Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, July 2000, p. 1818-1824, Vol. 44, No. 7
Center for Biofilm
Engineering,1 Department of Chemical
Engineering,2 and Department of
Microbiology,3 Montana State
University-Bozeman, Bozeman, Montana 59717-3980
Received 4 October 1999/Returned for modification 1 February
2000/Accepted 5 April 2000
The penetration of two antibiotics, ampicillin and ciprofloxacin,
through biofilms developed in an in vitro model system was investigated. The susceptibilities of biofilms and corresponding freely
suspended bacteria to killing by the antibiotics were also measured.
Biofilms of Klebsiella pneumoniae were developed on microporous membranes resting on agar nutrient medium. The
susceptibilities of planktonic cultures and biofilms to 10 times the
MIC were determined. Antibiotic penetration through biofilms was
measured by assaying the concentration of antibiotic that diffused
through the biofilm to an overlying filter disk. Parallel experiments
were performed with a mutant K. pneumoniae strain in which
Bacterial biofilms are frequently
observed on the surfaces of tissues (12, 30, 31) and
biomaterials (5, 10, 39, 42, 47) at the site of persistent
infections (8). Medical implants are particularly
susceptible to biofilm formation because immune responses are markedly
reduced in the proximity of foreign bodies (7, 42). In fact,
biofilm formation is a major cause of implant failure and often limits
the lifetimes of many indwelling medical devices (24). Once
in the biofilm, extracellular polymeric substances shield bacteria from
opsonization and phagocytosis (8, 23). In addition, in vitro
experiments have demonstrated that the bacteria in biofilms are
considerably less susceptible to antibiotics than their planktonic
counterparts (3, 10, 14, 19, 28, 41). Treatment of an
infection after the biofilm is established is frequently futile with
current remedies (8). Often, the only solution is mechanical
removal of the biofilm or implant, which is costly and traumatic to the
patient (8).
Two principal hypotheses have been formulated to explain the reduced
susceptibility of biofilms to antibiotics. The first hypothesis, which
could be termed penetration limitation, suggests that only the surface
layers of a biofilm are exposed to a lethal dose of the antibiotic due
to a reaction-diffusion barrier that limits transport of the antibiotic
into the biofilm (17, 19, 25, 26, 28, 34, 35, 38). This
mechanism has been demonstrated for reactive oxygen species such as
hypochlorite (11, 44) and hydrogen peroxide (26).
Synthesis of an antibiotic-degrading enzyme, such as a The objective of the work presented here was to evaluate the
significance of penetration limitation as a mechanism of biofilm resistance to two antibiotics, ampicillin and ciprofloxacin. The antibiotic susceptibilities of membrane-supported Klebsiella
pneumoniae biofilms were compared to the susceptibilities of
corresponding planktonic cultures. Permeation of active antibiotic
through membrane-supported biofilms was tracked by a novel bioassay
technique. The role of transport limitation was assessed for the two
antibiotics by comparing the susceptibility and permeability data.
Bacterial strains, media, and antimicrobials.
Pure cultures
of K. pneumoniae Kp1 and Kp102M were used in the
experiments. Kp1, an environmental isolate, was obtained from the
culture collection at the Center for Biofilm Engineering. Kp102M is a
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Role of Antibiotic Penetration Limitation in Klebsiella
pneumoniae Biofilm Resistance to Ampicillin and
Ciprofloxacin
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase activity was eliminated. For wild-type K. pneumoniae grown in suspension culture, ampicillin and
ciprofloxacin MICs were 500 and 0.18 µg/ml, respectively. The log
reductions in the number of CFU of planktonic wild-type bacteria after
4 h of treatment at 10 times the MIC were 4.43 ± 0.33 and
4.14 ± 0.33 for ampicillin and ciprofloxacin, respectively.
Biofilms of the same strain were much less susceptible, yielding log
reductions in the number of CFU of 
0.06 ± 0.06 and 1.02 ± 0.04 for ampicillin and ciprofloxacin, respectively, for the same
treatment. The number of CFU in the biofilms after 24 h of
antibiotic exposure was not statistically different from the number
after 4 h of treatment. Ampicillin did not penetrate wild-type
K. pneumoniae biofilms, whereas ciprofloxacin and a nonreactive tracer (chloride ion) penetrated the biofilms quickly. The
concentration of ciprofloxacin reached the MIC throughout the biofilm
within 20 min. Ampicillin penetrated biofilms formed by a
-lactamase-deficient mutant. However, the biofilms formed by this
mutant were resistant to ampicillin treatment, exhibiting a 0.18 ± 0.07 log reduction in the number of CFU after 4 h of exposure
and a 1.64 ± 0.33 log reduction in the number of CFU after
24 h of exposure. Poor penetration contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin. The increased resistance of the wild-type strain to ciprofloxacin and the
mutant strain to ampicillin and ciprofloxacin could not be accounted
for by antibiotic inactivation or slow diffusion since these
antibiotics fully penetrated the biofilms. These results suggest that
some other resistance mechanism is involved for both agents.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase
enzyme (17, 28, 35), or sorption of an antibiotic to biofilm
components (25, 35) could also give rise to such a
situation. The transport properties of numerous antibiotics, including
ciprofloxacin (33, 40, 41), levofloxacin (33),
ofloxacin (33, 44, 46), sparfloxacin (33),
gentamicin (25, 33, 45), imipenem (33),
piperacillin (19, 33), and vancomycin (10, 14),
through biofilms have been examined. Some investigators report retarded
penetration of antibiotics (19, 25, 33), while others
indicate rapid penetration (10, 14, 25, 33, 40, 41). The
second hypothesis for reduced biofilm susceptibility, which could be
termed physiological limitation, proposes that some microorganisms
within the biofilm exist in a more recalcitrant phenotypic state
(6, 9, 34). The roles of various physiological factors, such
as growth rate (13, 15, 16), biofilm age (2), and
starvation (22, 27), have been examined. Gradients in the
physiological status of cells within biofilms have been demonstrated
recently (20, 21, 37, 43).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase-deficient mutant isolated by nitrosoguanidine mutagenesis
of Kp1, followed by screening for ampicillin-sensitive mutants.
