- freely available
Mar. Drugs 2014, 12(5), 2422-2445; doi:10.3390/md12052422
Published: 30 April 2014
Abstract: Six antimicrobial-producing seaweed-derived Bacillus strains were evaluated in vitro as animal probiotics, in comparison to two Bacillus from an EU-authorized animal probiotic product. Antimicrobial activity was demonstrated on solid media against porcine Salmonella and E. coli. The marine isolates were most active against the latter, had better activity than the commercial probiotics and Bacillus pumilus WIT 588 also reduced E. coli counts in broth. All of the marine Bacillus tolerated physiological concentrations of bile, with some as tolerant as one of the probiotics. Spore counts for all isolates remained almost constant during incubation in simulated gastric and ileum juices. All of the marine Bacillus grew anaerobically and the spores of all except one isolate germinated under anaerobic conditions. All were sensitive to a panel of antibiotics and none harbored Bacillus enterotoxin genes but all, except B. pumilus WIT 588, showed some degree of β-hemolysis. However, trypan blue dye exclusion and xCELLigence assays demonstrated a lack of toxicity in comparison to two pathogens; in fact, the commercial probiotics appeared more cytotoxic than the majority of the marine Bacillus. Overall, some of the marine-derived Bacillus, in particular B. pumilus WIT 588, demonstrate potential for use as livestock probiotics.
In the past, sub-therapeutic doses of antibiotics were used in feed to promote growth and maintain health in farm animals, mainly pigs and poultry. However, due to concerns over increasing bacterial antibiotic resistance, the routine use of in-feed antibiotics was banned in the European Union (EU) in 2006. As a result, alternatives are now being sought. Probiotics, defined as “live microorganisms, which when administered in adequate amounts, confer a health benefit on the host”  are one such alternative. In fact, different probiotic strains have been shown to improve growth performance and to reduce enteric disease in pigs, poultry, ruminants and cultured fish [2,3].
In the EU, animal probiotics are controlled by Regulation EC 1831/2003, which establishes procedures for the authorization of feed additives . This regulation and associated European Food Safety Authority (EFSA) guidelines  make no recommendations on strain origin, apart from the fact that the origin must be stated. However, it is generally recommended that probiotics should be isolated from the gastrointestinal tract (GIT) , as it is considered to give them the best chance of surviving in and colonizing the intestine . Nonetheless, regardless of origin, Bacillus spores can tolerate the harsh environment of the GIT and are capable of germinating and proliferating within the intestine [8,9]. Furthermore, EU-authorized animal probiotics with proven efficacy are often not of intestinal origin. For example, six of the seven Bacillus-containing probiotic products currently licensed in the EU for use as feed additives, contain isolates from soil or soybean fermentations; in fact only one (Bacillus subtilis ATCC PTA-6737) is of intestinal origin [10,11,12,13,14,15,16].
Alternative probiotic sources should therefore not be overlooked. The marine environment is worth considering in this respect, given that it represents an untapped source of potentially novel microorganisms and that antimicrobial production, considered an important probiotic trait , is common amongst marine microflora . Furthermore, Bacillus, which offers advantages as an animal probiotic, in that its spores survive harsh conditions, such as those encountered during feed manufacture and in the GIT, is commonly isolated from marine sources [3,19]. However, while marine-derived bacteria (mainly isolated from shrimp or fish intestines) have demonstrated beneficial effects when used as probiotics for fish , to our knowledge, they have not been evaluated as livestock probiotics to date.
As with any probiotic candidate organisms, rigorous in vitro testing is required prior to progressing to in vivo trials. The objective of the present study was, therefore, to characterize six seaweed-derived Bacillus isolates in vitro, for potential use as animal probiotics, using two Bacillus isolates from an EU-authorized animal probiotic product for comparison. The isolates were selected as most promising from a previous screening study on the basis of their antimicrobial activity against Escherichia coli and Salmonella , and in the present study were investigated mainly for their potential as probiotics for pigs.
2.1. Molecular Fingerprinting and Growth of Marine Bacillus
The spontaneous rifampicin-resistant (Rifr) variants of the marine Bacillus yielded pulsed field gel electrophoresis (PFGE) fingerprints that were indistinguishable from those of the corresponding parent strains and their growth curves were also identical (data not shown). They were therefore used in all subsequent experiments. PFGE fingerprints of the marine Bacillus also differed from those of the two Bacillus isolates from the Bioplus 2B® animal probiotic product, indicating that they were different strains (data not shown).
2.2. Sporulation Efficiency
As Bacillus probiotics will most likely be used as spore preparations, it is important to obtain information on sporulation. Sporulation is assumed to start at the end of the exponential phase in exhaustion medium. This occurred 12 h post-inoculation for Bacillus pumilus WIT 582, 588 and 592 and the two Bioplus 2B® isolates, and after 15 h for B. pumilus WIT 584 and 590 and Bacillus licheniformis WIT 586 (data not shown). All of the marine isolates showed good sporulation efficiencies, with spore titres in the order of 107–108 spores/mL (data not shown). B. licheniformis WIT 586 and B. pumilus WIT 582 and WIT 590 had the highest sporulation rates (80%–82%), while B. pumilus WIT 588 had the lowest at 61%. In comparison, one of the commercial probiotics (B. licheniformis DSM 5749) had the highest sporulation efficiency (97%), while the other (B. subtilis DSM 5750) had one of the lowest (67%).
2.3. Growth and Spore Germination under Anaerobic Conditions
Anaerobic growth of the Bacillus isolates was investigated when either vegetative cells or spores were used as the inoculum (Figure 1), as anaerobic conditions prevail in the distal GIT. Using a spore inoculum, the increase in counts of B. pumilus WIT 590 was higher under aerobic than anaerobic conditions (p < 0.001). In fact, the increase in counts was very low under anaerobic conditions, indicating poor (if any) spore germination. Furthermore, the increase in counts was higher when vegetative cells were used as the inoculum, either under aerobic or anaerobic conditions, compared to spores (p < 0.001). However, all of the other Bacillus grew as well aerobically as they did anaerobically, both when spores and vegetative cells were used as the inoculum, and their spores all appear capable of germinating under anaerobic conditions.
