Division of Microbiology and Infectious Diseases, School of Clinical Laboratory Sciences, University of Nottingham, The City Hospital, Nottingham NG5 1PB, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biofilm-associated disease in humans is widespread and increasing, and occurs largely as a consequence of the increase in the use of indwelling medical devices, for example catheters and prosthetic devices.5 Biofilm cells typically have very slow growth rates and are under some sort of nutrient limitation, whereas cells grown in batch cultures in vitro are usually in nutrient-rich laboratory medium. Consequently the physiology of the two populations will differ greatly. Adherent biofilms persist in spite of active host immune defence systems and antimicrobial chemotherapy.58 For these reasons we have studied inhibitory/ bactericidal activity and recovery times of selected Gram-positive pathogens in a biofilm model using quinuprisitn/ dalfopristin and five comparator antibiotics. In clinical situations when intermittent antibiotic dosing is used, the organisms are subjected to decreasing concentrations of the drug, so experimental exposure of bacteria in vitro to constant levels of antibiotics does not reflect the true clinical situation.9 To compare in vitro activity and to simulate the situation in vivo, bacteria were exposed to a constant concentration of the drugs for 1 h and, using an in vitro pharmacokinetic model, to exponentially decreasing concentrations.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The following antibiotics were supplied as reference powders by their respective manufacturers: quinupristin/ dalfopristin (RhônePoulenc Rorer, Collegeville, PA, USA), ciprofloxacin (Bayer, Wuppertal, Germany), vancomycin (Eli Lilly, Basingstoke, UK), teicoplanin (Hoechst Marion Roussel, Uxbridge, UK), flucloxacillin (SmithKline Beecham, Worthing, UK) and erythromycin (Abbott, Queensborough, UK). Stock solutions were prepared and stored according to the recommendations of The British Society for Antimicrobial Chemotherapy.10
MIC and MBC determinations
MICs and MBCs were determined using the standard broth dilution method10 and MuellerHinton broth (Oxoid, Basingstoke, UK).
Biofilm studies
The biofilm model used in this study was a modification of the Sorbarod model,11 in which quarter Sorbarods were used to reduce the number of bacteria forming the biofilms. A Sorbarod (Ilacon Ltd, Kent, UK) consists of a cylindrical paper sleeve encasing a compacted concertina of cellulose fibres. Cells attach to the cellulose fibre plug and are subsequently perfused with medium (MuellerHinton broth) from one side; cells are shed from the opposite side. After initial loss of loosely attached cells into the eluted medium, a steady state is achieved where the adherent biomass and the rate of cell release from the membrane remain constant. The number of cells eluted from the biofilm reflects the number of actively dividing cells in the biofilm, and so it can be be used to quantify the effects of antibiotics on the biofilm cells.
Exposure to constant antibiotic concentrations for 1 h
Test and control biofilms were set up and perfused with MuellerHinton broth. When the biofilms reached a steady state, i.e. when a constant number of cells were being eluted from them (as determined by performing viable counts on the eluate), the test biofilms were perfused for 1 h with MuellerHinton broth containing the appropriate concentration of antibiotic. The concentrations used were the MIC and MBC for the bacteria being tested. After this exposure, perfusion continued in the absence of the drug. The flow rate of the medium (and antibiotic) was 15 mL/h throughout all experiments. Viable counts were performed on the eluted cells at hourly intervals, 2 h before exposure, during exposure and up to 7 h after exposure to the antibiotics. The viable counts of bacteria were determined by spiral plating (Spiral Systems, Cincinnatti, OH, USA) of serially diluted samples of eluate on nutrient agar (Oxoid).
