Journal of Antimicrobial Chemotherapy (1991) 27, 177-184
Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate D . J. Evans", D . G. Allison', M . R. W. Brown* and P. Gilbert**
Methods of cell culture which enable the control of specific growth rate and expression of iron-regulated membrane proteins within Gram-negative biofilms were employed for various clinical isolates of Pseudomonas aeruginosa taken from the sputum of cystic fibrosis patients and a laboratory strain of Escherichia coli. Susceptibility towards ciprofloxacin was assessed as a function of growth-rate for intact biofilms, cells resuspended from the biofilms and also for newly formed daughter cells shed from the biofilm during its growth and development. Patterns of susceptibility with growth rate were compared to those of suspended cultures grown in a chemostat. In all instances the susceptibility of chemostat cultures was directly related to growth rate. Whilst little difference was observed in the susceptibility pattern for P. aeruginosa strains with different observed levels of mucoidness, such populations were generally more susceptible towards ciprofloxacin than those of E. coli. At fast rates of growth P. aeruginosa cells resuspended from biofilms were significantly more resistant than chemostat grown cells. Intact P. aeruginosa biofilms were significantly more resistant than cells resuspended from them. This is in contrast to E. coli, where cells resuspended from biofilm and intact biofilms were, at the slower rates of growth, equivalent and significantly more susceptible than chemostat-grown cells. At high growth rates all methods of E. coli culture produced cells of equivalent susceptibility. For all strains, daughter cells dislodged from the biofilms demonstrated a high level of susceptibility towards ciprofloxacin which was unaffected by growth rate. This sensitivity corresponded to that of the fastest grown cells in the chemostat.
Introduction In patients with cystic fibrosis (CF), chronic lung infection by mucoid strains of Pseudomonas aeruginosa is commonly associated with progressive pulmonary infection and mortality (Hoiby, 1974). Under such conditions the organism is often found growing slowly, under iron limitation (Anwar et al., 1984), as a biofilm (Costerton et al., 1987). Such chronic infection is particularly recalcitrant to antibiotic treatment. Resistance has been variously attributed to, (i) failure to penetrate an exopolysaccharide matrix associated with the biofilm (Nichols et al., 1989), (ii) changes in outermembrane proteins (Aronoff, 1988), (iii) binding of cationic antibiotics (Peterson, Hancock & McGroaty, 1985) and/or intrinsic physiological properties of the biofilm. 'Corresponding author. 0305-7453/91/020177+08 $02.00/0
177 © 1991 The British Society for Antimicrobial Chemtherapy
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"Department of Pharmacy, University of Manchester, Oxford Road, Manchester; b Pharmaceutical Sciences Research Institute, Aston University, Birmingham, UK
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The present study utilises these techniques to investigate the susceptibility of P. aeruginosa biofilms towards ciprofloxacin. Use of clinical isolates taken from the sputum of infected patients with cystic fibrosis allow the results to be extrapolated to the clinical situation. Materials and methods Ciprofloxacin was the kind gift of Dr Zeiler (Bayer, Wuppertal, FDR). Nutrient Agar (Oxoid CM3) was employed for maintenance of cultures and viable count determinations. All other reagents were of the purest available grade and were obtained either from Sigma or from BDH. Micro-organisms used Escherichia coli ATCC 8739, together with two clinical isolates of P. aeruginosa taken from the sputum of infected cystic fibrosis patients were used throughout, P. aeruginosa PaWH is a mucoid isolate whereas P. aeruginosa PaTM is non-mucoid. These phenotypes were maintained on a number of culture media and retained after culture in the chemostat. Cultures were maintained on Nutrient Agar slants at room temperature, in a darkened cupboard, following incubation at 37°C. Continuous culture Continuous cultures were established, at 37°C, utilizing small volume, all-glass chemostats (Gilbert & Stuart, 1977). For E. coli cultures the carbon-depleting, simple salts
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Bacterial biofilms are particularly common with infections involving prosthetic devices and medical implants, and also those of the heart, lung and bladder (Costerton, Marrie & Cheng, 1985; Costerton et al., 1987). Sessile biofilm populations have many important properties which are distinct from their planktonic counterparts and which might contribute towards their survival within the infected host (Baltimore & Mitchell, 1980; Brown, Anwar & Lambert, 1984). Growth rate per se and nutrient deprivation, particularly that of iron (Griffiths, 1983; Anwar et al., 1984; Brown & Williams, 1985a, b; Shand et al., 1985) are important in bacterial pathogenesis, since the cells characteristically grow at rates much reduced from that experienced in typical laboratory media (Brown, Allison & Gilbert, 1988). Few direct studies of the antibiotic susceptibility of bacterial biofilms have been reported. These demonstrate tobramycin resistance of P. aeruginosa and Staphylococcus epidermidis and vancomycin resistance of S. epidermidis to be increased 20-100-fold for biofilms relative to 'equivalent' planktonic populations (Nickel et al., 1985; Gristina et al, 1987; Prosser et al., 1987). Whilst the importance of the biofilm mode of growth in-vitro is well documented, observations, such as these concerning antibiotic susceptibility, have generally been made in experimental systems where growth-rate is uncontrolled (Evans, & Holmes, 1987; Brown, et al., 1988;). Recent studies (Allison et al., 1990a, b; Evans et al., 1990a, b) have utilized a technique of biofilm growth (Gilbert et al., 1989) which allows growth rate control. Such studies have enabled the separation of those effects associated with the rate of growth of the cells and those associated with formation and maintenance of a biofilm. Such studies have also strongly implicated the involvement of cell division cycle in dispersal of cells from biofilms and in the apparent antibiotic recalcitrance of biofilms in-vivo.
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minimal media described by Gilbert & Brown (1978a) were employed. This media, whilst limiting growth through carbon-source availability also causes expression of iron regulated membrane proteins associated with iron-starvation (Wright & Gilbert, 1987). For the P. aeruginosa isolates an iron-depleting chemically defined medium (CDM-Fe) was employed as previously described (Shand et al., 1985). Setting up and equilibration of the chemostats has been described previously (Gilbert & Brown, 19786). Samples taken from the chemostat, at the various steady-states, were randomized with respect to growth-rate. Each study commenced and finished at the same slow rates of growth. Properties of these samples were identical in all cases indicating that the effects of growth rate reported are phenotypic rather than genotypic.