2.4. Antimicrobial Activity on Solid Media
Although the marine Bacillus have been shown to have antimicrobial activity against a range of Gram-positive and some Gram-negative bacteria due, at least in part, to the production of bacteriocins , more targeted testing against pathogens of relevance to pigs was performed in the present study. Activity was determined against Salmonella Typhimurium and Derby (Table 1), as Salmonella transmission to humans via carcass contamination at slaughter is a major food safety issue and these serotypes are amongst those most commonly carried by pigs . They are also capable of causing clinical disease. Similarly, the enterotoxigenic and enterohemorrhagic E. coli used as targets (Table 1) are pathogenic to animals, mainly pigs, and/or humans.
B. pumilus WIT 582 had the best antimicrobial activity, with activity against all of the E. coli and Salmonella strains against which it was tested (Table 1, Figure 2). B. licheniformis WIT 586 and B. pumilus WIT 588 were active against all of the E. coli strains, and the latter also showed consistent activity against one S. Typhimurium isolate (Table 1, Figure 2). The other three marine bacteria had only weak antimicrobial activity, showing consistent activity against only one E. coli strain (WIT 584) or only variable activity against S. Typhimurium (WIT 590 and 592) (Table 1, Figure 2). In comparison, of the two Bacillus strains from the commercially available Bioplus 2B® animal probiotic product, only B. licheniformis DSM 5749 showed activity and it was only consistently active against one S. Typhimurium strain (Table 1). To investigate potential activity against the marine pathogen Vibrio, the potential probiotics were also tested against Vibrio cholerae and fischeri. B. pumilus WIT 582, WIT 588 and B. licheniformis WIT 586 were active against the former, but none inhibited the latter (data not shown). In general, antimicrobial activity against Gram-negative bacteria (both those tested in the present and previous studies ) was detected only from growing cultures and not in the cell-free supernatant (data not shown). All of the marine isolates also showed activity against four or five Lactobacillus species likely to be found in the porcine intestine but this activity was detected in the cell-free supernatants (Supplementary Information, Table S1. In comparison, the Bioplus 2B® isolates showed greater activity against Lactobacillus, inhibiting nine and 14 species, respectively out of a total of 19 (Supplementary Information, Table S1).
|Table 1. Antimicrobial activity 1 against porcine E. coli and Salmonella of marine Bacillus compared with that of Bacillus isolated from a commercially available animal probiotic product.|
|Test strain||E. coli||Salmonella|
|K88||F18ab||F2S2||F3P3||F1L3||F15OF3||Typhimurium DT104 (DPC 6046)||Typhimurium PT12 (DPC 6465)||Typhimurium DT17 (WIT 396)||Typhimurium DT104 (WIT 387)||Derby (WIT 411)|
|B. pumilus WIT 582||+||+++||++||++||++||+||++||+||+||++||++|
|B. pumilus WIT 584||-||-||-||++||-||+/−||-||-||-||-||-|
|B. licheniformis WIT 586||+||+||+||++||++||++||+/−||+/−||-||-||+/−|
|B. pumilus WIT 588||+++||+++||++||+++||+++||++||++||+/−||-||-||-|
|B. pumilus WIT 590||-||-||-||-||-||-||+/−||-||-||-||-|
|B. pumilus WIT 592||-||-||-||-||-||-||-||+/−||-||-||-|
|B. licheniformis DSM 5749 2||-||-||-||-||-||-||+/−||++||-||-||-|
|B. subtilis DSM 5750 2||-||-||-||-||-||-||-||-||-||-||-|
1 Mean radii of zones of inhibition from triplicate deferred antagonism assays. + = 0.1–1 mm, ++ = 1.1–2 mm, +++ = 2.1–3 mm, ++++ >3 mm; - = no antimicrobial activity; +/− = variable activity; 2 Isolated from Bioplus 2B®.
2.5. Antimicrobial Activity in Liquid Culture
Firstly, the effects of co-incubation of spores of the marine Bacillus with either S. Typhimurium or E. coli were studied in liquid culture anaerobically (to simulate intestinal conditions). No reduction in Salmonella or E. coli counts was observed compared to controls without Bacillus spores (p > 0.05; data not shown), even though the Bacillus grew as well with or without E. coli/Salmonella (p > 0.05; data not shown). The co-culture experiments were repeated with a lower inoculum of Salmonella/E. coli (i.e., ~102–103 instead of ~106–107 CFU/mL). However, no reductions in either Salmonella or E. coli counts were obtained, even though the Bacillus isolates again grew as well with or without the Gram-negative bacteria (p > 0.05; data not shown). The co-incubation of Bacillus vegetative cells with Salmonella (data not shown) and E. coli (Figure 3) was also studied under aerobic conditions to overcome possible issues with spore germination and to provide the Bacillus isolates with more favorable growing conditions. Again, no reductions in Salmonella counts were obtained, even though the Bacillus grew as well when co-cultured with Salmonella as when grown alone (p > 0.05; data not shown). However, when the same experiment was conducted with E. coli, the increase in E. coli counts was ~10-fold less when co-cultured with B. pumilus WIT 588 (p < 0.001; Figure 3A). The Bacillus grew as well with and without E. coli, except for B. pumilus WIT 584 and WIT 592 which had less of an increase in counts when co-cultured (p < 0.0001; Figure 3B).
2.6. Bile Tolerance and Resistance to Simulated Gastrointestinal Conditions
Ability to survive the harsh environment in the stomach and small intestine is one of the properties required of probiotics. Bile tolerance was first investigated, as bile is a bactericidal agent found in the small intestine. Three of the marine isolates (B. pumilus WIT 582 and 588 and B. licheniformis 586) and one of those isolated from the commercial probiotic product (B. subtilis DSM 5750) grew at concentrations of up to 1% bile, while B. licheniformis DSM 5749 was capable of growth up to 2% (Table 2). Given the origin of the isolates, salt tolerance was also investigated and compared with that of the commercial probiotics. Even though it is not particularly relevant for animal probiotics, salt tolerance may be important for probiotic survival in certain foods. All of the isolates, except B. pumilus WIT 584 and B. licheniformis WIT 586, were capable of growing at concentrations of up to 10% NaCl (data not shown) and can therefore, be considered salt tolerant. Interestingly, this included the commercial probiotics which are not of marine origin. However, none of the isolates was capable of growth at the maximum concentration investigated (15%).
