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A total of 393 NTM isolates were identified at species level using multilocus sequence analysis. M. intracellulare (132, 33.6%) was the most abundant organism, followed by the M. abscessus group (100, 25.4%), M. kansasii (82, 20.9%), M. avium (41, 10.4%), M. gordonae (26, 6.6%), and M. fortuitum (12, 3.1%). In total, 173 MAC isolates were used for further study, including 132 M. intracellulare and 41 M. avium isolates.
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Antimicrobial susceptibility testing was performed on M. avium and M. intracellulare isolates. The range of MICs of each antimicrobial agent for M. avium and M. intracellulare is shown in Table 1. Clarithromycin and amikacin were the two most effective agents against both M. avium (97.6% and 97.6%, respectively) and M. intracellulare isolates (96.2% and 94.7%, respectively), with no significant difference between the species (P < 0.05). Compared with M. intracellulare isolates, lower rates of drug resistance to LZD, RIF, MEM, IMP, CFX, GAT, and SFX were observed in M. avium isolates (48.8% vs. 63.2%, P = 0.018 for LZD; 41.5% vs. 78.7%, P < 0.001 for RIF; 75.6% vs. 97.7%, P < 0.001 for MEM; 78.0% vs. 99.2%, P = 0.005 for IMP; 73.0% vs. 99.2%, P = 0.007 for CFX; 41.5% vs. 74.2%, P < 0.001 for GAT, and 48.8% vs. 87.1%, P < 0.001 for SFX), with a significant difference between these two species (P < 0.05). In addition, MOX (34.1% vs. 33.1%), AZM (70.7% vs. 60.6%), and RFB (36.6% vs. 26.5%) exhibited higher activity against M. intracellulare than against M. avium isolates, but there was no statistical difference between these two species (P > 0.05).
Antimicrobial Agent M. avium (n = 41) M. intracellulare (n = 132) χ2 P MIC50a (μg/mL) MIC90a (μg/mL) %Resistant Strainsb MIC50a (μg/mL) MIC90a (μg/mL) %Resistant Strainsb CLA 2 16 2.4 1 16 3.8 0.096 0.756 AMK 16 32 2.4 16 32 5.3 0.673 0.412 MOX 2 4 34.1 2 4 33.1 1.186 0.276 LZD 16 64 48.8 32 64 63.2 5.570 0.018 RIF 4 64 41.5 8 64 78.7 29.360 0.000 EMB 4 64 36.6 4 16 45.6 1.668 0.196 CAP 4 32 39.0 4 64 50.0 1.511 0.219 TOB 4 16 36.6 4 32 41.7 0.655 0.418 MEM 64 256 75.6 256 256 97.7 15.111 0.000 IMP 256 256 78.0 256 256 99.2 7.800 0.005 CFX 256 256 73.0 256 256 99.2 7.352 0.007 AZM 32 64 70.7 32 64 60.6 1.376 0.241 LFX 16 32 82.9 16 32 90.9 1.182 0.277 GAT 2 8 41.5 4 8 74.2 12.623 0.000 MIN 8 16 73.1 16 32 82.6 1.808 0.179 TIG 64 64 97.6 64 64 99.2 0.774 0.379 SFX 32 256 48.8 128 256 87.1 26.829 0.000 SM 16 64 90.2 16 64 92.4 0.095 0.758 CFM 1 16 39.0 1 32 43.2 0.222 0.638 RFB 0.125 32 36.6 0.25 32 26.5 1.544 0.214 Note. aMIC50 represents the concentration required to inhibit the growth of 50% of the strains; MIC90 represents the concentration required to inhibit the growth of 90% of the strains. bThe breakpoints to establish susceptibility and resistance were followed as recommended by the Clinical and Laboratory Standards Institute (CLSI-M24-A2). Table 1. In Vitro Antibiotic Susceptibility of 41 M. avium and 132 M. intracellulare Isolates by Broth Microdilution Method
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A total of 41 M. avium strains were genotyped by 13 M. avium tandem repeat (MATR) loci combinations recommended by Kenji, except MATR-9 and MATR-12, which cannot be amplified for most DNA extracts, even when different conditions were attempted, and these two loci were excluded for further analysis. Using this method, the 41 isolates were differentiated into six clusters (two isolates per cluster) and 29 unique genotypes. As shown in Figure 1, of the 13 MATR loci, six loci (MATR-2, -3, -4, -6, -7, and -8) had a high diversity index (h ≥ 0.5); five loci (MATR-1, -5, -11, -13, and -16) achieved a medium diversity index (0.5 > h ≥ 0.1); and MATR-14 and -15 had a low diversity index (h < 0.1). The cumulative cluster rate was 14.6%, and the HGDI value for the VNTR typing of M. avium isolates was 0.993 as shown in Table 2, indicating that the VNTR analysis was capable of high discriminatory power.
Organism Total Isolates (n) Clustered Isolates (n) Isolates in Each Cluster (n) Clustering Rate/% HGDIa M. avium 41 12 2 14.6 0.993 M. intracellulare 132 48 2-5 22.0 0.995 Note. aHGDI = Hunter-Gaston Discriminatory Index. Table 2. Discriminatory Index and Clustering Rate of VNTR Applied to MAC Strains
The 132 M. intracellulare isolates were classified into 88 genotypes by 16-locus MIRU-VNTR as recommended by Kenji, including 48 isolates from 19 clusters (2-5 isolates per cluster) and 84 isolates with unique patterns; the cumulative cluster rate was 22.0%, and the HGDI value was 0.995 as shown in Table 2. As shown in Figure 2, of the 16 VNTR loci, VNTR-7 and VNTR-11 were classified as lowly discriminating (h < 0.1), and the remaining 14 loci were defined as highly discriminating (h ≥ 0.5).
