Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection
Mongkol Thirabunyanon
a,⇑, Narin Thongwittaya
baBiotechnology Section, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand
bFaculty of Animal Science and Technology, Maejo University, Chiang Mai 50290, Thailand
a r t i c l e i n f o
Article history:
Received 30 September 2010 Accepted 12 August 2011
Keywords:
Bacillus subtilis Disease Epithelial cells Infection Probiotic
SalmonellaEnteritidis
a b s t r a c t
The activity of 240 bacterial isolates screened from the gastrointestinal tracts of native chickens were evaluated for use as a potential probiotic in food animal production in order to protect against animal diseases and reduce pathogenic contamination of human food products. In observing the antagonistic activity of 117 bacilli isolates, 10 of these isolates exhibited higher growth inhibition of seven foodborne pathogens, includingSalmonella Enteritidis, Salmonella Typhimurium, Escherichia coli,Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, andVibrio cholerae. Beneficial probiotic criteria from these isolates – which included non-pathogenicity, acid and bile salt tolerance, hydrophobicity, and adhesion to intestinal epithelial cells – exhibited that one isolate of NC11 had the most potential as a probiotic. 16S rRNA gene sequencing showed that this NC11 isolate wasBacillus subtilis. ThisB. subtilisNC11 was sen- sitive to all antibiotics and was not cytotoxic to intestinal epithelial cells. Reduction ofS.Enteritidis attachment to the surfaces of intestinal epithelial cells via action of a cultured medium fromB. subtilis NC11 was observed by scanning electron microscopy.B. subtilisNC11 cells, as well as the bacterial cul- tured medium or the cultured medium adjusted to pH 7, significantly inhibitedS.Enteritidis invasion (P< 0.01) of intestinal epithelial cells. This study indicates thatB. subtilisNC11 has characteristics of a potential probiotic, and exhibits strong inhibition activity againstS.Enteritidis infection to intestinal epithelial cells.
Ó2011 Elsevier Ltd. All rights reserved.
1. Introduction
Salmonellais a major foodborne pathogen that is found in poul- try products and can cause severe illness in humans. Diseases and syndromes such as enteric fever, bacteremia, focal infection, and enterocolitis are caused by this type of pathogenic bacterium.
Human salmonellosis has become an important international pub- lic health and economic issue (Duguid and North, 1991; Velge et al., 2005). Human health protection through the elimination of foodborne pathogens from food animals and their products has been an increasing concern for all sectors of the food production chain (La Ragione and Woodward, 2003; La Ragione et al., 2001).
The widespread use of antibiotics as therapeutic agents and growth promoters in animal husbandry has led to a worldwide in- crease in the antibiotic resistance of bacteria, an imbalance of nor- mal microflora, and drug residues in food products. Consequently these compounds have been banned as animal feed additives by the European Union (Perreten, 2003). One alternative method that has been recommended due to its successful application is the use of probiotics (Reuter, 2001). Our previous study (Samanya and
Yamauchi, 2002) suggested that due to its probiotic potential, Bacillus subtilisvar.nattois one strain that could be promoted as a biological product intended for humans or animals.
There are many criteria that must be investigated before estab- lishing that a new strain of bacteria is a probiotic. These criteria must include the non-pathogenicity of the microorganism, its abil- ity to inhibit the growth of harmful organisms, its tolerance for acid and bile salt conditions, and its ability to adhere to intestinal epithelial cells (Salminen et al., 1998). Competitive exclusion is one of the modes of action of a beneficial probiotic that is exhibited to protect against infection from any pathogen in the intestinal epi- thelial cells of animal host. However, it is unclear whether the effective action of competitive exclusion by using probioticBacillus spp. will result in solo or combined actions in the gastrointestinal tract of the host, including immunomodulation, competition for adhesion sites, and production of antimicrobial agents (Patterson and Burkholder, 2003). Lactic acid bacteria (LAB) have been found to inhibit infection bySalmonellaspp. to the intestinal epithelial cells (Tsai et al., 2005; Golowczyc et al., 2007). There was no report, however, of any effective action of probioticBacillusspp. in inhib- iting the infection ofSalmonellain the intestinal epithelial cells.
The aims of this study were to investigate the activity of isolates of Bacillus spp. for possible use as potential probiotics, and 0034-5288/$ - see front matterÓ2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rvsc.2011.08.008
⇑Corresponding author. Tel.: +66 5387 3535; fax: +66 5387 8225.
E-mail address:[email protected](M. Thirabunyanon).
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their protective inhibition activity against Salmonella Enteritidis infection.
