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A total of 183 (55.29%, 183/331) strains were classified into Beijing family with a single point mutation in the Rv2952 gene, and 148 (44.71%, 148/331) non-Beijing family strains without a single point mutation in Rv2952 were defined (Table 1). Following the 8 SNPs based on the 3R genes, the Beijing family strains were grouped into eight sub-lineages (Table 1). The most predominant sub-lineage was Bmyc10 (39.34%, 72/183), followed by Bmyc25 (20.77%, 38/183), Bmyc210 (17.49%, 32/183), Bmyc2 (5.46%, 10/183) and Bmyc4 (5.46%, 10/183), Bmyc26 (4.37%, 8/183), Bmyc13 (3.83%, 7/183) and Bmyc6 (3.78%, 6/183). Among all the sub-lineages, Bmyc10, Bmyc13, and Bmyc210 were defined as modern Beijing strains (60.66%, 111/183) according to the mutation of mutT2_58 gene[32]. Strains without mutations were defined as ancient Beijing strains. Bmyc10 and Bmyc25 were the most prevalence sub-lineage of modern and ancient Beijing strains, respectively.
Sublineage SNPs No. (%) Subgroup recR (codon 44) mutT4 (codon 48) recX (codon 59) mutT2 (codon 58) uvrD1 (codon 462) adhE2 (codon 124) ligD (codon 580) ogt (codon 37) Bmyc2 W W W W W W M W 10 (5.46) Ancient Bmyc4 M W W W W W W W 10 (5.46) Ancient Bmyc6 M M W W W W W W 6 (3.78) Ancient Bmyc25 M M W W W W W M 38 (20.77) Ancient Bmyc26 M M M W W W W W 8 (4.37) Ancient Bmyc10 M M M M W W W W 72 (39.34) Modern Bmyc13 M M M M M W W W 7 (3.83) Modern Bmyc210 M M M M W M W W 32 (17.49) Modern Table 1. Distribution of 183 Beijing Strains in Each Sub-lineage (W = wildtype, M = mutant)
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We performed 15-loci VNTR to all 331 isolates to study the transmission clusters and population diversity of MTB in remote mountainous areas of southwest China. two hundred sixty-one MTB strains (78.61%; 261/331) had complete VNTR genotyping data that were included in this study. The 261 isolates were classified into 15 clusters (2 to 5 strains per cluster) and 224 unique genotypes, showing a clustering rate of 14.18% (37/261) and a discriminatory index of 0.9990 (Figure 2). The 148 Beijing isolates and 113 non-Beijing strains both contained 7 clusters. In addition, we also calculated the HGI for Beijing lineage and non-Beijing lineage. The Beijing family contained 129 unique genotypes, showing a clustering rate of 12.84% (19/148) and a discriminatory index of 0.9981. Additionally, the non-Beijing family isolates showed a clustering rate of 14.16% (16/113) and a discriminatory index of 0.9983. No significant difference between the Beijing and non-Beijing strains was observed with respect to the clustering rate (χ2 = 0.096, P = 0.756). There is a trend toward higher resolution for Beijing strains compared with non-Beijing strains (0.9983 vs. 0.9981); however, the trend was not significant. From the minimal spanning tree of the 261 strains based on VNTR-15 data, Beijing strains were mainly concentrated in one complex (shadowed with red color), and the genetic distance was relatively close, and non-Beijing strains were more dispersed (Figure 2). There were three isolates that were defined as non-Beijing strains (marked with fluorescent green color) located on the Beijing strains complex (Figure 2).
Figure 2. Minimal spanning tree of the 261 strains based on VNTR data. Each circle corresponds to a certain VNTR type. The size of the circle is proportional to the number of the isolates. The shadow zones in different colors correspond to different clonal complexes, and the color within the cycles represents different sub-lineages. The dotted line separates Beijing and non-Beijing strains.
