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Role of pncA and rpsA Gene Sequencing in Diagnosis of 1
Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates 2
from Southern China 3
Running title: Pyrazinamide Resistance Diagnosis with pncA and rpsA 4
Yaoju Tan,a Zuqiong Hu,
a Tianyu Zhang,
b# Xingshan Cai,
a Haobin Kuang,
a Yanwen 5
Liu, a Junyu Chen,
a Feng Yang,
b Ke Zhang,
b Shouyong Tan,
a# and Yanlin Zhao
c# 6
State Key Laboratory of Respiratory Diseases, Department of Clinical Laboratory, 7
Guangzhou Chest Hospital, Guangzhou, Chinaa; State Key Laboratory of Respiratory 8
Diseases, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese 9
Academy of Sciences (CAS), Guangzhou, Chinab; National Tuberculosis Reference 10
Laboratory, China Center for Disease Control and Prevention, Beijing, Chinac 11
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23 1 Address correspondence to Tianyu Zhang, [email protected]; Shouyong Tan,
[email protected], or Yanlin Zhao, [email protected]
JCM Accepts, published online ahead of print on 16 October 2013J. Clin. Microbiol. doi:10.1128/JCM.01903-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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24
ABSTRACT 25
We sequenced pncA and rpsA genes plus flanking regions of 161 Mycobacterium 26
tuberculosis isolates and found 10 new pncA and 3 novel rpsA mutations in 27
pyrazinamide-resistant strains determined by the Bactec MGIT 960. The 3’ of rpsA 28
might be added as the target of molecular diagnosis of pyrazinamide susceptibility. 29
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Pyrazinamide (PZA) is an indispensable first-line antituberculosis drug which 48
exhibits unique sterilizing activity for both drug sensitive and multidrug-resistant 49
tuberculosis. It can kill semi-dormant bacilli which persist in acidic-pH environments 50
inside macrophages (1), where other drugs may not act so well. It is a prodrug that 51
needs to be converted into its active form, pyrazinoic acid (POA), by the 52
Mycobacterium tuberculosis (MTB) pyrazinamidase (PZase) which is encoded by the 53
561 nucleotide (nt) pncA gene (2, 3). In 1996, the pncA gene was confirmed to be 54
strongly associated with the PZA resistance in MTB (3), and in the following year, 55
investigators found that pncA mutations constituted the primary mechanism of PZA 56
resistance (4). In 2011, through systematic review with meta-analyses, Chang KC et 57
al drew the conclusion that molecular assays based on the pncA mutations were 58
probably the way forward for detecting pyrazinamide resistance in MTB (5). 59
Molecular detection of PZA resistance-related mutations in pncA gene is indeed more 60
rapid than traditional mycobacterial susceptibility testing methods that depend on the 61
growth of MTB, and are hampered by the complication of testing PZA activity (3). 62
Shi et al recently confirmed that the ribosomal protein S1 (RpsA), encoded by rpsA 63
gene, was a target of POA which might be associated with the PZA resistance in 64
clinical MTB isolates (6), so it is worth investigating the molecular characterization of 65
rpsA gene mutations in both PZA-resistant and PZA-susceptible MTB clinical isolates. 66
However some reports suggested that rpsA gene sequencing might not play a role in 67
detecting PZA susceptibility with molecular method (7). 68
Rapid and accurate diagnosis of drug resistance contributes to early optimization of 69
MTB treatment, which is an effective way to further avoid the emergence of other 70
drug resistance and the potential PZA toxicity. However, the mutations in pncA are 71
highly diverse in different regions (8-10), which hampers the development and 72
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application of molecular assays that are always based on the molecular 73
characterization of gene mutations. In southern China, there are no data so far 74
published describing pncA mutations in PZA-resistant clinical strains of MTB, so it is 75
necessary to analyze the profile of pncA and rpsA mutations of clinical MTB strains in 76
this region for the convenience of rapid and accurate diagnosis of PZA resistance. 77
Drug susceptibility profile of clinical isolates. It is well known that testing for the 78
