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Vol. 2, 2003COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 101 2003 Insti tute of Food Technologists

Sporeformers asHuman Probiotics:

Bacillus,Sporolactobacillus,andBrevibacillus

M.E. Sanders, L. Morelli, and T.A. Tompkins

IntroductionConsumption of certain live microor-

ganisms has been shown in some circum-stances to have a beneficial impact onboth man and animals. A diverse group ofmicrobes has been evaluated for suchprobiotic activity, including many speciesof the genera Lactobacillusand Bifidobac-teria. These genera are the most abundant

in probiotic-containing food products.Less commonly, species of Enterococcus,Saccharomyces, Escherichia, and thesporeformers Sporolactobacillus, Breviba-cillus, and Bacillushave been suggestedfor probiotic effects. An electron micro-graph of a commercial Bacillusprobioticstrain is shown in Figure 1. It is often as-serted that the ideal probiotic targeted to-ward functionality in the gastrointestinaltract should be isolated from the gas-trointestinal tract of the species to which itwill be administered. But a more pragmat-ic approach recognizes that probiotic ac-tivity may result from action at a variety of

sites (including nonintestinal sites such asthe mouth, stomach, or vaginal tract) anda variety of mechanisms, some of whichmay not require the attributes associatedwith native flora such as adherence to epi-thelial cells or colonization of the gas-trointestinal tract. One example of this isthe use of Saccharomycesboulardii, a mi-crobe that is not a normal member of hu-man gastrointestinal flora, for the preven-tion of recurrence of Clostridiumdifficile-induced pseudomembranous colitis. Oneproposed mechanism for effectiveness of

this probiotic strain is a protease ex-pressed by S. boulardiithat cleaves the C.difficiletoxin A receptor sites from the sur-face of intestinal epithelial cells (Czeruckaand Rampal 2002). Therefore, there ap-pears to be a rationale for the use of mi-crobes of nonintestinal origin for probiot-ics.

The ability of some bacteria to form

spores (endospores) is an attribute of someaerobic and anaerobic rods and a few coc-ci. In the case of probiotic sporeformers,only the aerobic rods are used. Sporeform-ers are capable of growth and metabolicactivity only when in the vegetative state,and resort to sporulation when conditionsof inadequate nutrition or other challengeto survival is experienced. As spores, cellsare metabolically inactive and more resis-tant to the lethal effects of heat, drying,freezing, toxic chemicals, and radiation.Some commercial products containingsporeformers are listed in Table 1. Althoughuse of sporeformers as probiotics has not

been well studied, the inherent resistanceof spores to environmental stress is an at-tractive attribute for commercial applica-tion, especially in the animal agriculture in-dustry. Unlike other probiotic bacteria,sporeformers are present in commercialproducts as spores and are not consumedas vegetative cells.

The oral consumption of large numbersof viable microbes that are not normal in-habitants in the gastrointestinal tract doesraise additional questions about safety. Thisis especially true with the use of genera and

ABSTRACT: The value of exogenously supplied live bacteria for the maintenance of health in humans has been recog-nized both scientifically in the published literature and commercially in the availability of probiotic products. Al-though many bacteria characterized as probiotics are strains of Lactobacillusor Bifidobacterium, sporeforming bacte-ria, primarily of the genus Bacillusand related genera, have also been studied and commercialized as probiotics. Thisarticle reviews the characterization, efficacy, and safety of sporeformers used as probiotics.

species that do not have a history of safeuse in foods, such as many sporeformers.Even normal intestinal inhabitants can attimes act as opportunistic pathogens.

Nomenclature ofBacillus,

Sporolactobacillus, andBrevibacillus

Although there is no official classifica-tion of bacteria, there is regulation of their

nomenclature. As summarized by J.P. Euz-by (www.bacterio.cict.fr ), the InternationalCode of Nomenclature of Bacteria (1990)governs the ways in which the names ofbacteria are used. According to this code, abacterial name is valid if: It is cited in the Approved Lists of Bac-

terial Names (Int J Syst Bacteriol, 1980,30:225420); or, if it is published in the Intl J of Syst

and Evolutionary Microbiol (or, prior to2000, the Int J of Syst Bacteriol) and con-forms to the Bacteriological Code (1975 re-vision); or, if it is announced in a Validation

List, which validates bacterial names pub-lished elsewhere. The Validation List is pub-lished in the Intl J of Sys and EvolutionaryMicrobiol.