Biofilms of E. coli and P. aeruginosa were established on cellulose acetate membranes as described by Gilbert et al. (1989) and Allison et al. (1990a) for the two species respectively. These were perfused with the identical media to those used in the continuous culture studies, maintained at 37°C and left for 40 h, to achieve va'rious steady-state growth rates, Newly formed daughter cells were collected in the perfusate at each steady state.
Susceptibility testing Preliminary experiments, utilizing mid-log batch cultures of the test organisms, were conducted to establish those concentrations of ciprofloxacin which gave appropriate levels of killing (1-2 log cycles) within a 1 h, contact period at 37°C. Levels of survival in all cases decreased with increasing drug concentration. There was little or no sign of the paradoxical dose-response reported by Smith (1986) for P. aeruginosa or E. coli after this contact period. Marked paradoxical dose-responses were however observed for E. coli at 15-30 min but not 1 h exposures for ciprofloxacin concentrations > 50 mg/1. Appropriate concentrations were used subsequently to assess the susceptibility of resuspended biofilms (5 x 108 cells/ml in normal saline), perfusates from the biofilms containing newly divided cells at a density of c. 5 x 107 cells/ml, and samples removed directly from the chemostat (5 x 108 cells/ml). Procedures were devised which minimised any time delay between sampling and testing which might alter growth rate of the cells. Thus, resuspended biofilm cell suspension (01 ml), chemostat cultures (01 ml) and perfusates (1 ml) were diluted directly into sterile saline (9-9, 9-9 and 9 ml respectively at 37°C) to give final ciprofloxacin concentrations of 50, 5, and 0-5 mg/1. After incubation at 37°C for 1 h, with gentle agitation, samples (0-1 ml) were removed to solutions of sterile normal saline (9-9 ml). Further serial dilutions were made in sterile normal saline and viable counts made onto the surfaces of triplicate pre-dried nutrient agar plates. Additionally, samples of intact biofilm, of equivalent cell density, were exposed to antibiotic and viability determined, as before, following resuspension in saline after the 1 h contact period. All plates were subsequently incubated for 16 h at 37°C. Each exposure to antimicrobial agent was performed in triplicate. Results were expressed as percentage reductions in viability relative to appropriate unexposed controls. Control experiments in the absence of ciprofloxacin showed there to be little multiplication of the cells during the exposure period for any of the inocula.
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Figure 1. Influence or specific growth rate upon the susceptibility of (a) P. aeruginosa PaWH (Mucoid) and (b) P. aeruginosa PaTM (non-mucoid) towards exposure to ciprofloxacin (05 mg/1) for 1 h at 37°C. Suspensions of cells were either grown in suspended, chemostat culture ( • ) , or as biofilms. Intact biofilms were exposed to ciprofloxacin ( • ) as were cells, resuspended from the biofilms (O) or eluted as newly divided daughter cells from them ( • ) .
Results Results for P. aeruginosa, illustrate the influence of specific growth rate upon the sensitivity towards ciprofloxacin (0-5 mg/1) of the non-mucoid (PaTM) and the mucoid isolates (PaWH) (Figure 1). The susceptibility of chemostat-grown (planktonic) cells increased in proportion with growth rate and was not significantly different for the two phenotypes. Susceptibility of the non-mucoid strain (Figure l(b)) showed a similar, but reduced, growth-rate dependency for the resuspended and intact biofilm populations as that for planktonic cultures. At slow rates of growth biofilm cultures appeared to be slightly more sensitive than those grown in the chemostat. At faster rates of growth (ji > 015/h) biofilm derived cells were significantly more resistant than their planktonic counterparts. In this instance intact biofilms were much less susceptible than cell suspensions prepared from them. For the P. aeruginosa mucoid strain (Figure l(a)), whilst resuspended biofilms demonstrated similar orders of susceptibility and growth rate dependency as those of the non-mucoid strain, intact biofilm populations were virtually unaffected by ciprofloxacin treatment. This was apparent even at the fastest rates of growth. Higher doses of ciprofloxacin (5 mg/1) were therefore employed against this strain (Figure 2). A similar pattern of growth-rate dependence emerged but intact biofilms remained less sensitive even at these dose levels. In both instances (Figures 1 and 2) newly-divided cells eluted from the biofilms, at the various steady-state growthrates were especially susceptible to the agent. Such sensitivity, unlike that for the heterogenous populations, did not alter significantly with specific-growth rate. Results for E. coli (Figure 3) were similar in some respects to those for P. aeruginosa, in that chemostat populations showed significant increases in sensitivity with increases in growth rate. Cells resuspended from the biofilms, however, did not. The most slowly growing cells resuspended from biofilms appeared to be the more sensitive to the agent. Only at growth rates greater than 0-5/h did the sensitivities of the biofilm and planktonic cultures correspond (Figure 3). Heightened, uniform susceptibility of the
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0.-2 Specific growth rate (div/h)
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Figure 2. Influence of specific growth rate upon the susceptibility of P. aeruginosa PaWH (mucoid) towards exposure to ciprofloxacin (5 mg/1) for 1 h at 37°C. Suspensions of cells were either grown in suspended, chemostat culture ( # ) , or as biofilms. Intact biofilms were exposed to ciprofloxacin ( • ) as were cells, resuspended from the biofilms (O) or eluted as newly divided daughter cells from them (D).
