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Antimicrobial Agents and Chemotherapy, April 1998, p. 939-941, Vol. 42, No. 4
First Department of Propedeutic Medicine, Laiko General
Hospital, Athens University School of Medicine, Athens, Greece
Received 7 July 1997/Returned for modification 20 November
1997/Accepted 6 February 1998
A novel in vitro semiquantitative method was developed to
investigate the influence of staphylococcal slime on the activities of
22 antimicrobial agents. Pefloxacin, teicoplanin, and vancomycin demonstrated remarkable decreases in efficacy: 30, 52, and 63%, respectively. The activity of rifampin was not significantly reduced (0.99%), whereas all other agents tested were modestly affected (<15% decrease). These data could be influential in the treatment of
implant-associated infections caused by slime-producing staphylococci.
The use of synthetic materials for
temporary or permanent implantation has been accompanied by the
emergence of a new challenging entity, namely, implant-associated
infection. Staphylococcus epidermidis strains with the
ability to form biofilms are the predominant pathogens (17, 20,
22, 30). Biofilm consists of multilayered cell clusters embedded
in a matrix of extracellular polysaccharide (referred to as slime)
(6). It is now well documented that efforts to eradicate
biofilm bacteria are often unsuccessful and removal of the infected
device is required (3, 6, 12, 29). The mechanism of
resistance of biofilm bacteria to antimicrobial agents still remains
unclear but appears to depend on both diffusion limitation (11,
16, 18, 19) and altered physiology associated with low growth
rates and atypical phenotypes in these cells (1-3, 6, 23).
The present study was designed to address the issue of reduced
antibiotic penetration through staphylococcal slime due to trapping.
For this purpose, a novel in vitro semiquantitative method was
developed. To our knowledge, this is the first study to assess the
effect of slime on 22 antistaphylococcal agents, by a uniform method.
(Part of this work was presented at the 31st Interscience Conference on
Antimicrobial Agents and Chemotherapy [13].)
Twenty-six clinical isolates of S. epidermidis were used in
the study. Strains were collected at Laiko General Hospital in Athens,
Greece, over a 6-month period. The collection consisted of isolates
from pus (13 strains), catheter tips (7 strains), blood (3 strains),
and cerebrospinal fluid shunts (3 strains). All strains were identified
as slime producers (4). Two additional strains of S. epidermidis, ATCC 35983 and ATCC 35984 (American Type Culture
Collection, Rockville, Md.), were used as slime-producing standard
strains for quality control. The antimicrobial agents used and their
final concentrations are shown in Table
1.
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Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Slime Produced by Clinical Isolates of
Coagulase-Negative Staphylococci on Activities of Various
Antimicrobial Agents
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TABLE 1.
Concentration of each antimicrobial agent in the 40-µl
solution used to fill the wells
All strains were cultured on staphylococcus agar no. 110 (Becton Dickinson, Cockeysville, Md.), which promotes slime production (4, 31). Single colonies were inoculated in tryptic soy broth (Gibco BRL, Paisley, United Kingdom), and after a 24-h incubation, adherent bacteria from the biofilm formed on the inner surface of each tube were released by vortexing. Forty microliters of this suspension was inoculated on 13-mm-pore-size sterile cellulose filters (Millipore Corporation, Bedford, Mass.) placed on staphylococcus agar no. 110 plates. An adherent biofilm was visible on the surface of each filter after 24 h of incubation. To remove nonadherent bacteria, each filter was eluted in tryptic soy broth. The remaining slime was collected with a sterile loop, mixed with 40 µl of each antimicrobial solution, and transferred into wells of specially prepared Mueller-Hinton agar (Becton Dickinson) plates preseeded with Bacillus subtilis spores (MD32SDR). An equal volume (40 µl) of each antimicrobial solution without slime was placed in an additional well and used as a relevant control. Since the strains were exposed to the same environmental conditions across all experiments, the quantity of slime produced by each strain was considered to be constant (4). The suspensions used in each experiment were of standard volume, and all the wells were filled to the rim. Following a 24-h incubation, inhibition zone diameters were measured. Each experiment was simultaneously done in triplicate, and each time, samples (antibiotic with slime from each strain) and the relevant controls were placed in random places on the plate in order to eliminate the edge effect and other possible gradients across the plates.
