Spoligotyping and Drug Resistance Analysis of Mycobacterium tuberculosis Strains from National Survey in China

Background Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is one of the major causes of death in the world today. Although China has the second largest global case rate of tuberculosis, a systematic study of TB prevalence in China has not been completed. From 2006 to 2007, the base line surveillance of tuberculosis was carried out by Ministry of Health, and more than 4000 representative strains were selected from 31 provinces in China. Methodology/Principal Findings The aim of the present research was to survey the genotypes of representative Mycobacterium tuberculosis (M. tuberculosis) strains from China using spacer oligonucleotide typing (spoligotyping), and to analyze the relationship between genotype and drug resistance for the first time. A total of 4017 clinical isolates were collected from 2007 to 2008 throughout China. Among those M. tuberculosis isolates, 2500 (62.2%) isolates were Beijing genotypes. The percentage of Beijing genotypes in northern China was higher than in southern China (76.5% vs. 53.2%). Additionally, the frequencies of rifampin-resistant, ofloxacin-resistant and multidrug-resistant isolates were significantly higher in Beijing genotype strains than non-Beijing strains. Furthermore, a novel genotype named “China Southern genotype (CS)” was only isolated from Fujian and Guangdong provinces. Hence, it is very practical to uncover the reason for prevalence of the CS type in southern China. Conclusions/Significance In conclusion, Beijing family genotypes were still the predominant genotype throughout China, which exhibited a greater correlation with rifampin-resistance, ofloxacin-resistance and MDR phenotypes than other TB spoligotypes, and some regions of China showed several unique characters in the distribution of M. tuberculosis genotypes. Our research represents an important contribution for the TB control and research community, which completes broad pictures on drug resistance levels and distribution of M. tuberculosis strain types over China.


Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is one of the major causes of death in the world today [1,2]. It is estimated that one-third of the world's population has been infected by Mycobacterium tuberculosis. The situation in developing countries is worse. According to the World Health Organization (WHO), about 95% of the eight million new cases each year occur in middle-and low-income countries [2]. It is well-known that China, where 80% of TB patients live in rural areas [3], has the second largest population of tuberculosis patients [4]. Tuberculosis is not only a disease harming people's health, but also a social-economic issue [3].
Genotyping of M. tuberculosis isolates has significantly improved knowledge of the epidemiology of tuberculosis (TB) [5]. With the use of molecular tools, it is possible to detect unsuspected transmission, to identify false-positive cultures, and to distinguish between reinfection and relapse [6]. Spacer oligonucleotide typing (spoligotyping) is a reliable and informative technology for characterizing the genetic structure of tuberculosis populations, especially Beijing family genotype strains [7,8]. Using PCR technology, spoligotyping analyzes genomic polymorphisms in the short direct repeat (DR) of M. tuberculosis, a genetic marker consisting of identical 36-bp DRs interspersed with 35-to 41-bp non-repetitive spacer sequences [8].
Molecular typing of M.tuberculosis strains from several Asian countries revealed that a specific family of M. tb strains, named the Beijing genotype, were the dominant pattern [9].The Beijing family genotype is also common in the countries of the former Soviet Union [10], but rare in other regions such as Finland and India [11]. In previous reports, the Beijing genotype was found responsible for outbreaks of multidrug-resistant TB (MDR-TB), and some study indicate that Beijing genotype is associated with drug resistance [7,12]. Although China has the second largest global case rate of tuberculosis (TB), a systematic study of TB prevalence in China, especially Beijing family, has not been completed. Hence, it is meaningful to analyze the situation of Beijing genotype in tuberculosis isolates.
In China, the base line surveillance of tuberculosis was carried out by Ministry of Health recently, and representative strains selected from 31 provinces in China. In this study, we used spoligotyping to classify the 4017 representative strains mentioned above. Our aim was to find the prevalent genotype in China, especially the prevalence of Beijing genotyping. In addition, the relationship between genotype and drug-resistance was also analyzed.

Predominant spoligotypes in China
Among the 4017 typed isolates, a total of 568 spoligotypes were identified. Of these, 98 spoligotypes were previously represented as Shared International Types (STs) according to SpolDB4.0, while the other 470 were reported for the first time ( Table 2). After clustering with BioNumerics software, 3610 (89.87%) isolates were classified into 161 groups containing 2 or more strains. The mean number of isolates per cluster was 22.42. Additionally, 407 (10.13%) strains did not form clusters ( Figure 1, Table 2).

