Skip to main content
SpringerLink
Log in

Two Strains of Endophytic Bacillus velezensis Carrying Antibiotic-Biosynthetic Genes Show Antibacterial and Antibiofilm Activities Against Methicillin-Resistant Staphylococcus aureus (MRSA)

  • ORIGINAL RESEARCH ARTICLE
  • Published:
Indian Journal of Microbiology Aims and scope Submit manuscript

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is considered a priority pathogen causing high mortality that requires effective control measures. This study aimed to detect the presence of antibiotic-biosynthetic genes and to evaluate the anti-MRSA activity of two strains of endophytic Bacillus velezensis isolated from Archidendron pauciflorum. PCR-based screening showed that B. velezensis strains, such as DJ4 and DJ9 possessed six antibiotic-biosynthetic genes, namely MlnA, DhbE, BacD, DfnD, SrfA, and BaeR. According to the preliminary test conducted using disc-diffusion assay, metabolite extracts from these strains have anti-MRSA activity with clear zone diameters of 13.00 ± 0.82 mm, and 17.33 ± 0.47 mm, respectively. Extract from DJ9 strain was more active to MRSA, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 62.50 µg/mL and 250 µg/mL, respectively. Furthermore, a bactericidal effect was observed, as evidenced by MBC/MIC ratio of four. Both DJ9 and DJ4 extracts showed a dose-dependent inhibitory effect on MRSA biofilm formation. Furthermore, a maximum inhibition percentage of 60.12 ± 2.5% was shown by DJ9 extract in two-fold MIC. The corresponding extract disrupted MRSA mature biofilms most effectively at 55.74 ± 1.4%. In conclusion, crude extract, particularly the DJ9 strain had significant potential in inhibiting MRSA cell growth, MRSA biofilm formation, and disrupting MRSA mature biofilm in vitro.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

MRSA:

Methicillin-resistant Staphylococcus aureus

MIC:

Minimum inhibitory concentration (MIC)

MBC:

Minimum bactericidal concentration

NRPS:

Non-ribosomal peptide synthetase

PKS:

Polyketide synthase

BaeR :

Gene for bacillaene synthesis

SrfA :

Gene for surfactin synthesis

DfnD :

Gene for difficidin synthesis

BacD :

Gene for bacilysin synthesis

DhbE :

Gene for bacillibactin synthesis

MlnA :

Gene for macrolactin synthesis

ItuA :

Gene for iturin A synthesis

FenA :

Gene for fengycin synthesis

References

  1. Ellabaan MMH, Munck C, Porse A et al (2021) Forecasting the dissemination of antibiotic resistance genes across bacterial genomes. Nat Comm 12:1–10. https://doi.org/10.1038/s41467-021-22757-1

    Article  CAS  Google Scholar 

  2. World Health Organization (WHO) (2021) Record response to WHO’s call for antimicrobial resistance surveillance reports in 2020. https://www.who.int/news/item/09-06-2021-record-response-to-who-s-call-for-antimicrobial-resistance-surveillance-reports-in-2020. Accessed 23 September 2023.

  3. World Health Organization (WHO) (2017) Prioritization of pathogens to guide discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis. https://www.who.int/publications/i/item/WHO-EMP-IAU-2017.12. Accessed 23 September 2023.

  4. World Health Organization (WHO) (2021) Antimicrobial resistance. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance. Accessed 23 September 2023.

  5. Okwu MU, Olley M, Akpoka AO et al (2019) Methicillin-resistant Staphylococcus aureus (MRSA) and anti-MRSA activities of extracts of some medicinal plants: a brief review. AIMS Microbiol 5(2):117–137. https://doi.org/10.3934/microbiol.2019.2.117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Singh M, Kumar A, Singh R et al (2017) Endophytic bacteria: a new source of bioactive compounds. 3 Biotech 7:1–14. https://doi.org/10.1007/s13205-017-0942-z

    Article  Google Scholar 

  7. Martinez-Klimova E, Rodríguez-Peña K, Sánchez S (2017) Endophytes as sources of antibiotics. Biochem Pharmacol 134:1–17. https://doi.org/10.1016/j.bcp.2016.10.010

    Article  CAS  PubMed  Google Scholar 

  8. Lopes R, Tsui S, Gonçalves PJRO et al (2018) A look into a multifunctional toolbox: endophytic Bacillus species provide broad and underexploited benefits for plants. World J Microbiol Biotechnol 34:1–10. https://doi.org/10.1007/s11274-018-2479-7