Bacillus spores and vegetative cells were also tested for resistance to simulated gastric juice (pH 2.2) and simulated ileum juice (pH 7) (Figure 4 and Figure 5). After only 30 min of exposure to simulated gastric juice, vegetative cells of B. pumilus WIT 582, WIT 588, WIT 590 and WIT 592 were undetectable (Figure 4A). On the other hand, B. pumilus WIT 584, although reduced by ~4 log CFU/mL, was detectable after 1 h at a survival rate of 0.2%, but not at 90 min. B. licheniformis WIT 586 was the most tolerant of the marine isolates and, again although the count was reduced dramatically, it was capable of surviving for up to 2 h under simulated gastric conditions, albeit at a low rate (0.3%). However, the Bacillus isolated from Bioplus 2B® (B. licheniformis DSM 5749 and B. subtilis DSM 5750) were the most resistant; after an initial 1.4–1.6 log CFU/mL decline, counts remained relatively stable over the remainder of the 2-h incubation period, with a survival rate of 9% obtained for both strains. Only those isolates that demonstrated some degree of resistance to simulated gastric juice (B. pumilus WIT 584, B. licheniformis WIT 586 and DSM 5749 and B. subtilis DSM 5750) were selected for study in simulated ileum juice. Counts of all isolates declined by 1–2 log CFU/mL after 2.5 h but increased or remained stable thereafter (Figure 5A). As a result, final counts recovered after the 5-h incubation period were only slightly lower than initial counts, with survival rates ranging from 4% to 81%. In comparison to vegetative cells, spores of all isolates were more resistant to both simulated gastric and ileum juices. This was evidenced by the fact that counts remained relatively stable during the 2- and 5-h treatment periods, respectively, with maximum survival rates of 63% and 98% obtained (Figure 4B and Figure 5B).
|Table 2. In vitro assessment of probiotic properties of marine Bacillus compared with Bacillus isolated from a commercially available animal probiotic product and a Lactobacillus probiotic.|
|Test strain||Swarming motility 1||Bile tolerance 2||Adherence to intestinal epithelial cells 3|
|B. pumilus WIT 582||+||1%||17.4 ± 8.3 a|
|B. pumilus WIT 584||−||<0.3%||12.1 ± 10.6 a|
|B. licheniformis WIT 586||+||1%||11.9 ± 5.1 a|
|B. pumilus WIT 588||−||1% 4||9.3 ± 4.0 a|
|B. pumilus WIT 590||−||<0.3%||11.0 ± 10.5 a|
|B. pumilus WIT 592||+||<0.3%||268.4 ± 239.7 b|
|B. licheniformis DSM 5749||+||2% 4||116.5 ± 79.9 b|
|B. subtilis DSM 5750||+||1% 4||196.6 ± 87.1 b|
|Lb. rhamnosus GG||ND 5||ND||705.6 ± 182.8 b|
1 + = swarming motility (indicated by growth of the isolate over the entire surface of the plate); − = absence of swarming motility (from triplicate assays); 2 maximum concentration of bile at which growth was observed. Growth was assessed in triplicate on BHI agar containing 0.3, 0.5, 1 and 2% (w/v) porcine bile after 48 h; 3 bacterial cells/100 HT-29 intestinal epithelial cells (mean of triplicate assays ± SE) after 4 h of co-incubation; 4 B. pumilus WIT 588 and B. subtilis DSM 5750 grew at a concentration of up to 0.5% bile after 24 h and 1% after 48 h. B. licheniformis DSM 5749 grew at a concentration of up to 0.5% after 24 h and 2% after 48 h; 5 ND = not determined; a, b values within a column without a common letter are significantly different (p < 0.05).
2.7. Biofilm Formation, Swarming Motility and Adherence to Intestinal Epithelial Cells
Biofilm formation was assessed in all Bacillus isolates, as it may facilitate adherence to intestinal epithelial cells  and could therefore, be considered a probiotic property. All isolates, both marine-derived and those isolated from Bioplus 2B®, were capable of forming biofilms in vitro (data not shown). Swarming motility was also investigated as it may also assist in intestinal colonization . Swarming motility was observed in three of the marine isolates and both of those isolated from the Bioplus 2B® product (Table 2).
Adherence of the marine and Bioplus 2B® isolates to intestinal epithelial cells was also investigated and compared to an adherent Lactobacillus probiotic (Lactobacillus rhamnosus GG) (Table 2). When presented as the number of bacterial cells adhering to 100 epithelial cells, adherence rates did not differ between B. pumilus WIT 592, the Bioplus 2B® isolates (B. subtilis DSM 5750, B. licheniformis DSM 5749) and the positive control Lb. rhamnosus GG (p > 0.05). Furthermore, these isolates demonstrated higher rates of adherence than the other five marine-derived Bacillus strains (p < 0.05). When expressed as percent adherence (i.e., the number of bacterial cells adhering to the epithelial cells relative to the total number added), rates of 0.001%–0.06% were obtained for the Bacillus isolates and no strain differences were observed (p > 0.05). However, Lb. rhamnosus GG displayed a higher adherence rate (0.305%; p < 0.05; data not shown).
2.8. In Vitro Safety Testing of Bacillus Isolates
2.8.1. Hemolytic Activity, Antibiotic Resistance and Presence of Enterotoxin Genes
Data on hemolytic activity of the Bacillus isolates, as determined on sheep blood agar, are presented in Table 3. Only two of the marine isolates (B. pumilus WIT 582 and B. licheniformis WIT 586) showed complete β-hemolysis and three others (B. pumilus WIT 584, 590 and 592) were only weakly or very weakly β-hemolytic. B. pumilus WIT 588 and the two Bioplus 2B® isolates did not display any hemolytic activity. All of the Bacillus isolates were sensitive to the panel of nine antibiotics against which they were tested, as the minimum inhibitory concentration (MIC) values were lower than the breakpoints set by EFSA  (data not shown). We also tested for the presence of genes encoding Bacillus cereus enterotoxins, as although none of the isolates are B. cereus, Fakhry et al.  showed that other species can harbor these genes. All of the isolates were negative in the PCR analysis (data not shown), demonstrating that they did not harbor genes for any of these known Bacillus enterotoxins.
