Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay
Abstract
:1. Introduction
2. Results
2.1. Limit of Detection (LoD)
2.2. Specificity
2.3. Comparison of LoD between the EndoPep-MS Method and the MBA
2.4. Diagnostic Sensitivity and Specificity
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Materials and Reagents
5.2. Coupling of Antibodies with Magnetic Beads
5.3. Enrichment Culture for Detection of Neurotoxin-Producing CLOSTRIDIA
5.4. Sample Preparation for EndoPep-MS Detection
5.5. Sample Preparation to Determine the LoD of the EndoPep-MS Method
5.6. Toxin Concentration and Endo-Peptidase Activity Assay
5.7. MALDI-TOF MS Detection
5.8. Limit of Detection (LoD) of the EndoPep-MS Method
5.9. Specificity of the EndoPep-MS Method
5.10. Comparison of the LoD of EndoPep-MS and MBA
5.11. Evaluation of Diagnostic Sensitivity and Specificity of EndoPep-MS
5.12. Multiplex Real-Time PCR to Detect BoNT/C and D and Their Mosaic Forms.
5.13. Ethic Approval
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Weigand, M.R.; Pena-Gonzalez, A.; Shirey, T.B.; Broeker, R.G.; Ishaq, M.K.; Konstantinidis, K.T.; Raphael, B.H. Implications of Genome-Based Discrimination between Clostridium Botulinum Group I and Clostridium Sporogenes Strains for Bacterial Taxonomy. Appl. Environ. Microbiol. 2015, 81, 5420–5429. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mansfield, M.J.; Adams, J.B.; Doxey, A.C. Botulinum Neurotoxin Homologs in Non-Clostridium Species. FEBS Lett. 2015, 589, 342–348. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhang, S.; Lebreton, F.; Mansfield, M.J.; Miyashita, S.I.; Zhang, J.; Schwartzman, J.A.; Tao, L.; Masuyer, G.; Martínez-Carranza, M.; Stenmark, P.; et al. Identification of a Botulinum Neurotoxin-Like Toxin in a Commensal Strain of Enterococcus Faecium. Cell Host Microbe 2018, 23, 169–176.e6. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wentz, T.G.; Muruvanda, T.; Lomonaco, S.; Thirunavukkarasu, N.; Hoffmann, M.; Allard, M.W.; Hodge, D.R.; Pillai, S.P.; Hammack, T.S.; Brown, E.W.; et al. Closed Genome Sequence of Chryseobacterium Piperi Strain CTM(T)/ATCC BAA-1782, a Gram-Negative Bacterium with Clostridial Neurotoxin-Like Coding Sequences. Genome Announc. 2017, 5, e01296-17. [Google Scholar] [CrossRef] [Green Version]
- Pirazzini, M.; Rossetto, O.; Eleopra, R.; Montecucco, C. Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology. Pharmacol. Rev. 2017, 69, 200–235. [Google Scholar] [CrossRef]
- Peck, M.W.; Smith, T.J.; Anniballi, F.; Austin, J.W.; Bano, L.; Bradshaw, M.; Cuervo, P.; Cheng, L.W.; Derman, Y.; Dorner, B.G.; et al. Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature. Toxins 2017, 9, 38. [Google Scholar] [CrossRef]
- Poulain, B.; Popoff, M.R. Why are Botulinum Neurotoxin-Producing Bacteria so Diverse and Botulinum Neurotoxins so Toxic? Toxins 2019, 11, 34. [Google Scholar] [CrossRef] [Green Version]
- Zhang, S.; Masuyer, G.; Zhang, J.; Shen, Y.; Lundin, D.; Henriksson, L.; Miyashita, S.I.; Martínez-Carranza, M.; Dong, M.; Stenmark, P. Identification and Characterization of a Novel Botulinum Neurotoxin. Nat. Commun. 2017, 8, 14130. [Google Scholar] [CrossRef]
- Bano, L.; Drigo, I.; Tonon, E.; Pascoletti, S.; Puiatti, C.; Anniballi, F.; Auricchio, B.; Lista, F.; Montecucco, C.; Agnoletti, F. Identification and Characterization of Clostridium Botulinum Group III Field Strains by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Anaerobe 2017, 48, 126–134. [Google Scholar] [CrossRef]
- Lindstrom, M.; Korkeala, H. Laboratory Diagnostics of Botulism. Clin. Microbiol. Rev. 2006, 19, 298–314. [Google Scholar] [CrossRef] [Green Version]