    Article  Google Scholar 

  9. Caulier S, Nannan C, Gillis A et al (2019) Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group. Fronti Microbiol 10:1–19. https://doi.org/10.3389/fmicb.2019.00302

    Article  Google Scholar 

  10. Rasiya KT, Sebastian D (2021) Iturin and surfactin from the endophyte Bacillus amyloliquefaciens strain RKEA3 exhibits antagonism against Staphylococcus aureus. Biocat Agricult Biotechnol 36:1–11. https://doi.org/10.1016/j.bcab.2021.102125

    Article  CAS  Google Scholar 

  11. Numan M, Shah M, Asaf S et al (2022) Bioactive compounds from endophytic bacteria Bacillus subtilis strain EP1 with their antibacterial activities. Metabolites 12:1–10. https://doi.org/10.3390/metabo12121228

    Article  CAS  Google Scholar 

  12. Li FZ, Zeng YJ, Zong MH et al (2020) Bioprospecting of a novel endophytic Bacillus velezensis FZ06 from leaves of Camellia assamica: production of three groups of lipopeptides and the inhibition against food spoilage microorganisms. J Biotechnol 323:42–53. https://doi.org/10.1016/j.jbiotec.2020.07

    Article  CAS  PubMed  Google Scholar 

  13. Gokulan K, Khare S, Cerniglia C (2014) Production of secondary metabolites of bacteria. Encycl Food Microbiol 2:561–569. https://doi.org/10.1016/B978-0-12-384730-0.00203-2

    Article  Google Scholar 

  14. Hemmerling F, Piel J (2022) Strategies to access biosynthetic novelty in bacterial genomes for drug discovery. Nat Rev Drugs Discov 21:359–378. https://doi.org/10.1038/s41573-022-00414-6

    Article  CAS  Google Scholar 

  15. Kumar A, Jaiswal V, Kumar V et al (2018) Functional redundancy in Echinocandin B in-cluster transcription factor ecdB of Emericella rugulosa NRRL 11440. Biotechnol Rep 19:1–8. https://doi.org/10.1016/j.btre.2018.e00264

    Article  Google Scholar 

  16. Priyanto JA, Prastya ME, Astuti RI et al (2023) The Antibacterial and antibiofilm activities of the endophytic bacteria associated with Archidendron pauciflorum against multidrug-resistant strains. Appl Biochem Biotechnol 195:6653–6674. https://doi.org/10.1007/s12010-023-04382-4

    Article  CAS  PubMed  Google Scholar 

  17. Hazarika DJ, Goswani G, Gautom T et al (2019) Lipopeptide mediated biocontrol activity of endophytic Bacillus subtilis against fungal phytopathogens. BMC Microbiol 19(71):1–13. https://doi.org/10.1186/s12866-019-1440-8

    Article  Google Scholar 

  18. Mamarasulov B, Davranov K, Umruzaqov A et al (2023) Evaluation of the antimicrobial and antifungal activity of endophytic bacterial crude extracts from medicinal plant Ajuga turkestanica (Rgl.) Brig (Lamiaceae). J King Saud Univ-Sci 35:1–11. https://doi.org/10.1016/j.jksus.2023.102644

    Article  Google Scholar 

  19. NCCLS (National Committee for Clinical Laboratory Standard) (2020) Performance standard for antimicrobial susceptibility testing. Ninth informational supplement. 30th edition, NCCLS, Malvern, 40(1): 1–294.

  20. Wintachai P, Paosen S, Yupanqui CT et al (2019) Silver nanoparticles synthesized with Eucalyptus critriodora ethanol leaf extract stimulate antibacterial activity against clinically multidrug-resistant Acinetobacter baumannii isolated from pneumonia patients. Microbial Pathogen 126:245–257. https://doi.org/10.1016/j.micpath.2018.11.018

    Article  CAS  Google Scholar 

  21. Tran C, Cock IE, Chen X et al (2022) Antimicrobial Bacillus: metabolites and their mode of action. Antibiotics 11(1):1–25. https://doi.org/10.3390/antibiotics11010088

    Article  CAS  Google Scholar 

  22. Rabbee MF, Baek KH (2020) Antimicrobial activities of lipopeptides and polyketides of Bacillus velezensis for agricultural applications. Molecules 25(21):1–16. https://doi.org/10.3390/molecules25214973