2. Materials and methods 2.1. Bacterial isolation
Thirty-three native chickens (12–24 weeks old, weight 1–2 kg) were collected. They had been raised under natural farming and feeding conditions in different areas of Thailand. All animals were handled in accordance with institutional guidelines for the care and use of animals in research, and in compliance with the ethical principles and guidelines for the use of animals for scientific pur- poses of the National Research Council of Thailand. Fresh intestinal content weighing 1 g, obtained after dissection of the chicken small intestine, was collected from each chicken and then tenfold diluted with 0.85% NaCl in sterilized distilled water. Suitable dilutions of 100
l
l were spread on nutrient agar (NA; Merck, Darmstadt, Ger- many) and incubated at 37°C in aerobic conditions for 24 h. Mor- phologically different colonies, about 6–9 colonies per chicken, were collected. In order to obtain pure isolation, restreaking on agar of the same media was done. A total of 240 bacterial isolates were obtained. Each isolate was stored at80°C in nutrient broth (NB; Merck), supplemented with 20% glycerol (Merck) until further study.2.2. Bacterial morphology and catalase assay
The fundamental morphological determination ofBacillusgen- era – Gram and spore-staining – was performed as previously de- scribed byBenson (1998). Gram-staining of each isolate was done in order to separate Gram-positive, Gram-negative, rod, and cocci forms. Only isolates that were Gram-positive and rod shaped were further examined by Schaeffer-Fulton staining method to confirm that the isolates were endospore-forming after bacterial culture for 24 h. For the catalase assay, the method of Barbosa et al.
(2005)was used; bacterial isolates which were grown in NB were incubated at 37°C for 18 h. The resuspension of a colony in a 3%
solution of hydrogen peroxide was evaluated. The isolates exhib- ited the formation of gas bubbles, indicating a positive test (cata- lase-positive). This assay was done in duplicate.
2.3. Antagonistic activity of bacilli isolates against foodborne pathogens
A total of 117 bacilli isolates – Gram-positive rods, endospore- forming and catalase-positive – were further studied for their effect against foodborne pathogens includingS.Enteritidis DMST 15676, S. Typhimurium TISTR 292, Escherichia coli TISTR 780, Bacillus cereusTISTR 687,Staphylococcus aureusTISTR 118,Listeria monocytogenesDMST 1783, andVibrio choleraeDMST 2873. A mod- ification of the paper disc (Durchmesser: 6 mm; Macherey–Nagel, Duren, Germany) diffusion method was used for triplicate tests, as previously suggested bySansawat and Thirabunyanon (2009).
Pathogenic and tested bacteria were grown in NB, and incubated at 37°C for 18 h in aerobic conditions at an adjusted approximate concentration of 108CFU/ml. The only exception was pathogenicL.
monocytogenes, for which brain heart infusion (Criterion, Santa Maria, USA) was used. This was incubated at 37°C for 18 h in anaerobic conditions. Sterilized paper discs were impregnated with 20
l
l of diluted tested bacterial isolates and placed on the surface of agar plates which were already inoculated with indicator pathogens at a concentration of about 107CFU. Next, the plates were incubated at 37°C for 24 h, and inhibition zones around the paper discs were recorded.2.4. Acid and bile salt tolerance assay
Acid tolerance was evaluated by a modified method ofPennac- chia et al. (2004). Bacterial cultures were grown in NB at 37°C for 18 h. One milliliter of bacterial suspension was then transferred into 9 ml of phosphate buffered saline (PBS) with pH value ad- justed to 2.5 using 5 M HCl (Merck). The suspensions were again incubated at 37°C and later examined for survival rate after expo- sure at 0 and 3 h. The number of viable counts was determined after incubation at 37°C for 24 h on NA. This method was done in triplicate. For bile salt tolerance, the methods ofGilliland et al.
(1984)andPennacchia et al. (2004)were performed. One milliliter of bacteria cultured for 18 h was inoculated into 9 ml of NB pre- pared with bile salt (Sigma, St Louis, MO, USA). Bile salt at a con- centration of 0.3% was applied. The suspension was incubated at 37°C and the viable bacteria were counted after exposure at 0 and 24 h on NA. This method was performed in triplicate.
2.5. Hydrophobicity assay
The method according toSavage (1992)was used. Bacterial iso- lates were grown in NB at 37°C for 18 h. After centrifugation at 5000g for 15 min, the pellets were washed twice with PBS and the absorbance at 450 nm adjusted to 0.5 with an approximate concentration of 107CFU/ml. About 1 ml of bacterial suspension was added to 60
l
l of hydrocarbonsn-hexadecane (Fluka, Buchs, Germany), xylene (Fisher, Loughborough, England), and toluene (Merck), and vortexed for 1 min. The suspensions were left undis- turbed for 1 h, and the OD of the water phase was measured. The method was conducted as duplicate. Hydrophobicity was calcu- lated according to the equation: [(OD450 beforeOD450 after/OD450before100) = % hydrophobicity].
