# Patent

## Title
Antibiotic sensitive Bacillus strains having antimicrobial effect against E. coli and Clostridium perfringens and having high sporulation capacity

## Abstract

A Bacillus strain characterized by (i): sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol; (ii) antimicrobial activity against E. coli and Clostridium perfringens; and (iii) a sporulation percentage of at least 80 when measured after 2 days of incubation. The invention further relates to a method for selecting such strains. Many of the identified strains according to the invention are of the species Bacillus amyloliquefaciens. Some of the Bacillus amyloliquefaciens were further identified as Bacillus amyloliquefaciens subsp. amyloliquefaciens whereas others were identified as amyloliquefaciens subsp. plantarum. A Bacillus strain of the invention may be used as a feed additive to animal feed where it has a probiotic effect.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is the U.S. National Stage of International Application No. PCT/EP2013/057590, filed on Apr. 11, 2013, which claims the benefit of European Patent Application No. 12164087.4, filed on Apr. 13, 2012.
FIELD OF THE INVENTION
Bacillus spp are used for probiotic solutions in the animal feed industry and positive effects of Bacillus based probiotics on production and health in production animals are well known (Spiehs et al., 2008; Cutting, 2011). Their usage is related to the ability of Bacillus to replace or reduce the use of antibiotics, which are used as growth promoters in the animal feed industry.
However, there is an unmet need for Bacillus strains which do not have antibiotic resistance against antibiotics which are commonly used for humans. The present invention provides isolated Bacillus strains which are characterized by sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol and which also have antimicrobial activity against major pathogens such as E. coli and Clostridium perfringens. The strains further have a sporulation percentage of at least 80 when measured on day 2 making it possible to efficiently produce safe and useful Bacillus spores for animal feed production.
The invention further relates to use of the spores of the Bacillus strains of the invention for production of animal feed additives, in particular products for pigs and poultry, where the strains have a probiotic (health, feed utilization and growth promoting) effect.
BACKGROUND OF THE INVENTION
Pigs, especially piglets, suffer from scours, that is, diarrhea, which can be caused by bacteria such as Escherichia coli (E. coli) and Clostridium perfringens Types A and C (C. perfringens). Scours can cause death losses and severe production losses, including weight loss, if left untreated.
E. coli is the primary cause for diarrhea in piglets and 50-75% of the antibiotic used on farms is used against weaning diarrhea, primarily caused by E. coli. Diarrhea is the biggest problem in weaners and growers (up to 40 kg) and E. coli is the most important pathogen causing diarrhea (Klose et al., 2010).
Enteric clostridial infections in swine occur predominantly in the preweaning period but are also associated with hemorrhagic bowel syndrome affecting pigs in the finishing period. Although immunization against C. perfringens type C has greatly reduced pre-weaning mortality, no commercial vaccines are currently available for C. perfringens type A. C. perfringens type A infections are now recognized with increasing frequency in preweaning pigs and approaches to diagnosis and prophylaxis are both different and more complex than those for type C infections.
Several infections and diseases in poultry are caused by pathogenic bacteria, including E. coli and Clostridium perfringens. Infections and diseases caused by pathogens result in increased mortality, decreased performance, and increased production costs. In addition, many of these pathogens can be transmitted to humans. Avian colibacillosis is a systemic infection caused by E. coli and occurs most commonly in young broilers and poults.
Probiotics are used in animal health applications in order to maintain healthy gut microflora, including a reduction in detrimental bacteria such as Clostridia and E. coli and an increase in beneficial bacteria such as Lactobacillus spp. and Bifidobacterium. Probiotics are well-suited to maintaining a healthy balance between pathogenic and beneficial bacteria because, unlike antibiotics, they do not destroy bacteria indiscriminately nor do they lead to antibiotic resistant strains of pathogenic bacteria. There are many mechanisms by which probiotics are thought to maintain healthy gut microflora: competitive exclusion of pathogenic bacteria, reduction of pathogenic bacteria through production of antimicrobial substances, enhancing growth and viability of beneficial gut microflora, and stimulating a systemic immune response in the animal.
In view of the foregoing, it would be desirable to have one or more Bacillus strains to treat or prevent diseases due to infections with E. coli and/or Clostridium in pigs and poultry.
Guo et al., 2006, describes screening of Bacillus strains as potential probiotics and a test of Bacillus subtilis MA139 in pigs. 124 samples were collected from broiler, pigs, soils, fermented foods and Chinese herbs. 750 aerobic spore-forming strains were isolated from these samples. Inhibitory activity against E. coli K88 and K99, Salmonella and Staphylococcus aureus was tested using a disc plate diffusion assay. 6 Bacilli with best activity were tested for their survival within simulated GIT conditions (pH 2 and 0.3% bile salt). B. subtilis MA139 was the best candidate and was tested in vivo in piglets in a 28 days feeding trial with 90 piglets. ADG and feed utilization was improved. Lactic acid bacteria were increased, E. coli in feces was decreased. However, antimicrobial activity against Clostridium perfringens and sensitivity to antibiotics were not tested.
Barbosa et al., 2005 describes isolation of 237 Bacillus from feces from organically (contact to soil) reared broilers. 31 isolates were characterized. B. subtilis and B. licheniformis were among these. Several B. subtilis strains showed inhibition to C. perfringens and S. aureus. B. licheniformis also showed effect against C. perfringens. However, none of the selected Bacillus isolates exhibited antimicrobial activity against E. coli as defined in the present application. One selected Bacillus isolate shows reduction in growth intensity but not complete inhibition against E. coli strain O78:K80 and no effect against the other E coli strain tested (see Table 5). No data is provided on the sporulation percentage after 2 days of incubation or on the sensitivity to vancomycin, kanamycin, streptomycin, and clindamycin.
Chaiyawan et al., 2010, discloses a Bacillus strain sp. T3-1, which is susceptible to antibiotics widely used in medical treatment and which shows antimicrobial activity against C. perfringens ATCC 15191. The strain has no antimicrobial activity against E. coli O157. No data on the sporulation percentage after 2 days of incubation provided.
Benitez et al., 2011 has recently described that the presence of intact or inactivated E. coli enhanced the synthesis of antimicrobial peptides by Bacillus amyloliquefaciens LBM 5006 strain.
U.S. Pat. No. 7,754,469 relates to microorganisms and methods for treating poultry and U.S. Pat. No. 8,021,654 relates to methods of treating pigs with Bacillus strains.
However, in none of these articles or patents there is any description or suggestion to select for Bacillus strains that are sensitive for antibiotics which are commonly used for humans, have antimicrobial activity against both Clostridium perfringens and E. coli and have a high sporulation percentage in order to make the strain useful for efficient sporulation and thus Bacillus probiotic production.
None of the prior art documents e.g. Barbosa et al., 2005, Chaiyawan et al., 2010, and Guo et al., 2006 disclose strains having sensitivity for antibiotics which are commonly used for humans, antimicrobial activity in the sense of inhibition of growth against both Clostridium perfringens and E. coli, and a high sporulation percentage.
In summary, the prior art relating to screening of Bacillus strains does not provide the three distinguishing features of the present invention, i.e. sensitivity for antibiotics which are commonly used for humans, antimicrobial activity against E. coli and Clostridium perfringens and a high sporulation percentage. Nor does the prior art provide Bacillus strains fulfilling these three criteria.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to provide a Bacillus strain, which is characterized by sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol; antimicrobial activity against E. coli and Clostridium perfringens; and a sporulation percentage of at least 80 when measured on day 2.
The solution is based on a selection method developed by the present inventors for the identification of improved Bacillus strains having these improved properties.
A first essential step of the selection method is to specifically screen for Bacillus strains which are sensitive towards the antibiotics which are commonly used for humans. More specifically, the strains are screened for sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol.
Further, the strains are screened for antimicrobial activity against E. coli and Clostridium perfringens and for having a sporulation percentage of at least 80 when measured on day 2.
Out of 261 isolates from soil and feces and food sources investigated by the present inventors, 161 isolates were antibiotic resistant in the pre-screening test described in the examples. Of the 100 isolates that were sensitive to antibiotics 56 had antimicrobial effect against Clostridium perfringens and only 22 had effect against both E. coli and Clostridium perfringens. Of these were 12 isolates from the species B. amyloliquefaciens. Other representative strains were from the species B. subtilis and B. mojavensis. Tables 2 and 3 summarize the results of Chr. Hansen proprietory strains (22 of the 32 strains selected for the secondary screening).
The selection process focused on (i) safety, (ii) effect and (iii) high sporulation in media suitable for production. The safety aspect is mainly based on the absence of antibiotic resistance that is important due to the increased cases of resistant bacteria in human. These bacteria have resulted in well known diseases that no longer can be treated with antibiotics as the pathogen bacteria have become resistant.
It is well known that Bacillus can produce substances that may have antimicrobial activity as i.e. bacteriocins, bacteriolytic enzymes or surfactins. The second selection criterium, effect against E. coli and Clostridium perfringens, is important as both pathogens are main causes for diarrhea in pigs and poultry. The effect is tested against three strains of E. coli and against Clostridium perfringens type A, but it is contemplated that the results are indicative for a general effect against E. coli and for an effect against also other types of Clostridia such as Clostridium perfringens type C.
The third selection criterion is important for the production of the probiotic. The production process takes place in fermentors growing the Bacillus and at the end of the process a high sporulation rate is needed for a high production efficacy. The sporulation process of Bacillus has been investigated for many years but there are still a lot of questions. It is thus well known among persons of skill in the art working with the production and process development of Bacillus that some Bacillus strains have a very low sporulation rate. It has been suggested that Bacillus differentiates into subpopulations of specialized cells as i.e. communities that sporulate, communities that produce enzymes for the degradation of complex nutrients and communities that die (Lopez and Kolter, 2010). This differentiation seems to be regulated by extracellular signals, most of these produced by the Bacillus itself. It has thus been hypothesized that a high production of enzymes or antimicrobial substances may result in a low sporulation efficacy. For the person of ordinary skill in the art it is thus unusual and surprising for a Bacillus strain to have both an antimicrobial activity and a high sporulation percentage.



