School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK
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Abstract |
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Introduction |
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The mar operon is a global regulator that controls the expression of various genes in Escherichia coli that constitute the mar regulon. Upregulation of mar in planktonic cultures leads to a multi-drug resistance phenotype, which includes resistance towards structurally unrelated antibiotics (penicillins, cephalosporins, rifampicin, nalidixic acid and fluoroquinolones), oxidative stress agents and organic solvents.16 mar can be induced by subinhibitory concentrations of compounds such as tetracycline, chloramphenicol, salicylate or paracetamol18,19 that are frequently used in therapeutics. The expression of the acrAB efflux pump is upregulated in mar mutants20 and this accounts for most of the multidrug resistance phenotype of mar mutant isolates. The acrAB system can be induced under various stress conditions20,21 and its upregulation also leads to multiple drug resistance.22 In this respect, biofilms can be seen as a community within which physico-chemical gradients develop, creating in this way microenvironments with stressful conditions for growth. Additionally, low levels of mar inducers can be found at various locations within the biofilms after biofilm exposure to those agents. The possibility of mar and acrAB involvement in biofilm resistance to antibiotics was therefore investigated.
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Material and methods |
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Ciprofloxacin was the kind gift of Bayer (Wuppertal, Germany). Luria-Bertani (LB) agar (Difco, Detroit, MI, USA) was used for maintenance of cultures and tryptone soya agar (Oxoid CM131, Unipath, Basingstoke, UK) for viable count determinations. The chemically defined medium (CDM) consisted of glycerol (5 mM), ammonium sulphate (6 mM), magnesium sulphate heptahydrate (0.5 mM), potassium chloride (13.4 mM), potassium dihydrogen orthophosphate (28 mM), dipotassium hydrogen orthophosphate 3-hydrate (72 mM), arginine (1.15 mM), thiamine (0.3 mM) and distilled water to 1 L. The pH was adjusted to 7.4. All reagents were of the purest available grade and were obtained from either Sigma (Poole, UK) or BDH (Poole, UK).
Microorganisms
The isolates of E. coli AG100, AG102, MCH164, AG100-A and AG100-B were the kind gift of Professor Stuart B. Levy, Tufts Medical School, Boston, MA, USA. Cultures were maintained on LB agar plates at 4°C, in the dark, after incubation at 37°C. Plates of E. coli AG102 and AG100A/B were supplemented with tetracycline (5 mg/L) and kanamycin (25 mg/L), respectively.
Biofilm culture
Biofilms were grown using the Sorbarod model (Figure 1) and published protocols.23 A diagrammatic representation of the Sorbarod experimental setup is given in Figure 1
. A Sorbarod (Ilacon Ltd, Tonbridge, UK) consists of a cylindrical paper sleeve encasing a compacted concertina of cellulose fibres. Single Sorbarods were inserted into lengths of clear polyvinylchloride (PVC; 40 mm length, 10 mm diameter) and sterilized by autoclaving. Each Sorbarod was inoculated with an exponential-phase culture of E. coli in CDM (approximately 1 x 109 cfu in 6 mL). The plunger was withdrawn from a sterile, disposable 2 mL syringe with a central outlet (Monoject, Sherwood Medical, Crawley, UK), leaving only the rubber seal within the syringe lumen. The syringe was introduced into the PVC tubing containing the Sorbarod and a sterile, disposable needle (0.8 mm x 40 mm) inserted through the rubber seal. Multiples of these filter units were assembled and placed in a 37°C incubator. Media inlet tubing was attached to the needles and sterile CDM was delivered to each unit at a controlled flow rate of 0.25 mL/min via a peristaltic pump (Watson-Marlow, Model 505 S, Falmouth, UK). To monitor the growth of the biofilms the perfusates were collected over the study period and viable counts performed. The number of cells within each Sorbarod (bioburden) was assessed following the removal of the filter from the PVC tubing. This was sliced open and placed in a tube (2.54 cm diameter) containing sterile saline (10 mL). The tube was vortexed for 1 min to disperse the fibres. A tight-fitting silicone plunger (2.54 cm diameter, 2 mm thickness) was forced into the tube to consolidate the fibre. The supernatant fluids were decanted, the plunger removed and the consolidated fibre resuspended and revortexed. This washing and resuspension procedure was repeated four times in total and the fluids were bulked. Preliminary experimentation had shown that such processing removed >99.999% of attached cells. The number of cells that had been adherent to the Sorbarod fibre was calculated by performing viable counts on the combined cell suspensions obtained after the four washes.