2.6. Cell culture
Cells of the intestinal epithelial cell line (Caco-2) were routinely grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Sigma–Aldrich, USA) supplemented with 10% fetal calf serum (Hyclone, Logan, Utah, USA) inactivated at 56°C for 30 min, 1% (v/v) non-essential amino acid (Hyclone), and 1% (v/v) pen- icillin–streptomycin (10,000 IU/ml and 10,000
l
g/ml; Hyclone).Cells were incubated at 37°C in 5% CO2atmosphere. For bacilli adhesion, andSalmonellaattachment or invasion assays, intestinal epithelial cells were seeded with 1 ml of culture medium contain- ing 106viable cells/well in 24-well tissue culture plates. The cul- ture medium was changed every 48 h. The intestinal epithelial cells were used at 15 days post-confluence after becoming fully differentiated. The medium of non-supplemented DMEM was replaced at least 1 h before these assays.
2.7. Adhesion of bacilli isolates to intestinal epithelial cells
Testing of the ability of bacilli to adhere to intestinal epithelial cell culture monolayers was performed in 24-well tissue plates, as previously described byGagnon et al. (2004)andBogovic Matijasic´
et al. (2006). Bacilli isolates from 18 h cultures in NB were har- vested by centrifugation and washed twice with PBS. These bacilli isolates were then resuspended in non-supplemented DMEM to achieve a concentration of 108CFU/ml. After washing the intestinal epithelial cell culture monolayer twice with PBS, 0.5 ml of bacilli suspension was added to each well and then incubated at 37°C for 1 h in 5% CO2in air. Removing unattached bacteria was per- formed by washing with PBS three times while intestinal epithelial cells were lysed with 0.1% Triton X-100 (Merck) for 5 min. Viability of adherent bacilli was done by plate counting in triplicates on NA.
This analysis was done in five replicate.
2.8. Molecular identification by 16S rRNA sequencing
Total genomic DNA was extracted from an isolate of NC11 using an Isoplant DNA extraction kit (Nippon Gene, Tokyo, Japan). The six oligonucleotide primers, as described byNiamsup et al. (2003)– including 27F (50-AGAGTTTGATCCTGGCTCAG-30), 520R (50-ACCGCG GCKGCTGGC-30), 357F (50-CTACGGGAGGCAGCAG-30), 1080R (50- CCCAACATCTCACGAC-30), 920F (50-AAACTCAAAGGAATTGACGG-30), and 1522R (50-AAGGAGGTGATCCARCCGCA-30) (Operon, Cologne, Germany) – were used to amplify 16S rRNA gene sequences of the isolate. Amplification was carried out using a PCR with a reac- tion volume of 50
l
l. One microliter of genomic DNA sample was added to a mixture containing 25l
l of MasterMix (1.5 mM MgCl2, 200l
M dNTPs, 1.25 U-Taq polymerase; Eppendorf, New York, USA); 2l
l of each primer; and 20l
l of distilled water. The PCR program was performed using a denaturing step at 94°C for 5 min, followed by 25 cycles (94°C for 1 min, 55°C for 1 min, 72°C for 1 min) plus one additional cycle at 72°C for 5 min. The presence of PCR product was verified by agarose gel electrophore- sis. Five microliter of PCR product was mixed with 2l
l of loading dye (Fermentas, Maryland, USA) and loaded onto 1.5% agarose gel.DNA fragment sizes were compared using a 100 bp DNA ladder (Fermentas). The PCR product of the 16S rRNA gene was then se- quenced. Percentage of similarity was compared with the GenBank database by using BLAST (http://www.ncbi.nlm.nih.gov).
2.9. Antibiotic resistance assay
Antibiotic susceptibility of theB. subtilisNC11 strain was per- formed by the disc diffusion method recommended by the National Committee for Clinical Laboratory Standards (NCCLS, 1997). Bacte- ria from 18 h cultures were used at approximately 108CFU/ml. The suspensions were seeded onto Mueller–Hinton agar (Criterion) plates using a swab. Antibiotic-impregnated discs (Oxoid, Basing- stoke, Hampshire, England) were placed on the seeded plates and incubated at 37°C for 24 h. The inhibition zone around the antibi- otic discs was recorded. The assay was performed in triplicate.