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Among all 331 clinical strains, 19.64% (65/331) isolates were resistant to any drugs involved in this study. The resistance rates from high-to-low were INH (10.57%), SM (9.97%), OFX (8.16%), RFP (5.44%), PAS (3.32%), KAM (2.11%), CPM (1.81%), EMB (1.21%), and PTO (0.30%). Seventeen [4.98% (17/331)] MTB isolates were detected as MDR-TB, and of these, 15 [88.24% (15/17)] were Beijing family MTB, and 2 [11.76% (2/17)] were non-Beijing strains (Table 2). Beijing strains had significantly higher rates of resistance to INH (χ2 = 5.715, P = 0.017) and RFP (χ2 = 6.057, P = 0.014; Table 2) compared with non-Beijing strains. Extremely significant differences were observed regarding PAS resistance (χ2 = 7.426, P = 0.006) and MDR (simultaneous INH and RFP resistance; χ2 = 7.870, P = 0.005) between the Beijing strains and non-Beijing strains (Table 2); however, there was no significant difference in drug resistance rates when comparing modern Beijing isolates with ancient Beijing isolates (Table 2). In addition, no significant differences were obtained between the drug resistance rates of Bmyc10 and Bmyc25, which accounted for the dominant sub-lineage of modern and ancient Beijing isolates, respectively (Table 2). We tested the relationships between Beijing lineage/modern Beijing sub-lineage and drug resistance. No significant correlation was observed (Beijing lineage, P = 0.326; modern Beijing sub-lineage, P = 0.311).
Drug Resistance Total N = 331 Beijing (n = 183) non-Beijing (n = 148) Beijing vs. non-Beijing Modern Beijing vs. Ancient Beijing Bmyc10 vs. Bmyc25 Ancient Beijing (n = 72) Modern Beijing (n = 111) Total Total Bmyc25 n = 38 Total Bmyc10 n = 72 χ2 P Values χ2 P Values χ2 P Values INH 35 (10.57%) 8 (11.11%) 6 (15.79%) 18 (16.22%) 10 (13.89%) 26 (14.21%) 9 (6.08%) 5.715 0.017 0.934 0.334 0.072 0.788 EMB 4 (1.21%) 2 (2.78%) 1 (2.63%) 1 (0.90%) 0 (0%) 3 (1.64%) 1 (0.68%) 0.085 0.770 0.145 0.703 a 0.345b RFP 18 (5.44%) 6 (8.33%) 6 (15.79%) 9 (8.11%) 5 (6.94%) 15 (8.20%) 3 (2.03%) 6.057 0.014 0.003 0.957 1.291 0.256a SM 33 (9.97%) 7 (9.72%) 4 (10.53%) 16 (14.41%) 11 (15.28%) 23 (12.57%) 10 (6.76%) 3.079 0.079 0.875 0.350 0.477 0.490 KAM 7 (2.11%) 3 (4.17%) 2 (5.26%) 3 (2.70%) 1 (1.39%) 6 (3.28%) 1 (0.68%) 1.568 0.210a 0.014 0.906 a 0.326 0.568a OFX 27 (8.16%) 4 (5.56%) 3 (7.89%) 14 (12.61%) 8 (11.11%) 18 (9.84%) 9 (6.08%) 1.540 0.215 2.452 0.117 0.040 0.841a CPM 6 (1.81%) 4 (5.56%) 1 (2.63%) 2 (1.80%) 1 (1.39%) 6 (3.28%) 0 (0%) 3.272 0.070 a 0.937 0.333 a 1.000b PTO 1 (0.30%) 0 (0%) 0 (0%) 1 (0.90%) 0 (0%) 1 (0.55%) 0 (0%) 1.000b 1.000b 1.000b PAS 11 (3.32%) 7 (9.72%) 3 (7.89%) 4 (3.60%) 1 (1.39%) 11 (6.01%) 0 (0%) 7.426 0.006 a 1.912 0.167 a 1.435 0.231 a MDR 17 (5.14%) 6 (8.33%) 5 (13.16%) 9 (8.11%) 5 (6.94%) 15 (8.20%) 2 (1.35%) 7.870 0.005 0.003 0.957 0.532 0.466 a Note. INH, isoniazid; RFP, rifampin; EMB, ethambutol; SM, streptomycin; KAN, kanamycin; OFX, ofloxacin; CPM, capreomycin; PAS, para-aminosalicylic acid; PTO, protionamide; a, evaluated with continuity correction chi-square; b, evaluated with Fisher's exact test. Table 2. Drug Resistance in Beijing Lineage MTB and Non-Beijing Lineage MTB
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Based on the VNTR-15 genotyping results combined with drug resistance, as well as SNP genotyping results, we compared the characteristics of a cluster strain versus a unique type strain. The two groups did not differ with respect to drug resistance, lineage, or sub-lineage. The order of the clustering rate of different drug-resistant strains was OFX-resistant, MDR, RFP-resistant, any drug-resistant, INH-resistant, SM-resistant, and PAS-resistant. We tested for associations between clustering rates and the different drug resistant strains. No significant differences in the different drug resistance (except PTO) results were obtained between cluster strains versus unique type strains (P = 0.925; Table 3).