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Figure 3. Influence of specific growth rate upon the susceptibility of E. coli ATCC 8739 (rough-LPS) towards exposure to ciprofloxacin (50 mg/1) for 1 h at 37°C. Suspensions of cells were either grown in suspended, chemostat culture ( • ) , or as biofilms. Intact biofilms were exposed to ciprofloxacin ( • ) as were cells, resuspended from the biofilms (O) or eluted as newly divided daughter cells from them (D)-
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daughter cell population was observed throughout, as for P. aeruginosa. Unlike the results for P. aeruginosa the sensitivity of intact E. coli biofilms to ciprofloxacin was not significantly altered from that of resuspended biofilms (Figure 3). Discussion
In P. aeruginosa infections of patients with cystic fibrosis emergence of mucoid phenotypes is usually accompanied by reduced response to antibiotic therapy (Hoiby, 1974). Ciprofloxacin has been effective in the therapy of chest infections due to Haemophilus influenzae and S. aureus, but like other agents, does not eradicate P. aeruginosa from the lungs of patients with cystic fibrosis (Davies et al., 1986; Fass, 1987; Kobayashi, 1987). At slow rates of growth the susceptibilities of E. coli cells derived from the biofilms were significantly greater than for their planktonic counterparts. This indicated major differences in physiology for suspended and adherent populations of E. coli which were not as apparent for P. aeruginosa. The concentrations of ciprofloxacin required to exert these effects against E. coli were 10-100-times higher than those required for P. aeruginosa and significantly higher than those considered normal for enteric bacteria. Such concentrations are unlikely to be achieved in vivo, except perhaps in the urine. Such concentrations were required in the present study in that lower concentrations did not differentiate between the susceptibilities at various growth rates in the chemostat. Smith (1986) reported that at concentrations of ciprofloxacin greater than 10 x MIC the activity of ciprofloxacin reduces. The extent of the paradoxical doseresponse observed in our preliminary experiments with this organism was minimal and insufficient to account for the observed variations in susceptibility. In all instances the susceptibility of newly-formed daughter cells eluted from the biofilms was higher than for any other populations type and was unaffected by the growth rate of the parent cultures. This suggests some dependence of the action of ciprofloxacin upon the cellular division cycle. This is perhaps not surprising since DNA replication and transcription are highly regulated events with respect to cell division. Recently, Evans et al. (1990a) showed that the susceptibility of E. coli towards tobramycin varied throughout the division cycle. Enhanced sensitivity was observed for newly formed daughter cells with periods of resistance immediately preceeding cell
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Ciprofloxacin susceptibility of intact biofilms was, for P. aeruginosa, significantly less than that for cells resuspended from them (Figure 1). This suggested that organization of the cells within an exopolysaccharide containing glycocalyx confers some level of resistance. This was particularly evident for the mucoid isolate where higher levels of ciprofloxacin (5 mg/1) were relatively ineffective towards intact biofilms (Figure 2). Since MICs of > 2 mg/1 ciprofloxacin are associated with resistance (Scully, 1989), the results suggest that possession of a mucoid phenotype might increase resistance particularly when the cells are organized into an adherent biofilm. No apparent differences were observed for the sensitivities of intact and resuspended biofilm populations of E. coli (Figure 3). In this instance the presence of the glycocalyx would seem to be of much reduced significance. The E. coli strain employed was of a roughLPS phenotype. Possibly as the extent of LPS polysaccharide associated with cells is increased or as they adopt a mucoid phenotype then physical association and organization of the cells into a biofilm becomes the overriding factor associated with antibiotic resistance.
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division. It has also been suggested that the division cycle plays an important role in the dispersal of planktonic cells from adherent biofilms. Newly formed daughter cells are relatively hydrophilic and often shed from such surfaces (Allison et al., 1990a, b). Shedding of newly-formed daughter cells from a biofilm might therefore by a primary means of dispersal. Since such cells are especially sensitive to ciprofloxacin then its proven effectiveness in the management of CF-lung infections (Raeburn et al., 1987; Scully et al., 1987; Scully, 1989) might be associated with reductions in the spread of infection within the lung and enhanced bactericidal activity as cells undergo division. Acknowledgements
References Allison, D. G., Brown, M. R. W , Evans, D. J. & Gilbert, P. (1990a). Surface hydrophobicity and dispersal of Pseudomonas aeniginosa from biofilms. FEMS Microbiology Letters 71, 101-4. Allison, D. G., Evans, D. J., Brown, M. R. W. & Gilbert, P. (19906). Possible involvement of the division cycle in dispersal of Escherichia coli from biofilms. Journal of Bacteriology 172, 1667-9. Anwar, H., Brown, M. R. W., Day, A. & Weller, P. H. (1984). Outer membrane antigens of mucoid Pseudomonas aeniginosa isolated directly from the sputum of a cystic fibrosis patient. FEMS Microbiology Letters 24, 235-9. AronofF, S. C. (1988). Outer membrane permeability in Pseudomonas cepacia: diminished porin content in a /Mactam-resistant mutant and in resistant cystic fibrosis isolates. Antimicrobial Agents and Chemotherapy 32, 1636-9. Baltimore, R. S. & Mitchell, M. (1980). Immunologic investigations of mucoid strains of Pseudomonas aeniginosa: comparison of susceptibility to opsonic antibody in mucoid and non-mucoid strains. Journal of Infectious Diseases 141, 238-47. Brown, M. R. W., Allison, D. G. & Gilbert, P. (1988). Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? Journal of Antimicrobial Chemotherapy 22, 777-80. Brown, M. R. W., Anwar, H. & Lambert, P. A. (1984). Evidence that mucoid Pseudomonas aeniginosa in the cystic fibrosis lung grows under iron-restricted conditions. FEMS Microbiology Letters 21, 113-7. Brown, M. R. W. & Williams, P. (1985a). Influence of substrate limitation and growth phase on sensitivity to antimicrobial agents. Journal of Antimicrobial Chemotherapy 15, Suppl. A, 7-14. Brown, M. R. W. & Williams, P. (19856). The influence of environment on envelope properties affecting survival of bacteria in infections. Annual Review of Microbiology 39, 527-56. Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, T. I. Nickel, J. C , Dasgupta, M. et al. (1987). Bacterial biofilms in nature and disease. Annual Review of Microbiology 41, 435-64. Costerton, J. W., Marrie, T. J. & Cheng, K.-J. (1985). Phenomena of bacterial adhesion. In Bacterial Adhesion Mechanisms and Physiological Significance (Savage, D. C. & Fletcher, M., Eds), pp. 3-43. Plenum Press, New York. Davies, B. I., Maesen, F. P. V., Teengs, J. P. & Baur, C. (1986). The quinolones in chronic bronchitis. Pharmaceulisch Weekblad 8, 53-9. Evans, R. C. & Holmes, C. J. (1987). Effect of vancomycin hydrochloride on Staphylococcus epidermidis biofilm associated with silicone elastomer. Antimicrobial Agents and Chemotherapy 31, 889-94. Evans, D. J., Allison, D. G., Brown, M. R. W. & Gilbert, P. (1990A). Growth rate and the resistance of Gram-negative biofilms towards Cetrimide USP. Journal of Antimicrobial Chemotherapy 26, 473-8.
