Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA
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Abstract |
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Introduction |
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Methods |
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Nine strains of S. typhi that were blood culture isolates from patients in Egypt and India were used.1 Bacteria were grown in trypticase soy broth (TSB) for 24 h at 37°C to reach log phase growth. Tubes of cation-adjusted Mueller Hinton broth (CAMHB) were inoculated with bacteria at estimated concentrations of 107 and 103 bacteria/mL for large and small inocula, respectively, using dilutions of broth that corresponded to a 0.5 McFarland standard (Remel, Lenexa, KS, USA). Actual cfu were determined by plating dilutions of broth onto MacConkey agar (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). Azithromycin (Lot 25381-087-02; Pfizer Inc., Groton, CT, USA) was dissolved in ethanol. Two-fold dilutions of azithromycin from 2 to 32 mg/L were prepared in CAMHB. Tubes were incubated at 37°C and at 4, 8, 24 and 48 h aliquots were diluted and plated for cfu. Aliquots of turbid broth were placed onto microscope slides, fixed by heating, and stained with Gram's stain (Becton Dickinson). For clear tubes containing small inocula, bacteria were concentrated by centrifugation at 2000g for 15 min, and the pellet resuspended in 0.1 mL of 0.9% NaCl for placement onto slides.
Susceptibility of bacteria to azithromycin after exposure
Broths were subcultured onto MacConkey agar plates to determine the numbers of cfu at 48 h of incubation. Colonies that survived in the presence of 16 mg/L were compared with colonies that were not exposed to antibiotic. Colonies were grown to log phase in TSB and a standard inoculum of 5 x 105/mL used in CAMHB for determination of MIC in a series of two-fold dilutions of antibiotic.3
Assays for inactivation and removal of azithromycin by large inocula of S. typhi
CAMHB was inoculated with c. 107 cfu S. typhi/mL and incubated for 24 h at 37°C. Bacteria were removed by centrifugation at 2500g for 30 min and the supernatant sterilized with a 0.2 µm syringe filter (Gelman Sciences, Ann Arbor, MI, USA). For assays of removal of antibiotic, CAMHB containing 4 and 8 mg/L was inoculated with c. 107 cfu S. typhi/mL and incubated at 37°C for 24 h. Bacteria were removed by centrifugation and sterile filtration. Bacteria used for MIC assays using filtered broth were Staphylococcus aureus ATCC 29213 and Streptococcus agalactiae ATCC 13183.
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Results |
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Observing the tubes for growth curves at 24 h, large inocula showed MICs of 32 mg/L for seven strains and 16 mg/L for two strains. Small inocula showed MICs of 8 mg/L for five strains and 4 mg/L for four strains, indicating an overall more than four-fold increase in MIC for the large inocula compared with the small inocula. Growth to turbidity with cfu >108/mL occurred in tubes without azithromycin after 4 h with large inocula and at 24 h with small inocula (Figure 1). In the presence of 4 mg/L the large inocula grew to turbidity at 4 h although cfu did not reach the same levels as culture tubes without antibiotic. In the presence of 8 mg/L large inocula increased about 1 log at 4 h with faint turbidity visible in the tubes, and further increases in cfu after 24 h, whereas small inocula were suppressed throughout 48 h without development of turbidity. Large inocula in the presence of 16 mg/L showed a decrease in mean cfu of about 1.5 log by 24 h, but turbidity appeared in seven out of nine strains at 824 h, indicating an increase in bacterial mass during bacteriostasis; at 48 h there were increases in cfu of
1 log in four strains, resulting in a significant rise in mean cfu at 48 h. Large inocula in the presence of 32 mg/L showed consistent decreases in cfu without development of turbidity (Figure 1
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Large inocula in the presence of 416 mg/L developed elongated and widened forms as early as 2 and 4 h after exposure, with higher proportions of abnormal morphology evident at 24 and 48 h. Tubes that became turbid during growth inhibition, indicated by the number of cfu remaining unchanged or decreasing, also showed abnormal forms (Figure 2). Cultures exposed to 32 mg/L of azithromycin contained fewer bacteria than lower concentrations, with up to 50% showing widened, slightly elongated or beaded forms as early as 2 h after exposure, but greater elongation did not proceed after 24 h of exposure. Small inocula in tubes containing 8 and 16 mg/L that did not develop turbidity also produced elongated forms.
