Skip to main content

Deconstruction of a multi-strain Bacillus-based probiotic used for poultry: an in vitro assessment of its individual components against C. perfringens

Abstract

Objective

Probiotics have been used in poultry production to improve the performance and health of chickens raised without antibiotics. The combination of different probiotic strains has been used with the hope of conferring multiple benefits to the host. However, the inclusion of several strains does not necessarily boost benefits. There is a lack of studies that compare the efficacy of multi-strain probiotics to their individual components. In this study, the effects of a Bacillus-based probiotic product mix containing B. coagulans, B. licheniformis, B. pumilus, and B. subtilis against Clostridium perfringens were tested in vitro using a co-culture method. The individual strains and different combinations of the strains used in the product were also tested against C. perfringens.

Results

The probiotic product mix tested in this study did not show effects against C. perfringens (P = 0.499). When tested individually, the strain of B. subtilis was the most efficient strain to decrease C. perfringens concentrations (P ≤ 0.01), and the addition of other Bacillus species strains significantly decreased its efficacy against C. perfringens. We concluded that the probiotic mix of Bacillus strains used in this study (B. coagulans, B. licheniformis, B. pumilus and B subtilis) was not effective in decreasing C. perfringens concentrations in vitro. However, when deconstructing the probiotic, the strain of B. subtilis alone or combined with the strain of B. licheniformis were effective against C. perfringens. This suggests that the anticlostridial properties of the particular strains of Bacillus used in this study were negatively affected when combined with other Bacillus spp. strains.

Peer Review reports

Introduction

Probiotics are microorganisms that impart benefits to the host. Probiotics as single species or as a combination of multiple species can be used as feed additives to improve health and performance in poultry [1, 2]. The mode of action of probiotics is not entirely understood [3]. Studies have shown that probiotics used in poultry could prevent the growth of pathogenic bacteria, improve the gastrointestinal structure, and modulate the immune system [4]. Probiotics can be used as an alternative to antibiotics for the control of necrotic enteritis, a disease caused by toxin-producing C. perfringens that decreases growth performance in poultry, leading to economic losses [5].

Species of Bacillus, such as, B. subtilis, B. licheniformis, B. coagulans, B. clausii, B. pumilus, and B. cereus, have been considered good probiotic candidates because they are spore-forming and can survive in high temperatures and at low pH. These features increase the survivability of beneficial bacteria during feed processing and storage, and during their passage through the gastrointestinal tract [6].

Bacillus spp. probiotics have shown antagonistic effects against selected bacteria in vitro and are effective alternatives to antibiotics for the control of necrotic enteritis in vivo [7, 8]. In particular, B. subtilis is probably the best-characterized species for the control of C. perfringens and has been shown to be an effective probiotic in in vitro and in vivo experiments [9, 10]. B. subtilis has been used individually or combined with other species, however, it is not clear whether the inclusion of other Bacillus species results in enhanced probiotic efficiency against pathogens. Studies that compare the efficacy of a particular probiotic strain used as single-strain versus a multi-strain product are often missing [11].

The objectives of this study were to evaluate the inhibitory effect of a proprietary probiotic mix containing four strains of Bacillus spp. against C. perfringens in vitro, and to test all its individual strains and some of their possible combinations against C. perfringens in vitro.

Materials and methods

Experimental design

A proprietary probiotic product developed to improve poultry performance was tested for its ability to inhibit Clostridium perfringens in vitro following a modified method previously published [12]. The anti-clostridial effects of Bacillus subtilis (BS), Bacillus licheniformis (BL), Bacillus coagulans (BC) and Bacillus pumilus (BP) were assessed individually or in the combinations shown in Table 1, using a co-culture method.

A total of three experiments were performed in triplicates. For each experiment, a control treatment, consisting of a pure culture of C. perfringens was included to compare bacterial counts between the probiotic treatment and the control.