PZA susceptibility (3) in vitro is very hard and the CLSI-recommended Bactec 460TB 79
radiometric system (460TB, Becton Dickinson, Sparks, MD) was replaced recently in 80
many laboratories by the nonradiometric Bactec MGIT 960 system (BT960, Becton 81
Dickinson, Sparks, MD) (11). In the present study, 161 clinical MTB strains isolated 82
from January 2011 to October 2012 from southern China, mostly (115 of 161) from 83
Guangdong province, were selected to determine their susceptibilities to PZA by 84
BT960 as recommended by the manufacturer with modified Middle brook 7H9 broth 85
(pH5.9) containing 100ȝg/ml PZA. Mycobacterium bovis BCG ATCC34540 and 86
MTB H37Rv ATCC27294 were used as PZA-resistant and PZA-susceptible controls, 87
respectively. The susceptibility of these isolates to rifampicin (RIF), isoniazide (INH), 88
ethambutol (EMB) and streptomycin (STR) were previously performed by BT960 89
when the patients were hospitalized in Guangzhou Chest Hospital, the biggest 90
tuberculosis treatment center located in southern China (12). Of the 161 MTB clinical 91
isolates, 109 isolates were PZA-susceptible and 52 were PZA-resistant.Their 92
susceptibilities to RIF, INH, EMB and STR are summarized in Table 1 and Table 2. 93
Sequencing pncA and rpsA genes of the clinical isolates. As pncA and rpsA genes 94
are the only known genes related to the PZA susceptibility and their corresponding 95
flanking regions (FR) may contain regulatory elements which may affect gene 96
expression, it is reasonable that mutation in FR may alter the mutants’ susceptibility 97
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to PZA. We analyzed the pncA and rpsA genes and their corresponding FR. No 98
typical promoter was found in upstream of pncA gene from -1 to -358 nt including its 99
upstream gene Rv2044c. Though there are only 162 nts between rpsA and its upstream 100
gene (Rv1629), a typical putative prokaryotic promoter was found as: 101
CCGAGTTTGTCCAGCGTGTACCCGTCGAGTAGCCTCGTCAGGTACCAATC 102
(-90 to -41 nt of rpsA) which was predicted with online program 103
(http://www.fruitfly.org/seq_tools/promoter.html). The score was 0.86 with the total 104
score 1.0. The boxed G was supposed to be the transcription starting site. There were 105
only 25 nts between rpsA and its downstream gene and no regulatory element was 106
found. We designed primers to amplify and sequence the whole pncA and rpsA 107
genes+FR of the clinical strains according to sequences of the MTB H37Rv 108
(GenBank Accession No. NC000962) using the software Primer Premier 5.0 (Premier, 109
Canada). F1 and R1 for pncA gene+FR, F2 and R2 for rpsA gene+FR. Primers F1, 110
5'-TGCCACTCGCCGGTAACCGG (321 to 340 nt downstream of pncA) and R1, 111
5'-GGTGGCCGCCGCTCAGCTGG (-119 to -100 nt of pncA); primers F2, 112
5'-GGCCGCAGCTGGGACGCGGC (-192 to -173 nt of rpsA) and R2, 113
5'-CGGTCCAGCGCTCCGTCTGC (203 to 222 nt downstream of rpsA).The 114
additional rpsA sequencing primers (HC-0815-3-R1, 5'-GTCCTCATTGGCTTGC; 115
HC-0815-3-R2, 5'-CGTTGTTGCGGTTCTTGTC) and all other primers in this study 116
were synthesized at BGI,China, where DNA sequencing was performed. All the 117
PZA-resistant isolates that had no pncA+FR mutation were repeated for their 118
susceptibility testing and their rpsA genes and pncA genes were resequenced in a 119
double-blind form. The results were exactly the same as the first time. 120
Transcription of MTB pncA gene in a polycistron. Only 40 nts exist between pncA 121
and Rv2044c and 1 nt overlapped between pncA and its downstream gene (Rv2042c), 122
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so we infered that pncA might be co-transcribed with its surrounding genes in a 123
polycistron (Fig. 1A). We thus carried out a reverse transcription PCR (RT-PCR) 124
experiment to prove this suggestion. Four pairs of primers were designed: 125
Af(5’-ATCCGCATCAGCACCG)-Ar(5’-CCCTCGCAGAAGTCGTTC),Cf(5’-GGC126
ACGCCACTGCTGAA)-Cr(5’-CGAACCCACCGGGTCTT) and 127
Df(5’-ATGACTTTAGGCGAGGATGA)-Dr(5’-TCGAACGGCTTATTGACC) for 128
amplifying the fragments spanning Rv2045c-Rv2044c-pncA, pncA-Rv2042c and 129
Rv2042c-Rv2041c, respectively; 130
Bf(5’-CAATCGAGGCGGTGTTC)-Br(5’-CAATGATCGGTGGCAATAC) were 131
used to amplify a fragment in pncA as a positive control. Total RNA was extracted 132
