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Chicken embryos are a valuable model for the selection of Bacillus subtilis for probiotic purposes

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Abstract

Bacillus subtilis (BS) has been used as an excellent probiotic; however, some BS strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacteria, so the characterization of BS strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of BS, which can inhibit Salmonella spp., avian pathogenic Escherichia coli (APEC), and Campylobacter jejuni (CJ) using a chicken embryo as a model. We tested nine (9) strains of BS isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (CE) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight CEs. At the 17th DPI we checked CE mortality, gross lesions, CE weight, and whether BS strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the CE blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in CEs, we tested the inhibition capacity of the selected BS strains for diverse strains of Salmonella  Heidelberg (SH), S. Typhimurium (ST), S. Enteritidis (SE), S. Minnesota (SM), S. Infantis (SI), Salmonella var. monophasic (SVM), APEC and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed β-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of BS were viable in CEs at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in CEs or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the CE. From the tests in CEs, we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of SH, ST, SI, APEC, and two (2) strains of CJ. In this study, we selected BS strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of CE as an in vivo experimental model for assessing BS’s safety and possible benefits for poultry and other animals.

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Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

BS:

Bacillus subtilis

APEC:

Avian pathogenic Escherichia coli

CJ:

Campylobacter jejuni

DPI:

Day postincubation

SH:

Salmonella Heidelberg

ST:

Salmonella Typhimurium

SE:

Salmonella Enteritidis

SM:

Salmonella Minnesota

SI:

Salmonella Infantis

SVM:

Salmonella Var. monophasic

DSM 17299:

Commercial probiotic of BS

CLSI:

Clinical and Laboratory Standards Institute

MIC:

Minimal inhibitory concentration

EFSA:

European Food Safety Authority

NC:

Negative control

PC:

Positive control

YP:

Yo Pro-01

PI:

Propide iodate

DAPI:

4′,6-Diamidino-2-phenylindole

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Acknowledgements

The authors thank Luiz Ricardo Goulart Filho for all support, research partnership, and teachings. Your departure left us with a vast sadness, but your brilliance and generosity reached all who had the honour to learn from you. You live within us.

Funding

This research was funded by the National Institute of Science and Technology in Theranostics and Nanobiotechnology-INCT-Teranano (CNPq/CAPES/FAPEMIG, Grant # CNPq-403193/2022-2, FAPEMIG Processo nº C.B.B.-APQ-03613-17)).

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Author notes

  1. Thais Fernanda Martins dos Reis and Patricia Giovana Hoepers share the first authorship as they have contributed equally to the article.

Authors and Affiliations

  1. School of Veterinary Medicine, Federal University of Uberlândia, Uberlândia, Brazil

    Thais Fernanda Martins dos Reis, Patricia Giovana Hoepers, Gabriela Ribeiro da Silva, Fabiana Oliveira Notário, Maria Cecília Soares, André Eduardo Schlemper, Ícaro Mendonça Costa, Simone Sommerfeld, Alessandra Aparecida Medeiros-Ronchi, Daise Aparecida Rossi, Igor Paula de Castro & Belchiolina Beatriz Fonseca

  2. Graduate Program in Genetics at the Federal University of Minas Gerais, Belo Horizonte, Brazil

    Vasco Ariston de Carvalho Azevedo & Nina Dias Coelho-Rocha

  3. Graduate Program in Genetics and Biochemistry at the Federal University of Uberlândia, Uberlândia, Brazil

    Mylla Spirandelli da Costa & Belchiolina Beatriz Fonseca

Authors
  1. Thais Fernanda Martins dos Reis
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  2. Patricia Giovana Hoepers
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  3. Vasco Ariston de Carvalho Azevedo
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  4. Gabriela Ribeiro da Silva
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  8. Ícaro Mendonça Costa
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  9. Nina Dias Coelho-Rocha
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Contributions

BBF, NDC, SS, TFMR, GRS, IMC, DAR, and PGH conducted the in vitro experiment. TFMR, GRS, MCS, AES, SS, and IMC made the collections and analyses in embryos. FON, AAMR, and IPC prepared the histopathological slides and performed the analysis. MSC performed the ELISA. PGH wrote the manuscript. BBF and VAC idealized, designed, analysed the data, and edited the article.

Corresponding authors

Correspondence to Patricia Giovana Hoepers or Belchiolina Beatriz Fonseca.

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Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

The Ethics Committee on Animal Use of the U.F.U. certified under protocol number 11/2022/CEUA/PROPP/REITO, PROCESSO N°º23117.023808/2022-77.

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Communicated by Erko Stackebrandt.

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dos Reis, T.F.M., Hoepers, P.G., Azevedo, V.A. et al. Chicken embryos are a valuable model for the selection of Bacillus subtilis for probiotic purposes. Arch Microbiol 204, 715 (2022). https://doi.org/10.1007/s00203-022-03307-9

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