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Stock solutions of ciprofloxacin (1.6 g/L) were sterilized by filtration and serially diluted in sterile CDM to produce series of concentrations from 0.0016 mg/L to 0.16 mg/L. Aliquots (100 µL) of each were placed in the wells of a microtitre plate, which was inoculated by adding midlogarithmic phase culture (100 µL, 5 x 105 cfu) resuspended in fresh CDM. Plates were incubated at 37°C and growth was monitored spectrometrically for 40 h using an Anthos htIII microtitre plate reader (Anthos Labtec Instruments, Salzburg, Austria). From the growth inhibition curves obtained tangents were drawn to the growth curves at mid-log phase. The slopes of such curves were calculated and used to obtain the growth rates. Decreases in growth rate were expressed as a percentage of the control and then plotted against the concentration of ciprofloxacin. Growth rates were then extrapolated to determine the MIC as that which inhibits growth completely. MIC values of ciprofloxacin, together with those of tetracycline and ampicillin for comparison, are presented in the Table for the five test isolates. In all instances the MIC was reduced to a minor extent by deletion of mar, yet substantially increased through overexpression of acrAB or mar. mar-constitutive isolates were less susceptible than were acrAB constitutive ones, suggesting that mechanisms additional to acrAB induction are associated with mar-mediated resistance.
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Steady-state biofilms (48 h) were perfused for 42 h with CDM containing ciprofloxacin at 0.004, 0.015 or 0.1 mg/L. At this point the ciprofloxacin-containing medium was replaced with fresh CDM, and the eluted cell and biofilm populations were allowed to recover for a further 24 h. Samples of both the bioburden (total cells/Sorbarod) and perfusate (cells released/mL) were taken throughout the experiment and estimates of cfu made. Five replicate Sorbarods were set up for each experiment and incubated for 48 h. Three of these were treated with antibiotic and two were used as controls and perfused with antibiotic-free CDM. Samples of the perfusate were taken from all of the available Sorbarods at the time of sampling and the data averaged. Sampling for bioburden is destructive of the Sorbarod filter. Bioburden estimates therefore reflect the results from a single experiment. Estimates of the viable counts in the perfusates are averages of the available replicates. Accordingly, for t = 4872 h, n = 3 for the antibiotic-exposed biofilms and n = 2 for the controls, and for t = 9096 h, n = 2 for antibiotic-exposed biofilms and n = 1 for the controls. Preliminary experiments23 had shown that reproducibility between eluates and bioburdens for replicate Sorbarod experiments is better than ± 5%.
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Results |
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Discussion |
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From our results and those of earlier workers,27,28 it seems clear that biofilms of E. coli display a much decreased susceptibility towards ciprofloxacin over that of planktonic populations. Perfusion of pseudo steady-state biofilms with concentrations of ciprofloxacin of approximately 20 x MIC caused significant reductions (99%) in both bioburden and perfusate, but failed to completely eradicate the biofilm, which subsequently recovered rapidly. Resistance to therapeutic doses of ciprofloxacin did not appear to be related to the mar status of the population. Similar exposures of planktonic cells would have resulted in eradication of the exposed cells. Resistance is therefore likely to relate to some other aspects of the biofilm phenotype that outweigh the advantages of mar expression.
Although constitutive expression of mar protects biofilms towards the lowest concentration of ciprofloxacin used (0.004 mg/L), such protective effect was lost when the concentration of ciprofloxacin was further increased to 0.1 mg/L. mar mutants of E. coli are resistant to fluoroquinolones through a combined decrease in cell influx and an intrinsic efflux system. These results can be explained if we assume that the ciprofloxacin concentrations of 0.1 mg/L are already saturating the acrAB efflux pump, upregulation of which constitutes the major mechanism for making mar mutants resistant to agents such as antibiotics.22
Lack of differences in susceptibility to low concentrations of ciprofloxacin for the wild-type and mar-deleted isolates suggests that ciprofloxacin was not an inducer of mar, but that mar induction might give low-level resistance to the agent. Such failure of ciprofloxacin to induce mar within biofilms was not unexpected. mar mutants of E. coli can be selected with ciprofloxacin at much lower frequencies (1091011) than with other more accepted inducers such as tetracycline or chloramphenicol. Ciprofloxacin is therefore considered as a non-inducer of mar.29
Perfusion of AG100-A (acrAB-deleted) biofilms with ciprofloxacin at 0.004 mg/L caused reductions in viability of eluted cells associated within Sorbarods that were similar to those observed when biofilms of wild-type and mar-deleted isolates were challenged with this concentration of ciprofloxacin. From these data it can be concluded that the acrAB efflux pump is not upregulated per se within biofilms. The possibility exists that one or more efflux pumps other than acrAB could be implicated in the resistance of biofilms towards antimicrobial agents. In E. coli, 29 proven and putative drug pumps have been identified, nine of which have been shown to be capable of pumping drugs out of E. coli when expressed at high levels.30 Another likely possibility is that the global regulator HSL may contribute to aspects of the biofilm phenotype such as biofilm resistance and formation. In this respect, bacteria in biofilms accumulate chemical signals that can sense the local proximity and influence multicellular developmental patterns.15 Biofilm control and understanding of resistance development might therefore in the future hinge on the role of such signals and on our ability to interfere with such signalling events.
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Acknowledgments |
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Notes |
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References |
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Received 13 July 1999; returned 29 November 1999; revised 13 January 2000; accepted 28 January 2000