- Perry, M.J.; Centurioni, D.A.; Davis, S.W.; Hannett, G.E.; Musser, K.A.; Egan, C.T. Implementing the Bruker MALDI Biotyper in the Public Health Laboratory for C. Botulinum Neurotoxin Detection. Toxins 2017, 9, 94. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bano, L.; Tonon, E.; Drigo, I.; Pirazzini, M.; Guolo, A.; Farina, G.; Agnoletti, F.; Montecucco, C. Detection of Clostridium Tetani Neurotoxins Inhibited in Vivo by Botulinum Antitoxin B: Potential for Misleading Mouse Test Results in Food Controls. Toxins 2018, 10, 248. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dezfulian, M.; Bartlett, J.G. Detection of Clostridium Botulinum Type B Toxin in the Presence of a Lethal Substance Interfering with Toxin Neutralization. Diagn. Microbiol. Infect. Dis. 1985, 3, 105–112. [Google Scholar] [CrossRef]
- Kalb, S.R.; Krilich, J.C.; Dykes, J.K.; Luquez, C.; Maslanka, S.E.; Barr, J.R. Detection of Botulinum Toxins A, B, E, and F in Foods by Endopep-MS. J. Agric. Food Chem. 2015, 63, 1133–1141. [Google Scholar] [CrossRef] [PubMed]
- Kalb, S.R.; Moura, H.; Boyer, A.E.; McWilliams, L.G.; Pirkle, J.L.; Barr, J.R. The use of Endopep-MS for the Detection of Botulinum Toxins A, B, E, and F in Serum and Stool Samples. Anal. Biochem. 2006, 351, 84–92. [Google Scholar] [CrossRef] [PubMed]
- Wang, D.; Baudys, J.; Hoyt, K.; Barr, J.R.; Kalb, S.R. Sensitive Detection of Type G Botulinum Neurotoxin through Endopep-MS Peptide Substrate Optimization. Anal. Bioanal Chem. 2019, 411, 5489–5497. [Google Scholar] [CrossRef] [Green Version]
- Boyer, A.E.; Gallegos-Candela, M.; Lins, R.C.; Kuklenyik, Z.; Woolfitt, A.; Moura, H.; Kalb, S.; Quinn, C.P.; Barr, J.R. Quantitative Mass Spectrometry for Bacterial Protein toxins—A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis. Molecules 2011, 16, 2391–2413. [Google Scholar] [CrossRef] [Green Version]
- CNRB. Metodo per la Ricerca di Clostridi Produttori di Tossine Botuliniche Mediante Multiplex Real-Time PCR 2017, 1–29. Available online: https://www.iss.it/documents/20126/0/CNRB31.011.pdf/59a7faf1-15f5-dd54-ae13-d066de9dcea2?t=1582362668086 (accessed on 1 September 2020).
- Kostrzewa, M.; Nagy, E.; Schröttner, P.; Pranada, A.B. How MALDI-TOF Mass Spectrometry Can Aid the Diagnosis of Hard-to-Identify Pathogenic Bacteria—The Rare and the Unknown. Expert Rev. Mol. Diagn. 2019, 19, 667–682. [Google Scholar] [CrossRef]
- Hou, T.Y.; Chiang-Ni, C.; Teng, S.H. Current Status of MALDI-TOF Mass Spectrometry in Clinical Microbiology. J. Food Drug Anal. 2019, 27, 404–414. [Google Scholar] [CrossRef]
- Wang, D.; Krilich, J.; Baudys, J.; Barr, J.R.; Kalb, S.R. Enhanced Detection of Type C. Botulinum Neurotoxin by the Endopep-MS Assay through Optimization of Peptide Substrates. Bioorg. Med. Chem. 2015, 23, 3667–3673. [Google Scholar] [CrossRef] [Green Version]
- Hansbauer, E.M.; Skiba, M.; Endermann, T.; Weisemann, J.; Stern, D.; Dorner, M.B.; Finkenwirth, F.; Wolf, J.; Luginbühl, W.; Messelhäußer, U.; et al. Detection, Differentiation, and Identification of Botulinum Neurotoxin Serotypes C, CD, D, and DC by Highly Specific Immunoassays and Mass Spectrometry. Analyst 2016, 141, 5281–5297. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rasetti-Escargueil, C.; Lemichez, E.; Popoff, M.R. Public Health Risk Associated with Botulism as Foodborne Zoonoses. Toxins 2019, 12, 17. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Moriishi, K.; Koura, M.; Abe, N.; Fujii, N.; Fujinaga, Y.; Inoue, K.; Ogumad, K. Mosaic Structures of Neurotoxins Produced from Clostridium Botulinum Types C and D Organisms. Biochim. Biophys. Acta 1996, 1307, 123–126. [Google Scholar] [CrossRef]
- Wang, D.; Baudys, J.; Kalb, S.R.; Barr, J.R. Improved Detection of Botulinum Neurotoxin Type A in Stool by Mass Spectrometry. Anal. Biochem. 2011, 412, 67–73. [Google Scholar] [CrossRef]
- CNRB. Metodo Per La Ricerca Di Clostridi Produttori Di Tossine Botuliniche e Per La Ricerca Di Tossine Botuliniche (Metodo Colturale e Mouse Test). 2017, pp. 1–29. Available online: https://www.iss.it/documents/20126/0/CNRB30.011.pdf/59a7faf1-15f5-dd54-ae13-d066de9dcea2?t=1582362668086 (accessed on 1 September 2020).