from MTB H37Ra or the clinical -11nt mutant at their log phases by the RNAiso Plus 133
kit. Then the samples were treated with recombinant DNase I to remove the possible 134
contaminated genomic DNA. RT-PCR was performed using the kit DRR036S. PCR 135
products amplified with the above 4 pairs of primers using the RT-PCR products from 136
MTB H37Ra as templates were electrophorised. All the reagents for these 137
experiments were from Takara, China and the corresponding procedures were carried 138
out according to the manufacture’s instructions. The sizes of the PCR products with 139
these 4 pairs of primers were all exactly as expected: Af-Ar, 543 bp; Bf-Br, 148 bp; 140
Cf-Cr: 356 bp and Df-Dr: 255 bp (Fig. 1B). The samples with templates from 141
RT-PCR produced without adding reverse transcriptase all produced no bands, so 142
there was no genomic DNA contamination after the treatment with recombinant 143
DNase I (Fig. 1B). We verified that the pncA gene was transcribed in a polycistron in 144
MTB. So accurate description of the mutations at -11 and nearby region should be 145
that the mutation is at putative upstream regulatory region of pncA gene, but not at its 146
promoter. 147
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pncA mutations. There was no pncA mutation in all 109 PZA-susceptible isolates 148
(0%), but 44 out of 52 (84.6%) PZA-resistant isolates showed pncA mutations (Table 149
1), which showed the strong correlation between mutations in pncA and phenotypic 150
resistance to PZA, and supported the finding that pncA mutation could cause PZA 151
resistance in MTB (2). The pncA gene mutation frequency in the PZA phenotypic 152
resistant strains here is much higher than those reported previously in other provinces 153
of China (27 to 48.1%) (13-17), but similar to those from other countries (69% to 154
98.8%) in which the PZA phenotypic resistance was determined by BT960 or 460TB 155
system (18-21). It is well known that testing for the susceptibility of PZA in vitro is 156
very hard (3) because PZA only has very weak activity even in acid pH environment, 157
in which even the control without PZA could not grow well. Many factors, such as the 158
inoculum size and the metabolic state of the culture, can have a great influence on the 159
susceptibility testing results. In the previously published papers from China, absolute 160
concentration method was used to determine the PZA phenotypic susceptibility. This 161
method may have both higher false-positive and false-negative resistance results than 162
the BT960/460TB systems do, which may contribute to the disparities in mutation 163
frequencies in pncA, especially in the PZA phenotypic sensitive ones from different 164
regions. This is partially supported by the finding that obvious discrepancies existed 165
between BT960 and 460TB and even when they were manipulated by the same 166
persons working on the same samples (7, 11). Furthermore, a 90.5% pncA mutation 167
rate in the PZA-resistant strains in eastern China determined using BT960 system was 168
reported very recently, which was very similar to our finding here (22). 169
Among the 44 mutant isolates, 8 harbored deletions, 3 exhibited double point 170
mutations, while the remaining 33 isolates had single point mutations. The mutations 171
were randomly distributed almost along the entire 588 bp-long pncA gene, from nt 23 172
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to nt 525 in the clinical isolates. This was similar to other reports (23-25) and similar 173
to the “all pncA mutations” collected and analyzed in the TB Drug Resistance 174
Database (http://www.tbdreamdb.com/PZA_Rv2043c_AllMutations.html) in which 175
mutations were randomly distributed from nt1 to nt 554. Only 13 of 44 (29.5%) 176
mutants in this study located in the clustering of mutations of G132-T142, P69-L85 177
found by previous investigators (25) , which showed the location of mutations in pncA 178
gene was more variable than expected before (25) and not clustered as in other genes 179
associated with drug resistance, such as the rpoB gene (12). None of the isolates with 180
identical pncA mutations epidemiologically linked, so the PZA resistant strains might 181
