3-log decrease at 0.5 µg/ml) and poor killing of the
overexpression strain (Fig. 1). It was interesting to learn whether the
very prominent difference between strain susceptibility to tetracycline
would be observed in the biofilm. This was not the case; the results
showed a dramatic increase in the MexAB-OprM deletion strain's
resistance to tetracycline in a biofilm. A difference in strain
susceptibility was observed only at the very high range of
concentrations (Fig. 1).
Tobramycin is apparently not a substrate of the MexAB-OprM pump (or a
very poor one), and in planktonic cells the efficient killing with this
antibiotic was similar for both strains. Complete eradication was
achieved at around 2 µg/ml (Fig. 1). The biofilms showed very high
resistance to tobramycin, and the strains behaved similarly. At a very
high concentration (1 mg/ml), the deletion mutant was more sensitive
than the overproducing strain.
Contribution of slow growth to antibiotic resistance.
The
dramatic increase in resistance of the deletion MexAB-OprM strain to
tetracycline upon transition from the planktonic to the biofilm state
could be due largely to slow growth. Bactericidal action generally
decreases with lower growth rate, and slow growth is considered to be
one of the components of biofilm resistance (3, 8). We
wanted to learn how slow growth contributed to resistance of P. aeruginosa to antibiotics used in this study.
For these model experiments, we chose conditions that would provide
minimal or no growth. Dose-response killing of stationary-phase planktonic cells was studied in simple phosphate buffer (Fig. 1).
Almost identical killing dynamics were observed for planktonic cells in
phosphate buffer and for biofilm cells in growth medium in the case of
tetracycline. Thus, a decrease in growth rate could largely explain
biofilm resistance to tetracycline, a poorly lethal antibiotic. By
contrast, cells in phosphate buffer were very sensitive to tobramycin.
Biofilm cells exhibited approximately 100-fold-higher resistance than
planktonic cells in phosphate buffer. Thus, another unknown
mechanism(s) is responsible for high resistance of P. aeruginosa to aminoglycosides. Slow growth evidently increased resistance of planktonic cells to quinolones. Very high resistance to
quinolones in phosphate buffer was observed with the strain overexpressing MexAB-OprM. This suggests that slow growth and MDRs
might contribute to ofloxacin resistance of biofilms.
Antibiotic resistance of bacterial cells recovered from a
biofilm.
Whether increased resistance derives primarily from the
biofilm structure (decreased diffusion or binding of antibiotics) or is
an intrinsic property of biofilm cells remains an important question.
What has been firmly established is that cells derived from the biofilm
are not mutants. After regrowth, planktonic cells from a biofilm are
similar to cells of the parent strain. We confirmed this by examining
the sensitivity of cells taken from biofilms after antibiotic treatment
and regrown in rich medium (data not shown). What has also been
established is that MICs for cells taken from the biofilm and
immediately exposed to antibiotic are the same as MICs for the wild
type (42). This is to be expected: once a potentially
resistant cell starts growing in the presence of an antibiotic,
resistant mechanisms will be "diluted" after several divisions, and
the sample will be scored as having no growth. There seemed to be a
simple way to probe the issue of intrinsic resistance of biofilm cells.
The rationale is to test the ability of cells to resist killing rather
than to study their ability to grow. Biofilm cells can be dislodged,
and their dose-response killing can be studied. In this format, there
is no growth due to the presence of high concentrations of antibiotic,
and resistance mechanisms would not be "diluted." However,
disruption of a biofilm and transfer of cells into fresh growth medium
could change the cell properties (activate them from possible dormancy,
for example) and lead to susceptibility. This suggests that only a
positive result (preservation of increased resistance of liberated
cells) would provide useful information. According to our results with planktonic cells in buffer, only tobramycin produced effective killing.
Using this antibiotic would therefore allow us to observe possible
resistance of liberated planktonic cells that was not due to slow
growth alone.
Resistance of liberated cells to tobramycin was substantially greater
than that of planktonic cells in PBS and unexpectedly was similar to
resistance of biofilms in growth medium (Fig.