FIGURES
FIG. 1 shows schematically the antimicrobial activity of 261 Bacilli strains. It is surprising that many Bacillus amyloliquefaciens strains have antimicrobial effect.



DETAILED DESCRIPTION OF THE INVENTION
The phase-out of antibiotic growth promoters in the European Union in 2006 has resulted in an increased need for cost-effective feed additives with high efficacy and thus the need for new probiotics. Bacillus-based probiotic feed additives are known for their positive effects on health and production in pigs and poultry. These products are relevant for the feed industry because spores are heat stable and can survive the pelletizing process at temperatures up to 90-95° C.
Probiotics for pigs need to be safe for animals, humans and the environment and should increase growth and feed utilization of the animal. The objective of the present invention was to screen in three steps a wide range of aerobic endosporeforming bacteria (AEB) from different sources for their probiotic effect in pigs. The AEB were isolated from fermented food (Kantong, and Gergoush primary starters), pig feces, soil and different culture collections. 261 AEB isolates were identified by sequencing of 16S rDNA genes, and investigated for relevant antibiotic resistance by determination of the minimal inhibitory concentration (MIC) of several relevant antibiotics.
Further analyses included bile and acid tolerance, pathogen inhibition, growth in different media, sporulation as well as interactions with animal cell lines to assess the likelihood of positive effects on tight junctions in the intestinal system. Results show a high difference between both species and strains. The isolated species were primarily of the genus Bacillus including B. amyloliquefaciens, B. subtilis and B. safensis from food sources, B. subtilis, B. pumilus, B. amyloliquefaciens, B. licheniformis, B. megaterium from feces and B. licheniformis and B. simplex from soil.
Many of the isolates showed undesirable antibiotic resistance above breakpoints defined by EFSA and were discarded due to safety concerns. Good growth was observed for most of the strains when grown overnight in veal infusion broth, whereas 16% had unsatisfactory growth in a medium suitable for fermentation. In step 2 of the screening process, 32 selected strains with no antibiotic resistance were identified by sequencing the gyrB gene, and PFGE fingerprinting. In addition, their antimicrobial effect on selected pathogens was tested and considerable variation was observed between isolates. Several of the isolates showed inhibition of Clostridium perfringens while only a few isolates inhibited E. coli. The results of the present invention thus confirm that inhibition of growth of both Clostridium perfringens and E. coli is only rarely combined. Step 3 of the screening process involved 10 strains with high pathogen inhibition and included determination of the heat stability of spores, genome sequencing and further in vitro studies showing their effect on tight junctions.
The present invention provides Bacillus strains characterized by (i) sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol.
By the term “sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol” is meant that a strain, to be considered as sensitive to a particular antibiotic, must not grow at the breakpoint level given by EFSA (EFSA, 2008) outlined in Table 1.
The MIC values outlined in Table 1 are based upon the guidelines issued by EFSA (Technical guidance prepared by the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on the update of the criteria used in the assessment of bacterial resistance to antibiotics of human and veterinary importance. The EFSA Journal (2008) 732, 1-15) provides a list of antibiotics and acceptable cut-off values for the genus Bacillus. There is no breakpoint given by EFSA for ampicillin for Bacillus, however a breakpoint exist for several other bacteria, i.e. Lactobacillus spp. Thus this sensitivity of Bacillus strains against ampicillin has been chosen as a breakpoint for the present invention.