Characteristic Cluster Strains (%) Unique Type Strains (%) Cluster vs. Unique Type χ2 P Values Drug resistance 0.925b Yes 9 (17.65%) 42 (82.35%) 0.628 0.428 INH 4 (14.81%) 23 (85.19%) 0.000 1.000a EMB 0 (0%) 3 (100%) 1.000b RFP 3 (21.43%) 11 (78.57%) 0.165 0.685a SM 4 (14.81%) 23 (85.19%) 0.000 1.000a KAM 0 (0%) 4 (100%) 1.000b OFX 6 (30%) 14 (70%) 3.160 0.075a CPM 0 (0%) 4 (100%) 1.000b PTO 0 (0%) 0 (%) 1.000b PAS 1 (12.5%) 7 (87.5%) 0.000 1.000a MDR 3 (23.08%) 10 (76.92%) 0.287 0.592a Lineage 0.123 0.725 Beijing 20 (13.51%) 128 (86.49%) non-Beijing 17 (15.04%) 96 (84.96%) Sublineage 2.241 0.134 Ancient 4 (7.41%) 50 (92.59%) Modern 15 (15.96%) 79 (84.04%) Note. a, evaluated with continuity correction chi-square; b, evaluated with Fisher's exact test. Table 3. Factors Associated with Cluster Strains
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To explore the relevance between VNTR genotypes and SNP-defined sub-lineages in our study, we constructed the MST based on VNTR-15 and SNP genotyping results, which was mapped onto the network (Figure 2). In light of the MST map, the Beijing genotype strains in this study were divided into one major complex (shadowed in red). On the whole, the MST for the part of Beijing strains can be divided into three main branches, among which the modern Beijing strains were mainly concentrated on branches Ⅱ and Ⅲ, and the genetic distances among ancient strains were relatively greater than modern Beijing strains. Non-Beijing strains were mainly concentrated on the right of the dotted line, with the exception of three strains (Figure 2). The modern Beijing strains are mainly concentrated in the trunk part of the MST map with a closer genetic distance between each other. The results of VNTR-15 were consistent with the sub-lineage defined by SNP at a lower resolution.
From the dendrogram constructed of VNTR-15 by BioNumerics 5.0 (Figure 3) with the additional SNP-based sub-type information for strains which had both VNTR-15 and 8-loci SNP data [80.88% (148/183)], we found that strains belonging to the same cluster by VNTR were differentiated into different sub-lineages (e.g., cluster Ⅰ contained Bmyc10 and Bmyc13; cluster Ⅱ contained Bmyc10 and Bmyc210; cluster Ⅶ contained Bmyc25 and Bmyc26); however, the other four clusters were composed by the same sub-lineage strains. There were discordances between VNTR-15 and SNP sub-lineages if we set a higher resolution. The SNP genotyping results were a supplement to VNTR-15, which showed a high level of homoplasy.
Figure 3. Dendrogram of VNTR-15 patterns of 148 Beijing strains. The dendrogram was constructed using UPGMA by BioNumeric 5.0. The corresponding isolate ID and sub-lineage were shown alongside the dendrogram on the right.
In addition, we constructed a composite tree based on 8-loci SNP and VNTR-15 to further study the evolution and diversity of each sub-lineage, as previously reported (Figure 4). The skeleton of the composite tree was based on the phylogenetic relationship defined by the SNPs in the 3R gene[27], and each branch was based on the MST of VNTR-15. With the exception of Bmyc6 and Bmyc13, the other six branches of the MST were expressed as a star network structure. This finding is the same as a previous study[8], and the sub-lineages may be caused by the recent funder effect, whereas the VNTR-15 genotype at each MST center is the founder genotype of each sub-lineage. The combination of SNPs and VNTRs can improve the accuracy of epidemiologic analysis of these strains.