The gut microbiota is crucial for maintaining host health by supplying essential nutrients, regulating energy balance, modulating immune responses and protecting against pathogens. Consequently, restoring and maintaining its stability with beneficial bacteria – rather than antibiotics – has been proposed as a strategy to prevent adverse effects on gut health [1].

A census of the phylogenetic diversity of bacteria identified in the intestinal tract of healthy poultry conducted over the past decade shows that up to 60% of the genera present in the gut microbiome contain spore-forming bacteria, and these genera account for 30% of the total gut microbiota [2][3].

In microbiome research, spore-forming bacteria are considered a distinct functional group within the broader microbiota. The term “sporobiota” has been proposed to describe the total collection of these bacteria in a given microbial community [4].

Endospores are formed by members of Firmicutes, a large, diverse and morphologically complex bacterial phylum [5]. Within it, species of the genus Bacillus (family Bacillaceae) have attracted the most scientific attention due to their significant role in modulating the intestinal microbiota [6].

While spore-forming bacteria of the genus Bacillus are primarily associated with the soil microbiome [7], their presence in the intestine is attributed to the consumption of feed and water. It has been conventionally assumed that Bacillus species enter the intestine exclusively in their spore form [8]. However, accumulating evidence suggests that some Bacillus species exhibit a bimodal life cycle, capable of both growth and sporulation not only in the environment but also within the gastrointestinal tract [9][10].

Spore-forming bacteria of the genus Bacillus are widely used in animal feed to promote animal growth and inhibit pathogens [11][12]. The probiotic mechanisms of these microorganisms involve synthesizing antimicrobial compounds and lactic acid [13], enhancing non-specific immunity [14], and producing enzymes such as amylase, lipase, protease, pectinase and cellulose [15]. Specifically, Bacillus subtilis stimulates growth of beneficial gut microbiota, increases intestinal microbiome diversity [16], modulates regulatory systems for intestinal epithelium renewal and immune cell activity [17], and mitigates the adverse effects of various factors, particularly antibiotic therapy [18][19].

It has also been shown that B. subtilis plays an important role in stimulating the development of gut-associated lymphoid tissue and that sporulation of live bacilli is considered critical for this process [20][21].

Survival and colonization within the digestive tract are essential for the microbiota to exert its physiological functions [22]. For spore-forming bacteria, a key prerequisite for intestinal colonization is the adhesion of spores to the intestinal mucosa and subsequent biofilm formation, a process that enables the transition of planktonic cells to attached forms [21][23].

The term “biofilm” is used to describe a structured community of bacterial cells embedded in a self-produced polymeric extracellular matrix and adhered to an inert or living surface [24]. This polymeric extracellular matrix is a complex system composed of exopolysaccharides, the TasA protein, lipids, nucleic acids and various heteropolymers, all of which are secreted by microorganisms into the extracellular environment [25]. Studies have shown that the TasA protein and exopolysaccharides are key components of the Bacillus biofilm matrix. TasA is an amyloid protein that has been implicated in the formation of the biofilm matrix by various microorganisms, including spore-forming bacteria [26][27][28][29].

Bacterial biofilms can protect bacteria from environmental stressors, host immune responses and antimicrobial agents, including antibiotics [30].

The use of Congo red dye to assess bacterial amyloid synthesis, and thus identify mucus or biofilm-forming organisms like Staphylococcus sp., was first proposed by D. J. Freeman et al. [31]. This method distinguishes biofilm-forming Staphylococcus colonies from non-forming ones based on their morphology and color on Congo red agar. Subsequent studies confirmed the utility of this assay for detecting potential biofilm formation in a broad spectrum of gram-positive and gram-negative microorganisms, including spore-forming bacteria of the genus Bacillus [32][33][34].

Recent data indicates that Bacillus sporulation is more active within biofilms than in planktonic cultures [35].

The growing threat of antibiotic-resistant pathobiont spread has brought to the forefront research focused on assessing the antibiotic resistance of the commensal gut microbiota as potential donors of resistance determinants.