Escherichia coli ATCC 25922 (provided by Barbara Hudson, Montana State University) served as the antibiotic-sensitive
microorganism in antibiotic bioassays.
Biofilm preparation. Overnight planktonic cultures of K. pneumoniae were diluted to an optical density (600 nm, 1-cm path length) of 0.200 in PBW. One 5-µl drop (for susceptibility experiments) or five 10-µl drops (for permeability and antibiotic degradation studies) of diluted planktonic culture were used to inoculate individual sterile, black, polycarbonate membrane filters (diameter, 25 mm; pore size, 0.2 µm; Poretics Corp., Livermore, Calif.) resting on agar culture medium. The membranes were sterilized by UV exposure (15 min per side) prior to inoculation. The plates were inverted and incubated at 35°C for 48 h, with the membrane-supported biofilms transferred to fresh culture medium every 8 to 10 h.
Planktonic susceptibility. Overnight planktonic cultures of K. pneumoniae were diluted to an optical density (600 nm) of 0.200 with PBW. One milliliter of diluted culture was used to inoculate 100 ml of culture broth for a final population of ca. 106 CFU per ml. After removing the time-zero sample, 0.500 g of ampicillin or 180 µg of ciprofloxacin, dissolved in sterile nanopure water, was added to the subculture to attain antibiotic concentrations of 5,000 and 1.8 µg/ml, respectively. The culture was placed on a 35°C orbital shaker and was sampled every 30 min for 4 h. The sampling procedure was as follows. A 1.5-ml sample of the culture was pipetted into a 2.0-ml conical microcentrifuge tube (Fisher Scientific, San Francisco, Calif.). The bacteria were pelleted at 10,000 rpm for 7.5 min at room temperature with a Micro14 microcentrifuge (Fisher Scientific). The supernatant was removed with a pipette. The bacteria were washed with 1.5 ml of PBW and were repelleted as described above, and the supernatant was removed and discarded. The bacteria were suspended in 1.5 ml of PBW and were then serially diluted in PBW. Viable bacteria were enumerated as described below.
The MICs of ampicillin and ciprofloxacin for Kp1 and Kp102M were estimated as follows. Aliquots of culture broth containing 5 × 105 CFU/ml were treated with 50 to 700 µg of ampicillin per ml or 0.02 to 0.35 µg of ciprofloxacin per ml and were incubated for 20 h at 35°C. The optical density was measured at 600 nm with a Spectronic Genesys spectrophotometer (Spectronic Instruments, Inc., Rochester, N.Y.), and viable bacteria were quantified as described below. The MIC was taken as the minimum antibiotic concentration at which no increase in the number of viable bacteria after treatment was observed.Biofilm susceptibility. The biofilms were transferred to antibiotic-containing agar, and the agar plates were incubated at 35°C. The biofilms were sampled every 30 min for 4 h and after 24 h. When sampled, each membrane-supported biofilm was placed in 9.0 ml of PBW, and the mixture was vortexed at high speed for 2.0 min with a Maxi Mix II Vortex mixer (Barnstead/Thermolyne, Dubuque, Iowa) and then serially diluted in PBW. The viable bacteria were enumerated as described below.
Viable bacteria enumeration and comparison. Serially diluted samples were plated onto R2A agar (Difco Laboratories) by the drop plating method (18, 32), and the plates were incubated at 35°C for 18 to 20 h. The change in the number of CFU was normalized for each experiment by converting the measurements of the number of viable bacteria to the log reduction of the number of CFU. The log reduction of the number of CFU, or simply log reduction, at a particular sampling time was defined as the negative log10 of the quotient of the number of CFU at that time and the number of CFU prior to treatment. A positive log reduction represents a decrease in the number of CFU. The log reduction values at each time point for identical experiments were averaged, and the standard error of the mean was calculated. The mean log reductions for the various experiments were compared by a two-tailed, two-sample t test with the assumption of unequal variances.
Antibiotic penetration.
Black polycarbonate membrane filters
(diameter, 13 mm; pore size, 0.2 µm; Poretics Corp.) were placed on
top of 48-h-old K. pneumoniae biofilms. A concentration disk
(catalog no. 1599-33-6; Difco Laboratories) was moistened with 24 µl
of culture broth prior to placement on top of the 13-mm-diameter
membranes. Wetting of the disk helped prevent capillary action of the
antibiotic medium through the biofilm. The biofilm sandwiched between
membranes and the moistened disk was transferred to
antibiotic-containing agar culture medium (Fig.
1). The disk was removed after specified exposure times, sealed in parafilm (American National Can, Chicago, Ill.), and stored at 4°C. After collection of all samples, the disks
were placed on Mueller-Hinton (Difco Laboratories) plates spread with
100 µl of planktonic E. coli. The E. coli
strain was grown in LB broth at 35°C, and the growth was diluted with
LB broth to an optical density of 0.050 prior to plating. The plates were incubated at 35°C for 18 to 20 h. The zone of inhibited
growth was measured and was used to determine the concentration of
active antibiotic in the disk. The effective bulk agar antibiotic
concentration was estimated by averaging the equilibrated concentration
observed in disks placed on the two-membrane system, i.e., the unit
without the bacterial biofilm. The concentration that penetrated the
biofilms was divided by the bulk agar concentration to provide a
normalized penetration curve.
|
Chloride penetration. Black polycarbonate membrane filters (diameter, 13 mm; pore size, 0.2 µm; Poretics Corp.) were placed on top of 48-h-old K. pneumoniae biofilms grown on chloride-free culture medium. Concentration disks were moistened with 24 µl of chloride-free culture broth and were placed on top of the 13-mm-diameter membranes. The entire units were transferred to chloride-containing agar culture medium. The disks were removed after specified exposure times and were placed in 1.0 ml of sterile nanopure water. The resulting chloride concentration in the water was measured with a DX-500 ion chromatograph (Dionex, Sunnyvale, Calif.). The bulk agar chloride concentration was estimated by averaging the equilibrated concentration observed in the disks placed on the two-membrane system exposed to chloride-containing agar. The concentration that penetrated the biofilms was divided by the bulk agar concentration to provide a normalized penetration curve.