Table 1 Experiments performed with different combinations of Bacillus spp. against Clostridium perfringens ATCC 13,124™ using an in vitro co-culture method

Preparation of C. perfringens inoculum

A primary culture of C. perfringens ATCC 13,124 ™ was started by inoculating a fresh colony into 10 mL of Tryptic Soy Broth (TSB, Neogen®, United States) followed by incubation for 24 h at 37 °C under anaerobic conditions. Anaerobiosis was achieved by adding a pouch of the AnaeroPack® System (Mitsubishi Gas Chemical America®, United States) into a hermetic chamber. The cultures of C. perfringens were standardized to reach a concentration of 1 × 108 CFU/mL.

An inoculum of C. perfringens was created by diluting the primary culture one thousand-fold (1:1,000) to reach a target concentration of 1 × 105 CFU/mL. Tenfold serial dilutions were completed and plated onto Shahidi-Ferguson Perfringens (SFP) agar (Millipore®, Germany) for the determination of bacterial counts.

Preparation of Bacillus spp. inoculum

The product mix and the isolates of Bacillus spp. strains were provided by the manufacturer in a lyophilized form. The probiotics were suspended in sterile saline for their use in the co-culture model. Bacterial concentrations were determined by performing tenfold dilutions, plating onto Anaerobic Blood Agar (Remel™, United States), and incubating at 37 °C for 24 h under anaerobic conditions.

Co-cultures

The co-cultures were created by adding 1 mL of the C. perfringens inoculum and 5 µL each Bacillus spp. inoculum into 3 mL of TSB. For the commercial product, 20 µL of the inoculum were used. The final target concentration of Bacillus in the co-culture was 2.5 × 105 CFU/mL of each Bacillus strain to resemble the in-feed concentration of the probiotic recommended by the manufacturer. The final concentration of C. perfringens in the co-culture was 2.5 × 104 CFU/mL to represent the normal range of C. perfringens in the gastrointestinal tract of healthy birds (102-104 CFU/g of digesta) [13]. Co-cultures were incubated at 37 °C for 24 h under anaerobic conditions. In every experiment, a control treatment was created using the same procedure, with the Bacillus spp. inoculum replaced with sterile saline.

After incubation, ten-fold serial dilutions of the co-cultures and the control treatment were used to determine the concentrations (CFU/mL) of C. perfringens. The dilutions were plated onto SFP agar plates.

Statistical analysis

The one-way ANOVA was used to test the effects of the treatments on the concentration of C. perfringens in Minitab® 20 (Minitab Inc., United States). Treatment was assumed to be a fixed effect. Log base ten transformations were performed for the response variable (C. perfringens concentration) to stabilize the variances. Fisher’s least significant difference (LSD) was used to separate the means, and a difference in the mean of C. perfringens concentrations was claimed when P ≤ 0.05. Results are presented as mean ± standard error (SE).

Results

In Experiment 1, the commercial probiotic product containing B. coagulans, B. licheniformis, B. pumilus and B. subtilis did not reduce the concentration of C. perfringens (7.35 log10 CFU/mL ± 0.165) compared to the control (7.07 log10 CFU/mL ± 0.02) (P = 0.499).

In Experiment 2, B. coagulans, B. licheniformis, B. pumilus and B. subtilis produced different effects against C. perfringens (P < 0.01) (Table 2). The most effective species against C. perfringens was B. subtilis, with a 6-Log10 reduction (102) compared to the control (108). Individually, B. coagulans, B. pumilus, and B. licheniformis produced a 1-Log10 reduction (107) compared to the control treatment.

Table 2 The in vitro effect of single strains of Bacillus spp. on the concentration of C. perfringens using a co-culture method (Experiment 2)

In Experiment 3, a significant difference was observed in the treatments containing Bacillus subtilis (P < 0.01) (Table 3). The treatments B. subtilis + C. perfringens (BS + CP) and B. subtilis + B. licheniformis + C. perfringens (BS + BL + CP) were the most effective against C. perfringens with a 5-Log10 reduction (102) and a 3-Log10 reduction (104), respectively, compared to the control (107).

