Effect of serum from different patient populations on the serum bactericidal test

Michael B. Kaysa,*, Roger L. Whiteb and Lawrence V. Friedrichb

a Department of Pharmacy Practice, School of Pharmacy and Pharmaceutical Sciences, Purdue University, Indianapolis, IN; b Anti-infective Research Laboratory, College of Pharmacy, Medical University of South Carolina, Charleston, SC, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We evaluated the effect of serum from normal and uraemic volunteers, neutropenic patients and burn patients on the serum bactericidal test. Serum samples were spiked with ceftazidime to mimic in vivo peak (75 mg/L) and trough (5 mg/L) concentrations. Serum inhibitory and bactericidal titres (SIT and SBT) were performed in triplicate using Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. For E. coli, the trough SIT and SBT were significantly higher in serum from burn patients compared with normal volunteers (P <= 0.024). The trough SIT was significantly higher in serum from burn patients compared with neutropenic patients (P = 0.022) and in uraemic patients compared with normal volunteers (P = 0.04). No significant differences between subject populations were found for P. aeruginosa.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The serum bactericidal test has been used for many years to guide antimicrobial therapy and to perform ex vivo comparisons between antimicrobial agents. Previous studies have attempted to identify a bactericidal titre that can predict clinical outcome in patients with a given infection.1,2 However, the ability of a single titre to predict accurately clinical success or failure in a variety of patients with different levels of immune function is questionable. Many factors are responsible for the antimicrobial activity of serum, including complement, antibodies and other humoral substances.3 Therefore, the titre may vary depending on the concentration of factors present in the patient's serum. The objective of this study was to determine whether there are differences in serum bactericidal activity between different patient populations when the antibiotic concentrations are the same. Ceftazidime was the chosen antibiotic.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ten normal volunteers, eight neutropenic patients, seven burn patients and 10 uraemic subjects with chronic renal failure who were undergoing outpatient haemodialysis thrice weekly were recruited. The neutropenic (absolute neutrophil count <500/mm3) and burn patients were recruited from the in-patient services at the Medical University of South Carolina Hospital. Subjects were eligible for participation if they were not receiving antibacterial therapy at the time of enrolment, had not received antibacterial therapy within the previous month, and had no clinical and/or laboratory evidence of infection. Blood was collected in non-anticoagulant tubes, and serum was separated and stored at –70°C. For uraemic subjects, blood was drawn immediately before a dialysis session. The study was approved by the Institutional Review Board at the Medical University of South Carolina, and informed consent was obtained from each subject.

Stock ceftazidime solution (Eli Lilly & Co., Indianapolis, IN, USA) was prepared using laboratory grade standard powder (849 µg/mg). MICs and MBCs were determined in triplicate by broth microdilution for Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 in cation-supplemented Mueller–Hinton broth.4 The inoculum was 5 x 105 cfu/mL, and trays were incubated at 35°C for 24 h. MBCs were determined by subculturing 10 µL from each clear well onto antibiotic-free Mueller–Hinton agar and incubating as described above. Standard definitions for MIC and MBC were used.

Serum samples from each subject were spiked with ceftazidime to achieve concentrations of 75 and 5 mg/L, which approximate 45 min peak and 8 h trough concentrations, respectively, following administration of 2 g iv.5 Unspiked serum was also tested. The volume of ceftazidime stock solution added to the serum samples was <5% of the total volume. Serum inhibitory and bactericidal titres (SIT and SBT) were determined in triplicate using microdilution methodology.6 For each subject, 100 µL of serum spiked with ceftazidime was added to the first well of a microtitre tray and serially diluted in 50 µL of pooled human serum that had been heated at 56°C for 1 h and shown to be negative when screened for antimicrobial activity. The microtitre plates were inoculated with 50 µL of the test organisms in broth so that the starting inoculum was 5 x 105 cfu/mL (verified in duplicate by colony count determination). The final volume in each well was 100 µL, and the dilution range was 1:2–1:1024.

After incubation at 35°C for 24 h, the SIT was defined as the highest dilution that prevented visual turbidity. For SBT determinations, a 20 µL sample was removed from each clear well, plated onto trypticase soy agar, and incubated at 35°C for 24 h. The SBT was defined as the highest dilution that resulted in >=99.9% killing of the starting inoculum. All of the titres for each organism were performed at the same time.