|Table 3. Safety testing of marine Bacillus compared with Bacillus isolated from a commercially available animal probiotic product and a Lactobacillus probiotic.|
|Test strain||Viability of intestinal epithelial cells (%) 1||Hemolysis 2|
|B. pumilus WIT 582||65.9 ± 3||β|
|B. pumilus WIT 584||53 ± 10||very weak β|
|B. licheniformis WIT 586||80.7 ± 4||β|
|B. pumilus WIT 588||69.8 ± 3||γ|
|B. pumilus WIT 590||86.2 ± 7||very weak β|
|B. pumilus WIT 592||91.2 ± 6||weak β|
|B. licheniformis DSM 5749||74.9 ± 6||γ|
|B. subtilis DSM 5750||92.4 ± 10||γ|
|Lb. rhamnosus GG||68.6 ± 5||ND|
1 Viability of HT-29 intestinal epithelial cells (mean of triplicate assays ± SE) compared to the control without bacteria (set at 100% via7bility) as measured by trypan blue staining following 5 h of co-incubation; 2 Incubated on 5% sheep blood agar at 30 °C for 3 days. β = complete hemolysis, with clear zones around each colony; γ = no hemolysis (from triplicate assays).
2.8.2. Cytotoxic Activity against Intestinal Epithelial Cells
All of the Bacillus isolates grew to concentrations of up to 107–109 CFU/mL in the cell culture (CC) medium after 24 h, demonstrating that they were capable of growing well in this medium. The effect of the marine-derived and probiotic Bacillus and the probiotic Lactobacillus on viability of the HT-29 intestinal epithelial cells, as measured by trypan blue staining are shown in Table 3. Two of the marine Bacillus (WIT 590 and WIT 592) and one of the Bacillus probiotics (DSM 5750) caused only minimal (7.6%–13.8%) reductions in cell viability. On the other hand, the other marine Bacillus, one of the probiotic Bacillus and the Lactobacillus probiotic caused reductions of 19.3%–47%.
The xCELLigence real-time cell analysis system was then used to obtain more detailed data on any potential toxic effects of the candidate probiotics on intestinal epithelial cells. The optimal concentration of HT-29 cells was first determined. As illustrated in Supplementary Figure S1, a density of 20,000 cells/mL had the greatest slope in combination with the shortest doubling time, indicating that the cells were in the exponential phase of growth. As the HT-29 cells must be growing exponentially prior to bacterial exposure, this seed number was therefore used in subsequent cytotoxicity assays.
Cytotoxicity of the marine Bacillus was assessed by taking impedance (cell index; CI) measurements over time. Proliferation of cells or cell spreading results in enhanced electrical impedance resulting in a higher cell index (CI) value . The addition of the bacteria produced an initial increase in the CI, followed by a decrease at different rates, depending on the bacterial strain (Supplementary Information, Figure S2). The pathogenic methicillin sensitive Staphylococcus aureus (MSSA) and E. coli strains began to reduce the CI 6 h post-addition, while one of the animal probiotics (B. licheniformis DSM 5749) resulted in a decline after 9 h and the other (B. subtilis DSM 5750), together with the human probiotic Lb. rhamnosus GG, yielded reductions after 15 h (Supplementary Information, Figure S2). On the other hand, it was 22 h post-addition before any of the marine bacteria produced decreases in CI (Supplementary Information, Figure S2).
When the CI of the HT-29 cells co-incubated with the bacteria was calculated relative to that of control HT-29 cells without bacteria, the results showed that after 12 h of co-incubation the MSSA strain decreased the CI compared to the control and all other bacterial strains (Figure 6; p < 0.001) in agreement with the data reported above. The E. coli pathogen as well the B. licheniformis DSM 5749 probiotic also decreased the CI but only when compared with the probiotic B. subtilis and Lb. rhamnosus strains (Figure 6; p < 0.001). After 12 h the marine bacteria did not produce any differences in the CI of the HT-29 cells compared to the control without bacteria (Figure 6; p > 0.05). On the other hand, Lb. rhamnosus GG increased the CI in comparison to all except three treatments (p < 0.001; Figure 6).
After 24 h of co-incubation, the MSSA pathogen together with the B. subtilis probiotic decreased the CI relative to the control and all except two of the bacterial treatments (p < 0.001; Figure 6). In addition, the pathogenic E. coli strain as well as the marine bacteria, B. pumilus WIT 590 and WIT 592, and the probiotic B. licheniformis and Lb. rhamnosus strains decreased the CI relative to the control (p < 0.001; Figure 6). The remaining marine bacteria did not cause any differences compared to the control (p > 0.05; Figure 6).
Bacillus, well-known for antimicrobial production, have other advantages as probiotics, as their spores survive harsh conditions, such as those encountered in the GIT and during product manufacture . Although it is recommended, but not a prerequisite, that probiotics are sourced from the GIT, Bacillus are not considered endogenous intestinal microbiota. Indeed, Bacillus from sources as diverse as soil, fermented soybeans and Chinese herbs have proven efficacious as animal probiotics [10,11,12,14,16,27,28]. The marine environment is an untapped source of novel microorganisms in which antimicrobial production is a common trait  and from which Bacillus is commonly recovered [20,29]. However, although marine-derived Bacillus have been explored as probiotics for aquaculture [3,19], this is the first study to investigate their potential as livestock probiotics.
While the efficacy of animal probiotics must ultimately be demonstrated in vivo , initial in vitro tests are important for strain selection. The six seaweed-derived Bacillus investigated in the present study were selected based on antimicrobial activity against Gram-negative pathogens . This may be due to the production of bacteriocins , as although Bacillus bacteriocins are reportedly more active against Gram-positive bacteria, some have a broader spectrum that also includes Gram-negatives [30,31]. However, additional probiotic properties and safety must be evaluated prior to in vivo use. This was the purpose of the present study, which used two proven Bacillus animal probiotics from an EU-authorized product (Bioplus 2B®) for comparison. Antimicrobial activity against a range of serotypes of Salmonella and E. coli (both zoonotic and animal pathogens) was first demonstrated on solid media for the majority of the marine isolates. More activity was observed against E. coli, in agreement with preliminary findings . The reason for the poor anti-Salmonella activity is not known but might be due to the fact that Salmonella may be resistant to the particular bacteriocins or antimicrobials produced. Most of the marine isolates were more active against Gram-negatives than the isolates from the animal probiotic product but showed less activity against Lactobacillus and Bifidobacterium. The latter is desirable given that these genera are considered beneficial in the intestinal tract.