Table 3 The in vitro effect of different combinations of Bacillus subtilis with other Bacillus spp. on the concentration of C. perfringens using a co-culture method (Experiment 3)

Discussion

Under the conditions used in this study, the tested multi-strain probiotic product containing BC, BS, BL and BP did not have significant anticlostridial effects in vitro. It is pertinent to underscore that this product was not designed to suppress the growth of C. perfringens. In this context, it is not surprising that the product did not offer anticlostridial activity. However, it is interesting that, some of the strains utilized in this probiotic mix were indeed effective against C. perfringens when individually tested. Notably, the strain of B. subtilis contained in the tested probiotic mix was the most effective species in decreasing C. perfringens concentrations in vitro. This result is in accordance with previous studies that tested the efficacy of B. subtilis strains against C. perfringens in vitro [8]. However, the efficacy of B. subtilis against C. perfringens was significantly reduced when B. subtilis was combined with the other Bacillus species, possibly because of antagonistic interactions between these strains.

Bacteriocins are peptides produced by bacteria that can inactivate other bacterial species, including species within the same genus [14]. Bacillus spp. are known to produce bacteriocins, such as mersacidin and sublancin [15], that are part of the lantibiotic family of peptides. This family of peptides can form pores in the bacterial cell membrane and inhibit cell wall formation, leading to cell death [16,17,18]. Although mechanisms of action were not investigated in the present study, we speculate that the addition of other Bacillus species strains in a co-culture with the B. subtilis strain may have suppressed the latter. Moreover, bacteria can modulate their development through quorum sensing systems when the availability of nutrients is scarce and bacterial populations are growing [19, 20]. It is also possible that competition for nutrients may have developed and led to a decreased concentration of B. subtilis in the co-culture or changed the metabolism of B. subtilis, leading to a reduction in the production of antibacterial factors.

When tested individually, the strains of B. coagulans, B. pumilus and B. licheniformis contained in the tested probiotic mix were not as effective as the B. subtilis strain in reducing C. perfringens concentrations. These findings do not imply that other strains of these Bacillus species may not be effective against C. perfringens, as some have been reported effective reducing the numbers of C. perfringens [21]. The lack of efficacy of the tested strains of B. coagulans, B. licheniformis and B. pumilus, individually or in combination, against C. perfringens in vitro suggests that they may not be ideal candidates for the control of C. perfringens.

Probiotic mixtures have been reported to be more effective than individual probiotics against gastrointestinal disorders in vitro and in vivo [22, 23]. However, meaningful comparisons between multi-strain and single-strain probiotics are scarce. Studies often compare multi-strain probiotics to single strains products that do not share the same bacterial species. In addition, comparisons are sometimes made with products containing different concentrations of bacteria, which generate results that can be difficult to interpret [24, 25]. A standardized method for comparing the efficacy of single versus multiple-strain probiotics is currently needed.

Compatibility between probiotic strains should be considered when designing multi-strain products for the control of C. perfringens. It is important to consider that different strains of probiotics may inhabit different segments of the intestinal tract and that the microbial interactions that limited the anticlostridial performance of B. subtilis in vitro may not occur in vivo. In addition, it is likely that interactions with the gut microbiome could also modify the behavior of these probiotics in vivo. Therefore, in vivo experimentation is necessary to confirm our observations.

In conclusion, the multi-strain probiotic product tested in this study did not decrease C. perfringens concentration in vitro using a co-culture method. Deconstruction of the probiotic blend showed that some of the individual probiotic strains used in this product were effective against C. perfringens. Among the tested species, B. subtilis was the most efficacious strain against C. perfringens. Combining other strains with B. subtilis, significantly decreased its anticlostridial efficacy.

