Journal of Antimicrobial Chemotherapy (1999) 43, 281-284
© 1999 The British Society for Antimicrobial Chemotherapy
Brief reports |
Relationship between genetic alteration of the rpsL gene and streptomycin susceptibility of Mycobacterium tuberculosis in Japan
The Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We have investigated the effect of genetic alterations in the rpsLgene on the MICs of streptomycin for Mycobacterium tuberculosis strains. Direct DNA sequencing showed a point mutation in 23/121 strains; in 18 strains the mutation was associated with an amino acid change. The MICs of streptomycin in 22 out of 23 point-mutated strains were
256 mg/L.
Restriction fragment length polymorphism (RFLP) analysis showed mutations at codon 43 in all
18 strains with point mutations in the same codon. Our results suggest that both RFLP and base
sequencing analysis of the rpsLgene are useful for the rapid prediction of highly
streptomycin- resistant strains of M. tuberculosis. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Several new methods have been developed for screening drug-resistant strains of Mycobacterium tuberculosis, 1,2,3 but these methods can only be applied to clinical isolates from clinical specimens. Genes that confer drug-resistance of M. tuberculosisare gradually being identified, and gene mutations in mycobacteria that confer resistance to isoniazid and rifampicin have already been identified. A relationship between MIC and type of rpoB gene mutation in rifampicin-resistant strains of M. tuberculosishas also been reported. 4,5 We evaluated here the relationship between genetic alterations in rpsLand MICs of streptomycin in a large number of M. tuberculosis strains isolated from Japanese patients. We also compared two methods, namely direct DNA sequence analysis and restriction fragment length polymorphism (RFLP) analysis, for their ability to detect mutations in rpsL in streptomycin- resistant M. tuberculosisstrains.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We used 121 clinical isolates of M. tuberculosis. Resistance to one or more antituberculous drugs was based on the clinical course and/or results of susceptibility tests. We also used an M. tuberculosis standard strain, H37Rv, as a control. Susceptibility tests were performed by a broth microdilution method. 4 A few colonies of M. tuberculosis, grown on Ogawa egg-based medium, were inoculated into 15 mL ADC-supplemented Middlebrook 7H9 broth (Difco, Detroit, MI, USA) and cultured at 37°C for 1 week. The concentration of bacteria was adjusted to that of a McFarland 0.5 standard (
106 cfu/mL). One
hundred microlitres of a solution of streptomycin (Meiji Seika Co., Tokyo, Japan) in
Middlebrook 7H9 broth containing ADC supplement was dispensed into each well of a 96-well
microplate (Corning Glass Works, New York, NY, USA). The concentration of streptomycin
ranged from 0.5 to 4096 mg/L. Plates were stored at -70°C until use. Each well was
inoculated with 100 µL of bacterial suspension (106 cfu/mL) and cultured in
a plastic container. The final concentration of streptomycin in the wells ranged from 0.25 to 2048
mg/L. When growth was observed in streptomycin-free wells, the MIC was taken as the lowest
concentration of streptomycin resulting in absence of visible mycobacterial growth. After
incubation for 21 days the MICs were determined again to exclude falsely susceptible strains. A
nitrate reduction assay was also performed to confirm the presence or absence of bacterial
growth. DNA was extracted from a few colonies that had been grown on Ogawa egg-based medium using the phenol/ chloroform method. 4,5 Ten nanograms of each DNA was used for specific PCR of M. tuberculosis targeting protein antigen b (pab) gene. 4 PCR products were electrophoresed in a 2% agarose gel (Seakem ME agarose, FMC BioProducts, Rockland, ME, USA), stained with ethidium bromide, and visualized by UV transillumination. We designed two primers, based on the sequence of the rpsLgene which encodes the M. tuberculosis ribosomal protein S12, 6 to amplify a 360 bp internal fragment of rpsL. These primers, 5'-ATGCCAACCATCCAGCAGCT-3' and 5'-CTTAGCGCCGTAACGGCTGC-3', were synthesized using a Model 380B DNA synthesizer (Applied Biosystems, Foster City, CA, USA). Amplification was performed by using a Perkin-Elmer 9600 DNA Thermal Cycler (Perkin-Elmer Medical Instruments, Pomona, CA, USA). All PCR reactions comprised 40 cycles of 1 min at 94°C for denaturation, 1 min at 60°C for annealing and 1 min at 72°C for extension. After the final cycle, samples were maintained at 4°C until analysis. PCR products were electrophoresed in 2% agarose gel and confirmed by ethidium bromide staining as described above. The presence or absence of amplicons representing the 419 bp fragment of pab and the 360 bp fragment of rpsLwas determined.
