Journal of Antimicrobial Chemotherapy (2001) 48, 594-595
© 2001 The British Society for Antimicrobial Chemotherapy
Correspondence |
Enhanced decrease of blood colony counts by specific anti-pneumococcal antibodies in the presence of sub-inhibitory concentrations of amoxicillin
a Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid; b Medical Department, GlaxoSmithKline, Madrid, Spain
Sir,
Several studies have demonstrated the combined activity of ß-lactams and non-specific immunity (opsonophagocytosis) in the decrease of a Streptococcus pneumoniae initial inoculum in vitro or ex vivo.1–3 On the other hand, with respect to specific immunity, a combined effect of the presence of specific anti-capsular antibodies and penicillins has been demonstrated in animal models using mortality as end-point.4–6 This study investigates the combined effect of the presence of specific antibodies and amoxicillin sub-inhibitory concentrations on the bacteraemic profile (blood colony counts over time) in a mouse sepsis model using a penicillin-resistant, amoxicillin non-susceptible serotype 6B S. pneumoniae (MIC/MBC = 2/4 mg/L of penicillin and 4/4 mg/L of amoxicillin).
Hyperimmune serum was obtained by immunizing 8- to 12-week-old female BALB/c mice with the heatinactivated strain, as described previously.6 Twenty animals were infected intraperitoneally with the pneumococcal challenge dose (108 cfu/mL) as described previously6 and monitored for 21 days. Five were used as unprotected and untreated controls and received placebo (pyrogenfree distilled water). Five were treated with amoxicillin 3.12 mg/kg (amoxicillin-treated group), administered 100 µL subcutaneously tds for 48 h, starting treatment 1 h after the pneumococcal challenge. Five received one single intraperitoneal 200 µL injection of a 0.25 dilution of the hyperimmune serum (seroprotection group), 1 h before the challenge dose, and five received both the hyperimmune serum and the antibiotic treatment (seroprotection + amoxicillin group) as described above.
Blood samples were obtained daily (except on day 1 when they were collected at 2, 6 and 24 h) over the 21 day follow-up period to study the bacteraemic profile. Tails were disinfected and locally anaesthetized, and the end portion of the tail was cut off with scissors, taking 0.008 mL of blood with a calibrated loop which was resuspended in Todd–Hewitt broth containing 10% trisodium citrate and plated on to blood agar for colony counting, for the first sample. Plates were incubated at 37°C in 5% CO2 air for 24 h. To obtain the following blood samples, the crust was removed and the anaesthetized tail was pressed to collect the 0.008 mL blood sample. The lowest detectable count was 1.25 x 102 cfu/mL.
Mortality over the follow-up period was recorded. Survival was 0% in control mice (three dead within the first 24 h, one at 48 h and one at 72 h), and in the amoxicillin-treated group (four dead at 48 h and one at 120 h). In the seroprotection group, hyperimmune serum protected 60% of the animals (one dead at 192 h and one at 264 h) while in the seroprotection + amoxicillin group, all mice survived.
The Figure
shows the mean colony counts over time for the four study groups, dividing the seroprotection group into two subgroups: dead and surviving. As can be seen, mean bacterial counts were 
107 cfu/mL until death in groups of dead mice (all mice in the control and the amoxicillin-treated groups and two in the seroprotection group), while surviving mice (all mice in the seroprotection + amoxicillin group and three in the seroprotection group) had mean colony counts <107 cfu/mL over time. Mean colony counts were <106 cfu/mL over time in the seroprotection + amoxicillin group. A minimum of 3 log decrease (from 106 cfu/mL to <103 cfu/mL) is seen in surviving mice included in the seroprotection group between day 12 and day 17; with the addition of amoxicillin, this figure was achieved between day 10 and day 12. Disappearance of bacteraemia (colony counts under the detection limit) was obtained at day 20 in surviving mice in the seroprotection group and at day 17 when amoxicillin was added, thus indicating an acceleration of bacterial killing, which resulted in a decrease in mortality. The synergy of a ß-lactam agent and the immune system (resulting in an earlier bactericidal killing) has been demonstrated in vitro with non-specific immunity.1,2 In this study, the role of non-specific immunity could be ruled out, since complement and polymorphonuclear neutrophils (PMN) were also present in mice included in the group that only received amoxicillin and had a 100% mortality. Therefore, the results obtained in the seroprotection + amoxicillin group can be attributed mainly to the effect of amoxicillin sub-therapeutic doses and passive specific immunity, in vivo presence of specific anti-capsular serotype 6 antibodies. These results raise the possibility of a way forward in treating infections caused by penicillin-resistant pneumococci.
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, inoculum; —
---, serum 0.25 (survival); —•—, amoxicillin 3.12 mg/kg + serum 0.25.Notes
* Corresponding author. Tel: +34-91-509-7975; Fax: +34-91-509-7966; E-mail: jcasal{at}isciii.es 
References
1
.
Gómez-Lus, M. L., Aguilar, L., Martín, M., Giménez, M. J., Martínez, P. & Prieto, J. (1997). Intracellular and extracellular killing of a penicillin-resistant, serotype-9 strain of Streptococcus pneumoniae by polymorphonuclear leucocytes in the presence of sub-inhibitory concentrations of clavulanic acid. Journal of Antimicrobial Chemotherapy 40, 142–4.
2 . Martín, M., Gómez-Lus, M. L., Aguilar, L., Martínez, P., Giménez, M. J. & Prieto, J. (1997). Effect of clavulanic acid and/or polymorphonuclear neutrophils on amoxicillin bactericidal activity against Streptococcus pneumoniae. European Journal of Clinical Microbiology and Infectious Diseases 16, 512–6.[Web of Science][Medline]
3 . Gómez-Lus, M. L., Giménez, M. J., Prieto, J., Martín, M., Frías, J. & Aguilar, L. (1998). Effect of polymorphonuclear neutrophils on serum bactericidal activity against Streptococcus pneumoniae after amoxicillin administration. European Journal of Clinical Microbiology and Infectious Diseases 17, 40–3.[Web of Science][Medline]
4 . Chudwin, D. S. (1989). Prophylaxis and treatment of pneumococcal bacteremia by intravenous immune globulin in a mouse model. Clinical Immunology and Immunopathology 50, 62–71.[Medline]
5
.
De Hennezel, L., Ramisse, F., Binder, P., Marchal, G. & Alonso, J. (2001). Effective combination therapy for invasive pneumococcal pneumonia with ampicillin and intravenous immunoglobulins in a mouse model. Antimicrobial Agents and Chemotherapy 45, 316–8.
6 . Casal, J., Jado, I., Aguilar, L., Yuste, L., Giménez, M. J., Gómez-Lus, M. L., Prieto, J. & Fenoll, A. (2001). Increase of the amoxicillin in vivo efficacy by hyperimmune serum therapy: A dose-ranging pneumococcal sepsis mice model. In Twenty-second International Congress of Chemotherapy, Amsterdam, 2001. Electronic Poster EP 1.12. [On-line.] http://eurocongres.com/icc/scientific.html
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