- Hedeland, M.; Moura, H.; Baverud, V.; Woolfitt, A.R.; Bondesson, U.; Barr, J.R. Confirmation of Botulism in Birds and Cattle by the Mouse Bioassay and Endopep-MS. J. Med. Microbiol. 2011, 60, 1299–1305. [Google Scholar] [CrossRef]
BoNT | Matrix | Biological Replicates | ||
---|---|---|---|---|
1 | 2 | 3 | ||
LoD (mLD50/500 µL) | LoD (mLD50/500 µL) | LoD (mLD50/500 µL) | ||
C | Water | 1 | 2 | 2 |
Sera | 2 | 1 | 2 | |
Broth culture | 2 | 2 | 1 | |
Feces | N/A | N/A | N/A | |
D | Water | 1 | 0.5 | 0.5 |
Sera | 0.5 | 0.5 | 0.25 | |
Broth culture | 2 | 1 | 2 | |
Feces | 2 | 1 | 2 |
Strains | Reference N. | EndoPep-MS | qPCR * | MBA ** | |
---|---|---|---|---|---|
Peptide C | Peptide D | ||||
C. botulinum type A | ATCC 19397 | - | - | A | Expired |
C. botulinum type B | CCUG 7969 | - | - | B | Expired |
C. butyricum type E | DSM 10702 | - | - | E | Expired |
C. botulinum type F | NCTC 10281 | - | - | F | Expired |
C. botulinum type C | 9877/1/12 | + | - | C | Expired |
C. botulinum type C/D | 6503/1/13 | + | - | C/D | Expired |
C. botulinum type D/C | 3522/5/13 | - | + | D/C | Expired |
C. botulinum type D | 4150/24/18 | - | + | D | Expired |
Clostridium sordellii | ATCC 9714 | - | - | Negative | Survived |
Clostridium tetani | ATCC 10779 | - | - | Negative | Survived |
Clostridium sporogenes | DSM 795 | - | - | Negative | Survived |
Clostridium difficile | ATCC 9689 | - | - | Negative | Survived |
Clostridium haemolyticum | ATCC 9650 | - | - | Negative | Survived |
Clostridium novyi | ATCC 25758 | - | - | Negative | Survived |
Concentration | EndoPep-MS | MBA | ||||
---|---|---|---|---|---|---|
(mLD50/500 µL) | Replicate 1 | Replicate 2 | Replicate 3 | Mouse 1 | Mouse 2 | |
BoNT/C | ||||||
Serum | 4 | Positive | Positive | Positive | Expired | Expired |
2 | Positive | Positive | Positive | Expired | Expired | |
1 | Positive | Positive | Positive | Expired | Expired | |
0.5 | Negative | Negative | Negative | Expired | Expired | |
0.25 | Negative | Negative | Negative | Survived | Survived | |
Negative | Negative | Negative | Negative | NT | NT | |
Broth culture | 4 | Positive | Positive | Positive | Expired | Expired |
2 | Positive | Positive | Positive | Expired | Survived | |
1 | Negative | Negative | Negative | Survived | Survived | |
0.5 | Negative | Negative | Negative | Survived | Survived | |
0.25 | Negative | Negative | Negative | Survived | Survived | |
Negative | Negative | Negative | Negative | NT | NT | |
Feces (1:1 PGB) | 4 | N/A | N/A | N/A | Expired | Expired |
2 | N/A | N/A | N/A | Survived | Survived | |
1 | N/A | N/A | N/A | Survived | Survived | |
0.5 | N/A | N/A | N/A | Survived | Survived | |
0.25 | N/A | N/A | N/A | Survived | Survived | |
Negative | N/A | N/A | N/A | NT | NT | |
BoNT/D | ||||||
Serum | 4 | Positive | Positive | Positive | Expired | Expired |
2 | Positive | Positive | Positive | Expired | Expired | |
1 | Positive | Positive | Positive | Survived | Survived | |
0.5 | Positive | Positive | Negative | Survived | Survived | |
