Background
Methods
Extraction and purification of Exo-MS from M. smegmatis mc2155
Determination of in vitro effect of Exo-MS and DFO-B on MDR and PZA-resistant Mtb isolates by MGIT-DST method
No | Drug | Initial Concentration of the drug preparation | Volume added to MGIT for Test | Final Concentration in MGIT tube except GC | No. of Middle Brook tubes | |
---|---|---|---|---|---|---|
GC | MDR | |||||
1 |
INH
| 8.3 μg/mL | 100 μL | 0.1 μg/mL | 1 | 1 |
2 |
RIF
| 83 μg/mL | 100 μL | 1.0 μg/mL | 1 | |
3 | Exo-MS + INH
| 19 mg/mL + 8.3 μg/mL | 50 μL + 100 μL | 0.125 mg/mL and 0.1 μg/mL | 1 | |
4 | Exo-MS + INH
| 19 mg/mL + 8.3 μg/mL | 100 μL + 100 μL | 0.25 mg/mL and 0.1 μg/mL | 1 | |
5 | Exo-MS + INH
| 19 mg/mL + 8.3 μg/mL | 200 μL + 100 μL | 0.5 mg/mL and 0.1 μg/mL | 1 | 1 |
6 | Exo-MS + RIF
| 19 mg/mL + 83 μg/mL | 50 μL + 100 μL | 0.125 mg/mL and 1.0 μg/mL | 1 | |
7 | Exo-MS + RIF
| 19 mg/mL + 83 μg/mL | 100 μL + 100 μL | 0.25 mg and 1.0 μg/mL | 1 | |
8 | Exo-MS + RIF
| 19 mg/mL + 83 μg/mL | 200 μL + 100 μL | 0.5 mg and 1.0 μg/mL | 1 | |
9 | Exo-MS | 19 mg/mL | 50 μL | 0.125 mg/mL | 1 | 1 |
10 | Exo-MS | 19 mg/mL | 100 μL | 0.25 mg/mL | 1 | |
11 | Exo-MS | 19 mg/mL | 200 μL | 0.5 mg/mL | 1 | |
12 | Exo-MS+ FeCl3
| 19 mg/mL + 80 mg/mL | 200 μL + 50 μL | 0.5 mg/mL and 0.5 mg/mL | 1 |
No | Drug | Initial Concentration of the drug preparation | Volume added to MGIT for Test | Final Concentration in MGIT tube except GC | No. of PZA tubes | |
---|---|---|---|---|---|---|
GC |
PZA
R
| |||||
1 |
PZA
| 8 mg/mL | 100 μL | 0.1 mg/mL | 1 | 1 |
2 | Exo-MS + PZA
| 19 mg/mL + 8 mg/mL | 50 μL + 100 μL | 0.125 mg/mL and 0.1 mg/mL | 1 | |
3 | Exo-MS + PZA
| 19 mg/mL + 8 mg/mL | 100 μL + 100 μL | 0.25 mg/mL and 0.1 mg/mL | 1 | |
4 | Exo-MS + PZA
| 19 mg/mL + 8 mg/mL | 200 μL + 100 μL | 0.5 mg/mL and 0.1 mg/mL | 1 | |
5 | Exo-MS | 19 mg/mL | 50 μL | 0.125 mg/mL and 0.1 mg/mL | 1 | 1 |
6 | Exo-MS | 19 mg/mL | 100 μL | 0.25 mg/mL | 1 | |
7 | Exo-MS | 19 mg/mL | 200 μL | 0.5 mg/mL | 1 | |
8 | Exo-MS+ FeCl3
| 19 mg/mL + 80 mg/mL | 200 μL + 50 μL | 0.5 mg/mL and 0.5 mg/mL | 1 |
No | Drug | Initial Concentration of the drug preparation | Volume added to MGIT for Test | Final Concentration in MGIT tube except GC | No. of Middle Brook tubes | |
---|---|---|---|---|---|---|
GC | MDR | |||||
1 |
INH
| 8.3 μg/mL | 100 μL | 0.1 μg/mL | 1 | 1 |
2 |
RIF
| 83 μg/mL | 100 μL | 1.0 μg/mL | 1 | |
3 | DFO-B + INH