TABLE 1







EFSA breakpoints for various antibiotics


commonly used for humans













EFSA





breakpoint



Antibiotic type
Antibiotic
mg/L







B-lactam
Ampicillin
4



Glycopeptide
Vancomycin
4



Aminoglycosides
Gentamicin
4




Kanamycin
8




Streptomycin
8



Macrolide
Erythromycin
4



Lincosamide
Clindamycin
4



Tetracycline
Tetracycline
8



Chloramphenicol
Chloramphenicol
8










To be within the scope of the present invention the strain has to be sensitive towards all of the above antibiotics. In practice this means that no growth of the strain is observed at the breakpoint level when tested by a microdilution method (minimum inhibitory concentration (MIC)).
According to the present invention the MIC is measured by a broth microdilution method as outlined by the standard of CLSI (Clinical and Laboratory Standards Institute M07-A8 and M45-A2) performed as follows:
A suspension of an over-night growth of the strain to be tested is inoculated in ISO-SENSITEST Broth (Oxoid CM0473) in microtitre plates at an approximate concentration of 105 cfu/ml (colony-forming units/ml) in two-fold serial dilutions of the antibiotic to be tested (total volume 100 μl/well) and incubated aerobically for 20-24 hours at 37° C. The prefabricated panels VetMIC Lact-1 & Lact-2 comprising the antibiotics ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol can be used. The results are recorded after 24 hours as the lowest concentration of the antibiotic to inhibit visible growth.
The first part of aspect (ii) of the invention relates to a Bacillus strain which exhibits antimicrobial activity against E. coli. According to the present invention this is measured by the E. coli agar spot test performed as follows:
9 ml of Veal Infusion Broth (VIB) is inoculated with the Bacillus culture to be tested and incubated at 37° C. and 175 rpm overnight. Concurrently, 9 ml of Brain Heart Infusion (BHI) broth is inoculated with an E. coli strain selected from E. coli O149 (O149:k91,k88a), E. coli O147 (O147:K89 F4), and E. coli O101 (O101, F5) and incubated overnight at 37° C.
Overnight cultures of E. coli are added in a volume of 2 ml each into 200 ml liquid VIB agar at 50° C., and poured into each bioassay dish. Dishes are dried in a sterile bench. The over-night Bacillus culture to be tested is spotted onto the surface of the VIB agar mixed with E. coli and incubated at 37° C. for 2 days. Radii of the inhibition zones around the spots and spots diameters are recorded.
A Bacillus strain is considered to exhibit an antimicrobial activity towards E. coli if the inhibition zone is at least 1.5 mm. Preferably, the inhibition zone is at least 2.0 mm, such as at least 2.5 mm, more preferably at least 3 mm, most preferably at least 3.5 mm and even more preferably at least 4 mm. The inhibition zone may be different for the various E. coli strains. For a strain to be considered to exhibit an antimicrobial activity against E. coli according to the present invention it should exhibit an inhibition zone of at least 1.5 mm for all of the E. coli strains tested. Preferably, the inhibition zone of two or even more preferably the inhibition zone of all three of the E. coli strains is at least 2 mm. A Bacillus strain of the invention is characterized by inhibition of growth of E. coli, in particular inhibition of growth of the tested species. As evidenced by the prior art and confirmed by the present inventors, no inhibition of growth of one E. coli species is often combined with no inhibition of growth of another E. coli species (Table 5, Barbosa et al., 2005) and vice versa, i.e. inhibitory activity of one E. coli species is often combined with inhibitory activity of other E. coli species (Table 1, Guo et al., 2006).
The second part of aspect (ii) of the invention relates to a Bacillus strain which exhibits antimicrobial activity against Clostridium perfringens. According to the present invention this is measured by the Clostridium perfringens agar spot test performed as follows:
9 ml of VIB is inoculated with the Bacillus culture to be tested and incubated at 37° C. and 175 rpm overnight. Concurrently, 9 ml of BHI broth is inoculated with Clostridium perfringens Type A, DSM 756, and incubated overnight at 37° C. in an anaerobic jar.
Bacillus cultures are spotted onto the surface of the VIB agar in petri dishes and incubated at 37° C. overnight. C. perfringens overnight culture in a volume of 2 ml is mixed with 200 ml liquid BHI agar, and poured onto VIB agar with grown Bacillus spots. The dishes are incubated anaerobically at 37° C. for 1 day. Radii of clarified inhibition zones round the spots are measured.
A Bacillus strain is considered to exhibit an antimicrobial activity towards Clostridium perfringens if the inhibition zone is at least 5 mm. Preferably, the inhibition zone is at least 6 mm, more preferably at least 7 mm. A Bacillus strain of the invention is characterized by inhibition of growth of Clostridium perfringens, in particular inhibition of growth of the tested species. As known by the person of skill in the art, no inhibition of growth of one species is often combined with no inhibition of growth of other Clostridium perfringens species and vice versa, i.e. inhibition of growth of one Clostridium perfringens species is often combined with inhibition of growth of other Clostridium perfringens species.
Bacillus cells exist as bacillus spore cells and bacillus vegetative cells. When reference is made herein to Bacillus cells, this relates to both.
The term “Bacillus spore” in relation to a Bacillus spore cell relates herein to a spore that according to the art may be characterized as a dormant, tough, non-reproductive structure produced by Bacillus bacteria. The primary function of spores is generally to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to ultraviolet and gamma radiation, desiccation, lysozyme, temperature, starvation, and chemical disinfectants. Spores are commonly found in soil and water, where they may survive for long periods of time. The spore coat is impermeable to many toxic molecules and may also contain enzymes that are involved in germination. The core has normal cell structures, such as DNA and ribosomes, but is metabolically inactive. When a bacterium detects that environmental conditions are becoming unfavorable it may start the process of sporulation, which takes about eight hours.
The term “Bacillus vegetative cell” relates to functional vegetative Bacillus cells, which can divide to produce more vegetative cells.
In aspect (iii), the invention relates to a Bacillus strain which exhibits a sporulation percentage of at least 80 when measured on day 2. According to the present invention, the sporulation percentage is assayed as follows:
A Bacillus strain to be tested is added in a volume of 50 μl into 700 μl VIB in a Deep well (DW) plate and incubated at 37° C. and 175 rpm overnight. The Bacillus overnight culture in a volume of 50 μl is transferred to 700 μl of a sporulation medium comprising (w/w) 95% water; 1.5% nitrogen source (i.e. yeast); 3% sucrose; 0.06% microminerals; dipotassiumhydrogenphosphate 0.1% in DW plates. The plate is incubated at 37° C. and 175 rpm for 3 days.
Sporulation is followed microscopically and spore percentage (number of spores compared to the total number of Bacillus cells) is determined by visual evaluation after 1 day (24 hours), 2 days (48 hours) and 3 days (72 hours) of incubation.
By the term “having a sporulation percentage of at least 80 when measured on day 2” is meant that at least 80% of the cells have sporulated after 2 days of incubation. The sporulation percentage may preferably be higher such as at least 85%, at least 90%, at least 95% or at least 99%. An important object of the present invention is to select for Bacillus strains having cells with a high sporulation percentage in order to make the strain useful for animal feed production. A high sporulation percentage which may also be termed a high sporulation rate is needed for a high production efficacy as described above.
As described above, the prior art has described methods for selecting Bacillus strains, but the prior art screening methods have not focused on the sporulation percentage. Accordingly, the prior art selected Bacillus strains are not likely to sporulate to a sufficient degree to comply with the sporulation percentage as described herein.
Three strains having the combined ability of high growth properties and sporulation, no antibiotic resistance and high antimicrobial activity have been selected and deposited. But also other strains, in particular strains of the species Bacillus amyloliquefaciens (see strains D-J in Tables 2 and 3) fulfil the criteria outlined in the claims and thus included within the scope of the present invention.
As evident from FIG. 1 in particular many strains of the species Bacillus amyloliquefaciens are within the scope of the present invention. It is surprising that many Bacillus amyloliquefaciens strains have antimicrobial effect, as it has been assumed up to date that antimicrobial effect of the genus Bacillus is strain-specific and not related to species.
Based on the detailed assay descriptions the person of ordinary skill in the art is able to repeat these assays to determine whether a specific Bacillus strain complies with the sensitivity of item (i), the antimicrobial activity of item (ii) and the sporulation percentage of item (iii) of the various aspects of the invention. In this manner the person of ordinary skill in the art will be able to consistently produce strains with the stated properties. Preferably, the selection method will also include (iv) assaying for sensitivity of the vegetative cells at pH 4, and (v) assaying for bile resistance to ensure that the strains are able to survive to a sufficient degree in the gastrointestinal tract. Evidently, these assays can be performed in any order and some strains may be excluded during the process if they do not fulfill the criteria.
It is known from the literature that bile has some negative influences on the survival and germination and outgrowth of bacillus spore cells to vegetative cells in the GIT of animals.
Therefore probiotic bacteria shall generally be able to survive and proliferate in the gut of animals by being able to tolerate a low pH and resistant to bile salt in order to be useful as probiotic bacillus compositions for the addition to animal feed. The examples provide useful in vitro tests in this regard. The test for sensitivity to low pH (simulating gastric conditions) focuses on the resistance of vegetative cells to pH 4. It is well known that spores are resistant at pH values of 2-3 and that vegetative cells will die at pH 2. However, gastric pH may have pH values of up to 4 especially in feeding conditions. This may result in germination of the spores and it is thus relevant to test the sensitivity of vegetative cells at pH 4. Selected strains should preferably be resistant to pH at 4. Results for selected strains are presented in Table 2.
The strain of the invention is of the genus Bacillus, preferably one of the species Bacillus amyloliquefaciens, such as Bacillus amyloliquefaciens subsp. amyloliquefaciens or Bacillus amyloliquefaciens subsp. plantarum, Bacillus simplex, Bacillus licheniformis, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, Bacillus safensis, Bacillus simplex, Bacillus subtilis, Bacillus atrophaeus, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis or Bacillus tequilensis. 
The initial method of identification of the 261 strains was based on 16S. This method cannot distinguish between some closely related species of Bacillus. Thus, a strain may be identified as related to the group consisting of the species Bacillus amyloliquefaciens/atrophaeus/methylotrophicus/siamensis/vallismortis or the group consisting of the species Bacillus mojavensis/subtilis/tequilensis. Both groups contain many strains which fulfill the criteria of the invention and these groups thus represent important embodiments of the invention.
Where considered appropriate, the strains were further identified by a more detailed method (gyr B)). The data shown in Tables 2 and 3 are primarily based on Bacillus amyloliquefaciens (identified by gyr B). Selected Bacillus amyloliquefaciens isolates were further identified by RNA polymerase beta subunit (rpo B) gene sequence analysis and the subspecies identified and presented in Tables 4, 5 and 6.
It is desirable that the strain exhibits heat stability. Results for selected strains are presented in Table 4. The heat stability at 99.5° C. is measured in cfu as reduction after 2, 5 and 10 min in relation to time 0 (log/log). A reduction below 2 is achieved with common commercial Bacilllus spore formulations. For strains within the scope of the present invention the reduction should preferably be 0.5 or less after 2 min, more preferably 0.25 or less, most preferably 0.05 or less. In preferred embodiments the reduction after 5 min should preferably be 2.5 or less, more preferably 1 or less, most preferably 0.5 or less and after 10 min the reduction should also preferably be 2.5 or less, more preferably 1 or less, most preferably 0.5 or less. All of the strains in the table exhibit an appropriate heat stability. As evident from the table strains B, D and F have a very high heat stability even after 10 min. It is noteworthy that both B and F are Bacillus amyloliquefaciens subsp. amyloliquefaciens strains making this subspecies a preferred embodiment of the present invention.
Enzyme production has been investigated in Example 4. The present findings show for all investigated strain a cellulase activity of 50 mU/ml or more. It is contemplated that such an activity will be a beneficial property for a Baccillus strain of the invention. For certain embodiment it may be preferred that the strain has an even higher cellulase activity, such as 100 mU/ml or more, as found for the B. amyloliquefaciens subsp. plantarum strains making this subspecies a preferred embodiment of the present invention. For strains within the scope of the present invention the cellulase activity should preferably be 50 mU/ml or more, more preferably 100 mU/ml or more, most preferably 250 mU/ml or more, even more preferably 400 mU/ml or more.
Some strains show a high xylanase acticity of 70 mU/ml or more. Table 5 shows that for the investigated strains high cellulase activity is not necessarily combined with high xylanase or high protease activity defined as 40000 RFU/OD or more. Strains G, I and J which are all B. amyloliquefaciens subsp. plantarum are examples of strains demonstrating high activity for all three enzymes.
In a preferred embodiment the Bacillus strain is a Bacillus subtilis, a Bacillus mojavensis or a Bacillus amyloliquefaciens. Most preferably, the strain is selected from the group consisting of (a) the Bacillus mojavensis strain with accession number DSM 25839; (b) the Bacillus amyloliquefaciens strains with accession number DSM 25840, accession number DSM 27032 or accession number DSM 27033, and (c) the Bacillus subtilis strain with accession number DSM 25841; and mutant strains thereof.
Another aspect of the invention relates to a method for obtaining a mutant strain of

