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1 JCM Accepts, published online ahead of print on 2 May 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved Molecular characterization of multidrug and extensively drug-resistant Mycobacterium tuberculosis strains in Jiangxi, China Xiaoliang Yuan 1, Tiantuo Zhang 1 *, Kazuyoshi Kawakami 2, Jiaxin Zhu 1, Hongtao Li 1, Jianping Lei 3, Shaohua Tu 3 Division of Respiratory Diseases, Department of Internal Medicine, The 3rd Affiliated Hospital of Sun Yat-sen University, 600 Tianhe Road, Guangzhou , China 1 ;Department of Medical Microbiology, Mycology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Miyagi , Japan 2 ;Center for Tuberculosis Diagnosis and Therapy, Jiangxi Chest Hospital, Nanchang , China 3 * Corresponding author. Mailing address: Division of Respiratory Diseases, Department of Internal Medicine, The third Affiliated Hospital of Sun Yat-sen University, 600 Tianhe Road, Guangzhou , China; Phone: Fax: zhtituli@163.com. Downloaded from on September 22, 2018 by guest 1

2 17 18 ABSTRACT In this study, a total of 77 multidrug and extensively drug-resistant (MDR and XDR) isolates of 19 Mycobacterium tuberculosis were characterized among samples from patients living in Jiangxi province, China. The following approaches were used: (i) genotyping all drug-resistant isolates by the 15 loci MIRU-VNTR, and identifying the Beijing family genotype using the RD105 deletion targeted multiplex PCR; (ii) determining the mutation profiles associated with the resistance to the first-line antituberculous drugs rifampicin (RIF), isoniazid (INH) and the second-line drugs ofloxacin (OFX), kanamycin (KAN), amikacin (AMK), and capreomycin (CAP) with DNA sequencing. Six loci were examined: rpob (for resistance to RIF), katg and maba-inha (INH), gyra and gyrb (OFX), rrs (KAN, AMK, and CAP). It is shown that the Beijing genotype was predominant (80.5%) among these strains and the selected drug-resistant strains were genetically diverse, suggesting that they probably had independently acquired drug resistance. In comparison with the phenotypic data, the sensitivity for detection of RIF, INH, OFX, and KAN and AMK and CAP resistance by DNA sequencing was 94.8%, 80.5%, 84.6%, and 78.9% respectively. The most prevalent mutations involved in RIF, INH, OFX, and KAN and AMK and CAP resistance were Ser531Leu in rpob (44.2%), Ser315Thr in katg (55.8%)and C-15T in maba-inha (11.7%), Asp94Gly in gyra (48.7%), and A1401G in rrs (73.7%) respectively. Five novel katg mutants (Trp191Stop, Thr271Pro, Trp328Phe, Leu546Pro and Asp695Gly), and six new alleles (Ile569Val, Ile572Met, Phe584Ser, Val615Met, Asp626Glu and Lys972Thr) in the rpob gene, were identified. 2

3 INTRODUCTION With an estimated 9.4 million new cases and 1.7 million deaths in 2009,tuberculosis (TB) remains to be one of the major life-threatening diseases worldwide (49). The emergence of drug-resistant strains of M. tuberculosis, especially those that are multidrug-resistant (MDR) and extensively drug-resistant (XDR), has posed a serious threat to global TB control. MDR tuberculosis is defined as resistance to both isoniazid (INH) and rifampicin (RIF), XDR is defined as MDR tuberculosis with additional resistance to any fluoroquinolone (FQ) and at least one of the three second-line injectable drugs: kanamycin (KAN), amikacin (AMK) and capreomycin (CAP) (36).Comparing to drug-susceptible tuberculosis, the management of MDR is more complex, costly, time-consuming and less effective (23). However, the regimen for XDR is more expensive and difficult than MDR, and the prognosis is much worse,particularly in those infected with human immunodeficiency virus (24). As of February 2011, a total of 69 countries worldwide have reported identifying at least one case of XDR tuberculosis (47). The accurate and rapid detection of drug resistance is imperative for prompt implement of effective regimens to interrupt the transmission of MDR and XDR isolates. Any delay in the identification of resistant strains jeopardizes the efforts to control the disease. Exploring the molecular genetic basis of drug resistance will contribute to the establishment of efficient methods for the rapid identification of drug-resistant M. tuberculosis strains. However,the frequency,location, and type of resistance-associated mutations varies in different geographical areas (1, 30, 43, 52, 54). Jiangxi Province,a resource-limited area with about 44.5 million inhabitants in 2010, is situated in the southeast part of China. In the province, the prevalence of bacteriological positive pulmonary tuberculosis was recorded to be cases per 100,000 inhabitants ( in 2000, which is higher than the national average (160 cases per 100,000 inhabitants in China in 2000) (17). Unfortunately, so far little information has been obtained with regard to the molecular mutations of MDR and XDR strains isolated from this province. 3