2.10. Cytotoxicity assay
A trypan blue exclusion assay for measuring survival or death of the Caco-2 cells, as previously described byRamarao and Lereclus (2006)andThirabunyanon et al. (2009), was performed. Intestinal epithelial cells were seeded at a density of 106cells/well, with 1 ml of cell suspension being added into 24 well plates. The suspensions were incubated at 37°C in 5% CO2atmosphere for 24 h. Cells were infected with 1 ml of each cultured medium (cell-free superna- tants) of: (1) NB obtained by filtration of NB through a 0.2
l
m syr- inge filter (Sartorius, Goettingen, Germany) as a control; (2) B.subtilisNC11; (3)B. cereusas a pathogenic control; and (4)S. Ente- ritidis as a pathogenic control. These cultured media were added into the wells and incubated further for 24 h. Cultured media were obtained by centrifugation and then filtration through a 0.2
l
m syringe filter of 18 h bacterial cultures in NB. After incubation, the cell exposures were examined using trypan blue (Sigma) exclu- sion staining with a Neubauer hemocytometer. The analysis was evaluated in three independent studies, each conducted in five rep- licates [% cytotoxicity = (death cell count/total cell count)100].2.11. Scanning electron microscopy (SEM) observation of the inhibition activity of B. subtilis NC11 against S. Enteritidis attachment to intestinal epithelial cells
The procedures were performed as follows. Intestinal epithelial cell culture in 24-well tissue culture plates was used, and 1 ml of non-supplemented DMEM was replaced at least 1 h before attach-
ment assay. Then, 1 ml of each of the following was mixed into the wells: (1) NB obtained by filtration of NB through a 0.2
l
m syringe filter, as a control; (2)S. Enteritidis cells obtained by centrifugation of this culture in NB, followed by resuspension of the cells in NB at a concentration of 108CFU/ml, as an infection group; and (3)S.Enteritidis cells obtained by centrifugation of this culture in NB, followed by resuspension of the cells at a concentration of 108CFU/ml in a cultured medium fromB. subtilisNC11, as a com- petition group. Samples were then incubated at 37°C for 1 h. After removing the media, intestinal epithelial cell samples were washed twice with PBS and then fixed with 2.5% (v/v) glutaraldehyde (EMS, Pennsylvania, USA) in 0.1 M phosphate buffer (pH 7.4) at 4°C for 1 h. Samples were then washed twice with PBS and post-fixed with 1% (v/v) osmium tetraoxide (EMS) in 0.1 M phosphate buffer (pH 7.4) at room temperature for 2 h. After washing twice with PBS, samples were dehydrated in ethanol (Merck) graded series (30–
100%). Consequently, the dried samples were treated by a critical point drier in liquid CO2, and coated with gold. Cell specimens were later examined with a SEM (JEOL JSM-5410LV). This analysis was performed as five replicates.
2.12. Infection inhibition activity of B. subtilis NC11 against S. Enteritidis invasion of intestinal epithelial cells
The methods of Hudault et al. (1997) and Golowczyc et al.
(2007)were used, and the examination was performed as follows.
After non-supplemented DMEM was replaced at least 1 h before the invasion assay, the intestinal epithelial cells were washed twice with PBS. One milliliter ofS. Enteritidis at approximately 108CFU/ml was loaded into the intestinal epithelial cell culture monolayer in 24-well tissue culture plates, plus 1 ml each of: (1) NB obtained by filtration of NB through a 0.2
l
m syringe filter, as a control group; (2)B. subtilisNC11 cells obtained by centrifugation of this culture in NB, followed by resuspension of the cells in NB at a concentration of 108CFU/ml; (3) a cultured medium obtained by centrifugation of theB. subtilisNC11 culture in NB, which was then filtered through a 0.2l
m syringe filter; and (4) a cultured medium adjusted to pH 7. These were mixed into the wells, and later incu- bated at 37°C for 1 h. After washing the cells with PBS, 0.5 ml of DMEM containing 100l
g/ml gentamicin (Gibco, Grand Island, USA) was added to each well and incubated for another 1 h. Intes- tinal epithelial cells were then washed with PBS three times before being lysed with 0.1% Triton X-100. The appropriate dilution was spread on brilliant green agar (Criterion) and incubated at 37°C for 24 h. Viability ofS.Enteritidis was used to calculate the inva- sion rates using five replicate analyses.2.13. Statistical analysis
A one-way analysis of variance (ANOVA) was applied, using SPSS (version 15.0) for significant differences of experimental data.
Differences between groups were analyzed using Duncan’s multi- ple range test, with statistical significance accepted atP< 0.05.
3. Results
3.1. Bacterial isolates
The morphology of bacterial isolates was exhibited in terms of their diversity, such as being Gram-positive or Gram-negative, and with a rod or cocci form. From a total of 240 bacterial isolates col- lected, only 117 isolates were generally indicated as species of Bacillus due to their being Gram-positive, rod-shaped, endo- spore-forming, and catalase-positive. These isolates were further analysed.
3.2. Antagonistic activity of bacilli isolates against foodborne pathogens
In studying the antimicrobial activity of the 117 bacilli isolates, only 15 isolates were found to have the ability to inhibit the growth of foodborne pathogenic bacteria. Higher inhibiting activity against seven pathogenic bacteria was observed only among 10 isolates, as shown by their inhibition zone of more than 10 mm (Table 1).