The fact that the marine Bacillus showed only weak activity against Salmonella in agar plate assays likely explains the lack of anti-Salmonella activity in liquid culture. However, all of the marine isolates previously showed activity against E. coli DSM 10720 on solid media  but only one (B. pumilus WIT 588) reduced counts of this E. coli strain in liquid culture. This may be explained by the fact that the antimicrobial(s) seems not to be released from the bacterial cells. Similarly, Ruiz-Ponte et al.  observed that a marine Roseobacter isolate with activity against Vibrio pectenicida did not reduce counts in co-incubation studies.
Two of the marine isolates were resistant to simulated gastric transit in the vegetative state, although the level of resistance was low and neither was as tolerant as the commercial probiotics. However, if vegetative cells were to be used as probiotics, delivery in a feed matrix would likely provide protection, as previously demonstrated for food . Moreover, the spores of all isolates survived well, which is not surprising considering their resilience. In any case, it appears that once the marine Bacillus have transited the stomach, they will survive, at least in the small intestine, regardless of whether vegetative cells or spores are used. This seems to be the case even if the vegetative cells do not tolerate physiological concentrations of bile (0.3%) , as was the case for B. pumilus WIT 584.
The fact that all of the isolates formed biofilms in vitro and that some exhibit swarming motility may mean that they will be protected from harsh conditions in the GIT. These properties could also prevent pathogen attachment and facilitate intestinal colonization [23,25,35]. The latter is backed up by the fact that all of the marine Bacillus isolates were capable of adhering to colonic epithelial cells in vitro, although all except one were less adherent than the commercially available probiotics. Furthermore, our isolates showed low percent adherence rates, in agreement with those observed for probiotic B. cereus strains , but in contrast to those reported for B. polyfermenticus potential probiotics .
All of the marine Bacillus were also capable of growing anaerobically, in agreement with the fact that B. pumilus and licheniformis are facultative anaerobes. Anaerobic growth from vegetative cells was as good as that of the Bioplus 2B® isolates, which have been shown to grow in the porcine GIT . However, Bacillus feed additives are usually used as spores, mainly due to their resilience to gastrointestinal and manufacturing conditions and their extended shelf-life . All of the marine Bacillus sporulated efficiently (at least as well as the Bioplus 2B® isolates which are used commercially as a spore preparation). It is generally thought that Bacillus spores must be capable of germinating in the intestine in order to obtain a probiotic effect . However, although spore germination is most likely necessary for certain probiotic effects, e.g., antimicrobial production, it may not be a prerequisite, as benefits such as immunomodulation and competitive exclusion could potentially be achieved by spores. Nonetheless, spores of all except one of the marine isolates were capable of germinating under anaerobic conditions, albeit cell numbers did not increase as much as for vegetative cells. This may be explained by the fact that spores need more time to germinate, and then grow than vegetative cells. In any case, anaerobic outgrowth from spores of some of the marine isolates was as good as that of the Bioplus 2B® isolates, which have proven capable of germinating in the porcine GIT . Taken together, these in vitro data suggest that at least some of the marine isolates would be able to survive gastrointestinal transit and subsequently germinate and grow in the mammalian GIT. However, animal feeding trials are required in order to definitively evaluate intestinal survival, as well as spore germination and outgrowth in vivo and these have been reported in a follow-on study published by our group .
However, even if micro-organisms demonstrate probiotic potential, they cannot be used in practice unless they are safe. Particular care is necessary when selecting Bacillus strains for use as probiotics, as there are specific concerns regarding the use of this genus, namely toxin production and dissemination of antibiotic resistance genes . This is highlighted by the fact that toxigenic potential, hemolytic activity and high levels of antibiotic resistance have been found in commercial human Bacillus probiotics [41,42]. Although, B. licheniformis and B. pumilus are considered by EFSA to qualify for “qualified presumption of safety” (QPS) status, a generic qualification for all QPS bacterial taxonomic units is that strains should not harbor any acquired antimicrobial resistance genes to clinically relevant antibiotics . For Bacillus, absence of toxigenic activity should also be demonstrated . When tested in accordance with EFSA guidelines for Bacillus spp. used in animal nutrition  none of the marine isolates were found to harbor enterotoxin genes. Neither were they resistant to any of the antibiotics of human or veterinary importance set out in EFSA guidelines for microbial feed additives . Hemolytic activity was also tested, as it has been found in commercial Bacillus probiotics  and it is a test recommended by EFSA to detect toxigenic potential . B. pumilus WIT 588 was the only marine isolate that showed a complete lack of hemolytic activity, but three other isolates displayed only weak or very weak activity. While in vitro hemolytic activity does not always lead to negative effects in vivo, as evidenced by studies in fish and pigs [36,45], EFSA guidelines state that Bacillus strains proven to be hemolytic are not recommended for use as feed additives . Therefore, it would be preferable to select only the non-hemolytic or perhaps weakly/very weakly hemolytic isolates from the present study for probiotic use.
Cytotoxicity testing is also recommended by EFSA to detect toxigenic potential of Bacillus feed additives . Preliminary assays using the trypan blue dye exclusion assay showed that at least half of the marine Bacillus strains demonstrated some toxic effects against colonic epithelial cells in vitro. However, the dynamic real-time xCELLigence cell analysis system, used here for the first time to evaluate potential probiotics, demonstrated a lack of toxicity of any of the marine bacteria when compared with two known pathogens. Interestingly, the commercial strains (in particular the B. licheniformis) which have a safe history of use as probiotics [12,38] appeared to display greater cytotoxicity than the majority of the marine Bacillus. The greatest toxic effects were observed 24 h post-addition; however, effects observed at this time point could be due to nutrient exhaustion and may be unrelated to toxicity of the bacteria, highlighting the limitations of in vitro assays.