Antibiotic degradation. Antibiotic degradation by the bacteria within the biofilms was examined qualitatively. Forty-eight-hour-old Kp1 and Kp102M biofilms were placed on culture medium plates containing 500 µg of ampicillin per ml or 0.18 µg of ciprofloxacin per ml, and the plates were incubated at 35°C. After 24 h, the biofilms were removed and the plates were spread with 100 µl of a planktonic Kp1 culture (~108 CFU/ml). The spread plates were incubated at 35°C for 48 h and were then analyzed for zones of growth.
The rates of antibiotic degradation for Kp1 and Kp102M were quantified in planktonic cultures. Antibiotics were added to 100 ml of turbid overnight planktonic cultures (~109 CFU/ml) for a final antibiotic concentration of 1,000 µg of ampicillin per ml or 1.8 µg of ciprofloxacin per ml. A 1.5-ml sample from each culture was taken every 30 min for 4 h and was placed in a 2.0-ml microcentrifuge tube. The bacteria were pelleted at 7,000 × g for 7.5 min at room temperature. A concentration disk was inoculated with 24 µl of the supernatant. The antibiotic concentration was estimated by the zone-of-inhibition bioassay described above. The natural log of the antibiotic concentration was plotted with respect to time. An apparent first-order rate coefficient of antibiotic degradation was estimated from least-squares linear regression of the data. Analysis of variance was used to determine whether the estimated reaction rates were statistically different from zero.| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Antibiotic susceptibility.
The observed MICs of ampicillin for
planktonic Kp1 and Kp102M were 500 and 2 µg/ml, respectively. The MIC
of ciprofloxacin was 0.18 µg/ml for both strains. Planktonic cultures
and biofilms of both the wild-type and mutant strains were challenged
with 10 times the MIC of each antibiotic for the wild-type strain: 5,000 µg/ml for ampicillin and 1.8 µg/ml for ciprofloxacin.
Planktonic bacteria were rapidly killed by these treatments. The log
reductions in the number of CFU were 4.43 ± 0.33 and 4.14 ± 0.33 after 4 h of ampicillin and ciprofloxacin treatment,
respectively, for planktonic wild-type bacteria (Fig.
2a and c). Planktonic cultures of the
-lactamase-deficient mutant experienced a 4.62 ± 0.20 log
reduction after exposure to 5,000 µg of ampicillin per ml for 4 h (Fig. 2b). However, the number of CFU in biofilms challenged with
ampicillin remained virtually unchanged after 4 h of treatment. Kp1 (Fig. 2a) and Kp102M (Fig. 2b) biofilms exhibited 0.06 ± 0.06 and 0.18 ± 0.07 log reductions, respectively. Both log
reductions were statistically different from those for the analogous
planktonic treatments (P = 0.001 and P = 0.030, respectively). The log reduction observed for Kp1 biofilms
was not statistically different from that for the untreated control
(P = 0.63). Challenge of Kp1 biofilms with
ciprofloxacin for 4 h resulted in a 1.02 ± 0.04 log
reduction in the number of CFU, a result statistically different
(P = 0.011) from that for the treated planktonic
culture (Fig. 2c). The response of Kp102M biofilms treated with
ciprofloxacin for 4 h was not statistically different from that of
the wild type (P = 0.37) (data not shown).
|
, untreated controls;
,
antibiotic-challenged cultures. Error bars reflect ±1 standard error
of the mean.
0.05; Fig. 2a). Kp102M biofilms exhibited a 1.64 ± 0.33 log reduction in the number of CFU after 24 h (Fig. 2b),
which indicated that they were statistically significantly less
susceptible than planktonic cultures treated for only 4 h (P = 0.016). A log reduction of 1.07 ± 0.18 was
observed for Kp1 biofilms challenged with ciprofloxacin for 24 h
(Fig. 2c). This reduction was statistically different from that for 4-h
planktonic treatment (P = 0.004). Kp102M biofilms
treated with ciprofloxacin for 24 h responded the same as the
wild-type bacteria (data not shown).
Antibiotic penetration.
Chloride acted as a nonreactive
tracer, allowing visualization and qualitative comparison of solute
transport properties within Kp1 and Kp102M biofilms. Chloride quickly
penetrated biofilms of either strain with a penetration profile
characteristic of one described by Fick's second law (Fig.
3a). In less than 30 min, the chloride
concentration at the distal edges of biofilms of either strain was at
least 50% of the bulk concentration of 6.7 mM. The transport dynamics
of chloride were nearly identical for Kp1 and Kp102M biofilms.
|
) and Kp102M (
) biofilms. Error bars reflect
±1 standard error of the mean. The relatively large error bars in
panel b were due to the poor sensitivity of the bioassay above an
ampicillin concentration of 1,000 µg/ml. C, concentration on distal
edge of biofilm; C0, applied concentration.
-lactamase-deficient Kp102M biofilms. The ampicillin
concentration exceeded 500 µg/ml, or 250 times the MIC for the
mutant, at the distal edge of the biofilm in less than 10 min. Within
40 min, the ampicillin concentration was approximately 50% of the bulk
concentration, i.e., over 1,000 times the MIC for the mutant. The
sensitivity of the bioassay used in this study was poor at ampicillin
concentrations above 1,000 µg/ml, resulting in the relatively large
error bars.