3.3. Acid and bile salt tolerance of bacilli isolates
The tolerance of the isolates to pH 2.5 is shown inFig. 1. There were three levels of growth rates: very good tolerance (NC11, NC26, and NC132), exhibiting a survival rate greater than that of the initial bacteria; good tolerance (NC14, NC15, NC19, NC38, and NC39), showing a slight decline in the survival rate; and low tolerance (NC12 and NC111), expressing a low survival rate. Bacilli isolates exposed to 0.3% bile salt for 24 h exhibited different ranges of survival rates. Bacteria that had good tolerance (NC11, NC14, NC15, NC93, NC111, and NC132) exhibited a slightly decreased survival rate as compared to that of the initial bacteria; bacteria with moderate tolerance (NC19, NC26, and NC38) showed a de- creased survival rate; and those with low tolerance (NC12) had a low survival rate (Fig. 2).
3.4. Hydrophobicity of bacilli isolates
The use ofn-hexadecane, xylene, and toluene in order to evalu- ate the hydrophobic cell surface properties of tested bacilli isolates is shown inTable 2. Results showed rather constant data of each isolate from different tested hydrocarbons. The percentages of hydrophobicity were: n-hexadecane at 17–57%; xylene at 31–
62%; and toluene at 29–59%. Only four isolates, NC11, NC15, NC38, and NC93, had higher rates at about 50–60% hydrophobicity.
3.5. Adhesion to epithelial cells of bacilli isolates
The adhesion ability of bacilli isolates to intestinal epithelial cells is shown in Fig. 3. Among the isolates, different adherent ranges were found, beginning from 2.8–4.9 log CFU/well. Higher rates of adhesion efficiency were exhibited by NC11, NC14, NC15, NC26, NC19, and NC93 isolates, respectively. Meanwhile, lower adherent abilities were found in NC12, NC38, NC111, and NC132 isolates.
3.6. Identification of Bacillus probiotic isolate
Molecular identification of 16S rRNA sequencing gene of the NC11 isolate exhibited a 99% identity of 16S rRNA sequence. Based on database entries, the species could beB. subtilis(Accession No:
EF428253.2).
3.7. Antibiotic resistance
The profile of antibiotic susceptibility for theB. subtilis NC11 strain exhibited that it was sensitive to all clinically important antibiotics in both human and veterinary use, including amikacin, amoxicillin, ampicillin, cephalothin, chloramphenicol, ciprofloxa- cin, colistin sulfate, enrofloxacin, erythromycin, gentamicin, kana- mycin, lincomycin, methicillin, norfloxacin, ofloxacin, oxacillin, oxolinic acid, penicillin, streptomycin, tetracycline, and vancomy- cin (Table 3).
Table 1
Antimicrobial activity of bacilli isolates against foodborne pathogens.
Indicator strains Growth inhibition by activity of bacilli isolates (mm)
NC11 NC12 NC14 NC15 NC19 NC26 NC38 NC93 NC111 NC132
SalmonellaEnteritidis DMST 15676 12 ± 0 11 ± 1 12 ± 0 11 ± 0 11 ± 1 11 ± 0 11 ± 0 12 ± 0 12 ± 0 12 ± 1
SalmonellaTyphimurium TISTR 292 15 ± 2 11 ± 1 13 ± 2 15 ± 1 14 ± 3 13 ± 2 14 ± 2 14 ± 2 15 ± 2 13 ± 3
Escherichia coliTISTR 780 13 ± 1 14 ± 1 12 ± 1 11 ± 1 11 ± 1 10 ± 1 12 ± 1 14 ± 1 15 ± 1 14 ± 1
Bacillus cereusTISTR 687 12 ± 1 12 ± 1 13 ± 1 10 ± 0 10 ± 1 10 ± 1 10 ± 1 14 ± 1 14 ± 1 13 ± 0
Staphylococcus aureusTISTR 118 19 ± 0 16 ± 1 18 ± 2 20 ± 1 19 ± 1 17 ± 2 18 ± 1 19 ± 0 19 ± 1 18 ± 2
Listeria monocytogenesDMST 1783 15 ± 1 14 ± 1 16 ± 1 15 ± 1 – – 19 ± 1 16 ± 2 15 ± 1 –
Vibrio choleraeDMST 2873 22 ± 1 22 ± 0 21 ± 1 23 ± 1 23 ± 1 24 ± 1 24 ± 1 22 ± 1 23 ± 2 22 ± 2
Values are mean with SD of three replications. (–) = No inhibition.
2 3 4 5 6
NC11 NC12 NC14 NC15 NC19 NC26 NC38 NC93 NC111 NC132
log CFU/ml
Fig. 1.Acid tolerance activity of bacilli isolates. Survival of bacilli isolates was compared by plate counting after exposure to low pH 2.5 in PBS for 0 (j) and 3 ( ) h. Values are mean with SD of three replications.
2 3 4 5 6
NC11 NC12 NC14 NC15 NC19 NC26 NC38 NC93 NC111 NC132
log CFU/ml
Fig. 2.Bile salt tolerance activity of bacilli isolates. Survival of bacilli isolates was compared after exposure to bile salt 0.3% in NB for 0 (j) and 24 ( ) h. Values are mean with SD of three replications.