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This work was supported by a project grant from the Medical Research Council, UK to PG and MRWB and partly from support from the Cystic Fibrosis Research Trust (UK) and Bayer (FDR). DJE acknowledges receipt of a studentship from the Royal Pharmaceutical Society of Great Britain.
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(Received 4 July 1990; revised version accepted 11 October 1990)
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Evans, D. J., Brown, M. R. W., Allison, D. G. & Gilbert, P. (1990a). Susceptibility of bacterial biofilms to tobramycin: role of specific growth rate and phase in the division cycle. Journal of Antimicrobial Chemotherapy 25, 585-91. Fass, R. J. (1987). Efficacy and safety of oral ciprofloxacin in the treatment of serious respiratory infections. American Journal of Medicine 82, Suppl. 4A, 202-7. Gilbert, P., Allison, D. G., Evans, D. J., Handley, P. S. & Brown, M. R. W. (1989). Growth rate control of adherent bacterial populations. Applied and Environmental Microbiology 55, 1308-11. Gilbert, P. & Brown, M. R. W. (1978a). Influence of growth rate and nutrient limitation on the gross cellular composition of Pseudomonas aeruginosa and its resistance to 3- and 4-chlorophenol. Journal of Bacteriology 133, 1066-72. Gilbert, P. & Brown, M. R. W. (1978*). Effect of R-plasmid RP1 and nutrient depletion on the. gross cellular composition of Escherichia coli and its resistance to some uncoupling phenols. Journal of Bacteriology 133, 1062-5. Gilbert, P., Brown, M. R. W. & Costerton, J. W. (1987). Inocula for antimicrobial sensitivity testing: a critical review. Journal of Antimicrobial Chemotherapy 20, 147-54. Gilbert, P. & Stuart, A. (1977). Small-scale chemostat for the growth of mesophilic and thermophilic microorganisms. Laboratory Practice 26, 627—8. Griffiths, E. (1983). Availability of iron and survival of bacteria in infection. In Medical Microbiology, Volume 3. Role of the Envelope in the Survival of Bacteria in Infection (Easman, C. S. F., Jeljaszewicz, J., Brown, M. R. W. & Lambert, P. A., Eds), pp. 153-77. Academic Press, London. Gristina, A. G., Hobgood, C. D., Webb, L. X. & Myrvik, Q. N. (1987). Adhesive colonization of biomaterials and antibiotic resistance. Biomaterials 8, 423-6. Hoiby, N. (1974). Pseudomonas aeruginosa infection in cystic fibrosis. Relationship between mucoid strains of Pseudomonas aeruginosa and the humoral immune response. Ada Pathologica et Microbiologica Scandinavica, Section B, 82, 551-8. Kobayashi, H. (1987). Clinical efficacy of ciprofloxacin in the treatment of patients with respiratory tract infections in Japan. American Journal of Medicine 82, Suppl. 4A, 169-73. Nichols, W. W., Evans, M. J., Slack, M. P. E. & Walmsley, H. L. (1989). The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa. Journal of General Microbiology 135, 1291-303. Nickel, J. C , Ruseska, I., Wright, J. B. & Costerton, J. W. (1985). Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary tract catheter material. Antimicrobial Agents and Chemotherapy 27, 619-24. Peterson, A. A., Hancock, R. E. W. & McGroaty, E. J. (1985). Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa. Journal of Bacteriology 164, 1256-61. Prosser, B. T., Taylor, D., Dix, B. A. & Cleeland, R. (1987). Method of evaluating effects of antibiotics on bacterial biofilm. Antimicrobial Agents and Chemotherapy 31, 1502-6. Raeburn, J. A., Govan, J. R. W., McCrae, W. M., Greening, A. P., Collier, P. S., Hodson, M. E. et al. (1987). Ciprofloxacin therapy in cysticfibrosis.Journal of Antimicrobial Chemotherapy 20, 295-6. Scully, B. E. (1989). Therapy of respiratory tract infections with quinolone antimicrobial agents. In Quinolone Antimicrobial Agents (Wolfson, J. S. & Hooper, D. C, Eds), pp. 143-65. American Society for Microbiology, Washington, DC. Scully, B. E., Nakatomi, M., Ores, C, Davidson, S. & Neu, H. C. (1987). Ciprofloxacin therapy in cystic fibrosis. American Journal of Medicine 82, Suppl. 4A, 196-201. Shand, G. H., Anwar, H., Kadurugamuwa, J., Brown, M. R. W., Silverman, S. H. & Melling, J. (1985). In vivo evidence that bacteria in urinary tract infection grow under iron-restricted conditions. Infection and Immunity 48, 35-9. Smith, J. T. (1986). The mode of action of 4-quinolones and possible mechanisms of resistance. Journal of Antimicrobial Chemotherapy 18, Suppl. D, 21-9. Wright, N. E. & Gilbert, P. (1987). Influence of specific growth rate and nutrient-limitation upon the sensitivity of Escherichia coli towards polymyxin B. Journal of Antimicrobial Chemotherapy 20, 303-12.
Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate D . J. Evans", D . G. Allison', M . R. W. Brown* and P. Gilbert**
Methods of cell culture which enable the control of specific growth rate and expression of iron-regulated membrane proteins within Gram-negative biofilms were employed for various clinical isolates of Pseudomonas aeruginosa taken from the sputum of cystic fibrosis patients and a laboratory strain of Escherichia coli. Susceptibility towards ciprofloxacin was assessed as a function of growth-rate for intact biofilms, cells resuspended from the biofilms and also for newly formed daughter cells shed from the biofilm during its growth and development. Patterns of susceptibility with growth rate were compared to those of suspended cultures grown in a chemostat. In all instances the susceptibility of chemostat cultures was directly related to growth rate. Whilst little difference was observed in the susceptibility pattern for P. aeruginosa strains with different observed levels of mucoidness, such populations were generally more susceptible towards ciprofloxacin than those of E. coli. At fast rates of growth P. aeruginosa cells resuspended from biofilms were significantly more resistant than chemostat grown cells. Intact P. aeruginosa biofilms were significantly more resistant than cells resuspended from them. This is in contrast to E. coli, where cells resuspended from biofilm and intact biofilms were, at the slower rates of growth, equivalent and significantly more susceptible than chemostat-grown cells. At high growth rates all methods of E. coli culture produced cells of equivalent susceptibility. For all strains, daughter cells dislodged from the biofilms demonstrated a high level of susceptibility towards ciprofloxacin which was unaffected by growth rate. This sensitivity corresponded to that of the fastest grown cells in the chemostat.
Introduction In patients with cystic fibrosis (CF), chronic lung infection by mucoid strains of Pseudomonas aeruginosa is commonly associated with progressive pulmonary infection and mortality (Hoiby, 1974). Under such conditions the organism is often found growing slowly, under iron limitation (Anwar et al., 1984), as a biofilm (Costerton et al., 1987). Such chronic infection is particularly recalcitrant to antibiotic treatment. Resistance has been variously attributed to, (i) failure to penetrate an exopolysaccharide matrix associated with the biofilm (Nichols et al., 1989), (ii) changes in outermembrane proteins (Aronoff, 1988), (iii) binding of cationic antibiotics (Peterson, Hancock & McGroaty, 1985) and/or intrinsic physiological properties of the biofilm. 'Corresponding author. 0305-7453/91/020177+08 $02.00/0
177 © 1991 The British Society for Antimicrobial Chemtherapy
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"Department of Pharmacy, University of Manchester, Oxford Road, Manchester; b Pharmaceutical Sciences Research Institute, Aston University, Birmingham, UK
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The present study utilises these techniques to investigate the susceptibility of P. aeruginosa biofilms towards ciprofloxacin. Use of clinical isolates taken from the sputum of infected patients with cystic fibrosis allow the results to be extrapolated to the clinical situation. Materials and methods Ciprofloxacin was the kind gift of Dr Zeiler (Bayer, Wuppertal, FDR). Nutrient Agar (Oxoid CM3) was employed for maintenance of cultures and viable count determinations. All other reagents were of the purest available grade and were obtained either from Sigma or from BDH. Micro-organisms used Escherichia coli ATCC 8739, together with two clinical isolates of P. aeruginosa taken from the sputum of infected cystic fibrosis patients were used throughout, P. aeruginosa PaWH is a mucoid isolate whereas P. aeruginosa PaTM is non-mucoid. These phenotypes were maintained on a number of culture media and retained after culture in the chemostat. Cultures were maintained on Nutrient Agar slants at room temperature, in a darkened cupboard, following incubation at 37°C. Continuous culture Continuous cultures were established, at 37°C, utilizing small volume, all-glass chemostats (Gilbert & Stuart, 1977). For E. coli cultures the carbon-depleting, simple salts
Downloaded from http://jac.oxfordjournals.org/ at Aston University on September 4, 2014
Bacterial biofilms are particularly common with infections involving prosthetic devices and medical implants, and also those of the heart, lung and bladder (Costerton, Marrie & Cheng, 1985; Costerton et al., 1987). Sessile biofilm populations have many important properties which are distinct from their planktonic counterparts and which might contribute towards their survival within the infected host (Baltimore & Mitchell, 1980; Brown, Anwar & Lambert, 1984). Growth rate per se and nutrient deprivation, particularly that of iron (Griffiths, 1983; Anwar et al., 1984; Brown & Williams, 1985a, b; Shand et al., 1985) are important in bacterial pathogenesis, since the cells characteristically grow at rates much reduced from that experienced in typical laboratory media (Brown, Allison & Gilbert, 1988). Few direct studies of the antibiotic susceptibility of bacterial biofilms have been reported. These demonstrate tobramycin resistance of P. aeruginosa and Staphylococcus epidermidis and vancomycin resistance of S. epidermidis to be increased 20-100-fold for biofilms relative to 'equivalent' planktonic populations (Nickel et al., 1985; Gristina et al, 1987; Prosser et al., 1987). Whilst the importance of the biofilm mode of growth in-vitro is well documented, observations, such as these concerning antibiotic susceptibility, have generally been made in experimental systems where growth-rate is uncontrolled (Evans, & Holmes, 1987; Brown, et al., 1988;). Recent studies (Allison et al., 1990a, b; Evans et al., 1990a, b) have utilized a technique of biofilm growth (Gilbert et al., 1989) which allows growth rate control. Such studies have enabled the separation of those effects associated with the rate of growth of the cells and those associated with formation and maintenance of a biofilm. Such studies have also strongly implicated the involvement of cell division cycle in dispersal of cells from biofilms and in the apparent antibiotic recalcitrance of biofilms in-vivo.
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minimal media described by Gilbert & Brown (1978a) were employed. This media, whilst limiting growth through carbon-source availability also causes expression of iron regulated membrane proteins associated with iron-starvation (Wright & Gilbert, 1987). For the P. aeruginosa isolates an iron-depleting chemically defined medium (CDM-Fe) was employed as previously described (Shand et al., 1985). Setting up and equilibration of the chemostats has been described previously (Gilbert & Brown, 19786). Samples taken from the chemostat, at the various steady-states, were randomized with respect to growth-rate. Each study commenced and finished at the same slow rates of growth. Properties of these samples were identical in all cases indicating that the effects of growth rate reported are phenotypic rather than genotypic.