not be obtained from transmission. For the mutations in pncA, 23 types of point 182
mutations had been reported previously (8, 9, 21, 26-29) and, to our best knowledge, 183
10 novel types of mutations (13 strains) were first found in this study. The latter 184
included all the six types of the deletion mutations (L27, 91E, 111E, 122Q, 185
130-132VVG and 131V) and four types of amino acid substitutions (D8A, S18P, 186
F58S, and E174G+M175R double mutation) in PZases as summarized in Table 1. 187
Such novel mutants accounted for 25% (13 of 52) of all the PZA-resistant isolates. 188
The PCR-single-strand conformational polymorphism (PCR-SSCP) and line probe 189
assay (LiPA) developed previously (30, 31) could detect DNA mutations quickly, but 190
they could not tell if the mutation is synonymous or nonsynoonymous. No silent 191
mutations were found in pncA gene in our study, which indicated that PCR-SSCP and 192
LiPA might be used to detect PZA resistance quickly with high successful rate by 193
detecting the pncA mutation in southern China. 194
There were still 8 PZA-resistant MTB isolates that had no mutation in pncA gene. 195
Four of them didn’t harbor any mutation including FR, three of them showed an rpsA 196
single point mutation (see below), and one other strain had a point mutation in the 197
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putative regulatory region (A-11G) of pncA gene which was a very common mutation 198
found in isolates from other countries (10, 23-25). To explore the possible reason why 199
this can affect PZA resistance, we checked the transcription levels of pncA genes in 200
this clinical isolate and the labortory control MTB H37Ra strain. RT-PCR products 201
were prepared as described in the section demonstrating transcription of pncA gene in 202
a polycistron. Real-time PCR with SYBR Green dye was used for detecting the initial 203
concentration of pncA mRNA with sigA as inner reference. The primers used were 204
Bf-Br for pncA and sigAF 205
(5’-CTCGACGCTGAACCAGACCT)-sigAR(5’-AGGTCTTCGTGGTCTTCGTC) 206
for sigA(32). Levene’s test for equality of variances and t-test for equality of means 207
were calculated using the software SPSS13.0 (SPSS Inc., Chicago, USA). This A-11G 208
mutation may not affect pncA expression at the transcription level, because it can still 209
be transcribed in a polycistron with flanking genes in the clinical -11 mutant (data not 210
shown) and its expression level showed no difference with MTB H37Ra. This 211
mutation however may disturb the translation of pncA gene by affecting the ribosome 212
movement, because the -11 nt is close to the ribosome binding site. 213
These new findings displayed the geographically regional disparities in pncA 214
mutations and the high diverse patterns in pncA mutations (4,5,22), which partially 215
make molecular diagnosis of PZA susceptibility difficult to be adopted worldwide. 216
Therefore, systematic establishment of the relationship between the mutation 217
characterization of PZA resistance related genes and the PZA-resistance phenotype is 218
highly needed for development and application of rapid molecular assays. 219
68.4% (39/57) of MDR strains were PZA-resistant, but only 12.5% (13/104) of 220
non-MDR strains harbored such resistance. The former was significantly higher than 221
the latter (P<<0.01), which implied that the MDR strains were more likely to be 222
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PZA-resistant than the drug sensitive ones. This result is similar to that in South 223
Africa and Thailand (4, 27). The importance of PZA in treating MDR-TB has been 224
proved recently in a murine model in that all PZA containing regimens tested showed 225
better activities than those corresponding regimens without PZA when combined with 226
second-line drugs (33). All these findings protruded rapid diagnosis of PZA resistance 227
in controlling MDR-TB. 228
rpsA mutations. Three PZA-resistant clinical isolates with wild type pncA (pncAWT
) 229
harbored single mutation of R474L, R474W and E433D, respectively. One 230
PZA-susceptible clinical isolates also contained one single mutation of Q162R. The 231
frequency of rpsA mutations showed no significant statistic difference (P=0.099) 232