In light of this problem, it has been suggested that spore-forming bacteria may play a significant role in the dissemination and accumulation of antibiotic resistance genes due to their ability to withstand antibiotic treatment [36][37]. However, the extent of antibiotic resistance and the biofilm-forming capacity among members of the intestinal sporobiota remain largely unknown.

Previous studies on the spread of antibiotic resistance and the biofilm-forming ability of Bacillus bacteria were focused on a limited number of species and did not include isolates derived from poultry gut microbiota [38][39][40][41].

The prevalence of hemolytic activity within the intestinal population of aerobic Bacillus bacteria remains insufficiently studied. Furthermore, the role of their hemolysins in maintaining intestinal mucosal integrity is unclear, and it has not been established that hemolysin production is harmless to the host. Non-hemolytic (γ-hemolytic) strains are generally considered safe for their hosts, while strains exhibiting hemolytic activity are deemed pathogenic [42][43].

Given the modern understanding of the gut microbiota’s role in maintaining gut colonization resistance, this knowledge gap leads to a lack of understanding regarding the true role of aerobic Bacillus spore-formers in the poultry gut microbiome and the specificity required for their selection and application as probiotics [44][45].

The relevance of this work is that the safety assessment of aerobic spore-forming Bacillus bacteria, which are widely used in the production of probiotic feed additives, is associated not only with species identification but also with the individual characteristics of the strains.

The novelty of the obtained data lies in the study of specific biological properties of isolates from the ceca of commercial poultry, thereby evaluating the prospects of using Bacillus bacteria for developing probiotic feed supplements.

The aim of this work is to investigate the biological properties of Bacillus isolates (hemolytic activity, potential for biofilm formation, and antibiotic resistance) obtained from the large intestine of poultry, with the goal of selecting promising strains for use in the biotechnology of probiotic production.

Spore-forming bacteria used in this study were isolated from samples of the cecal content of clinically healthy poultry obtained from farms in the Moscow Oblast that are free from infectious diseases. Birds were euthanized in the animal facility using CO₂ and subjected to necropsy according to a standard protocol. All animal procedures complied with the ethical standards set by European Convention ETS No. 123.

The ceca were isolated by applying ligatures at the junction with the rectum, excised, placed in separate containers and transported to the laboratory under a cold chain (2–8 °C). Within maximum 30 minutes of obtaining the isolated ceca, the contents of each were extruded into sterile disposable dishes and labeled. The resulting aliquots of each chyme sample were diluted 1:100 with buffered peptone water and resuspended by vigorous mixing to obtain a homogeneous suspension.

To isolate spore-forming bacteria, the aliquots were heated in a water bath at 65 °C for 45 minutes [46].

To obtain isolated colonies, the heated aliquots were streaked onto Tryptic Soy Agar (SCD agar; HiMedia Laboratories Pvt Ltd., India). Cultivation was carried out under aerobic conditions at (37 ± 1) °C. The cultures were examined after 18, 24, and 36 hours of incubation.

Colonies exhibiting morphological characteristics of the Bacillus genus were selected and purified by repeated streaking on SCD agar. After 18 and 24 hours of cultivation, following visual assessment of growth and microscopy of gram-stained smears, isolates of spore-forming bacteria were subcultured into Tryptic Soy Broth (TSB; SCD broth; HiMedia Laboratories Pvt Ltd., India), and broth cultures of vegetative cells were used for further work.

Phenotypic characterization. A phenotypic characterization of the spore-forming bacterial isolates was performed based on morphological properties, Gram staining,hemolytic activity, catalase activity, biofilm-forming capacity and antibiotic susceptibility. All tests were performed in duplicate.

Isolate identification. Phenotypic identification of the spore-forming bacterial isolates was conducted using commercial disposable biochemical diagnostic test panels API 50CHB (bioMérieux, France). This test classifies bacterial strains based on their ability to ferment 49 different carbohydrates. Results were analyzed using the APIWEB™ software (bioMérieux, France).

