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Identification of bacterial endospores and targeted detection of foodborne viruses in industrially reared insects for food

Abstract

With edible insects being increasingly produced, food safety authorities have called for the determination of microbiological challenges posed to human health. Here, we find that the bacterial endospore fraction in industrially reared mealworm and cricket samples is largely comprised of Bacillus cereus group members that can pose insect or human health risks. Hepatitis A virus, hepatitis E virus and norovirus genogroup II were not detected in the sample collection, indicating a low food safety risk from these viral pathogens.

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Fig. 1: Microbial counts of samples assessed for bacterial endospores.
Fig. 2: Identification and characterization of bacterial endospore isolates.

Data availability

Sequencing data obtained in this study have been deposited in GenBank (National Center for Biotechnology Information) with the accession codes MN508485 to MN508527 (Supplementary Table 1). All other data that support the findings of this study are available from the corresponding author upon request.

References

  1. 1.

    EFSA Scientific Committee. Risk profile related to production and consumption of insects as food and feed. EFSA J. 13, 4257 (2015).

  2. 2.

    Garofalo, C. et al. Current knowledge on the microbiota of edible insects intended for human consumption: A state-of-the-art review. Food Res. Int. 125, 108527 (2019).

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Vandeweyer, D., Lenaerts, S., Callens, A. & Van Campenhout, L. Effect of blanching followed by refrigerated storage or industrial microwave drying on the microbial load of yellow mealworm larvae (Tenebrio molitor). Food Control 71, 311–314 (2017).

    CAS  Article  Google Scholar 

  4. 4.

    Wynants, E. et al. Microbial dynamics during production of lesser mealworms (Alphitobius diaperinus) for human consumption at industrial scale. Food Microbiol. 70, 181–191 (2018).

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Vandeweyer, D. et al. Microbial dynamics during industrial rearing, processing, and storage of tropical house crickets (Gryllodes sigillatus) for human consumption. Appl. Environ. Microbiol. 84, e00255-18 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Vandeweyer, D., Crauwels, S., Lievens, B. & Van Campenhout, L. Metagenetic analysis of the bacterial communities of edible insects from diverse production cycles at industrial rearing companies. Int. J. Food Microbiol. 261, 11–18 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. 7.

    Garofalo, C. et al. The microbiota of marketed processed edible insects as revealed by high-throughput sequencing. Food Microbiol. 62, 15–22 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8.

    Fasolato, L. et al. Edible processed insects from e-commerce: food safety with a focus on the Bacillus cereus group. Food Microbiol. 76, 296–303 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  9. 9.

    Stoops, J. et al. Minced meat-like products from mealworm larvae (Tenebrio molitor and Alphitobius diaperinus): microbial dynamics during production and storage. Innov. Food Sci. Emerg. Technol. 41, 1–9 (2017).

    Article  Google Scholar 

  10. 10.

    Petrović, T. & D’Agostino, M. in Antimicrobial Food Packaging (ed. Barros-Velázquez, J.) 65–79 (Academic, 2016).

  11. 11.

    Da Silva, A. K. et al. Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II. Appl. Environ. Microbiol. 73, 7891–7897 (2007).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  12. 12.

    De Smet, J. et al. Stability assessment and laboratory scale fermentation of pastes produced on a pilot scale from mealworms (Tenebrio molitor). LWT 102, 113–121 (2019).

    Article  CAS  Google Scholar 

  13. 13.

    Vandeweyer, D., Crauwels, S., Lievens, B. & Van Campenhout, L. Microbial counts of mealworm larvae (Tenebrio molitor) and crickets (Acheta domesticus and Gryllodes sigillatus) from different rearing companies and different production batches. Int. J. Food Microbiol. 242, 13–18 (2017).

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Stoops, J. et al. Microbial community assessment of mealworm larvae (Tenebrio molitor) and grasshoppers (Locusta migratoria migratorioides) sold for human consumption. Food Microbiol. 53B, 122–127 (2016).

    Article  Google Scholar 

  15. 15.

    FASFC Table With Regulatory Criteria and Action Limits. Action Limits for Microbiological Contaminants in Foods (2020); http://www.favv.be/professionelen/publicaties/thematisch/actiegrenzen/

  16. 16.

    Rasigade, J. P., Hollandt, F. & Wirth, T. Genes under positive selection in the core genome of pathogenic Bacillus cereus group members. Infect. Genet. Evol. 65, 55–64 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Liu, Y. et al. Genomic insights into the taxonomic status of the Bacillus cereus group. Sci. Rep. 5, 14082 (2015).

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Liu, Y. et al. Proposal of nine novel species of the Bacillus cereus group. Int. J. Syst. Evol. Microbiol. 67, 2499–2508 (2017).