We used two methods for detecting streptomycin-resistant strains based on analysis of the rpsLgene: direct DNA sequencing and RFLP analysis using a restriction enzyme. For the former method, 10 ng of M. tuberculosisDNA was used. Sequencing was performed using the ABI PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing FS kit (Perkin-Elmer). The reaction mixture was prepared as follows: 1.5 µL PCR templates were purified using Suprec 02 (Takara Shuzo Co., Shiga, Japan), 3.2 pmol primer, 4 µL 5x Terminator Ammonium Cycle Sequencing buffer, 1 µL dNTP, 1 µL DyeDeoxy terminator which labels each of the four different bases, and 4 U Taq polymerase. The sequencing reaction was performed on both strands, using the following primers: 5'-ATGCCAACCATCCAGCAGCT-3' and 5'-CTTAGCGCCGTAACGGCTGC-3'. The reaction mixture was subjected to 25 cycles of 15 s at 96°C for denaturation, 5 s at 50°C for annealing, and 4 min at 60°C for extension. Then 74 µL of 70% ethanol was added to the PCR samples before they were incubated on ice for 30 min and centrifuged at 10,000g for 20 min. The supernatant was discarded and the precipitate was dried in a Speed-vac for 5 min, resuspended in 6 µL of loading buffer (5:1 ratio of deionized formamide: 25 mM EDTA) and denatured at 90°C for 2 min before loading on to a 5% Long Ranger gel (Takara Shuzo Co.). Reactions were analysed using an ABI PRISM 377 DNA sequencer (Applied Biosystems).
For rapid detection of mutations at codon 43, RFLP analysis was used with the restriction enzyme MboII. For this purpose, 1 µg of PCR product, 10 U of MboII, 5 µL of a reaction buffer (10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.5), and 24 µL deionized distilled water were placed into a new microcentrifuge tube and mixed. The reaction was allowed to proceed for 1 h at 37°C, and was then electrophoresed in a 3% agarose gel (Nusieve 3:1 agarose, FMC BioProducts) and stained with ethidium bromide. The patterns of DNA bands were compared with those of the control H37Rv strain.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
All 121 strains of M. tuberculosis were positive for the amplification of pab and rpsL, confirming that they were M. tuberculosis 4 (data not shown). The relationship between MICs of streptomycin against 121 strains and the position and type of genetic alteration in the rpsL gene are shown in the Table. The presence of bacterial growth at streptomycin concentrations >10 mg/L was considered as an index of streptomycin resistance in liquid medium; using this criterion, 49 (41%) of the 121 isolates were streptomycin resistant. A total of 23 genetic alterations were detected in the 121 strains examined, with mutations being detected in 47% of the resistant strains. No alteration was observed in the remaining 98 strains and double point mutations were not present in any strain. Mutations at codon 43 (AAG
AGG; resulting in LysArg amino acid substitution) were found in 18 of the
121 strains; mutations at codon 88 were found in five strains, of which four had a LysArg
(AAGAGG) change and the other had a LysGln (AAGCAG) change. Thus
78% of the substitutions detected were in codon 43 and 22% in codon 88. The MICs of
streptomycin for all these 23 strains containing a point mutation were 256 mg/L, except for
one streptomycin-resistant strain with mutation at codon 88 (LysArg) with an MIC of 16
mg/L. In 98 strains with no genetic alterations, 72 (74%) of strains had a
streptomycin-susceptible phenotype with MICs 
10 mg/L, but the remaining 26 strains were
streptomycin-resistant.
|
Results of the RFLP analysis of the rpsLgene are shown in the Figure. Two types of RFLP pattern were observed. The 360 bp amplified products from the rpsL gene were digested by MboII into two fragments, of 138 bp and 222 bp, in 103 strains and in the H37Rv strain without mutation at codon 43. In the remaining 18 strains, which had a mutation at codon 43 and were resistant to streptomycin (MICs
256 mg/L), the
360 bp product was not digested by MboII.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Our results showed that almost all of the highly streptomycin-resistant strains (MICs
256
mg/L) had an alteration in the rpsL gene. The mutations detected were all in either
codon 43 (LysArg) or codon 88 (LysArg, or LysGln). Thus, all strains with
point mutations in codons 43 or 88 were streptomycin resistant, consistent with other reports.