Ciprofloxacin readily penetrated the biofilms of either strain (Fig.
3c). In less than 20 min, the ciprofloxacin concentration at the distal
edge of the biofilm exceeded the MIC (1.8 µg/ml). The ciprofloxacin
concentration was roughly 50% of the bulk concentration within 120 min. Ciprofloxacin permeation rates were similar for both strains.
Antibiotic degradation.
Degradation of the antibiotics by Kp1
and Kp102M biofilms was investigated qualitatively (Fig.
4). The control plates, not exposed to
biofilms, demonstrated that antibiotic instability was not sufficient
to significantly decrease the antibiotic concentration in the plates
and permit growth. The zone of growth directly under the biofilm's
former location on an ampicillin plate exposed to a Kp1 biofilm
suggested that the wild-type bacteria were capable of neutralizing
enough ampicillin to enable growth of a susceptible strain. The lack of
such a zone on the ampicillin plate exposed to a Kp102M biofilm
suggested that the mutant strain had little capacity to degrade
ampicillin. The experiment also indicated that neither strain was
capable of appreciably degrading ciprofloxacin.
|
1, a rate
statistically different (P = 0.007) from zero (Fig.
5a). The rate of ampicillin degradation
by Kp102M cultures was not statistically different (P = 0.655) from zero (Fig. 5b). Neither Kp1 (P = 0.992) nor Kp102M (P = 0.364) could degrade
ciprofloxacin at a rate statistically different from zero (data not
shown).
|
) were not
statistically different from zero (b).
Mutant characteristics.
Despite the nonspecific procedure used
to generate the -lactamase-deficient mutant, Kp102M appeared to
behave much like Kp1 with the exception of ampicillin-degrading
activity. The MIC of ciprofloxacin was nearly identical for both
strains. Kp102M responded to ciprofloxacin treatment in a manner
statistically indistinguishable from that for Kp1 when in planktonic or
biofilm state. The two strains also had very similar properties for
chloride and ciprofloxacin penetration. In addition, the maximum growth
rates of the two organisms were within 20% of each other, with the
mutant having a slightly faster growth rate (data not shown).
Furthermore, the structures and densities of Kp1 and Kp102M biofilms
were virtually indistinguishable via microscopy (data not shown).
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The biofilms formed by wild-type K. pneumoniae resisted killing by ampicillin and ciprofloxacin. This simple model system, while surely an imperfect representation of a real biofilm, does capture the characteristic antibiotic resistance of biofilm bacteria. Membrane-supported biofilms are attractive as in vitro systems for investigation of biofilm resistance because many biofilm samples can be cultured in parallel with relative ease. A particular advantage of this system is that it permits physical access to both sides of the biofilm, a feature that allowed us to directly measure the penetration of solutes through the model biofilms. Although not intended to simulate a particular disease, these colony biofilms might be considered primitive models of some infections.
While ciprofloxacin and chloride ion readily penetrated the biofilm, ampicillin was unable to penetrate a wild-type K. pneumoniae biofilm in the 4-h treatment period (Fig. 3). This result showed that the biofilm matrix did not pose an inherent barrier to solute mobility, an interpretation that is consistent with experimentally measured effective diffusion coefficients in biofilms (36). We hypothesized that the failure of ampicillin to penetrate biofilms was instead due to its deactivation in the surface layers of the biofilm faster than it could diffuse in. This reaction-diffusion mechanism of penetration limitation has previously been mathematically modeled and has been experimentally demonstrated for reactive oxidants such as hypochlorous acid (11, 35, 38, 44) and hydrogen peroxide (26, 35, 38).
To further test the role of reactive neutralization in limiting biofilm
penetration of ampicillin, we isolated a -lactamase-deficient mutant
of our wild-type K. pneumoniae strain. The wild-type strain degraded ampicillin in a batch experiment (Fig. 5a) at a statistically significant rate (P = 0.007). In the same batch test
with the mutant strain, there was no statistically discernible
degradation of ampicillin (P = 0.655) (Fig. 5b).
Furthermore, in a qualitative visualization of the potential of
biofilms to deplete the ampicillin in agar medium, the wild-type
K. pneumoniae strain locally neutralized ampicillin, whereas
the mutant strain did not (Fig. 4). The wild-type and
-lactamase-deficient mutant strains exhibited similar specific growth rates, ciprofloxacin susceptibilities, and microscopic biofilm
structures, and the chloride and ciprofloxacin penetrations were
similar for both strains. These measurements and observations suggest
that the mutant strain was deficient in -lactamase activity or
synthesis but was not otherwise altered in any way that would significantly affect our experiments.
Ampicillin penetrated biofilms of the -lactamase-deficient mutant
strain (Fig. 3b). Its penetration through biofilms of the mutant strain
was similar to the penetration of chloride ion through biofilms of both
the mutant and wild-type strains. The restoration of ampicillin
penetration by deletion of -lactamase activity supported the
contention that penetration breakdown in the wild-type results from a
reaction-diffusion interaction. This mechanism was further supported by
the Thiele modulus estimate of 6.7 for the ampicillin-challenged
wild-type biofilm. This value was calculated by using equation 20 of
Stewart (35), with values of 0.727/h for the first-order
reaction rate constant, 3.86 × 1010
m2/s for the effective diffusion coefficient of ampicillin,
and 0.465 mm for the biofilm thickness. The Thiele modulus is a
dimensionless parameter that compares the relative rates of reaction
and diffusion (4, 38). When the Thiele modulus exceeds 1, the diffusion rate is slower than the reaction rate and the system can
be considered transport limited. A Thiele modulus of 6.7 is sufficient
to account for the observed magnitude of reduced biofilm susceptibility
according to mathematical analysis of the
reaction-diffusion-disinfection problem (38).