    (a) the Bacillus mojavensis strain with accession number DSM 25839;
    (b) the Bacillus amyloliquefaciens strains with accession number DSM 25840, accession number DSM 27032 or accession number DSM 27033; or
    (c) the Bacillus subtilis strain with accession number DSM 25841;

the method comprising optionally subjecting the strain to mutagenization treatment and selecting for mutant strains having the following properties

    (i): sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol;
    (ii) antimicrobial activity against E. coli and Clostridium perfringens, and
    (iii) a sporulation percentage of at least 80 when measured on day 2.


The strain may be subjected to a mutagenization treatment as described in further detail below to obtain mutant strains and afterwards a selection process is performed. Alternatively, a selection is performed for spontaneously occurring mutants.
The method for obtaining a mutant strain may also include (iv) assaying for sensitivity of the vegetative cells at pH 4, and (v) assaying for bile resistance to ensure that the strains are able to survive to a sufficient degree in the gastrointestinal tract. Evidently, these assays can be performed in any order and some strains may be excluded during the process if they do not fulfill the criteria.
A bacterial “strain” as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied. The multiplicity of identical bacteria are included.
“Wild type strain” refers to the non-mutated form of a bacterium, as found in nature.
A “mutant bacterium” or a “mutant strain” refers to a natural (spontaneous, naturally occurring) mutant bacterium or an induced mutant bacterium comprising one or more mutations in its genome (DNA) which are absent in the wild type DNA. An “induced mutant” is a bacterium where the mutation was induced by human treatment, such as treatment with any conventionally used mutagenization treatment including treatment with chemical mutagens, such as a chemical mutagen selected from (i) a mutagen that associates with or become incorporated into DNA such as a base analogue, e.g. 2-aminopurine or an interchelating agent such as ICR-191, (ii) a mutagen that reacts with the DNA including alkylating agents such as nitrosoguanidine or hydroxylamine, or ethane methyl sulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UV- or gamma radiation etc. In contrast, a “spontaneous mutant” or “naturally occurring mutant” has not been mutagenized by man.
A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening/selection steps) are carried out. In a presently preferred mutant less than 1%, less than 0.1, less than 0.01, less than 0.001% or even less than 0.0001% of the nucleotides in the bacterial genome have been replaced with another nucleotide, or deleted, compared to the mother strain.
Mutant bacteria as described above are non-GMO, i.e. not modified by recombinant DNA technology. As an alternative to above preferred method of providing the mutant by random mutagenesis, it is also possible to provide such a mutant by site-directed mutagenesis, e.g. by using appropriately designed PCR techniques or by using a transposable element which is integratable in bacterial replicons.
When the mutant is provided as a spontaneously occurring mutant the above wild-type strain is subjected to the selection step without any preceding mutagenization treatment.
Several species of Bacillus have GRAS status, i.e., they are generally recognized as safe. All B. subtilis strains are GRAS. The Bacillus strains described herein are aerobic and facultative spore formers. Bacillus species are the only spore formers that are considered GRAS. Feeding microorganisms that have GRAS status to livestock is an acceptable practice amongst producers, veterinarians, and others in the livestock industry.
Accordingly, in a further aspect the invention relates to a Bacillus composition comprising cells of a Bacillus strain of the invention. The composition may comprise cells of at least one, at least two, at least three, at least four or even more Bacillus strains chosen from at least one of the strains of the invention. Preferably, the cells of the Bacillus composition are spore cells.
The relevant Bacillus strains of the composition may be present in a commercially relevant form known to the skilled person. Accordingly, in an embodiment the Bacillus strains of the composition are present as dried (e.g. spray dried) cells or as frozen cells. The composition may be provided in any suitable form such as in the form of a liquid, a slurry, a powder or a pellet.
In a preferred embodiment the Bacillus composition comprises from 105 to 1012 CFU/g, more preferably from 106 to 1012 CFU/g, and most preferably from 107 to 1012 CFU/g.
The term “CFU/g” relates to the gram weight of the composition as such, including suitable relevant additives present in the composition. As known to the skilled person a commercially relevant bacterial composition generally also comprises other relevant additives such as e.g. one carrier/ingredient of the group belonging to whey, whey permeate, calcium carbonate/limestone and anti caking agents such as aluminum silicates and kieselgur (diatomaceous earth). It does not include the weight of a suitable container used to package the Bacillus composition. An embodiment relates to a composition packaged into a suitable container.
Compositions of the present invention may include a Bacillus strain of the invention including mutants, and carriers that make these compositions suitable for feeding to animals as a feed additive or as an additive for drinking water. Alternatively, the Bacillus strain of the invention including mutants may be formulated with animal feed ingredients, including feed protein and/or feed carbohydrates. Such combinations may be in the form of pellets that are extruded through standard pelleting processes.
The Bacillus composition as described herein may be used as a probiotic additive to animal feed. The invention also provides a method for producing an animal feed or premix comprising adding a Bacillus composition of the invention to an animal feed.
As used herein the term “premix” refers to a Bacillus strain added to a carrier to make a premix which is then added to the feed at a desired inclusion rate and fed to the animal.
Another aspect of the invention relates to a method for feeding an animal comprising administering a Bacillus composition of the invention or an animal feed or premix produced according to the invention to an animal.
Example 5 describes feeding trials with strains B and C and shows that both Bacillus strains probiotic products supplemented to nursery diets numerically improved productive performance compared with a negative control group. Significant effect on production parameters could be observed during the trial. Mortality percentage was reduced in both Bacillus groups and in both trials.
In one of the sites, the number of animals treated per pen with Enrofluxacin to overcome a severe diarrhea was significantly higher (P>0.05) in those animals fed the control diet than those fed Bacillus. 
This example thus demonstrates that administration of the Bacillus composition of the invention can be used for treating and preventing diseases e.g. by inhibiting pathogens, such as E. coli and Clostridium, in the animal. The Bacillus composition can be fed as a direct-fed microbial or as a feed additive to animal feed. The compositions of the present invention are administered or fed to an animal in an amount effective to decrease the growth of pathogenic bacteria such as Clostridia and Escherichia coli in the animal gut.
The animal may be selected from the group consisting of poultry, ruminants, calves, pigs, rabbits, horses, fish and pets. In a preferred embodiment, the animal is a farm animal, which is raised for consumption, such as pigs, or as food-producers, such as broilers and egg-producing chickens.
Methods of administering one or more Bacillus strains of the invention to a piglet are also provided. Such methods may include feeding one or more Bacillus strains of the invention to a mother of a piglet. The strain(s) may be fed during gestation, lactation, or both. The one or more Bacillus strain may also be fed to nursery pigs and to grow-finish pigs.
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “having”, “including” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Deposited Strains
The Bacillus mojavensis strain CHCC 15510 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 25839 with a deposit date of Apr. 3, 2012 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
The Bacillus amyloliquefaciens strain CHCC 15516 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 25840 with a deposit date of Apr. 3, 2012 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
The Bacillus amyloliquefaciens strain CHCC 15536 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 27032 with a deposit date of Mar. 21, 2013 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
The Bacillus amyloliquefaciens strain CHCC 15539 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 27033 with a deposit date of Mar. 21, 2013 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
The Bacillus subtilis strain CHCC 15541 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) under the accession number DSM 25841 with a deposit date of Apr. 3, 2012 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
For all of the above-identified deposited microorganisms, the following additional indications apply:
As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn
Embodiments of the present invention are described below, by way of non-limiting examples.
EXAMPLES
Materials


