a Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, Km. 2, 28220 Majadahonda, Madrid; b Medical Department, GlaxoSmithkline, Tres Cantos, Madrid, Spain
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Abstract |
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Introduction |
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Bacterial virulence is the minimal bacterial mass capable of producing injury to a given host4 and can be defined as the competence of an infectious agent to produce pathological effects, as indicated by case fatality rates and/or the ability to invade the host.5 This study explores the modification by ß-lactam therapy of the bacteraemic profile (representative of ability to invade the host) produced by a bacterial mass (representative of virulence) and its relationship with mortality rates (representative of competence to produce pathological effects). A serotype 6 penicillin-resistant isolate of Streptococcus pneumoniae was used as infecting strain, as this is one of the most frequently isolated serotypes in bacteraemia and respiratory-tract infections in Spain.6,7
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Materials and methods |
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A serotype 6B S. pneumoniae strain isolated from a blood culture [MIC and minimum bactericidal concentration (MBC) of penicillin 2 and 4 mg/L, respectively] was used. The microorganism was grown until an absorbance of 0.3 (UV-VIS spectrophotometer, Shiimadzu UV-1203, Japan) was obtained in ToddHewit broth supplemented with 0.5% yeast extract (THYB) (Difco, Detroit, MI, USA), and aliquots were stored at -70°C in 15% glycerol.
In vitro studies
MICs and MBCs of amoxicillin and cefotaxime were determined by broth dilution following NCCLS procedures.8 Modal values of five separate determinations were considered.
Animals
Eight- to 12-week-old female BALB/c mice weighing 1922 g were used. The study was approved by the Spanish Central Laboratory of Public HealthInstituto de Salud Carlos III.
Determination of minimal lethal dose
Groups of 10 mice per dilution were injected intraperitoneally with 0.2 mL of different inocula, 102, 104, 106 and 108 cfu/mL (spectrometrically measured), to determine the minimal dose that produced a 100% mortality over a 15 day follow-up period (minimal lethal dose, MLD). Bacteria in a logarithmic phase of growth in THYB were centrifuged and the pellets washed three times and resuspended in PBS pH 7.2 to reach the desired turbidity. The inoculum was confirmed by the culture of serial dilutions onto blood MuellerHinton agar incubated at 37°C in 5% CO2 air. Mouse mortality was recorded daily. The MLD was determined from the results obtained in three independent experiments.
Dose-ranging treatment
Survival and the bacteraemic profile of inoculated animals over a 15 day follow-up period were determined in a dose-ranging study with amoxicillin and cefotaxime doses ranging from 6.25 to 50 mg/kg. Animals were inoculated intraperitoneally with 200 µL of the MLD, and antibiotic treatment was initiated 1 h after bacterial inoculation. Groups of five animals per dose were treated with 100 µL subcutaneously tds for 48 h. The five animals of the control group received placebo (apyrogen sterile distilled water). Animals were observed and deaths were recorded for 15 days.
Blood samples were obtained daily (except on day 1 when they were collected at 2, 6 and 24 h) over the 15 day follow-up period, from five animals per antibiotic dose, to study the bacteraemic profile. Tails were disinfected and anaesthetized, and the end portion of the tail was amputated with scissors. Using a calibrated loop, 0.008 mL of blood were taken and resuspended in ToddHewitt broth containing 10% trisodium citrate and plated onto blood agar for colony counting to obtain the first sample. Plates were incubated at 37°C in 5% CO2 air for 24 h. To obtain the subsequent blood samples, the crust was removed and the anaesthetized tail was pressed to collect the 0.008 mL. The lower limit of detection was 1.25 x 102 cfu/mL.
Determination of antibiotic concentrations in serum
Amoxicillin and cefotaxime concentrations in serum were determined in healthy animals after a single subcutaneous dose of the dose that obtained 100% survival or of the maximum dose tested if 100% survival was not achieved. Blood samples were collected 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h and 8 h after dose administration from groups of five animals per dose and antibiotic. Concentrations were measured by bioassay using Micrococcus luteus ATCC 4698 for amoxicillin and Escherichia coli ATCC 25922 for cefotaxime as reference organisms in 9 cm diameter plates with 14 mL of antibiotic agar no. 2 (Difco) for amoxicillin and MuellerHinton agar for cefotaxime, containing a final inoculum of 8 x 108 cfu/mL. Aliquots (30 mL) of each sample were deposited into 6 mm diameter wells in inoculated plates that were incubated at 36.5°C for 18 h. Standards containing 0.0121.6 and 0.450 mg/L were prepared in apyrogen distilled water for amoxicillin and cefotaxime, respectively, to determine the assay regression line (standard curve) and to extrapolate the antibiotic concentrations from the corresponding inhibition zone diameters.