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As shown in Table 3, of the 12 clustered and 29 unclustered isolates of M. avium, the proportion of isolates resistant to CAP (41.7% vs. 37.9%), AZM (75.0% vs. 69.0%), LFX (100.0% vs. 75.9%), SM (91.7% vs. 62.1%), MEM (83.3% vs. 72.4%), and CFX (75.0% vs. 72.4%) was higher than that of unclustered isolates, but there was no significant difference (P > 0.05). The resistance rate of other drugs in unclustered isolates was higher than that in clustered isolates, and the difference was not significant (P > 0.05).
Drug Clustered (12 isolates) Unclustered (29 isolates) χ2 P No.(%) of Sensitive Strains No.(%) of Resistant Strains No.(%) of Sensitive Strains No.(%) of Resistant Strains CLA 10 (83.3) 0 (0.0) 23 (79.3) 1 (3.4) 0.429 0.512 AMK 10 (83.3) 0 (0.0) 26 (89.7) 1 (3.4) 0.381 0.537 MOX 4 (33.3) 3 (25.0) 10 (34.5) 11 (37.9) 0.190 0.663 LZD 4 (33.3) 4 (33.3) 9 (31.0) 16 (55.2) 0.498 0.481 CAP 7 (58.3) 5 (41.7) 18 (62.1) 11 (37.9) 0.050 0.823 AZM 3 (25.0) 9 (75.0) 9 (31.0) 20 (69.0) 0.149 0.699 LFX 0 (0.0) 12 (100.0) 4 (33.3) 22 (75.9) 2.063 0.151 GAT 4 (33.3) 4 (33.3) 10 (34.5) 13 (44.8) 0.102 0.750 SM 1 (8.3) 11 (91.7) 11 (37.9) 18 (62.1) 3.592 0.058 CFM 9 (75.0) 3 (25.0) 16 (55.2) 13 (44.8) 1.402 0.236 TOB 6 (50.0) 4 (33.3) 10 (34.5) 11 (37.9) 0.416 0.519 MEM 0 (0.0) 10 (83.3) 4 (33.3) 21 (72.4) 1.806 0.179 IMP 1 (8.3) 6 (50.0) 1 (3.4) 26 (89.7) 1.124 0.289 CFX 3 (25.0) 9 (75.0) 8 (27.6) 21 (72.4) 0.029 0.865 MIN 0 (0.0) 8 (66.7) 2 (6.9) 22 (75.9) 0.711 0.399 RFB 8 (66.7) 4 (33.3) 18 (62.1) 11 (37.9) 0.077 0.781 SFX 9 (75.0) 3 (25.0) 12 (41.4) 17 (58.6) 3.840 0.050 RIF 8 (66.7) 4 (33.3) 16 (55.2) 13 (44.8) 0.462 0.497 EMB 8 (66.7) 4 (33.3) 18 (62.1) 11 (37.9) 0.077 0.781 Table 3. Drug Susceptibility Profiles of Clustered and Unclustered Strains of M. avium
As shown in Table 4, among the 48 clustered and 84 unclustered strains of M. intracellulare, CLA (4.2% vs. 3.6%), LZD (75.0% vs. 57.1%), GAT (77.1% vs. 72.6%), SM (95.8% vs. 90.5%), and CFM (50.0% vs. 39.3%) showed higher resistance rates in clustered isolates than that in unclustered isolates, with no significant difference (P > 0.05). Statistical analysis revealed that the percentage of LFX-resistant isolates was significantly higher in unclustered isolates than that in clustered isolates (P = 0.004).
Drug Clustered (48 isolates) Unclustered (84 isolates) χ 2 P No.(%) of Sensitive Strains No.(%) of Resistant Strains No.(%) of Sensitive Strains No.(%) of Resistant Strains CLA 45 (93.8) 2 (4.2) 77 (91.7) 3 (3.6) 0.020 0.888 AMK 40 (83.3) 1 (2.1) 66 (78.6) 6 (7.1) 1.562 0.211 MOX 9 (18.8) 15 (31.3) 18 (21.4) 29 (34.5) 0.004 0.948 LZD 4 (8.3) 36 (75.0) 16 (19.0) 48 (57.1) 3.566 0.059 CAP 29 (60.4) 19 (39.6) 37 (44.0) 47 (56.0) 3.274 0.070 AZM 21 (43.8) 27 (56.3) 31 (36.9) 53 (63.1) 0.599 0.439 LFX 6 (12.5) 39 (81.3) 1 (1.2) 81 (96.4) 8.187 0.004 GAT 6 (12.5) 37 (77.1) 12 (14.3) 61 (72.6) 0.127 0.721 SM 2 (4.2) 46 (95.8) 8 (9.5) 76 (90.5) 1.252 0.263 CFM 24 (50.0) 24 (50.0) 51 (60.7) 33 (39.3) 1.429 0.232 TOB 15 (31.3) 20 (41.7) 27 (32.1) 35 (41.7) 0.004 0.947 CFX 0 (0.0) 48 (100.0) 1 (1.2) 83 (98.8) 0.576 0.448 MIN 0 (0.0) 42 (87.5) 2 (2.4) 67 (80.0) 1.240 0.266 RFB 35 (72.9) 13 (27.1) 62 (73.8) 22 (26.2) 0.012 0.911 SFX 6 (12.5) 42 (87.5) 11 (13.1) 73 (86.9) 0.010 0.922 RIF 14 (29.2) 34 (70.8) 13 (15.5) 71 (84.5) 3.519 0.061 EMB 28 (58.3) 20 (41.7) 42 (50.0) 42 (50.0) 0.852 0.356 Table 4. Drug Susceptibility Profiles of Clustered and Unclustered Strains of M. intracellulare