    Veal Infusion Broth (VIB) (Difco, 234420)
    Veal Infusion Broth (VIB) agar (VIB+1.5% Agar bacteriological (Agar no. 1), Oxoid LP0011) T3 agar plates (per liter: 3 g of tryptone, 2 g of tryptose, 1.5 g of yeast extract, 0.05 M sodium dihydrogen phosphate and 0.005 g of MnCl2 [pH 6.8], and 15 g agar)
    Laura-Bertani (LB) broth (g/L: Bacto tryptone 10 (Difco 0123), Yeast extract 5 (Oxoid L21),
    NaCl 10 (Merck nr. 106404))
    Brain Heart Infusion (BHI) Broth (Oxoid CM225)
    Brain Heart Infusion (BHI) agar (Oxoid CM375)
    Bile salts (Bile extract, porcine; Sigma B8631)
    Bioassay dishes (Nunc 240845)
    Petri dishes (Procudan 140096, petridish with ribs)
    Sporulation medium: %(w/w) 95% water; 1.5% nitrogen source (i.e.yeast); 3% saccharide; 0.06% microminerals; dipotassiumhydrogenphosphate 0.1%.
    Physiological saline solution with peptone (0.9% sodium chloride, 1% peptone) FKP
    VetMIC Lact-1 & Lact-2 (SVA, Uppsala, Sweden)
    ISO-SENSITEST Broth (Oxoid CM0473)

Cultures:



Bacillus strains were isolated from feces, soil, food sources and collected from strain bank collections and maintained in VIB with 20% glycerol in MTP master plates at −80° C.
Antibiotics:


    Ampicillin (Sigma, A9518-5G)
    Vancomycin (Sigma, V1764-250MG)
    Gentamicin (Sigma, G1264-50MG)
    Kanamycin (Sigma, K1377-1G)
    Streptomycin (Sigma, S6501-5G)
    Erythromycin (Sigma E-5389)
    Clindamycin (Sigma, C2569-10MG)
    Tetracycline (Sigma T-7660)
    Chloramphenicol (Sigma, C0378-5G)

Pathogens:

    E. coli 0101 F5 (State Serum Institute, Copenhagen, Denmark)
    E. coli 0147:K89 F4 (State Serum Institute, Copenhagen, Denmark)
    E. coli 0149:k91,k88a (NCTC 10650) National Collection of Type Cultures
    E. coli strains were maintained in LB with 20% glycerol in MTP master plates at −80° C. Clostridium perfringens Type A, DSM 756, was maintained in BHI with 20% glycerol at −80° C.


Example 1
Pre-Screening
261 isolates from soil and feces and food sources were subjected to a pre-screening for antibiotic sensitivity, pathogen inhibition, bile resistance and sensitivity to low pH.
1.1 Antibiotic Sensitivity
Bacillus strains were added in a volume of 50 μl from MTP master plates into 700 μl VIB in DW plates and incubated at 37° C. and 175 rpm overnight. ISO test samples, supplemented with the antibiotics listed in Table 1 at 2 concentrations and ISO controls without antibiotics were added in MTP plates in a volume of 180 μl. Overnight Bacillus cultures were diluted 100-fold and transferred in aliquots of 20 μl to ISO test samples and controls. MTP plates were incubated at 37° C. Optical density (OD) at 620 nm was measured in the inoculum and in MTP test plates after 24 and 48 hours of incubation. Antibiotic sensitivity of bacteria was estimated as percentage of OD in ISO test samples to the OD in ISO controls.
1.2 Screening of Bacillus Strains for Pathogen Inhibition
Bacillus strains were added in a volume of 50 μl from MTP master plates into 700 μl VIB in DW plates and incubated at 37° C. and 175 rpm overnight.
Before the assay E. coli strains were grown in LB overnight at 30° C. C. perfringens CHCC14372 was grown in BHI overnight in an anaerobic jar at 37° C.
1.2.1 E. coli Inhibition by Agar Spot Test
2 ml of E. coli overnight culture was mixed with 200 ml liquid VIB agar at 50° C., and poured into each bioassay dish. The dishes were dried in a sterile bench. Overnight Bacillus cultures, 2 μl of each, were spotted onto the surface of the VIB agar mixed with E. coli and incubated at 37° C. for 2 days. Radii of clarified inhibition zones round the spots were measured and recorded as “high”—radius more than 2 mm, “medium”—radius between 0.5-2 mm and “low”—radius less than 0.5 mm.
1.2.2 C. perfringens Inhibition by Agar Spot Test
VIB agar was poured into the bioassay dishes (200 ml per dish) and dried thoroughly in a sterile bench. Overnight Bacillus cultures, 2 μl of each, were spotted onto the surface of the VIB agar dishes and incubated at 37° C. overnight. C. perfringens Type A CHCC14372 was added in a volume of 2 ml to 200 ml liquid BHI agar, mixed and overlaid gently into the bioassay dishes with Bacillus spots. The dishes were incubated anaerobically at 37° C. for 1 day. Radii of clarified inhibition zones round the spots were measured and recorded as “high” more than 2 mm, “medium”—between 1-2 mm, “low”—less than 1 mm.
1.2.3 C. perfringens Inhibition by Well Diffusion Test
2 ml of C. perfringens CHCC14372 overnight culture was mixed with 200 ml liquid BHI agar and poured into each bioassay dish. After solidification, the wells of 10 mm were made in BHI agar with a sterile borer and 80 μl of overnight Bacillus cultures were transferred into each well. Dishes were incubated anaerobically at 37° C. for 1 day. Radii of clarified inhibition zones round the wells were measured and recorded as “high”—more than 2 mm, “medium”—between 0.5-2 mm and “low”—less than 0.5 mm.
1.3 Bile Resistance Assay
Bacillus strains were added in a volume of 50 μl from MTP master plates into 700 μl VIB in DW plates and incubated at 37° C. and 175 rpm overnight. Bacillus overnight cultures in a volume of 50 μl were transferred to 800 μl VIB supplemented with 0.3% bile salts (test samples) and VIB without bile (controls) in DW plates. Plates were incubated at 37° C. and 175 rpm. Optical density at 620 nm (OD620) was measured at time 0, 6 and 24 hours in VIB with bile and subtracted from the corresponding OD620 values in VIB controls. Cultures were ranked according to the differences in OD620 values into groups A (OD<0.1), B (OD=0.1-0.4) and C (OD>0.4). Strains in the group A were considered as most resistant to bile salts. Selected strains should be in group A or B. Results for selected strains are presented in Table 2.
1.4 Sensitivity to Low pH (Simulating Gastric Conditions)
VIB aliquots of 800 μl adjusted to pH4 with 1.0 M hydrochloric acid and VIB controls (pH7) were distributed in DW plates. Bacillus strains were added in a volume of 50 μl from MTP master plates into DW plates and incubated at 37° C. and 175 rpm for 24 hours. Optical density (OD620) was measured in 200 μl cell suspensions at time 0 and after 24 hours of incubation. Bacterial growth at pH4 was defined by subtraction of OD620 values before incubation from the corresponding values after incubation. Cultures showing increase in OD620 of more than 0.1 were considered as resistant to pH4 (indicated with an R in Table 2), while the cultures with values OD620 less than 0.1 (no growth or negative values) were considered as sensitive to pH4 (indicated with an S in Table 2).
1.5 Results of Pre-Screening:
Based on the pre-screening test 32 strains were selected for the secondary screening. The selected strains should be sensitive for the described antibiotics and show antimicrobial effect against both E. coli and Clostridium perfringens and perform reasonably in the other performed tests.
Of the 261 isolates from soil and feces and food sources, 161 isolates were antibiotic resistant in the pre-screening test. Of the 100 isolates that were sensitive to antibiotics 56 has antimicrobial effect against Clostridium perfringens and only 22 had effect against both E. coli and Clostridium perfringens. Of these were 12 isolates from the species B. amyloliquefaciens. Other representative strains were from the species B. subtilis and B. mojavensis. Tables 2 and 3 summarize the results of Chr. Hansen proprietory strains (22 of the 32 strains selected for the secondary screening).
Example 2
Secondary screening
Based upon the results of the primary screening 40 selected isolates and reference strains were tested for inhibition of pathogens by repetition of the agar spot tests and for sporulation. 32 strains were also tested for antibiotic sensitivity by the MIC test.
2.1 Screening of Bacillus Strains for Pathogen Inhibition
Before the assay 9 ml of VIB was inoculated with Bacillus cultures and incubated at 37° C. and 175 rpm overnight. Concurrently, 9 ml of BHI broth was inoculated with E. coli strains, and incubated overnight at 37° C. Clostridium perfringens was grown at the same conditions in an anaerobic jar.
2.1.1 Inhibition of E. coli by Agar Spot Test:
Overnight cultures of pathogens were added in a volume of 2 ml each into 200 ml liquid VIB agar at 50° C., and poured into each bioassay dish. Dishes were dried in a sterile bench. Overnight Bacillus cultures were spotted onto the surface of the VIB agar mixed with pathogens and incubated at 37° C. for 2 days. Radii of the inhibition zones around the spots and spots diameters were recorded.
2.1.2 Inhibition of C. perfringens by Agar Spot Test
Bacillus cultures were spotted onto the surface of the VIB agar in petri dishes and incubated at 37° C. overnight. C. perfringens overnight culture in a volume of 2 ml was mixed with 200 ml liquid BHI agar, and poured into VIB agar with grown Bacillus spots. The dishes were incubated anaerobically at 37° C. for 1 day. Radii of clarified inhibition zones round the spots were measured.
2.2 Growth in VIB and in Sporulation Medium
Bacillus strains were added in a volume of 50 μl from MTP master plates into 700 μl VIB or sporulation medium in DW plates and incubated at 37° C. and 175 rpm for 24 hours. Bacterial growth was determined by optical density at 620 nm (OD620) in 200 μl suspensions. Because of a high turbidity of the sporulation medium and its variation between the wells, OD620 values before incubation were subtracted from the OD620 values in the corresponding wells after incubation. Cultures showing OD620 more that 0.4 were ranked to the group A (high growth) and OD620 less than 0.4—to the group B (low growth).
2.3 Sporulation in Sporulation Medium
Bacillus strains were added in a volume of 50 μl from MTP master plates into 700 μl VIB in DW plates and incubated at 37° C. and 175 rpm overnight. Bacillus overnight cultures in a volume of 50 μl were transferred to 700 μl of a sporulation medium (SM) in DW plates. Plates were incubated at 37° C. and 175 rpm for 3 days. Sporulation was followed microscopically and spore percentage (number of spores in relation to total cells) was determined by visual evaluation after 1 day (24 hours), 2 days (48 hours) and 3 days (72 hours) of incubation.
2.4 Antibiotic Sensitivity Measured by MIC
The strains were analyzed for antibiotic sensitivity by measuring the minimum inhibitory concentration (MIC) for a number of antibiotics. The method used was a broth microdilution method as outlined by the standard of CLSI (Clinical and Laboratory Standards Institute M07-A8 and M45-A2).
A suspension of an over-night growth of the strain to be tested is inoculated in ISO-SENSITEST Broth (Oxoid CM0473) in microtitre plates at an approximate concentration of 105 cfu/ml (colony-forming units/ml) in two-fold serial dilutions of the antibiotic to be tested (total volume 100 μl/well) and incubated aerobically for 20-24 hours at 37° C. The prefabricated panels VetMIC Lact-1 & Lact-2 comprising the antibiotics ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol can be used. The results are recorded after 24 hours as the lowest concentration of the antibiotic to inhibit visible growth. The test was performed twice as two independent biological replicates.
2.5 Results
Results from the 32 selected strains showed that Bacillus amyloliquefaciens had the best combined properties of antibiotic sensitivity, antimicrobial effect and sporulation cf. Table 2. Only data relating to Chr. Hansen proprietary strains (22 of the 32 strains) are included.