between PZA-resistant and PZA-susceptible isolates, which may be because of the 233
small sample size of PZA-resistant isolates. The low frequency of rpsA mutation 234
proved again that the rpsA mutation was not the main mechanism of PZA resistance 235
in MTB, though it can affect the susceptibility to PZA (6). Alexander et al could not 236
find any rpsA mutation in PZA-resistant isolates but one RpsA protein mutation 237
(A364G) in 13 PZA sensitive strains was discovered. In addition, the rpsA gene+FR is 238
long and not very convenient for amplification and sequencing, so they concluded that 239
the analysis of the rpsA gene had no role in the diagnosis of pyrazinamide resistance 240
(7). This suggestion was further supported by Sami O. Simons and colleagues who 241
reported only 1 of 5 PZA-resistant isolates with pncAWT
had RpsA (V260I) mutation 242
(34) and that this so called mutation was even questionable according to the disparity 243
of the reported mutated codon and the amino acid it coded. However, in the current 244
study, 3 of 7 phenotypically PZAr but genotypically pncA
WT+FR
WT isolates had rpsA 245
mutations which were all clustered in the C terminal of RpsA protein (3’ of rpsA 246
gene). This was concordant with the findings by Shi et al (6) who reported that 247
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deletion of amino acid 438 in the C-terminus of RpsA (Fig.2A) could cause PZA 248
resistance in a MTB clinical isolate with pncAWT
. We noticed that rpsA mutations in 249
the PZA sensitive strains found until now were mostly away from the C-terminal of 250
RpsA. We then made an alignment of C-terminal amino acid sequences of ribosomal 251
protein S1 (RpsA) from 33 strains of 28 mycobacterial species (Fig. 2A) and found 252
the same phenomenon as described by Shi et al (6) that, unlike other parts, 253
C-terminals of RpsA proteins were highly variable. At the same time, we drew a 254
phylogentic tree according to the C-terminal amino acid sequences of RpsAs from 255
these mycobacterial species (Fig. 2B) using Vector NTI Suite 7.0 (Invitrogen, USA). 256
It is similar to the phylogentic trees based on either nearly complete 16S rRNA gene 257
sequence or the concatenated hypervariable sequences of 16S rRNA, rpoB and hsp65 258
genes (35). Two different species, for example, the M. marinum and M. ulcerans have 259
identical RpsAs not only in the C terminal, while different strains of the same species, 260
for example, M. smegmatis MC2 155 and M. smegmatis JS263, or M. rhodesiae NBB3 261
and another strain, have very different RpsA C terminals but are even more close to 262
other species in the phylogentic tree. This is accordant with the fact that the genus 263
Mycobacterium has very limited interspecies genetic variability (35). In the current 264
study, we found that all the 3 mutated sites (E433, A438 and R474) were highly 265
conserved among all the Mycobacterium species. It is interesting that A438 deletion 266
that took place at AAA (436-438) had the same effect as A436 which seemed more 267
conserved than A438 did (Fig.2A). If E433 and R474 mutations did cause PZA 268
resistance as A438 deletion did, sequencing the C terminal of rpsA gene in isolates 269
with pncAwt
may be needful for more rapid and accurate diagnosis of PZA 270
susceptibility, especially in southern China, though the role of this region is modest. 271
86.5 % (45 of 52) phenotypically PZA- resistance could be predicted by only 272
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sequencing pncA+FR and 92.3% (48 of 52) could be predicted if sequencing 3’ 273
terminal of rpsA was added in our study. In the meantime, it is needful to point out 274
that not all mutations at the C-terminals of RpsA may cause PZA resistance. For 275
example, M. bovis BCG has a nonfunctional pncA and an rpsA gene encoding mutated 276
RpsA (A440T) and is naturally resistant to PZA. M. smegmatis pncA and pzaA encode 277
proteins both with PZase activity and can transform PZA into POA. Introduction of M. 278
smegmatis pncA or pzaA into M. bovis BCG can restore its sensitivity to PZA (36). 279
Therefore, the resistance of M. bovis BCG to PZA is possibly because of the pncA 280
mutation but not rpsA mutation, and so the RpsA (A440T) may not play a role in PZA 281
