a Department of Medical Microbiology, Cantonal Hospital, Lucerne; b Institute for Infectious Diseases, University of Bern, Bern, Switzerland
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of this study was to assess the phenotypic reproducibility and comparability of three quantitative susceptibility testing methods for the detection of clarithromycin resistance in M. avium isolates and to compare these with the detection of resistance mutations within the 23S rRNA gene.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MICs were determined in triplicate. The methods used were Bactec 460-TB (Becton Dickinson, Franklin Lakes, NJ, USA), broth microdilution assay and Etest (AB Biodisk, Solna, Sweden).57
Clarithromycin was kindly provided by the manufacturer (Abbott Laboratories, Cham, Switzerland). A stock solution of 2048 mg/L clarithromycin dissolved in methanol was prepared according to the manufacturer's instructions.
Serial two-fold dilutions of the stock solution were prepared with 0.1 mol/L phosphate buffer pH 6.5. Dilutions ranged from 256 to 0.25 mg/L (Bactec) and from 512 to 0.125 mg/L (broth microdilution). The Etest strips cover a clarithromycin gradient from 256 to 0.016 mg/L.
Bactec 460-TB
The test strains were inoculated into a Bactec 12B vial and incubated until they attained a growth index (GI) of 999. The strains were then diluted 1:100 in sterile distilled water. Bacterial density was specified as 104105 cfu/mL. A control vial and the vials containing clarithromycin were each inoculated with 0.1 mL of this bacterial suspension diluted 1:100. The MIC was defined as the lowest drug concentration that inhibited >99% of the bacterial population within 8 days of culture, as described by Heifets.5
Broth microdilution assay
Each strain was inoculated in 4 mL of 7H9SF broth (Difco Laboratories, Detroit, MI, USA), and incubated at 35°C for 7 days. On the day before the MIC test was performed, this culture was diluted in 1:20 fresh 7H9SF broth, and incubated overnight at 35°C. The following day this diluted culture was mixed by tilting it 10 times, and then diluted 1:50 in 7H9SF broth. This produced a concentration of 106 cfu/mL. A clarithromycin-containing microtitre plate was inoculated with 100 µL of the 1:50 diluted bacterial suspension. The microtitre plates were sealed in a plastic bag and incubated aerobically at 35°C. Readings were made after 2, 3, 4, 6 and 7 days of incubation. The MIC was taken to be the lowest antibiotic concentration at which no growth could be determined by visual inspection.6
Etest
MuellerHinton agar plates enriched with 10% OADC (Difco Laboratories) were used for the Etest. Each of the plates was inoculated with 100 µL of the same bacterial suspension (1:50 dilution) employed for the broth microdilution assay according to the manufacturer' instructions. After pre-incubation for 18 h at 35°C in 5% CO2, the Etest strips were applied. The plates were then sealed in a plastic bag (permeable to CO2), and incubated at 35°C in 5% CO2. The Etest strips used gave rise to a gradient representing a range of 0.016256 mg/L of clarithromycin. After a 7 day incubation period, the MIC was read according to the manufacturer's instructions.7
In all three phenotypic methods Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used for quality control purposes.5,6
Detection of resistance mutations within the 23S rRNA gene
In order to define mutations conferring resistance to macrolides, the partial sequence of the 23S rRNA gene was determined in all isolates. For this purpose mycobacterial DNA was extracted as described previously.8 The sequencing of the 23S rRNA gene was performed according to the protocol described by Kirschner & Böttger.9
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All strains recovered prior to anti-mycobacterial therapy had a wild-type genotype, i.e. no mutation was detected within the 23S rRNA gene, and the respective MICs were 4 mg/L by either the radiometric method (range 0.54 mg/L), the broth microdilution assay (range 14 mg/L) or the Etest (range 0.0944 mg/L). The follow-up isolates recovered during treatment had one of the following mutations within the 23S rRNA gene: 2058 A
C/G (n = 3) or 2059 A
C (n = 2). The respective MICs were
128 mg/L by all three phenotypic methods (Table
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present work has demonstrated that mutationassociated resistance to clarithromycin in M. avium may be detected with each of the three phenotypic methods evaluated. Thus, additional considerations such as resources, in terms of personnel and facilities, as well as turnaround time and cost of a given assay will guide the choice of method for the detection of clarithromycin-resistant M. avium. While access to sequencing facilities may generally be limited, each of the three phenotypic assays evaluated can be introduced to any diagnostic mycobacteriology laboratory. In our hands, the turnaround times for the unambiguous identification of resistance were 7, 8 and 8 days, respectively, for the Etest, broth microdilution assay and radiometric method. The radiometric method proved to be the most expensive,
costing an estimated
52 per test, as compared with
23 and
10 per test for the broth microdilution method and Etest, respectively.
On the basis of these considerations the implementation of Etest for the detection of mutation-associated resistance to clarithromycin in M. avium may prove to be a good choice for many diagnostic mycobacteriology laboratories, because of the ease of use and cost-effectiveness.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Young, L. S., Inderlied, C. B., Berlin, O. G. & Gottlieb, M. S. (1986). Mycobacterial infections in AIDS patients, with an emphasis on the Mycobacterium avium complex. Reviews of Infectious Diseases 8, 102432.[ISI][Medline]
3 . Inderlied, C. B. & Salfinger, M. (1999). Antimicrobial agents and susceptibility tests: Mycobacteria. In Manual of Clinical Microbiology, 7th edn (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 160123. American Society for Microbiology, Washington, DC.
4 . Heifets, L. B., Mor, N. & Vanderkolk, J. (1993). Mycobacterium avium strains resistant to clarithromycin and azithromycin. Antimicrobial Agents and Chemotherapy 37, 236470.[Abstract]
5 . Heifets, L. B. (1991). Dilemmas and realities in drug susceptibility testing of M. aviumM. intracellulare and other slowly growing nontuberculosis mycobacteria. In Drug Susceptibility in the Chemotherapy of Mycobacterial Infections, (Heifets, L. B., Ed.), pp. 12346. CRC Press, Boca Raton, FL, USA.
6 . Yajko, D. M., Nassos, P. S. & Hadley, W. K. (1987). Broth microdilution testing for susceptibilities to 30 antimicrobial agents of Mycobacterium avium strains from patients with acquired immune deficiency syndrome. Antimicrobial Agents and Chemotherapy 31, 157984.[ISI][Medline]
7 . Lebrun, L., Onody, C., Vincent, V. & Nordmann, P. (1996). Evaluation of the Etest for rapid susceptibility testing of Mycobacterium avium to clarithromycin. Journal of Antimicrobial Chemotherapy 35, 9991003.
8 . Telenti, A., Marchesi, F., Balz, M., Bally, F., Böttger, E. C. & Bodmer, T. (1993). Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. Journal of Clinical Microbiology 31, 1758.[Abstract]
9 . Kirschner, P. & Böttger, E. C. (1996). Resistance mutations in mycobacteria. In PCR Protocols for Emerging Infectious Diseases (Pershing, D. H., Ed.), pp. 1307. ASM Press, Washington, DC.
10 . Nash, K. A. & Inderlied, C. B. (1996). Rapid detection of mutations associated with macrolide resistance in Mycobacterium avium complex. Antimicrobial Agents and Chemotherapy 40, 174850.[Abstract]
Received 25 July 2001; returned 8 October 2001; revised 23 November 2001; accepted 17 December 2001