In performing the statistical analysis, we calculated the difference between the average of the three measurements for each strain and the average control value and then tested whether these differences were statistically significant by the Wilcoxon signed rank test. In order to summarize the effects of slime on various antibiotics, we calculated the percent difference of each sample (antibiotic with slime from each strain) from the relevant control and took the average across the 26 strains as the percent reduction in the effectiveness of the antibiotic.
The results of the study are summarized in Table 2. Although the literature contains reports that argue against the presence of an antibiotic diffusion barrier in biofilms (7, 24), our results suggest that staphylococcal slime is responsible for a significant decrease in the efficacy of certain antimicrobial agents, whereas its effect is minimal for others.
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Vancomycin is the drug of choice for the treatment of infections caused by methicillin-resistant staphylococci (14). However, our findings as well as data from previous reports (7, 9-11, 28, 31) suggest that the glycopeptides may not be the optimal antimicrobial agents for the treatment of foreign-body infections. A possible explanation could be the entrapment of vancomycin and teicoplanin by the extracellular mucopolysaccharide because of their high molecular weights (MWs) (1,450 and 1,600 to 1,900, respectively). These agents have higher MWs than most of the agents used in this study (range of MWs, 360 to 830). However, this explanation should exclude daptomycin, which also shares a rather high MW (1,619) but was shown to be less influenced by staphylococcal slime. Our efforts to correlate the hydrophobicities or the charges of the antibiotic molecules tested with the reductions in effectiveness produced by slime were unsuccessful, although these physicochemical properties could partly explain the reduced permeabilities of certain antibiotics through biofilms (12, 19).
We have shown that the presence of slime did not influence the activity
of rifampin and had minimal influence on the activities of clindamycin
and the macrolides. There is evidence to suggest that slime itself
interferes with local host defenses and creates conditions of local
immunosuppression (15, 26). Therefore, bacteriostatic agents
such as the macrolides may not be the agents of choice for the
treatment of these infections. Rifampin is a bactericidal agent with a
high level of intrinsic activity against staphylococci and could be
active against methicillin-resistant staphylococci (14). It
also remains very potent in the presence of slime, as confirmed by
other related in vitro and in vivo studies (8, 9, 21, 28,
31), suggesting that it would be an indispensable agent in
combination regimens for the treatment of prosthetic-device infections.
In view of the data presented in Table 2, we could conclude that among
the tested agents the 
-lactams, trimethoprim-sulfamethoxazole, the
aminoglycosides, and the quinolones, with the exception of pefloxacin,
would be potent alternatives in combination regimens for the treatment of prosthetic-device infections.
Recently, several publications have focused on the in vitro use of electric fields to enhance the penetration of antibiotics through microbial biofilms (5, 25, 27). Until this approach finds its application in clinical practice, the successful treatment of device-related infections will depend on the prudent use of antibiotics and surgery. The findings of this in vitro study may be influential in the appropriate use of antibiotics for the treatment of implant-associated infections caused by slime-producing staphylococci. This is of the utmost importance, especially for debilitated patients who cannot undergo surgical removal of the infected foreign body.
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ACKNOWLEDGMENTS |
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We are indebted to Robert A. Parker, Associate Professor of Medicine (Biostatistics) at Harvard Medical School and Director of the Biometrics Center at Beth Israel Deaconess Medical Center, for his valuable assistance in conducting the statistical analysis.
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FOOTNOTES |
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* Corresponding author. Mailing address: First Department of Propedeutic Medicine, Laiko General Hospital, 17 Agiou Thoma str., GR 115 27 Athens, Greece. Phone: (301) 7790802. Fax: (301) 7709447. E-mail: vatmsidl{at}hol.gr.
Present address: Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA 02115.