TABLE 2







Basic data for selected Bacillus strains


Source, species by gyrB, growth in VIB and sporulation


medium as well as sporulation percentage.


Strain A = 15510; Strain B = 15516; Strain C = 15541;


Strain H = 15536; Strain I 15539











Antibiotic
Growth
Sporulation, %















Strains
Source
Species
Resistance
VIB
SM
Day 1
Day 2
Day 3


















A
Feces
B. mojavensis
Sensitive
A
B
5
95
90


B
Feces
B. amyloliquefaciens
Sensitive
A
A
10
99
99


C
Feces
B. subtilis
Sensitive
B
A
0
80
95


D
Feces
B. amyloliquefaciens
Sensitive
B
A
40
99
99


E
Feces
B. amyloliquefaciens
Sensitive
B
A
20
99
99


F
Feces
B. amyloliquefaciens
Sensitive
B
A
50
99
99


G
Feces
B. amyloliquefaciens
Sensitive
B
A
95
95
95


H
Feces
B. amyloliquefaciens
Sensitive
A
A
99
99
99


I
Feces
B. amyloliquefaciens
Sensitive
B
A
80
99
99


J
Feces
B. amyloliquefaciens
Sensitive
B
A
50
99
99


K
LMG
B. amyloliquefaciens
Sensitive
A
A
2
99
99


L
Feces
B. licheniformis
Sensitive
B
A
90
99
NA


M
Soil
B. licheniformis
Resistant
B
A
0
1
80


N
Soil
B. megaterium
Resistant
B
A
0
0
0


O
Feces
B. subtilis
Sensitive
A
B
0
60
10


P
Feces
B. pumilus
Resistant
A
A
0
10
99


Q
Feces
B. licheniformis
Resistant
A
A
0
1
5


R
Feces
B. licheniformis
Resistant
A
A
0
few
40


S
Feces
B. pumilus
Sensitive
A
A
0
10
95


T
Soil
B. megaterium
Resistant
B
A
0
0
0


U
Soil
B. licheniformis
Resistant
A
A
0
few
70


V
Feces
B. subtilis
Resistant
A
B
95
99
90
















TABLE 3







Inhibition of E. coli and Clostridium perfringens (mm) as well as


resistance against bile and acid by selected Bacillus strains


Strain A = CHCC15510; Strain B = 15516; Strain C = 15541;


Strain H = 15536; Strain I = 15539


O149, O147 and O101 are the three E. coli


swine pathogens mentioned under Pathogens














E. coli, mm
Cl. perfringens,



Bile

inhibition
mm















Strains
Species
6 h
24 h
Acid
O149
O147
O101
Mm


















A
B. mojavensis
C
A
S
1.5
1.7
2.3
7


B
B. amyloliquefaciens
A
A
S
2.5
2.3
3.5
8


C
B. subtilis
A
B
R
1.8
1.5
2.7
7


D
B. amyloliquefaciens
A
A
R
2
1.7
2.5
6


E
B. amyloliquefaciens
B
A
S
3
3
3.5
6


F
B. amyloliquefaciens
B
A
S
2.5
2.5
3.3
7


G
B. amyloliquefaciens
B
B
R
2
1.7
2
7


H
B. amyloliquefaciens
A
A
R
3.5
3
4.5
7


I
B. amyloliquefaciens
B
A
R
2
2
2
8


J
B. amyloliquefaciens
B
A
S
2
2.5
2.5
8


K
B. amyloliquefaciens
B
A
S
0.3
<1
1
4


L
B. licheniformis
A
B
S
<1
<1
1.3
8


M
B. licheniformis
B
A
S
0
0
0
4


N
B. megaterium
B
A
S
0
0
0
0


O
B. subtilis
B
A
S
1
<1
1.3
7


P
B. pumilus
A
A
R
1
0
1.7
8


Q
B. licheniformis
B
B
S
0
0
0
2


R
B. licheniformis
B
B
S
0
0
0
2


S
B. pumilus
A
A
R
<1
0
<1
6


T
B. megaterium
A
A
S
0
0
0
0


U
B. licheniformis
B
B
S
0
0
0
3


V
B. subtilis
C
A
S
2.5
1
2.5
10









Example 3
Heat Stability
3.1 Method for Heat Stability Test
Bacillus strains were grown overnight in VIB at 37° C. and 220 rpm. Overnight cultures were spread on T3 agar plates (100 μl of 105-106 dilutions) and incubated at 37° C. for 1-2 days until sporulation was complete. Spores were scraped from the plates, suspended in FKP solution and incubated at 80° C. for 15 min in order to inactivate vegetative cells. Spore suspensions were placed on ice immediately after heating. Spores preparations were washed twice in FKP, re-suspended in FKP with 20% glycerol and kept at 80° C. before use. Heat resistance of bacterial spores was accessed by holding eppendorf tubes with 500 μl of spore suspensions in FKP (at concentration of approximately 1×106 CFU per ml) at 99.5° C. for 2, 5 and 10 min. CFU counts were determined in dilutions of heated suspensions plated onto VIB agar after incubation at 37° C. overnight.
3.2 Results of Test for Heat Stability
Heat stability data are shown in Table 4.







