1 Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, UK; 2 Bristol Centre for Antimicrobial Research and Evaluation, Department of Medical Microbiology, Southmead Hospital, Bristol BS10 5NB, UK
Received 28 April 2005; returned 30 June 2005; revised 8 July 2005; accepted 24 July 2005
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Abstract |
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Methods: The Sorbarod continuous perfusion culture system was used for the cultivation of biofilms of a self-bioluminescent strain of P. aeruginosa PAO1. Biofilms were challenged with ciprofloxacin (5 mg/L) in the perfusing medium for 3 h and allowed to recover to pre-challenge population levels before initiation of a second 3 h challenge. In addition to determining eluate and biofilm cell survival by conventional viable plate counts, light output was monitored via a luminometer and a low-light-level ICCD camera, to give an indication of metabolism. The effect of drug challenge on biofilm structure was investigated using an environmental scanning electron microscope, which allowed discernment of changes to the three-dimensional biofilm architecture.
Results: On challenge with ciprofloxacin, eluate light output measurements declined to a lesser extent than viable counts for the same samples and also indicated that post-challenge recovery of the biofilm metabolism did not occur as rapidly as suggested by viable count data. Photon detection by ICCD camera allowed real-time, non-invasive imaging of metabolic activity within intact biofilms.
Conclusions: The application of a bioluminescent reporter strain to biofilm research provides valuable real-time positional data on the efficacy of anti-biofilm treatment strategies.
Keywords: P. aeruginosa , biofilms , bioluminescence
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Introduction |
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A range of in vitro laboratory models of biofilm growth is used by the biofilm research community, with various models (e.g. flow cells, continuous perfusion systems, microtitre plates), and the conditions under which they are used, often providing different snapshots of life in a biofilm.47 An inherent difficulty in the study of biofilms is that it is often necessary to physically disrupt them in order to investigate features of their constituent cell populations. This creates the potential disadvantage of biofilm-specific characteristics being lost. In attempts to avoid this, biofilm growth, activity and survival have been directly visualized with fluorescent probes and reporter genes.8 Recently, there have been several studies utilizing bacterial bioluminescence as a tool to study surface-associated growth, both in vitro and in vivo.913 An attractive feature of this approach is that cellular metabolic activity can be monitored in real-time, in a non-invasive manner, without the addition of an exogenous substrate. Light emission is catalysed by the enzyme luciferase, whereby molecular oxygen oxidizes both reduced flavin mononucleotide (FMNH2) and a long-chain aldehyde to produce oxidized flavin (FMN), the corresponding long-chain fatty acid as well as bluegreen light.11 FMNH2 depends upon functional electron transport, thus only metabolically active cells produce light.14
We report here a study of a bioluminescent strain of Pseudomonas aeruginosa in an in vitro biofilm model, challenged with ciprofloxacin, a commonly used anti-pseudomonal fluoroquinolone. The effect on the biofilm was also monitored by conventional plate counts and by environmental scanning electron microscopy (ESEM).
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Materials and methods |
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P. aeruginosa PAO1 SEI was transformed with a recombinant plasmid containing the luxCDABE gene cassette of Photorhabdus luminescens. The recombinant plasmid, pMCS-5 lite, was constructed by insertion of the 7 kb EcoRI fragment from plasmid pLite2715 into the broad-host-range plasmid pBBR1MCS-516 with the luxCDABE genes under the control of the constitutive p lac promoter. P. aeruginosa PAO1 SEI MCS5-lite was maintained on nutrient agar (NA) containing gentamicin (10 mg/L), the selective agent for the recombinant plasmid.
