Haiyan Dong, Zhiguang Liu, Bing Lv, Yuanyuan Zhang, Jie Liu, Xiuqin Zhao, Jinghua Liu and

JCM Accepts, published online ahead of print on August 0 J. Clin. Microbiol. doi:./jcm.00- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China Haiyan Dong, Zhiguang Liu, Bing Lv, Yuanyuan Zhang, Jie Liu, Xiuqin Zhao, Jinghua Liu and Kanglin Wan* National Institute for Communicable Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Beijing, China Haiyan Dong, Zhiguang Liu, Bing Lv and Yuanyuan Zhang contributed equally to this study. *Corresponding author. Mailing address: National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, P.O. Box, Changping, Beijing 0, P. R. China. Tel/Fax: 00 00. E-mail: wankanglin@icdc.cn Downloaded from http://jcm.asm.org/ on December, 01 by guest 1 1

ABSTRACT 1 1 1 1 A total of Mycobacterium tuberculosis isolates from provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 1 isolates were grouped into clusters, and the remaining isolates were orphans. Comparison with the SpolDB.0 database revealed that spoligotypes had shared international type numbers in the database and the other 10 were novel. These 10 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (.0%) followed by T family (.%). CAS family strains were only found in the Xinjiang and Tibet regions, while EAI family strains were only found in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study. INTRODUCTION Tuberculosis (TB) continues to be a major public health problem in China. Based on the data of a nationwide random survey of the epidemiology of TB in China in 000, there were probably.1 million active TB patients in the country, including 1.0 million smear-positive cases, which were the infectious sources (1). From 00 to 00, more than 1 million new TB cases emerged each year. Consequently, the task of controlling TB in China remains difficult. Downloaded from http://jcm.asm.org/ on December, 01 by guest 1 The genotyping of Mycobacterium tuberculosis (M. tuberculosis) strains is important for TB

1 1 1 1 1 0 control because it allows for the detection of suspected outbreaks and the tracing of transmission chains. It is also important to monitor species diversity as well as identify secondary infections (,,, 1). The insert sequence 1 restriction fragment length polymorphism (IS1-RFLP) is thought of as the gold standard genotyping method for M. tuberculosis strain genotyping identification (,, 1). However, this method is time-consuming, labor intensive, and costly. Furthermore, it is difficult to compare results between laboratories. Spacer oligonucleotide typing (Spoligotyping), which is based on the analysis of polymorphisms of direct repeat (DR) regions comprised of bp DRs interspersed with to 1 bp unique spacer sequences, is a good alternative to traditional IS1-RFLP fingerprinting because of its simplicity, speed, and reliability (, ). Spoligotyping is useful for classifying M. tuberculosis strains into spoligotype families and subfamilies according to the presence or absence of spacer regions (). Brosch et al. report that M. tuberculosis can be divided into ancestral or modern strains based on M. tuberculosis specific deletion 1 (TbD1) region analysis. The TbD1 region is present in ancestral M. tuberculosis strains, but is absent from modern ones. These ancestral strains predominantly originated from endemic foci, whereas modern M. tuberculosis represent epidemic M. tuberculosis strains that were introduced into the same geographical regions more recently as a consequence of the worldwide spread of the tuberculosis epidemic (). Presently, an international spoligotype database, SpolDB.0, has been established. Although the updated SpolDB.0 version regards the global distribution of M. tuberculosis spoligotypes, it contains little information regarding M. tuberculosis strains in China (). In this study, we typed Downloaded from http://jcm.asm.org/ on December, 01 by guest

