a Department of Biology, University College London b Department of Clinical Microbiology, University College Hospital, London, UK
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Abstract |
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Introduction |
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An in-vitro study was conducted to examine the efficiency of blood cultures in recovering Staphylococcus aureus in the presence of teicoplanin and to determine if bacteria released into the blood during manipulation of the burn wound were likely to have been killed during in-vitro incubation rather than in vivo. Blood cultures with and without resin were compared.
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Materials and method |
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In the second experiment, the culture of S. aureus (0.8 mL) and the solution of teicoplanin (4.2 mL, 50 mg/L) were injected without earlier mixing, but at the same time into the blood culture bottle. Both experiments were repeated using a mixture of equal parts of pooled human serum and broth (instead of nutrient broth alone) to make the teicoplanin solution. Finally, all the experiments were repeated again using resin-containing blood culture bottles (Becton Dickinson). Controls were prepared using the same inocula of staphylococci but with no teicoplanin added.
Repeatability was assessed using one of the strains (301) in five resin bottles and five non-resin bottles at five inocula from 10 to 10 5 cfu/mL, following either incubation of organism with teicoplanin or simultaneous injection of S. aureus and teicoplanin into the bottles.
The proportion of organisms surviving exposure to teicoplanin (50 mg/L) for 30 min outside blood culture bottles was assessed. The two strains of S. aureus (10 4 cfu/mL) were incubated with antibiotic and agitated in a water bath at 37°C. Aliquots of 10 µL were removed at 0, 10, 20 and 30 min and plated for quantitative counts.
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Results |
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After a 30 min incubation with teicoplanin (50 mg/L) in nutrient broth at 37°C, 27% and 26% of the original inoculum of each strain of S. aureus remained (301 and 359). In the absence of antibiotic, 2.4- and 4.2-fold increases in bacterial numbers occurred.
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Discussion |
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Bacteria isolated from blood cultures may be present in the blood or introduced by subsequent manipulation, e.g. as contaminants from the patient's skin. Incubation of organisms and teicoplanin in vitro before inoculation of blood culture bottles was used to simulate the effect on true bacteraemia. Simultaneous inoculation of the bottles with antibiotic and organisms was used to mimic contamination.
Teicoplanin had continued to kill the staphylococci despite dilution (to 7 mg/L), exposure to SPS, and binding to proteins in human serum and broth in the blood culture bottle. The bactericidal activity of teicoplanin can be reduced up to 10-fold in the presence of serum because it is highly protein bound (88 - 91%). 5 ,6 ,7 However, no difference was observed between cultures with or without serum. Resin-containing bottles increased the recovery of the organisms but still required a minimum initial inoculum of 10 2- 10 4 cfu/mL, depending on the strain and experimental conditions, to allow growth in the presence of teicoplanin.
In-vitro conditions did bear resemblance to those in vivo. In both cases, teicoplanin concentrations would fall, protein binding would occur and bacteria would continue to be exposed to teicoplanin at 37°C beyond the critical hours following the procedure. Despite some degradation, the concentration of teicoplanin in the blood culture bottle was likely to exceed the MBC of most Gram-positive organisms for at least 1 week. 2,4 In burns patients, serum concentrations of teicoplanin would be expected to exceed 1 mg/L for 6 days following a single dose of 12 mg/kg 4 and bacterial killing is independent of concentration once the MBC is exceeded. 8 Bacteraemic organisms surviving in the blood during the operation would be expected to have been killed by the time the clinical outcome was assessed.
Patients with high plasma IL-6 concentrations during surgery are more likely to have Gram-positive bacteraemia than patients with low concentrations. 3 They are also more likely to have fever, graft failure and poor clinical and bacteriological outcomes. The use of teicoplanin during surgery reduced the proportion of patients in whom Gram-positive bacteraemia was detected from 38% (20/52) to 4% (2/52) but did not significantly affect the final IL-6 serum concentration or outcome. 2,3 Bacteria or their components may have been present in the blood long enough to initiate a cytokine cascade. Teicoplanin has been reported to be slowly bactericidal but a direct comparison of teicoplanin with flucloxacillin in serum in vitro showed no significant difference in bactericidal rate at the concentrations used for prophylaxis. 7
Efficacy of a prophylactic antibiotic may not be adequately assessed by failure to grow organisms from blood taken during surgery. The use of resin increases bacterial yield but does not reflect the continued exposure of organisms to antibiotic in vivo. If two antibiotics are compared, differences in the bactericidal activity may occur after inoculation into the blood culture bottle. Despite the problems defining a clinical endpoint, it seems this is the only reliable measure of efficacy in prophylactic trials.
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Notes |
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References |
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2 . Steer, J. A., Papini, R. P. G., Wilson, A. P. R., McGrouther, D. A., Nakhla, L. S. & Parkhouse, N. (1997). Randomized placebo controlled trial of teicoplanin in the antibiotic prophylaxis of infection following manipulation of burns. British Journal of Surgery 84 , 848 53.[ISI][Medline]
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Papini, R. P., Wilson, A. P. R., Steer, J. A., Hill, G., McGrouther, D. A. & Parkhouse, N. (1997). Plasma concentrations of tumour necrosis factor- and interleukin-6 during
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4 . Steer, J. A., Papini, R. P. G., Wilson, A. P. R., Dhillon, S., Hichens, M. F., McGrouther D. A. et al . (1996). Pharmacokinetics of a single dose of teicoplanin in burn patients. Journal of Antimicrobial Chemotherapy 37 , 545 53.[Abstract]
5 . Assandri, A. & Bernareggi, A. (1987). Binding of teicoplanin to human serum albumin. European Journal of Clinical Pharmacology 33 , 191 5.[ISI][Medline]
6 . Bailey, E. M., Rybak, M. J. & Kaatz, G. W. (1991). Comparative effect of protein binding on the killing activities of teicoplanin and vancomycin. Antimicrobial Agents and Chemotherapy 35 , 1089 92.[ISI][Medline]
7 . Steer, J. A., Hill, G. B., Robbins, M. J., Newton, J. C. & Wilson, A. P. R. (1997). In vitro modelling of the bactericidal activity of teicoplanin versus flucloxacillin as used in surgical prophylaxis, against Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 39 , 477 81.[Abstract]
8 . Maple, P. A. C., Hamilton-Miller, J. M. T. & Brumfitt, W. (1989). Comparative in-vitro activity of vancomycin, teicoplanin, ramoplanin (formerly A16686), palimycin, DuP 721 and DuP 105 against methicillin and gentamicin resistant Staphylococcus aureus . Journal of Antimicrobial Chemotherapy 23 , 517 25.[Abstract]
Received 30 June 1998; accepted 15 September 1998