Biofilms of E. coli and P. aeruginosa were established on cellulose acetate membranes as described by Gilbert et al. (1989) and Allison et al. (1990a) for the two species respectively. These were perfused with the identical media to those used in the continuous culture studies, maintained at 37°C and left for 40 h, to achieve va'rious steady-state growth rates, Newly formed daughter cells were collected in the perfusate at each steady state.
Susceptibility testing Preliminary experiments, utilizing mid-log batch cultures of the test organisms, were conducted to establish those concentrations of ciprofloxacin which gave appropriate levels of killing (1-2 log cycles) within a 1 h, contact period at 37°C. Levels of survival in all cases decreased with increasing drug concentration. There was little or no sign of the paradoxical dose-response reported by Smith (1986) for P. aeruginosa or E. coli after this contact period. Marked paradoxical dose-responses were however observed for E. coli at 15-30 min but not 1 h exposures for ciprofloxacin concentrations > 50 mg/1. Appropriate concentrations were used subsequently to assess the susceptibility of resuspended biofilms (5 x 108 cells/ml in normal saline), perfusates from the biofilms containing newly divided cells at a density of c. 5 x 107 cells/ml, and samples removed directly from the chemostat (5 x 108 cells/ml). Procedures were devised which minimised any time delay between sampling and testing which might alter growth rate of the cells. Thus, resuspended biofilm cell suspension (01 ml), chemostat cultures (01 ml) and perfusates (1 ml) were diluted directly into sterile saline (9-9, 9-9 and 9 ml respectively at 37°C) to give final ciprofloxacin concentrations of 50, 5, and 0-5 mg/1. After incubation at 37°C for 1 h, with gentle agitation, samples (0-1 ml) were removed to solutions of sterile normal saline (9-9 ml). Further serial dilutions were made in sterile normal saline and viable counts made onto the surfaces of triplicate pre-dried nutrient agar plates. Additionally, samples of intact biofilm, of equivalent cell density, were exposed to antibiotic and viability determined, as before, following resuspension in saline after the 1 h contact period. All plates were subsequently incubated for 16 h at 37°C. Each exposure to antimicrobial agent was performed in triplicate. Results were expressed as percentage reductions in viability relative to appropriate unexposed controls. Control experiments in the absence of ciprofloxacin showed there to be little multiplication of the cells during the exposure period for any of the inocula.
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Figure 1. Influence or specific growth rate upon the susceptibility of (a) P. aeruginosa PaWH (Mucoid) and (b) P. aeruginosa PaTM (non-mucoid) towards exposure to ciprofloxacin (05 mg/1) for 1 h at 37°C. Suspensions of cells were either grown in suspended, chemostat culture ( • ) , or as biofilms. Intact biofilms were exposed to ciprofloxacin ( • ) as were cells, resuspended from the biofilms (O) or eluted as newly divided daughter cells from them ( • ) .
Results Results for P. aeruginosa, illustrate the influence of specific growth rate upon the sensitivity towards ciprofloxacin (0-5 mg/1) of the non-mucoid (PaTM) and the mucoid isolates (PaWH) (Figure 1). The susceptibility of chemostat-grown (planktonic) cells increased in proportion with growth rate and was not significantly different for the two phenotypes. Susceptibility of the non-mucoid strain (Figure l(b)) showed a similar, but reduced, growth-rate dependency for the resuspended and intact biofilm populations as that for planktonic cultures. At slow rates of growth biofilm cultures appeared to be slightly more sensitive than those grown in the chemostat. At faster rates of growth (ji > 015/h) biofilm derived cells were significantly more resistant than their planktonic counterparts. In this instance intact biofilms were much less susceptible than cell suspensions prepared from them. For the P. aeruginosa mucoid strain (Figure l(a)), whilst resuspended biofilms demonstrated similar orders of susceptibility and growth rate dependency as those of the non-mucoid strain, intact biofilm populations were virtually unaffected by ciprofloxacin treatment. This was apparent even at the fastest rates of growth. Higher doses of ciprofloxacin (5 mg/1) were therefore employed against this strain (Figure 2). A similar pattern of growth-rate dependence emerged but intact biofilms remained less sensitive even at these dose levels. In both instances (Figures 1 and 2) newly-divided cells eluted from the biofilms, at the various steady-state growthrates were especially susceptible to the agent. Such sensitivity, unlike that for the heterogenous populations, did not alter significantly with specific-growth rate. Results for E. coli (Figure 3) were similar in some respects to those for P. aeruginosa, in that chemostat populations showed significant increases in sensitivity with increases in growth rate. Cells resuspended from the biofilms, however, did not. The most slowly growing cells resuspended from biofilms appeared to be the more sensitive to the agent. Only at growth rates greater than 0-5/h did the sensitivities of the biofilm and planktonic cultures correspond (Figure 3). Heightened, uniform susceptibility of the
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Figure 2. Influence of specific growth rate upon the susceptibility of P. aeruginosa PaWH (mucoid) towards exposure to ciprofloxacin (5 mg/1) for 1 h at 37°C. Suspensions of cells were either grown in suspended, chemostat culture ( # ) , or as biofilms. Intact biofilms were exposed to ciprofloxacin ( • ) as were cells, resuspended from the biofilms (O) or eluted as newly divided daughter cells from them (D).