6,7,8,9,10 Our results also showed that RFLP analysis using MboII was useful in
detecting certain point mutations at codon 43. Although direct sequencing is necessary for
detailed information on the nature of the point mutation, our RFLP test is cost-effective and
faster. Therefore, we propose that RFLP analysis is a useful screening test for detecting highly
streptomycin-resistant strains, particularly when analysing a large number of clinical samples.
In addition to rpsL, the rrs gene (which encodes 16S ribosomal RNA) has been
implicated in streptomycin resistance with a frequency of about 10%.
6,7,8,9 Recent studies have also shown that
low-level streptomycin resistance (MIC 10 mg/L) is determined by different genes from
high-level streptomycin resistance (MIC 160 mg/L).
8,10 All 23 highly
resistant strains with point mutations in rpsL were quickly detected in our study.
However, 26 resistant strains without genetic alterations could not be predicted by such a
molecular technique. Further studies are necessary to identify additional mechanisms conferring
streptomycin resistance. Such studies should be extended to the development of new methods for
detecting streptomycin-resistance genes directly from clinical specimens.
|
Notes |
|---|
* Corresponding author. Tel:+81-95-849-7271; Fax:+81-95-849-7285; E-mail: sk1227{at}net.nagasaki-u.ac.jp
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 . Jacobs, W. R., Barletta, R. G., Udani, R., Chan, J., Kalkut, G., Sosne, G. et al. (1993). Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science 260, 819–22.
2
.
Van der Vliet, G. M., Schepers, P., Schukkink, R. A. F., Van Gemen, B. & Klatser, P. R. (1994). Assessment of mycobacterial viability by RNA amplification. Antimicrobial Agents and Chemotherapy 38,1959
–65.
3 . Miyamoto, J., Koga, H., Kohno, S., Tashiro, T. & Hara, K. (1996). New drug susceptibility test for Mycobacterium tuberculosis using the hybridization protection assay. Journal of Clinical Microbiology 34, 1323–6.[Abstract]
4 . Ohno, H., Koga, H., Kuroita, T., Tomono, K., Ogawa, K., Yanagihara, K. et al . (1997). Rapid prediction of rifampin susceptibility of Mycobacterium tuberculosis. American Journal of Respiratory and Critical Care Medicine155 , 2057–63.[Abstract]
5 . Ohno, H., Koga, H., Kohno, S., Tashiro, T. & Hara, K. (1996). Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrobial Agents and Chemotherapy 40, 1053–6.[Abstract]
6
.
Meier, A., Kirschner, P., Bange, F.-C., Vogel, U. & Bottger, E. C. (1994). Genetic
alterations in streptomycin-resistant Mycobacterium tuberculosis : mapping of
mutations conferring resistance. Antimicrobial Agents and Chemotherapy 38, 228–33.
7 . Honore, N., Marchal, G. & Cole, S. T. (1995). Novel mutation in 16S rRNA associated with streptomycin dependence in Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 39, 769–70.[Abstract]
8 . Cooksey, R. C., Morlock, G. P., McQueen, A., Glickman, S. E. & Crawford, J. T. (1996). Characterization of streptomycin resistance mechanisms among Mycobacterium tuberculosis isolates from patients in New York City. Antimicrobial Agents and Chemotherapy 40, 1186–8.[Abstract]
9 . Sreevatsan, S., Pan, X., Stockbauer, K. E., Williams, D. L., Kreiswirth, B. N. & Musser, J. M. (1996). Characterization of rpsL and rrs mutations in streptomycin-resistant Mycobacterium tuberculosis isolates from diverse geographic localities. Antimicrobial Agents and Chemotherapy 40, 1024–6.[Abstract]
10 . Meier, A., Sander, P., Schaper, K.-J., Scholz, M. & Bottger, E. C. (1996). Correlation of molecular resistance mechanisms and phenotypic resistance levels in streptomycin-resistant Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 40, 2452–4.[Abstract]
Received 11 May 1998; returned 29 June 1998; revised 31 July 1998; accepted 15 September 1998
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  F. S. Spies, P. E. Almeida da Silva, M. O. Ribeiro, M. L. Rossetti, and A. Zaha Identification of Mutations Related to Streptomycin Resistance in Clinical Isolates of Mycobacterium tuberculosis and Possible Involvement of Efflux Mechanism Antimicrob. Agents Chemother., August 1, 2008; 52(8): 2947 - 2949. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||