Promising Anti-MRSA Activity of Brevibacillus sp. Isolated from Soil and Strain Improvement by UV Mutagenesis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection and Bacterial Isolation
2.2. Determination of Antibacterial Activity
2.2.1. Cross-Streak Method
2.2.2. Agar Well Diffusion Assay
2.3. Bacterial Identification by 16S rRNA Sequencing
2.4. Production Kinetics of Anti-MRSA Substances
2.5. Stability Study of Antibacterial Substances
2.5.1. Thermal Stability
2.5.2. pH Sensitivity
2.5.3. Catalytic Enzyme and Surfactant Stability
2.6. Strain Improvement by UV Mutagenesis
2.7. Scanning Electron Microscope (SEM)
2.8. Strain Stability of the Mutant on the Antibacterial Activity
2.9. Susceptibility Study of the Mutant Strain
3. Results
3.1. Bacterial Isolation and Antibacterial Activity
3.2. Bacterial Identification and Phylogenetic Tree Construction
3.3. Production Kinetics of Anti-MRSA Substances from Brevibacillus sp. SPR-20
3.4. Stability Study of Antibacterial Substances
3.5. Strain Improvement of SPR-20 by UV Mutagenesis
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sampling Location | S. aureus TISTR 517 | E. coli TISTR 887 |
---|---|---|
8°33′55.4″ N, 99°31′12.1″ E | 1 | 0 |
8°46′03.3″ N, 99°48′09.3″ E | 5 | 2 |
12°25′07.3″ N, 99°58′58.3″ E | 29 | 20 |
9°05′22.6″ N, 99°53′39.9″ E | 4 | 5 |
9°00′50.3″ N, 99°46′18.0″ E | 7 | 1 |
8°33′33.4″ N, 99°46′55.6″ E | 7 | 6 |
8°38′31.7″ N, 99°53′38.7″ E | 13 | 8 |
8°38′55.6″ N, 99°52′51.4″ E | 6 | 0 |
8°38′49.7″ N, 54°45′50″ E | 5 | 8 |
8°38′18.3″ N, 54°45′48.5″ E | 17 | 17 |
Total | 94 | 67 |
Isolates | S. aureus TISTR 517 (mm) | MRSA Isolate 142 (mm) | MRSA Isolate 1096 (mm) | MRSA Isolate 2468 (mm) | E. coli TISTR 887 (mm) | P. aeruginosa TISTR 357 (mm) | C. albicans TISTR 5554 (mm) |
---|---|---|---|---|---|---|---|
AK-74 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 10.89 ± 0.16 | 0.00 ± 0.00 | 0.00 ± 0.00 |
B6-28 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 11.95 ± 0.27 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Play-88 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 11.26 ± 0.53 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Pr-18 | 15.41 ± 0.61 | 16.01 ± 0.42 | 16.12 ± 0.35 | 16.07 ± 0.24 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Pr-82 | 15.75 ± 0.33 | 16.38 ± 0.13 | 17.14 ± 0.32 | 17.09 ± 0.27 | 12.65 ± 0.39 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Pr-83 | 13.12 ± 0.92 | 10.99 ± 0.26 | 13.09 ± 0.35 | 11.82 ± 0.57 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Pr-117 | 14.02 ± 0.11 | 13.70 ± 0.55 | 14.60 ± 0.27 | 14.02 ± 0.47 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Pr-122 | 15.08 ± 0.05 | 15.46 ± 0.16 | 14.00 ± 0.44 | 15.97 ± 0.26 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
SPR-02 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 13.34 ± 0.34 | 0.00 ± 0.00 | 0.00 ± 0.00 |
SPR-14 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 10.42 ± 0.50 | 0.00 ± 0.00 | 0.00 ± 0.00 |
SPR-20 | 17.52 ± 0.60 | 18.28 ± 0.58 | 18.03 ± 0.36 | 19.05 ± 0.84 | 13.29 ± 0.12 | 0.00 ± 0.00 | 0.00 ± 0.00 |