In contrast to the situation with ampicillin, transport limitation
probably played a very minor role in protecting the biofilms from
ciprofloxacin. The concentration of ciprofloxacin reached the MIC
throughout the biofilm in less than 20 min and reached 50% of the bulk
agar concentration within 2 h (Fig. 3c). These measurements were
consistent with literature reports of relatively facile penetration of
fluoroquinolone agents into biofilms (25, 33, 40, 41). We
attribute the ability of ciprofloxacin to penetrate a biofilm to its
low reactivity. Enzymes that chemically deactivate fluoroquinolones,
analogous to the -lactamases that cleave penicillins, have not been
described. Fluoroquinolones can be exported from the cell by efflux
pumps (29), but this would not be expected to change the
ability of the antibiotic to penetrate the biofilm.
In summary, antibiotics can fail to penetrate microbial biofilms. The ability of an antibiotic to penetrate a biofilm depends on the rate at which it is deactivated in the biofilm. Agents that experience reactive neutralization in the biofilm are prone to penetration failure. On the other hand, agents that are not deactivated in the biofilm will penetrate a biofilm in a matter of minutes or hours. Mathematical models of the underlying reaction-diffusion interaction exist (28, 34, 35, 38) and could be used as quantitative frameworks to test this mechanism of antimicrobial resistance of biofilms with other systems.
The data reported here provide evidence for a mechanism of biofilm
resistance other than incomplete antimicrobial penetration. Ciprofloxacin penetrated biofilms within a few hours, but bacteria were
not killed even after 24 h of treatment (log reduction, 1.07 ± 0.18). A biofilm formed by a -lactamase-deficient mutant was fully penetrated by ampicillin but, likewise, was not effectively killed. A planktonic -lactamase-deficient mutant K. pneumoniae strain exposed to ampicillin experienced a 4.62 ± 0.20 log reduction in the number of CFU within 4 h of treatment.
Biofilms, on the other hand, were reduced by only 1.64 ± 0.33 log
CFU after 24 h of antibiotic exposure, even though the ampicillin
concentration was over 1,000 times the MIC for the mutant for at least
23 of the 24 h. Localized regions of slow growth or starvation
within the biofilm interior may be at the root of this resistance.
Slowly growing or nongrowing bacteria are known to be less susceptible to many antibiotics (13, 15, 16, 27). Spatial variation in
the physiological status of bacteria within biofilms has recently been
demonstrated by fluorescence staining approaches (20, 21, 37,
43). Membrane-supported biofilms of K. pneumoniae
similar to those used in this study also exhibit pronounced
physiological heterogeneity over distances as short as 10 µm
(20, 43). Ongoing studies in our laboratory are using the
membrane-supported biofilm model system to investigate the role
of nutrient-limited physiology in mediating K. pneumoniae biofilm resistance to ampicillin and ciprofloxacin.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported through cooperative agreement EEC- 8907039 between the National Science Foundation and Montana State University and by the industrial partners of the Center for Biofilm Engineering.
We thank Matthew Jackson for helping with the isolation of a
-lactamase-deficient K. pneumoniae mutant.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Center for Biofilm Engineering, Montana State University-Bozeman, Bozeman, MT 59717-3980. Phone: (406) 994-2890. Fax: (406) 994-6098. E-mail: phil_s{at}erc.montana.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | American Public Health Association, American Water Works Association, and Water and Environment Federation. 1995. Standard methods for the examination of water and wastewater. American Public Health Association, Washington, D.C. |
| 2. |
Anwar, H.,
J. L. Strap, and J. W. Costerton.
1992.
Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy.
Antimicrob. Agents Chemother.
36:1347-1351 |
| 3. |
Anwar, H.,
J. L. Strap, and J. W. Costerton.
1992.
Kinetic interaction of biofilm cells of Staphylococcus aureus with cephalexin and tobramycin in a chemostat system.
Antimicrob. Agents Chemother.
36:890-893 |
| 4. | Bailey, J. E., and D. F. Ollis. 1986. Biochemical engineering fundamentals. McGraw-Hill Book Co., New York, N.Y. |
| 5. | Blaser, J., P. Vergeres, A. F. Widmer, and W. Zimmerli. 1995. In vivo verification of in vitro model of antibiotic treatment of device-related infection. Antimicrob. Agents Chemother. 39:1134-1139[Abstract]. |
| 6. | Brown, M. R. W., and P. Gilbert. 1993. Sensitivity of biofilms to antimicrobial agents. J. Appl. Bacteriol. 74(Suppl.):87S-97S. |
| 7. | Christensen, G. D., L. M. Baddour, D. L. Hasty, G. H. Lowrance, and W. A. Simpson. 1989. Microbial and foreign body factors in the pathogenesis of medical device infections, p. 27-59. In A. L. Bison, and F. A. Waldvogel (ed.), Infections associated with indwelling medical devices. American Society for Microbiology, Washington, D.C. |
| 8. |
Costerton, J. W.,
P. S. Stewart, and E. P. Greenberg.
1999.
Bacterial biofilms: a common cause of persistent infections.
Science
284:1318-1322 |
| 9. | Costerton, J. W., K.-J. Cheng, G. G. Geesey, T. J. Ladd, J. C. Nickel, M. Dasgupta, and T. J. Marrie. 1987. Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 41:435-464[CrossRef][Medline]. |
| 10. | 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]. |
| 11. |
DeBeer, D.,
R. Srinivasan, and P. S. Stewart.
1994.
Direct measurement of chlorine penetration into biofilms during disinfection.
Appl. Environ. Microbiol.
60:4339-4344 |
| 12. |
Deretic, V.,
M. J. Schurr,
J. C. Boucher, and D. W. Martin.
1994.
Conversion of Pseudomonas aeruginosa to mucoidy in cystic fibrosis: environmental stress and regulation of bacterial virulence by alternative sigma factors.
J. Bacteriol.
176:2773-2780 |
| 13. |
Duguid, I. G.,
E. Evans,
M. R. W. Brown, and P. Gilbert.
1992.
