Microbiology Systems, Cockeysville, Md.) (9, 13). ... (9) and peritoneal dialysis catheter (2) infections. ... soy broth ina Falcon 2052 tube (Falcon, Oxnard, Calif.).
Vol. 28, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1990, p. 2578-2579
0095-1137/90/112578-02$02.00/0 Copyright © 1990, American Society for Microbiology
Differential Production of Slime under Aerobic and Anaerobic Conditions BARKER,1,2*
SIMPSON,"2 3 AND G. D. CHRISTENSEN1 2'3 Research Service, Harry S Truman Memorial Veterans Hospital,' and Departments of Molecular Microbiology and Immunology2 and Medicine,3 University of Missouri, Columbia, Missouri 65201 L. P.
W. A.
Received 22 February 1990/Accepted 6 August 1990
A series of 37 clinical isolates of coagulase-negative staphylococci previously identified as negative for slime production by the tube test were reexamined by the tissue culture plate test under aerobic and anaerobic conditions. None of the strains produced slime under anaerobic conditions; however, five strains (13%) produced slime under aerobic conditions.
Coagulase-negative staphylococci are a major source of nosocomial infections, particularly in the setting of intravenous catheters or other indwelling medical devices (2, 4, 8, 14). Many strains of coagulase-negative staphylococci also form an adherent bacterial film, or slime, on the walls of the culture vessel when grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) (9, 13). Slime production has been implicated as a virulence factor (7) and is postulated to be a mechanism by which bacteria attach to and colonize indwelling medical devices (5). The production of slime is generally determined by one of two methods: a tube test, in which the slimy film lining a culture tube is visually scaled, and a tissue culture plate method, in which the optical density (OD) of the bacterial film is determined spectrophotometrically (10). Using the tissue culture plate method, our group has reported the association of slime production with intravascular catheter (9) and peritoneal dialysis catheter (2) infections. These reports have been substantiated by some investigators (3, 11, 15), but not by others (1, 12). The source of this discordance is unknown, but it may be attributable to the different assay systems used to test for slime production. We examined a series of coagulase-negative staphylococci recently isolated from patients at the Harry S Truman Memorial Veterans Hospital, Columbia, Mo. These strains were designated HST 1 to HST 116. An overnight culture of each isolate was diluted 1:100 into 2.0 ml of fresh Trypticase soy broth in a Falcon 2052 tube (Falcon, Oxnard, Calif.). Slime production was scaled by the visible accumulation of material on the culture tube walls after 24 h of growth at 37°C. When the clinical isolates negative for slime by the tube test were retested by the tissue culture plate assay for slime production (see below), we found that five isolates thought to be slime negative produced slime in the tissue culture plate system. Because the surface-to-volume ratio of the tube test was approximately one-fourth the ratio for the tissue culture plate method (0.43 and 1.66 cm2/cm3, respectively), we reasoned that the greater exposure of the medium to oxygen in the tissue culture plates could be responsible for the discrepancies between the two test systems. In order to determine the role of oxygen in slime production, the tissue culture plate asssay was performed under aerobic and anaerobic conditions. Each of the 37 HST isolates that were negative for slime *
Corresponding author. 2578
production by the tube test was examined by using the tissue culture plate method as described previously (10), except that the OD was determined on an EIA reader (model 2550; Bio-Rad Laboratories, Richmond, Calif.). The OD for each strain was performed in eight wells, and duplicate plates were run in parallel under aerobic and anaerobic conditions. Anaerobic conditions were generated by the GasPak System (BBL Microbiology Systems). Values represent the means of two separate experiments of eight wells per strain, with 2.0 being the maximum OD. We used Hi and H4A as control stains; these are slimepositive and slime-negative derivatives of the pathogenic isolate RP62A (ATCC 35984), respectively (6). In addition, we used strain HAM 892, which is a putative slime-negative deletion mutant of Hi (L. P. Barker, L. M. Baddour, G. D. Christensen, and W. A. Simpson, unpublished data). A paired Student's t test was performed by comparing aerobic and anaerobic slime production within each isolate and between each isolate and the Hi control. The results of this experiment are shown in Fig. 1. We observed two phenotypes of slime production, class I and class Il. In the collection of 37 tube test-negative coagulasenegative staphylococcal isolates, 32 strains were designated as having the class Il phenotype. These class Il strains produced significantly less slime under both aerobic and anaerobic conditions than the slime-producing control strain Hi did (P < 0.001). The remaining five strains (13%) were positive for slime production under aerobic conditions, but not under anaerobic conditions. Strains HST 7, HST 60, o
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Aerobic S1ime Production
(Optical Density) FIG. 1. Aerobic versus anaerobic slime production. Values are ODs determined by the tissue culture plate method. All points are HST isolates, except for Hi (-), H4A (O), and HAM 892 (*).
NOTES
VOL. 28, 1990 2.5
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CONTROLS
HST 7
HST HST HST HST 64 65 60 62 CLASS I ISOLATES
FIG. 2. Aerobic and anaerobic slime production of class I isolates. Values are ODs determined by the tissue culture plate method. Bars represent slime production under aerobic (solid) and anaerobic (shaded) culture conditions.