M. tuberculosis clinical isolates from different provinces across China between 00 and 00 using spoligotyping to study the M. tuberculosis diversity in China. 1 1 1 1 1 MATERIALS AND METHODS M. tuberculosis strains and DNA isolation. A total of M. tuberculosis isolates were randomly collected between 00 and 00 from patients at different provincial tuberculosis hospitals across China; provincial tuberculosis hospitals are responsible for the delivery of health care to TB patients in each province (Fig. 1). Demographic, epidemiologic, and clinical information were obtained from the medical records of all patients, including sex, age, and contact (family/close contact) data, as well as results of mycobacterial smears, signs, symptoms, previous TB history, present address, and associated medical data from local doctors working in TB hospitals using unitive epidemiological methods. Mycobacterial genomic DNA was extracted from mycobacterial colonies grown on Löwenstein Jensen medium by resuspending one loopful of mycobacterial colonies in 00 µl TE buffer ( mm Tris-Cl, 1 mm EDTA) and incubated at for 0 min. Then, the supernatant containing the DNA was collected by centrifugation,000 g for min and stored at 0 for further use (). Spoligotyping. In order to investigate the population structure of the M. tuberculosis strains, Spoligotyping was carried out using a homemade membrane with covalently bound oligonucleotides derived from the spacer sequences of M. tuberculosis HRv and M. Bovis BCG P as previously described by Kamerbeek et al. (). Briefly, the DR region was amplified by Downloaded from http://jcm.asm.org/ on December, 01 by guest

PCR using primers DRa and DRb. The amplified DNA was subsequently hybridized to a set of oligonucleotide probes by reverse line blotting. 1 1 1 1 1 TbD1 analysis. TbD1 analysis was performed using the methods described by Brosch et al. (). Briefly, the presence or absence of TbD1 was analyzed by PCR assays using primers complementary to the sequences of the deleted region or the internal sequences of the intact region. PCR products were analyzed by electrophoresis on % agarose gel. For isolates containing the TbD1 region (TbD1+) or lacking the TbD1 region (TbD1 ), a PCR product was obtained with either internal or flanking primers, respectively. Database comparison. The spoligotyping results were entered in octal and binary formats in Microsoft Excel spreadsheets; spoligotype patterns were designated as -character-long strings consisting of black and white squares representing the presence or the absence of an individual spacer, respectively. Spoligotype designations were determined by comparing the spoligotyping results with already existing designations in the international database, SITVIT (Institut Pasteur de Guadeloupe) an updated version of the international spoligotyping database, SpolDB.0 () (http://www.pasteur-guadeloupe.fr:01/sitvitdemo). At the time of matching analysis, the updated SpolDB.0 contained 0 patterns distributed into 1 shared types in 1 countries. Patterns that were not found in SpolDB.0 were assigned to families and subfamilies using the SpotClust program (), which was built on the SpolDB database (http://www.pasteur-guadeloupe.fr/tb/spoldb). Downloaded from http://jcm.asm.org/ on December, 01 by guest

RESULTS 1 1 1 1 1 Genetic diversity and family assignment. Among the typed isolates, (1.0%) were classified into one of the shared international types (SITs) according to SpolDB.0 (data was summarized in Table 1& detailed in supplemental Table ). The remaining 1 isolates generated 10 different spoligotypes that had not been previously described in the database (data was shown in supplemental Table ). 1 isolates were grouped into clusters containing to 10 isolates, while the remaining did not form clusters. Out of the isolates that did not form clusters, represented true orphan patterns that did not previously exist in SpolDB.0; pseudo-orphans were found in SpolDB.0, but were present as singles in this study. The main results of the spoligotyping analysis are summarized in Table. Among the isolate clusters, we found minor spoligotypes (including to isolates) and major spoligotypes (> isolates). Isolates ST1, ST, ST, ST, ST0, ST, ST, ST, and ST represent more than 0% of the total number of isolates in this study (Table 1). Family assignment revealed that the most frequent strain was the Beijing family ST1 (.1%), followed by ST (.%) of the T1 family and ST (1.%) of the Beijing family. The Beijing family was the most prevalent genotype in China. These novel spoligotypes were assigned to families and subfamilies using the SpotClust program. Based on the results of these new spoligotypes, the most prevalent strains were the T1 family, followed by family and the Beijing family. Out of all the studied isolates, the most prevalent family was the Beijing family Downloaded from http://jcm.asm.org/ on December, 01 by guest