0.25 | Negative | Negative | Negative | Survived | Survived | |
Negative | Negative | Negative | Negative | NT | NT | |
Broth culture | 4 | Positive | Positive | Positive | Expired | Expired |
2 | Positive | Positive | Positive | Expired | Expired | |
1 | Negative | Negative | Negative | Expired | Expired | |
0.5 | Negative | Negative | Negative | Survived | Survived | |
0.25 | Negative | Negative | Negative | Survived | Survived | |
Negative | Negative | Negative | Negative | NT | NT | |
Feces (1:1 PGB) | 4 | Positive | Positive | Positive | Expired | Expired |
2 | Positive | Positive | Positive | Expired | Survived | |
1 | Negative | Negative | Negative | Survived | Survived | |
0.5 | Negative | Negative | Negative | Survived | Survived | |
0.25 | Negative | Negative | Negative | Survived | Survived | |
Negative | Negative | Negative | Negative | NT | NT |
Sample ID | Origin | BoNTs | EndoPep-MS | ||
---|---|---|---|---|---|
MBA | qPCR | BoNT/C | BoNT/D | ||
GPB extracts | |||||
8/4/15 | Vegetables + meat | NEG | / | NEG | NEG |
8/3/15 | Vegetables + meat | NEG | / | NEG | NEG |
8/6/14 | Vegetables + meat | BoNT/E | / | NEG | NEG |
8/7/14 | Vegetables + meat | BoNT/A | / | NEG | NEG |
8/8/15 | Vegetables + meat | BoNT/A | / | NEG | NEG |
8/10/15 | Vegetables + meat | BoNT/A | / | NEG | NEG |
8/9/15 | Vegetables + meat | NEG | / | NEG | NEG |
8/1/15 | Vegetables + meat | NEG | / | NEG | NEG |
3/1/15 | Vegetables + meat | BoNT/B | / | NEG | NEG |
8/2/15 | Vegetables + meat | BoNT/A | / | NEG | NEG |
Enrichment cultures | |||||
45/2/12 | Mushrooms in oil | NEG | / | NEG | NEG |
3/1/15 | Eggplants in oil | BoNT/B | B | NEG | NEG |
43/1/12 | Human feces | NEG | / | NEG | NEG |
19/10/11 | Rectal swab | BoNT/E | E | NEG | NEG |
1/4/15 | Mushrooms in oil | BoNT/B | B | NEG | NEG |
2/2/15 | Human feces | NEG | / | NEG | NEG |
8/8/08 | Human feces | BoNT/B | B | NEG | NEG |
48/4/12 | Human feces | NEG | / | NEG | NEG |
18/5/12 | Olives | BoNT/B | B | NEG | NEG |
18/5/12 | Sausages | NEG | / | NEG | NEG |
21/19/12 | Tuna in oil | NEG | / | NEG | NEG |
55/6/11 | Eggplants in oil | NEG | / | NEG | NEG |
153/113 | Truffle cream | NEG | / | NEG | NEG |
20/3/12 | Chili pepper in oil | NEG | / | NEG | NEG |
16/3/12 | Human feces | BoNT/B | B | NEG | NEG |
166/2/13 | Human feces | NEG | / | NEG | NEG |
1556/15 | Tuna | BoNT/B | B | NEG | NEG |
16/4/12 | Mushrooms in oil | BoNT/B | B | NEG | NEG |
22/2/14 | Human feces | BoNT/B | B | NEG | NEG |
22/4/14 | Human feces | BoNT/B | B | NEG | NEG |
46/3/14 | Human feces | BoNT/B | B | NEG | NEG |
152/1/10 | Truffle cream | BoNT/B | B | NEG | NEG |
47/2/15 | Human feces | BoNT/B | B | NEG | NEG |
11/3/15 | Mushrooms in oil | BoNT/B | B | NEG | NEG |
18/2/15 | Human feces | BoNT/B | B | NEG | NEG |
42/2/15 | Human feces | BoNT/B | B | NEG | NEG |
4871/15 | Human feces | NEG | / | NEG | NEG |
49/2/15 | Human feces | BoNT/B | B | NEG | NEG |
5271715 | Chili pepper + tuna in oil | NEG | / | NEG | NEG |