resistance. Another strain, MTB CTRI-2 contained a mutated RpsA (M432T) at its 282
C-terminal and was PZA sensitive (37). In this regard therefore, the actual role of 283
E433 and R474 mutations of RpsA found here in PZA resistance need to be verified 284
further. 285
In conclusion, the pncA mutations were randomly dispersed along with the entire 286
gene and observed with high ratio in PZA-resistant clinical isolates in southern China. 287
28.9 % of clinical isolates with pncA+FR mutations (13 of 45) were new, which 288
displayed the high diversity of mutations in pncA gene in different regions. The pncA 289
gene was transcribed in a polycistron and the A-11G mutation of pncA did not affect 290
its expression at transcription level, which were first proved here. The 3’ of rpsA gene, 291
if not the whole rpsA gene +FR, should be added as the target for a two-step approach, 292
where only pncAWT
isolates from southern China are subjected to sequencing this 293
fragment. However, the influence of these rpsA mutations on the PZA susceptibility 294
needs to be proved further in the future as that of many pncA mutations. For more 295
accurate results, the phenotypic PZA susceptibility testing, especially using BT960 296
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system, is needed for MTB, and in particular MDR MTB clinical isolates with no 297
known gene mutations. 298
299
ACKNOWLEDGMENTS. We are grateful to BGI for assistance in DNA 300
sequencing. This work was supported by the National Great Research Program of 301
China (2008zx10003-009). TZ was supported by the Chinese Academy of Sciences 302
'One Hundred Talents Program' (Category A) and the key layout program of the 303
Chinese Academy of Sciences (KSZD-EW-Z-006). We thank Dr. Wing Wai Yew at 304
The Chinese University of Hong Kong for helpful discussion. 305
306
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FIG.2. A. Alignment of C-terminal amino acid sequences of ribosomal protein S1 452 (RpsA) from different mycobacterial species. Species are represented as below with 453 GenBank accession numbers indicated in the brackets: MTB1, M. tuberculosis H37Rv 454 (NP_216146.1); MTB2, M. tuberculosis CTRI-2 (YP_005916702.1); MCT1, M. 455 canettii CIPT 140070008 (YP_007287527.1); MBV, M. bovis AF2122/97 456 (NP_855309); BCG, M. bovis BCG Pasteur 1173P2 (NP_855309.1); MCT2, M. 457 canettii CIPT 140070010 (YP_007264352.1) ; MLP, M. leprae TN (NP_301983.1); 458 MMR, M. marinum M (YP_905584.1); MUR, M. ulcerans Agy99 (YP_905584.1); 459 MAV, M. avium subsp. paratuberculosis K-10 (NP_960259); MIC1, M. 460 intracellulare 13950 (YP_005338525.1); MIC2, M. intracellulare 461 (WP_009954955.1); MCL, M. colombiense CECT 3035 (WP_007776137.1); MPF, M. 462 parascrofulaceum (WP_007169711.1); MAB, M. abscessus (YP_001703031.1); 463 WP_007169711.1MML, M.massiliense GO 06 (YP_006520985.1); MIG, M. 464 immunogenum MTCC 9506 (YP_006730099.1); MFL, M. franklinii (AEI54876.1); 465 MCN, M. chelonae (AEI54878.1); MRS2, M. rhodesiae NBB3 (YP_005002313.1); 466 MFT, M. fortuitum (WP_003884756); MSM1, M. smegmatis MC
2 155 467
(YP_888124.1); MVB, M. vanbaalenii PYR-1 (YP_954159.1); MPL, M. phlei 468 (WP_003884756.1); MTR, M. thermoresistibile (WP_003923841.1); MRS1, M. 469 rhodesiae (WP_005149060); MSM2, M. smegmatis JS623 (YP_007292760.1); 470 MCB1, M.chubuense NBB4 (YP_006453027.1); MGV, M. gilvum PYR-GC 471 (YP_001134823.1); MTC, M. tusciae (WP_006246193.1); MVC, M. vaccae 472 (WP_003931928.1); MHC, M. hassiacum (WP_005627902.1) and MXP, M. xenopi 473 (WP_003920384.1). Amino acid numbers are indicated on the top according to the 474 order of RpsA from MTB. The identical amino acids from all species are highlighted 475 in dark gray and the similar resides are highlighted in light gray, respectively. B. The 476 phylogentic tree drawn according to the C-terminal amino acid sequences of 477 RpsA from above mycobacterial species. t, type strains; n, non-type strains;?, Not 478 sure if they are type stains or not according to the web sources 479 (http://www.bacterio.net/( updated and expanded on 09-Jul-2013) and 480 http://www.atcc.org). 481
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TABLE 1 Drug susceptibility and genotypic characteristics of PZA-resistant clinical isolates.
