Relationship between genetic alteration of the rpsL gene and streptomycin susceptibility of Mycobacterium tuberculosis in Japan
Miho Fukuda,
Hironobu Koga,
Hideaki Ohno,
Bing Yang,
Yoichi Hirakata,
Shigefumi Maesaki,
Kazunori Tomono,
Takayoshi Tashiro and
Shigeru Kohno*
The Second Department of Internal Medicine, Nagasaki University School of Medicine,
1-7-1 Sakamoto, Nagasaki 852-8501, Japan
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Abstract
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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.
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Introduction
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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.
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Materials and methods
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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.
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Results
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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 Lys
Arg 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 Lys
Arg
(AAG
AGG) change and the other had a Lys
Gln (AAG
CAG) 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 (Lys
Arg) 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.

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Figure. Detection of mutation by RFLP analysis. Note the two patterns of digestion of the
360 bp amplified fragment of the rpsL gene by MboII. MW, 100 bp molecular
weight standard; C, control: (H37Rv) wild-type pattern consisting of two fragments (138 and 222
bp); lane 1, strain with MIC of streptomycin of 0.25 mg/L without mutation; lane 2, strain with
MIC of 16 mg/L and mutation at codon 88 (Lys Arg), lane 3, strain with MIC of 1012
mg/L
and mutation at codon 88 (LysR Arg), lane 4, strain with MIC of 1012 mg/L and mutation
at codon 88 (Lys Gln). Lanes 2-4 show a pattern similar to that of the control. Lanes 5 and
6
show strains with MICs of 256 mg/L and 2048 mg/L, respectively; both have a mutation at
codon
43 (Lys Arg) which destroys the MboII restriction site, leaving the 360 bp
fragment undigested.
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Discussion
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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 (Lys
Arg) or codon 88 (Lys
Arg, or Lys
Gln). 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.
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Notes
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* Corresponding author. Tel:+81-95-849-7271;
Fax:+81-95-849-7285; E-mail: sk1227{at}net.nagasaki-u.ac.jp 
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Received 11 May 1998;
returned 29 June 1998; revised 31 July 1998;
accepted 15 September 1998