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MICs of colonies obtained after exposure of large inocula were 1632 mg/L, with a geometric mean of 21.1 mg/L, whereas colonies of the same strains not exposed to antibiotic showed MICs of 416 mg/L, with a mean of 9.8 mg/L. This two-fold greater MIC was consistently observed in all experiments and was persistent when a second passage was tested in colonies obtained from MacConkey agar plates used to subculture the strains.
Assays for inactivation and removal of azithromycin by large inoculum of S. typhi
MICs of azithromycin for S. aureus and S. agalactiae in broth that had been exposed to a large inoculum of S. typhi for 24 h were two- to four-fold lower than control broth, indicating no inactivation of antibiotic by substances secreted by S. typhi into the broth. Similarly, broth containing 4 and 8 mg/L exposed to a large inoculum of S. typhi for 24 h did not show loss of antibiotic, with MICs remaining unchanged or changed by less than two-fold.
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Discussion |
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The effect of a large inoculum in decreasing azithromycin activity against S. typhi differs from the result of Odenholt et al.,5 who found that bactericidal activity against S. pyogenes and H. influenzae was not affected by inoculum size. The reasons for the difference may be the use of 10 times the MIC in assays reported by Odenholt et al.,5 whereas the present studies used concentrations that were within two to four times the MIC. Additionally, there may be differences in responses to variations of inoculum size because of lower MICs for S. pyogenes and H. influenzae.
Other Gram-negative bacilli, Pseudomonas aeruginosa and Enterobacter spp., were shown to exhibit an inoculum effect with cefepime, other ß-lactam antibiotics and ciprofloxacin.6 This inoculum effect was attributed to greater cumulative activity of ß-lactamase and not to the emergence of resistant mutants during exposure to antibiotic.6 The inoculum effect of azithromycin is different from that of cefepime, because inactivating enzymes have not been described and after exposure to azithromycin S. typhi developed low-grade resistance.
Quantitative cultures of blood and bone marrow in patients with typhoid fever revealed <102 bacteria/mL in most cases.7 The clinical implication of the inoculum effect is that azithromycin may perform better in patients than indicated by the MIC using the standard inoculum of 5 x 105/mL.3
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Acknowledgements |
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Notes |
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References |
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2
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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 MIC and reduction of MIC by alkaline pH and small inocula. Journal of Antimicrobial Chemotherapy 47, 4558.
3 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Testing for Bacteria that Grow Aerobically: Approved Standard M7-A4. NCCLS, Wayne, PA.
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Pankuch, G. A., Jueneman, S. A., Davies, T. A., Jacobs, M. R. & Appelbaum, P. C. (1998). In vitro selection of resistance to four ß-lactams and azithromycin in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 29148.
5 . Odenholt, I., Lowdin, E. & Cars, O. (1997). Studies of the killing kinetics of benzylpenicillin, cefuroxime, azithromycin, and sparfloxacin on bacteria in the postantibiotic phase. Antimicrobial Agents and Chemotherapy 41, 25226.[Abstract]
6 . Johnson, C. C., Livornese, L., Gold, M. J., Pitsakis, P. G., Taylor, S. & Levison, M. E. (1995). Activity of cefepime against ceftazidime-resistant gram-negative bacilli using low and high inocula. Journal of Antimicrobial Chemotherapy 35, 76573.[Abstract]
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Wain, J., Bay, P. V. B., Vinh, H., Duong, N. M., Diep, T. S., Walsh, A. L. et al. (2001). Quantitation of bacteria in bone marrow from patients with typhoid fever: relationship between counts and clinical features. Journal of Clinical Microbiology 39, 15716.
Received 30 January 2001; returned 29 May 2001; revised 26 June 2001; accepted 20 August 2001