4. Experimental Section
4.1. Bacterial Strains and Culture Conditions and Chemicals
All chemicals and cell culture media were purchased from Sigma Aldrich (Dublin, Ireland) unless otherwise stated. Six seaweed-derived Bacillus isolates, B. pumilus WIT 561, WIT 563, WIT 572,WIT 573 and WIT 574 and B. licheniformis WIT 565, were selected as potential probiotics from a previous screening study on the basis of their antimicrobial activity against E. coli and Salmonella . It should be noted that the WIT 561, 563, 573 and WIT 574 strains referred to as B. pumilus in fact belong to the B. pumilus group, as they cannot be distinguished from other members of this group (Bacillus altitudinis, Bacillus aerophilus, acillus. safensis, Bacillus stratosphericus) by 16S rRNA gene sequencing . The WIT 572 strain has been confirmed as B. pumilus on the basis of sequencing of the gyrB and pyrE housekeeping genes . Rifr variants of each of the Bacillus isolates were generated in order to facilitate subsequent enumeration in vivo according to the method of Gardiner et al.  and were used in all experiments. The Rifr variants were designated B. pumilus WIT 582, WIT 584, WIT 588, WIT 590 and WIT 592 and B. licheniformis WIT 586, respectively. To confirm that the Rifr variants were identical to the parent strains, growth curves were performed by inoculating overnight cultures of each of the isolates (1%) into 250 µL of Brain Heart Infusion (BHI) broth (Oxoid Ltd., Basingstoke, Hampshire, UK) in 96-well plates and incubating at 37 °C with agitation. The OD600 was measured every 10 min for 24 h using a 96-well plate reader (BioTek Instruments, Winooski, VT, USA). Assays were performed in triplicate. Molecular fingerprinting by PFGE was also performed, as described previously . B. licheniformis and B. subtilis were isolated from the Bioplus 2B® animal probiotic product (Chr. Hansen, Hørsholm, Denmark) and their identity confirmed by 16S rRNA gene sequencing, as outlined previously . PFGE fingerprints of the Rifr variants of the marine bacteria were also compared with those of the Bioplus 2B® isolates. The marine Bacillus and the Bacillus isolated from Bioplus 2B® were routinely grown in/on BHI broth/agar at 37 °C, with agitation used for broth cultures. Bacterial strains used as indicators for detection of antimicrobial activity and as controls for hemolysis, enterotoxin gene, adhesion and cytotoxicity assays, and their growth conditions, are listed in Supplementary Information, Table S2.
4.2. Determination of Time Taken to Sporulate and Sporulation Efficiency and Preparation of Spores
The Bacillus cultures were induced to sporulate by the nutrient exhaustion method outlined by Nicholson and Setlow  except that 250 µL of cell suspension was added to a 96-well plate, and the OD600 was measured every 30 min for 48 h using a 96-well plate reader. The start of sporulation is arbitrarily defined as the time at which the cultures cease to grow exponentially in the exhaustion medium and was noted for each isolate. Cultures were harvested by centrifugation (10,000× g, 10 min) 48 h after inoculation and spores were purified using lysozyme treatment and salt and detergent washes as described by Nicholson and Setlow . Spores were suspended in sterile deionized water at 4 °C and a final purification step was added, which involved heating the spore suspensions to 80 °C for 10 min to ensure elimination of all vegetative cells. Total viable counts of spore suspensions were determined by diluting 10-fold in maximum recovery diluent (MRD; Merck, Darmstadt, Germany) and spread plating on BHI agar incubated at 37 °C for 24 h. Purified spores were centrifuged (10,000× g, 10 min) and re-suspended in sterile deionized water at 4 °C. Sporulation efficiency was determined by inducing sporulation, as outlined above and, 48 h after inoculation, performing viable counts before and after heating to 80 °C for 20 min. Sporulation efficiency was calculated as the percentage of survivors remaining after heating.
4.3. Evaluation of Bacillus Growth and Spore Germination under Anaerobic Conditions
The ability of the Bacillus isolates to grow anaerobically was evaluated by inoculating an overnight culture of each (1%) into 5 mL BHI broth and incubating at 37 °C for 24 h aerobically with shaking at 200 rpm and anaerobically in anaerobic jars with CO2-generating kits (Anaerocult A™; Merck). Counts were performed initially (0 h) and after 24 h by diluting 10-fold in MRD, spread plating appropriate dilutions on BHI agar and incubating at 37 °C for 24 h. The ability of the spores to germinate under anaerobic conditions was also evaluated using the same procedure except that spore suspensions were used as the inoculum. Increases in Bacillus counts were calculated as follows:
All experiments were conducted in triplicate.
4.4. Assessment of Antimicrobial Activity of Bacillus Isolates on Solid Media
Antimicrobial activity against the Salmonella, E. coli and Vibrio strains listed in Supplementary Information, Table S2 was determined in triplicate using the deferred antagonism assay as outlined previously , with the Bacillus isolates spotted onto BHI agar and overlaid with the appropriate soft agar seeded with a 0.25% (v/v) inoculum of each of the indicator bacteria. Novobiocin (0.001% w/v) was added to the overlay to prevent overgrowth of Bacillus. Antimicrobial activity against these Gram-negative bacteria as well as the Lactobacillus, Weissella and Bifidobacterium strains listed in Supplementary Information, Table S2 was also determined by the well diffusion assay (WDA), as previously described .
4.5. Assessment of Antimicrobial Activity of Bacillus Isolates in Liquid Culture
In the first experiment, 10 mL BHI broth was inoculated to a final concentration of ~106–107 CFU/mL of Salmonella PT12 (DPC 6465) or E. coli DSMZ 10720 from an overnight culture. Each of the marine Bacillus isolates was then added as a spore suspension at a final concentration of ~106–107 spores/mL and the bacteria were co-incubated anaerobically at 37 °C. In the second experiment, the conditions were the same, except that the initial concentration of Salmonella/E. coli was ~102–103 CFU/mL. The third experiment was conducted in the same way but with an inoculum of ~107 CFU/mL vegetative cells of Bacillus and ~102–103 CFU/mL Salmonella or E. coli incubated aerobically at 37 °C with agitation (200 rpm). In each experiment control cultures were inoculated with Bacillus spores/vegetative cells or Salmonella/E. coli alone, depending on the experiment. Samples were taken at 0 and 24 h. Salmonella, E. coli and Bacillus were enumerated by performing 10-fold serial dilutions in MRD and spread-plating appropriate dilutions on xylose lysine deoxycholate agar (XLD; Oxoid), ChromoCult® tryptone bile X-glucuronide (CTBX; Merck) and BHI agar containing 100 μg/mL rifampicin, respectively. All plates were incubated at 37 °C for 24 h. Increases in Salmonella, E. coli and Bacillus counts were calculated as outlined in Section 4.3. All assays were performed either in duplicate or triplicate.