| 160 mg/mL + 8.3 μg/mL | 12.5 μL + 100 μL | 0.25 mg/mL and 0.1 μg/mL | 1 | |
4 | DFO-B + INH
| 160 mg/mL + 8.3 μg/mL | 25 μL and 100 μL | 0.5 mg/mL and 0.1 μg/mL | 1 | |
5 | DFO-B + INH
| 160 mg/mL + 8.3 μg/mL | 50 μL and 100 μL | 1.0 mg/mL and 0.1 μg/mL | 1 | 1 |
6 | DFO-B + RIF
| 160 mg/mL + 83 μg/mL | 12.5 μL + 100 μL | 0.25 mg/mL and 1.0 μg/mL | 1 | |
7 | DFO-B + RIF
| 160 mg/mL + 83 μg/mL | 25 μL + 100 μL | 0.5 mg/mL and 1.0 μg/mL | 1 | |
8 | DFO-B + RIF
| 160 mg/mL + 83 μg/mL | 50 μL + 100 μL | 1.0 mg/mL and 1.0 μg/mL | 1 | |
9 | DFO-B | 160 mg/mL | 12.5 μL | 0.25 mg/mL | 1 | 1 |
10 | DFO-B | 160 mg/mL | 25 μL | 0.5 mg/mL | 1 | |
11 | DFO-B | 160 mg/mL | 50 μL | 1.0 mg/mL | 1 | |
12 | DFO-B + FeCl3
| 160 mg/mL + 80 mg/mL | 50 μL + 50 μL | 1.0 mg/mL and 0.5 mg/mL | 1 |
No | Drug | Initial Concentration of the drug preparation | Volume added to MGIT for Test | Final Concentration in MGIT tube except GC | No. of PZA tubes | |
---|---|---|---|---|---|---|
GC |
PZA
R
| |||||
1 |
PZA
| 8 mg/mL | 100 μL | 0.1 mg/mL | 1 | 1 |
2 | DFO-B + PZA
| 160 mg/mL + 8 mg/mL | 12.5 μL + 100 μL | 0.25 mg/mL and 0.1 mg/mL | 1 | |
3 | DFO-B + PZA
| 160 mg/mL + 8 mg/mL | 25 μL + 100 μL | 0.5 mg/mL and 0.1 mg/mL | 1 | |
4 | DFO-B + PZA
| 160 mg/mL + 8 mg/mL | 50 μL + 100 μL | 1.0 mg/mL and 0.1 mg/mL | 1 | |
5 | DFO-B | 160 mg/mL | 12.5 μL | 0.25 mg/mL | 1 | 1 |
6 | DFO-B | 160 mg/mL | 25 μL | 0.5 mg/mL | 1 | |
7 | DFO-B | 160 mg/mL | 50 μL | 1.0 mg/mL | 1 | |
8 | DFO-B + FeCl3
| 160 mg/mL + 80 mg/mL | 50 μL + 50 μL | 1.0 mg/mL and 0.5 mg/mL | 1 |
Results
Extraction and purification of Exo-MS from M. smegmatis mc2155
Inhibitory effect of Exo-MS alone and in combination with INH and RIF on MDR-Mtb isolates in vitro
MDR- Mtb (INH
R
+ RIF
R) Isolate | Exo-MS (mg/mL) | Exo-MS (mg/mL) + INH 0.1 μg/mL | Exo-MS (mg/mL) + RIF 1.0 μg/mL | Ferric citrate + Exo-MS (mg/mL) | ||||||
---|---|---|---|---|---|---|---|---|---|---|
0.125 | 0.25 | 0.5 | 0.125 | 0.25 | 0.5 | 0.125 | 0.25 | 0.5 | 0.5 | |
1 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 |
2 | 400 | 400 | 0 | 400 | 400 | 0 | 400 | 400 | 0 | 400 |
3 | 400 | 400 | 0 | 400 | 400 | 0 | 400 | 400 | 12 | 400 |
4 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 |
5 | 400 | 400 | 253 | 400 | 400 | 105 | 400 | 400 | 131 | 400 |
INFERENCE | R | R | S n = 2 | R | R | S n = 2 | R | R | S n = 2 | R |