    Article  CAS  Google Scholar 

  23. Tabbene O, Kalai L, Slimene IB et al (2011) Anti-Candida e¡ect of bacillomycin D-like lipopeptides from Bacillus subtilis B38. FEMS Microbiol Lett 316:108–114. https://doi.org/10.1111/j.1574-6968.2010.02199.x

    Article  CAS  PubMed  Google Scholar 

  24. Romero-Tabarez M, Jansen R, Sylla M et al (2006) 7-O-malonyl macrolactin A, a new macrolactin antibiotic from Bacillus subtilis active against methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and a small-colony variant of Burkholderia cepacia. Antimicrob Agents Chemother 50:1701–1709. https://doi.org/10.1128/AAC.50.5.1701-1709.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chakraborty K, Kizhakkekalam VK, Joy M et al (2022) Bacillibactin class of siderophore antibiotics from a marine symbiotic Bacillus as promising antibacterial agents. Appl Microbiol Biotechnol 106:329–340. https://doi.org/10.1007/s00253-021-11632-0

    Article  CAS  PubMed  Google Scholar 

  26. Islam T, Rabbee MF, Choi J et al (2022) Biosynthesis, molecular regulation, and application of Bacilysin produced by Bacillus Species. Metabolites 12:1–13. https://doi.org/10.3390/metabo12050397

    Article  CAS  Google Scholar 

  27. Wu L, Wu H, Chen L et al (2015) Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens. Sci Rep 5:1–9. https://doi.org/10.1038/srep12975

    Article  CAS  Google Scholar 

  28. Müller S, Strack SN, Hoefler BC et al (2014) Bacillaene and sporulation protect Bacillus subtilis from Predation by Myxococcus xanthus. Environ Microbiol 80:5603–5610. https://doi.org/10.1128/AEM.01621-14

    Article  CAS  Google Scholar 

  29. Baharudin MMA, Ngalimat MS, Mohd Shariff F et al (2021) Antimicrobial activities of Bacillus velezensis strains isolated from stingless bee products against methicillin-resistant Staphylococcus aureus. PLoS ONE 16:1–20. https://doi.org/10.1371/journal.pone.0251514

    Article  CAS  Google Scholar 

  30. Ren Z, Xie L, Okyere SK et al (2022) Antibacterial activity of two metabolites isolated from endophytic bacteria Bacillus velezensis Ea73 in Ageratina Adenophora. Front Microbiol 13:1–14. https://doi.org/10.3389/fmicb.2022.860009

    Article  Google Scholar 

  31. Francis A, Chakraborty K (2021) Marine macroalga-associated heterotroph Bacillus velezensis as prospective therapeutic agent. Archive Microbiol 203:1671–1682. https://doi.org/10.1007/s00203-020-02169-3

    Article  CAS  Google Scholar 

  32. Saeloh D, Visutthi M (2021) Efficacy of thai plant extracts for antibacterial and anti-biofilm activities against pathogenic bacteria. Antibiotics (Basel) 10:1–9. https://doi.org/10.3390/antibiotics10121470

    Article  CAS  Google Scholar 

  33. Mogana R, Adhikari A, Tzar MN et al (2020) Antibacterial activities of the extracts, fractions and isolated compounds from Canarium patentinervium Miq. against bacterial clinical isolates. BMC Complement Med Ther 20:1–11. https://doi.org/10.1186/s12906-020-2837-5

    Article  Google Scholar 

  34. Wei G, He Y (2022) Antibacterial and antibiofilm activities of novel cyclic peptides against methicillin-resistant Staphylococcus aureus. Int J Mol Sci 23:1–16. https://doi.org/10.3390/ijms23148029

    Article  CAS  Google Scholar 

  35. Kamer AMA, Abdelaziz AA, Al-Monofy KB et al (2023) Antibacterial, antibiofilm, and anti-quorum sensing activities of pyocyanin against methicillin-resistant Staphylococcus aureus: in vitro and in vivo study. BMC Microbiol 23:1–19. https://doi.org/10.1186/s12866-023-02861-6

    Article  CAS  Google Scholar 

  36. Silva V, Almeida L, Gaio V et al (2021) Biofilm formation of multidrug-resistant MRSA strains isolated from different types of human infections. Pathogens 10(8):1–18. https://doi.org/10.3390/pathogens10080970

    Article  CAS  Google Scholar 

  37. Boudet A, Sorlin P, Pouget C et al (2021) Biofilm formation in methicillin-resistant Staphylococcus aureus isolated in cystic fibrosis patients is strain-dependent and differentially influenced by antibiotics. Front Microbiol 12:1–15. https://doi.org/10.3389/fmicb.2021.750489