1 1 (/,.0%), followed by the T family (.%) and the Haarlem family (.%). In addition, other family strains, such as the CAS family, EAI family, S family, MANU family, and X family, were found in China. It is interesting to note that CAS family strains were only found in the Xinjiang and Tibet regions, while EAI family strains were only found in Fujian Province. Geographical distribution of Beijing family strains in provinces. The geographical distribution of the Beijing family strains in provinces is shown in Table. The mean percentage of Beijing family strains was.0%, which varied greatly among the provinces (~.0.%). The highest prevalence was found in Beijing and its surrounding areas, while a lower prevalence was found in Central and Southern China. The geographical distribution of Beijing family strains provides evidence for the recent clonal expansion of this particular family strain. TbD1 analysis. All isolates were analyzed for the presence or absence of the TbD1 region by PCR. Seven isolates had this region intact, whereas all other isolates lacked this region. Based on data from SpolDB.0 and SpotClust, these isolates belonged to the EAI family ( isolates had SIT numbers and were true orphans). DISCUSSION Downloaded from http://jcm.asm.org/ on December, 01 by guest 1 1 1 The present study aims to describe the genetic diversity of M. tuberculosis strains collected from provinces in China using the spoligotyping method. Although these strains are not representative of all strains present in these areas, they provide a preliminary insight into the

1 1 1 1 1 0 population structure of M. tuberculosis spoligotypes in China. Our study identified previously described (according to SpolDB.0) spoligotypes and 10 novel spoligotypes. The most prevalent M. tuberculosis strains were ST1 (.1%; Beijing family), followed by ST (.%; T1 family) and ST (1.%; Beijing family). Based on the SpolDB.0 spoligotype database and SpotClust results, the most prevalent lineage was the Beijing family (.0%). This family strains are genetically closely related, have a characteristic spoligotype pattern, and have been identified in many countries worldwide (,, ) since they were first described by van Sooligen et al. in the Beijing area in 1 (). However, there is little available information regarding whether the observations made in the Beijing area are representative of the whole country. This study may provide a preliminary insight into the population structure of M. tuberculosis genotypes in China. The prevalence of the Beijing genotype of the strains analyzed exhibited geographical variation ranging from.0% to.% the highest prevalence was found in Northern China, followed by Central and Southern China. However, Xinjiang might represent a special case because its particular situation may be related to geographic, climatic, or ethnic differences. Rates of infection of Beijing family strains in the regions neighboring Beijing are higher than those in the most distant regions this supports the hypothesis that this family strains might originate from the Beijing area. Beijing family strains are aggressively expanding clones that are found in a number of populations worldwide (). Beijing family strains are dominant in neighboring countries of China, such as Thailand, South Korea, Mongolia, and Vietnam. Beijing family Downloaded from http://jcm.asm.org/ on December, 01 by guest

1 1 1 1 1 strains also been found in Europe, Africa, and the United States. Based on epidemiologic data, Beijing family strains may have a selective advantage over other strains, enabling better communication among the population (,, 1). The low diversity of the highly prevalent Beijing family in this study may indicate that the Beijing family is spreading rapidly in China. It may also reflect a slower evolution of the DR region in the Beijing family strains. In addition to the prevalent Beijing family strains, in this study, we also detected other strain family: Haarlem, CAS, EAI, LAM, X, S, and MANU family strains. The remaining strains belonged to the T family, which was the second most frequently occurring of all tested strains and 10 novel spoligotypes. Although the T family is one of the most prevalent strains, it remains an ill-defined family of M. tuberculosis that is found worldwide (1, 1). It has been suggested that this family strains have started spreading across China; of course, this requires further testing using more extended typings of more clinical isolates across more provinces of China. One very interesting finding of this study is that of the strains tested belong to the CAS family, which was previously found primarily in India (, 1) these CAS family strains were only found in Tibet and Xinjiang. Among the CAS family strains, and strains originated from patients of Tibetan and Uyghur ethnicity, respectively. This finding suggests that CAS family might have biogeographical specificity. Moreover, Tibet and Xinjiang share geographic borders with India where the CAS family is dominant. It has also been suggested that this family strains might be transported by trade, tourism, or migration from India. Downloaded from http://jcm.asm.org/ on December, 01 by guest