Drug susceptibility profiles of predominant spoligotypes in China
The five most predominant spoligotypes as well as the newly described CS spoligotype were selected to determine drug susceptibility profiles. The ST1 type, a member of Beijing family, was designated as the control, for comparison with other types. As shown in Table 5, relative to the ST1 profile, ST53 and ST54 presented bigger difference than other types. As the second most frequent strain, ST53 displayed lower INH (14.0% vs. 21.7%, x 2 = 7.55, P = 0.006), RIF (6.0% vs. 13.2%, x 2 = 8.76, P = 0.002) and OFLX (0% vs. 4.6%, x 2 = 8.22, P = 0.001) resistance. The frequency of MDR isolates with the ST53 genotype was significantly lower than that of ST1 (6.0% vs. 11.2%, x 2 = 5.24, P = 0.020), which contributed greatly to low drug resistant percentage of non-Beijing family. On the contrary, the frequency of drug resistance was higher in ST54 than ST1, and the proportions of EMB (15.9% vs. 8.6%, x 2 = 7.58, P = 0.010), INH (31.8% vs. 21.7%, x 2 = 6.88, P = 0.011) and SM (42.9% vs. 27.8%, x 2 = 13.26, P = 0.001) resistant were significantly higher. Besides, there were no RIF resistant and MDR isolate in ST52 type, therefore, the ratio of them is lower than those of ST1 significantly (Table 5).