Growth-rate-independent killing by ciprofloxacin of biofilm-derived Staphylococcus epidermidis; evidence for a cell-cycle dependency.
J. Antimicrob. Chemother.
30:791-802 |
| 14. |
Dunne, W. M., Jr.,
E. O. Mason, and S. L. Kaplan.
1993.
Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilm.
Antimicrob. Agents Chemother.
37:2522-2526 |
| 15. |
Evans, D. J.,
D. G. Allison,
M. R. W. Brown, and P. Gilbert.
1991.
Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate.
J. Antimicrob. Chemother.
27:177-184 |
| 16. |
Gilbert, P.,
P. J. Collier, and M. R. Brown.
1990.
Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response.
Antimicrob. Agents Chemother.
34:1865-1868 |
| 17. |
Giwercman, B.,
E. 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 |
| 18. | Hoben, H. J., and P. Somasegaran. 1948. Comparison of the pour, spread, and drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized peat. Appl. Environ. Microbiol. 44:1246-1247. |
| 19. |
Hoyle, B. D.,
J. Alcantara, and J. W. Costerton.
1992.
Pseudomonas aeruginosa biofilm as a diffusion barrier to piperacillin.
Antimicrob. Agents Chemother.
36:2054-2056 |
| 20. |
Huang, C.-T.,
K. D. Xu,
G. A. McFeters, and P. S. Stewart.
1998.
Spatial patterns of alkaline phosphatase expression within bacterial colonies and biofilms in response to phosphate starvation.
Appl. Environ. Microbiol.
64:1526-1531 |
| 21. | Huang, C.-T., F. P. Yu, G. A. McFeters, and P. S. Stewart. 1995. Nonuniform spatial patterns of respiratory activity with biofilms during disinfection. Appl. Environ. Microbiol. 61:2252-2256[Abstract]. |
| 22. |
Jenkins, D. E.,
J. E. Schultz, and A. Matin.
1988.
Starvation-induced cross protection against heat or H2O2 challenge in Escherichia coli.
J. Bacteriol.
170:3910-3914 |
| 23. |
Jensen, E. T.,
A. Kharazmi,
K. Lam,
J. W. Costerton, and N. Hoiby.
1990.
Human polymorphonuclear leukocyte response to Pseudomonas aeruginosa grown in biofilms.
Infect. Immun.
58:2383-2385 |
| 24. | Khardori, N., and M. Yassien. 1995. Biofilms in device-related infections. J. Ind. Microbiol. 15:141-147[CrossRef][Medline]. |
| 25. | Kumon, H., K. Tomochika, T. Matunaga, M. Ogawa, and H. Ohmori. 1994. A sandwich cup method for the penetration assay of antimicrobial agents through Pseudomonas exopolysaccharides. Microbiol. Immunol. 38:615-619[Medline]. |
| 26. | Liu, X., F. Roe, A. Jesaitis, and Z. Lewandowski. 1998. Resistance of biofilms to the catalase inhibitor 3-amino-1,2,4-triazole. Biotechnol. Bioeng. 59:156-162[CrossRef][Medline]. |
| 27. |
McLeod, G. I., and M. P. Spector.
1996.
Starvation- and stationary-phase-induced resistance to the antimicrobial peptide polymyxin B in Salmonella typhimurium is RpoS ( S) independent and occurs through both phoP-dependent and -independent pathways.
J. Bacteriol.
178:3683-3688 |
| 28. |
Nichols, W. W.,
M. J. Evans,
M. P. 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 |
| 29. | Nikaido, H. 1998. Multiple antibiotic resistance and efflux. Curr. Opin. Microbiol. 1:516-523[CrossRef][Medline]. |
| 30. |
Potera, C.
1999.
Forging a link between biofilms and disease.
Science
283:1837-1839 |
| 31. |
Rayner, M. G.,
Y. Zhang,
M. C. Gorry,
Y. Chen,
J. C. Post, and G. D. Ehrlich.
1998.
Evidence of bacterial metabolic activity in culture-negative otitis media with effusion.
JAMA
279:296-299 |
| 32. | Reed, R. W., and G. B. Reed. 1948. "Drop plate" method of counting viable bacteria. Can. J. Res. 26:317-326. |
| 33. | Shigeta, M., G. Tanaka, H. Komatsuzawa, M. Sugai, H. Suginaka, and T. Usui. 1997. Permeation of antimicrobial agents through Pseudomonas aeruginosa biofilms: a simple method. Chemotherapy (Basel) 43:340-345. |
| 34. |
Stewart, P. S.
1994.
Biofilm accumulation model that predicts antibiotic resistance of Pseudomonas aeruginosa biofilms.
Antimicrob. Agents Chemother.
38:1052-1058 |
| 35. | Stewart, P. S. 1996. Theoretical aspects of antibiotic diffusion into microbial biofilms. Antimicrob. Agents Chemother. 40:2517-2522[Abstract]. |
| 36. | Stewart, P. S. 1998. A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms. Biotechnol. Bioeng. 59:261-271[CrossRef][Medline]. |
| 37. |
Stewart, P. S.,
T. Griebe,
R. Srinivasan,
C.-I. Chen,
F. P. Yu,
D. DeBeer, and G. A. McFeters.
1994.
Comparison of respiratory activity and culturability during monochloramine disinfection of binary population biofilms.
Appl. Environ. Microbiol.
60:1690-1692 |
| 38. | Stewart, P. S., and J. B. Raquepas. 1995. Implications of reaction-diffusion theory for the disinfection of microbial biofilms by reactive antimicrobial agents. Chem. Eng. Sci. 50:3099-3104[CrossRef]. |
| 39. |
Stickler, D. J.,
N. S. Morris,
R. J. C. McLean, and C. Fuqua.
1998.
Biofilms on indwelling urethral catheters produce quorum-sensing signal molecules in situ and in vitro.
Appl. Environ. Microbiol.
