In Vitro Assessment of Marine Bacillus for Use as Livestock Probiotics
Abstract
:1. Introduction
2. Results
2.1. Molecular Fingerprinting and Growth of Marine Bacillus
2.2. Sporulation Efficiency
2.3. Growth and Spore Germination under Anaerobic Conditions
2.4. Antimicrobial Activity on Solid Media
Test strain | E. coli | Salmonella | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
K88 | F18ab | F2S2 | F3P3 | F1L3 | F15OF3 | Typhimurium DT104 (DPC 6046) | Typhimurium PT12 (DPC 6465) | Typhimurium DT17 (WIT 396) | Typhimurium DT104 (WIT 387) | Derby (WIT 411) | |
B. pumilus WIT 582 | + | +++ | ++ | ++ | ++ | + | ++ | + | + | ++ | ++ |
B. pumilus WIT 584 | - | - | - | ++ | - | +/− | - | - | - | - | - |
B. licheniformis WIT 586 | + | + | + | ++ | ++ | ++ | +/− | +/− | - | - | +/− |
B. pumilus WIT 588 | +++ | +++ | ++ | +++ | +++ | ++ | ++ | +/− | - | - | - |
B. pumilus WIT 590 | - | - | - | - | - | - | +/− | - | - | - | - |
B. pumilus WIT 592 | - | - | - | - | - | - | - | +/− | - | - | - |
B. licheniformis DSM 5749 2 | - | - | - | - | - | - | +/− | ++ | - | - | - |
B. subtilis DSM 5750 2 | - | - | - | - | - | - | - | - | - | - | - |
2.5. Antimicrobial Activity in Liquid Culture
2.6. Bile Tolerance and Resistance to Simulated Gastrointestinal Conditions
Test strain | Swarming motility 1 | Bile tolerance 2 | Adherence to intestinal epithelial cells 3 |
---|---|---|---|
B. pumilus WIT 582 | + | 1% | 17.4 ± 8.3 a |
B. pumilus WIT 584 | − | <0.3% | 12.1 ± 10.6 a |
B. licheniformis WIT 586 | + | 1% | 11.9 ± 5.1 a |
B. pumilus WIT 588 | − | 1% 4 | 9.3 ± 4.0 a |
B. pumilus WIT 590 | − | <0.3% | 11.0 ± 10.5 a |
B. pumilus WIT 592 | + | <0.3% | 268.4 ± 239.7 b |
B. licheniformis DSM 5749 | + | 2% 4 | 116.5 ± 79.9 b |
B. subtilis DSM 5750 | + | 1% 4 | 196.6 ± 87.1 b |
Lb. rhamnosus GG | ND 5 | ND | 705.6 ± 182.8 b |
2.7. Biofilm Formation, Swarming Motility and Adherence to Intestinal Epithelial Cells
2.8. In Vitro Safety Testing of Bacillus Isolates
2.8.1. Hemolytic Activity, Antibiotic Resistance and Presence of Enterotoxin Genes
Test strain | Viability of intestinal epithelial cells (%) 1 | Hemolysis 2 |
---|---|---|
B. pumilus WIT 582 | 65.9 ± 3 | β |
B. pumilus WIT 584 | 53 ± 10 | very weak β |
B. licheniformis WIT 586 | 80.7 ± 4 | β |
B. pumilus WIT 588 | 69.8 ± 3 | γ |
B. pumilus WIT 590 | 86.2 ± 7 | very weak β |
B. pumilus WIT 592 | 91.2 ± 6 | weak β |
B. licheniformis DSM 5749 | 74.9 ± 6 | γ |
B. subtilis DSM 5750 | 92.4 ± 10 | γ |
Lb. rhamnosus GG | 68.6 ± 5 | ND |
2.8.2. Cytotoxic Activity against Intestinal Epithelial Cells
3. Discussion
4. Experimental Section
4.1. Bacterial Strains and Culture Conditions and Chemicals
4.2. Determination of Time Taken to Sporulate and Sporulation Efficiency and Preparation of Spores
4.3. Evaluation of Bacillus Growth and Spore Germination under Anaerobic Conditions
4.4. Assessment of Antimicrobial Activity of Bacillus Isolates on Solid Media
4.5. Assessment of Antimicrobial Activity of Bacillus Isolates in Liquid Culture
4.6. Bile, Salt Tolerance and Gastric and Ileum Juice Assays
4.7. Determination of Biofilm Formation and Swarming Motility
4.8. Evaluation of Adherence to Intestinal Epithelial Cells
4.8.1. Cell Culture
4.8.2. Assessment of Bacterial Growth in Cell Culture Medium
4.8.3. Adherence Assays
4.9. Analysis of Virulence Factors and Antibiotic Resistance
4.10. Evaluation of Cytotoxicity against Intestinal Epithelial Cells
4.11. Statistical Analysis
5. Conclusions
Supplementary Files
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Prieto, M.L.; O'Sullivan, L.; Tan, S.P.; McLoughlin, P.; Hughes, H.; Gutierrez, M.; Lane, J.A.; Hickey, R.M.; Lawlor, P.G.; Gardiner, G.E. In Vitro Assessment of Marine Bacillus for Use as Livestock Probiotics. Mar. Drugs 2014, 12, 2422-2445. https://doi.org/10.3390/md12052422
Prieto ML, O'Sullivan L, Tan SP, McLoughlin P, Hughes H, Gutierrez M, Lane JA, Hickey RM, Lawlor PG, Gardiner GE. In Vitro Assessment of Marine Bacillus for Use as Livestock Probiotics. Marine Drugs. 2014; 12(5):2422-2445. https://doi.org/10.3390/md12052422
Chicago/Turabian StylePrieto, Maria Luz, Laurie O'Sullivan, Shiau Pin Tan, Peter McLoughlin, Helen Hughes, Montserrat Gutierrez, Jonathan A. Lane, Rita M. Hickey, Peadar G. Lawlor, and Gillian E. Gardiner. 2014. "In Vitro Assessment of Marine Bacillus for Use as Livestock Probiotics" Marine Drugs 12, no. 5: 2422-2445. https://doi.org/10.3390/md12052422