Hemolytic activity test. The hemolytic properties of the spore-forming bacterial isolates were determined using Columbia agar (HiMedia Laboratories Pvt Ltd., India) supplemented with 5% sterile defibrinated sheep blood. Twenty-four-hour cultures of the isolates were stab-inoculated onto the agar surface. The reaction was recorded 24 hours after incubation at (37 ± 1) °C. An isolate was considered α-hemolytic if colonies caused a green or brown discoloration of the surrounding medium, β-hemolytic if true lysis of erythrocytes resulted in a clear, transparent zone surrounding the colonies, and γ-hemolytic (non-hemolytic) if no reaction was observed in the surrounding medium [47].

Catalase test. The catalase activity of bacterial isolates was determined using a 10% hydrogen peroxide solution [48]. The liberation of oxygen, evident by the formation of gas bubbles, indicated catalase production.

Antibiotic susceptibility test. The antibiotic susceptibility of Bacillus sp. isolates was tested using the disk diffusion method [49]. Standard antibiotic-impregnated discs (HiMedia Laboratories Pvt Ltd., India) with the following concentrations (5 to 30 μg/disk) were used: ciprofloxacin (Cip 5), rifampicin (Rif 5), enrofloxacin (Ex5), doxycycline (Do10), gentamicin (Gen10), neomycin (N30), cefazolin (Cz30), norfloxacin (Nx10), benzylpenicillin (P100), pefloxacin (Pf5), kanamycin (K30), lincomycin (L15), azithromycin (AZM15), nalidixic acid (NA30), chloramphenicol (C30), oxytetracycline (O30), imipenem (Ipm10), oleandomycin (Ol15), clindamycin (Cd2), clarithromycin (Clr15), oxacillin (Ox1), ampicillin (Amp25).

Eighteen-hour agar cultures of spore-forming bacteria were adjusted to a 0.5 McFarland standard; 250 μL of the culture was swabbed onto the surface of SCD agar plates, and antibiotic discs were dispensed (8 per plate) using a dispenser (HiMedia Laboratories Pvt Ltd., India). Plates were then incubated at (37 ± 1) °C for 20–24 hours.

Strains were classified as resistant (R) if the inhibition zone diameters for the antibiotics listed in EFSA (European Food Safety Authority) documents exceeded the threshold level for Bacillus strains [50]. For antibiotics lacking reliable interpretive criteria, Bacillus strains exhibiting inhibition zones of less than 12 mm around the antibiotic disc were considered resistant.

Determination of biofilm producers. Screening of spore-forming bacteria as potential biofilm producers was performed using a qualitative method by detecting extracellular amyloid proteins on Brain Heart Infusion Agar (BHI agar; HiMedia Laboratories Pvt Ltd., India) supplemented with Congo red indicator and 5% sucrose [31].

A 24-hour broth culture of the test isolate was stabinoculated onto Congo red BHI agar and incubated for 48 hours at (37 ± 1) °C. The color and morphology of the resulting colonies were assessed visually.

The interaction of Congo red with amyloid proteins of the biofilm produces a compound that gives colonies a dark red or dark brown color with a black base. Weak biofilm producers typically remain pink, though some darkening in the center may be observed. Isolates incapable of forming biofilms produce white or very light pink colonies [51].

During the study, a total of 68 strains of aerobic spore-forming bacteria of the genus Bacillus were isolated and identified to the species level.

Phenotypic data confirmed that the isolates obtained from the contents of the ceca of the large intestine of clinically healthy birds belonged to spore-forming bacteria.

The identified isolates of spore-forming bacteria were represented by 5 species: Bacillus licheniformis, Bacillus subtilis/amyloliquefaciens, Bacillus megaterium, Bacillus cereus and Bacillus mucoides. A number of isolates could not be identified using the standardized API 50CHB system.

Microscopic examination of the isolated cells revealed a diverse collection of rod-shaped bacteria producing endospores of various sizes and shapes.