1 1 1 1 1 The TbD1 analysis employed in this study shows that the majority of isolates (.0%) belonged to the TbD1 modern strains, whereas only strains (0.0%) were TbD1+/EAI isolates. These TbD1+/EAI strains were only found in Fujian Province, which is located in Southeastern China. TbD1 was specifically lacking from the mmpl genes of nearly all M. tuberculosis strains. On the other hand, TbD1 is present in all other members of the M. tuberculosis complex. However, the few M. tuberculosis strains that contained the TbD1 region were designated as ancestral M. tuberculosis strains, because they belong to a lineage that split from all other M. tuberculosis strains before the deletion of TbD1 occurred. EAI strains have been the focus of attention for some time due to their presumed low virulence characteristics and links to ancestral African predecessors (1,, 0). The predominance of modern M. tuberculosis strains and relatively poor representation of ancestral strains in China supports the hypothesis that China has a relatively modern endemic focus of TB. Since the ancestral TbD1+/EAI strains were only found in Fujian Province, this suggests that they are endemic there. This finding corroborates the biogeographical specificity of most of the spoligotype clades. In conclusion, this study provides a preliminary insight into the population structure of M. tuberculosis circulating in China as well as the distribution of Beijing family strains in various provinces. This study will be continued and extended to other provinces in China in order to provide a better estimation of the Beijing family as a risk factor for the development of TB in China. Downloaded from http://jcm.asm.org/ on December, 01 by guest 0 ACKNOWLEDGMENTS

1 We would like to thank Lishui Zhang, Xiujun Yang, Chongxiang Tong, Li Shi, Feiying Liu, Yingcheng Qi, Qing Wang, Xuanmin Zhang, Xiaohui Cao, Yunhong Tan, Haitao Li, Xiaomeng Wang, and Jun Yang for supplying strains. We would also like to thank Christine Pourcel, Gilles Vergnaud, Philippe Le Fleche, Christophe Sola, Dick van Sooligen, and Kristin Kremer for their helpful advice. Lastly, we would like to thank the hospital staff of the provinces for their invaluable contributions to this study. This work was financially supported by the Transmission Mode of Tuberculosis project of the National Key Programme of Mega Infectious Diseases (00ZX0/0-0-0) and the Study on the genetic polymorphism and strain level identification of Mycobacterium tuberculosis in China of the National Natural Science Funding of China (). APPENDICES A TABLE. Spoligotypes of isolates with a shared international type (SIT) number in SpolDB.0 A TABLE. Spoligotypes of 1 isolates that had not been identified in SpolDB.0 FIGURE LEGENDS Downloaded from http://jcm.asm.org/ on December, 01 by guest 1 1 Figure 1. A map of China showing the distribution of Mycobacterium tuberculosis isolates included in this study (the numbers indicate the absolute number of isolates per province).

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Nguyen, M. A. Behr, T. C. Victor, and P. D. van Helden. 00. Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data. J Clin Microbiol 0:-. 1 1 1. Ahmed, N., N. Z. Ehtesham, and S. E. Hasnain. 00. Ancestral Mycobacterium tuberculosis genotypes in India: implications for TB control programmes. Infect Genet Evol :-.. Bhanu, N. V., D. van Soolingen, J. D. van Embden, L. Dar, R. M. Pandey, and P. Seth. 00. Predominace of a novel Mycobacterium tuberculosis genotype in the Delhi region of India. Tuberculosis (Edinb) :-.. Bifani, P. J., B. Mathema, N. E. Kurepina, and B. N. Kreiswirth. 00. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol :-.. Brosch, R., S. V. Gordon, M. Marmiesse, P. Brodin, C. Buchrieser, K. Eiglmeier, T. Garnier, C. Gutierrez, G. Hewinson, K. Kremer, L. M. Parsons, A. S. Pym, S. Samper, D. van Soolingen, and S. T. Cole. 00. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A :-. Downloaded from http://jcm.asm.org/ on December, 01 by guest 1 1. Brudey, K., J. R. Driscoll, L. Rigouts, W. M. Prodinger, A. Gori, S. A. Al-Hajoj, C. Allix, L. Aristimuno, J. Arora, V. Baumanis, L. Binder, P. Cafrune, A. Cataldi, S. Cheong, R. 1