3.8. Cytotoxicity
Fig. 4shows a cytotoxicity comparison ofB. subtilisNC11 with pathogenicB. cereusandS. Enteritidis. No toxicity to intestinal epi- thelial cells was found in the control andB. subtilisNC11 groups,
with a low percentage of cytotoxicity rates. High toxicity to intes- tinal epithelial cells was observed in both pathogenicB. cereusand S. Enteritidis groups, with significantly increased cytotoxicity (P< 0.01) compared to that of the control and B. subtilis NC11 groups. Cytotoxicity percentages of the control,B. subtilis NC11, B. cereus, and S. Enteritidis groups were 12.3%, 9.4%, 72.4%, and 80.3%, respectively.
3.9. SEM photographs of the inhibition activity of B. subtilis NC11 against S. Enteritidis attachment to intestinal epithelial cells
Inhibition of the beginning of theS. Enteritidis infection process was shown in SEM photographs. Smooth surfaces of intestinal epi- thelial cells were observed in the control group, which was not in- fected byS. Enteritidis (Fig. 5A). Cells infected withS. Enteritidis were found to be attached to the surfaces of intestinal epithelial cells (Fig. 5B). Whereas infection inhibition by a cultured medium ofB. subtilisNC11 was demonstrated by a decrease in the number ofS. Enteritidis attached to the surfaces of intestinal epithelial cells (Fig. 5C).
3.10. Infection inhibition activity of B. subtilis NC11 against S.
Enteritidis invasion of intestinal epithelial cells
The activity ofB. subtilisNC11 cells, or their cultured medium or cultured medium adjusted to pH 7, againstS.Enteritidis invasion of intestinal epithelial cells is shown inFig. 6. This was compared to the incubation ofS.Enteritidis alone with intestinal epithelial cells, which resulted in an invasion rate of 4.8 log CFU/well. Significantly reduced invasion rates (P< 0.01) were observed in the co-incuba- tion ofS. Enteritidis and B. subtilis NC11 cells (or their cultured medium or cultured medium adjusted to pH 7) with intestinal epi- thelial cells, with invasive inhibition rates of 3.9, 4.1, and 4.0 log C- FU/well, respectively.
4. Discussion
Concerns about food safety and the reduction of foodborne pathogens from food animals and their products have become a Table 2
Ability of bacilli isolates on hydrophobic activity.
Bacilli isolates Hydrophobicity (%)
Hexadecane Xylene Toluene
NC11 55 ± 6 56 ± 8 54 ± 2
NC12 39 ± 8 55 ± 1 54 ± 4
NC14 37 ± 3 50 ± 0 49 ± 1
NC15 50 ± 2 51 ± 0 50 ± 5
NC19 22 ± 1 33 ± 2 33 ± 1
NC26 17 ± 2 31 ± 2 29 ± 0
NC38 57 ± 4 62 ± 3 59 ± 1
NC93 45 ± 6 58 ± 2 57 ± 7
NC111 27 ± 2 31 ± 1 36 ± 2
NC132 45 ± 4 48 ± 1 44 ± 2
Values are mean with SD of two replications.
2 3 4 5 6 7 8
NC11 NC12 NC14 NC15 NC19 NC26 NC38 NC93 NC111 NC132
log CFU/well
Fig. 3.Ability of bacilli adhesion to intestinal epithelial cells. The adherence activity of each isolate was compared by plate counting between initial (j) and adhered ( ) bacterial concentrations. Values are mean with SD of five replications.
Table 3
Antibiotic resistance forB. subtilisstrain NC11.
Antibioticsa Zone diameter (mm) Result
Amikacin (30lg) 33 ± 2 S
Amoxicillin (10lg) 30 ± 1 S
Ampicillin (10lg) 30 ± 0 S
Cephalothin (30lg) 51 ± 1 S
Chloramphenicol (30lg) 31 ± 1 S
Ciprofloxacin (5lg) 33 ± 1 S
Colistin sulphate (10lg) 10 ± 1 S
Enrofloxacin (5lg) 33 ± 1 S
Erythromycin (15lg) 30 ± 1 S
Gentamicin (10lg) 28 ± 3 S
Kanamycin (30lg) 28 ± 0 S
Lincomycin (10lg) 31 ± 1 S
Methicillin (5lg) 32 ± 2 S
Norfloxacin (10lg) 31 ± 1 S
Ofloxacin (5lg) 29 ± 1 S
Oxacillin (1lg) 21 ± 1 S
Oxolinic acid (2lg) 27 ± 1 S
Penicillin (10lg) 30 ± 1 S
Streptomycin (10lg) 18 ± 0 S
Tetracycline (30lg) 21 ± 0 S
Vancomycin (30lg) 25 ± 1 S
aAntibiotic-impregnated discs (6 mm) with amount, inlg shown in brackets.