4 To characterize the prevalence and pattern of mutations occurred to the drug target loci, a collection of MDR and XDR strains were isolated from patients in Jiangxi Province. In this study, we screened the drug target genes for RIF, INH, FQ, and KAN, AMK or CAP, which are the first and second-line drugs commonly used for treating TB in China. The genetic loci studied were rpob (RNA polymerase B subunit), katg (catalase-peroxidase), maba-inha (inha regulatory region), rrs (16S rrna), gyra and gyrb (DNA gyrase). Additionally, a 15 loci MIRU-VNTR genotyping was performed to identify the genetic lineage of these TB isolates. Downloaded from on September 22, 2018 by guest 4

5 MATERIALS AND METHODS M. tuberculosis isolates. From January 2010 to June 2011, a total of 97 M. tuberculosis strains were isolated as monocultures from single patients (69 males and 28 females; aged 14 to 78 years) with pulmonary tuberculosis. These patients were epidemiologically unlinked and all living in Jiangxi Province. Among the 97 isolates, 77 were drug resistant strains which included 35 simple MDR strains (resistance to INH and RIF, but not a FQ and one of three second-line injectable drugs), 26 Pre-extensively drug resistant strains (pre-xdr, defined as resistance to INH and RIF, and either a FQ or one of three second-line injectable drugs, but not both) (55), and 16 XDR strains. However, the remaining 20 isolates were pan-susceptible strains and therefore used as negative controls. Meanwhile, the M. tuberculosis H37Rv (ATCC27294) strain was used as a reference. All the isolates were cultured on Lowenstein Jensen solid medium and grown colonies were identified using p-nitrobenzoic acid and thiophene carboxylic acid hydrazine resistance tests. The first-passage of all the clinical strains was used in testing for the first-line drug susceptibility, while the second-passage was used in two testings for the second-line drug susceptibility and for the mutations in the target genes. These were done in the Jiangxi Chest Hospital, a tertiary care hospital and the sole specialized center for the diagnosis and treatment of TB in Jiangxi province. The study was approved by the Institutional Review Board of the Third Affiliated Hospital of Sun Yat-Sen University. Drug susceptibility testing. The first-line drug susceptibility testing (DST) was routinely performed on Lowenstein-Jensen medium using 1% indirect proportion method. For MDR isolates, four second line drugs were chosen to test. These were done according to the WHO guidelines (48) based on the following drug concentrations: isoniazid (0.2μg/ml), rifampin (40.0μg/ml), streptomycin (STR, 4μg/ml), ethambutol (EMB, 2.0μg/ml), ofloxacin (OFX, 2.0μg/ml), kanamycin (30.0μg/ml), capreomycin (40.0μg/ml) and amikacin (40.0μg/ml) (48, 55). Periodic external quality assessment of the performance of DST results was conducted by the Tuberculosis Reference Laboratory at the Beijing Research Institute for Tuberculosis Control. Genomic DNA isolation, PCR amplification and DNA sequencing. Genomic DNA 5