Exposure to exponentially decreasing concentrations of antibiotics
Perfusing biofilms with media via a dilution vessel into which drugs are added allows biofilm cells to be exposed to exponentially decreasing concentrations of antibiotic. This method of perfusion is achieved by using an uninoculated flask as a dilution vessel for the antibiotic (see Figure 1). Biofilms were established as described above, except that the tubing carrying the medium from the medium reservoir to the biofilms went via the dilution vessel. Once the biofilms reached a steady state, the antibiotic was added directly to the dilution vessel. The biofilm cells were, thus, exposed to an exponentially decreasing concentration of the drug. The dilution rate of the antibiotics was matched to their half-lives and the volume of medium in the dilution vessel was calculated from the equation t1/2 = 0.6931 V/r, based on first-order decay kinetics (see Appendix). The half-lives of the antibiotics used in these experiments are as follows: quinupristin/dalfopristin, 1.36 h; ciprofloxacin, 3.5 h; vancomycin, 8.0 h; teicoplanin, 50 h; flucloxacillin, 1.0 h; and erythromycin, 1.25 h. The flow rate remained at 15 mL/h throughout all experiments. Viable counts were performed on the cells eluted from the biofilms before and during perfusion with the antibiotics, as described above for the constant drug concentration exposure. All experiments were performed in triplicate.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The lack of any significant difference between the effects of the antibiotics, inhibitory or bactericidal and recovery times following both methods of exposure, was unexpected. Owing to the method of perfusion, the concentrations of the drugs in the bacterial biofilms in the exponentially decreasing model were reduced only by dilution at the rate of the half-lives of the antibiotics. The recovery times of the bacterial biofilms in the presence of decreasing antibiotic concentrations were therefore compared with recovery times for the 1 h constant concentration model which were in the absence of any drugs. The bacteria in the exponentially decreasing concentration model would have been exposed to subinhibitory concentrations of the antibiotics. Subinhibitory concentrations of antimicrobial agents, including those used in this study,2225 often influence the growth rate of bacteria, especially after exposure to suprainhibitory levels of the drug. Thus bacteria exposed to decreasing concentrations of antibiotics would, therefore, have been expected to take longer to recover than those exposed to constant concentrations. Although the recovery times for the two methods of exposure were not significantly different, the times for exponentially decreasing exposure, especially to ciprofloxacin, are slightly longer than those for the constant concentration exposure. For several organismantibiotic combinations, no recovery was seen within the 420 min of monitoring and further differences may have been apparent if recovery had been followed for longer.
Given the increase in biofilm-associated infections, it is important to study how antimicrobial agents affect biofilms in vitro. In vitro determination of MICs and post-antibiotic effect (PAE) are still usually carried out using batch cultures of bacteria grown in nutrient-rich media. There is very little information on PAEs in biofilms. The effect of exposing biofilm cells to a constant concentration of antibiotic for a fixed time could be considered a measure of the PAE. The equation used to calculate PAE in batch culture is said to represent the time taken for the cells to return to the normal rate of growth.26 When a biofilm is at steady state, cells are shed from it at a constant rate, which could be considered as the normal rate. The number of cells shed from a biofilm will decrease when the biofilm is exposed to an inhibitory concentration of a drug, and will increase on removal of the drug, eventually returning to the original steady state. Thus the recovery time of the biofilms could be considered as representing a return to normal growth as with the batch culture PAE.
In a similar study using a different biofilm model, the Swinnex model, which uses a much denser filter matrix, we previously investigated the effects of ciprofloxacin on Escherichia coli (S. Gander and P. Gilbert, unpublished). The effects of exposure to constant and exponentially decreasing concentrations of the antibiotic were investigated. Although the inhibitory/bactericidal effects were similar for both methods of exposure, cells exposed to a decreasing concentration of ciprofloxacin took longer to recover (24 h) than cells exposed to a constant concentration (20 h).
The two methods of exposure used in our study, constant for 1 h and exponentially decreasing, could be seen as analogous to administering antibiotics by infusion and parenteral dosing. If this were the case, the results of this study would suggest that there are no significant differences between these two methods of dosing. Rybak et al.27 performed a similar study, looking at the effects of quinupristin/dalfopristin administered by continuous infusion and parenterally, using S. aureus-infected fibrinplatelet clots in an in vitro infection model. They also found no significant differences between the bactericidal effects produced by the two dosing regimens.