Differences between groups in age, white blood cell count and absolute neutrophil count were determined using a one-way analysis of variance (Scheffe post hoc test). Geometric mean, median and range for the SIT and SBT were calculated for each organism and subject population. Following logarithmic transformation of the reciprocal titre, differences in SIT and SBT between groups were determined using a one-way analysis of variance (Scheffe post hoc test). Statistical significance for all analyses was defined as P < 0.05. Observed SIT and SBT were compared with predicted titres calculated by dividing the drug concentration by the median MIC and MBC, respectively, and rounding to the next lowest dilution. For example, the predicted SIT is 1:32 for a ceftazidime concentration of 75 mg/L and an MIC of 2 mg/L, since a titre of 1:37.5 cannot be achieved and the drug concentration is insufficient for an SIT of 1:64.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient demographics and serum characteristics are shown in Table 1Go. The age of the uraemic volunteers was higher than the normal volunteers (P = 0.019). The WBC and absolute neutrophil counts for the neutropenic patients were lower than the other groups (P < 0.0001). Also, the absolute neutrophil count for the normal volunteers was lower than the uraemic volunteers (P = 0.016). The SIT and SBT from all unspiked (ceftazidime-free) subjects' sera were <1:2.


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Table 1. Patient demographics and serum characteristics (values represent mean ± s.d.)
 
The reciprocal geometric means, medians and ranges for the SIT and SBT for spiked sera are shown in Table 2Go. For E. coli, the trough SIT for burn patients was higher than for normal volunteers (P = 0.014) and neutropenic patients (P = 0.022). The trough SIT for uraemic volunteers and the trough SBT for burn patients were higher than for normal volunteers (P = 0.04 and P = 0.024, respectively). For P. aeruginosa, no differences were found between groups for SIT and SBT at either peak or trough concentrations.


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Table 2. Reciprocal SIT and SBT
 
For every study population, the predicted and observed peak SIT for P. aeruginosa were the same (1:32), but the observed peak SBT was two-fold lower than the predicted value. Conversely, the observed trough SIT and SBT titres were two-fold higher than the predicted titres. In the burn patients, there was a strong correlation between the percent total body surface area burn and the log of the trough SBT for P. aeruginosa (r = –0.879).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study was designed to determine whether incorporation of serum from different patient populations into the serum bactericidal test would affect the SIT and SBT for E. coli ATCC 25922 and P. aeruginosa ATCC 27853. Serum samples were spiked with ceftazidime to control for the potential pharmacokinetic variability between the subject populations. Using the geometric mean of the reciprocal titre, the only differences were seen in the trough SIT and SBT for E. coli. However, these differences would not be detected if the median two-fold dilution titres were used. For P. aeruginosa, the median trough titres were also identical for the subject populations. The explanation for differences in trough titres for E. coli but not P. aeruginosa is unknown. Neither organism appeared to be serum-sensitive, since all titres of the subject's serum without ceftazidime were <1:2.

Patients with neutropenia and burn injury are highly susceptible to bacterial infections. In our study, serum from neutropenic and burn patients does not appear to be less bactericidal than normal volunteers. Sculier & Klastersky1 reported that neutropenic patients required higher SBT than non-neutropenic patients for a favourable response in Gram-negative bacillary bacteraemia (>=1:16 versus >=1:8, P < 0.0001). However, it is not known whether this requirement was because of inadequate host defences in neutropenic serum, pharmacokinetic differences between groups or higher MICs for pathogens encountered in neutropenic patients. A previous study showed that the bactericidal activity of blood and plasma from burn patients was comparable to normals.7 Although trough titres for P. aeruginosa in burn patients were not different from other groups, there was a correlation between extent of the burn injury and trough SBT (r = –0.879).

No differences among subject populations were seen in peak SIT and SBT for either organism. This may be owing to the fact that, at higher dilutions, the patient's serum has been diluted so that serum factors may be present in insufficient concentrations to produce a demonstrable antibacterial effect. Only 6.25% of a patient's serum remains at a titre of 1:16, and the bactericidal activity of the alternate complement pathway has been shown to be insignificant when the essential proteins of this pathway are present in concentrations equivalent to a 1:16 dilution of serum.3 Therefore, the bactericidal activity at peak antibiotic concentrations may be attributed primarily to the drug, with little to no contribution of serum factors, at higher dilutions.

Some investigators have suggested that serum bactericidal activity can be predicted by dividing the drug concentration by the MBC.8,9 Similarly, serum inhibitory activity should be predicted by dividing the drug concentration by the MIC. With a ceftazidime concentration of 5 mg/L, an MIC of 2 mg/L and an MBC of 4 mg/L, the predicted SIT and SBT would be 1:2 and <1:2, respectively, for P. aeruginosa. However, in our study, the observed trough SIT and SBT were two-fold higher than the predicted titres for each subject population. This effect may be due to the additive and/or synergic activity of the subject's serum and ceftazidime. Dividing the drug concentration by the MIC or MBC may not accurately predict the titres, since MIC tests utilize two-fold dilutions and the actual MIC may be between two concentrations. With an MIC of 2 mg/L, the actual MIC is >1 mg/L and <=2 mg/L. At a drug concentration of 5 mg/L, the predicted SIT would be 1:4 if the actual MIC is between 1 and 1.25 mg/L, but the predicted SIT would be 1:2 if the actual MIC is between 1.25 and 2 mg/L.