TABLE 4







Heat stability at 99.5° C. of selected Bacillus strains; measured in cfu


as reduction after 2, 5 and 10 min in relation to time 0 (log/log)


Strain B = 15516; Strain C = 15541, Strain H = 15536









Reduction in cfu after











Strains
Species
2 min
5 min
10 min














B
B. amyloliquefaciens subsp
0.05
0
0



amyloliquefaciens


C
B. subtilis
0.25
2.5
3.8


D
B. amyloliquefaciens/siamensis
0
0
0.05



related


E
B. amyloliquefaciens subsp.
0.34
3.8
5.3



plantarum


F
B. amyloliquefaciens subsp.
0
0
0



amyloliquefaciens


H
B. amyloliquefaciens subsp.
0.19
2.5
5



plantarum









In general a reduction of less than 2 (log/log) cfu after 2 min in relation to time 0 is appropriate for spores to be included in feed for pelletizing and results below 2 are achieved with common commercial Bacillus spores preparations. Thus all strains tested and shown in Table 4 have good heat stability. Some strains also showed good heat stability after 5 and 10 minutes, i.e. strain B and strain F, both B. amyloliquefaciens subsp. amyloliquefaciens, that showed no cfu reduction.
Example 4
Enzyme Production
4.1 Method for Cellulase Assay
Bacillus strains were grown in carboxymethyl cellulose (CMC) medium (Abou-Taleb et al. 2009) (per l: 10.0 g carboxymethyl cellulose (C9481), 2.0 g Bacto Tryptone (cat. 211705, Becton Dickinson A/S, Denmark), 4 g KH2PO4, 4.0 g Na2HPO4, 0.2 g MgSO4 7H2O, 0.001 g CaCl2 2H2O, 0.004 g FeSO4 7H2O, pH 7) at 37° C. and vigorous magnetic agitation for 24 hours. Cellulase production was determined using the EnzChek Cellulase Substrat kit (cat. E33953, Life Technologies) according to the manufacturer's instructions. Shortly, culture supernatants were collected by centrifugation and distributed in MTPs (200 μl per well) in serial dilutions. Standard curves were constructed using cellulase from Aspergillus niger (C1184) starting from 2 U ml−1. EnzChek substrate solution was added to the culture supernatants in Nunc 96 well Black FluoroNunc plates (cat. 237105, Thermo Fisher Scientific, NUNC Inc.). Fluorescence was recorded at excitation 360 nm/emission 420 nm after 30 min incubation (Enspire 2300 Multilabel Reader, Perkin Elmer Inc.). Cellulase activity was calculated from standard curves in two independent experiments and expressed as means (U ml−1).
4.2 Method for Xylanase Assay
Bacillus cultures were grown in medium containing beech wood xylan (Cordeiro et al. 2002) (per l: 5.0 g xylan (X4252), 2.0 g Yeast Extract (cat. 288620, Becton Dickinson A/S, Denmark), 5.0 g Bacto Peptone (cat. 211677, Becton Dickinson A/S, Denmark), 0.5 g NaCl, 0.5 g MgSO4 7H2O, 0.15 g CaCl2 2H2O, pH 7.5) at 37° C. and vigorous magnetic agitation for 24 hours. The xylanase assay was performed with the use of the EnzChek Ultra Xylanase Assay Kit (cat. E33650, Life Technologies) according to the manufacturer's instructions. Briefly, culture supernatants were collected by centrifugation, distributed in MTPs (200 μl per well), in serial dilutions and added xylanase substrate working solution. Fluorescence in culture supernatants was measured at excitation 360 nm/emission 420 nm after incubation for 30 min (Enspire 2300 Multilabel Reader, Perkin Elmer Inc.). Thermomyces lanuginosis (X2753) was used as standard enzyme and loaded in MTPs in serial dilutions, starting from 25 mU ml−1. Xylanase activity of the Bacillus strains was calculated from the standard curves and expressed as means (mU ml−1) of two independent assays.
4.3 Method for Protease Assay
Bacillus overnight cultures (grown in VIB at 37° C.) were transferred to a reaction mixture with Fluorescein Isothiocyanate-casein (FITC-C) as substrate (Sigma C3777) and incubated at 37° C. 3 hours. After precipitation the amount of soluble peptides was measured by fluorescence, at excitation 497 nm, emission 515 nm. This assay detects a wide range of proteases (serine, aspartic, cysteine and metalloproteases).
4.4 Methods for Biofilm Production
Bacillus strains were added in VIB (about 107 CFU ml−1), distributed into Polypropylene (PP) MTPs (96 Well Conical Btm PP Plt Natural; NUNC Inc., Denmark) and incubated at 37° C. for 24 hours without shaking. Growth was controlled by measurements of the optical density (OD) at 620 nm. Biofilm formation was assessed by crystal violet staining with as described previously (Auger et al. 2009). Briefly, after washing the wells with distilled water, crystal violet solution of 0.1% (w v−1) was added to PP-MTPs and the plates were incubated for 30 min. Then, the washing procedure was repeated and ethanol 96% (v v−1) was added to the plates. The absorbance at 570 nm was measured after 15 min incubation (Wallac Victor2 spectrophotometer, Perkin Elmer Inc.). The Bacillus spp. strains were classified as either high (Abs570 nm>2.0), medium (Abs570 nm=1.0-2.0) or low (Abs570 nm<1.0) biofilm producers. The assay was performed twice in duplicates.
4.5. Results for Enzyme Assays







TABLE 5







Enzyme production and Biofilm


(RFU = relative fluorescence unit)


Strain A = 15510; Strain B = 15516; Strain C = 15541;


Strain H = 15536; Strain I = 15539














Cellulase,
Xylanase,
Protease,



Strains
Species
mU/ml
mU/ml
RFU/OD
Biofilm















A
B. mojavensis
1734
50
142117
+


B
B. amyloliquefaciens subsp.
67
47
599919
+



amyloliquefaciens


C
B. subtilis
1037
24
445091
+


D
B. amyloliquefaciens/siamensis
2196
70
291908
+++



related


E
B. amyloliquefaciens subsp.
612
28
381459
+++



plantarum


F
B. amyloliquefaciens subsp.
54
57
400456
+++



amyloliquefaciens


G
B. amyloliquefaciens subsp.
371
71
453158
+++



plantarum


H
B. amyloliquefaciens subsp.
631
30
252377
++



plantarum


I
B. amyloliquefaciens subsp.
466
121
411206
+++



plantarum


J
B. amyloliquefaciens subsp.
469
72
421338
+++



plantarum









The table shows that strains E, G, H, I and J which are all B. amyloliquefaciens subsp. plantarum have high cellulase activity whereas strains B and F which are B. amyloliquefaciens subsp. amyloliquefaciens have low cellulase activity.
Example 5
Piglet Trials
Two selected Bacillus strains (Strain B or C) were supplemented to nursery diets to assess their effect on growth performance and mortality of post-weaned piglets. As E. coli is one of the main pathogens in the nursery period with great impact on production parameters and mortality these trials can give information about the effect on E. coli inhibition in the animals. The trials were performed at 2 different sites; site 1 was a research farm and site 2 a university. The trial set up was similar at both sites.







TABLE 6







Overview of sites used in piglet trials












Total no.
No. of

Days in



of pigs
replicates
Weighings
trial

















Site 1
576
96
28, 35, 42, 63
35



Site 2
720
24
28, 35, 49, 63
35










5.1.1 Experimental Set Up at Site 1

The day of weaning, 576 piglets (28 days old) from two consecutive weaning batches (288 piglets each), originating from the experimental farm, were used in the experiment. Piglets were crossbred piglets (ACMC×Pietrain). Selected piglets were healthy with good general aspect and did not receive any vaccination in the nursery phase. The experimental farm was positive to Porcine Reproductive & Respiratory Syndrome (PRRS), but under control, and had some problems of colibacillosis in the post-weaning phase. The piglets were sorted according to body weight and then allocated to 48 pens in both weaning batches (96 pens in total) such that each block of pens contained 6 piglets, 3 entire males and 3 females, of similar body weight in both treatments. The treatments were allocated to the pens of light and heavy piglets by block, so that each treatment was applied to 24 pens of 6 piglets (6 pens per treatment and room; 12 pens per treatment and weaning batch). Bacillus products were added to the feed at 400 g/ton of feed or 1.28*10 7 CFU g/feed.
Pigs were individually weighed at 28 (day 0; weaning day), 35, 42 and 63 days of age to calculate the average daily weight gain (ADWG). Average daily feed intake (ADFI) and feed conversion ratio (FCR) were measured by pen in the same phases (28-35; 35-42; 42-63) and for main periods (prestarter: 28-42 days of age; starter: 42-63 days of age; and total nursery period). Mortality and incidence of pathologies were controlled daily, including registration of individual antibiotic treatments applied.