1 1 1 1 Diel, C. Ellermeier, J. T. Evans, M. Fauville-Dufaux, S. Ferdinand, D. Garcia de Viedma, C. Garzelli, L. Gazzola, H. M. Gomes, M. C. Guttierez, P. M. Hawkey, P. D. van Helden, G. V. Kadival, B. N. Kreiswirth, K. Kremer, M. Kubin, S. P. Kulkarni, B. Liens, T. Lillebaek, M. L. Ho, C. Martin, I. Mokrousov, O. Narvskaia, Y. F. Ngeow, L. Naumann, S. Niemann, I. Parwati, Z. Rahim, V. Rasolofo-Razanamparany, T. Rasolonavalona, M. L. Rossetti, S. Rusch-Gerdes, A. Sajduda, S. Samper, I. G. Shemyakin, U. B. Singh, A. Somoskovi, R. A. Skuce, D. van Soolingen, E. M. Streicher, P. N. Suffys, E. Tortoli, T. Tracevska, V. Vincent, T. C. Victor, R. M. Warren, S. F. Yap, K. Zaman, F. Portaels, N. Rastogi, and C. Sola. 00. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB) for classification, population genetics and epidemiology. BMC Microbiol :.. Cave, M. D., K. D. Eisenach, P. F. McDermott, J. H. Bates, and J. T. Crawford.. IS1: conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting. Mol Cell Probes :-0.. Dale, J. W., H. Al-Ghusein, S. Al-Hashmi, P. Butcher, A. L. Dickens, F. Drobniewski, K. J. Forbes, S. H. Gillespie, D. Lamprecht, T. D. McHugh, R. Pitman, N. Rastogi, A. T. Smith, C. Sola, and H. Yesilkaya. 00. Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS1. J Bacteriol 1:-. Downloaded from http://jcm.asm.org/ on December, 01 by guest 1. Glynn, J. R., J. Whiteley, P. J. Bifani, K. Kremer, and D. van Soolingen. 00. Worldwide 1

occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg Infect Dis :-. 1 1. Groenen, P. M., A. E. Bunschoten, D. van Soolingen, and J. D. van Embden.. Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol Microbiol :-.. Gutierrez, M. C., S. Brisse, R. Brosch, M. Fabre, B. Omais, M. Marmiesse, P. Supply, and V. Vincent. 00. Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS Pathog 1:e.. Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van Embden. 1. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol :0-.. Kremer, K., J. R. Glynn, T. Lillebaek, S. Niemann, N. E. Kurepina, B. N. Kreiswirth, P. J. Bifani, and D. van Soolingen. 00. Definition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers. J Clin Microbiol :00-. Downloaded from http://jcm.asm.org/ on December, 01 by guest 1 1. Kremer, K., D. van Soolingen, R. Frothingham, W. H. Haas, P. W. Hermans, C. Martin, P. Palittapongarnpim, B. B. Plikaytis, L. W. Riley, M. A. Yakrus, J. M. Musser, and J. D. van 1