64:3486-3490 |
| 40. |
Suci, P. A.,
M. W. Mittelman,
F. P. Yu, and G. G. Geesey.
1994.
Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms.
Antimicrob. Agents Chemother.
38:2125-2133 |
| 41. | Vrany, J. D., P. S. Stewart, and P. A. Suci. 1997. Comparison of recalcitrance to ciprofloxacin and levofloxacin exhibited by Pseudomonas aeruginosa biofilms displaying rapid-transport characteristics. Antimicrob. Agents Chemother. 41:1352-1358[Abstract]. |
| 42. |
Ward, K. H.,
M. E. Olson,
K. Lam, and J. W. Costerton.
1992.
Mechanism of persistent infection associated with peritoneal implants.
J. Med. Microbiol.
36:406-413 |
| 43. | Wentland, E. J., P. S. Stewart, C.-T. Huang, and G. A. McFeters. 1996. Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilms. Biotechnol. Prog. 12:316-321[CrossRef][Medline]. |
| 44. | Xu, X., P. S. Stewart, and X. Chen. 1996. Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads. Biotechnol. Bioeng. 49:93-100. |
| 45. |
Yasuda, H.,
Y. Ajiki,
T. Koga,
H. Kawada, and T. Yokota.
1993.
Interaction between biofilms formed by Pseudomonas aeruginosa and clarithromycin.
Antimicrob. Agents Chemother.
37:1749-1755 |
| 46. |
Yasuda, H.,
Y. Ajiki,
T. Koga, and T. Yokota.
1994.
Interaction between clarithromycin and biofilms formed by Staphylococcus epidermidis.
Antimicrob. Agents Chemother.
38:138-141 |
| 47. | Younger, J. J., G. D. Christensen, D. L. Bartley, J. C. H. Simmons, and F. F. Barrett. 1987. Coagulase-negative staphylococci isolated from cerebrospinal fluid shunts: importance of slime production, species identification, and shunt removal to clinical outcome. J. Infect. Dis. 156:548-554[Medline]. |
Antimicrobial Agents and Chemotherapy, July 2000, p. 1818-1824, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
May, T., Ito, A., Okabe, S.
(2009). Induction of Multidrug Resistance Mechanism in Escherichia coli Biofilms by Interplay between Tetracycline and Ampicillin Resistance Genes. Antimicrob. Agents Chemother.
53: 4628-4639
[Abstract] [Full Text] -
Verma, V., Harjai, K., Chhibber, S.
(2009). Restricting ciprofloxacin-induced resistant variant formation in biofilm of Klebsiella pneumoniae B5055 by complementary bacteriophage treatment. J Antimicrob Chemother
0: dkp360v1-dkp360
[Abstract] [Full Text] -
Singh, R., Ray, P., Das, A., Sharma, M.
(2009). Role of persisters and small-colony variants in antibiotic resistance of planktonic and biofilm-associated Staphylococcus aureus: an in vitro study. J Med Microbiol
58: 1067-1073
[Abstract] [Full Text] -
Tetz, G. V., Artemenko, N. K., Tetz, V. V.
(2009). Effect of DNase and Antibiotics on Biofilm Characteristics. Antimicrob. Agents Chemother.
53: 1204-1209
[Abstract] [Full Text] -
Kvist, M., Hancock, V., Klemm, P.
(2008). Inactivation of Efflux Pumps Abolishes Bacterial Biofilm Formation. Appl. Environ. Microbiol.
74: 7376-7382
[Abstract] [Full Text] -
Lenz, A. P., Williamson, K. S., Pitts, B., Stewart, P. S., Franklin, M. J.
(2008). Localized Gene Expression in Pseudomonas aeruginosa Biofilms. Appl. Environ. Microbiol.
74: 4463-4471
[Abstract] [Full Text] -
Zhang, L., Mah, T.-F.
(2008). Involvement of a Novel Efflux System in Biofilm-Specific Resistance to Antibiotics. J. Bacteriol.
190: 4447-4452
[Abstract] [Full Text] -
Webster, P., Wu, S., Gomez, G., Apicella, M., Plaut, A. G., St. Geme, J. W. III
(2006). Distribution of Bacterial Proteins in Biofilms Formed by Non-typeable Haemophilus influenzae. J. Histochem. Cytochem.
54: 829-842
[Abstract] [Full Text] -
Burton, E., Gawande, P. V., Yakandawala, N., LoVetri, K., Zhanel, G. G., Romeo, T., Friesen, A. D., Madhyastha, S.
(2006). Antibiofilm Activity of GlmU Enzyme Inhibitors against Catheter-Associated Uropathogens.. Antimicrob. Agents Chemother.
50: 1835-1840
[Abstract] [Full Text] -
Chambless, J. D., Hunt, S. M., Stewart, P. S.
(2006). A three-dimensional computer model of four hypothetical mechanisms protecting biofilms from antimicrobials.. Appl. Environ. Microbiol.
72: 2005-2013
[Abstract] [Full Text] -
Marques, C. N. H., Salisbury, V. C., Greenman, J., Bowker, K. E., Nelson, S. M.
(2005). Discrepancy between viable counts and light output as viability measurements, following ciprofloxacin challenge of self-bioluminescent Pseudomonas aeruginosa biofilms. J Antimicrob Chemother
56: 665-671
[Abstract] [Full Text] -
Cerca, N., Martins, S., Cerca, F., Jefferson, K. K., Pier, G. B., Oliveira, R., Azeredo, J.
(2005). Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry. J Antimicrob Chemother
56: 331-336
[Abstract] [Full Text] -
Putnam, S. D., Sanders, J. W., Tribble, D. R., Rockabrand, D. R., Riddle, M. S., Rozmajzl, P. J., Frenck, R. W.
(2005). Posttreatment Changes in Escherichia coli Antimicrobial Susceptibility Rates among Diarrheic Patients Treated with Ciprofloxacin. Antimicrob. Agents Chemother.