6 was extracted from samples using cetyltrimethyl ammonium bromide (CTAB-NaCl) method as described previously (44). Expected fragment were amplified each in a standard 50μl reaction volume,containing 20~50ng of genomic DNA, 1.25U of Taq DNA polymerase (Takara Bio, Dalian, China), 1μl of 20μM each forward and reverse primers, 5μl of 10 Taq PCR buffer, 4μl of 2.5 mm each deoxynucleotide triphosphate, and 36.5μl distilled H 2 O. The amplification was performed on a Veriti 96-well Thermal cycler (Applied Biosystems) under the following condition: initial denaturation at 94 C for 5 min that was followed by 35 cycles each consisting of denaturation at 94 C for 1 min, annealing at the primer-dependent temperature for 45sec, and extension at 72 C for 1 min. The final extension was at 72 C for 10 min. The primer sequences and the amplicons size were shown in Table 1. Amplified products were detected by 1.5% agarose gel electrophoresis and sent to BGI for sequencing service (Beijing Genomics Institute-Shenzhen, China). The amplified products were sequenced in both directions using the same forward and reverse primers as PCR amplification. Nucleotide and amino acid sequences of the amplified fragments were aligned with the corresponding sequences of the reference M. tuberculosis H37Rv strain using the Blast 2 software on the NCBI website ( All the mutations found were compared with those included in the TB Drug Resistance Mutation Database ( (41). In addition, we performed complete sequencing of rpob katg gyra gyrb and rrs (whole) genes respectively for the corresponding resistant strains in which no mutations were identified within the specific regions of these genes. Primers, used in complete sequencing of the five target genes, were presented in Table 2. Long fragments were amplified using a high fidelity DNA polymerase (PrimeSTAR HS, Takara). Genotyping method. Seventy-seven MDR isolates were genotyped using the 15 loci MIRU-VNTR technique as described previously (45). Identification of the Beijing family strains was carrying out using the RD105 deletion targeted multiplex PCR (DTM-PCR) method (13). The MIRU-VNTR genotyping data, transformed into a distance matrix on 6

7 the web site MIRU-VNTRplus ( by default setting, were treated as categorical variables and the phylogenetic analysis of the distance data was conducted with the neighbor-joining algorithm using MEGA version 5.05 (3, 46). 7

8 RESULTS General profile of drug resistance and genotyping. Among the 97 isolates obtained, 77 were drug resistant. These included 35 MDR, 26 pre-xdr, and 16 XDR. However, the remaining 20 were susceptible to all the drugs tested. Table S1 showed the profiles of the 77 resistant strains against 8 drugs: INH, RIF, EMB, STR, OFX, KAN, AMK, and CPM. Of these isolates, 42(54.5%), 51(66.2%), 39(50.6%), 18(23.4%), 16(20.8%) and 13(16.9%) were resistant to EMB, STR, OFX, KAN, AMK, and CPM, respectively. They each showed a unique genotype pattern but no cluster was formed, as analyzed using the 15 loci MIRU-VNTR method (Figure 1). Most isolates (62/77; 80.5%) were identified as Beijing family strains by DTM-PCR. Mutations identified in the rpob, katg, maba-inha, gyra, gyrb and rrs genes. Sequences were obtained for specific regions of the rpob, katg, gyra, gyrb, rrs genes and for the maba-inha operon as well. Mutations identified in the 77 resistant strains were summarized in Table 3 and 4. Rifampin and rpob. Since most RIF-resistant strains harbor mutations within the 81-bp rifampin resistance determining region (RRDR) (9, 39), a 557-bp fragment of the rpob gene comprising this region and its flanking sequences was analyzed. In this study, the majority (72/77; 93.5%) of MDR strains harbored at least one mutation within the RRDR of rpob, while 5 of the RIF-resistant strains lacked this mutation (Table 3). Moreover, a mutation Lys972Thr was detected outside the RRDR of rpob in one pre-xdr isolate with complete sequencing of rpob gene for the five strains (Table 4). A total of 26 missense mutations, one 3-bp insertion, and two silent mutations were observed (Table 3 and 4). One MDR isolate had a silent mutation Phe505Phe. Another MDR isolate showed a silent mutation Thr525Thr plus a substitution Ser531Leu. Eleven isolates had two missense mutations represented by 9 unique base changes. Five double mutants each showed a mutation within and a mutation outside the RRDR. Two isolates had triple mutations: Ser531Leu, Val615Met, and Asp626Glu were observed in one MDR isolate, while Met515Val, His526Asn, and Pro535Ser were found in one pre-xdr isolate. Of the 26 missense mutations, six, (Ile569Val, Ile572Met, Phe584Ser, 8