Pharmacokinetic study
Concentrationtime curves for each antibiotic were analysed by a non-compartmental approach using the Win-Nonlin program (Pharsight, Mountainview, CA, USA). The areas under the serum concentrationtime curves 0 h to (AUC0
) were calculated from the equation AUC0
= AUC0480min + AUC480min
. The values for AUC0480min were calculated from plots of serum concentrations versus time by using the trapezoidal rule. The values for AUC480min
were calculated from the expression AUC480min
= C480min/ß, where ß is the slope obtained from least-square regression of the terminal elimination phase. The value of ß was calculated for each antibiotic using at least the last three sample time values of serum concentrations. The theoretical concentration at time 0 (obtained by back-extrapolation to the origin of the elimination regression line) was considered the maximum concentration in serum (Cmax). Time above MIC (
T > MIC) was calculated graphically from the semi-logarithmic plot representing the concentrationtime data.
Statistical analysis
Survival curves were obtained by the KaplanMeier method. A dose-adjusted Cox regression analysis was used to compare survival with each antibiotic. A Probit regression analysis was carried out to calculate the 50% efficacy dose (EC50) in the theoretical model using the dose as covariable and the antibiotic as factor. A binary logistic regression analysis was used to study the relationship between the maximum cfu/mL and outcome (death/survival) per individual.
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Results and discussion |
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Figures 1 and 2 show the mean bacteraemic profile (blood culture cfu/mL over 360 h) with the different amoxicillin and cefotaxime doses, with animals divided in each study group by outcome: death and survival. As can be seen, mean blood colony counts in surviving mice were
106 cfu/mL over the 360 h. In contrast, maintained mean bacteraemic colony counts >106 cfu/mL over the first 144 h were found in those animals that died. This can be clearly seen with the amoxicillin 12.5 mg/kg and cefotaxime 50 mg/kg regimens, where some animals survived and some died, and where both bacteraemic profiles were present. A statistically significant (P = 0.012) relationship was found between the maximum cfu/mL in blood and mortality, with ß0 = -15.21 and ß1 = 2.5944 as logistic regression coefficients. In the theoretical model, a maximum log cfu/mL of 6.5 was associated with an 84% probability of death.
In contrast to previous studies, where pneumococcal counts in blood were poorly related to the outcome of infection in penicillin-treated animals,10 in the present study, using a penicillin-resistant strain, bacterial counts were good indicators of mouse survival and death. The two ß-lactams (amoxicillin to a higher degree) reduced fatality rates of the serotype 6B pneumococcal strain by decreasing colony counts of the bacteraemic profile.
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Acknowledgements |
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Notes |
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References |
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2 . Frimodt-Moller, N. (1993). The mouse peritonitis model: present and future use. Journal of Antimicrobial Chemotherapy 31 Suppl. D, 5560.[ISI][Medline]
3 . Amsterdam, D. (1990). Assessing cidal activity of antimicrobial agents: problems and pitfalls. The Antimicrobic Newsletter 7, 4956.
4 . Lorian, V. & Gemmell C. G. (1991). Effect of low antibiotic concentrations on bacteria: effects on ultrastructure, virulence, and susceptibility to immunodefenses. In Antibiotics in Laboratory Medicine, 3rd edn, (Lorian, V., Ed.), pp. 493555. Williams & Wilkins, Baltimore, MD.
5 . Gemmell, C. G. & Lorian, V. (1996). Effects of low concentrations of antibiotics on bacterial ultrastructure, virulence, and susceptibility to immunodefenses: clinical significance. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed.), pp. 397452. Williams & Wilkins, Baltimore, MD.
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.
Fenoll, A., Jado, I., Vicioso, D., Pérez, A. & Casal, J. (1998). Evolution of Streptococcus pneumoniae serotypes and antibiotic resistance in Spain. An update: 19901996. Journal of Clinical Microbiology 36, 344754.
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.
Marco, F., Bouza, E., García-de-Lomas, J., Aguilar, L. & the Spanish Surveillance Group for Respiratory Pathogens. (2000). Streptococcus pneumoniae in community-acquired respiratory tract infections in Spain: the impact of serotypes, geographical, seasonal and clinical factors on its susceptibility to the most commonly prescribed antibiotics. Journal of Antimicrobial Chemotherapy 46, 55764.
8 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility TestingEleventh Informational Supplement: Document M100-S11. NCCLS, Wayne, PA.
9 . Craig, W. A. (1998). Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clinical Infectious Diseases 26, 110.[ISI][Medline]
10 . Frimodt-Moller, N. & Frolund Thomsen, V. (1986). The pneumococcus and the mouse protection test: inoculum, dosage and timing. Acta Pathologica Microbiologica et Immunologica Scandinavica Sect. B 94, 337.
Received 15 June 2001; returned 25 September 2001; revised 17 October 2001; accepted 12 November 2001