Discussion
China has one of the highest tuberculosis burdens in the world. In previous reports, genotyping was performed on some clinical TB isolates collected from several areas of China, which provided us the knowledge about the distribution of dominant TB genotypes. However, due to incomplete representation, these strains could not reveal prevalence and epidemic trend of tuberculosis in China. In this study, 4017 strains were enrolled from the National Drug Resistance Base-Line Surveillance of Tuberculosis. Of these, 62.2% were representatives of the Beijing genotype. Early research based on clinical isolates of different region showed that the percentage of Beijing genotype varied from 67.1% to 92% [7,11,[13][14][15][16][17], which was higher than our research. The explanation of this obvious difference may be that the strains used in previous study were obtained from clinical hospital instead of epidemiological survey. In comparison with surrounding countries, prevalence of the Beijing genotype of this study is less than Hong Kong (70%) [18], but more than Taiwan (44.4%) [19], Vietnam (54%) [20], Thailand (44%) [21] and Russia (44.5%) [22]. Hence, this study demonstrates that the Beijing genotype is a dominant TB genotype of China. {Isolates resistant to isonazid (n = 744; 20.5%), rifampin (n = 412; 11.3%), ethambutol (n = 287; 7.9%), streptomycin (n = 1073; 29.5%), kanamycin (n = 85; 2.3%), ofloxacin (n = 145; 4.0%), multiple drugs (MDR) (n = 62; 9.9%) or extensively drugs (XDR) (n = 27; 0.7%). {MDR, multidrug resistant, represents isolates resistant to at least isonazid and rifampin. 1XDR, extensively drug resistant, represents isolates resistant to at least rifampicin and isoniazid, as well as any member of the quinolone family and at least one of the following second-line anti-TB injectable drugs. *: P,0.05 (significant); **: P,0.01 (highly significant); ***: P,0.001 (extremely significant). doi:10.1371/journal.pone.0032976.t004  The spoligotying profiles of different geographical areas including north and south China showed different characteristics. The main diversity was that the proportion of Beijing genotype in the north area was higher than that in the south area. However, the T family genotype, one of the prevalent genotypes in Africa, Central and South America and Europe [23], was significantly higher in southern China than in the north. In addition, more novel spoligotypes were identified in southern China. Overall, the genetic variability of the DR locus was more diversified in the south of China, and several reasons may be responsible for it. First of all, the economic state and traffic condition of southern is better than that of northern, causing more frequent movement of the population between either different provinces or different countries in south area. Additional, the dissimilar customs adapted to the each climate may the other possible reason for this diversity. People lived in the north China will share longer indoor time because of cold winter months, which may be related with the prevalence of Beijing genotype. However, the proof of this hypothesis is difficult since the more population-based epidemical results are necessary. Interestingly, CAS1-DEHL1 type was almost exclusively detected in northern China (29/30). Of the 29 isolates, 26 were collected from Xinjiang Uygur Autonomous Region. In a previous report, the Middle East, and Central and Southern Asia were ''high incidence'' areas of CAS1-DEHL1 family [23][24][25]. Obviously, the Xinjiang region is adjacent to several Central Asian countries and they share similar culture traditions. Hence, frequent movement of ethnic groups may be the major factor to explain the prevalence of the CAS1-DEHL1 genotype in Xinjiang [26].
According to subtype analysis with Shared International Types (SITs), a novel CS genotype containing 22 strains was all indentified among the isolates from Fujian and Guangdong provinces, which were all located in the south area of China. Hence, it is very practical to uncover the reason for prevalence of the CS type in southern China. In general, as the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a host population, isolates with the CS genotype need to be studied carefully.
According to literature reports, the Beijing genotype has significant associations with drug-resistance and might be responsible for the spread and emergence of MDR-TB. Studies in several countries including Vietnam, Germany, Cuba and America, have confirmed these results. Because of the high incidence of the Beijing genotype in many regions, some researchers have proposed that the Beijing genotype may be correlated with high virulence and easy transmission. In order to define any association of drug resistance with the Beijing genotype, we determined the frequency of the Beijing genotype among bacterial isolates exhibiting different categories of drug resistance. In this study, comparison between Beijing genotype and non-Beijing genotype, the resistance to RIF and OFLX was both significantly higher in Beijing, In agreement with previous reports, the proportion of MDR-TB was also higher in Beijing genotypes compared to non-Beijing families. In 2006, Nikolayevskyy et al. revealed that the Beijing genotype was closely related to RIF and INH resistance as well as to MDR in the Ukraine [27]. Research in Central Asia showed similar results, including higher RIF, INH, SM and MDR resistance in Beijing family [12]. Referred to OFLX, Duong et al. reported that gyrA mutations were more common in Beijing type, which resulted in higher levels of OFLX resistance [28]. In contrast to previous studies [20], we found that the non-Beijing genotype presented higher SM resistance than Beijing. Our results are in agreement with studies performed by Niemann et al. [29] and Tanveer et al. [30], respectively, which showed that some non-Beijing genotypes represented higher percentage of SM-resistance. It is obvious that non-Beijing genotype comprised of a large number of sub-genotypes. Hence, the conflicting results observed in different studies may be due to the different distribution of non-Beijing genotypes. While the association between Beijing genotype and levels of drug resistance appear to arise from high mutation rates in drug resistanceassociated genes, it was not determined whether virulence and infectivity of the Beijing family was more critical than other genotypes. That is because the discriminatory power of spoligotyping is not ideal, and the strains selected as the Beijing type may not represent for Beijing family well.
Some published studies have revealed several families of non-Beijing genotypes, such as Haarlem family [31] and LAM family [32], have also shown affinity to drug resistance. Therefore, it is meaningful to analyze the relationship between spoligotype and drug-resistant phenotypes in China. After analysis towards the fifth most popular and new found (CS) types, we can find that ST1, the main member of Beijing genotype in China, was responsible for the strong association between the Beijing genotype and resistance to several drugs as well as with MDR-TB. In this investigation we also found a high proportion of drug resistance in ST54 genotypes from the MANU2 family. The level of INH, EMB and SM resistance for ST54 was even greater than ST1. The MANU2 family has a low prevalence worldwide, so there are few reports on an association between phenotype and the MANU2 genotype. In a study from South Africa, 2 isolates were enrolled, one of which was resistant to RIF, and the other of which was INH resistant. As this is the first report of a strong association between drug resistance and the MANU2 family genotype, further confirmatory research is needed. In addition, the drug resistance of CS type displayed similar patterns with ST1, which also indicated that CS type was a member of Beijing family, and might derive from ST1.
This study shows the prevalence of different TB genotypes in China for the first time, which completes broad pictures on drug resistance levels and distribution of M. tuberculosis strain types over China, and Beijing genotype (62.2%) is still the predominant strains in China, while the percentage of the type is lower than previous report. The distribution of Beijing family represents that the proportion of north is significant higher than that of south. Due to the characters of population mobility in China, the composition of other spoligotypes also shows distinct features. Because of the limitation of spoligotyping, future studies are needed to perform genotyping with more powerful technologies to consummate the genotype data, such as MIRU-VNTR and RFLP. Moreover, Beijing family is found to be significantly associated with RIF and OFLX resistance, as well as with MDR-TB, and it is essential to carry out a systematic study on relationship between genotype and phenotype of Beijing genotype.