Limitations

The Bacillus strains reported in our experiment presented satisfactory growth under anaerobic conditions, however, the optimal growth of Bacillus is under aerobic conditions. This may affect interactions between bacterial species. Additionally, we tested the efficacy of the probiotics against Clostridium perfringens (ATCC 13,124 ™) to increase the reproducibility of the experiments. Therefore, different observations may be seen when testing different strains of C. perfringens, as well as different strains of Bacillus.

Data availability

Raw data and statistical analyses can be requested from Vinicius Buiatte (vub172@psu.edu) and Alberto Gino Lorenzoni (agl20@psu.edu). Experimental protocols can be requested from Vinicius Buiatte.

Abbreviations

ATCC:

American Type Culture Collection

BS:

Bacillus subtilis

BC:

Bacillus coagulans

BL:

Bacillus licheniformis

BP:

Bacillus pumilus

References

  1. Ramlucken U, Lalloo R, Roets Y, Moonsamy G, van Rensburg CJ, Thantsha MS. Advantages of Bacillus-based probiotics in poultry production. Livest Sci. 2020;241(January):104215.

    Article  Google Scholar 

  2. Food and Agriculture Organization of the United Nations. Probiotics in food. Rome, Italy: FAO/WHO; 2006.

    Google Scholar 

  3. Williams NT, Probiotics. Am J Health-System Pharm. 2010;67(6):449–58.

    Article  CAS  Google Scholar 

  4. Jha R, Das R, Oak S, Mishra P. Probiotics (Direct-Fed Microbials) in poultry nutrition and their effects on nutrient utilization, growth and laying performance, and gut health: a systematic review. Animals. 2020;10:1–18.

    Article  Google Scholar 

  5. Adhikari P, Kiess A, Adhikari R, Jha R. An approach to alternative strategies to control avian coccidiosis and necrotic enteritis. Journal of Applied Poultry Research [Internet]. 2020;29(2):515–34. Available from: https://doi.org/10.1016/j.japr.2019.11.005.

  6. Cutting SM. Bacillus probiotics. Food Microbiol. 2011;28(2):214–20.

    Article  PubMed  Google Scholar 

  7. Fernández SM, Cretenet M, Bernardeau M. In vitro inhibition of avian pathogenic Enterococcus cecorum isolates by probiotic Bacillus strains. Poult Sci. 2019;98(6):2338–46.

    Article  Google Scholar 

  8. Caly DL, D’Inca R, Auclair E, Drider D. Alternatives to antibiotics to prevent necrotic enteritis in broiler chickens: a microbiologist’s perspective. Front Microbiol. 2015;6(12):1–12.

    Google Scholar 

  9. Teo AYL, Tan HM. Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis isolated from the gastrointestinal tracts of healthy chickens. Appl Environ Microbiol. 2005;71(8):4185–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Jayaraman S, Thangavel G, Kurian H, Mani R, Mukkalil R, Chirakkal H. Bacillus subtilis PB6 improves intestinal health of broiler chickens challenged with Clostridium perfringens-induced necrotic enteritis. Poult Sci. 2013;92(2):370–4.

    Article  CAS  PubMed  Google Scholar 

  11. McFarland LV. Efficacy of single-strain Probiotics Versus Multi-Strain Mixtures: systematic review of strain and Disease specificity. Digestive Diseases and Sciences. Volume 66. Springer; 2021. pp. 694–704.

  12. Šimunović K, Stefanic P, Klančnik A, Erega A, Mandic Mulec I, Smole Možina S. Bacillus subtilis PS-216 antagonistic activities against Campylobacter jejuni NCTC 11168 are modulated by temperature, Oxygen, and Growth Medium. Microorganisms. 2022;10(2):1–.

    Article  Google Scholar 

  13. Emami NK, Dalloul RA, Centennial, Review. Recent developments in host-pathogen interactions during necrotic enteritis in poultry. Poultry Science. Volume 100. Elsevier Inc.; 2021.