9 Val615Met, Asp626Glu and Lys972Thr) were first reported (Table 3 and 4). Among them, Ile569Val was found in one MDR isolate, while Ile572Met and Phe584Ser were observed in two XDR isolate respectively. In addition, the most highly changed codons were 531, 526, 516, and 511, which had frequencies of 44.2%, 24.7%, 13.0%, and 10.4%, respectively (Table 3). When all the mutations were considered, regardless they are single, double and triple, a total of 27 rpob genotype patterns were identified (Table 3 and 4). The three most frequently observed mutations accounted for 58.4% of the RIF-resistant isolates (Table 5). In contrast, 20 pan-susceptible strains and the reference H37Rv strain contained no mutations within the target fragment of rpob gene. Detection of nonsynonymous single nucleotide polymorphisms (nsnps) in rpob gene exhibited a sensitivity of 94.8% and a specificity of 100% for predicting the phenotypic RIF results (Table 6). Isoniazid and katg and inha. It has been well established that most INH-resistant isolates possess mutations in katg or maba-inha or the both (9, 39-40, 54). Therefore, a 593-bp region of katg encompassing codon 315 and a 520-bp region of maba-inha were sequenced. A total of 77 INH-resistant isolates were identified, of which, 61 (79.2%) were found to contain mutation(s) in katg and/or maba-inha (Table 3). Among the 61 mutants, 7 (11.5%) showed mutations in both katg and maba-inha, 48 (78.7%) showed mutations only in katg, and 6 (9.8%) showed mutations only in maba-inha. In contrast, the remaining 16 (20.8%) isolates had no mutations within the target fragments of the both loci. As anticipated, the most prevalent katg alteration was the substitution of threonine for serine at amino acid 315. This amino acid substitution resulted from a codon change from AGC to ACC in 40 (51.9%) isolates and to ACA in 3 (3.9%). Other codon 315 mutations of katg included a substitution with asparagine (AAC), a substitution with arginine (CGC), and a substitution with isoleucine (ATC), each found in one isolate. Two resistant strains showed a single katg missense mutation at codon 271, resulting in the substitution of proline (CCT) for threonine (ACT). Three resistant isolates had a single katg missense mutation at codon 299, leading to the substitution of cysteine (TGC; n =1) and serine (AGC; n =2) for glycine (GGC). Besides, one resistant isolate carried an alteration from TGG to TGA at codon 9

10 , resulting in a pseudogene formation. Totally, eleven different kinds of missense mutations were detected in five distinct codons (191, 271, 299, 315, and 328), and a silent mutation (Lys327Lys) was found within the 593-bp region of katg gene examined. Furthermore, complete sequencing of katg gene was performed for the 23 INH-resistant strains, in which no mutations were found within the specific region of katg gene. Consequently, one silent mutation (Val47Val) and 5 missense mutations were detected (Table 4). One MDR isolate harbored the mutation Ala110Val, and the C insertion at nucleotide position 133 of katg gene was detected in one XDR strain, Leu546Pro and Asp695Gly were found in one MDR isolate and one XDR strain respectively, while Arg463Leu, a natural polymorphism, was identified in these strains (Table 4). Collectively, 5 novel missense mutations were detected in katg: Trp191Stop, Thr271Pro, Trp328Phe, Leu546Pro and Asp695Gly (Table 3 and 4). Thr271Pro was found in one pre-xdr isolate and one XDR strain respectively, while Trp191Stop and Trp328Phe were observed in two pre-xdr isolate respectively. However, no katg mutations were found in the 20 pan-susceptible isolates. Detection of an nsnp within the region of katg analyzed exhibited a sensitivity of 75.3% and a specificity of 100% (Table 6). Thirteen of the 77 INH-resistant isolates had a mutation within the maba-inha regulatory region. Among them, 9 (69.2%) had a C-to-T transition at position 15 upstream of the start site of maba (Table 3), 3 had a transition from T to C at position 8 upstream of the maba start site, and one had a transition from T to A at the nucleotide positioned 8 bases upstream of the maba initiation codon. Of the 9 isolates with a C-15T mutation, 2 also had a katg Ser315Thr substitution, and 2 also had a katg Thr271Pro substitution. Of the three isolates with a T-8C mutation, one also had a katg Ser315Asn substitution, and 2 also had a katg Ser315Thr subtitution. In contrast, none of the 20 pan-susceptible isolates possessed a mutation in the target fragment of maba-inha. Detection of an inha promoter mutation was 16.9% sensitive and 100% specific. When the results for both katg and inha are considered together, the assay sensitivity improves from 75.3% to 80.5% and the specificity remains 100% (Table 6). 10