Inhibitory effect of DFO-B alone and in combination with INH and RIF on MDR-Mtb isolates in vitro
MDR- Mtb (INH
R
+ RIF
R) Isolate | DFO-B (mg/mL) | DFO-B (mg/mL) + INH 0.1 μg/mL | DFO-B (mg/mL) + RIF 1.0 μg/mL | Ferric citrate + DFO-B (mg/mL) | ||||||
---|---|---|---|---|---|---|---|---|---|---|
0.25 | 0.5 | 1.0 | 0.25 | 0.5 | 1.0 | 0.25 | 0.5 | 1.0 | 1.0 | |
1 | 400 | 400 | 0 | 400 | 400 | 0 | 400 | 400 | 0 | 400 |
2 | 400 | 400 | 0 | 400 | 400 | 0 | 400 | 400 | 0 | 400 |
3 | 400 | 0 | 0 | 400 | 0 | 0 | 400 | 0 | 0 | 400 |
4 | 400 | 0 | 0 | 400 | 0 | 0 | 400 | 0 | 0 | 400 |
5 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 | 400 |
6 | 400 | 400 | 0 | 400 | 0 | 0 | 400 | 0 | 0 | 400 |
7 | 400 | 400 | 0 | 400 | 400 | 0 | 400 | 400 | 0 | 400 |
8 | 400 | 0 | 0 | 400 | 0 | 0 | 400 | 0 | 0 | 400 |
9 | 400 | 0 | 0 | 400 | 0 | 0 | 400 | 0 | 0 | 400 |
10 | 400 | 400 | 0 | 400 | 400 | 0 | 400 | 400 | 0 | 400 |
INFERENCE | R | S n = 4 | S n = 9 | R | S n = 5 | S n = 9 | R | S n = 5 | S n = 9 | R |
Inhibitory effect of Exo-MS and DFO-B alone and in combination with PZA on PZA-resistant Mtb isolates in vitro
Mtb Isolate | Exo-MS (mg/mL) | Exo-MS (mg/mL) + PZA 100 μg/mL | Ferric citrate + Exo-MS (mg/mL) | ||||
---|---|---|---|---|---|---|---|
0.125 | 0.25 | 0.5 | 0.125 | 0.25 | 0.5 | 0.5 | |
PZA
R
isolate 1 | 0 | 0 | 0 | 0 | 0 | 0 | 400 |
PZA
R
isolate 2 | 0 | 0 | 0 | 0 | 0 | 0 | 400 |
MDR + PZA
R
isolate 3 | 400 | 0 | 0 | 400 | 0 | 0 | 400 |
MDR + PZA
R
isolate 4 | 400 | 400 | 400 | 400 | 400 | 400 | 400 |
MDR + PZA
R
isolate 5 | 400 | 25 | 0 | 400 | 0 | 0 | 400 |
MDR + PZA
R
isolate 6 | 400 | 317 | 0 | 400 | 237 | 0 | 400 |
MDR + PZA
R
isolate 7 | 400 | 400 | 400 | 400 | 386 | 253 | 400 |
INFERENCE | S n = 2 | S n = 4 | S n = 5 | S n = 2 | S n = 4 | S n = 5 | R |
Mtb Isolate | DFO-B (mg/mL) | DFO-B (mg/mL) + PZA 100 μg/mL | Ferric citrate + DFO-B (mg/mL) | ||||
---|---|---|---|---|---|---|---|
0.25 | 0.5 | 1.0 | 0.25 | 0.5 | 1.0 | 1 | |
PZA
R
isolate 1 | 209 | 41 | 0 | 0 | 0 | 0 | 400 |
PZA
R
isolate 2 | 129 | 0 | 0 | 0 | 0 | 0 | 400 |
MDR + PZA
R
isolate 3 | 400 | 400 | 0 | 7 | 0 | 0 | 400 |
MDR + PZA
R
isolate 4 | 400 | 400 | 179 | 400 | 400 | 400 | 400 |
MDR + PZA
R
isolate 5 | 400 | 400 | 0 | 0 | 0 | 0 | 400 |
MDR + PZA
R
isolate 6 | 400 | 400 | 400 | 400 | 400 | 400 | 400 |
MDR + PZA
R
isolate 7 | 400 | 400 | 0 | 400 | 400 | 0 | 400 |
INFERENCE | R | S n = 2 | S n = 5 | S n = 4 | S n = 4 | S n = 5 | R |