" In vitro " Evaluation of Some Properties in Spore Former Bacteria Isolated from Calves Faeces

Aims: To isolate and evaluate spore-former bacteria for being used as probiotic additives in animal nutrition by their technological features. Study Design: The study was experimental, by using calves’ faeces for spore-forming identification and further evaluation of their “in vitro” probiotic-related properties. Place and Duration of Study: Laboratory of Preventive Microbiology, Centro de Referencia para Lactobacilos (CERELA-CONICET), between June 2013 and November 2013. Methodology: In this work, some Bacillus strains were isolated from calves’ faeces and evaluated for their “in vitro” beneficial characteristics: Surface properties, biosurfactant and emulsification production, and inhibition of pathogens. The antibiotic sensibility was also assayed. Short Research Article Maldonado et al.; BMRJ, 11(3): 1-8, 2016; Article no.BMRJ.21943 2 Results: Two Bacillus strains were selected, identified by phenotypic and molecular techniques as Bacillus subtilis strains M14 and M12. Spores resulted to be more hydrophobic than vegetative cells. The M14 strain showed biosurfactant and emulsifying properties. Inhibition assays against pathogenic bacteria indicated they inhibit gram-positive microorganisms. The antibiotic susceptibility showed that the two strains were sensitive to the antibiotics assayed, except Bacillus M12 that was resistant to Kanamycin. Conclusion: The results indicate these strains can be further studied for their inclusion in the design of a probiotic product for newborn calves.


INTRODUCTION
Probiotic additives in beef and dairy farms are alternative adjuncts to be used for animal-feed to replace antibiotics -as promoting growth factors for animals-mainly in newborn animals [1]. Most of the probiotics, defined as "live microorganisms administered to the host to produce a beneficial physiological effect" [2] applied to farm animals are formulated with Lactic Acid Bacteria (LAB) [3]. However, some Bacillus species are considered as a promising alternative for their incorporation in animal-feed [4]. Bacillus species have shown to be effective in helping to a favorable balance of the microbiota of the Gastrointestinal Tract (GIT) and a further improvement of the animal performance [5].
One of the main disadvantages of LAB probiotic products is their short shelf-life and the low resistance to the production process, in contrast to the spore former bacteria that are stable and resistant. According to the scientific reports of the European Food Safety Authority Journal (EFSA) to assess the safety of Bacillus [6], the strains must be included into the Qualified Presumption of Safety (QPS) category. Even though certain species of the Bacillus genera as B. subtilis group could be potentially included, the absence of some toxigenic activity must be verified.
As there is not a wide consensus on the more adequate beneficial characteristics of the sporeformer bacteria to be included in a probiotic formula, and supported by our previous experience, our research group has applied some "in vitro" assays formerly used for LAB to perform the screening and selection of those spore-former strains that could be later used for the design of a probiotic additive for calves [7][8][9]. Then, the objective of this work was to isolate spore-former bacteria from calves, based on the host specificity that supports the selection of probiotic microorganisms [10]. Also, to evaluate the beneficial and safety properties of bacteria considered QPS, including the antibiotic resistance profile, by employing "in vitro" assays related to the capability of the bacteria to adhere to the intestinal epithelium [11] or the production of antagonistic substances against pathogenic bacteria.

Isolation and Storage of the Sporeformer Bacteria from Calves' Faeces
The samples were obtained from rectum of healthy young calves 0-4 months age, collected with sterile swabs in LAPT (1% yeast extract, 1.5% peptone, 1% tryptone, 1% glucose, 0.1% Tween 80) broth [12] supplemented with 0.7% agar (wt/vol) as transport medium and stored at 4ºC until processed. For spore isolation, the cultures were heated for 15 min at 85ºC and maintained for 3 h at 37ºC and later spread onto Nutrient Agar (NA). Plates were incubated for 24 h in aerobic conditions at 37ºC. The quantification of the spore-forming bacteria was not performed, because the main interest was to isolate and select those that could be screened by their beneficial properties. One to three colonies were subcultured and stored in Brain Heart Infusion (BHI, Britania, Capital Federal, Argentine) containing 20% glycerol at -20ºC.
The study protocol was approved by the CERELA-CONICET Bioethics Committee.

Phenotypic characterization
The phenotypic identification was performed by standard tests for Bacillus species described in Prokaryotes [13]. Also, green malachite (Britania, Capital Federal, Argentine) staining technique was applied to check sporulation.