11 Ofloxacin and gyra and gyrb. Mutations in the quinolone resistance determining region (QRDR) of gyra, largely clustered in codons 90, 91, and 94, are the most important mechanism to confer FQ resistance(12, 15, 25). In this study, we analyzed a 427-bp region of gyra comprising the QRDR. Twenty pan-susceptible and 38 of 39 OFX-resistant isolates(23 pre-xdr and 16 XDR strains)were found to harbor the Glu21Gln and Ser95Thr mutations in gyra (Table 2). Thirty-two OFX-resistant isolates displayed 6 different types of single-point mutations at codons 94, 90, and 91, with frequencies of 69.2%, 7.7%, and 5.1%, respectively (Table 3). The most common mutations were observed at codon 94 (n =27), where the wild-type aspartic acid (GAC) was replaced with a glycine (Asp94Gly; n = 19), an alanine (Asp94Ala; n =2), an asparagine (Asp94Asn; n =5), or a tyrosine (Asp94Tyr; n =1) (Table 2). In addition, 3 OFX-resistant isolates harbored the Ala90Val mutation. Ser91Pro was another mutation found to associate with the OFX-resistant isolates (n=2). Seven OFX-resistant and 20 pan-susceptible isolates were determined to be wild type for the QRDR of gyra. Further, a mutation Gly668Asp was detected in six of the seven OFX-resistant strains by means of complete sequencing for gyra gene (Table 4). When the genotypic data for gyra were compared with the drug susceptibility results for the OFX, the sensitivity and specificity values were determined to be 82.1% and 100%, respectively (Table 6). One pre-xdr strain carried the Asp500His mutation and another XDR isolate carried the Glu540Asp mutation, both in gyrb. The isolate with the latter mutation also had an Asp94Ala mutation in gyra (Table 3). In contrast, other 37 OFX-resistant and 20 pan-susceptible isolates did not have any mutations in the examined 413-bp gyrb segment. Additionally, no mutations were detected with complete sequencing of gyrb gene for the 37 OFX-resistant strains (Table 4). To determine the OFX phenotypes, the nsnps of gyrb was analyzed, which showed a sensitivity of 5.1% and specificity of 100% (Table 6). When the gyra and gyrb data were combined, the overall sensitivity increased from 82.1%to 84.6% (Table 6). Amikacin, kanamycin, capreomycin and rrs. Amikacin, kanamycin, and capreomycin are key drugs for treating MDR tuberculosis, and the most common 11

12 mutations associated with a resistance of these drugs are located within the rrs gene coding for 16S rrna(4, 19-20). We therefore analyzed a 481-bp region of this gene. Fourteen of nineteen resistant isolates (3 pre-xdr and 16 XDR strains) showed an A-to-G transition at nucleotide position 1401, one pre-xdr isolate resistant to AMK, KAN, and CAP had a C-to-T transition at nucleotide position 1402, while the remaining four strains had no mutation within the specific region (Table 3). And further, no mutations were detected in complete sequencing of rrs gene for the four resistant isolates (Table 4). Generally, when the genotype data were compared with the susceptibility results, the sensitivity and specificity values were determined to be 78.9% and 100% (Table 6). Downloaded from on September 22, 2018 by guest 12

