Safety Assessment of Bacillus subtilis G8 Isolated from Natto for Food Application

https://doi.org/10.22146/jtbb.87079

Nathania Calista Putri(1), Hans Victor(2), Vivian Litanto(3), Reinhard Pinontoan(4), Juandy Jo(5*)

(1) Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan. Jl. M.H. Thamrin Boulevard 1100, Tangerang 15811, Banten, Indonesia.
(2) Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan. Jl. M.H. Thamrin Boulevard 1100, Tangerang 15811, Banten, Indonesia.
(3) Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan. Jl. M.H. Thamrin Boulevard 1100, Tangerang 15811, Banten, Indonesia.
(4) Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan. Jl. M.H. Thamrin Boulevard 1100, Tangerang 15811, Banten, Indonesia.
(5) Department of Biology, Faculty of Science and Technology, Universitas Pelita Harapan. Jl. M.H. Thamrin Boulevard 1100, Tangerang 15811, Banten, Indonesia; Mochtar Riady Institute for Nanotechnology, Jl. Boulevard Jendral Sudirman No.1688, Tangerang 15811, Banten, Indonesia.
(*) Corresponding Author

Abstract


Various bacteria are widely used as food-fermenting agents, including Lactobacillus, Bifidobacterium, and Bacillus. Despite they are generally recognized as safe to be consumed by humans, those bacteria could potentially cause antibiotic resistance as they could acquire and transfer antibiotic resistance genes from or to other microbes within the human gastrointestinal tract. Profiling antibiotic resistance pattern in those bacteria is therefore important to control the spread of antibiotic resistance. In this study, antibiotic resistance profile of Bacillus subtilis G8 was assessed. B. subtilis G8 had been isolated from commercialised Japanese natto in Indonesia and had been previously reported for its fibrinolytic characteristics. The antibiotic resistance phenotype and genotype of B. subtilis G8 were assessed through the Kirby-Bauer disk diffusion method and whole-genome analysis, respectively. B. subtilis G8 exhibited resistance towards Oxacillin, Lincomycin and Tiamulin-Lefamulin. The bioinformatics analysis indicated several responsible genes mediating those resistance, i.e., ybxI (for Oxacillin), lmrB (for Lincomycin) and vmlR (for Lincomycin and Tiamulin-Lefamulin). All identified genes were found in the chromosomal DNA. Further analysis found no mobile genetic elements within the genome, therefore reducing a risk of resistance gene transfer via plasmid and subsequently supporting safety profile of B. subtilis G8 in food fermentation usage.


Keywords


Bacillus subtilis G8; natto; antibiotic resistance; disk diffusion method; whole-genome sequencing

Full Text:

PDF


References

Alcock, B.P. et al., 2020. CARD 2020: Antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res, 48, pp.D517–D525. doi: 10.1093/nar/gkz935.

Amin, F.A.Z. et al., 2020. Probiotic Properties of Bacillus Strains Isolated from Stingless Bee (Heterotrigona itama) Honey Collected across Malaysia. Int J Environ Res Public Health, 17, pp.1–15. doi: 10.3390/ijerph17010278

Ammor, M.S., Belén Flórez, A. & Mayo, B., 2007a. Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria. Food Microbiol, 24, pp.559–70. doi: 10.1016/j.fm.2006.11.001.

Ammor, M.S. et al., 2007b. Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria. J Mol Microbiol Biotechnol, 14(1-3), pp.6–15. doi: 10.1159/000106077.

Andrews, S., 2010, ‘FastQC: A Quality Control Tool for High Throughput Sequence Data’, in Babraham Bioinformatics, viewed 16 July 2021, from https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.

Antunes, N.T. & Fisher, J.F., 2014. Acquired class D β-Lactamases. Antibiotics, 3, pp.398–434. doi: 10.3390/antibiotics3030398.

Bankevich, A. et al., 2012. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol, 19, pp.455–77. doi: 10.1089/cmb.2012.0021.

Broekema, N.M. et al., 2009. Comparison of cefoxitin and oxacillin disk diffusion methods for detection of mecA-mediated resistance in staphylococcus aureus in a large-scale study. J Clin Microbiol, 47(1), pp.217–219. doi: 10.1128/JCM.01506-08.