Iron-deplete chemically defined medium (CDM) consisted of glucose (40 mM), KCl (0.74 mM), NaCl (0.60 mM), (NH4)2SO4 (48 mM), MgSO4.7H2O (0.48 mM), MOPS (60 mM) and K2HPO4.3H2O (3.84 mM). Trace elements were included to give final concentrations as follows: CaCl2.6H2O (0.50 µM), H3BO3 (0.50 µM), CoCl2.6H2O (0.05 µM), CuSO4.7H2O (0.05 µM), ZnSO4.7H2O (0.05 µM), MnSO4.4H2O (0.10 µM) and (NH4)6Mo7O24.4H2O (0.005 µM). Ciprofloxacin solution (200 mg/mL), included in the CDM for drug perfusion experiments, was the gift of Southmead Hospital (Bristol, UK).
Biofilm culture
Biofilms were grown using the Sorbarod in vitro biofilm culture system described previously.17,18 Briefly, 10 Sorbarods, pre-wetted with 5 mL of 0.85% saline, were inoculated with 1 mL of an exponential phase culture of P. aeruginosa PAO1 SEI MCS5-lite (1 x 108 cfu). CDM with gentamicin (10 mg/L), to maintain the plasmid, was delivered into the system at a controlled flow rate of 15 mL/h. Samples of eluate (eluted medium together with dispersed cells) were collected over time and quantified via viable counts [spiral plating onto NA + gentamicin (10 mg/L); spiral plater Autoplate model 3000, Spiral Biotech, Bethesda, MD, USA]. To assess the bioburden (number of viable cells within each Sorbarod), individual Sorbarod biofilms were broken down at various times and processed by vortexing as described previously.17 All measurements were made in triplicate and statistical analyses were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, USA).
Bioluminescence was measured either in mV/mL using a 1250001 luminometer (BioOrbit, Finland) at 37°C, or in photons/s using an ICCD photon counting camera (model 225, Photek, UK). Using the latter technique, photons emitted by P. aeruginosa PAO1 SEI MCS5-lite were quantified and analysed using IFS32 imaging software (Photek, UK). A 1% detector filter was employed for high population densities, to prevent camera damage. Biofilm and eluate samples were processed differently. Sorbarods, still intact in their PVC housings, were disconnected temporarily from the flow system for observation, while eluate samples (1 mL) were collected in microfuge tubes. Both samples were imaged by the ICCD camera mounted on a 37°C incubator. Photon emission data from samples were collected every second over a 30 s integration period, with the detector filter set at 1% and 100% for biofilm and eluate samples, respectively. All measurements were performed in triplicate and analysed statistically, as described previously.
Antibiotic perfusion of biofilms
The MIC of ciprofloxacin for P. aeruginosa PAO1 SEI MCS5-lite was determined by the broth macrodilution method.19 In addition to use of standard Iso-Sensitest broth, the MIC was also determined in CDM. MIC determinations carried out in CDM were incubated for 24 h at 37°C, instead of the standard 18 h, due to the slower growth rate of P. aeruginosa in this minimal medium.
Sorbarod biofilms perfused with CDM and gentamicin (10 mg/L) for 24 h were deemed to be in quasi-steady-state (a constant number of cells/mL shed from the biofilm). Biofilms were then perfused with CDM (minus gentamicin) containing ciprofloxacin at 5 x MIC (5 mg/L, as determined both in Iso-Sensitest broth and in CDM), at a rate of 15 mL/h for 3 h, after which the medium was replaced with fresh CDM and gentamicin (10 mg/L). It should be noted that prior batch culture of P. aeruginosa in CDM without gentamicin had demonstrated that bioluminescence was not diminished over a 24 h period and also that chequerboard assay showed no interaction between gentamicin and ciprofloxacin (data not shown). Eluate cell numbers (cfu/mL) were allowed to recover to original levels before biofilms were challenged with ciprofloxacin (5 mg/L) for a second 3 h period. Bioluminescence levels and viable counts of eluate samples were monitored regularly throughout, while biofilm bioburdens were assessed less often due to the requirement for biofilm sacrifice to obtain population viable cell plate counts. NA plates contained 1% MgCl2.6H2O to inactivate any remaining ciprofloxacin in samples for viable counts.20 Biofilms were run for a total of 100 h and the experiment carried out on three separate occasions.