Resistance toa pncA+FR
b mutation(s) rpsA+FR
b mutation No. of Isolates
INH,RIF D8Ac (GACൺGCC) — 1
INH,RIF Q10P(CAGൺCCG) — 1
INH,RIF F13L(TTCൺCTC) — 1
INH,RIF S18Pc (TCGൺCCG) /P54L(CCGൺCTG) — 1
INH,RIF G24D(GGCൺGAC) — 2
INH,RIF L27c deletion — 1
INH,RIF T47A(ACCൺGCC) — 1
INH,RIF H57Y (CACൺTAC) — 2
INH,RIF F58Sc (TTCൺTCC) — 1
INH,RIF S66P(TCGൺCCG) — 1
INH,RIF T76P(ACTൺCCT) — 2
INH,RIF F81V (TTCൺGTC) — 1
INH,RIF H82R(CATൺCGG) — 1
INH,RIF E91 deletionc — 1
INH,RIF F94L(TTCൺCTC) — 2
INH,RIF F94S(TTCൺTCC) — 1
INH,RIF K96E(AAGൺGAG) — 2
INH,RIF E111 deletionc — 1
INH,RIF Q122 deletionc — 2
INH,RIF VVG130-132 deletionc — 1
INH,RIF V131 deletionc — 2
INH,RIF G132A(GGTൺGCT) — 1
INH,RIF T135P(ACCൺCCC) — 2
INH,RIF D136G(GATൺGGT) — 2
INH,RIF V139G(GTGൺGGG) — 1
INH,RIF V139A(GTGൺGCG) — 1
INH,RIF V139L(GTGൺCTG) — 1
STR,RIF Q141P(CAGൺCCG) — 1
INH,RIF L159R(CTGൺCGG) — 1
INH,STR,EMB G162D(GGTൺGAT) — 1
INH T168P(ACCൺCCC) — 3
INH,RIF E174Gc (GAGൺGGG)/ — 2
M175Rc (ATGൺAGA)
INH,RIF — R474W(CGGൺTGG) 1
INH — R474L(CGGൺCTG) 1
STR ᥊11(AൺG) — 1
INH,STR,EMB — E433D(GAGൺGAC) 1
STR — — 4
Total 45 3 48+4 a INH, isoniazid; RIF, refampin; STR, streptomycin; EMB, ethambutol.
b FR, flanking region, which means the upstream and downstream regions of the gene. —, means no mutation.
c first found nonsynonymous mutations of pncA.
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TABLE 2 Drug susceptibility and genotypic characteristics of PZA-sensitive clinical isolates.
Resistance to a pncA+FR
b mutation(s) rpsA+FR
b mutation No. of Isolates
— — — 57
INH,RIF — — 17
INH,RIF — Q162R(CAGൺCGG) 1
INH,STR — — 8
INH — — 6
RIF — — 4
STR — — 14
EMB — — 1
INH,STR,EMB — — 1
Total 108 1 109
a INH, isoniazid; RIF, refampin; STR, streptomycin; EMB, ethambutol.
b FR, flanking region, which means the upstream and downstream regions of the gene. —, means no mutation.
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