2). When cells are liberated from the
biofilm by sonication, one would expect the biofilm matrix to be
liberated as well. This matrix could bind some of the tobramycin,
contributing to higher resistance of liberated cells. We examined this
possibility by studying dose-response killing of liberated cells
compared to killing in a 10-fold- and a 150-fold-diluted suspension of
liberated cells. The decline of the cell number with an increase in
tobramycin was more pronounced in the suspension diluted 10-fold than
in undiluted cells, suggesting that the undiluted suspension might
contain a factor protecting cells from the antibiotic. The patterns of
decline in cell counts in a 10-fold- and 150-fold-diluted suspensions
were comparable. This means that in diluted cells, only intrinsic
resistance of cells determines the pattern of killing. Even the diluted
biofilm cells were substantially more resistant than stationary-phase planktonic cells, as judged both by population decline (Fig. 1K) and by
the concentrations required for eradication. This experiment suggests
that resistance to tobramycin is due in part to intrinsic resistance
that is acquired by cells growing in a biofilm.
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FIG. 2.
Dose-response killing of P. aeruginosa
wild-type cells liberated from a biofilm. Cells were dislodged from a
biofilm by sonication, placed in Mueller-Hinton growth medium, and
treated with tobramycin (Tobra) (diamonds). Similarly prepared cells
were diluted prior to tobramycin treatment 10-fold (squares) and
150-fold (triangles).
|
|
Antibiotic resistance of a biofilm deficient in quorum-sensing
factor.
Recently, it was shown that a P. aeruginosa
strain deficient in the production of
N-(3-oxododecanoyl)-L-homoserine lactone (HSL)
quorum-sensing factor forms a dense biofilm that is dislodged from the
surface by 0.2% SDS, unlike the wild-type biofilm (10). It
was also reported that MexAB-OprM extrudes HSL (11, 36), raising the question of the interrelation between HSL, MexAB-OprM, and
antibiotic resistance. In order to probe directly for a possible role
of HSL in biofilm resistance to antibiotics, we used the lasI mutant strain defective in HSL production. We found no
difference in susceptibilities of the biofilms formed by the
lasI strain and the wild type in resistance to ofloxacin and
tobramycin (Fig. 3) or in resistance to
SDS (not shown). In order to verify that the method we are using allows
us to distinguish between cells with different capabilities to form
biofilms, a pilB strain defective in pilus synthesis and
biofilm formation (32) was tested. Biofilms formed by the
pilB strain had 100-fold fewer cells than the wild-type PAO1
or the K766 and K1119 strains (data not shown). The apparent discrepancy between our results and the above-cited study is probably due to the difference in the substratum used. The surface material has
a strong effect on biofilm adherence (26). We employed
polystyrene, which the MBEC device is made of, while glass was used in
the other study (10).
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FIG. 3.
Killing of P. aeruginosa biofilms by
ofloxacin (Oflx) and tobramycin (Tobra). Conditions were as described
for Fig. 1. PAO-JP1 is a lasI mutant defective in the
production of HSL, and PAO1 is the wild-type parent strain.
|
|
| |
DISCUSSION |
Role of MDRs.
Since MDRs play a significant role in
multidrug resistance of planktonic cells, it might seem obvious that
they should have a role in biofilm resistance as well. However, our
results show that this is not necessarily the case. There was no
difference in biofilm resistance to ciprofloxacin between cells
overexpressing the MexAB-OprM pump and those lacking it. On the other
hand, a notable (50- to 100-fold) difference in susceptibility between the strains was observed in the case of ofloxacin at a relatively low
concentration range. These differences do not follow from the very
similar and large differences between the susceptibilities of two
strains to these antibiotics in planktonic cells. One interesting possibility that would account for the unexpected lack of a role for
MexAB-OprM in biofilm resistance to ciprofloxacin is that another
MDR(s), perhaps with preferential selectivity for ciprofloxacin, is
being expressed in the biofilm. Many MDRs are regulated by environmental factors: EmrAB of E. coli is induced by its
drug substrates (4, 23), as are BMR of B. subtilis (1) and QacA of S. aureus
(16). In E. coli, the RND pump AcrAB is induced by stress (24, 31). Two of these RND pumps, MexCD-OprJ
(38) and MexEF-OprN (19), have substrate spectra
similar to that of MexAB-OprM, and mutants overexpressing these pumps
have been identified in resistant clinical isolates (12, 18,
43). MexCD-OprJ and MexEF-OprN are not expressed in planktonic
cells. There are 10 additional putative RND pumps in the genome of
P. aeruginosa (Pseudomonas Genome Project,
http://www.pseudomonas.com). Examination of mutants with disruptions in
the MDRs will provide a definitive answer as to whether and which MDRs
have an important role in biofilm resistance.
Intrinsic resistance of biofilm cells.
Cells liberated from a
biofilm into growth medium were considerably more resistant to
tobramycin than planktonic cells. This experiment suggests that cells
become intrinsically more resistant when growing in the biofilm and
retain part of this resistance even outside the biofilm. This is the
first observation that shows increased resistance of cells liberated
from the biofilm and provides the bases for searches for genes that
might be specifically induced in and responsible for biofilm resistance
to antibiotics.