Susceptibility testing of eluate and resuspended biofilm cells
To ascertain whether there were any changes in ciprofloxacin susceptibility of eluate and resuspended biofilm cells over the course of the study, MICs were assessed using antimicrobial strips (Etest®, AB BIODISK, Sweden). Resuspended biofilm samples were adjusted to give a 0.5 McFarland turbidity inoculum density, inoculated onto Iso-Sensitest plates and incubated at 35°C for 1618 h. Eluate samples were pooled, allowed to grow to reach 0.5 McFarland turbidity, inoculated onto Iso-Sensitest plates and incubated as above.
Environmental scanning electron microscopy (ESEM)
Intact Sorbarod biofilms, pre- and post-ciprofloxacin perfusion (24, 75, and 120 h), were fixed with 4% glutaraldehyde in 0.1 M phosphate buffer at pH 7.0 for 1 h. A non-inoculated Sorbarod, perfused with CDM for 24 h, was also prepared for analysis. Each Sorbarod was washed three times for 1 h with 0.1 M phosphate buffer at pH 7.0, then washed in sterile distilled water for 15 min and cut vertically to 2.5 mm thickness. Samples were examined using a Philips XL 30 ESEM together with a gaseous secondary electron detector. Samples were cooled to 5°C and initially viewed at 7.5 Torr. The pressure was reduced to evaporate water and expose the biofilm and fibres in conditions of high relative humidity.
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Results |
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The results of perfusion of 24 h quasi-steady-state biofilms with ciprofloxacin for 3 h, followed by a period of recovery before a second 3 h challenge, are presented in Figure 1(b). Bioluminescence levels (mV/mL and photon counts/s/mL) and viable counts (cfu/mL) were followed throughout. After the first 3 h of ciprofloxacin perfusion, eluate bioluminescence levels had decreased 1.61 log (mV/mL) and 1.52 log (photons/s/mL) in comparison with their respective controls, whereas viable counts had declined 3.88 log in comparison with controls. Eluate viable counts appeared to indicate that recovery began 3 h after cessation of drug perfusion, whereas bioluminescence (mV/mL) only began to increase 20 h later. Eluate photon counts indicated a similar prolonged recovery time. Following the second ciprofloxacin perfusion, it was again noted that eluate viable counts were increasing while both types of bioluminescence measurements were still indicating a decline in cellular metabolic activity.
Resuspended biofilm viable cell counts and intact biofilm bioluminescence ICCD data (photons/s/mL) for control Sorbarod biofilms (Figure 2) showed increases in the biofilm bioburdens of 0.5 log over the 130 h time period studied (i.e. biofilms were in quasi-steady-state). Challenge of the biofilms with ciprofloxacin resulted in a drop in biofilm bioburden of at least 2 log cfu/mL, while bioluminescence (photons/s) of the intact biofilms declined less than 1 log, for those time points monitored. As with eluate samples, viable counts indicated recovery of the biofilm population, while bioluminescence levels were still in decline. However, bioluminescence dropped little following the second challenge with ciprofloxacin, for those time points monitored, in contrast to the 2 log decrease observed for viable counts.
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Photon emission analyses of intact biofilms were carried out over an integration time of 30 s. During that time, an image was recorded that allowed spatial distribution of levels of bioluminescence within the Sorbarod filter to be visualized (Figure 3). Images were analysed at different time points: quasi-steady-states (time points 24, 72.5 and 125 h), immediately after ciprofloxacin challenges (time points 27 and 75.5 h) and 20 h after cessation of both challenges (47 and 95.5 h). At 24 h, photon emission was most intense at the top of the Sorbarod (Figure 3a). A similar distribution, although higher photon output, was observed in the second quasi-steady-state (72.5 h) (Figure 3d). Twenty hours following the first ciprofloxacin perfusion (Figure 3c), photon emission was clearly most intense in the upper third of the Sorbarod, with relatively sparse output in the lowest third. Twenty hours after the second drug challenge (Figure 3f), photon output was again greatest in the upper third of the Sorbarod, but in comparison to Figure 3(c), photon intensity was far greater throughout. Immediately following ciprofloxacin perfusions (Figure 3b and e), photon emission was higher (compared with previous quasi-steady-states) and distributed uniformly throughout the Sorbarod. Photon emission from a final quasi-steady-state biofilm (Figure 3g) and from a control, non-challenged biofilm of the same age (Figure 3h) was identical in terms of overall numbers of photons. However, while emission from the control, was homogeneously distributed throughout the Sorbarod, this was not the case for the drug-challenged Sorbarod, where emission was more intense at the top of the filter.