Spore Suspension
The

Hydrophobicity and auto-aggregation characterization
The hydrophobic nature of the surface of vegetative and spore cells was studied by the method of microbial adhesion to hydrocarbons as described by Ocaña et al. [7]. Autoaggregation was assessed according to the technique published by Ocaña and Nader Macías [14]. Briefly, OD 600nm variation of cellular and spore suspensions in saline solution was monitored for 2 hours.

Screening of exopolysaccharide (EPS)
The production of EPS in the Bacillus isolates was determined in 24 h cells grown in 10% sucrose medium at 37ºC. The capsular polysaccharides were evidenced by the Indian ink negative staining technique [15].

Biosurfactant detection and emulsifying properties
The biosurfactant production was screened by the oil spreading technique, as described by Youssef et al. [16]. The emulsification activity and the interphase stability were evaluated by using the modified method of Cooper and Goldenberg [17]. One ml Bacillus cell suspension obtained from a 24 h culture in NB was added to 1.5 ml of n-hexadecane (Merck, Darmstadt, Germany) or Kerosene. The mixture was vortexed for 2 min and the emulsion index obtained after 24 h by the size of the emulsion layer divided by the size of the complete suspension, multiplied by 100.

Hydrogen peroxide
The production of H 2 O 2 was qualitatively determined by the technique described by Juárez Tomás et al. [18]. The isolates were inoculated in the TMB-Nutrient Agar (1%) plates and incubated for 24 h at 37ºC.

Quantification of organic acids
The organic acids production was quantified in the supernatants from the microorganisms grown in NB for 12 h with agitation at 37ºC, by using HPLC with a column for organic acids (HPX-87H 300 x 7.8 mm, BioRad Laboratories, Hercules, USA) at 41ºC 11 [9], the detection was carried out with an RI detector Knauer K-2301 (Knauer, Berlin, Germany).

Inhibition of pathogenic bacteria
The production of inhibitory substances was evaluated by the plate diffusion technique in the supernatant of the selected isolates [19]. Aliquots (35 µl) of cell-free supernatant centrifuged for 15 min 9,300xg and sterilized by filtration from the second subculture of Bacillus grown in NB with agitation at 32ºC were placed into holes performed in plates containing the pathogens in 0.75% agar NA. The plates were incubated 48 h at 37°C. An inhibition zone of at least 6mm diameter was considered positive.

Compatibility between Strains
The assays to evaluate the compatibility between the selected strains were performed by using the plate diffusion technique [20]. Aliquots (35 µl) of cell-free sterile supernatant (12 h incubation centrifuged for 15 min at 9,300xg) of the strains grown in NB with agitation at 32ºC was placed into holes of NA supplemented-0.75% agar plates containing the Bacillus strains as indicator (5x10 7 CFU ml -1 ). The plates were incubated for 24-48 h and the presence of inhibitory halos was considered as positive.

Haemolytic activity and lecithinase production
Hemolytic activity of the strains in BHI sheepblood agar (5%) was evaluated. Lecithinase activity was determined in Cereus Selective Agar (CSA, Merck, Darmstadt, Germany) added with egg yolk emulsion (Britania, Capital Federal, Argentine). Plates were incubated for 24 h in aerobic conditions at 37ºC to detect the production of hemolysis and lecithinase [24].

Genetic Identification of the Strains
The genetic identification of the two strains was performed by 16S ribosomal RNA gene sequencing. Amplifications of DNA were carried out by colony PCR. Strains were cultured in NA; three colonies were selected and heated for 3 min at 85ºC, primers were used for PCR amplifications [25]. The amplified products were electrophoresed in 0.8% agarose gels and DNA sequencing service was performed by Ruralex-Fagos (Capital Federal, Argentine). The identification was obtained from the Genbankdatabase by using The Basic Local Alignment Search Tool (BLAST) algorithm.