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Ehling-Schulz, M., Lereclus, D. & Koehler, T. M. The Bacillus cereus group: Bacillus species with pathogenic potential. Microbiol. Spectr. 7, GPP3-0032-2018 (2019).

    Article  Google Scholar 

  20. 20.

    Guinebretière, M.-H. et al. Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus group occasionally associated with food poisoning. Int. J. Syst. Evol. Microbiol. 63, 31–40 (2013).

    PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Stenfors, L. P., Mayr, R., Scherer, S. & Granum, E. Pathogenic potential of fifty Bacillus weihenstephanensis strains. FEMS Microbiol. Lett. 215, 47–51 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Osimani, A. et al. Insight into the proximate composition and microbial diversity of edible insects marketed in the European Union. Eur. Food Res. Technol. 243, 1157–1171 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Bravo, A., Gill, S. S. & Soberón, M. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49, 423–435 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Martínez, S., Borrajo, R., Franco, I. & Carballo, J. Effect of environmental parameters on growth kinetics of Bacillus cereus (ATCC 7004) after mild heat treatment. Int. J. Food Microbiol. 117, 223–227 (2007).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  25. 25.

    Ahmed, I., Yokota, A., Yamazoe, A. & Fujiwara, T. Proposal of Lysinibacillus boronitolerans gen. nov. sp. nov., and transfer of Bacillus fusiformis to Lysinibacillus fusiformis comb. nov. and Bacillus sphaericus to Lysinibacillus sphaericus comb. nov. Int. J. Syst. Evol. Microbiol. 57, 1117–1125 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Berry, C. The bacterium, Lysinibacillus sphaericus, as an insect pathogen. J. Invertebr. Pathol. 109, 1–10 (2012).

    PubMed  Article  PubMed Central  Google Scholar 

  27. 27.

    Lücking, G., Stoeckel, M., Atamer, Z., Hinrichs, J. & Ehling-Schulz, M. Characterization of aerobic spore-forming bacteria associated with industrial dairy processing environments and product spoilage. Int. J. Food Microbiol. 166, 270–279 (2013).

    PubMed  Article  PubMed Central  Google Scholar 

  28. 28.

    De Jonghe, V. et al. Toxinogenic and spoilage potential of aerobic spore-formers isolated from raw milk. Int. J. Food Microbiol. 136, 318–325 (2010).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  29. 29.

    Wang, Y. & Zhang, Y. Investigation of gut-associated bacteria in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae using culture-dependent and DGGE methods. Ann. Entomol. Soc. Am. 108, 941–949 (2015).

    CAS  Article  Google Scholar 

  30. 30.

    Osimani, A. et al. The bacterial biota of laboratory-reared edible mealworms (Tenebrio molitor L.): from feed to frass. Int. J. Food Microbiol. 272, 49–60 (2018).

    PubMed  Article  Google Scholar 

  31. 31.

    Wang, L. T., Lee, F. L., Tai, C. J. & Kasai, H. Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group. Int. J. Syst. Evol. Microbiol. 57, 1846–1850 (2007).

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    De Vos, P. et al. Bergey’s Manual of Systematic Bacteriology Vol. 3 (Springer Science+Business Media, 2009).

  33. 33.

    Kramer, J. M. & Gilbert, R. J. in Foodborne Bacterial Pathogens (ed. Doyle, M.) 21–70 (Marcel Dekker, 1989).

  34. 34.

    From, C., Pukall, R., Schumann, P., Hormazábal, V. & Granum, P. E. Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group. Appl. Environ. Microbiol. 71, 1178–1183 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Serrano, L., Manker, D., Brandi, F. & Cali, T. The use of Bacillus subtilis QST 713 and Bacillus pumilus QST 2808 as protectant fungicides in conventional application programs for black leaf streak control. Acta Hortic. 986, 149–156 (2013).

    Article  Google Scholar 

  36. 36.

    Tactacan, G. B., Schmidt, J. K., Miille, M. J. & Jimenez, D. R. A Bacillus subtilis (QST 713) spore-based probiotic for necrotic enteritis control in broiler chickens. J. Appl. Poult. Res. 22, 825–831 (2013).

    Article  Google Scholar 

  37. 37.

    Ruiu, L. Brevibacillus laterosporus, a pathogen of invertebrates and a broad-spectrum antimicrobial species. Insects 4, 476–492 (2013).

    PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Osimani, A. et al. Occurrence of transferable antibiotic resistances in commercialized ready-to-eat mealworms (Tenebrio molitor L.). Int. J. Food Microbiol. 263, 38–46 (2017).