There are cases where two or more valid-ly published names remain in use, but no-menclature committees do not considerpurely commercial motives adequate to ap-prove this practice.

Bacillus. Sporeforming, catalase-posi-tive, aerobic bacteria have been tradition-ally classified in the genus Bacillus. Sporesand vegetative cells of Bacillussubtilisare
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102 COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 2, 2003 www.ift.org/publications/crfsfs

evident in Figure 1. It is noteworthy that B.subtilis and B. cereus have been docu-mented to grow anaerobically undersome conditions (Nakano and Zuber1998), which is an important observationregarding in vivointestinal function. Cur-rently, there are 77 recognized species ofthe genus Bacillus(Table 2). This group of

bacteria is quite diverse, with a range ofmole % G+C content from 32 to 69%(Sneath 1986). Of these 77 species, thefollowing have been evaluated for probi-otic functionality, with several currentlybeing sold worldwide as components ofproducts for human and animal use: co-agulans, subtilis, clausii, cereus, toyoi(nota valid species name and thought to beequivalent to cereus). Identification ofmembers of the genus Bacillusbased on

phenotypic traits has always been diffi-cult, but the genetic approach to identifi-cation is also facing problems. The 5groups of species obtained on the basisof 16S rRNA sequences (Ash and others1991) contain clusters of species soclosely related they are indistinguishableby a single test.

In some cases, commercial productscontaining Bacillus coagulansuse the in-valid name Lactobacillus sporogenes on

Table 1Commercial products containing sporeforming bacteria

Product name Manufacturer Microbe listed Comments

Lactospore Sabinsa Corp., Piscataway, NJ Lactobacillus sporogenes Human use, contains Bacillus coagulans

Lacbon, Lacris Uni-Sankyo Lactobacillus sporogenes Human, Approved by the JapaneseMinistry of Health and Welfare

Enterogermina Sanofi-Winthrop SpA, Milan, Italy Bacillus clausii Human use

Bactisubtil Marion-Merril-Down Laboratories, B. cereusLevallois-Perret, France

Biosubtyl Biophar Co. Ltd., Nha Trang, Vietnam B. pumulisLactopure Pharmed Medicare Lactobacillus sporogenes Human and animal use

Flora-Balance Flora-Balance, Montana, USA Brevibacillus laterosporus Human useBOD

Medilac Hanmi Pharmaceutical Co., Ltd., B. subtilisR0179 (and Human use, clinically documented,Korea and Beijing, China Enterococcus faecium) approved by the Chinese State Drug

Authority, also sold OTC in Korea.

Biosporin, Subalin, Gynesporin D. K. Zabolotny Institute of B. subtilisand recombinant Human and animal useand others Microbiology and Virology, Ukraine Bacillus strains

Natures First Food Natures First Law, San Diego, CA 42 species listed as Pro- Human usebiotic Complex Ingredients,including B. laterosporus,B. polymyxa, B. subtilis,B. pumulis

Table 2RecognizedB.species. Fromwww.bacterio.cict.fr, updated January 2001.Subspecies not included.