S-STD-1 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 11.18 ± 0.44 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Su-7 | 11.80 ± 0.76 | 13.62 ± 0.19 | 14.25 ± 0.57 | 13.60 ± 1.08 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
WUL-02 | 11.49 ± 0.81 | 12.10 ± 0.05 | 0.00 ± 0.00 | 12.06 ± 0.11 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
WUL-10 | 14.59 ± 0.06 | 15.91 ± 0.58 | 15.11 ± 0.43 | 14.66 ± 0.06 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Cefoxitin (30 µg) | 35.98 ± 0.53 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 34.71 ± 0.73 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Oxacillin (1 µg) | 30.82 ± 1.40 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Vancomycin (30 µg) | 25.48 ± 0.15 | 25.74 ± 0.15 | 25.74 ± 0.29 | 25.99 ± 0.29 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Conditions | % Activity | |||
---|---|---|---|---|
S. aureus TISTR 517 | MRSA Isolate 2468 | |||
SPR-20 | Nisin | SPR-20 | Nisin | |
Untreated Sample | 100.00 ± 4.46 | 100.00 ± 3.78 | 100.00 ± 4.93 | 100.00 ± 1.95 |
Thermal Stability | ||||
Sample 60 °C, 1 h | 100.00 ± 1.37 | 89.13 ± 3.77 * | 99.31 ± 3.17 | 92.43 ± 2.49 * |
Sample 80 °C, 1 h | 96.44 ± 1.81 * | 73.91 ± 2.49 * | 97.92 ± 1.59 | 88.05 ± 1.38 * |
Sample 100 °C, 1 h | 95.65 ± 1.81 * | 0.00 ± 0.00 * | 96.54 ± 1.04 * | 69.72 ± 1.38 * |
Sample 121 °C, 15 psi, 15 min (autoclave) | 91.30 ± 3.14 * | 72.28 ± 3.39 * | 92.39 ± 1.80 * | 92.43 ± 0.69 * |
Enzyme Stability | ||||
Sample + Proteinase K (1 mg/mL) | 96.08 ± 0.68 * | 0.00 ± 0.00 * | 95.90 ± 2.33 | 0.00 ± 0.00 * |
Sample + Trypsin (1 mg/mL) | 98.00 ± 1.83 | 0.00 ± 0.00 * | 96.72 ± 0.63 * | 0.00 ± 0.00 * |
Sample + α-Chymotrypsin (1 mg/mL) | 95.20 ± 1.83 * | 0.00 ± 0.00 * | 94.89 ± 3.34 | 68.95 ± 3.20 * |
Proteinase K (1 mg/mL) | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * |
Trypsin (1 mg/mL) | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * |
α-Chymotrypsin (1 mg/mL) | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * |
Surfactant Stability | ||||
Sample + 1% SDS | 108.81 ± 2.76 * | 130.86 ± 0.99 * | 102.95 ± 1.28 * | 95.14 ± 1.40 * |
Sample + 1% Triton X-100 | 109.24 ± 0.75 * | 148.00 ± 1.98 * | 106.36 ± 1.94 * | 121.05 ± 4.27 * |
1% SDS | 105.29 ± 0.75 * | 137.71 ± 1.98 * | 102.95 ± 2.53 | 99.60 ± 1.21 |
1% Triton X-100 | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * |
pH Stability | ||||
pH 2 | 102.51 ± 1.92 | 98.86 ± 0.99 | 102.33 ± 1.16 | 97.98 ± 2.42 |
pH 4 | 100.84 ± 2.90 | 100.57 ± 0.99 | 102.33 ± 0.00 | 97.18 ± 1.85 |
pH 5 | 100.84 ± 3.16 | 97.71 ± 1.71 | 102.33 ± 2.01 | 98.39 ± 1.85 |
pH 6 | 100.00 ± 2.90 | 96.57 ± 1.98 | 101.55 ± 2.93 | 100.00 ± 1.85 |
pH 7 | 100.42 ± 2.17 | 97.71 ± 1.71 | 100.39 ± 0.67 | 97.98 ± 1.21 |
pH 8 | 100.00 ± 1.45 | 101.14 ± 2.97 | 100.00 ± 1.16 | 99.19 ± 1.21 |
pH 9 | 101.67 ± 1.26 | 98.29 ± 1.98 | 101.57 ± 0.00 | 99.18 ± 0.71 |
pH 11 | 101.26 ± 0.72 | 96.00 ± 1.71 * | 100.00 ± 0.68 | 97.95 ± 1.42 |
pH 12 | 102.93 ± 1.26 | 0.00 ± 0.00 * | 100.39 ± 1.18 | 78.69 ± 2.13 * |
pH 13 | 102.51 ± 1.45 | 0.00 ± 0.00 * | 101.97 ± 1.36 | 0.00 ± 0.00 * |