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Madigan, M. T., Martinko, J. M., Dunlap, P. V. & Clark, D. P. Brock Biology of Microorganisms (Pearson/Benjamin Cummings, 2009).

  40. 40.

    Ceuppens, S., Boon, N. & Uyttendaele, M. Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles. FEMS Microbiol. Ecol. 84, 433–450 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  41. 41.

    Agata, N., Ohta, M. & Yokoyama, K. Production of Bacillus cereus emetic toxin (cereulide) in various foods. Int. J. Food Microbiol. 73, 23–27 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Finlay, W. J. J., Logan, N. A. & Sutherland, A. D. Bacillus cereus produces most emetic toxin at lower temperatures. Lett. Appl. Microbiol. 31, 385–389 (2000).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    Rouzeau-Szynalski, K., Stollewerk, K., Messelhäusser, U. & Ehling-Schulz, M. Why be serious about emetic Bacillus cereus: cereulide production and industrial challenges. Food Microbiol. 85, 103279 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  44. 44.

    EFSA Panel on Biological Hazards Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs. EFSA J. 14, 93 (2016).

    Google Scholar 

  45. 45.

    Ehling-Schulz, M., Frenzel, E. & Gohar, M. Food-bacteria interplay: pathometabolism of emetic Bacillus cereus. Front. Microbiol. 6, 704 (2015).

    PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Hoton, F. M. et al. Family portrait of Bacillus cereus and Bacillus weihenstephanensis cereulide-producing strains. Environ. Microbiol. Rep. 1, 177–183 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Diez-Valcarce, M., Kovač, K., Cook, N., Rodríguez-Lázaro, D. & Hernández, M. Construction and analytical application of internal amplification controls (IAC) for detection of food supply chain-relevant viruses by real-time PCR-based assays. Food Anal. Methods 4, 437–445 (2011).

    Article  Google Scholar 

  48. 48.

    Lievens, B. et al. Design and development of a DNA array for rapid detection and identification of multiple tomato vascular wilt pathogens. FEMS Microbiol. Lett. 223, 113–122 (2003).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  49. 49.

    Yoon, S. H. et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613–1617 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Zhuang, K. et al. Typing and evaluating heat resistance of Bacillus cereus sensu stricto isolated from the processing environment of powdered infant formula. J. Dairy Sci. 102, 7781–7793 (2019).

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Guinebretière, M. H. et al. Ecological diversification in the Bacillus cereus group. Environ. Microbiol. 10, 851–865 (2008).

    PubMed  Article  CAS  Google Scholar 

  52. 52.

    Fricker, M., Messelhäußer, U., Busch, U., Scherer, S. & Ehling-Schulz, M. Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks. Appl. Environ. Microbiol. 73, 1892–1898 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Ueda, S., Yamaguchi, M., Iwase, M. & Kuwabara, Y. Detection of emetic Bacillus cereus by real-time PCR in foods. Biocontrol Sci. 18, 227–232 (2013).

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Diez-Valcarce, M. et al. Occurrence of human enteric viruses in commercial mussels at retail level in three European countries. Food Environ. Virol. 4, 73–80 (2012).

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    JMP Pro, Version 14.0.0 (SAS Institute Inc., Cary, NC, 1989–2019).

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Acknowledgements

Virus references and IACs were provided by I. Di Bartolo from the Italian Istituto Superiore di Sanità (ISS) and N. Cook from Fera Science Ltd. We thank A. Paeleman (Scientia Terrae Research Institute) for her expertise and assistance in designing and optimizing the qPCR protocols, S. Crauwels (KU Leuven) for processing the sequencing results and R. Smets (KU Leuven) for help with the statistics. J. Franciotti, L. De Vrindt, N. Huybrechts, M. Gerits, S. Machtajiw, J. Plas and E. Van Vossole are acknowledged for their assistance in the lab. This research was financially supported by Flanders Innovation & Entrepreneurship (VLAIO) (Project 141129) as well as Internal Funds KU Leuven (grant number PDM/18/159).

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D.V. designed, prepared and executed all experiments, including sample collection and preparation, microbiological analyses, DNA and RNA extractions and PCR and RT-qPCR reactions, and analysed the data, constructed tables and figures and wrote the main text. B.L. and L.V.C. supervised the study, provided additional insight in data analysis and revised the manuscript.

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Correspondence to Leen Van Campenhout.

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Reporting Summary

Supplementary Table 1

Identification of isolated spore-forming bacteria harboured by yellow mealworms or house crickets.

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Vandeweyer, D., Lievens, B. & Van Campenhout, L. Identification of bacterial endospores and targeted detection of foodborne viruses in industrially reared insects for food. Nat Food 1, 511–516 (2020). https://doi.org/10.1038/s43016-020-0120-z

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