Buchfink, B., Xie, C. & Huson, D.H., 2014. Fast and sensitive protein alignment using DIAMOND. Nat Methods, 12, pp.59–60. doi: 10.1038/nmeth.3176.

Camacho, C. et al., 2009. BLAST+: Architecture and applications. BMC Bioinformatics, 10, 421. doi: 10.1186/1471-2105-10-421.

Carver, T. et al., 2012. Artemis: An integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics, 28(4), pp.464–469. doi: 10.1093/bioinformatics/btr703.

Chukiatsiri, K. et al., 2012. Serovar Identification, Antimicrobial Sensitivity, and Virulence of Avibacterium paragallinarum Isolated from Chickens in Thailand. Avian Dis Dig, 7(2). doi: 10.1637/10120-988112-digest.1.

Clinical and Laboratory Standards Institute (CLSI), 2021. Performance Standards for Antimicrobial Susceptibility Testing: CSLI Supplement M100. 31st editi. USA: Clinical and Laboratory Standards Institute.

Colombo, M.L. et al., 2004. The ybxI Gene of Bacillus subtilis 168 Encodes a Class D β-Lactamase of Low Activity. Antimicrob Agents Chemother, 48, pp.484–490. doi: 10.1128/AAC.48.2.484-490.2004.

Crowe-McAuliffe, C. et al., 2018. Structural basis for antibiotic resistance mediated by the Bacillus subtilis ABCF ATPase VmlR. Proc Natl Acad Sci U S A, 115(36), pp.8978–8983. doi: 10.1073/pnas.1808535115.

Darling, A.C.E. et al., 2004. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res, 14(7), pp.1394–1403. doi: 10.1101/gr.2289704.

Dikson, D. et al., 2022. Whole-genome analysis of Bacillus subtilis G8 isolated from natto. Biodiversitas, 23, pp.1293–1300. doi: 10.13057/biodiv/d230313.

Grant, J.R. et al., 2023. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res, 51, pp.W484-W492. doi: 10.1093/nar/gkad326.

Gueimonde, M. et al., 2013. Antibiotic resistance in probiotic bacteria. Front Microbiol, 4, 202. doi: 10.3389/fmicb.2013.00202.

Guo, M. et al., 2017. Bacillus subtilis improves immunity and disease resistance in rabbits. Front Immunol, 8, 354. doi: 10.3389/fimmu.2017.00354.

Hua, X. et al., 2021. BacAnt: A Combination Annotation Server for Bacterial DNA Sequences to Identify Antibiotic Resistance Genes, Integrons, and Transposable Elements. Front Microbiol, 12, 649969. doi: 10.3389/fmicb.2021.649969.

Hyatt, D. et al., 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics, 11, 119. doi: 1471-2105/11/119.

Irkitova, A.N., Grebenshchikova, A.V. & Dudnik, D.E., 2019. Antibiotic susceptibilty of bacteria from the Bacillus subtilis group. Ukr J Ecol, 9, pp.363–366.

Islam, K.M.S., Klein, U. & Burch, D.G.S., 2009. The activity and compatibility of the antibiotic tiamulin with other drugs in poultry medicine - A review. Poult Sci, 88, pp.2353–2359. doi: 10.3382/ps.2009-00257.

Jorgensen, J.H. & Turnidge, J.D., 2015. Susceptibility Test Methods: Dilution and Disk Diffusion Methods. Man. Clin. Microbiol, 11th edition. Wiley, pp.1253–73. doi: 10.1128/9781555817381.

Kamada, M. et al., 2015. Whole-Genome Sequencing and Comparative Genome Analysis of Bacillus subtilis Strains Isolated from Non-Salted Fermented Soybean Foods. PLoS One, 10(10), e0141369. doi: 10.1371/journal.pone.0141369.

Larsen, J. et al., 2022. Emergence of methicillin resistance predates the clinical use of antibiotics. Nature, 602(7895), pp.135-141. doi: 10.1038/s41586-021-04265-w.

Lucy, J. et al., 2019. Clot Lysis Activity of Bacillus subtilis G8 Isolated from Japanese Fermented Natto Soybeans. Appl Food Biotechnol, 6(2), pp.101–109. doi: 10.22037/afb.v6i2.22479.

Marco, M.L. et al., 2017. Health benefits of fermented foods: microbiota and beyond. Curr Opin Biotechnol, 44, pp.94–102. doi: 10.1016/j.copbio.2016.11.010.