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REFERENCES |
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Abstract
Text
References
|
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| 1. |
Anwar, H.,
J. L. Strap, and J. W. Costerton.
1992.
Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy.
Antimicrob. Agents Chemother.
36:1347-1351 |
| 2. | Brown, M. R. W., R. E. Courcol, S. Gander, and P. Gilbert. 1994. Influence of growth rate on susceptibility of biofilm-grown Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli to ciprofloxacin, p. 296-297. In J. Einhorn, C. E. Nord, and S. R. Norby (ed.), Recent advances in chemotherapy. American Society for Microbiology, Washington, D.C. |
| 3. | Brown, M. R. W., and P. Gilbert. 1993. Sensitivity of biofilms to antimicrobial agents. J. Appl. Bacteriol. 74(Suppl. 1):S87-S97. |
| 4. |
Christensen, G. D.,
W. A. Simpson,
A. L. Bisno, and E. H. Beachey.
1982.
Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces.
Infect. Immun.
37:318-326 |
| 5. |
Costerton, J. W.,
B. Ellis,
K. Lam,
F. Johnson, and A. E. Khoury.
1994.
Mechanism of electrical enhancement of efficacy of antibiotics in killing biofilm bacteria.
Antimicrob. Agents Chemother.
38:2803-2809 |
| 6. | Costerton, J. W., Z. Lewandowski, D. E. Caldwell, D. R. Korber, and H. M. Lappin-Scott. 1995. Microbial biofilms. Annu. Rev. Microbiol. 49:711-745[Medline]. |
| 7. | Darouiche, R. O., A. Dhir, A. J. Miller, G. C. Landon, I. I. Raad, and D. M. Musher. 1994. Vancomycin penetration into biofilm covering infected prostheses and effect on bacteria. J. Infect. Dis. 170:720-723[Medline]. |
| 8. |
Drancourt, M.,
A. Stein,
J. N. Argenson,
A. Zannier,
G. Curvale, and D. Raoult.
1993.
Oral rifampin plus ofloxacin for treatment of Staphylococcus-infected orthopedic implants.
Antimicrob. Agents Chemother.
37:1214-1218 |
| 9. |
Dunne, W. M.,
E. O. Mason, and S. L. Kaplan.
1993.
Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilm.
Antimicrob. Agents Chemother.
37:2522-2526 |
| 10. |
Evans, R. C., and C. J. Holmes.
1987.
Effect of vancomycin hydrochloride on Staphylococcus epidermidis biofilm associated with silicone elastomer.
Antimicrob. Agents Chemother.
31:889-894 |
| 11. | Farber, B. F., M. H. Kaplan, and A. G. Glogston. 1990. Staphylococcus epidermidis extracted slime inhibits the antimicrobial action of glycopeptide antibiotics. J. Infect. Dis. 161:37-40[Medline]. |
| 12. |
Gander, S.
1996.
Bacterial biofilms: resistance to antimicrobial agents.
J. Antimicrob. Chemother.
37:1047-1050 |
| 13. | Giamarellou, H., M. Souli, and M. Papapetropoulou. 1991. Diffusion of various antibiotics through slime produced by clinical isolates of coagulase-negative staphylococci (CNS), abstr. 1310, p. 317. In Program and abstracts of the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
| 14. | Graninger, W., C. Wenisch, and M. Hasenhundl. 1995. Treatment of staphylococcal infections. Curr. Opin. Infect. Dis. 8(Suppl. 1):20-28. |
| 15. | Gray, E. D., W. E. Regelmann, and G. Peters. 1987. Staphylococcal slime and host defences: effects on lymphocytes and immune function, p. 45-54. In G. Pulverer, P. G. Quie, and G. Peters (ed.), Pathogenicity and clinical significance of coagulase-negative staphylococci. Gustav Fischer Verlag, Stuttgart, Germany. |
| 16. |
Hoyle, B. D.,
J. Alcantara, and J. W. Costerton.
1992.
Pseudomonas aeruginosa biofilm as a diffusion barrier to piperacillin.