4.6. Bile, Salt Tolerance and Gastric and Ileum Juice Assays
Tolerance of the Bacillus isolates to porcine bile was assessed as described by Casey et al. , except that BHI agar with bile concentrations of 0.3, 0.5, 1.0 and 2.0% (w/v) was used and plates were incubated aerobically for 48 h. Salt tolerance was tested on BHI plates containing 5, 10 and 15% (w/v) NaCl, incubated at 37 °C for 48 h. For both bile and salt tolerance assays, plates were examined for evidence of bacterial growth after 24 h and 48 h and both assays were conducted in triplicate.
The survival of Bacillus spores and vegetative cells in simulated gastric and ileum juices was determined as described by Dobson et al.  with some modifications as follows; cells from overnight cultures were harvested by centrifugation at 14,000× g for 2 min, washed in MRD and adjusted to OD600 = 1. Spore suspensions were also prepared in this way. Each cell/spore suspension was added to simulated gastric (pH 2.2) or simulated ileum juice (pH 7.0) and incubated for 2 and 5 h, respectively. Samples were taken at regular intervals, and counts obtained by spread-plating on BHI agar incubated at 37 °C for 24 h. While the spores of all isolates were evaluated under simulated gastric and ileum conditions, only the vegetative cells of isolates that survived simulated gastric juice were evaluated in simulated ileum juice. All assays were performed in triplicate.
4.7. Determination of Biofilm Formation and Swarming Motility
Biofilm formation was tested as described by Barbosa et al.  except that overnight cultures of each of the Bacillus isolates were inoculated (1%) into 3 mL low nutrient medium  in 25 mL polypropylene tubes. A positive result was recorded as the presence of a ring of staining at the interface between air and medium. Swarming motility was tested as described by Connelly et al.  using Luria Bertani (LB) agar (Merck) containing 0.7% (w/v) agar. Each assay was performed in triplicate.
4.8. Evaluation of Adherence to Intestinal Epithelial Cells
4.8.1. Cell Culture
The HT-29 colonic epithelial cell line was obtained from the American Type Culture Collection (ATCC, LGC Standards, Middlesex, UK). Cells were routinely grown in McCoy’s 5A modified medium supplemented with 10% fetal bovine serum (CC medium). All cells were routinely maintained in 75 cm2 tissue culture flasks and incubated at 37 °C in a humidified atmosphere with 5% (v/v) CO2. Cells were passaged when they reached ~90% confluence.
4.8.2. Assessment of Bacterial Growth in Cell Culture Medium
Overnight cultures of each Bacillus isolate (1 mL) were centrifuged at 18,620× g for 10 min and the pellet was washed and re-suspended in 1 mL CC medium. Growth curves were then performed by inoculating this bacterial suspension (1%) into 5 mL CC medium, incubating at 37 °C with agitation and measuring the OD600 every 2 h for 10 h and then at 24 h.
4.8.3. Adherence Assays
Suspensions of each of the marine Bacillus, the Bacillus isolated from Bioplus 2B® and the Lb. rhamnosus GG probiotic were prepared in CC medium as outlined above and the OD600 was adjusted in order to obtain 108 CFU/mL. HT-29 cells were seeded into 12 well-plates at a density of 1 × 105 cells/well. Cells were allowed to grow for 48 h and the medium was changed at least 24 h prior to addition of bacteria. Each well was then inoculated with 500 µL bacterial suspension, yielding 5 × 107 CFU/well. A 500 µL aliquot of each bacterial suspension was also added to a 25 mL polypropylene tube containing 500 µL of CC medium to calculate the total CFU/mL after the incubation period. All plates and tubes were incubated in a humidified atmosphere with 5% (v/v) CO2 at 37 °C for 4 h. After the incubation period, the HT-29 cells were washed five times with phosphate buffered saline (PBS) to remove any unattached bacteria. The cell-associated bacteria were determined by lysing the HT-29 cells with Triton X-100 (0.1%) in PBS at 37 °C for 30 min. The number of bacterial cells per mL of CC medium was then determined by diluting the well and tube contents 10-fold in MRD and spread-plating appropriate dilutions on BHI agar incubated at 37 °C for 24 h. The experiment was conducted in triplicate. The number of bacterial cells adhering to 100 HT-29 cells was calculated. Percent adherence was also calculated as follows:
4.9. Analysis of Virulence Factors and Antibiotic Resistance
The Bacillus isolates were PCR-screened for the presence of the genes hbl (C, D, A, and B), nhe (A, B, and C), bceT and cytK, which encode the following B. cereus enterotoxins, respectively; hemolytic enterotoxin BL, non-hemolytic enterotoxin Nhe, bc-D-ENT enterotoxin and cytotoxin K. This was performed as described by Guinebretiere et al. , except that each 15 µL PCR reaction mixture contained 7.5 µL GoTaq® Green Master Mix (Promega, Southampton, UK), 25 ρmoles/µL of both primers and 1 µL of bacterial cells adjusted to OD600 = 1 (NA-1000 NanoDrop®, Thermo Scientific, Wilmington, DE, USA). B. cereus DSM 31 and B. cereus DSM 4384 were used as positive controls, and E. coli DSMZ 10720 was used as a negative control. Hemolytic activity was investigated by streaking isolates in triplicate onto Columbia agar (Sigma Aldrich) with 5% sheep blood (TCS Biosciences Ltd., Buckingham, UK). B. subtilis PY79 and B. cereus DSM 31 were used as negative and positive controls, respectively. Plates were incubated at 30 °C for 3 days and examined for β-hemolysis (clear zones), α-hemolysis (greening) or γ-hemolysis (no change).
The antibiotic susceptibility of the Bacillus isolates was tested by determining the MIC for a panel of nine antimicrobials using a broth dilution method with commercial microtitre plates (Sensititre, TREK Diagnostic Systems Ltd., East Grinstead, UK). The microtitre wells were inoculated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines  and incubated aerobically with agitation at room temperature for 20 h. The MIC was defined as the lowest concentration of antimicrobial that produced no visible growth. Isolates were tested with the following antimicrobials and testing ranges, as recommended by EFSA ; clindamycin (0.12–4 μg/mL), chloramphenicol (4–64 μg/mL), erythromycin (0.25–8 μg/mL), gentamicin (1–16 μg/mL), kanamycin (4–64 μg/mL), quinupristin/dalfopristin (0.5–4 μg/mL), streptomycin (4–32 μg/mL), tetracycline (0.5–16 μg/mL) and vancomycin (1–16 μg/mL). An isolate was considered resistant to a given antimicrobial if the MIC was higher than the breakpoint recommended by EFSA .