Superresistant cells.
The detailed dose-response killing
examination of biofilms reported in this study allowed us to detect the
presence of a subpopulation of cells that show little sensitivity to
quinolones and are responsible for a characteristic "plateau" on
the killing plot. Not much has been published on dose-response killing
of microbial biofilms due to the lack of a convenient susceptibility
test, but the few existing studies report a pattern of killing that is
very similar to our observations. In E. coli, increasing
concentrations of ciprofloxacin or imipenem caused an initial decrease
in live cells of a biofilm by 2 to 3 orders of magnitude, while the
remaining small population was essentially insensitive to a further
increase in drug concentration (3). This pattern was also
observed with amoxicillin and clindamycin in Lactobacillus
acidophilus, and with erythromycin and metronidazole in the case
of Gardnerella vaginalis biofilms, where initial rapid
killing was followed by a plateau of resistant cells (28).
To our knowledge, the phenomenon of superresistant cells in the biofilm
has not been discussed. With a number of other antibiotics, the initial
killing was more gradual due to high resistance of the bulk of the
cells, and no plateau was observed. This is to be expected
a
superresistant fraction will show up only if the majority of cells are
distinctly more sensitive. It appears that four different
microorganisms tested with a number of unrelated drugs behave very
similarly, showing a rapid decline of the bulk of the population in
response to an increase in a potent antibiotic and a resistant
subpopulation. It thus seems that the biofilm employs a dual strategy
for survivalexpression of energy-dependent resistance
mechanisms like MDRs in the active cells that make up the majority of
the population, and (possibly) passive resistance of a superresistant
subpopulation. Superresistant cells are ultimately responsible for the
very high levels of biofilm resistance, at least in the cases discussed
above. Unlike planktonic cells, where a small remaining population
would be eliminated by the immune system, biofilm cells are protected
by the matrix (17), and remaining superresistant cells will
be responsible for biofilm regrowth after treatment with an antibiotic.
Elucidating the nature of these superresistant cells is a formidable
challenge, since they make up only a small part of the total
population. However, their importance in biofilm survival will
undoubtedly drive the effort to understand the mechanisms of their
remarkable resistance.
| |
ACKNOWLEDGMENTS |
We thank M. Mittelman for helpful discussions and H. Ceri for
advice on biofilm cultivation. K. Poole, B. H. Iglewski, R. Kolter, and G. A. O'Toole are gratefully acknowledged for the kind gift of bacterial strains.
This research was supported by NIH grant GM54412-01.
| |
ADDENDUM |
After this paper was submitted, a study by Aires and coauthors
(2) reported that OprM is part of another MDR, MexXY, that extrudes aminoglycosides. We did not find significant differences in
killing by tobramycin between cells expressing and lacking OprM; one
substantial difference between our conditions and those of Aires et al.
was that large amounts of divalent metals were included in the media in
the latter study, which could have affected OprM and/or MexXY.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biotechnology
Center, Tufts University, 4 Colby St., Medford, MA 02155. Phone: (617) 627-3731. Fax: (617) 627-3993. E-mail: klewis{at}tufts.edu.
| |
REFERENCES |
| 1.
|
Ahmed, M.,
C. M. Borsch,
S. S. Taylor,
N. Vazquez-Laslop, and A. A. Neyfakh.
1994.
A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates.
J. Biol. Chem.
269:28506-28513[Abstract/Free Full Text].
|
| 2.
|
Aires, J. R.,
T. Kohler,
H. Nikaido, and P. Plesiat.
1999.
Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides.
Antimicrob. Agents Chemother.
43:2624-2628[Abstract/Free Full Text].
|
| 3.
|
Ashby, M. J.,
J. E. Neale,
S. J. Knott, and I. A. Critchley.
1994.
Effect of antibiotics on non-growing planktonic cells and biofilms of Escherichia coli.
J. Antimicrob. Chemother.
33:443-452[Abstract/Free Full Text].
|
| 4.
|
Brooun, A.,
J. J. Tomashek, and K. Lewis.
1999.
Purification and ligand binding of EmrR, a regulator of a multidrug transporter.
J. Bacteriol.
181:5131-5133[Abstract/Free Full Text].
|
| 5.
|
Ceri, H.,
M. E. Olson,
C. Stremick,
R. R. Read,
D. Morck, and A. Buret.
1999.
