Effect of increased inoculum of Salmonella typhi on MIC of azithromycin and resultant growth characteristics

Thomas Butler,*

Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was designed to investigate the effect of an increased inoculum on the MIC of azithromycin for Salmonella typhi. Growth curves of nine strains were obtained in cation-adjusted Mueller–Hinton broth containing azithromycin at concentrations of 0–32 mg/L, and comparisons made between inoculation with large inocula, estimated as 107 cfu/mL, and with small inocula of c. 103 cfu/mL. Turbidity developed only with large inocula after 4–8 h in the presence of 8 or 16 mg/L of azithromycin, thus correlating with microscopic appearance of elongated, curved and widened bacteria. Bacteria that had survived exposure to 16 mg/L for 48 h showed low-grade resistance in comparison with those not exposed to antibiotic. Thus, the mechanism of the inoculum effect was expressed as an enlarging bacterial mass during bacteriostasis, with survival of bacterial populations with low-grade resistance of about two-fold increase in MIC.


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Salmonella typhi has been reported to be susceptible to azithromycin with MICs in the range 4–16 mg/L. The antibiotic is clinically effective in the treatment of typhoid fever.1 Previous studies have found that inoculation of culture tubes with a large inoculum of about 106 bacteria/mL resulted in MICs of 16–32 mg/L, which was six times greater than MICs using a small inoculum of about 10 bacteria/mL.2 The following in vitro studies were carried out to elucidate the mechanism of the inoculum effect of S. typhi on the MIC of azithromycin.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Growth curves

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 2–4 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.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Effect of large inoculum on growth curves in the presence of azithromycin

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 1Go). 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 8–24 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 1Go).



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Figure 1. Growth curves of large inoculum (upper) and small inoculum (lower) of S. typhi in CAMHB without antibiotic ({diamondsuit}) and in concentrations of azithromycin in mg/L: 2 (), 4 ({square}), 8 ({triangleup}), 16 (x) and 32 (•). Each point is a geometric mean of nine strains tested and the bar is 1 s.e.m. At the start, large and small inoculations were estimated by McFarland standard at 107 and 103/mL, respectively. Actual cfu/mL were about 10-fold less.

 
Morphological changes in S. typhi after exposure to azithromycin

Large inocula in the presence of 4–16 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 2Go). 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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Figure 2. Elongated and curved morphologies of S. typhi exposed to azithromycin detected by Gram's stain and microscopy. (a) Large inoculum of 3 x 105 cfu/mL after 48 h in 16 mg/L showed light turbidity and increase of cfu to 8 x 106/mL. (b) Small inoculum of 2 x 103 cfu/mL in 8 mg/L after 18 h incubation did not develop turbidity but culture concentrated by centrifugation showed elongated forms. (c) Large inoculum of 9 x 105 cfu/mL, after 2 h exposure to 32 mg/L, bacteria showed mild elongation and bipolar patchy staining.

 
MICs of surviving bacteria in the presence of azithromycin when re-exposed to antibiotic

MICs of colonies obtained after exposure of large inocula were 16–32 mg/L, with a geometric mean of 21.1 mg/L, whereas colonies of the same strains not exposed to antibiotic showed MICs of 4–16 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.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
For large inocula of S. typhi, azithromycin concentrations of 4–16 mg/L that were partially inhibitory and bacteriostatic promoted elongation and widening of bacteria that increased their mass sufficiently to reach the threshold of turbid appearance in 4–8 h. Small inocula, however, were too far below the threshold of turbidity for any increase in mass during bacteriostasis to render tubes with 8–16 mg/L turbid. Also contributing to a greater MIC with large inocula was a delayed growth in some experiments between 24 and 48 h in tubes containing 16 mg/L. Bacterial colonies that had grown in azithromycin were about two-fold more resistant than colonies not exposed to antibiotic, and this low-grade resistance may have allowed increases in cfu after 24 h of exposure. High-grade resistance to azithromycin in S. pneumoniae was elicited by Pankuch et al.4 in multiple passages, but the present study obtained low-grade resistance after a single passage and the resistance was stable when re-tested.

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


    Acknowledgements
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Raymond Johnson contributed assistance and encouragement. This work was supported by an unrestricted educational grant from Pfizer Inc.


    Notes
 
* Tel: +1-806-743-3155; Fax: +1-806-743-3148; E-mail: medtcb{at}ttuhsc.edu Back


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Butler, T., Sridhar, C. B., Daga, M. K., Pathak, K., Pandit, R. B., Khakhria, R. et al. (1999). Treatment of typhoid fever with azithromycin vs chloramphenicol in a randomized multicentre trial in India. Journal of Antimicrobial Chemotherapy 44, 243–50.[Abstract/Free Full Text]

2 . 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, 455–8.[Abstract/Free Full Text]

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.

4 . 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, 2914–8.[Abstract/Free Full Text]

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, 2522–6.[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, 765–73.[Abstract]

7 . 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, 1571–6.[Abstract/Free Full Text]

Received 30 January 2001; returned 29 May 2001; revised 26 June 2001; accepted 20 August 2001