Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis
Abstract
:1. Introduction
2. Results
2.1. Antimicrobial Investigation of Bacterial Isolates from Mangrove Sediments
2.2. Production Kinetics of Antibacterial Compounds of NNS5-6
2.3. Purification of Antibacterial Compounds of NNS5-6
2.4. De Novo Amino Acid Sequence of the Purified AMP and Determination of Its Secondary Structure
2.5. Investigation of the Antibacterial Activities of the AMP
2.6. The Antibacterial Activity of NNS5-6 Derived AMP on Bacterial Pathogens Observed Using Scanning Electron Microscopy (SEM) Analysis
2.7. Time-Kill Assay of NNS5-6 AMP
2.8. Studies of Cell Permeability
2.9. Stability Studies of NNS5-6 AMP under Various Conditions
2.10. Phenotypic Characterization of NNS5-6
2.11. Genome Insight for Coding Sequence Annotation and Whole-Genome Phylogenetic Analysis
2.12. Comparative Analysis of Biosynthetic Gene Clusters in NNS5-6
2.13. Prediction of Antibiotic Resistance Genes in the NNS5-6 Genome and Determination of Antibiotic Susceptibility
3. Discussion
4. Materials and Methods
4.1. Sample Collection and Bacterial Isolation
4.2. Antibacterial Screening Using the Soft Agar Overlay Method against P. aeruginosa TISTR 357
4.3. Verification of Antibacterial Activity Using the Agar Well Diffusion Technique
4.4. Investigation of the Production Kinetics of Antimicrobial Compounds of NNS5-6
4.5. Purification of the Antimicrobial Peptide
4.6. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Agar Overlay Assay
4.7. Peptide Sequencing
4.8. Determination of the Peptide Secondary Structure
4.9. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of NNS5-6 AMP
4.10. Scanning Electron Microscopy (SEM) of Cells Treated with NNS5-6 AMP
4.11. Time-Kill Kinetics of NNS5-6 AMP
4.12. Stability Studies of NNS-5-6 AMP
4.13. Effect of the AMP on Cell Membrane Permeability
4.14. Characterization of Bacterial Morphology
4.15. Whole Genome Sequencing and Bioinformatic Analysis
4.16. Antibiotic Susceptibility Studies of NNS5-6
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Isolate | Zone of Inhibition (mm ± SD; n = 3) | ||||||
---|---|---|---|---|---|---|---|
S. aureus TISTR 517 | MRSA Strain 2468 | E. coli TISTR 887 | K. pneumoniae TISTR 1383 | P. aeruginosa TISTR 357 | S. typhimurium TISTR 1469 | V. parahaemolyticus TISTR 1596 | |
NNS5-6 | 0.00 ± 0.00 | 0.00 ± 0.00 | 14.24 ± 0.15 | 13.34 ± 0.53 | 14.84 ± 0.15 | 12.70 ± 0.25 | 13.09 ± 0.64 |
Vancomycin (30 µg) | 21.59 ± 0.51 | 21.76 ± 0.78 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Colistin (1 µg) | 0.00 ± 0.00 | 0.00 ± 0.00 | 18.13 ± 0.88 | 18.81 ± 0.39 | 18.05 ± 0.67 | 20.4 ± 0.89 | 18.8 ± 0.76 |
Purification Procedure | Volume (mL) | Total Dried Weight (mg) | Activity (AU/mL) | Total Activity (AU) | Specific Activity (AU/mg) | Purification Factor | %Yield |