The area under the inhibitory serum concentration–time curve (AUIC24) is a surrogate marker for clinical outcome, and an AUIC24 >= 125 has been associated with efficacy.10 Other investigators have measured the AUIC24 as the reciprocal SIT versus time (SIT–1•h),10 but the ability of the reciprocal SIT versus time to predict accurately the AUIC24 is questionable. If an antibiotic is administered every 12 h and produces 1 h peak and 12 h trough concentrations of 63 and 15 mg/L, respectively, the AUIC24 would be 117 for a pathogen with an MIC of 8 mg/L. At these concentrations and MIC, the predicted peak and trough SIT would be 1:4 and <1:2, respectively. Using a reciprocal titre of 1 for <1:2, the 24 h reciprocal SIT versus time would be 60, a value almost half of the calculated AUIC24. There are two problems with using this method to measure AUIC24. First, the two-fold nature of the dilutions limits the accurate assessment of the SIT. If intermediate titres are performed (e.g. 1:3, 1:6, etc.), the accuracy of the reciprocal SIT versus time curve to measure AUIC24 may be improved, but this is methodologically cumbersome. Secondly, the observed titre may be higher than the predicted titre because of the potential antibacterial activity of the serum. Additional studies are needed to determine the accuracy of estimating the AUIC24 using the SIT.

In summary, serum from different patient populations appears to have a minimal effect on the serum bactericidal test. Although differences were seen in the trough SIT and SBT for E. coli, the median SIT and SBT for both test organisms were very similar for each study population. Measured titres were not in full agreement with the mathematically predicted titres, and further studies are needed to assess the ability to predict titres using the antimicrobial concentration and the MIC or MBC for the organism.


    Notes
 
* Correspondence address. Purdue University School of Pharmacy, Department of Pharmacy Practice, D711 Myers Building, WHS, 1001 West Tenth Street, Indianapolis, IN 46202-2879, USA. Tel: +1-317-613-2315, ext. 319; Fax: +1-317-613-2316; E-mail: mkays{at}iupui.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Sculier, J. P. & Klastersky, J. (1984). Significance of serum bactericidal activity in gram-negative bacillary bacteremia in patients with and without granulocytopenia. American Journal of Medicine 76, 429–35.[ISI][Medline]

2 . Weinstein, M. P., Stratton, C. W., Ackley, A., Hawley, H. B., Robinson, P. A., Fisher, B. D. et al. (1985). Multicenter collaborative evaluation of a standardized serum bactericidal test as a prognostic indicator in infective endocarditis. American Journal of Medicine 78, 262–9.[ISI][Medline]

3 . Taylor, P. W. (1983). Bactericidal and bacteriolytic activity of serum against gram-negative bacteria. Microbiological Reviews 47, 46–83.[ISI]

4 . National Committee for Clinical Laboratory Standards. (1988). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-T2. NCCLS, Villanova, PA.

5 . Drusano, G. L., Standiford, H. C., Fitzpatrick, B., Leslie, J., Tangtatsawasdi, P., Ryan, P. et al. (1984). Comparison of the pharmacokinetics of ceftazidime and moxalactam and their microbiological correlates in volunteers. Antimicrobial Agents and Chemotherapy 26, 388–93.[ISI][Medline]

6 . Reller, L. B. & Stratton, C. W. (1977). Serum dilution test for bactericidal activity. II. Standardization and correlation with antimicrobial assays and susceptibility tests. Journal of Infectious Diseases 136, 196–203.[ISI][Medline]

7 . Ward, C. G., Spalding, P. B., Marcial, E. & Bullen, J. J. (1991). The bactericidal power of the blood and plasma of patients with burns. Journal of Burn Care and Rehabilitation 12, 120–6.[Medline]

8 . Stratton, C. W., Weinstein, M. P. & Reller, L. B. (1982). Correlation of serum bactericidal activity with antimicrobial agent level and minimal bactericidal concentration. Journal of Infectious Diseases 145, 160–8.[ISI][Medline]

9 . Drusano, G., Standiford, H., Ryan, P., McNamee, W., Tatem, B. & Schimpff, S. (1986). Correlation of predicted serum bactericidal activities and values measured in volunteers. European Journal of Clinical Microbiology 5, 8(–92.

10 . Hyatt, J. M., McKinnon, P. S., Zimmer, G. S. &+ Schentag, J. J. (1995). The importance of pharmacokinetic/pharmacodynamic surrogate markers to outcome. Focus on antibacterial agents. Clinical Pharmacokinetics 28, 143–60.[ISI][Medline]

Received 13 July 2000; returned 20 February 2001; revised 8 May 2001; accepted 29 May 2001