TABLE 7







Production parameters at site 1


P-values compared to control (AP = <0.1; a= <0.05)


Strain B = 15516; Strain C = 15541 SE = Standard Error












Body weight,
28-42 days of age





kg
(14 days in trial)
42-63 days of age
Total (28-63 days)



















28 d
63 d
ADG
ADFI
FCR
ADG
ADFI
FCR
ADG
ADFI
FCR






















Control
7.7
20.7
179
254
1.43
493
697
1.42
371
522
1.40


Strain B
7.7
21.1
  196A
264
1.37A
513
  723A
1.41
383
535
1.40


Strain C
7.8
21.0
  199a
  267A
1.37A
501
710
1.42
380
532
1.40


SE
0.150
0.254
    0.007
    0.006
0.024
0.010
    0.010
0.008
0.007
0.007
0.009









5.1.2 Results

Both Bacillus strains probiotic products supplemented to nursery diets numerically improved productive performance compared with a negative control group, without showing differences between them. Significant effect on both ADG and FU could be observed in the prestarter phase (28-42 days of age). Mortality percentage was reduced in both Bacillus groups (Mortality %: Control (3.47), Strain B (1.39%), Strain C (2.08))
5.2.1 Experimental Set UP at Site 2
Just after weaning all the piglets selected were housed in a weaning room of 24 pens with thirty animals per pen. The room is equipped with central heating and forced ventilation with cooling system and completely slatted floors. Each pen it is equipped with a commercial double-sided wet-dry feeder to ensure ad libitum feeding and free water access with a capacity for feeding three animals at the same time. Feed was distributed ad libitum throughout the entire experimental phase.
A total of seven hundred and twenty commercial crossing weanling piglets [Pietrain×(Landrace×Large White)] were used. The animals were obtained from the sows of the same farm on the day of weaning and moved to the experimental facility (without transport). Male and female 26 d-old piglets of 7.0 kg SD=1.64 kg of BW were used. Plastic ear tag identification with the animal's number was used. The animals were distributed into three blocks by initial body weight. Within each block piglets were distributed in pens for a balanced body weight distribution. Therefore, each block consisted of 8 pens of 30 animals to which the experimental diets were randomly assigned.
Trial started at weaning at 28 days of age. Animals were individually weighted on days 0 and 7 and group weighted on days 21 and 35 days in trial. Feed disappearance from each hopper was measured throughout the experiment. Average daily feed intake (ADFI), average daily gain (ADG) and feed:gain ratio (FCR) according to the total feed intake were therefore calculated. The health status of piglets was regularly assessed and any abnormal signs or medications given were recorded. Mortality rate and culling percentage were also calculated.







TABLE 8







Production parameters at site 2


least square means, P-values compared to control (AP = <0.1)


Strain B = 15516; Strain C = 15541











28-35 days of age
49-63 days of age
Total (28-63 days)

















ADG
ADFI
FCR
ADG
ADFI
FCR
ADG
ADFI
FCR




















Control
73.8
157
2.26
345
602
1.76
227
388
1.72


Strain B
80.9
174
2.26
  375A
584
1.58A
235
383
1.64


Strain C
87.1
166
1.96
379
586
1.60
232
389
1.69


SE
0.010
0.008
0.0002
    0.019
0.025
0.0001
0.0091
0.0066
0.00065









5.2.2 Results

Both Bacillus strains supplemented to nursery diets numerically improved productive performance compared with a negative control group, without showing differences between them. Significant effect on both ADG and FU could be observed in trial periods.
The number of animals treated per pen with Enrofluxacin to overcome a severe diarrhea was significantly higher (P>0.05) in those animals fed the control diet than those fed Bacillus during the first week post-weaning. The same results (P<0.05) were observed for the entire experimental period (0 to 35 days post weaning). Mortality percentage was reduced in both Bacillus groups (Mortality %: Control (4.17), Strain B (0.04%), Strain C (2.65))
REFERENCES


    Barbosa et al., 2005, Screening for Bacillus Isolates in the Broiler Gastrointestinal Tract, Applied and Environmental Microbiology, 968-978
    Benitez et al., 2011, Antimicrobial Activity of Bacillus amyloliquefaciens LBM 5006 is enhanced in the Presence of Escherichia Coli, Curr Microbiol 62, 1017-1022
    Chaiyawan et al., 2010, Characterization and probiotic properties of Bacillus strains isolated from broiler, The Thai Journal of Veterinary Medicine, 40, 2, 207-214
    Cutting, S. M. 2011. Bacillus probiotics. Food Microbiology 28 (2):214-20.
    EFSA 2008. Technical Guidance. Update of antibiotic resistance criteria. The EFSA Journal 732, 9-15
    Guo et al, 2006, Screening of Bacillus strains as potential probiotics and subsequent confirmation of the in vivo effectiveness of Bacillus subtilis MA139 in pigs, Antonie van Leeuwenhoek 90:139-146
    Klose et al., 2010. In vitro antagonistic activities of animal intestinal strains against swine-associated pathogens. Vet. Microbiology 144: 515-521.
    López, D., and R. Kolter. 2010. Extracellular signals that define distinct and coexisting cell fates in bacillus subtilis. FEMS Microbiol. Rev. 34(2): 134-149.
    Spiehs, M. J., G. C. Shurson, and L. J. Johnston. 2008. Effects of two direct-fed microbial on the ability of pigs to resist an infection with salmonella enterica serovar typhimurium. Journal of Swine Health and Production. 16(1): 27-36.

## Claims

1. A method for feeding an animal, comprising administering a Bacillus composition to an animal, wherein the Bacillus composition comprises cells of at least one Bacillus strain that (i) is sensitive to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline and chloramphenicol; (ii) inhibits growth of E. coli and Clostridium perfringens, and (iii) exhibits a sporulation percentage of at least 80 when measured after 2 days of incubation, wherein the cells are of at least one of the Bacillus strains selected from:
(a) the Bacillus mojavensis strain CHCC 15510 with accession number DSM 25839;
(b) the Bacillus amyloliquefaciens strain CHCC 15516 with accession number DSM 25840;
(c) the Bacillus amyloliquefaciens strain CHCC 15536 with accession number DSM 27032;
(d) the Bacillus amyloliquefaciens strain CHCC 15539 with accession number DSM 27033; and
(e) the Bacillus subtilis strain CHCC 15541 with accession number DSM 25841,

wherein the Bacillus composition comprises from 105 to 1012 CFU/g of the at least one Bacillus strains, and
wherein the animal is selected from poultry, ruminants, calves, pigs, rabbits, horses, and fish.

2. The method of claim 1, wherein the Bacillus composition comprises cells of at least two Bacillus strains selected from:
(a) the Bacillus mojavensis strain CHCC 15510 with accession number DSM 25839;
(b) the Bacillus amyloliquefaciens strain CHCC 15516 with accession number DSM 25840;
(c) the Bacillus amyloliquefaciens strain CHCC 15536 with accession number DSM 27032;
(d) the Bacillus amyloliquefaciens strain CHCC 15539 with accession number DSM 27033; and
(e) the Bacillus subtilis strain CHCC 15541 with accession number DSM 25841.

3. The method of claim 1, wherein the Bacillus composition comprises cells of at least three Bacillus strains selected from:
(a) the Bacillus mojavensis strain CHCC 15510 with accession number DSM 25839;
(b) the Bacillus amyloliquefaciens strain CHCC 15516with accession number DSM 25840;
(c) the Bacillus amyloliquefaciens strain CHCC 15536 with accession number DSM 27032;
(d) the Bacillus amyloliquefaciens strain CHCC 15539 with accession number DSM 27033; and
(e) the Bacillus subtilis strain CHCC 15541 with accession number DSM 25841.

4. The method of claim 1, wherein the cells of the at least one Bacillus strain are spore cells.

5. The method for feeding an animal of claim 1, wherein the animal is selected from poultry, ruminants, calves, pigs, rabbits, and horses.

6. The method of claim 1, wherein the Bacillus composition comprises cells of the Bacillus mojavensis strain CHCC 15510 with accession number DSM 25839.

7. The method of claim 1, wherein the Bacillus composition comprises cells of the Bacillus amyloliquefaciens strain CHCC 15516 with accession number DSM 25840.

8. The method of claim 1, wherein the Bacillus composition comprises cells of the Bacillus amyloliquefaciens strain CHCC 15536 with accession number DSM 27032.

9. The method of claim 1, wherein the Bacillus composition comprises cells of the Bacillus amyloliquefaciens strain CHCC 15539 with accession number DSM 27033.

10. The method of claim 1, wherein the Bacillus composition comprises cells of the Bacillus subtilis strain CHCC 15541 with accession number DSM 25841.

11. The method for feeding an animal of claim 1, wherein the cells of the at least one Bacillus strain inhibit growth of E. coli as determined by an inhibition zone of at least 1.5 mm when measured by an E. coli agar spot test.