Department of Microbiology, Freeman Hospital, Newcastle upon Tyne NE7 7DN, UK
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Abstract |
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Introduction |
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Although the requirement for bactericidal activity in the successful treatment of bacterial endocarditis is undisputed, routine MBC testing is not recommended, because of the technical difficulties associated with the test. 4,5 Numerous data about the in-vitro and in-vivo activity of vancomycin and teicoplanin in endocarditis are available in the literature; however, direct comparative information regarding the cidal activity of these two agents against a range of organisms is sparse. 6,7
To compare the in-vitro bactericidal activity of these agents we performed MIC and MBC testing against a range of Gram-positive organisms isolated from patients with proven endocarditis.
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Materials and methods |
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MIC and MBC tests were performed in Brain Heart Infusion (BHI) broth (Oxoid, Basingstoke, UK) as this was the only broth type able to support the growth of all the test organisms. All sub-cultures and colony counts were performed using Columbia agar (Lab M, Bury, UK) supplemented with 10% defribrinated horse blood (TCS, Buckingham, UK).
Bacterial isolates
A collection of 100 Gram-positive isolates that had been responsible for proven cases of endocarditis over the last 7 years were rejuvenated from freeze-dried storage and sub-cultured at least twice on Columbia blood agar before analysis.
Antibiotics
Teicoplanin was supplied by Hoechst Marion Roussel Ltd (Romainville, France) and vancomycin was obtained from the Sigma Chemical Company (Poole, UK). For each isolate a duplicate double-dilution range from 64 to 0.125 mg/L was prepared in BHI broth using the method described by Ericsson & Sherris. 8
MIC determination
Each test organism was cultivated on Columbia blood agar and incubated overnight at 35°C under suitable atmospheric conditions. Five colonies were harvested and inoculated into 10 mL BHI broth and incubated overnight at 35°C. After incubation, 100 µL of organism suspension was inoculated into 9.9 mL of fresh BHI broth. This was then incubated in a 35°C water bath until the turbidity of the suspension was equivalent to a McFarland 1.0 turbidity standard (approximately 3 x 10 8 cfu/mL). A 0.5 mL sample of this suspension was then added to 7 mL of fresh warm BHI broth to produce a logarithmic-phase suspension of approximately 2 x 10 7 cfu/mL. Aliquots of this suspension (25 µL) were then added to each tube in the duplicate antibiotic dilution ranges including growth control tubes. Care was taken to inoculate organisms beneath the meniscus of the broth without splashing the sides of the tubes. This procedure was calculated to produce a final inoculum in each tube of approximately 5 x 10 5 cfu/mL. Appropriate sterility and growth controls were included.
Immediately after the tubes were inoculated one of the growth control tubes was vortexed and sampled in duplicate to determine the exact number of colony forming units per mL. All tubes were then incubated for exactly 18 h at 35°C in suitable atmospheric conditions. After incubation all tubes were vortexed and the MIC was recorded as the lowest concentration of antimicrobial that resulted in inhibition of visible growth. For each batch of MIC determinations, a control isolate of Staphylococcus aureus (NCTC 6571) was tested in parallel.
MBC determination
For each tube (including control tubes) 20 µL of broth was removed and inoculated onto
the centre of a Columbia blood agar plate and allowed to dry in. Once dry the inoculum was
spread over the entire area of the plate to minimize the effects of antibiotic carry-over. All plates
were incubated for 48 h in suitable atmospheric conditions at 35°C. Also, the number of
colonies produced from each of the antibiotic-containing tubes was counted. The viable count of
the final inoculum was compared with the count of bacteria remaining at 18 h to calculate the
MBC. This was defined as the lowest concentration of antimicrobial that resulted in
99.9% kill of the organism under test. Isolates were defined as tolerant to an
antimicrobial if they demonstrated an MBC:MIC ratio of
32.
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Results |
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The mean MIC of teicoplanin for enterococci was at least six-fold lower than the mean MIC of vancomycin. One isolate, Enterococcus casseliflavus, demonstrated low-level resistance to vancomycin but was susceptible to teicoplanin. The level of tolerance displayed by the enterococci was extremely high for both vancomycin (100%) and teicoplanin (92%), and the MBC 90 of both antimicrobials was >64 mg/L.