13 DISCUSSION In this study, a detailed genetic analysis of the drug-resistance of Mycobacterium tuberculosis isolates was performed using the MIRU-VNTR approaches, which identified 77 resistant strains among patients in the Jiangxi province of China. The 77 strains showed a great genetic diversity among comparing with each other and with those reported elsewhere, suggesting these strains were epidemically unrelated and had evolved under varied conditions of selection. They could have independently acquired drug resistance, and the acquisition could have been more dependent on the selective pressure of treatment failure. The latter varied individually, as a result of either inappropriate chemotherapy or poor adherence to treatment or inadequate monitoring (23), although transmission is now thought to play an increasing role (16, 55). Of note, the Beijing genotype was found to be predominant (80.5%) among these 77 strains. Of the 77 resistant isolates, a high number (55.8%) had the Ser315Thr mutation in katg (Table 5). This mutation has shown different rates in different countries, correlating with the prevalence of tuberculosis (5). Where the prevalence is high, the rate is also high. This mutation has occurred to 59%, 71%, and 91% of the isolates from India (34), Vietnam (10), and Russia (1), respectively. In contrast, it accounted for INH resistance only in 22% of the strains in Japan (5), and 23% in Singapore (29). In addition, five novel katg mutants were identified. Among them, the Trp191Stop mutation confers an INH resistance since it results in a pseudogene formation in katg. However, it remains to be ascertained if the Thr271Pro, Trp328Phe, Leu546Pro and Asp695Gly mutations are associated with INH resistance. Further studies are needed to assess their specific effects on katg function. The present study showed that 15 resistant strains lacked missense mutation in the target fragments of katg and maba-inha. The INH-resistance of the 15 strains may have involved additional mechanism(s), possibly the mutations in inha structural gene or in other loci, such as ndh, msha, and ahpc (4, 9, 54). For rapid identification of MDR isolates, a detection of mutations within the RRDR 13

14 of rpob is of prime importance. This is based on the following lines of evidence. First, among the available antituberculosis drugs, the molecular mechanism of RIF resistance is the most completely understood. Second, mutations within the RRDR of rpob have been reported to occur in 95% or higher of the RIF-resistant isolates (9, 36, 54). Third, the RIF resistance is an excellent surrogate marker for MDR M. tuberculosis (26, 37). In the study, six novel rpob mutations were identified, and they all fell outside of the RRDR region of the rpob gene. Nevertheless, it is not clear whether these mutations are involved in the RIF resistance. Among them, five mutations (Ile572Met, Phe584Ser, Ile569Val, and Ala615Met and Asp626Glu) were detected in the four resistant strains, which each had another mutation within the RRDR of rpob (Table 3). Are they representing compensatory mutations (6, 22) or related to RIF resistance? Further studies are therefore required for characterizing their roles. In addition, rpob His526Asp was identified in pre-xdr (5/26, 19.2%) and XDR (2/16, 12.5%) strains, while absent in simple MDR (0/35, 0%). However, it is unclear whether rpob His526Asp becomes a marker for pre-xdr and XDR M. tuberculosis strains from Jiangxi province, because a relatively smaller number of samples were analyzed in this study, more samples are required for validating it. Fluoroquinolones are the mainstay of treatment for patients with MDR and XDR tuberculosis since their inclusion in therapeutic regimens improves treatment outcome (11). In contrast, resistance to FQ increases the risk of treatment failure and death (8). Hence, FQ resistance among patients means poor prognosis. In this study, 82.1% of the OFX-resistant isolates were identified by screening the QRDR region of the gyra gene. However, by analysis of nsnps in gyrb, only 5.1% of these isolates can be verified. The latter result indicates that the inclusion of gyrb may not considerably change the performance of the genotyping assay. This is in agreement with previous observations (21, 51) that gyrb mutations are less frequent than gyra mutations and it may reflect the fact that the phenotypic resistance to FQ is mainly due to mutations in the QRDR of gyra rather than gyrb, as suggested in a recent review and meta-analysis (12). In addition, the correlation between FQ resistance and the gyrb mutations (Asp500His and Glu540Asp), which was first reported by Duong et al in 14