49: 2571-2572
[Abstract] [Full Text] -
Samaranayake, Y. H., Ye, J., Yau, J. Y. Y., Cheung, B. P. K., Samaranayake, L. P.
(2005). In Vitro Method To Study Antifungal Perfusion in Candida Biofilms. J. Clin. Microbiol.
43: 818-825
[Abstract] [Full Text] -
Keren, I., Shah, D., Spoering, A., Kaldalu, N., Lewis, K.
(2004). Specialized Persister Cells and the Mechanism of Multidrug Tolerance in Escherichia coli. J. Bacteriol.
186: 8172-8180
[Abstract] [Full Text] -
Werner, E., Roe, F., Bugnicourt, A., Franklin, M. J., Heydorn, A., Molin, S., Pitts, B., Stewart, P. S.
(2004). Stratified Growth in Pseudomonas aeruginosa Biofilms. Appl. Environ. Microbiol.
70: 6188-6196
[Abstract] [Full Text] -
Al-Fattani, M. A., Douglas, L. J.
(2004). Penetration of Candida Biofilms by Antifungal Agents. Antimicrob. Agents Chemother.
48: 3291-3297
[Abstract] [Full Text] -
Fux, C. A., Wilson, S., Stoodley, P.
(2004). Detachment Characteristics and Oxacillin Resistance of Staphyloccocus aureus Biofilm Emboli in an In Vitro Catheter Infection Model. J. Bacteriol.
186: 4486-4491
[Abstract] [Full Text] -
Borriello, G., Werner, E., Roe, F., Kim, A. M., Ehrlich, G. D., Stewart, P. S.
(2004). Oxygen Limitation Contributes to Antibiotic Tolerance of Pseudomonas aeruginosa in Biofilms. Antimicrob. Agents Chemother.
48: 2659-2664
[Abstract] [Full Text] -
Houlihan, A. J., Russell, J. B.
(2003). The susceptibility of ionophore-resistant Clostridium aminophilum F to other antibiotics. J Antimicrob Chemother
52: 623-628
[Abstract] [Full Text] -
Conley, J., Olson, M. E., Cook, L. S., Ceri, H., Phan, V., Davies, H. D.
(2003). Biofilm Formation by Group A Streptococci: Is There a Relationship with Treatment Failure?. J. Clin. Microbiol.
41: 4043-4048
[Abstract] [Full Text] -
Mukherjee, P. K., Chandra, J., Kuhn, D. M., Ghannoum, M. A.
(2003). Mechanism of Fluconazole Resistance in Candida albicans Biofilms: Phase-Specific Role of Efflux Pumps and Membrane Sterols. Infect. Immun.
71: 4333-4340
[Abstract] [Full Text] -
Chole, R. A., Faddis, B. T.
(2003). Anatomical Evidence of Microbial Biofilms in Tonsillar Tissues: A Possible Mechanism to Explain Chronicity. Arch Otolaryngol Head Neck Surg
129: 634-636
[Abstract] [Full Text] -
Finelli, A., Gallant, C. V., Jarvi, K., Burrows, L. L.
(2003). Use of In-Biofilm Expression Technology To Identify Genes Involved in Pseudomonas aeruginosa Biofilm Development. J. Bacteriol.
185: 2700-2710
[Abstract] [Full Text] -
Anderl, J. N., Zahller, J., Roe, F., Stewart, P. S.
(2003). Role of Nutrient Limitation and Stationary-Phase Existence in Klebsiella pneumoniae Biofilm Resistance to Ampicillin and Ciprofloxacin. Antimicrob. Agents Chemother.
47: 1251-1256
[Abstract] [Full Text] -
Teitzel, G. M., Parsek, M. R.
(2003). Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa. Appl. Environ. Microbiol.
69: 2313-2320
[Abstract] [Full Text] -
Walters, M. C. III, Roe, F., Bugnicourt, A., Franklin, M. J., Stewart, P. S.
(2003). Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin and Tobramycin. Antimicrob. Agents Chemother.
47: 317-323
[Abstract] [Full Text] -
Aaron, S. D., Ferris, W., Ramotar, K., Vandemheen, K., Chan, F., Saginur, R.
(2002). Single and Combination Antibiotic Susceptibilities of Planktonic, Adherent, and Biofilm-Grown Pseudomonas aeruginosa Isolates Cultured from Sputa of Adults with Cystic Fibrosis. J. Clin. Microbiol.
40: 4172-4179
[Abstract] [Full Text] -
Chole, R. A., Faddis, B. T.
(2002). Evidence for Microbial Biofilms in Cholesteatomas. Arch Otolaryngol Head Neck Surg
128: 1129-1133
[Abstract] [Full Text] -
Stone, G., Wood, P., Dixon, L., Keyhan, M., Matin, A.
(2002). Tetracycline Rapidly Reaches All the Constituent Cells of Uropathogenic Escherichia coli Biofilms. Antimicrob. Agents Chemother.
46: 2458-2461
[Abstract] [Full Text] -
Zahller, J., Stewart, P. S.
(2002). Transmission Electron Microscopic Study of Antibiotic Action on Klebsiella pneumoniae Biofilm. Antimicrob. Agents Chemother.
46: 2679-2683
[Abstract] [Full Text] -
Zheng, Z., Stewart, P. S.
(2002). Penetration of Rifampin through Staphylococcus epidermidis Biofilms. Antimicrob. Agents Chemother.
46: 900-903
[Abstract] [Full Text] -
Spoering, A. L., Lewis, K.
(2001). Biofilms and Planktonic Cells of Pseudomonas aeruginosa Have Similar Resistance to Killing by Antimicrobials. J. Bacteriol.
183: 6746-6751
[Abstract] [Full Text] -
Lewis, K.
(2001). Riddle of Biofilm Resistance. Antimicrob. Agents Chemother.
45: 999-1007
[Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»