    Article  Google Scholar 

  38. Hawas S, Verderosa AD, Totsika M (2022) Combination therapies for biofilm inhibition and eradication: a comparative review of laboratory and preclinical studies. Front Cell Infect Microbiol 12:1–19. https://doi.org/10.3389/fcimb.2022.850030

    Article  CAS  Google Scholar 

  39. Kumari N, Signh S, Kumari V et al (2019) Ouabain potentiates the antimicrobial activity of aminoglycosides against Staphylococcus aureus. BMC Complement Altern Med 19:1–12. https://doi.org/10.1186/s12906-019-2532-6

    Article  CAS  Google Scholar 

  40. Liu J, Li W, Zhu X et al (2019) Surfactin effectively inhibits Staphylococcus aureus adhesion and biofilm formation on surfaces. Appl Microbiol Biotechnol 103:4565–4574. https://doi.org/10.1007/s00253-019-09808-w

    Article  CAS  PubMed  Google Scholar 

  41. Erega A, Stefanic P, Dogsa I et al (2021) Bacillaene mediates the inhibitory effect of Bacillus subtilis on Campylobacter jejuni biofilms. Appl Environ Microbiol 87(12):1–14. https://doi.org/10.1128/AEM.02955-20

    Article  Google Scholar 

Download references

Acknowledgements

The authors are grateful to the Directorate of Research and Innovation, IPB University, for supporting this study through “The National Collaborative Research Program 2023–2024” that received by Jepri Agung Priyanto, and the National Research and Innovation Agency (BRIN).

Funding

Grants were provided by Directorate of Research and Innovation, IPB University, through The National Collaborative Research Program (Grant Number: 505/IT3.D10/PT.01.03/P/B/2023) and awarded to Jepri Agung Priyanto. This work was also partly supported by the Research Health Organization of National Research and Innovation Agency (BRIN), Indonesia through “Rumah Program Purwarupa Bahan Baku Obat Terapi Terarah 2024” (Grant Number: B-11155/III.9/TK.02.02/12/2023).

Author information

Authors and Affiliations

  1. Division of Microbiology, Department of Biology, Faculty of Mathematics and Natural Sciences, IPB University, Agatis Street, IPB Dramaga Campus, Bogor, West Java, 16680, Indonesia

    Jepri Agung Priyanto & Egiyanti Nur Widhia Hening

  2. Research Center for Pharmaceutical Ingredients and Traditional Medicine, National Research and Innovation Agency (BRIN), Kawasan Sains Dan Teknologi (KST) B.J Habibie (PUSPIPTEK), Serpong, South Tangerang, Banten, Indonesia

    Muhammad Eka Prastya

  3. Department of Biology, Faculty of Sciences, Sumatera Institute of Technology, Lampung Selatan, Lampung, Indonesia

    Erma Suryanti

  4. Indonesian Marine Education and Research Organisation (MERO) Foundation, Br. Dinas Muntig, Bali, Indonesia

    Rhesi Kristiana

Authors
  1. Jepri Agung Priyanto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Muhammad Eka Prastya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Egiyanti Nur Widhia Hening
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Erma Suryanti
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rhesi Kristiana
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JAP and MEP contributed to designing the research framework. JAP and MEP conducted the antibacterial test and determination of MIC and MBC, as well as molecular genetics analysis. ENWH contributed to the bacterial genome extraction and PCR. ES and RK engaged in the anti-biofilm analysis. JAP, MEP, and ES performed the data analysis, verification, and interpretation. The primary draft of this article was written by JAP, and other authors revised the first version and approved the final version.

Corresponding author

Correspondence to Jepri Agung Priyanto.

Ethics declarations

Conflict of Interests

All authors stated no conflict of interest.

Data Availability

All experimental data collected were provided in this paper.

Ethics Approval

This is an observational investigation and does not comprise human participants or animals. Therefore, no ethical approval was required.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 19 kb)

Supplementary file1 (PDF 178 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Priyanto, J.A., Prastya, M.E., Hening, E.N.W. et al. Two Strains of Endophytic Bacillus velezensis Carrying Antibiotic-Biosynthetic Genes Show Antibacterial and Antibiofilm Activities Against Methicillin-Resistant Staphylococcus aureus (MRSA). Indian J Microbiol (2024). https://doi.org/10.1007/s12088-024-01262-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12088-024-01262-1

Keywords

Search

Navigation