---|---|---|---|---|---|---|---|
Crude product | 976.50 | 453.30 | 20.00 | 19,530.00 | 43.08 | 1.00 | 100.00 |
Salt precipitation | 72.78 | 102.40 | 80.00 | 5822.40 | 56.86 | 1.32 | 29.81 |
Cation-exchange chromatography | 42.67 | 37.92 | 80.00 | 3413.60 | 90.02 | 2.09 | 17.48 |
Size-exclusion chromatography | 12.88 | 3.63 | 160.00 | 2060.80 | 567.40 | 13.17 | 10.55 |
Active Compounds | Tested Strains | MIC (µg/mL) | MBC (µg/mL) |
---|---|---|---|
NNS5-6 AMP | P. aeruginosa TISTR 357 | 4 | 4 |
K. pneumoniae TISTR 1383 | 4 | 8 | |
Colistin | P. aeruginosa TISTR 357 | 1 | 1 |
K. pneumoniae TISTR 1383 | 1 | 1 |
Conditions | % Residual Activity of NNS5-6 AMP against P. aeruginosa TISTR 357 | ||
---|---|---|---|
1 h | 6 h | 12 h | |
Effect of Temperatures | |||
Non-treated NNS5-6 AMP | 100.00 ± 1.23 | 100.00 ± 0.70 | 100.00 ± 0.62 |
37 °C | 99.39 ± 1.98 | 99.39 ± 0.61 | 99.69 ± 0.95 |
40 °C | 99.80 ± 0.94 | 99.19 ± 0.93 | 99.07 ± 0.72 |
50 °C | 65.16 ± 3.07 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * |
60 °C | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * |
80 °C | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * |
100 °C | 0.00 ± 0.00 * | 0.00 ± 0.00 * | 0.00 ± 0.00 * |
121 °C, 15 psi, 15 min | 0.00 ± 0.00 * | ||
121 °C, 15 psi, 30 min | 0.00 ± 0.00 * | ||
Effect of Proteolytic enzymes | |||
Non-treated NNS5-6 AMP | 100.00 ± 0.95 | 100.00 ± 0.34 | 100.00 ± 0.58 |
NNS5-6 AMP with Proteinase K (1 mg/mL) | 96.08 ± 3.41 * | 86.58 ± 1.22 * | 83.62 ± 1.34 * |
NNS5-6 AMP with Trypsin (1 mg/mL) | 99.38 ± 1.99 | 90.47 ± 2.02 * | 88.90 ± 1.46 * |
NNS5-6 AMP with α-chymotrypsin (1 mg/mL) | 99.38 ± 0.95 | 99.42 ± 1.22 | 98.46 ± 0.88 |
Effect of Surfactants | |||
Non-treated NNS5-6 AMP | 100.00 ± 0.60 | 100.00 ± 0.93 | 100.00 ± 0.61 |
NNS5-6 AMP with 1% SDS | 116.77 ± 1.80 * | 122.52 ± 1.86 * | 119.19 ± 1.53 * |
NNS5-6 AMP with 1% Triton X-100 | 116.17 ± 0.60 * | 120.28 ± 1.53 * | 120.81 ± 1.53 * |
1% SDS alone | 121.76 ± 2.42 * | ||
1% Triton X-100 alone | 126.83 ± 2.72 * | ||
Effect of pH variation | |||
Non-treated NNS5-6 AMP | 100.00 ± 1.19 | 100.00 ± 1.28 | 100.00 ± 0.72 |
pH 1.2 | 89.62 ± 1.85 * | 83.83 ± 1.72 * | 81.14 ± 1.71 * |
pH 4.5 | 97.31 ± 0.33 | 95.69 ± 1.75 | 95.62 ± 1.19 |
pH 6.8 | 97.31 ± 3.33 | 96.46 ± 0.57 | 96.00 ± 0.57 |
pH 7.4 | 98.65 ± 0.58 | 98.18 ± 1.15 | 98.48 ± 0.66 |
pH 8.0 | 99.23 ± 1.15 | 97.22 ± 0.98 | 98.48 ± 0.87 |
pH 10.0 | 98.85 ± 1.20 | 97.42 ± 0.82 | 98.10 ± 1.44 |
pH 12.0 | 94.31 ± 0.68 * | 93.40 ± 0.78 * | 90.10 ± 2.38 * |
pH 14.0 | 90.77 ± 0.88 * | 84.40 ± 1.15 * | 79.05 ± 1.75 * |
Antibiotic Resistance Gene | Antibiotic Resistance Gene Family | Resistance Mechanism | Position in Genome | Identity (%) | Coverage Length (%) |
---|---|---|---|---|---|
vanY gene in vanB cluster | vanY, glycopeptide resistance gene cluster | antibiotic target alteration | 291,432 to 292,277 | 33.04 | 104.85 |