Embden. 1. Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J Clin Microbiol :0-1. 1 1. Li, W. M., S. M. Wang, C. Y. Li, Y. H. Liu, G. M. Shen, X. X. Zhang, T. G. Niu, Q. Gao, D. van Soolingen, K. Kremer, and H. J. Duanmu. 00. Molecular epidemiology of Mycobacterium tuberculosis in China: a nationwide random survey in 000. Int J Tuberc Lung Dis :-. 1. Mokrousov, I., H. M. Ly, T. Otten, N. N. Lan, B. Vyshnevskyi, S. Hoffner, and O. Narvskaya. 00. Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: clues from human phylogeography. Genome Res 1:-. 1. National Technic Steering Group Of The Epidemiological Sampling Survey For, T., and H. Duanmu. 00. [Report on fourth national epidemiological sampling survey of tuberculosis]. Zhonghua Jie He He Hu Xi Za Zhi :-. 1. Singh, U. B., N. Suresh, N. V. Bhanu, J. Arora, H. Pant, S. Sinha, R. C. Aggarwal, S. Singh, J. N. Pande, C. Sola, N. Rastogi, and P. Seth. 00. Predominant tuberculosis spoligotypes, Delhi, India. Emerg Infect Dis :-. Downloaded from http://jcm.asm.org/ on December, 01 by guest 1 1 1. Sola, C., I. Filliol, M. C. Gutierrez, I. Mokrousov, V. Vincent, and N. Rastogi. 001. Spoligotype database of Mycobacterium tuberculosis: biogeographic distribution of 0

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Mycobacterium tuberculosis complex strain families using spoligotypes. Infect Genet Evol :1-0.. Warren, R. M., E. M. Streicher, S. L. Sampson, G. D. van der Spuy, M. Richardson, D. Nguyen, M. A. Behr, T. C. Victor, and P. D. van Helden. 00. Microevolution of the direct repeat region of Mycobacterium tuberculosis: implications for interpretation of spoligotyping data. J Clin Microbiol 0:-.. Yates, M. D., F. A. Drobniewski, and S. M. Wilson. 00. Evaluation of a rapid PCR-based epidemiological typing method for routine studies of Mycobacterium tuberculosis. J Clin Microbiol 0:-. TABLES TABLE 1. Spoligotypes of most prevelent clades with a shared international type (SIT) number in SpolDB.0 TABLE. Number and frequencies of isolates clustered by spoligotyping TABLE. Beijing family strains distribution in provinces of China Downloaded from http://jcm.asm.org/ on December, 01 by guest

Downloaded from http://jcm.asm.org/ on December, 01 by guest

TABLE 1. Spoligotypes of most prevelent clades with a shared international type (SIT) number in SpolDB.0 Spoligotype description binary SIT a SpolDB ID b No. c Prevalence d 1 BEIJING 10. T1.0 BEIJING 1 1. T 1. 0 H 1. MANU 1. T 0. H 0. BEIJING 0. H 0. a Shared international type (SIT), international spoligotype database SpolDB.0 http://www.pasteur-guadeloupe.fr:01/sitvidemo/. b Representing spoligotype families as assigned in the international spoligotype database SpolDB.0. c Number of isolates with a common SIT. d Prevalence representing the number of isolates with a common SIT relative to the total number of isolates in this study. Downloaded from http://jcm.asm.org/ on December, 01 by guest

TABLE. Number and frequencies of isolates clustered by spoligotyping Isolates clustered by spoligotyping Number of isolates studied Number of clusters Mean number of isolates per cluster. Median number of isolates per cluster Number of clustered isolates (and %) 1 (1.%) Number of unclustered isolates (and %) (.%) Downloaded from http://jcm.asm.org/ on December, 01 by guest

TABLE. Beijing family strains distribution in provinces of China Province Number of Number of Prevalence(%) Number of Prevalence(%) strains tested Beijing family non-beijing strains family strians Beijing 0..1 Tibet 0 1 0. 0. Jilin.. Gansu 1.0. Anhui 1.. Henan 0.00 1 0.00 Shanxi 0.00 0.00 Xinjiang 0.0.0 Hunan 0.00.00 Zhejiang 1..1 Sichuan.. Guangxi 0..1 Fujian.0 1.0 Total.0 0. Downloaded from http://jcm.asm.org/ on December, 01 by guest