5509/1/20 | Wood shavings | BoNT/C + BoNT/D | C + D | POS | POS |
5509/2/20 | Litter | BoNT/C + BoNT/D | C + D | POS | POS |
4888/20 | Quail | BoNT/D | D/C | NEG | POS |
5291/1/20 | Water (drinking) | BoNT/D | D/C | NEG | POS |
5291/2/20 | Water (tank) | BoNT/D | D/C | NEG | POS |
5729/1/20 | Bovine | BoNT/D | D | NEG | POS |
5729/2/20 | Bovine | BoNT/D | D | NEG | POS |
6126/2/20 | Water | BoNT/D | D | NEG | POS |
6126/3/20 | Water | BoNT/D | D | NEG | POS |
6126/7/20 | Quail | BoNT/D | D | NEG | POS |
6126/8/20 | Quail | BoNT/D | D | NEG | POS |
6126/10/20 | Quail | BoNT/D | D | NEG | POS |
6126/11/20 | Quail | BoNT/D | D | NEG | POS |
Sera | |||||
5674/10 | Duck | BoNT/C | / | POS | NEG |
8103/2/09 | Broiler | BoNT/C | / | POS | NEG |
8103/10/09 | Broiler | NEG | / | NEG | NEG |
8103/13/09 | Broiler | BoNT/C | / | POS | NEG |
2659/2/10 | Broiler | NEG | / | NEG | NEG |
2659/4/10 | Broiler | NEG | / | POSw | NEG |
2659/5/10 | Broiler | BoNT/C | / | POS | NEG |
4691/11 | Turkey | BoNT/C | / | POS | NEG |
4863/13 | Pheasant | BoNT/C | / | POS | NEG |
474/14 | Dog | BoNT/C | / | POS | NEG |
5313/28/14 | Broiler | BoNT/C | / | POS | NEG |
5313/29/14 | Broiler | BoNT/C | / | POS | NEG |
5313/30/14 | Broiler | BoNT/C | / | POS | NEG |
6289/3/14 | Mallard | NEG | / | NEG | NEG |
6289/4/14 | Mallard | NEG | / | NEG | NEG |
5993/15 | Pheasant | BoNT/C | / | POS | NEG |
6353/15 | Broiler | BoNT/C | / | POS | NEG |
6660/1/15 | Broiler | BoNT/C | / | POS | NEG |
6660/2/15 | Broiler | BoNT/C | / | POS | NEG |
6841/2/15 | Bovine | NEG | / | NEG | NEG |
6841/3/13 | Bovine | NEG | / | POSw | NEG |
Feces | |||||
5641/12 | Swan | BoNT/C | / | POS | NEG |
Milk | |||||
9861/51/18 | Bovine | NEG | / | NEG | NEG |
9861/189/18 | Bovine | NEG | / | NEG | NEG |
9861/242/18 | Bovine | NEG | / | NEG | NEG |
9861/251/18 | Bovine | NEG | / | NEG | NEG |
9861/258/18 | Bovine | NEG | / | NEG | NEG |
Toxin Type | Peptide | m/z | Reference | ||
---|---|---|---|---|---|
Name | Sequence | ||||
BoNT/C | PepC | KGSNRTIDEANQRA/ATRMLGGK-biotin | [M + H]+ | 2912.3 | [27] |
[M + 2H]2+ | 1457.1 | ||||
CT | 1059.5 | ||||
NT | 1870.9 | ||||
BoNT/D | Pep D/F | TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQK/LSELDDRADAL | [M + H]+ | / | [14] |
[M + 2H]2+ | 2605.4 | ||||
CT | 1217.3 | ||||
NT | 4013.5 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Drigo, I.; Tonon, E.; Pascoletti, S.; Anniballi, F.; Kalb, S.R.; Bano, L. Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay. Toxins 2021, 13, 10. https://doi.org/10.3390/toxins13010010
Drigo I, Tonon E, Pascoletti S, Anniballi F, Kalb SR, Bano L. Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay. Toxins. 2021; 13(1):10. https://doi.org/10.3390/toxins13010010
Chicago/Turabian StyleDrigo, Ilenia, Elena Tonon, Simone Pascoletti, Fabrizio Anniballi, Suzanne R. Kalb, and Luca Bano. 2021. "Detection of Active BoNT/C and D by EndoPep-MS Using MALDI Biotyper Instrument and Comparison with the Mouse Test Bioassay" Toxins 13, no. 1: 10. https://doi.org/10.3390/toxins13010010