Other studies have investigated the effect of quinupristin/dalfopristin on adherent bacteria. Hamilton-Miller & Shah28 investigated the effects of quinupristin/dalfopristin and ciprofloxacin on Staphylococcus epidermidis biofilms. They found that both the antibiotics had the same bactericidal effects on the biofilms. Although they used much higher concentrations than we did, the results are in agreement. Berthaud & Desnottes29 looked at the effect of quinupristin/dalfopristin and vancomycin on S. aureus biofilms cultured on fibronectin-coated nylon membrane discs. They found that at 5, 10 and 50 x MIC, the two antibiotics had a similar bactericidal effect. These results do not agree with those of this study, probably because of the higher concentrations of antibiotics used. Our investigations indicate that quinupristin/dalfopristin would be effective for treating Gram-positive infections, including those caused by multidrug-resistant MRSA and VRE.
![]() |
Appendix: Derivation of half-life equation |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
Hence t1/2 = 0.6931V/r
|
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2
.
Hill, R. L. R., Smith, C. T., Seyed-Akhavani, M. & Casewell, M. W. (1997). Bactericidal and inhibitory activity of quinupristin/dalfopristin against vancomycin- and gentamicin-resistant Enterococcus faecium. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 238.
3
.
Kang, S. L. & Rybak, M. J. (1997). In-vitro bactericidal activity of quinupristin/dalfopristin alone and in combination against resistant strains of Enterococcus species and Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 339.
4 . Low, D. E. & Nadler, H. L. (1997). A review of in-vitro antibacterial activity of quinupristin/dalfopristin against methicillin-susceptible and -resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 538.[Abstract]
5 . Finch, R. G. (1994). Medical problems associated with biofilms. In Bacterial Biofilms and their Control in Medicine and lndustry, (Wimpenny, J., Nichols, W., Stickler, D. & Lappin-Scott, H., Eds), pp. 8892. Bioline, Chippenham.
6 . Dasgupta, M. K. & Costerton, J. W. (1989). Significance of biofilm-adherent bacterial microcolonies on Tenckhoff catheters of CAPD patients. Blood Purification 7, 14455.[ISI][Medline]
7 . Dickinson, G. M. & Bisno, A. L. (1989). Infections associated with indwelling devices: concepts of pathogenesis; infections associated with intravascular devices. Antimicrobial Agents and Chemotherapy 33, 597601.[ISI][Medline]
8 . Dickinson, G. M. & Bisno, A. L. (1989). Infections associated with indwelling devices: infections related to extravascular devices Antimicrobial Agents and Chemotherapy 33, 6027.[ISI][Medline]
9 . Lowdin, E., Odenholt, I., Bengtsson, S. & Cars, O. (1996). Pharmacodynamic effects of sub-MICs of benzylpenicillin against Streptococcus pyogenes in a newly developed in vitro kinetic model. Antimicrobial Agents and Chemotherapy 40, 247882.[Abstract]
10 . BSAC Working Party Report. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 150.[ISI][Medline]
11 . Hodgson, A. E., Nelson, S. M., Brown, M. R. W. & Gilbert, P. (1995). A simple in vitro model for growth control of bacterial biofilms. Journal of Applied Bacteriology 79, 8793.[ISI][Medline]
12
.
Bouanchaud, D. H. (1997). In-vitro and in-vivo antibacterial activity of quinupristin/dalfopristin. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 1521.
13
.
Mouton, J. W., Endtz, H. P., den Hollander, J. G., van den Braak, N. & Verbrugh, H.A. (1997). In-vitro activity of quinupristin/ dalfopristin compared with other widely used antibiotics against strains isolated from patients with endocarditis. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 7580.
14
.
Williams, J. D., Maskell, J. P., Whiley, A. C. & Sefton, A. M. (1997). Comparative in-vitro activity of quinupristin/dalfopristin against Enterococcus spp. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 416.