The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.
J. Clin. Microbiol.
37:1771-1776[Abstract/Free Full Text].
|
| 6.
|
Chen, H. Y.,
M. Yuan, and D. M. Livermore.
1995.
Mechanisms of resistance to beta-lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993.
J. Med. Microbiol.
43:300-309[Abstract].
|
| 7.
|
Costerton, J. W.,
Z. Lewandowski,
D. E. Caldwell,
D. R. Korber, and H. M. Lappin-Scott.
1995.
Microbial biofilms.
Annu. Rev. Microbiol.
49:711-745[CrossRef][Medline].
|
| 8.
|
Costerton, J. W.,
P. S. Stewart, and E. P. Greenberg.
1999.
Bacterial biofilms: a common cause of persistent infections.
Science
284:1318-1322[Abstract/Free Full Text].
|
| 9.
|
Darouiche, R. O.,
A. Dhir,
A. J. Miller,
G. C. Landon,
I. I. Raad, and D. M. Musher.
1994.
Vancomycin penetration into biofilm covering infected prostheses and effect on bacteria.
J. Infect. Dis.
170:720-723[Medline].
|
| 10.
|
Davies, G. D.,
M. R. Parsek,
J. P. Pearson,
B. H. Iglewski,
J. W. Costerton, and E. P. Greenberg.
1998.
The involvement of cell-to-cell signals in the development of a bacterial biofilm.
Science
280:295-298[Abstract/Free Full Text].
|
| 11.
|
Evans, K.,
L. Passador,
R. Srikumar,
E. Tsang,
J. Nezezon, and K. Poole.
1998.
Influence of the MexAB-OprM multidrug efflux system on quorum sensing in Pseudomonas aeruginosa.
J. Bacteriol.
180:5443-5447[Abstract/Free Full Text].
|
| 12.
|
Fukuda, H.,
M. Hosaka,
S. Iyobe,
N. Gotoh,
T. Nishino, and K. Hirai.
1995.
nfsC-type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
39:790-792[Abstract].
|
| 13.
|
Gander, S.
1996.
Bacterial biofilms: resistance to antimicrobial agents.
J. Antimicrob. Chemother.
37:1047-1050[Free Full Text].
|
| 14.
|
Giwercman, B.,
E. T. T. Jensen,
N. Hoiby,
A. Kharazmi, and J. W. Costerton.
1991.
Induction of -lactamase production in Pseudomonas aeruginosa biofilm.
Antimicrob. Agents Chemother.
35:1008-1010[Abstract/Free Full Text].
|
| 15.
|
Govan, J. R. W., and V. Deretic.
1996.
Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.
Microbiol. Rev.
60:539-574[Abstract/Free Full Text].
|
| 16.
|
Grkovic, S.,
M. H. Brown,
N. J. Roberts,
I. T. Paulsen, and R. A. Skurray.
1998.
QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA.
J. Biol. Chem.
273:18665-18673[Abstract/Free Full Text].
|
| 17.
|
Hoyle, B. D.,
J. Jass, and J. W. Costerton.
1990.
The biofilm glycocalyx as a resistance factor.
J. Antimicrob. Chemother.
26:1-5[Free Full Text].
|
| 18.
|
Jakics, E. B.,
S. Iyobe,
K. Hirai,
H. Fukuda, and H. Hashimoto.
1992.
Occurrence of the nfxB type mutation in clinical isolates of Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
36:2562-2565[Abstract/Free Full Text].
|
| 19.
|
Kohler, T.,
M. Michea-Hamzehpour,
U. Henze,
N. Gotoh,
L. K. Curty, and J. C. Pechere.
1997.
Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa.
Mol. Microbiol.
23:345-354[CrossRef][Medline].
|
| 20.
|
Lewis, K.
1994.
Multidrug resistance pumps in bacteria: variations on a theme.
Trends Biochem. Sci.
19:119-123[CrossRef][Medline].
|
| 21.
|
Li, X.-Z.,
H. Nikaido, and K. Poole.
1995.
Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
39:1948-1953[Abstract].
|
| 22.
|
Li, X. Z.,
L. Zhang,
R. Srikumar, and K. Poole.
1998.
Beta-lactamase inhibitors are substrates for the multidrug efflux pumps of Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
42:399-403[Abstract/Free Full Text].
|
| 23.
|
Lomovskaya, O.,
K. Lewis, and A. Matin.
1995.
EmrR is a negative upstream regulator of the E. coli multidrug resistance pump EmrA.
