Phylogenetic and lineage analyses
ML trees were constructed from the 169 M. tuberculosis isolates obtained from patients registered in the NESID in 2007 and 2013 (Fig. 1a and b). These ML trees were congruent with the trees constructed by the neighbor-joining method for each of these years (Fig. 2a and b). Phylogenetic analysis revealed four clades in good agreement with four of the lineages (Lineages 1–4) categorized by genotyping based on LSP (Figs. 1 and 2). Of the 169 isolates, 111 (65.7%) belonged to Lineage 2 (East Asian or Beijing), 43 (25.4%) to Lineage 4 (Euro-American), 13 (7.7%) to Lineage 1 (Indo-Oceanic), and two (1.2%) to Lineage 3 (East African-Indian) (Table 1). Of the 111 isolates belonging to Lineage 2, 32 (28.8%) belonged to the typical (modern) Beijing sub-genotype, and 79 (71.2%) to the atypical (ancestral) Beijing sub-genotype. Fifty-seven (65.5%) of the 87 isolates obtained in 2007, and 54 (65.9%) of the 82 isolated in 2013 belonged to Lineage 2, with 44 (50.6%) and 35 (42.7%), respectively, belonging to the atypical Beijing sub-genotype (Table 1). The percentage of isolates belonging to the typical Beijing sub-genotype was higher in 2013 (19/82, 23.2%) than in 2007 (13/87, 14.9%).
Three ML trees were constructed from isolates belonging to Lineages 1, 2 and 4, respectively (Fig. 3). The 13 isolates belonging to Lineage 1 did not form any sub-clades (Fig. 3a). The 111 isolates belonging to Lineage 2 could be divided into four sub-clades, containing 26, 22, 31 and 32 isolates, respectively (Fig. 3b). Isolates belonging to clades 1, 2 and 3 were of the atypical (ancestral) Beijing sub-genotype, with the isolates belonging to sub-clade 4 being of the typical Beijing sub-genotype (Fig. 3b). Forty-three isolates belonged to Lineage 4 (Fig. 3c). Both isolates belonging to Lineage 3 were detected in 2013 (Fig.1a), with none detected in 2007 (Fig. 1b).
Lineage comparison
Isolates from foreign- and Japanese-born patients
Of the 169 isolates, 21 (12.4%) were from foreign-born patients, with nine, four, two, and six belonging to Lineages 1, 2, Lineage 3, and 4, respectively (Table 1). Of the 13 Lineage 1 isolates nine (69.2%) were from foreign-born patients (Table 1 and Fig. 3a), including three patients from the Philippines, two each from Taiwan and Thailand, and one each from Nepal and Vietnam. Of the 111 isolates belonging to Lineage 2, 107 (96.4%) were from Japan-born patients and only four (3.6%) from foreign-born patients (Table 1), with one each of the latter belonging to each of the four sub-clades of Lineage 2 (Fig. 3b). One isolate, belonging to the typical sub-genotype of Lineage 2, was obtained from a patient born in China, whereas the other three were from patients born in Korea, Taiwan, and China, respectively. The two Lineage 3 isolates detected in 2013 (Table 1) were obtained from foreign-born patients, a man in his twenties from Nepal and a woman in her forties from the Philippines, both of whom were living in the southern region of Tochigi. Of the 43 isolates belonging to Lineage 4, six (14.0%) were from foreign-born patients, including two each from Korea and Peru and one each from Brazil and Thailand (Fig. 3c).
Elderly and nonelderly patients
Figure 4 shows the age distribution from which isolates of each lineage were obtained. Among patients infected with Lineage 2 isolates, those infected with Ancestral strains were significantly older than those infected with Modern strains (p = 0.0095). Of the 79 patients with Lineage 2-Ancestral isolates and the 22 with Lineage 4 isolates in 2007 (Table 1), 78 and 20, respectively developed TB at the indicated ages (Fig. 4). Japan-born patients with Lineage 1 isolates were significantly younger than patients with Lineage 2-Ancestral (p = 1.15E-06), Lineage 2-Modern (p = 0.0016), and Lineage 4 (p = 4.21E-05) isolates. Patients with isolates belonging to Lineage 1, both Japan- and foreign-born, were also significantly younger than either Japan-born patients with Lineage 2-Ancestral (p = 9.38E-04) or Lineage 4 (p = 1.56E-03) isolates. Both patients with Lineage 3 isolates were relatively young, one in his twenties and the other in her forties.
Residential regions of patients (northern, central and southern parts of Tochigi prefecture)
Both patients infected with Lineage 3 isolates lived in the southern part of Tochigi prefecture. There were no other relationships between the Lineages of isolates and residential regions (Additional file 4: Figure S2 and Additional file 2: Table S1).
Patient gender
There were twice as many male as female patients in 2007, with the numbers of males infected with Lineage 1, 2, and 4 isolates in that year being higher than those of females (Additional file 3: Table S2). The number of male patients was slightly higher than the number of females in 2013 (Additional file 3: Table S2).
Drug susceptibility
CASTB analysis showed that, of the 169 isolates, 11 (6.5%) were resistant to at least one drug, including five resistant to isoniazid, three resistant to streptomycin, and one each resistant to ciprofloxacin, isoniazid + streptomycin, and isoniazid + streptomycin + ciprofloxacin. Of the five isoniazid-resistant isolates, three belonged to Lineage 1 and two to Lineage 4. The three isolates belonging to Lineage 1 were from three patients born in the Philippines, whereas the two belonging to Lineage 4 were from two Japan-born patients. One isolate resistant to both isoniazid and streptomycin belonged to Lineage 2 and was from a patient born in China. The isolate resistant to ciprofloxacin belonged to Lineage 4 and was from a Japan-born patient. One of the isolates resistant to isoniazid, streptomycin and ciprofloxacin belonged to Lineage 2 and was from a Japan-born patient. Collectively, of the 11 drug-resistant isolates, four were from foreign-born patients, with the percentage of drug-resistant isolates being significantly higher in foreign-born than in Japan-born patients (p = 0.033).
NESID reports included the results of drug susceptibility tests for 86 (50.9%) of 169 isolates, not for the other 83. Of the 86 isolates, three were resistant to at least one drug, with one each resistant to isoniazid, isoniazid + ethionamide, and isoniazid + streptomycin + levofloxacin. The drug susceptibility profiles of these isolates essentially agreed with those of CASTB analysis (Additional file 5: Table S3).