Genetic diversity and drug resistance of Mycobacterium tuberculosis in Yunnan, China

Background China is a country with high burden of tuberculosis (TB), especially drug‐resistant TB (DR‐TB), which is still a serious health problem in Yunnan Province. Mycobacterium tuberculosis (MTB) is the pathogenic microorganism of TB. The epidemiological characteristics of MTB strains in local areas need to be described. Methods A total of 430 clinical MTB isolates were collected from Yunnan Province and genotyped through the method of 24‐locus mycobacterial interspersed repetitive unit‐variable number tandem DNA repeats (MIRU‐VNTR). Results The genotypes of the 24 loci showed abundantly genetic diversity, and allelic diversity index (h) of these loci varied from 0.012 to 0.817. Among the 430 strains, 30 clusters and 370 unique genotypes were identified. Beijing family was the predominant lineage (70.47%) in Yunnan MTB strains, and the other lineages contained T family (5.81%), MANU2 (0.70%), LAM (3.26%), CAS (0.23%), New‐1 (8.37%), and some unknown clades (11.16%). A total of 74 TB strains were identified as drug resistance through drug susceptibility testing (DST), including 38 multidrug‐resistant TB (MDR‐TB) and 36 single‐drug‐resistant TB (SDR‐TB). The frequency of MDR‐TB strains was significantly higher in Beijing family (10.89%) than that in non‐Beijing family (3.94%, P = 0.032). Conclusions Although MTB strains showed high genetic diversity in Yunnan, China, the Beijing family was still the dominant strain. A high frequency of MDR‐TB strains was recorded in the Beijing family.

increasing number of HIV-infected persons, drug-resistant TB cases, and healthcare delay. 3 The genotypes of MTB have proven to be a valuable tool in characteristics of strains, treatment effect of drug-resistant TB, tracking transmission, predicting outbreaks and pathological properties, and evaluating the immune response. 4,5 IS6110-RFLP is the first and classic method for genotyping MTB strains and is widely used since the early 1990s. 6 Many methods have been invented based on PCR. 7 Recently, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method is the prevailing clinical technique because of its rapid, better repeatability, high-throughput, and digitized analysis. 8,9 Among the different sets of MIRU-VNTR loci described for genotyping MTB isolates, 10,11 a system based on 12, 15, and 24 loci is currently the most widely used in TB control systems in China. 9,12,13 It aids in the elucidation of the phylogenetic relationships among clinical isolates and the identification of high-risk groups that are susceptible to TB infection. Beijing genotype is associated with drug resistance, showing an increased drug resistance level. It possibly explains the wide distribution of the Beijing family. 14 However, other studies did not show a relationship between drug resistance and strain clade. 15 Yunnan belongs to the regions in China, where TB is prevalent.
TB morbidity reached 6‰ in Yunnan population due to high incidence of various infectious diseases, large rural cohort, and limited medical condition. 16 In addition, the genetic diversity of MTB was more plenty in Yunnan than in the other regions. 12,17 This study aims to investigate the genetic diversity and molecular epidemiology of MTB in the Yunnan population.

| 24-locus MIRU-VNTR genotyping
Genomic DNA was extracted from all MTB isolates by using the Bacteria DNA Kit (TIANamp, China) according to the manufacturer's instructions. Fragments of 24-locus MIRU-VNTR were amplified and genotyped as previously described. 10 The PCR products were analyzed by using 3% agarose electrophoresis at 5 V/cm for 90 minutes. The 100-bp DNA ladder and the amplicons of standard strain H37Rv were used as markers to evaluate the results. 5% of the genotyping results were randomly identified through direct sequencing.

| Data analysis
The allelic diversity of the VNTR locus was calculated by using the

| Comparison among 12-, 15-, and 24-locus MIRU-VNTR
Considering that the genotyped 24 loci could cover all the loci of the

| Higher frequency of MDR-TB strains in Beijing family
When we divided 430 MTB strains into three groups (drug-sensitive TB, MDR-TB, and SDR-TB), the allelic diversity of each locus differed (Table 4). Consequently, the evaluation of each locus for allelic diversity varied. The statistic results showed significantly higher frequency of MDR-TB in Beijing family than in non-Beijing family (P = 0.032) (Table S1). However, no statistical difference in SDR-TB frequency was identified between them.

| D ISCUSS I ON
TB is an ancient infectious disease, which remains as a global health problem. More than one million deaths from MTB infection is recorded worldwide every year. 2 27 and Pakistan (3%) 28 and was similar as that in Russia (67.9%), 29 Japan (73.8%), 11 and Korea (80%). 30 The transmission of MTB strains between Yunnan Province and Southeast Asia might be limited. However, a strain belonging to the CAS lineage was first identified in this study.
The mainly prevalent region of the CAS family was India, 26 which was also reported in Tibet, 12 Xinjiang, 31 Jiangsu, 22  (38/430), which was higher than that in the national survey in 2007. 33 A significantly higher proportion of MDR-TB strains was identified in Beijing family (12.56%, 54/430) than that of non-Beijing family (4.65%, 20/430) (P = 0.032), but the SDR-TB strains showed a similar ratio. This result was consistent with a previous study. 21 The high frequency of MDR-TB in Beijing family might explain its easy global distribution. 34,35 The analysis of epidemic characteristics in MDR-TB strains will aid the prevention of TB infection in the Yunnan population.
In conclusion, MTB strains showed high genetic diversity in Yunnan, China, and Beijing family was the dominant lineage in both total MTB and MDR-TB strains. Although a MTB strain belonging to the CAS lineage was identified in this study, we could not interpret its transmission way. Thus, the more molecular genetic characteristics of MTB strains should be further studied.

ACK N OWLED G M ENTS
This study was supported by the National Natural Science