RESULTS AND DISCUSSION
Different Bacillus strains were isolated from the GIT of newborn calves, the same homologous host where the product would be applied, based on the host-specificity of the indigenous microbiota [10]. Nine spore-former bacterial strains were isolated (n=9) and their partial phenotypic characterization was performed. Based on the macroscopic characteristics of the colonies on agar plates, they were stored for further identification. Although Bacillus strains are frequently isolated from soil, some authors suggest that this genus is a natural habitat of the intestine with a close relationship with animals [26]. All the isolates were spore-former microorganisms, but only two strains were identified as B. subtilis M12 and B. subtilis M14 by phenotypic and genotypic assays and were used to later study their beneficial characteristics. The other spore-forming isolates were phenotypically identified as B. cereus and related bacteria, and were not included in the screening, because these are species that not qualify in the QPS category [22] for their incorporation to a probiotic additive.
The use of bacterial spores for animal feed has shown resistance to the technological processes and an easier storage [27]. As the spore germination of bacilli was evidenced in the small intestine [28], the evaluation of the beneficial characteristics of the vegetative cell and spores was performed in this work. The spores of B. subtilis M12 (5x10 7 spores ml -1 ) and M14 (1x10 9 spores ml -1 ) were collected after the sporulation procedure. The index of sporulation for B. subtilis M12 and M14 was 79% and 92% respectively, showing that the spore-forming capability is specific or different for each strain.
The results indicate that the hydrophobicity of the spores was higher than those of vegetative cells, and similar findings were published by others authors [29]. Neither the vegetative cells nor the spores showed auto-aggregation.
Emulsifying properties and biosurfactant production were detected in the strains in contact with hexadecane and kerosene (Table 1; Fig. 1), showing stable emulsions and a weak production of surfactant. The most common site for the isolation of microorganisms that produce biosurfactant is the hydrocarbon-contaminated sites; nevertheless some authors suggest that the application of a screening technique is the basis for the detection of those that produce biosurfactants [30].
The biosurfactant production assayed in the stationary phase of growth indicated that only B. subtilis M14 produced a weak biosurfactant by the oil spreading technique ( Table 2). The use of biosurfactants is related not only with bioremediation and pharmaceutical products, but also applied in the medical field as protection against pathogens [31]. Some authors [32] relate the emulsifying characteristics with the surface hydrophobicity and production of biosurfactants in Bacillus subtilis. This emulsificant technique applied to detect biosurfactants could be suggested as an "in vitro" method to evaluate the beneficial properties in the Bacillus genus. Also, antimicrobial properties should be evaluated when looking for surfactant activities, because the surfactancts have shown the ability to destabilize the biological membranes of pathogens by their pore forming capacity [33].

Fig. 1. Emulsions produced by B. subtilis M14 supernatant when added to hexadecane
Referred to the EPS production, the two strains showed to produce EPS in solid agar medium and a weak formation of capsules in the case of B. subtilis M14 by the Indian ink negative stain. B. subtilis M14 produced a hard floating biofilm when incubated in static and aerobic conditions. Different inhibitory substances produced by Bacillus were reported, most of them against gram-positive bacteria. The two B. subtilis strains inhibited the growth of all the grampositive pathogens assayed (Fig. 2, Table 2). Further studies are being performed to determine the chemical nature of the inhibitory substances, because a bacteriocin-like substance, as subtilin or a natural antibiotic could be involved [4]. On the other hand, they did not inhibit any of the gram-negative microorganisms assayed. Lactic acid production was not detected by HPLC. The two strains were compatible, because they did not inhibit each other, and were not able to produce H 2 O 2 in the medium assayed, then can be included in the design of a probiotic product. *The autoaggregation pattern, EPS and biofilm production was classified as: (-) negative or (+) positive. *The hydrophobic index was determined as described by Ocaña et al.[7] †The autoaggregation was performed according to  ‡EPS production was assayed by the method of Mozzi et al. [15]. §Macroscopic observation of the floating biofilms after incubation in nutritive agar. ||Emulsification index was determined by the method described by Cooper and Goldenberg [17]  *Analyzed by the plate diffusion method. †The lactic acid production was quantified by HPLC. ‡The production of H2O2 in TMB-plates was performed according to Juárez-Tomás et al. [18].

Fig. 2. Production of antagonistic substances by Bacillus subtilis M12 (12) and M 14 (14) by using Listeria sp as indicator
§ Biosurfactant detection was determined by the method described by Youssef et al. [16]   The safety assays (lecithinase and hemolysis tests) indicate that the two strains do not have cytotoxic phospholipases or produce a weak alpha-hemolysis in sheep blood, and did not showed lecithinase activity. Animal studies have shown that B. cereus IP 5832, a hemolytic strain, produced acute toxicity by the intra-peritoneal administration in mice, in contrast with B. subtilis 3 and B. licheniformis 31 with no hemolytic properties [23].

CONCLUSION
The results obtained in this paper show that some techniques applied to determine the beneficial properties of Lactic Acid bacteria could be used for the screening of this type of characteristics in spore-forming bacteria. The two Bacillus strains selected are being evaluated to define their inclusion in a product for calves. The results obtained support their use as probiotic according to the EFSA recommendations, and they could be combined with the LAB previously selected for their beneficial properties [9], to restore the intestinal microbioma of calves as multi-strains-hostspecific probiotic.