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A representative example of quasi-steady-state (24 h old) biofilm is shown in Figure 4(a). Clumps of cells and individual cells were visible at the surfaces of well-defined three-dimensional biofilm structuresthese structures presumably consisting of pseudomonal extracellular polymeric substances (EPS). No such well-defined structures were observed in biofilms sampled following ciprofloxacin challenge, a representative example of which is shown in Figure 4(b). Bacterial cells, elongated to a size of 4 or 5 µm, were visible within the mass of EPS. As biofilms recovered post-challenge and reached quasi-steady-state once again, they appeared less disaggregated and there were more three-dimensional structures composed of EPS and mixed populations of short and long rods (Figure 4c). The non-drug-challenged biofilm sample of the same age contained smooth, protruding, well-defined biofilm masses, an example of which is shown (Figure 4d). Bacterial cells of 2 µm in length were visible on the surface of these structures.
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Samples of eluate and biofilm cells were taken at various time points over the course of biofilm perfusion experiments, and ciprofloxacin MICs ascertained using Etest® strips (Table 1). Biofilm samples only showed a significant decrease in susceptibility to ciprofloxacin (MIC 6-fold greater than pre-exposure levels) following the second challenge with ciprofloxacin, when biofilms were being allowed to recover at 95.5 h. Eluate cell MICs increased immediately following the first drug perfusion, declined, then increased significantly (to eight times the original MIC value) immediately following the second perfusion. MIC values declined thereafter, although remained greater than those of control, non-challenged, eluate cells.
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Discussion |
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Monitoring the effect of 3 h exposures of ciprofloxacin on biofilms, using bioluminescence measurements, yielded some interesting differences when compared with viable count data for the same samples. The log decline in eluate viable cell counts was always greater than the decline in bioluminescence over the same time periods. Furthermore, when eluate viable counts began to increase, indicating recovery of the biofilm populations, bioluminescence measurements were still in decline. These findings are consistent with those reported previously, for fluoroquinolone-challenged planktonic and biofilm cultures.11,21 There are several possible explanations for these apparent differences. The direct target of ciprofloxacin in the bacterial cell is not that of energy-generating metabolic processes; therefore, the real-time measurements of bioluminescence would not be affected to a great extent on initial challenge with this agent. After cessation of drug-exposure, however, the cells could be directing their metabolism towards recovery and repair processes, rather than light emission. Moreover, unlike bioluminescence, viable counts do not give real-time measurements. Drug-challenged cells plated onto agar may either be irretrievably injured and unable to develop into observable colonies, or they may recover and ultimately be enumerated as cfu. The more rapid recovery of the biofilm indicated by eluate colony counts suggests that injured cells may indeed have been able to recover on agar. An alternative explanation might be that biofilm cells were elongated as a consequence of ciprofloxacin exposure.22 On removal of the drug, filaments divided into individual cells, with a resultant increase in cfu in the eluate but no increase in bioluminescence.