Values are mean with SD of three replications. (S) = susceptible.
a a
b
b
0 20 40 60 80 100
Control BNC BCER SALM
Cytotoxicity (%)
Fig. 4.Cytotoxicity ofB. subtilisNC11 (BNC) and a comparative pathogenic strain of B. cereus(BCER) orS. Enteritidis (SALM) on intestinal epithelial cells. Values are mean with SD of three independent studies of each five replicates.a,bMeans within each grouping with different letter designations differ (P< 0.01).
very important focus in human health protection. Alternative application of biological products such as probiotics also provides benefits for food animal production. Finding a novel probiotic strain which inhibited the infection activity ofS.Enteritidis was the focus of this study. Of the initial 240 bacterial isolates, 117 were indicated as species ofBacillusdue to their being Gram-posi- tive, rod-shaped, endospore-forming, and catalase-positive. This positive action of catalase is a characteristic ofBacillusspp. isolated from the chicken gastrointestinal tract, thus separating it distinctly from pathogenicClostridiumspp. ManyBacillusspecies have been isolated from the gastrointestinal tract of chickens, such asB. sub- tilis, B. licheniformis, B. pumilus, B. megaterium, B. firmus, B. clausii, andB. cereus(Teo and Tan, 2005; Barbosa et al., 2005).
Most of the 117 bacilli isolates exhibited no antagonistic activ- ity against pathogens. About 10 of these isolates showed higher antimicrobial activity that inhibited the growth of seven foodborne pathogens. Infections by some of these pathogens are associated with being the causes of human illnesses. S. Enteritidis is com- monly carried by chickens and poultry products, and is one of the major sources of human intestinal infections (Velge et al., 2005; Rostagno et al., 2006). It is proposed that antimicrobial agents be produced from the genera ofB. subtilis– as in the case of amicoumacin (Pinchuk et al., 2001) or bacteriocin (Teo and Tan, 2005) – which have an ability to inhibit foodborne pathogens.
Most of the 10 bacilli isolates were able to survive well in the envi- ronment of the gastrointestinal tract model, which included pH 2.5 and 0.3% bile salt. This correlates with the fact that these bacilli normally reside in the chicken gastrointestinal tract. A relationship between the high hydrophobicity ofLactobacillus acidophilusM92 and its ability to adhere to porcine ileal epithelial cells was re- ported byKos et al. (2003). The Caco-2 cell line has been used as anin vitromodel for intestinal epithelial cells (Tuomola and Salmi- nen, 1998). This adhesion to intestinal epithelial cells was an important component of probiotic screening, as was the case in selecting strains of LAB in our previous study (Thirabunyanon et al., 2009). The highest adherent ability was found in theBacillus NC11 isolate, suggesting that this isolate could be able to adhere to intestinal epithelial cells, where it could exert probiotic action or inhibition of pathogenic infection. Probiotic strain NC11 was iden- tified asB. subtilis.
In the United States, bacteria considered safe for human con- sumption are awarded GRAS status (‘‘Generally Regarded as Safe’’) by the Food and Drug Administration. As a similar system, the European Food Safety Authority (EFSA) proposed the concept of Qualified Presumption of Safety (QPS), in which selected groups of microorganisms are assessed for safety in human and animal foods. TheB. subtilisspecies can be considered to be a group that meets QPS criteria (EFSA, 2005). This is in line with the use ofB.
subtilisas an approved food supplement in at least one European country (Italy) as a probiotic (Hoa et al., 2000). In vitro and in vivosafety assessments of the commercial probiotic strainsB.
subtilisBS3 (Sorokulova et al., 2008),B. subtilisPY79 andB. subtilis Fig. 5.Attachment ofS.Enteritidis onto the surfaces of intestinal epithelial cells by
(A) non-infected cells (control), (B) cells infected withS.Enteritidis, and (C) a cultured medium ofB. subtilisNC11 against cells infected withS.Enteritidis. Arrows indicateS.Enteritidis attachment. It is evident that only smooth cell surfaces are present in non-infected cell groups. Several clusters ofS.Enteritidis attached to cell surfaces are evident (arrows in B) in cells infected withS. Enteritidis. There is a reduction ofS.Enteritidis attachment to cell surfaces (arrows in C) in the group ofS.
Enteritidis-infected cells in the cultured medium ofB. subtilisNC11.
Fig. 6.Infection inhibition activity ofB. subtilisNC11 probiotic cells (Cells) or their cultured medium (CM) or cultured medium adjusted pH 7 (CM pH 7) against S.Enteritidis invasion of intestinal epithelial cells. Values are mean with SD of five replications.a,bMeans within each grouping with different letter designations differ (P< 0.01).
var.natto(Hong et al., 2008), indicated that theseB. subtilisstrains are safe for human consumption. This was also confirmed for theB.
subtilisNC11 strain,in vitroandin vivo, although it is known that strains ofB. subtilisare generally safe for use both in human and animals.