J. Bacteriol.
177:2328-2334[Abstract/Free Full Text].
|
| 24.
|
Ma, D.,
M. Alberti,
C. Lynch,
H. Nikaido, and J. E. Hearst.
1996.
The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals.
Mol. Microbiol.
19:101-112[CrossRef][Medline].
|
| 25.
|
Masuda, N., and S. Ohya.
1992.
Cross-resistance to meropenem, cephems, and quinolones in Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
36:1847-1851[Abstract/Free Full Text].
|
| 26.
|
Mittelman, M. W.
1997.
Adhesion to biomaterials, p. 89-127.
In
M. Fletcher (ed.), The molecular and ecological diversity of bacterial adhesion. Wiley, New York, N.Y.
|
| 27.
|
Moore, R. A.,
D. DeShazer,
S. Reckseidler,
A. Weissman, and D. E. Woods.
1999.
Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei.
Antimicrob. Agents Chemother.
43:465-470[Abstract/Free Full Text].
|
| 28.
|
Muli, F. W., and J. K. Struthers.
1998.
The growth of Gardnerella vaginalis and Lactobacillus acidophilus in Sorbarod biofilms.
J. Med. Microbiol.
47:401-405[Abstract].
|
| 29.
|
Nichols, W. W.,
M. J. Evans,
M. P. E. Slack, and H. L. Walmsley.
1989.
The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa.
J. Gen. Microbiol.
135:1291-1303[Medline].
|
| 30.
|
Nikaido, H.
1996.
Multidrug efflux pumps of gram-negative bacteria.
J. Bacteriol.
178:5853-5859[Free Full Text].
|
| 31.
|
Okusu, H.,
D. Ma, and H. Nikaido.
1996.
AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants.
J. Bacteriol.
178:306-308[Abstract/Free Full Text].
|
| 32.
|
O'Toole, G. A., and R. Kolter.
1998.
Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.
Mol. Microbiol.
30:295-304[CrossRef][Medline].
|
| 33.
|
O'Toole, G. A., and R. Kolter.
1998.
Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis.
Mol. Microbiol.
28:449-461[CrossRef][Medline].
|
| 34.
|
Paulsen, I. T.,
M. H. Brown, and R. A. Skurray.
1996.
Proton-dependent multidrug efflux systems.
Microbiol. Rev.
60:575-608[Abstract/Free Full Text].
|
| 35.
|
Pearson, J. P.,
E. C. Pesci, and B. H. Iglewski.
1997.
Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes.
J. Bacteriol.
179:5756-5767[Abstract/Free Full Text].
|
| 36.
|
Pearson, J. P.,
C. Van Delden, and B. H. Iglewski.
1999.
Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals.
J. Bacteriol.
181:1203-1210[Abstract/Free Full Text].
|
| 37.
|
Poole, K.
1994.
Bacterial multidrug resistanceemphasis on efflux mechanisms and Pseudomonas aeruginosa.
J. Antimicrob. Chemother.
34:453-456[Free Full Text].
|
| 38.
|
Poole, K.,
N. Gotoh,
H. Tsujimoto,
Q. Zhao,
A. Wada,
T. Yamasaki,
S. Neshat,
J. Yamagishi,
X. Z. Li, and T. Nishino.
1996.
Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-type multidrug-resistant strains of Pseudomonas aeruginosa.
Mol. Microbiol.
21:713-724[CrossRef][Medline].
|
| 39.
|
Potera, C.
1999.
Forging a link between biofilms and disease.
Science
283:1837-1839[Free Full Text].
|
| 40.
|
Pratt, L. A., and R. Kolter.
1998.
Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili.
Mol. Microbiol.
30:285-293[CrossRef][Medline].
|
| 41.
|
Stewart, P. S.
1996.
Theoretical aspects of antibiotic diffusion into microbial biofilms.
Antimicrob. Agents Chemother.
40:2517-2522[Abstract].
|
| 42.
|
Wright, T. L.,
R. P. Ellen,
J. M. Lacroix,
S. Sinnadurai, and M. W. Mittelman.
1997.
Effects of metronidazole on Porphyromonas gingivalis biofilm.
J. Periodont. Res.
32:473-477[CrossRef][Medline].
|
| 43.
|
Yoshida, T.,
T. Muratani,
S. Iyobe, and S. Mitsuhashi.
1994.
Mechanisms of high-level resistance to quinolones in urinary tract isolates of Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
38:1466-1469[Abstract/Free Full Text].
|
Antimicrobial Agents and Chemotherapy, March 2000, p. 640-646, Vol. 44, No. 3
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Abstract
Introduction