Staphylococci
Using criteria defined by BSAC one isolate of Staphylococcus hominis and one of Staphylococcus epidermidis showed low-level resistance to teicoplanin. All isolates were susceptible to vancomycin. The MBC 90 for S. epidermidis was >64 mg/L of teicoplanin and 32 mg/L of vancomycin. The mean MIC of teicoplanin for S. aureus was half that of vancomycin. The level of tolerance among staphylococci was almost three times higher for teicoplanin (37%) than for vancomycin (13%). However, the MBC 90 of both agents was >64 mg/L for S. aureus.
Streptococci
MIC values for teicoplanin were generally at least four-fold lower than for vancomycin for all
streptococcal species tested. The frequency of tolerance was high amongst the streptococci for
both teicoplanin (78%) and vancomycin (57%). For example, the MBC
90 of both agents was >64 mg/L for all species of -haemolytic streptococci
with the exception of Streptococcus salivarius. Bactericidal activity for vancomycin and
not teicoplanin could be demonstrated in 8/15 isolates of Streptococcus bovis.
Propionibacterium granulosum
This isolate was highly sensitive to both glycopeptides but was also highly tolerant, with an MBC:MIC ratio of >512 for both antimicrobials.
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Discussion |
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As expected, MIC values for teicoplanin against nearly all the streptococci and enterococci were four- to six-fold lower than for vancomycin, 9 although there was greater variation in MBC values. For example, all the enterococci, with the exception of one isolate of Enterococcus faecaliswhich had an MIC:MBC ratio of 16 for teicoplanin, were highly tolerant to both agents.
For streptococci the results were less clear cut. Although teicoplanin MICs tended to be lower than those of vancomycin, more isolates demonstrated tolerance (78% with teicoplanin, 57% with vancomycin). Tolerance could, to a limited extent, be predicted by the species of streptococcus tested. For example, isolates of Streptococcus sanguis and Streptococcus oraliswere universally tolerant to teicoplanin whereas S. salivarius was generally killed effectively by both glycopeptides.
For staphylococci, despite slightly lower MICs of teicoplanin, 11/30 isolates were tolerant as compared with 4/30 isolates for vancomycin, all of which were S. aureus.
Our data suggest that, for Gram-positive bacteria isolated from patients with endocarditis,
bactericidal activity cannot always be achieved with either teicoplanin or vancomycin in
vitro, despite a low MIC. Together with the low diffusing capacity of glycopeptides into
vegetations, this may account for the disappointing results obtained using monotherapy in animal
models.
7 Some current guidelines do not specifically recommend
glycopeptide combination therapy as an alternative to penicillin plus aminoglycoside to treat
-haemolytic streptococci in patients with penicillin allergy.
10 Our data suggest that it may be prudent to add an
aminoglycoside (assuming absence of high-level resistance) unless MBCs are available.
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Acknowledgments |
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Notes |
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References |
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2 . Wilson, A. P. & Gaya, H. (1996). Treatment of endocarditis with teicoplanin: a retrospective analysis of 104 cases. Journal of Antimicrobial Chemotherapy 38, 50721.[Abstract]
3
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8 . Ericsson, H. M. & Sherris, J. C. (1971). Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathologica et Microbiologica Scandinavica: Section B, Suppl. 217, 190.
9 . Kenny, M. T., Dulworth, J. K. & Brackman, M. A. (1991). Comparative in vitro activity of teicoplanin and vancomycin against United States teicoplanin clinical trial isolates of Gram-positive cocci. Diagnostic Microbiology and Infectious Disease 14, 2931.
10 . Wilson, W. R., Karchmer, A. W., Dajani, A. S., Taubert, K. A., Bayer, A., Kaye, D. et al. (1995). Antibiotic treatment of adults with infective endocarditis due to streptococci, enterococci, staphylococci and HACEK microorganisms. American Heart Association. Journal of the American Medical Association 274, 170613.[Abstract]
Received 23 November 1998; returned 15 February 1999; revised 3 March 1999; accepted 18 March 1999