4.10. Evaluation of Cytotoxicity against Intestinal Epithelial Cells
Cytotoxicity of the bacterial strains against HT-29 cells was first evaluated using the trypan blue exclusion assay as follows; the HT-29 cells were grown as outlined above and suspensions of the marine and Bioplus 2B® Bacillus and Lb. rhamnosus GG containing 108 CFU/mL were prepared in CC medium as detailed above. One hundred microlitres of each bacterial suspension was then added to 10 mL CC medium, yielding a suspension containing 108–109 CFU/mL and this was added to the HT-29 cells which had been grown in 25 cm2 tissue culture flasks to ~90% confluence. HT-29 cells without bacteria served as a control. The flasks were then incubated in a humidified atmosphere with 5% CO2 at 37 °C for 5 h. After this incubation period the cells were washed five times with PBS and trypsinized to form a cell suspension. Trypan blue was added to an aliquot of this cell suspension and the number of dead (stained) and viable (unstained) cells were counted using a Countess® automated cell counter (Invitrogen, Bio-Sciences, Dun Laoghaire, Co Dublin, Ireland). The experiment was conducted in triplicate. Percent cell viability was calculated as follows:
The xCELLigence real-time cell analysis system (Roche Applied Science, West Sussex, UK) was then used to obtain more detailed data on any potential toxic effects against HT-29 cells. The optimal concentration of HT-29 cells for use in assays was first determined by serially diluting HT-29 cells to final concentrations of 40,000, 20,000, 10,000, 5000, 2500, 1250 and 625 cells/mL in CC medium and seeding in triplicate into xCELLigence E-plates. The E-plates were then inserted into the xCELLigence system and incubated in a humidified atmosphere with 5% CO2 at 37 °C for 24 h. Impedance (CI) measurements were taken every 30 min. CI values were plotted against time and the doubling time and slope of each curve was determined for each cell concentration.
HT-29 cells were then seeded into E-plates at a density of 2 × 104 cells/well (the optimal concentration determined above). The E-plates were inserted into the xCELLigence system and incubated for 20–24 h with 5% CO2 at 37 °C. Cell index readings were taken every 30 min. Cell pellets from overnight cultures of each of the marine Bacillus, the Bioplus 2B® Bacillus, Lb. rhamnosus GG and two pathogenic strains (MSSA and E. coli O157:H7 NCTC 12900) were washed twice with CC medium and re-suspended at a concentration of 108 CFU/mL in CC medium. Wells containing HT-29 cells were then inoculated (1%) with these bacterial cell suspensions in triplicate and the E-plates were incubated for a further 28 h, with CI readings taken every 10 min. The experiment was conducted in triplicate. For each bacterial strain, the CI relative to the control without bacteria (which was set at 100%) was calculated after 12 and 24 h as follows:
4.11. Statistical Analysis
For the anaerobic, antimicrobial activity in liquid culture and adhesion assays, the individual bacterium was considered the experimental unit. Bacterial counts were log-transformed to the base 10 prior to analysis in an attempt to ensure normal distribution. Only data which were normally distributed and with equal variances were analyzed as a one-factor analysis of variance (ANOVA) using the mixed models of SAS . Cytotoxicity data were analyzed as repeated measures using the mixed procedure of SAS with time (12 and 24 h) as the repeated variable and the individual well considered the experimental unit. The appropriate covariance structure, as indicated by the model fit statistics, was fitted to the data. The denominator degrees of freedom were computed using the Satterthwaite approximation. The fixed effect was strain and replication was included as a random effect. Simple main effects were obtained using the “slide” option in SAS. Least squares means were computed and p values were adjusted for multiple comparisons using the Tukey-Kramer adjustment. The level of significance for all tests was p < 0.05.
Taken together, the data from the present study indicate that at least one of the marine Bacillus (B. pumilus WIT 588) has potential for use as a livestock probiotic. The strain satisfied a number of the necessary in vitro criteria, including antimicrobial production (mainly anti-E. coli activity), tolerance to simulated intestinal transit and a lack of toxic effects. Some of the other marine isolates also look promising but hemolytic activity would most likely limit their use as probiotics. The work described in the present study is only the first step in characterizing these marine Bacillus for use as probiotics and, while it provides important information which can be used for strain selection purposes, safety and efficacy can only be definitively determined in animal-feeding trials. A preliminary feeding trial has recently been performed in pigs with the B. pumilus WIT 588 strain and the data published in Prieto et al. .
The authors would like to thank the Higher Education Authority/Institutes of Technology Ireland Technological Sector Research Strand III Programme for funding this research. We also acknowledge Mary Rea and Susan Crowley for technical assistance.
Planned the experiments: Maria Luz Prieto, Laurie O’Sullivan, Montserrat Gutierrez, Jonathan A. Lane, Rita M. Hickey and Gillian E. Gardiner. Performed the experiments: Maria Luz Prieto, Laurie O’Sullivan, Montserrat Gutierrez, Jonathan A. Lane. Analysed the data: Maria Luz Prieto, Laurie O’Sullivan, Jonathan A. Lane, Peadar G. Lawlor and Gillian E. Gardiner. Contributed reagents or other essential material: Maria Luz Prieto, Laurie O’Sullivan, Shiau Pin Tan, Peter McLoughlin, Helen Hughes, Montserrat Gutierrez, Jonathan A. Lane, Rita M. Hickey, Peadar G. Lawlor and Gillian E. Gardiner. Wrote the paper: Maria Luz Prieto and Gillian E. Gardiner. Read and approved the final manuscript: Maria Luz Prieto, Laurie O’Sullivan, Shiau Pin Tan, Peter McLoughlin, Helen Hughes, Montserrat Gutierrez, Jonathan A. Lane, Rita M. Hickey, Peadar G. Lawlor and Gillian E. Gardiner.
Conflicts of Interest
The authors declare no conflict of interest.
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