Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
Sir,
Typhoid fever is an intracellular infection of humans with Salmonella typhi that responds to treatment with azithromycin.1,2 To be effective the antibiotic must penetrate into a cellular milieu that has been affected by growth of S. typhi. Previous work showed that the MIC of azithromycin was lower in medium exposed to previous growth of S. typhi,3 suggesting that azithromycin could be more active in tissues than indicated by measurements of standard MIC. The following experiments were designed to examine medium exposed to previous growth of S. typhi for activity of azithromycin and the mechanism for reduction of MIC in the exposed medium.
Five strains of S. typhi from blood cultures of patients in Egypt and India who participated in clinical trials were used. Staphylococcus aureus ATCC 29213 was used for MIC assays. Bacterial colonies on MacConkey agar or blood agar plates (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) were inoculated into trypticase soy broth (TSB) and incubated for 24 h to reach log phase growth. Dilutions of TSB were adjusted to 0.5 MacFarland standard (Remel, Lenexa, KS, USA) to inoculate MuellerHinton II cation-adjusted broth (MHB) (Becton Dickinson Company) with 5 x 105 bacteria/mL.4 Azithromycin (047451-065-02) was obtained from Pfizer, Inc. (Groton, CT, USA), dissolved in ethanol and diluted in water for two-fold dilutions in MHB. Tubes were examined after 24 h of incubation at 37°C for turbidity. To examine broth exposed to bacterial growth, strains of S. typhi were inoculated into MHB for incubation at 37°C for 24 h. Bacteria were removed from broth by centrifugation at 2500g for 30 min, and the supernatant was sterilized with a 0.45 µm syringe filter (Gelman Sciences, Ann Arbor, MI, USA). The sterile supernatant was inoculated again with the same bacterial strain for incubation, centrifugation and sterile filtration for each of three additional passages. After the fourth passage, broth was designated as exposed to previous growth of S. typhi. Exposed broth was concentrated three-fold by lyophilizing broth and resuspending solids in one-third the original volume of water. Sterile water was added to the broth to obtain a 1:3 dilution.
Growth curves were made by inoculating MHB with estimated concentrations of 102 bacteria/mL. Actual cfu were determined by plating dilutions of broth on to MacConkey agar. Tubes were incubated at 37°C, and at 4, 8, 24 and 48 h aliquots were diluted and plated for cfu. The pH of media was measured with an electrode (G-P combo w/rj; Corning, NY, USA). Adjustment of exposed broth to c. pH 7.2 was achieved by adding 0.1 M citric acid (Fisher Scientific, Fair Lawn, NJ, USA) dropwise.
For five strains of S. typhi in seven experiments, MICs of azithromycin were four- to 32-fold lower in MHB exposed to previous growth of S. typhi than in MHB. The median of MICs in exposed broth was eight-fold lower than MICs in MHB. For S. aureus in five experiments, MICs were also four- to 32-fold lower in exposed MHB, with medians of MICs showing an eight-fold reduction (Table 1). Visual inspection of tubes without antibiotic in MIC assays revealed that turbidity of S. typhi growth was less dense in exposed broth than in MHB. Growth curves of S. typhi revealed slower growth in exposed broth after 4 and 8 h of incubation. After 24 h incubation, cfu in exposed broth were >10-fold less than in MHB, and after 48 h incubation cfu remained at a lower density.
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The median MIC in concentrated exposed media increased 12-fold above media that were not concentrated, and the MIC in diluted media decreased two-fold (Table 1). The lowest MIC of 0.5 mg/L for S. typhi occurred in diluted exposed broth. Against S. aureus, the same pattern of increased MIC in concentrated medium was obtained, and diluted medium gave the same MIC as undiluted medium (Table 1). These results did not indicate the presence of an inhibitor of bacterial growth in exposed medium.
These results indicated that MICs of azithromycin in medium exposed to previous growth of S.typhi were approximately eight-fold lower than in standard unexposed medium. The mechanism of the reduction of MIC was attributed to an effect of nutrient depletion shown by a slower rate of growth of S. typhi in exposed medium and inability of bacteria after 2448 h of incubation to attain the maximal density of organisms that occurred in unexposed medium. An additional mechanism was alkaline pH, which increased to >7.6 after four passages of S. typhi, because adjustment of pH back to <7.2 partially reversed the reduction in MIC in exposed medium. The contribution of alkaline pH to the reduction in MIC is consistent with the earlier report of a strong effect of buffered broth at a pH of >7.5 to reduce the MIC of azithromycin.5
High intracellular concentrations of azithromycin help to explain its clinical efficacy despite MICs that are higher than achievable concentrations of antibiotic in blood.6 In addition to the high intracellular concentrations, our results indicate that azithromycin may provide better antibacterial performance in infected tissues because growth of S. typhi modifies its environment to render it favourable for enhanced effect of the antibiotic.
Footnotes
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References
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2 . Frenck, R. W., Nakhla, I., Sultan, Y., Bassily, S. B., Girgis, Y. F., David, J. et al. (2000). Azithromycin vs ceftriaxone for the treatment of uncomplicated typhoid fever in children. Clinical Infectious Diseases 31, 11348.[ISI][Medline]
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Butler, T. (2001). Effect of increased inoculum of Salmonella typhi on MIC of azithromycin and resultant growth characteristics. Journal of Antimicrobial Chemotherapy 48, 9036.
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5
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Butler, T., Frenck, R. W., Johnson, R. B. & Khakhria, R. (2001). In vitro effects of azithromycin on Salmonella typhi: early inhibition by concentrations less than the MIC and reduction of MIC by alkaline pH and small inocula. Journal of Antimicrobial Chemotherapy 47, 4558.
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