The effect of ciprofloxacin on biofilm bioburden was not as great as that seen on eluted cell viable counts, for the same sample points. It was notable, though, that bioburden viable counts again declined significantly more than photon counts. Indeed, for those samples taken, the decline in photons as a result of the second drug challenge was relatively minor. The ciprofloxacin MICs for eluate and resuspended biofilm cells increased after both drug challenges and, although they subsequently declined, they did not return to pre-exposure levels within the time period studied. It is possible that the drug challenges selected for a resistant sub-population of cells that were less able to predominate under normal conditions. The presence of resistant sub-populations of cells within biofilms has been indicated previously,23 although in this present work, use of a range of ciprofloxacin concentrations would be required to identify the presence of markedly resistant cells.
It is also possible that ciprofloxacin was being removed from the cells via efflux, although the fluoroquinolones are not known inducers of efflux mechanisms in P. aeruginosa. Ciprofloxacin is, however, a substrate for the constitutively expressed MexAB-OprM efflux system,24 but this would not account for the transient increase in MICs. Furthermore, there is no evidence, to date, that efflux pump expression in biofilms is up-regulated.25,18
Using the colony biofilm technique,26 it has been shown that oxygen limitation plays an important protective role in antimicrobial-challenged P. aeruginosa biofilms.27,28 In this current study, oxygen levels within the biofilm were not monitored. Since bioluminescence is an oxygen-dependent phenomenon, and there is no decline in photon counts for 125 h control biofilms, it could be surmised that mature biofilms grown by this continuous perfusion method were not severely oxygen-limited. It has been reported that bioluminescence is detectable at oxygen concentrations as low as 10 nM29 and it is tempting to speculate that direct imaging of intact biofilms by ICCD camera may be able to distinguish aerobic from strictly anaerobic areas within a bioluminescent P. aeruginosa biofilm that is otherwise nutritionally complete.
There are relatively few published studies that use ESEM as a means to visualize biofilm structure. This current work has demonstrated that it allows informative visualization of the three-dimensional structure of biofilms. Ciprofloxacin challenge clearly resulted in loss of biofilm integrity, with ESEM images showing what appeared to be a morass of cells and EPS post-drug exposure, as opposed to the more defined structures seen pre-exposure and in control samples. Nevertheless, the biofilm architecture appeared to be rebuilt as the biofilms recovered to quasi-steady-state. Further evidence of biofilm recovery was provided by direct visualization of intact biofilms by ICCD camera and the photon data acquired. This demonstrated that, following the second ciprofloxacin perfusion, the extent of photon emission differed little from intact biofilms in quasi-steady-state, although the distribution of highly bioluminescent cells altered with time. This phenomenon warrants further investigation, with more frequent observation of the intact biofilms using the ICCD.
Bioluminescence and viable counts measure different aspects of cell physiology and neither should be regarded as the definitive indicator of cell viability. Bioluminescence, however, can provide important data regarding cellular activity within the biofilm. The bioluminescence readings from resuspended biofilms and the photon camera images of intact biofilms both measure the metabolically active biomass within a biofilm in real time and suggest that challenge with ciprofloxacin has less effect on metabolism than on viable counts. This is particularly apparent after the second ciprofloxacin challenge and the data could have clinical significance in the elucidation of the persistence of biofilms after antibiotic treatment.
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Footnotes |
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Acknowledgements |
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References |
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2
Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother 2001; 45: 9991007.
3 Stewart PS. Mechanisms of antibiotic resistance in bacterial biofilms. Int J Med Microbiol 2002; 292: 10713.[ISI][Medline]
4 Allison D, Maira-Litrán T, Gilbert P. Perfused biofilm fermenters. Methods Enzymol 1999; 310: 23248.[ISI][Medline]
5 Dibdin G, Wimpenny J. Steady-state biofilm: practical and theoretical models. Methods Enzymol 1999; 310: 296322.[CrossRef][ISI][Medline]
6 Heydorn A, Ersboll BK, Hentzer M et al. Experimental reproducibility in flow-chamber biofilms. Microbiology 2000; 146: 240915.[ISI][Medline]
7 O'Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annu Rev Microbiol 2000; 54: 4979.[CrossRef][ISI][Medline]
8 Geesey GG. Bacterial behaviour at surfaces. Curr Opin Microbiol 2001; 4: 296300.[CrossRef][ISI][Medline]
9
Kadurugamuwa JL, Sin L, Albert E et al. Direct continuous method for monitoring biofilm infection in a mouse model. Infect Immun 2003; 71: 88290.