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Figure 3. Influence of specific growth rate upon the susceptibility of E. coli ATCC 8739 (rough-LPS) towards exposure to ciprofloxacin (50 mg/1) for 1 h at 37°C. Suspensions of cells were either grown in suspended, chemostat culture ( • ) , or as biofilms. Intact biofilms were exposed to ciprofloxacin ( • ) as were cells, resuspended from the biofilms (O) or eluted as newly divided daughter cells from them (D)-
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daughter cell population was observed throughout, as for P. aeruginosa. Unlike the results for P. aeruginosa the sensitivity of intact E. coli biofilms to ciprofloxacin was not significantly altered from that of resuspended biofilms (Figure 3). Discussion
In P. aeruginosa infections of patients with cystic fibrosis emergence of mucoid phenotypes is usually accompanied by reduced response to antibiotic therapy (Hoiby, 1974). Ciprofloxacin has been effective in the therapy of chest infections due to Haemophilus influenzae and S. aureus, but like other agents, does not eradicate P. aeruginosa from the lungs of patients with cystic fibrosis (Davies et al., 1986; Fass, 1987; Kobayashi, 1987). At slow rates of growth the susceptibilities of E. coli cells derived from the biofilms were significantly greater than for their planktonic counterparts. This indicated major differences in physiology for suspended and adherent populations of E. coli which were not as apparent for P. aeruginosa. The concentrations of ciprofloxacin required to exert these effects against E. coli were 10-100-times higher than those required for P. aeruginosa and significantly higher than those considered normal for enteric bacteria. Such concentrations are unlikely to be achieved in vivo, except perhaps in the urine. Such concentrations were required in the present study in that lower concentrations did not differentiate between the susceptibilities at various growth rates in the chemostat. Smith (1986) reported that at concentrations of ciprofloxacin greater than 10 x MIC the activity of ciprofloxacin reduces. The extent of the paradoxical doseresponse observed in our preliminary experiments with this organism was minimal and insufficient to account for the observed variations in susceptibility. In all instances the susceptibility of newly-formed daughter cells eluted from the biofilms was higher than for any other populations type and was unaffected by the growth rate of the parent cultures. This suggests some dependence of the action of ciprofloxacin upon the cellular division cycle. This is perhaps not surprising since DNA replication and transcription are highly regulated events with respect to cell division. Recently, Evans et al. (1990a) showed that the susceptibility of E. coli towards tobramycin varied throughout the division cycle. Enhanced sensitivity was observed for newly formed daughter cells with periods of resistance immediately preceeding cell
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Ciprofloxacin susceptibility of intact biofilms was, for P. aeruginosa, significantly less than that for cells resuspended from them (Figure 1). This suggested that organization of the cells within an exopolysaccharide containing glycocalyx confers some level of resistance. This was particularly evident for the mucoid isolate where higher levels of ciprofloxacin (5 mg/1) were relatively ineffective towards intact biofilms (Figure 2). Since MICs of > 2 mg/1 ciprofloxacin are associated with resistance (Scully, 1989), the results suggest that possession of a mucoid phenotype might increase resistance particularly when the cells are organized into an adherent biofilm. No apparent differences were observed for the sensitivities of intact and resuspended biofilm populations of E. coli (Figure 3). In this instance the presence of the glycocalyx would seem to be of much reduced significance. The E. coli strain employed was of a roughLPS phenotype. Possibly as the extent of LPS polysaccharide associated with cells is increased or as they adopt a mucoid phenotype then physical association and organization of the cells into a biofilm becomes the overriding factor associated with antibiotic resistance.
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division. It has also been suggested that the division cycle plays an important role in the dispersal of planktonic cells from adherent biofilms. Newly formed daughter cells are relatively hydrophilic and often shed from such surfaces (Allison et al., 1990a, b). Shedding of newly-formed daughter cells from a biofilm might therefore by a primary means of dispersal. Since such cells are especially sensitive to ciprofloxacin then its proven effectiveness in the management of CF-lung infections (Raeburn et al., 1987; Scully et al., 1987; Scully, 1989) might be associated with reductions in the spread of infection within the lung and enhanced bactericidal activity as cells undergo division. Acknowledgements
References Allison, D. G., Brown, M. R. W , Evans, D. J. & Gilbert, P. (1990a). Surface hydrophobicity and dispersal of Pseudomonas aeniginosa from biofilms. FEMS Microbiology Letters 71, 101-4. Allison, D. G., Evans, D. J., Brown, M. R. W. & Gilbert, P. (19906). Possible involvement of the division cycle in dispersal of Escherichia coli from biofilms. Journal of Bacteriology 172, 1667-9. Anwar, H., Brown, M. R. W., Day, A. & Weller, P. H. (1984). Outer membrane antigens of mucoid Pseudomonas aeniginosa isolated directly from the sputum of a cystic fibrosis patient. FEMS Microbiology Letters 24, 235-9. AronofF, S. C. (1988). Outer membrane permeability in Pseudomonas cepacia: diminished porin content in a /Mactam-resistant mutant and in resistant cystic fibrosis isolates. Antimicrobial Agents and Chemotherapy 32, 1636-9. Baltimore, R. S. & Mitchell, M. (1980). Immunologic investigations of mucoid strains of Pseudomonas aeniginosa: comparison of susceptibility to opsonic antibody in mucoid and non-mucoid strains. Journal of Infectious Diseases 141, 238-47. Brown, M. R. W., Allison, D. G. & Gilbert, P. (1988). Resistance of bacterial biofilms to antibiotics: a growth-rate related effect? Journal of Antimicrobial Chemotherapy 22, 777-80. Brown, M. R. W., Anwar, H. & Lambert, P. A. (1984). Evidence that mucoid Pseudomonas aeniginosa in the cystic fibrosis lung grows under iron-restricted conditions. FEMS Microbiology Letters 21, 113-7. Brown, M. R. W. & Williams, P. (1985a). Influence of substrate limitation and growth phase on sensitivity to antimicrobial agents. Journal of Antimicrobial Chemotherapy 15, Suppl. A, 7-14. Brown, M. R. W. & Williams, P. (19856). The influence of environment on envelope properties affecting survival of bacteria in infections. Annual Review of Microbiology 39, 527-56. Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, T. I. Nickel, J. C , Dasgupta, M. et al. (1987). Bacterial biofilms in nature and disease. Annual Review of Microbiology 41, 435-64. Costerton, J. W., Marrie, T. J. & Cheng, K.-J. (1985). Phenomena of bacterial adhesion. In Bacterial Adhesion Mechanisms and Physiological Significance (Savage, D. C. & Fletcher, M., Eds), pp. 3-43. Plenum Press, New York. Davies, B. I., Maesen, F. P. V., Teengs, J. P. & Baur, C. (1986). The quinolones in chronic bronchitis. Pharmaceulisch Weekblad 8, 53-9. Evans, R. C. & Holmes, C. J. (1987). Effect of vancomycin hydrochloride on Staphylococcus epidermidis biofilm associated with silicone elastomer. Antimicrobial Agents and Chemotherapy 31, 889-94. Evans, D. J., Allison, D. G., Brown, M. R. W. & Gilbert, P. (1990A). Growth rate and the resistance of Gram-negative biofilms towards Cetrimide USP. Journal of Antimicrobial Chemotherapy 26, 473-8.