15 (18), has not yet been validated. Given this reason, we should preferentially screen the gyra gene rather than gyrb for FQ resistance in resource-constrained settings with high burdens of TB. Consistent with other reports (18, 50), in this study 84.6% of the OFX resistant isolates had gyra or gyrb mutations in the QRDR region, while 6 strains did not. The latter implies additional mechanism likely accounting for the OFX resistance. Possibilities include decreased cell wall permeability to the drug, increased expression of efflux pump, drug sequestration or drug inactivation (25). Of particular note, two rarely reported mutations gyra Glu21Gln (42) and Gly668Asp (28) were detected in this study. Similar to the gyra Ser95Thr mutation, the two mutations are not associated with FQ resistance, and they are characterized to be a natural polymorphism and a marker for evolutionary genetics, which has been validated by Lau et al recently (28). Therefore, in this study, we identified three natural polymorphisms in gyra gene: Glu21Gln, Ser95Thr and Gly668Asp. Recently, more novel mutations were identified in M. tuberculosis clinical isolates (14, 38). However, some strains with the mutations were phenotypically sensitive. For example, a study (35) demonstrated that the strain harboring substitution Gly551Arg in gyrb is susceptible to FQ, and 8 gyrb substitutions (Asp473Asn, Pro478Ala, Arg485His, Ser486Phe, Ala506Gly, Ala547Val, Gly551Arg, and Gly559Ala) are not responsible for quinolone resistance, although they have been observed previously in FQ-resistant strains. Therefore, the use of FQ should not be ruled out in the treatment of TB patients infected by stains harboring these mutations in gyrb. So it obviously is of importance for adopting appropriate chemotherapy regimens to know which mutations confer phenotypic resistance and which do not. Although amikacin and kanamycin are aminoglycoside antibiotics, and capreomycin is a macrocyclic peptide antibiotic, cross-resistance among them has been well documented (9, 19, 27, 31). Common rrs mutations associated with resistance to these drugs include the A1401G, C1402T and G1484T (9, 31). In this study, we observed two (A1401G and C1402T) of these mutations with A1401G (73.7%) more predominant (Table 5). It is worth noting that none of these isolates resistant to KAN, AMK and/or CAP harbored the G1484T substitution, which in most 15

16 cases is associated with high-level resistance to all three second-line injectable drugs (31). Some possible reasons may explain. First, a relatively smaller number of samples were analyzed in the present study, which may have limited the detection of the variety of variations. Second, different geographical regions may give rise to different pressure of artificial antibiotic selection on the corresponding strains. Third, the different regimes administrated may have induced different mechanisms of drug resistance. In this study, one pre-xdr isolate, resistant to KAN, AMK and CAP, was also found to contain the C1402T mutation in rrs (Table 3), but the A1401G and G1484T mutations associated with AMK resistance in the rrs gene were not detected. Since the C1402T mutation has not been shown to confer resistance to AMK (19), this stresses at least another unknown mechanism for AMK resistance in the isolate. Similar to other studies (9, 21), no mutation was detected in the rrs gene in four resistant strains. The resistance phenotypes are probably attributed to the alterations within other locus such as tlya (for CPM resistance) (32) or the promoter region of eis (KAN), which conferred low-level resistance to KAN (53), or an enhanced multi-drug efflux pump (54). Compared with the phenotypic data, the sensitivity for detection of RIF, INH, OFX, and KAN, AMK, and CAP resistance by DNA sequencing was 94.8%, 80.5%, 84.6%, and 78.9%, respectively, which are similar to those demonstrated by the majority of studies elsewhere (2, 7, 9). The most common genetic alterations related to RIF, INH, OFX, and KAN and AMK and CAP resistance were rpob Ser531Leu (44.2%), katg Ser315Thr (55.8%) and maba-inha C-15T (11.7%), gyra Asp94Gly (48.7%),and rrs A1401G (73.7%) respectively (Table 5). In addition, the RIF resistance is an excellent surrogate marker for MDR M. tuberculosis. Based on these facts, we think that detecting the RRDR of rpob alone is efficient for accurate and rapid identification of MDR from Jiangxi province, and screening the RRDR of rpob and the QRDR of gyra is economical and effective for prediction of pre-xdr, and examining three loci (rpob, gyra and rrs) simultaneouly is suitable for rapid identification of XDR in the region. 16