10
Kadurugamuwa JL, Sin LV, Yu J et al. Noninvasive optical imaging method to evaluate postantibiotic effects on biofilm infection in vivo. Antimicrob Agents Chemother 2004; 48: 22837.
11 Nelson SM, Marques CNH, Greenman J et al. Real-time monitoring of metabolic activity in biofilms. In: McBain A, Allison D, Brading, M et al., eds. Biofilm Communities: Order from Chaos? Cardiff: Bioline, 2003; 25768.
12 Parveen A, Smith G, Salisbury V et al. Biofilm culture of Pseudomonas aeruginosa expressing lux genes as a model to study susceptibility to antimicrobials. FEMS Microbiol Lett 2001; 199: 1158.[CrossRef][ISI][Medline]
13 Swanson B, Savel R, Szoka F. Development of a high throughput Pseudomonas aeruginosa epithelial cell adhesion assay. J Microbiol Methods 2003; 52: 3616.[CrossRef][ISI][Medline]
14
Alloush HM, Salisbury V, Lewis RJ et al. Pharmacodynamics of linezolid in a clinical isolate of Streptococcus pneumoniae genetically modified to express lux genes. J Antimicrob Chemother 2003; 52: 51113.
15 Marincs F, White DWR. Immobilisation of Escherichia coli expressing the lux genes of Xenorhabdus luminescens. Appl Environ Microbiol 1994; 60: 38623.[Abstract]
16 Kovach ME, Elzer PH, Hill DS et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 1995; 166: 1756.[CrossRef][ISI][Medline]
17 Hodgson AE, Nelson SM, Brown MRW et al. A simple in vitro model for growth control of bacterial biofilms. J Appl Bacteriol 1995; 79: 8793.[ISI][Medline]
18
Maira-Litrán T, Allison DG, Gilbert P. An evaluation of the potential of the multiple antibiotic resistance operon (mar) and the multidrug efflux pump acrAB to moderate resistance towards ciprofloxacin in Escherichia coli biofilms. J Antimicrob Chemother 2000; 45: 78995.
19
BSAC Working Party Report. Antimicrobial susceptibility testing. J Antimicrob Chemother 2001; 48 Suppl S1: 516.
20
Bowker KE, Wooton M, Rogers CA et al. Comparision of in-vitro pharmacodynamics of once and twice daily ciprofloxacin. J Antimicrob Chemother 1999; 44: 6617.
21
Salisbury V, Pfoestl A, Weisinger-Mayr H et al. Use of a clinical Escherichia coli isolate expressing lux genes to study the antimicrobial pharmacodynamics of moxifloxacin. J Antimicrob Chemother 1999; 43: 82932.
22 Smith JT. Awakening the slumbering potential of the 4-quinolone antibacterials. Pharmaceut J 1984; 233: 299305.
23
Brooun A, Liu S, Lewis K. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2000; 44: 6406.
24 Li X-Z, Nikaido H, Poole K. Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrob Agents Chemother 1995; 39: 194853.[Abstract]
25
De Kievit TR, Parkins MD, Gillis RJ et al. Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2001; 45: 176170.
26
Anderl JN, Franklin MJ, Stewart PS. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother 2000; 44: 181824.
27
Borriello G, Werner E, Roe F et al. Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrob Agents Chemother 2004; 48: 265964.
28
Walters MC III, Roe F, Bugnicourt A. Contributions of antibiotic penetration, oxygen limitation and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob Agents Chemother 2003; 47: 31723.
29 Bourgois J-J, Sluse FE, Baguet F et al. Kinetics of light emission and oxygen consumption by bioluminescent bacteria. J Bioenerg Biomemb 2001; 33: 35363.[CrossRef][ISI][Medline]
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