Study population and bacterial strains
A total of 4017 clinical strains of tuberculosis, isolated from the base-line surveillance of tuberculosis in 2006, were collected from 31 provinces in China excluding Taiwan, Hong Kong and Macao. The distribution of isolates was shown in Figure 2. The number of patients enrolled in surveillance was proportionally based on patient numbers in different provinces. The protocols applied in this study were approved by the Ethics Committee of the Chinese Center for Disease Control and Prevention. Patients were able to access the study if they signed an Informed Consent form. All strains, stored in Trypticase soy broth with glycerol ay 270uC freezer, were recovered on Lowenstein-Jensen medium for 4 weeks at 37uC.

Drug susceptibility testing and Mycobacterium species identification
Mycobacterium identification and testing of the drug susceptibility of these strains to four first-line anti-TB drugs (isonazid, rifampin, ethambutol and streptomycin) and two second-line anti-TB drugs (kanamycin and ofloxacin) were performed as recommended by WHO/IUATLD [33]. The concentrations of drugs in media were as following: isonazid 0.2 mg/mL, rifampin 40 mg/mL, ethambutol 2 mg/mL, streptomycin 4 mg/mL, kanamycin 30 mg/mL and ofloxacin 2 mg/mL. Strains resistance to isoniazid and rifampin were defined as MDR-TB. The strains was declared resistant to the specific drug when the growth rate was .1% compared to the control. In addition, XDR-TB was defined as isolates resistant to rifampicin and isoniazid, as well as any member of the quinolone family and at least one of the following second-line anti-TB injectable drugs. Media supplied separately with paranitrobenzoic acid (500 mg/mL) and thiophen-2-carboxylic acid hydrazide (5 mg/mL) was used to perform Mycobacterium species identification. All the drugs were purchased from Sigma-Aldrich (St. Louis, MO).

Molecular typing methods
Genomic DNA was extracted from freshly cultured bacteria. Cells were resuspended in 500 mL TE buffer(pH 8. 0), then heated in 95uC water bath for 1 hour. After centrifugation of cellular debris, DNA in the supernatent served as PCR template. Spoligotyping was used to identify the genotype of TB strain in the DR locus as described previously [10]. A commercially available kit (Isogen Bioscience BV, Maarssen, The Netherlands) was used as described by the manufacturer. All chromosomal DNA was amplified with primers DRa (59-CCGAGAGGG-GACGGAAAC-39) and DRb (59-GGTTTTGGGTCTGAC-GAC-39). Then the amplified products were hybridized with the membrane. After washing with 26SSPE solution (360 mM NaCl, 20 mM NaH 2 PO 4 , 2 mM EDTA [pH 7.2]) supplemented with 0.5% SDS, the membrane was hybridized with streptavidinperoxidase conjugate. The final image was detected with a chemiluminescence system, including the ECL detection liquid (Amersham, Buckinghamshire, United Kingdom) and ECL-Hyperfilm (Kodak, Rochester, NY). Beijing genotype strains were defined with the pattern that hybridized to all of the last nine spacer oligonucleotides (spacers 35 to 43), and Beijing-like genotype strains were ones that hybridized to only some of the last nine spacers.

Data analysis
The genotyping were expressed in binary and octal formats in Microsoft Excel spreadsheets. Drug resistance test results were arranged in binary formats in Excel. All spoligotype data was submitted to the international databases MIRU-VNTRplus (http://www.miru-vntrplus.org) [23] and SpolDB4.0 http://www. pasteur-guadeloupe.fr:8081/SITVITDemo) [13]. In addition, the resulting data was analyzed using BioNumerics (Version5. 0, Applied Maths, Sint-Martens-Latem, Belgium) software. Cluster analysis was performed and a dendrogram was generated in Bionumerics using the Dice similarity coefficient and UPGMA coefficient. In addition, statistical analyses between genotype and phenotype were performed in SPSS 11.5 (SPSS Inc.). Chi-square and Crosstab analysis were run to identify if the differences between two statistical datas were significant, and P value less than 0.05, 0.01 and 0.001 were defined as ''significant'', ''highly significant'' and ''extremely significant'' diversity, respectively. The extent of association was shown as an odds ratio (OR) and 95% confidence interval (95% C.I.).