pH 14 | 99.16 ± 2.17 | 0.00 ± 0.00 * | 101.97 ± 0.63 | 0.00 ± 0.00 * |
Isolates | S. aureus TISTR 517 (mm) | MRSA Isolate 142 (mm) | MRSA Isolate 1096 (mm) | MRSA Isolate 2468 (mm) |
---|---|---|---|---|
M5 | 20.56 ± 0.25 * | 20.85 ± 0.73 | 22.02 ± 1.21 | 21.93 ± 0.26 |
M6 | 21.41 ± 0.40 | 21.76 ± 0.27 | 21.70 ± 0.64 | 23.26 ± 0.56 |
M7 | 21.32 ± 0.25 | 21.67 ± 0.37 | 22.46 ± 0.30 | 22.53 ± 0.39 |
M8 | 22.27 ± 0.52 * | 22.20 ± 0.41 | 22.46 ± 0.29 | 23.53 ± 1.21 |
M10 | 20.99 ± 0.62 | 20.70 ± 0.93 | 19.89 ± 0.97 | 22.02 ± 1.07 |
M11 | 21.32 ± 0.53 | 21.00 ± 0.14 * | 21.54 ± 0.38 | 21.68 ± 0.53 |
M19 | 21.57 ± 0.50 | 22.01 ± 0.18 | 22.22 ± 0.42 | 22.70 ± 0.26 |
M56 | 20.90 ± 0.39 | 21.59 ± 0.43 | 21.96 ± 0.52 | 22.10 ± 0.44 |
M79 | 21.57 ± 0.29 | 21.76 ± 0.14 | 22.21 ± 0.29 | 22.70 ± 0.73 |
M83 | 20.73 ± 0.67 | 21.34 ± 0.00 * | 21.21 ± 0.52 | 21.34 ± 0.51 |
M142 | 21.92 ± 0.40 | 21.93 ± 0.77 | 21.61 ± 0.25 * | 22.99 ± 0.31 * |
M149 | 21.57 ± 1.09 | 21.88 ± 0.73 | 23.17 ± 1.36 | 23.04 ± 1.03 |
M151 | 20.87 ± 0.42 | 21.47 ± 0.58 | 22.67 ± 0.16 * | 23.03 ± 0.16 * |
M159 | 21.82 ± 0.50 | 22.13 ± 0.18 | 22.53 ± 0.24 | 22.45 ± 0.15 |
M178 | 21.42 ± 0.16 | 22.41 ± 0.28 * | 22.67 ± 0.16 * | 23.40 ± 0.75 |
M201 | 24.62 ± 0.67 * | 23.91 ± 0.52 * | 26.70 ± 1.02 * | 25.24 ± 0.61 * |
M203 | 21.49 ± 0.25 | 21.50 ± 0.52 | 22.12 ± 0.66 | 22.01 ± 0.52 |
M212 | 21.82 ± 0.64 | 21.67 ± 0.29 | 22.87 ± 0.66 | 22.79 ± 0.52 |
M214 | 21.51 ± 0.28 | 21.38 ± 0.16 * | 22.21 ± 0.16 * | 22.27 ± 0.00 |
M220 | 21.15 ± 0.58 | 22.09 ± 0.87 | 22.62 ± 0.43 | 21.93 ± 0.40 |
M228 | 21.57 ± 0.66 | 21.63 ± 0.54 | 21.80 ± 0.24 | 22.53 ± 0.15 |
M231 | 21.40 ± 0.29 | 22.00 ± 0.43 | 21.56 ± 0.37 | 23.14 ± 0.45 |
Wild-type | 21.23 ± 0.69 | 21.76 ± 0.82 | 22.21 ± 0.83 | 22.27 ± 0.83 |
Antibiotics | Wild-Type Strain (mm) | Mutant 201 Strain (mm) |
---|---|---|
Cefoxitin (30 µg) | 36.24 ± 0.96 | 36.58 ± 1.10 |
Ceftriaxone (30 µg) | 32.34 ± 0.24 | 36.91 ± 0.96 * |
Ciprofloxacin (5 µg) | 34.71 ± 0.63 | 35.90 ± 0.86 |
Erythromycin (15 µg) | 37.25 ± 0.86 | 38.78 ± 1.27 |
Gentamicin (10 µg) | 26.42 ± 1.10 | 26.25 ± 1.46 |
Imipenem (10 µg) | 36.58 ± 0.83 | 38.10 ± 0.41 |
Tetracycline (30 µg) | 28.79 ± 0.24 | 30.31 ± 1.04 |
Vancomycin (30 µg) | 19.64 ± 0.24 | 21.67 ± 0.86 |
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Songnaka, N.; Lertcanawanichakul, M.; Atipairin, A. Promising Anti-MRSA Activity of Brevibacillus sp. Isolated from Soil and Strain Improvement by UV Mutagenesis. Sci. Pharm. 2021, 89, 1. https://doi.org/10.3390/scipharm89010001
Songnaka N, Lertcanawanichakul M, Atipairin A. Promising Anti-MRSA Activity of Brevibacillus sp. Isolated from Soil and Strain Improvement by UV Mutagenesis. Scientia Pharmaceutica. 2021; 89(1):1. https://doi.org/10.3390/scipharm89010001
Chicago/Turabian StyleSongnaka, Nuttapon, Monthon Lertcanawanichakul, and Apichart Atipairin. 2021. "Promising Anti-MRSA Activity of Brevibacillus sp. Isolated from Soil and Strain Improvement by UV Mutagenesis" Scientia Pharmaceutica 89, no. 1: 1. https://doi.org/10.3390/scipharm89010001