Antimicrob. Agents Chemother.
36:2054-2056 |
| 17. |
Kloos, W. E., and T. L. Bannerman.
1994.
Update on clinical significance of coagulase-negative staphylococci.
Clin. Microbiol. Rev.
7:117-140 |
| 18. | Kumon, H., K. Tomochika, T. Matunaga, M. Ogawa, and H. Ohmori. 1994. A sandwich cup method for the penetration assay of antimicrobial agents through Pseudomonas aeruginosa exopolysaccharides. Microbiol. Immunol. 38:615-619[Medline]. |
| 19. | Nichols, W. W., S. M. Dorrington, M. P. E. Slack, and H. I. Walmsley. 1988. Inhibition of tobramycin diffusion by binding to alginate. Antimicrob. Agents Chemother. 32:1518-1523. |
| 20. | Peters, G., C. von Eiff, and M. Herrmann. 1995. The changing pattern of coagulase-negative staphylococci as infectious pathogens. Curr. Opin. Infect. Dis. 8(Suppl. 1):12-19. |
| 21. | Richards, G. K., R. F. Gagnon, and R. J. Morcos. 1994. An assay to measure antibiotic efficacy against Staphylococcus epidermidis biofilms on implant surfaces. ASAIO J. 40:M570-M575[Medline]. |
| 22. | Rupp, M. E., and G. D. Archer. 1994. Coagulase-negative staphylococci: pathogens associated with medical progress. Clin. Infect. Dis. 19:231-245[Medline]. |
| 23. | Shigeta, M., H. Komatsuzawa, M. Sugai, H. Suginaka, and T. Usui. 1997. Effect of the growth rate of Pseudomonas aeruginosa biofilms on the susceptibility to antimicrobial agents. Chemotherapy 43:137-141[Medline]. |
| 24. | Stewart, P. S. 1996. Theoretical aspects of antibiotic diffusion into microbial biofilms. Antimicrob. Agents Chemother. 40:2517-2522[Abstract]. |
| 25. | Stoodley, P., D. DeBeer, and H. M. Lappin-Scott. 1997. Influence of electric fields and pH on biofilm structure as related to the bioelectric effect. Antimicrob. Agents Chemother. 41:1876-1879[Abstract]. |
| 26. |
Stout, E. D.,
K. P. Ferguson,
Y. Li, and D. W. Lambe.
1992.
Staphylococcal exopolysaccharides inhibit lymphocyte proliferative responses by activation of monocyte prostaglandin production.
Infect. Immun.
60:922-927 |
| 27. | Wellman, N., S. M. Fortun, and B. R. McLeod. 1996. Bacterial biofilms and the bioelectric effect. Antimicrob. Agents Chemother. 40:2012-2014[Abstract]. |
| 28. | Widmer, A. F., R. Frei, Z. Rajacic, and W. Zimmerli. 1990. Correlation between in vivo and in vitro efficacy of antimicrobial agents against foreign body infections. J. Infect. Dis. 162:96-102[Medline]. |
| 29. | Younger, J. J., G. D. Christensen, D. L. Bartley, J. C. Simmons, and F. F. Barrett. 1987. Coagulase-negative staphylococci isolated from cerebrospinal fluid shunts: importance of slime production, species identification and shunt removal to clinical outcome. J. Infect. Dis. 156:548-554[Medline]. |
| 30. | Ziebuhr, W., C. Heilmann, F. Gotz, P. Meyer, K. Wilms, E. Straube, and J. Hacker. 1997. Detection of the intercellular adhesion gene cluster (ica) and phase variation in Staphylococcus epidermidis blood culture strains and mucosal isolates. Infect. Immun. 65:890-896[Abstract]. |
| 31. | Ziscovici, S., and V. Lorian. 1989. Slime from Staphylococcus epidermidis interrupts antibiotic diffusion, abstr. 216, p. 128. In Program and abstracts of the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. |
Antimicrobial Agents and Chemotherapy, April 1998, p. 939-941, Vol. 42, No. 4
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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