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This work was supported by a project grant from the Medical Research Council, UK to PG and MRWB and partly from support from the Cystic Fibrosis Research Trust (UK) and Bayer (FDR). DJE acknowledges receipt of a studentship from the Royal Pharmaceutical Society of Great Britain.
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(Received 4 July 1990; revised version accepted 11 October 1990)
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Evans, D. J., Brown, M. R. W., Allison, D. G. & Gilbert, P. (1990a). Susceptibility of bacterial biofilms to tobramycin: role of specific growth rate and phase in the division cycle. Journal of Antimicrobial Chemotherapy 25, 585-91. Fass, R. J. (1987). Efficacy and safety of oral ciprofloxacin in the treatment of serious respiratory infections. American Journal of Medicine 82, Suppl. 4A, 202-7. Gilbert, P., Allison, D. G., Evans, D. J., Handley, P. S. & Brown, M. R. W. (1989). Growth rate control of adherent bacterial populations. Applied and Environmental Microbiology 55, 1308-11. Gilbert, P. & Brown, M. R. W. (1978a). Influence of growth rate and nutrient limitation on the gross cellular composition of Pseudomonas aeruginosa and its resistance to 3- and 4-chlorophenol. Journal of Bacteriology 133, 1066-72. Gilbert, P. & Brown, M. R. W. (1978*). Effect of R-plasmid RP1 and nutrient depletion on the. gross cellular composition of Escherichia coli and its resistance to some uncoupling phenols. Journal of Bacteriology 133, 1062-5. Gilbert, P., Brown, M. R. W. & Costerton, J. W. (1987). Inocula for antimicrobial sensitivity testing: a critical review. Journal of Antimicrobial Chemotherapy 20, 147-54. Gilbert, P. & Stuart, A. (1977). Small-scale chemostat for the growth of mesophilic and thermophilic microorganisms. Laboratory Practice 26, 627—8. Griffiths, E. (1983). Availability of iron and survival of bacteria in infection. In Medical Microbiology, Volume 3. Role of the Envelope in the Survival of Bacteria in Infection (Easman, C. S. F., Jeljaszewicz, J., Brown, M. R. W. & Lambert, P. A., Eds), pp. 153-77. Academic Press, London. Gristina, A. G., Hobgood, C. D., Webb, L. X. & Myrvik, Q. N. (1987). Adhesive colonization of biomaterials and antibiotic resistance. Biomaterials 8, 423-6. Hoiby, N. (1974). Pseudomonas aeruginosa infection in cystic fibrosis. Relationship between mucoid strains of Pseudomonas aeruginosa and the humoral immune response. Ada Pathologica et Microbiologica Scandinavica, Section B, 82, 551-8. Kobayashi, H. (1987). Clinical efficacy of ciprofloxacin in the treatment of patients with respiratory tract infections in Japan. American Journal of Medicine 82, Suppl. 4A, 169-73. Nichols, W. W., Evans, M. J., Slack, M. P. E. & Walmsley, H. L. (1989). The penetration of antibiotics into aggregates of mucoid and non-mucoid Pseudomonas aeruginosa. Journal of General Microbiology 135, 1291-303. Nickel, J. C , Ruseska, I., Wright, J. B. & Costerton, J. W. (1985). Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary tract catheter material. Antimicrobial Agents and Chemotherapy 27, 619-24. Peterson, A. A., Hancock, R. E. W. & McGroaty, E. J. (1985). Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa. Journal of Bacteriology 164, 1256-61. Prosser, B. T., Taylor, D., Dix, B. A. & Cleeland, R. (1987). Method of evaluating effects of antibiotics on bacterial biofilm. Antimicrobial Agents and Chemotherapy 31, 1502-6. Raeburn, J. A., Govan, J. R. W., McCrae, W. M., Greening, A. P., Collier, P. S., Hodson, M. E. et al. (1987). Ciprofloxacin therapy in cysticfibrosis.Journal of Antimicrobial Chemotherapy 20, 295-6. Scully, B. E. (1989). Therapy of respiratory tract infections with quinolone antimicrobial agents. In Quinolone Antimicrobial Agents (Wolfson, J. S. & Hooper, D. C, Eds), pp. 143-65. American Society for Microbiology, Washington, DC. Scully, B. E., Nakatomi, M., Ores, C, Davidson, S. & Neu, H. C. (1987). Ciprofloxacin therapy in cystic fibrosis. American Journal of Medicine 82, Suppl. 4A, 196-201. Shand, G. H., Anwar, H., Kadurugamuwa, J., Brown, M. R. W., Silverman, S. H. & Melling, J. (1985). In vivo evidence that bacteria in urinary tract infection grow under iron-restricted conditions. Infection and Immunity 48, 35-9. Smith, J. T. (1986). The mode of action of 4-quinolones and possible mechanisms of resistance. Journal of Antimicrobial Chemotherapy 18, Suppl. D, 21-9. Wright, N. E. & Gilbert, P. (1987). Influence of specific growth rate and nutrient-limitation upon the sensitivity of Escherichia coli towards polymyxin B. Journal of Antimicrobial Chemotherapy 20, 303-12.
