Effect of teicoplanin on isolation of Staphylococcus aureus from blood culture media

I. F. Cherna, J. A. Steerb and A. P. R. Wilsonb,*

a Department of Biology, University College London b Department of Clinical Microbiology, University College Hospital, London, UK


    Abstract
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 Abstract
 Introduction
 Materials and method
 Results
 Discussion
 References
 
Intraoperative bacteraemia has been used as an indicator of the efficacy of prophylactic antibiotics. Two clinical isolates of Staphylococcus aureus in nutrient broth, with or without human serum, were exposed to teicoplanin (50 mg/L) and, either immediately or after 30 min, inoculated into blood culture bottles. Bottles with and without resin were used and the experiment was repeated five times with one strain. In the absence of teicoplanin, an inoculum of 10 cfu/mL produced growth in both resin and non-resin bottles. In the presence of teicoplanin, an inoculum of at least 10 5 cfu/mL was required in non-resin bottles to obtain growth, but this was reduced to 102 - 103 cfu/mL for resin bottles. Intraoperative blood cultures overestimate the efficacy of bacterial killing by prophylactic antibiotics during surgery.


    Introduction
 Top
 Abstract
 Introduction
 Materials and method
 Results
 Discussion
 References
 
Bacteraemia is a common occurrence during surgery and prophylactic antibiotics are used in the expectation that prevention of bacteraemia will improve the subsequent clinical condition of the patient. 1 In a randomized double-blind placebo-controlled trial of the use of teicoplanin for prevention of infection after burn wound excision, clinical outcome was not affected despite apparently preventing Gram-positive bacteraemia. 2 This was contrary to the observation that Gram-positive bacteraemia was associated with increased serum concentrations of interleukin 6 (IL-6) which in turn related to poor clinical outcome. 3

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.


    Materials and method
 Top
 Abstract
 Introduction
 Materials and method
 Results
 Discussion
 References
 
Two strains of S. aureus (301 and 359), isolated from blood cultures collected during or at the end of burns surgery, were inoculated into nutrient broth to produce an overnight broth culture which was then diluted to produce an estimated concentration of 10 5 cfu/mL (confirmed by quantitative plating). Teicoplanin (Hoechst Marion Roussel, Uxbridge, UK) was dissolved in nutrient broth to produce a solution of 50 mg/L. This was approximately the in-vivo serum concentration 1 h after a bolus iv injection of teicoplanin (12 mg/kg) in burn patients. 4 The broth culture of S. aureus (1 mL) was added to teicoplanin solution (5 mL) at 37°C and incubated for 30 min. A volume (5 mL) was then injected into an aerobic blood culture bottle (containing 30 mL of medium with sodium polyethole sulphonate (SPS) 0.035%) (Becton Dickinson, Sparks, MD, USA). The bottle was incubated for 7 days on the Bactec system (Becton Dickinson). The experiment was repeated at a range of bacterial inocula from 10 to 10 5 cfu/mL.

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.


    Results
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 Abstract
 Introduction
 Materials and method
 Results
 Discussion
 References
 
All the control bottles (which contained no teicoplanin) demonstrated bacterial growth including those at an inoculum of 10 cfu/mL. Cultures in non-resin blood culture bottles containing teicoplanin failed to show any bacterial growth of either strain, even when the initial inoculum was 10 5 cfu/mL (Table). Resin-containing bottles demonstrated growth at minimum inocula of 10 2 - 10 4 cfu/mL, with or without previous incubation with teicoplanin and regardless of the presence of human serum.


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Table. Culture of Staphylococcus aureus and teicoplanin (50 mg/L) in blood culture bottles
 
When the procedure was repeated five times for strain 301 after incubation with teicoplanin, growth was observed in all resin bottles at an inoculum of 10 3 cfu/mL, in two of five at 10 2 cfu/mL and in one of five at 10 cfu/mL. Without previous exposure to antibiotic, growth was again observed in all bottles at 10 3 cfu/mL and in one of five at 10 2 cfu/mL but not at 10 cfu/mL. Two of five non-resin bottles yielded growth but only at an inoculum of 10 5 cfu/mL without preincubation with teicoplanin. Plating of four aliquots of 10 µL of each inoculum showed actual concentrations to be slightly lower than expected: 10 5 cfu/mL: mean 3 x10 4 (range 2 - 5 x10 4) cfu/mL; 10 4 cfu/mL; mean 3 x 10 3 (range 2 - 5 x 10 3) cfu/mL; 10 3 cfu/mL: mean 5 x 10 2 (3- 8 x 10 2) cfu/mL.

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.


    Discussion
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 Abstract
 Introduction
 Materials and method
 Results
 Discussion
 References
 
At inocula of up to 10 4 cfu/mL, S. aureus was not recovered from non-resin blood culture bottles when teicoplanin was present in the sample inoculated into the bottle. At 10 5 cfu/mL, organisms were recovered in some cases but this would exceed the number of bacteria likely to be present clinically during intraoperative bacteraemia. The concentration of teicoplanin was that achieved intraoperatively following a preoperative intravenous dose of 12 mg/kg. Removal of teicoplanin by resin at the time of inoculation into the blood culture bottle reduced the inoculum necessary for bacterial recovery by 10 3 suggesting a 3-log kill of bacteria had occurred during in-vitro incubation of non-resin bottles. The effect was repeatable. Continued bacterial killing by teicoplanin following dilution in the blood culture bottle would result in falsely negative blood cultures but killing would also be expected to continue in vivo so the clinical significance of bacteraemia at low levels is uncertain.

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.


    Notes
 
* Corresponding address: Department of Clinical Microbiology, University College Hosptial, Grafton Way, London WC1E6DB, UK. Tel+44-171-380-9516; Fax +44-171-388-8514; E-mail: p.wilson{at}academic.uclh.nthames.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and method
 Results
 Discussion
 References
 
1 . Papini, R. P. G., Wilson, A. P. R., Steer, J. A., McGrouther, D. A. & Parkhouse, N. (1995). Wound management in burn centres in the United Kingdom. British Journal of Surgery 82, 505 –9.[ISI][Medline]

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]

3 . Papini, R. P., Wilson, A. P. R., Steer, J. A., Hill, G., McGrouther, D. A. & Parkhouse, N. (1997). Plasma concentrations of tumour necrosis factor-{alpha} and interleukin-6 during burn wound surgery or dressing. British Journal of Plastic Surgery 50 , 354 –61.[ISI][Medline]

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





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