17 To aid in rapid diagnosis of MDR and XDR-TB, two commercially available DNA strip assays, the GenoType MTBDRplus and GenoType MTBDRsl assay (Hain Lifescience, Nehren, Germany), have been recently developed to detect genetic mutations associated with resistance to the first-line drugs (INH and RIF), and the second-line injectable drugs and fluoroquinolones as well (7, 33). According to the preliminary sequencing results obtained in this study and the mutations involved in drug resistance identified by the two assays, it has been speculated that the GenoType MTBDRplus assay might have been able to identify about 90% of RIF-resistant isolates, about 70% of INH-resistant isolates, and about 65% of MDR isolates from Jiangxi region. Meanwhile, the GenoType MTBDRsl assay might have been able to detect about 80% of the OFX resistance and 78% of the resistance to KAN, AMK or CAP. In combination with the MTBDRplus assay, the GenoType MTBDRsl assay might have been able to identify 75% of XDR isolates (Table S1 in the supplemental material). Therefore, we conclude that the GenoType MTBDRplus assay might have performed equally well for the detection of RIF resistance in Jiangxi province. However, because the mechanisms of drug resistance in M. tuberculosis are complicated and not yet fully understood, and resistance-associated mutations vary geographically, the GenoType MTBDRplus assay might have demonstrated a relatively poor prediction for INH resistance in this area. The performance of the two assays for detecting MDR and XDR strains from the region are needed to evaluate in a real-world setting. In summary, our results indicate that most frequent mutations in the rpob, katg, inha, gyra, gyrb and rrs genes are consistent with those reported from other regions of the world(2, 9, 54), suggesting that these mutations have global ramifications. Five novel mutations in katg gene and six new alleles in rpob gene were identified, which will broaden current knowledge of molecular basis of drug resistance in M. tuberculosis. Further studies are needed to elucidate their actual roles in the drug resistance of M. tuberculosis. These new mutations together with existing mutations can be used to design diagnostic tests utilizing other mutation detection technologies such as the line probe assay or DNA microarrays. The molecular information obtained 17

18 from this study will be of particular value in the rapid clinical detection of MDR and XDR M. tuberculosis strains in the Jiangxi province in the near future. Nucleotide sequence accession numbers. The nucleotide sequences for the mutant genes obtained in this study were deposited in the GenBank database under the following accession numbers: JN037847, JN049495, and JN for the katg gene mutants Trp191stop, Trp328Phe, and Thr271Pro respectively, JN037845, JN037846, JN210554, and JN for the rpob gene mutants Ile572Met, Phe584Ser, Ile569Val, and Ala615Met and Asp626Glu respectively. Downloaded from on September 22, 2018 by guest 18

19 418 ACKNOWLEDGMENTS We thank Yijun Luo and Jianlin Yang, the Clinical Laboratory in the Fifth People's Hospital of Ganzhou, Ganzhou, China, for providing some study isolates. We are indebted to Rongyao Tu, Department of Clinical Mycobacteriology, Jiangxi Chest Hospital, Nanchang, China, for his collection of clinical isolates and technical assistance in the drug susceptibility testing. This work was supported by a Grant-in-Aid for Scientific Research (B) ( ) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Downloaded from on September 22, 2018 by guest 19

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25 604 TABLE 1 Primers used in this study by PCR method in the hot-spots of drug target genes Primer a Nucleotide sequence Annealing temp( ) Amplicon position b Product size (bp) rpob-f rpob-r katg-f katg-r (maba-inha) -F (maba-inha) -R gyra-f gyra-r gyrb-f gyrb-r rrs-f rrs-r 5'-TCAAGGAGTTCTTCGGCACC-3' 5'-CTGCATGTTTGCCCCCAT-3' 5'-TGGGCGGACCTGATTGTT-3' 5'-CCGTCCTTGGCGGTGTATT-3' 5'-AAGGCAGAAGCCGAGTAG-3' 5'-ACATTCGACGCCAAACAG-3' 5'-CCGGATCGAACCGGTTGAC-3' 5'-GTTAGGGATGAAATCGACTG-3' 5'-GTCGTTGTGAACAAGGCTGTG-3 5'-GTGGAAATATGTTGGCCGTC-3 5'-GTGAGATGTTGGGTTAAGTCC-3' 5'-TGGTGCTCCTTAGAAAGGAG-3' c a F:forward; R: reverse. 606 b according to the complete sequences of M. tuberculosis H37Rv genome (Accession No: NC_000962) except for katg. 607 c according to the sequence of M. tuberculosis H37Rv gene for catalase-peroxidase (Accession No: X68081). Reference This study This study This study Downloaded from on September 22, 2018 by guest 25