15 . Appelbaum, P. C. (1996). Emerging resistance to antimicrobial agents in Gram-positive bacteria. Pneumococci. Drugs 51, Suppl. 1, 15.[Medline]
16 . Finch, R. G. (1996). Antibacterial activity of quinupristin/dalfopristin. Rationale for clinical use. Drugs 51, Suppl. 1, 317.[ISI][Medline]
17 . Pechère, J. C. (1996). Streptogramins. A unique class of antibiotics. Drugs 51, Suppl. 1, 139.[ISI][Medline]
18
.
Boswell, F. J., Sunderland, J., Andrews, J. M. & Wise, R. (1997). Timekill kinetics of quinupristin/dalfopristin on Staphylococcus aureus with and without a raised MBC evaluated by two methods. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 2932.
19 . Andrews, J. M. & Wise, R. (1994). The in-vitro activity of a new semi-synthetic streptogramin compound, RP 59500, against staphylococci and respiratory pathogens. Journal of Antimicrobial Chemotherapy 33, 84953.[ISI][Medline]
20 . Fantin, B., Leclercq, R., Merle, Y., Saint-Julien, L., Veyrat, C., Duval, J. et al. (1995). Critical influence of resistance to streptogramin B-type antibiotics on activity of RP 59500 (quinupristin dalfopristin) in experimental endocarditis due to Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 39, 4005.[Abstract]
21 . Fantin, B., Leclercq, R., Merle, Y., Saint-Julien, L., Duval, J. & Carbon, C. (1994). Influence of constitutive resistance to erythromycin on the activity of RP 59500 (quinupristin/dalfopristin) in experimental endocarditis due to Staphylococcus aureus. In Program and Abstracts of the Thirty-Fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, FL, 1994, Abstract B79, p. 150. American Society for Microbiology, Washington, DC.
22 . Licata, L., Smith, C. E., Goldschmidt, R. M., Barrett, J. F. & Frosco, M. (1997). Comparison of the postantibiotic and postantibiotic sub-MIC effects of levofloxacin and ciprofloxacin on Staphylococcus aureus and Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 41, 9505.[Abstract]
23
.
Löwdin, E., Odenholt, I. & Cars, O. (1998). In vitro studies of pharmocodynamic properties of vancomycin against Staphylococcus aureus and Staphylococcus epidermidis. Antimicrobial Agents and Chemotherapy 42, 273944.
24
.
Pankuch, G. A., Jacobs, M. R. & Appelbaum, P. C. (1998). Postantibiotic effect and postantibiotic sub-MIC effect of quinupristindalfopristin against Gram-positive and -negative organisms. Antimicrobial Agents and Chemotherapy 42, 302831.
25
.
Spangler, S. K., Lin, G., Jacobs, M. R. & Appelbaum, P. C. (1998). Postantibiotic effect and postantibiotic sub-MIC effect of levofloxacin compared to those of ofloxacin, ciprofloxacin, erythromycin, azithromycin, and clarithromycin against 20 pneumococci. Antimicrobial Agents and Chemotherapy 42, 12535.
26 . Craig, W. A. & Gudmundsson, S. (1991). Postantibiotic effect. In Antibiotics in Laboratory Medicine, 3rd edn, (Lorian, V., Ed.), pp. 40331. Williams & Wilkins, Baltimore, MD.
27 . Rybak, M. J., Houlihan, H. H., Mercier, R.-C. & Kaatz, G. W. (1997). Pharmacodynamics of RP 59500 (quinupristindalfopristin) administered by intermittent versus continuous infusion against Staphylococcus aureus-infected fibrinplatelet clots in an in vitro infection model. Antimicrobial Agents and Chemotherapy 41, 135963.[Abstract]
28 . Hamilton-Miller, J. M. T. & Shah, S. (1997). Activity of quinupristin/dalfopristin against Staphylococcus epidermidis in biofilms: a comparison with ciprofloxacin. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 1038.[Abstract]
29 . Berthaud, N. & Desnottes, J.-F. (1997). In-vitro bactericidal activity of quinupristin/dalfopristin against adherent Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 39, Suppl. A, 99102.[Abstract]
Received 1 July 1999; returned 8 November 1999; revised 4 January 2000; accepted 8 February 2000