Otr(A) | tetracycline-resistant ribosomal protection protein of tetracycline antibiotics | antibiotic target protection | 1,037,370 to 1,039,346 | 45.40 | 99.25 |
vanW gene in vanI cluster | vanW, glycopeptide resistance gene cluster | antibiotic target alteration | 1,132,154 to 1,133,269 | 34.43 | 99.46 |
qacG | small multidrug resistance (SMR) antibiotic efflux pump | antibiotic efflux | 1,236,341 to 1,236,751 | 42.86 | 127.10 |
potxA | Miscellaneous ABC-F subfamily ATP-binding cassette ribosomal protection proteins of oxazolidinones antibiotics | antibiotic target protection | 1,668,816 to 1,670,789 | 36.48 | 121.22 |
norC | major facilitator superfamily (MFS) antibiotic efflux pump of fluoroquinolone antibiotics | efflux pump complex or subunit conferring antibiotic resistance | 3,712,623 to 3,714,038 | 59.69 | 101.95 |
vanW gene in vanI cluster | vanW, glycopeptide resistance gene cluster | antibiotic target alteration | 3,898,660 to 3,900,096 | 36.69 | 128.15 |
vanY gene in vanB cluster | vanY, glycopeptide resistance gene cluster | antibiotic target alteration | 5,289,399 to 5,290,199 | 33.56 | 99.25 |
vanT gene in vanG cluster | vanT, glycopeptide resistance gene cluster | antibiotic target alteration | 5,756,379 to 5,758,274 | 48.63 | 88.62 |
vanXY gene in vanG cluster | vanXY, glycopeptide resistance gene cluster | antibiotic target alteration | 5,758,264 to 5,759,118 | 43.70 | 111.81 |
vanG | Van ligase, glycopeptide resistance gene cluster | antibiotic target alteration | 5,759,115 to 5,760,176 | 53.47 | 101.15 |
Antibiotics | Zone of Inhibition (mm ± SD); n = 3 |
---|---|
Ciprofloxacin (5 µg) | 36.24 ± 0.63 |
Piperacillin (100 µg) and Tazobactam (10 µg) | 50.29 ± 0.41 |
Imipenem (10 µg) | 38.52 ± 0.52 |
Ceftriaxone (30 µg) | 42.50 ± 0.48 |
Cefoxitin (30 µg) | 25.40 ± 0.36 |
Doxycycline (30 µg) | 31.07 ± 0.73 |
Vancomycin (30 µg) | 21.51 ± 1.25 |
Erythromycin (15 µg) | 34.88 ± 0.73 |
Gentamicin (10 µg) | 25.57 ± 0.32 |
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Sermkaew, N.; Atipairin, A.; Krobthong, S.; Aonbangkhen, C.; Yingchutrakul, Y.; Uchiyama, J.; Songnaka, N. Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis. Antibiotics 2024, 13, 846. https://doi.org/10.3390/antibiotics13090846
Sermkaew N, Atipairin A, Krobthong S, Aonbangkhen C, Yingchutrakul Y, Uchiyama J, Songnaka N. Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis. Antibiotics. 2024; 13(9):846. https://doi.org/10.3390/antibiotics13090846
Chicago/Turabian StyleSermkaew, Namfa, Apichart Atipairin, Sucheewin Krobthong, Chanat Aonbangkhen, Yodying Yingchutrakul, Jumpei Uchiyama, and Nuttapon Songnaka. 2024. "Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis" Antibiotics 13, no. 9: 846. https://doi.org/10.3390/antibiotics13090846