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



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.


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.

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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.


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).


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)


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.


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 ( and Alberto Gino Lorenzoni ( Experimental protocols can be requested from Vinicius Buiatte.



American Type Culture Collection


Bacillus subtilis


Bacillus coagulans


Bacillus licheniformis


Bacillus pumilus


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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.

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Correspondence to Alberto Gino Lorenzoni.

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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).

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  • Probiotics
  • Poultry
  • Competitive exclusion
  • Co-culture