HST 62, HST 64, and HST 65 were designated as having the class I phenotype (Fig. 2). Class I isolates were able to produce slime at levels similar to those of the Hi control under aerobic conditions, but they produced significantly less slime than the Hi control under anaerobic conditions (P < 0.001). Isolates with the class I phenotype also produced significantly less slime anaerobically than they did aerobically (P < 0.001). We distinguished two phenotypes, class 1 and class II, by the effect of oxygenation on slime production. Since oxygen is subject to concentration fluctuations in human hosts, these findings could have important implications regarding the pathogenicity of individual strains of Staphylococcus epidermidis. Furthermore, our data also demonstrated that important differences can exist between the tube test and tissue culture plate methods for determining slime production. These findings could explain the discrepancies in slime production by previous investigators who used either the tube test or the tissue culture plate method. It is important, then, to consider the culture environment when assaying for the production of slime in both clinical and research laboratories. We thank J. Lowrance and L. Baddour for discussion and critical review of the manuscript, D. Cosby for technical assistance, J. Sheffield for secretarial assistance, and D. Benish for supplying HST strains. This work was supported by research funds from the Department of Veterans Affairs and the University of Missouri, Columbia. W.A.S. is the recipient of an Associate Career Scientist award from the Department of Veterans Affairs. LITERATURE CITED 1. Alexander, W., and D. Rimland. 1987. Lack of correlation of slime production with pathogenicity in continuous ambulatory peritoneal dialysis peritonitis caused by coagulase negative staphylococci. Diagn. Microbiol. Infect. Dis. 8:215-220.
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2. Baddour, L. M., D. L. Smalley, A. P. Kraus, Jr., W. J. Lamoreaux, and G. D. Christensen. 1986. Comparison of microbiologic characteristics of pathogenic and saprophytic coagulase-negative staphylococci from patients on continuous ambulatory peritoneal dialysis. Diagn. Microbiol. Infect. Dis. 5:197205. 3. Beaman, M., L. Solaro, D. Adu, and J. Michael. 1987. Peritonitis caused by slime-producing coagulase negative staphylococci in continuous ambulatory peritoneal dialysis. Lancet i:42. 4. Christensen, G. D. 1987. The confusing and tenacious coagulase-negative staphylococci. Adv. Intern. Med. 32:177-192. 5. Christensen, G. D., L. M. Baddour, D. L. Hasty, J. H. Lowrance, and W. A. Simpson. 1989. Microbiol and foreign body factors in the pathogenesis of medical device infections, p. 27-59. In A. L. Bisno and F. A. Waldvogel (ed.), Infections associated with indwelling medical devices. American Society for Microbiology, Washington, D.C. 6. Christensen, G. D., L. M. Baddour, B. M. Madison, J. T. Parisi, S. N. Abraham, D. L. Hasty, J. H. Lowrance, J. A. Josephs, and W. A. Simpson. 1990. Colonial morphology of staphylococci on Memphis agar: phase variation of slime production, resistance to beta-lactam antibiotics and virulence. J. Infect. Dis. 161: 1153-1169. 7. Christensen, G. D., L. M. Baddour, and W. A. Simpson. 1987. Phenotypic variation of Staphylococcus epidermidis slime production: in vitro and in vivo studies. Infect. Immun. 55:28702877. 8. Christensen, G. D., A. L. Bisno, J. T. Parisi, B. McLaughlin, M. G. Hester, and R. W. Luther. 1982. Nosocomial septicemia
due to multiply antibiotic resistant Staphylococcus epidermidis. Ann. Intern. Med. 96:1-10.
9. 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. 10. Christensen, G. D., W. A. Simpson, J. J. Younger, L. M. Baddour, F. F. Barrett, D. M. Melton, and E. H. Beachey. 1985. Adherence of coagulase negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J. Clin. Microbiol. 22:996-1006. 11. Davenport, D. S., R. M. Massanari, M. A. Pfaller, M. J. Bale, S. A. Streed, and W. J. Hierholzer, Jr. 1986. Usefulness of a test for slime production as a marker for clinically significant infections with coagulase negative staphylococci. J. Infect. Dis. 153:332-339. 12. Kristinsson, K. G., and R. C. Spencer. 1986. Slime production as a marker for clinically significant infections with coagulase negative staphylococci. J. Infect. Dis. 154:728. 13. Locci, R., G. Peters, and G. Pulverer. 1981. Microbial colonization of prosthetic devices. I. Microphotographical characteristics of intravenous catheters as detected by scanning electron microscopy. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I Orig. Reihe B 173:285-292. 14. Lowy, F. D., and S. M. Hammer. 1983. Staphylococcus epidermidis infections. Ann. Intern. Med. 99:834-839. 15. Needham, C. A., and W. Stempsey. 1984. Incidence, adherence, and antibiotic resistance of coagulase-negative staphylococcus species causing human disease. Diagn. Microbiol. Infect. Dis. 2:293-299.