  14. Ouwehand AC, Invernici MM, Furlaneto FAC, Messora MR. Effectiveness of Multistrain Versus single-strain Probiotics Current Status and Recommendations for the future. J Clin Gastroenterol. 2018;52(December):35–40.

    Article  Google Scholar 

  15. Simons A, Alhanout K, Duval RE. Bacteriocins, antimicrobial peptides from bacterial origin: overview of their biology and their impact against multidrug-resistant bacteria. Microorganisms. 2020;8(5).

  16. Moll GN, Konings WN, Driessen AJM. Bacteriocins: mechanism of membrane insertion and pore formation. Antonie van Leeuwenhoek. Int J Gen Mol Microbiol. 1999;76(1–4):185–98.

    CAS  Google Scholar 

  17. Hassan M, Kjos M, Nes IF, Diep DB, Lotfipour F. Natural antimicrobial peptides from bacteria: characteristics and potential applications to fight against antibiotic resistance. J Appl Microbiol. 2012;113(4):723–36.

    Article  CAS  PubMed  Google Scholar 

  18. Hasper HE, Kramer NE, Smith JL, Hillman JD, Zachariah C, Kuipers OP et al. An alternative bactericidal mechanism of action for lantibitioc peptides that target lipid II. Science (1979). 2006 Sep 15;313:1636–7.

  19. Arnaouteli S, Bamford NC, Stanley-Wall NR, Kovács ÁT. Bacillus subtilis biofilm formation and social interactions. Nat Rev Microbiol. 2021;19(9):600–14.

    Article  CAS  PubMed  Google Scholar 

  20. Shank EA, Klepac-Ceraj V, Collado-Torres L, Powers GE, Losick R, Kolter R. Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus. Proc Natl Acad Sci U S A. 2011;108(48):1236–43.

    Article  Google Scholar 

  21. Horng YB, Yu YH, Dybus A, Hsiao FSH, Cheng YH. Antibacterial activity of Bacillus species-derived surfactin on Brachyspira hyodysenteriae and Clostridium perfringens. AMB Express [Internet]. 2019;9(1):1–9. Available from: https://doi.org/10.1186/s13568-019-0914-2.

  22. Fijan S, Šulc D, Steyer A. Study of the in vitro antagonistic activity of various single-strain and multi-strain probiotics against Escherichia coli. Int J Environ Res Public Health. 2018;15(7).

  23. Sandvang D, Skjoet-Rasmussen L, Cantor MD, Mathis GF, Lumpkins BS, Blanch A. Effects of feed supplementation with 3 different probiotic Bacillus strains and their combination on the performance of broiler chickens challenged with Clostridium perfringens. Poult Sci. 2021;100(4):100982.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Chapman CMC, Gibson GR, Rowland I. Health benefits of probiotics: are mixtures more effective than single strains? Eur J Nutr. 2011;50(1):1–17.

    Article  CAS  PubMed  Google Scholar 

  25. Chapman CMC, Gibson GR, Rowland I. In vitro evaluation of single- and multi-strain probiotics: inter-species inhibition between probiotic strains, and inhibition of pathogens. Anaerobe. 2012;18(4):405–13.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This research received no external funding.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization, VB and AGL; Data curation: VB; Formal analysis: VB and MS; Investigation: VB and AGL; Methodology: VB; Project administration: VB and MS; Supervision: AGL; Writing – original draft: VB and MS; Writing – review & editing: VB, MS and AGL.

Corresponding author

Correspondence to Alberto Gino Lorenzoni.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

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

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Buiatte, V., Schultheis, M. & Lorenzoni, A.G. Deconstruction of a multi-strain Bacillus-based probiotic used for poultry: an in vitro assessment of its individual components against C. perfringens. BMC Res Notes 16, 117 (2023). https://doi.org/10.1186/s13104-023-06384-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13104-023-06384-1

Keywords

  • Probiotics
  • Poultry
  • Competitive exclusion
  • Co-culture