Noguchi, N., Sasatsu, M. & Kono, M., 1993. Genetic mapping in Bacillus subtilis 168 of the aadK gene which encodes aminoglycoside 6-adenylyltransferase. FEMS Microbiol Lett, 114, pp.47–52. doi: 10.1016/0378-1097(93)90140-w.

Okonechnikov, K., Conesa, A. & García-Alcalde, F., 2016. Qualimap 2: Advanced multi-sample quality control for high-throughput sequencing data. Bioinformatics, 32, pp.292–4. doi:10.1093/bioinformatics/btv566.

Patel, A.K. et al., 2009. Comparative accounts of probiotic characteristics of Bacillus spp. isolated from food wastes. Food Res Int, 42(4), pp.505–510. doi: 10.1016/j.foodres.2009.01.013.

Paukner, S. & Riedl, R., 2017. Pleuromutilins: Potent drugs for resistant bugs-mode of action and resistance. Cold Spring Harb Perspect Med, 7, pp.1–16. doi: 10.1101/cshperspect.a027110.

Pawlowski, A.C. et al., 2018. The evolution of substrate discrimination in macrolide antibiotic resistance enzymes. Nat Commun, 9(1), 112. doi: 10.1038/s41467-017-02680-0.

Penders, J. et al., 2013. The human microbiome as a reservoir of antimicrobial resistance. Front Microbiol, 4, 87. doi: 10.3389/fmicb.2013.00087.

Pinontoan, R. et al., 2021. Fibrinolytic characteristics of Bacillus subtilis G8 isolated from natto. Biosci Microbiota, Food Heal, 40(3), pp.144–149. doi: 10.12938/bmfh.2020-071.

Ramandinianto, S.C., Khairullah, A.R. & Effendi, M.H., 2020. Meca gene and methicillin-resistant Staphylococcus aureus (MRSA) isolated from dairy farms in East Java, Indonesia. Biodiversitas, 21, pp.3562–8. doi: 10.13057/biodiv/d210819.

Rolain, J.M., 2013. Food and human gut as reservoirs of transferable antibiotic resistance encoding genes. Front Microbiol, 4. doi: 10.3389/fmicb.2013.00173.

Tanizawa, Y. et al., 2016. DFAST and DAGA: Web-based integrated genome annotation tools and resources. Biosci Microbiota, Food Heal, 35(4), pp.173–84. doi: 10.12938/bmfh.16-003.

Tanizawa, Y., Fujisawa, T. & Nakamura, Y., 2018. DFAST: A flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics, 34, pp.1037–9. doi: 10.1093/bioinformatics/btx713.

The European Committee on Antimicrobial Susceptibility Testing, 2022, ‘New definitions of S, I and R from 2019 2022’, in EUCAST, viewed 11 April 2022, from https://www.eucast.org/newsiandr/.

Vincent, C. et al., 2010. Food reservoir for Escherichia coli causing urinary tract infections. Emerg Infect Dis, 16(1), pp.88–95. doi: 10.3201/eid1601.091118.

Wright, G.D., 2007. The antibiotic resistome: The nexus of chemical and genetic diversity. Nat Rev Microbiol, 5, pp.175–86. doi: 10.1038/nrmicro1614.

Yassin, N.A. & Ahmad, A.M., 2012. Incidence and Resistotyping Profiles of Bacillus subtilis Isolated from Azadi Teaching Hospital in Duhok City, Iraq. Mater Socio Medica, 24, 194. doi: 10.5455/msm.2012.24.194-197.

Yoshida, K.I.l et al., 2004. Bacillus subtilis LmrA is a repressor of the lmrAB and yxaGH operons: Identification of its binding site and functional analysis of lmrB and yxaGH. J Bacteriol, 186(17), pp.5640–5648. doi: 10.1128/JB.186.17.5640-5648.2004.



DOI: https://doi.org/10.22146/jtbb.87079

Article Metrics

Abstract views : 645 | views : 437

Refbacks

  • There are currently no refbacks.


Copyright (c) 2024 Journal of Tropical Biodiversity and Biotechnology

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Editoral address:

Faculty of Biology, UGM

Jl. Teknika Selatan, Sekip Utara, Yogyakarta, 55281, Indonesia

ISSN: 2540-9581 (online)