The colonies of spore-forming bacteria exhibited significant polymorphism. B. licheniformis isolates, after 18–24 hours of growth on SCD agar, formed raised, medium-sized colonies colored white or beige, with a flower-like shape.

When cultured on SCD agar, B. subtilis/amyloliquefaciens isolates grew into large white, medium-sized colonies with a lighter central point, or rough undulating colonies of a white-cream color with a dry, finely wrinkled structure, slightly raised above the agar surface.

Bacillus megaterium isolates on SCD agar formed sharply defined colonies of a dirty white color.

Morphologically, the colonies of B. mucoides and B. cereus isolates were identical and appeared as rough, spreading, rhizoid, greyish-white colonies with undulating edges, or as wrinkled, milky-colored colonies with wavy edges.

The isolated spore-forming bacteria were assessed for their safety based on hemolytic activity (Table 1).

All isolates of spore-forming bacteria obtained from the chyme samples of birds’ ceca lacked α-hemolytic activity. Growth on blood agar revealed a wide zone of hemolysis, characteristic of β-hemolytic bacteria, in 14 out of the 57 studied isolates of the species B. licheniformis, B. subtilis/amyloliquefaciens, B. megaterium, and B. cereus (Fig. 1). It was found that 9 isolates of spore-forming bacteria exhibited γ-hemolytic activity.

The majority of the studied isolates of spore-forming microorganisms grown on Congo red BHI agar were classified as potential biofilm producers based on their dark red colonies with a black base, with the exception of 3 isolates of B. licheniformis, 1 isolate of B. megaterium, and 2 isolates of B. cereus, which formed light pink colonies (Fig. 2).

Antibiotic susceptibility tests showed that the majority of the studied isolates of spore-forming bacteria were sensitive to all 22 antibiotics tested (Table 2).

Only 4 isolates of spore-forming bacteria were resistant to rifampicin, 7 – to cefazolin, 12 – to lincomycin, and 6 – to oxytetracycline. Some isolates were resistant to 2 antibiotics, but the majority of isolates exhibited resistance to maximum one antibiotic.

Based on the test results, the identified species of spore-forming bacteria of the genus Bacillus isolated from the ceca of commercial poultry included: B. licheniformis, B. subtilis/amyloliquefaciens, B. mycoides, B. megaterium and B. cereus; a number of isolates could not be identified.

All studied isolates were catalase-positive and lacked α-hemolytic activity. Some isolates exhibited β-hemolytic activity, which precludes their classification as non-pathogenic.

The potential ability of the isolated strains to form biofilms, which indirectly characterizes their capacity to survive in the intestine, was additionally investigated. The vast majority of isolates were classified as potential biofilm-forming phenotypes and demonstrated susceptibility to all 22 tested antibiotics.

Hemolytically active intestinal isolates of spore-forming bacteria cannot be considered safe until the effect of this virulence factor on the animal organism is elucidated.

The research results can be used for the selection of candidate strains of bacteria of the genus Bacillus chosen as probiotics.

Contribution of the authors: Malik N. I. – scientific supervision, research implementation, methodology, formal analysis, manuscript writing; Chupahina N. A. and Rusanov I. A. – investigation, formal analysis and data processing, table preparation, digital image preparation; Malik E. V. – formal analysis, data processing, article manuscript preparation; Malenkova L. A. and Samokhvalova N. S. – literature review and analysis; Surogin M. V. – text editing.

Вклад авторов: Малик Н. И. – научное руководство, проведение исследований, методология, формальный анализ, написание текста статьи; Чупахина Н. А. и Русанов И. А. – исследование, формальный анализ и обработка данных, составление таблиц, подготовка цифровых снимков; Малик Е. В. – формальный анализ, обработка данных, подготовка текста статьи; Маленкова Л. А. и Самохвалова Н. С. – подбор и анализ литературных источников по теме; Сурогин М. В. – редактирование текста.