Antimicrobial susceptibility tests have shown that B. subtilis NC11 is sensitive to all antibiotics of human or veterinary impor- tance, as listed in a report by the EFSA (EFSA, 2005). These results were similar to those regarding the three commercial probiotics of B. subtilisstrains of BS3, PY79, and Natto which were found to be sensitive to antibiotics, as proposed byHong et al. (2008)andSor- okulova et al. (2008). This also is an indication that resistance to an antibiotic is correlated with a bacterial species or genus – such as, resistance to clindamycin may be an intrinsic characteristic ofB.
licheniformisorBacillus indicus. The cytotoxicity on intestinal epi- thelial cells by action of a bacterial culture medium is one method of evaluating whether a bacterium strain is harmful or non-harm- ful. This has resulted in a finding that cytotoxicity on intestinal epi- thelial cells is caused byB. cereus, but that there is no toxic action byB. subtilis(Ramarao and Lereclus, 2006). In the case ofB. subtilis NC11, no toxicity was exhibited on these cells. These results are similar to the action of theB. subtilis strain PY79, which shows no toxicity to intestinal epithelial cells, but different from the Natto strain that is able to lyse the intestinal epithelial cells. Although the Natto strain could lyse the cells duringin vitrotests,in vivosafety assessments of acute and chronic dosing in guinea pigs and rabbits showed no such toxicity in the hematological indexes (Hong et al., 2008). For ourin vivoinvestigation, 288 broiler chicks were tested.
It was found that the potential ofB. subtilisNC11 as a spore-former could significantly increase growth performance, and did not affect blood biochemical or white blood cell changes (unpublished), sug- gesting that this probiotic strain of B. subtilis NC11 is safe for animals.
The activity ofB. subtilisNC11 to protect againstS.Enteritidis infection, as well as its ability to eliminate it by competitive exclu- sion from the originate route of food contamination, were investi- gated. An intestinal epithelial cell culture model using Caco-2 cells has proven to be a suitable system for the study of pathogenic invasion or infection (Coconnier et al., 1993; Bernet et al., 1994).
A key pathogenic factor of intestinal pathogens is their ability to at- tach to the surfaces of intestinal epithelial cells (Weinstein et al., 1998). Infection bySalmonellaspp. is via the fecal–oral route; the pathogens can then survive and colonize the gastrointestinal tract.
Attachment to intestinal epithelial cells is via fimbriae or pili that are located on the bacterial cell surface (Foley and Lynne, 2008).
The subsequent steps in pathogenesis lead to mucosal infection, systemic spread and disease (Darwin and Miller, 1999). Our results showed that the infection process ofS. Enteritidis attachment to intestinal epithelial cells was clearly demonstrated by SEM photo- graphs, and is considered to be evidence of infection. Conse- quently, S. Enteritidis invasion of the epithelial cells is the next step in the process of infection, as also found in this investigation.
B. subtilisNC11 is resident in the chicken gastrointestinal tract, indicating that it might have the ability to establish itself in the gastrointestinal tract environment by surviving in the low pH of the stomach and colonizing multiple sites, including the small intestine, colon and cecum. This correlates with the finding of a high tolerance of theB. subtilisNC11 strain to low pH and bile salt conditions. Importantly, this strain also had a greater ability to ad- here to epithelial cells than did other strains, as also found in this study. The adherence of LAB to the intestinal epithelial cells of the host may result in the competitive exclusion of pathogenic bacteria adhesion, as proposed byBernet et al. (1994)andLee et al. (2003).
The reduction ofS. Enteritidis attachment to the surfaces of intes- tinal epithelial cells (which is the initial evidence of the infection process) by action of a cultured medium from B. subtilis NC11
was observed by SEM photographs. This inhibition of the subse- quent infection process was not a direct effect ofB. subtilisNC11 cells only; their cultured medium or cultured medium adjusted to pH 7 also significantly inhibitedS. Enteritidis invasion of intes- tinal epithelial cells, as was found in this investigation. This means that even live cells which had already had their antimicrobial agents removed, or only the remaining antimicrobial agents, were found to exhibit an effective action, indicating that the protective mechanism inhibiting the infectiousness ofS.Enteritidis by theB.
subtilisNC11 probiotic strain is caused by both competitive exclu- sion at the adhesion site and the production of antimicrobial sub- stances. The inhibition of the invasion ofSalmonellainto epithelial cells is a process of disease protection, as suggested byTsai et al.
(2005)andGolowczyc et al. (2007).
In conclusion, a probiotic strain of B. subtilis, NC11, has a protective activity against S.Enteritidis infection, and is able to competitively exclude it from its original site in the gastrointesti- nal tract, which is the beginning of the route of food pathogenic contamination.
Acknowledgment
The authors would like to thank the Thailand Research Fund (TRF) for financial support of this project (MRG5080094).
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