Glycopeptide tolerance in bacteria causing endocarditis

John D. Perry*, Amanda L. Jones and Frances K. Gould

Department of Microbiology, Freeman Hospital, Newcastle upon Tyne NE7 7DN, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Glycopeptides have been recommended as therapy for endocarditis. MICs and MBCs of vancomycin and teicoplanin were compared for 100 isolates from patients with proven bacterial endocarditis. MICs were generally lower for teicoplanin and tolerance to both agents was common. Almost all isolates of enterococci were tolerant to both glycopeptides. Among the streptococci, 78% were tolerant to teicoplanin and 57% to vancomycin. Similar findings were demonstrated for staphylococci. Although isolates appear sensitive to glycopeptides, bactericidal activity cannot always be predicted. If a glycopeptide is indicated for treatment of endocarditis, combination therapy with a suitable aminoglycoside should be considered unless MBC testing can be performed.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The glycopeptide group of antibiotics have well-proven activity against a range of infections caused by Gram-positive bacteria. 1,2 As a result, they have been recommended by a number of authorities as second line agents, with or without an aminoglycoside, in the therapy of streptococcal and staphylococcal endocarditis. 3

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Media

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Table shows the MIC and MBC ranges for all of the species tested.


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Table. Comparison of MIC/MBC values for vancomycin and teicoplanin
 
Enterococci

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 {alpha}-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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A penicillin, with or without an aminoglycoside, remains the ‘gold standard’ therapy for Gram-positive bacterial endocarditis. There may be occasions, however, where resistance or allergy necessitates the use of alternative agents. 3 Historically, despite a paucity of clinical trials, second-line therapy has usually taken the form of a glycopeptide, initially vancomycin or more recently teicoplanin, but there is no consensus as to which agent is more effective. Although resistance to these agents is still unusual, it may be unrecognized if MIC testing is not performed, and lack of bactericidal activity may be of importance in the therapy of bacterial endocarditis. To compare the activity of these two agents we tested 100 Gram-positive isolates collected from proven cases of endocarditis referred to our centre.

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 {alpha}-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.


    Acknowledgments
 
We are grateful to Hoechst Marion Roussel Ltd, who supported this study.


    Notes
 
* Corresponding author. Tel: +44-191-2843111, Ext. 26291; Fax: +44-191-2231224; E-mail: homeresearch{at}compuserve.com Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Wood, M. J. (1996). The comparative efficacy and safety of teicoplanin and vancomycin. Journal of Antimicrobial Chemotherapy 37, 209–22.[Abstract]

2 . Wilson, A. P. & Gaya, H. (1996). Treatment of endocarditis with teicoplanin: a retrospective analysis of 104 cases. Journal of Antimicrobial Chemotherapy 38, 507–21.[Abstract]

3 . Working Party of the British Society for Antimicrobial Chemotherapy. (1998). Antibiotic treatment of streptococcal, enterococcal and staphylococcal endocarditis. Heart 79, 207–10.[Free Full Text]

4 . Carbon, C. (1993). Experimental endocarditis: a review of its relevance to human endocarditis. Journal of Antimicrobial Chemotherapy 31 , Suppl. D, 71–85.[ISI][Medline]

5 . Tuomanen, E., Durack, D. T. & Tomasz, A. (1986). Antibiotic tolerance among clinical isolates of bacteria. Antimicrobial Agents and Chemotherapy 30, 521–7.[ISI][Medline]

6 . Pittet, D. & Harding, I. (1998). Infective endocarditis and glycopeptides. Journal of Infection 37, 127–35.[ISI][Medline]

7 . McGrath, B. J., Kang, S. L., Kaatz, G. W. & Rybak, M. J. (1994). Bactericidal activities of teicoplanin, vancomycin, and gentamicin alone and in combination against Staphylococcus aureus in an in vitro pharmacodynamic model of endocarditis. Antimicrobial Agents and Chemotherapy 38, 2034–40.[Abstract]

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, 1–90.

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, 29–31.

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, 1706–13.[Abstract]

Received 23 November 1998; returned 15 February 1999; revised 3 March 1999; accepted 18 March 1999