B.agaradhaerens B. alcalophilus B. amyloliquefaciensB.anthracis B.atrophaeus B.azotoformansB. badius B. benzoevorans B. carboniphilusB.cereus B. chitinolyticus B. circulansB. clarkii B. clausii B.coagulansB.cohnii B.edaphicus B. ehimensisB. fastidiosus B. firmus B. flexusB. fumarioli B. fusiformis B. gibsoniiB. globisporus B.halmapalus B. haloalkaliphilusB. halodenitrificans B.halodurans B.halophilusB. horikoshii B.horti B. infernos

B. insolitus B. kaustophilus B. laevolacticusB.lentus B. licheniformis B.marinusB.megaterium B. methanolicus B.mojavensisB. mucilaginosus B.mycoides B.naganoensisB. niacini B. oleronius B.pallidusB.pasteurii B. pseudalcaliphilus B.pseudofirmusB.pseudomycoides B. psychrophilus B.psychrosaccharolyticusB.pumilus B. schlegelii B. silvestrisB. simplex B. siralis B. smithiiB. sphaericus B. sporothermodurans B. stearothermophilusB. subtilis B. thermoamylovorans B. thermocatenulatusB. thermocloaceae B. thermodenitrificans B. thermoglucosidasiusB. thermoleovorans B. thermosphaericus B. thuringiensisB. tusciae B. vallismortis B.vedderiB.vulcani B. weihenstephanensis

Figure 1Electron micrograph of a com-mercial preparation ofB. subtilisR0179demonstrating the presence of both veg-etative cells (approximately 2.0 to 2.5mm 0.5 mm) and ellipsoidal en-dospores (approximately 0.8-1.0 mm 0.4 mm). Magnification 15000 times.Image courtesy of Dr. Sandy Smith,Dept. of Food Sciences, Univ. of Guelph,Guelph, Canada.
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Sporeformers as human probiotics . . .

product labels (Sanders and others 2001).This name can be traced to a paper pub-lished in 1932 (Horowitz-Wlassowa andNowotelnow 1932). However, since thebacterium described in this paper was aspore-forming bacterium, it could not beconsidered a species of Lactobacillus. Al-though subsequent editions of BergeysManual make reference to the erroneous

name, this species was described as a mis-classification in Bergeys Manual (1939)and the name was acknowledged to referto the species Bacillus coagulans. Thename Lactobacillus sporogenes has noscientific validity.

The case of Lactobacillus sporogenesisnot the only example of misclassificationof sporeformer probiotics. Recent reportsdemonstrated that none out of 7 probioticproducts were labeled with the correct tax-onomic position of sporeformer bacteriacontained as active ingredients (Table 3).

Sporolactobacillus.The genus Sporolac-tobacillus (Kitahara and Suzuki 1963) iscomprised of 5 species of catalase-nega-tive, facultative anaerobic or microaero-philic endosporeformers (Table 4). The ge-nus has a mole % G+C content of 38 to40. Originally proposed as a componentof the genus Lactobacillus, Sporolactoba-cillus was subsequently elevated to genusstatus in the family Bacillaceae (Kitaharaand Toyota 1972). Its distinction from thegenus Lactobacilluswas shown by DNA toDNA hybridization studies by Dellaglioand others (1975). The type species forSporolactobacillus is Sporolactobacillusinulinus (Kitahara and Suzuki 1963; Ki-

tahara and Lai 1967). It produces D(-) lac-tic acid, but is unable to ferment lactose.Optimal growth temperature is 35 C(range 15 to 40 C). Species claimed toproduce L(+) or DL (Sporolactobacilluslaevusand Sporolactobacillus racemicus)isomers of lactic acid are not validly recog-nized. Fatty acid configuration and iso-prenoid quinone cell components areconsistent with the Bacillusgroup and dif-fer from those of Lactobacillus (Uchidaand Mogi 1973; Collins and Jones 1979;Hess and others 1979).

Brevibacillus. Brevibacillus is a genusestablished in 1996 of aerobic, en-

dospore-forming bacteria. This genus wasderived by a genetic reclassification ofstrains previously allotted to the Bacillusbrevis group. Results of gene sequenceanalyses (Shida and others 1996) demon-strated that strains grouped into the Bacil-lus brevis cluster formerly included 10species (Bacillus brevis, Bacillus agri, Bacil-lus centrosporus, Bacillus choshinensis,Bacillus parabrevis, Bacillus reuszeri, Ba-cillus formosus, Bacillus borstelensis, Ba-cillus laterosporus, andBacillus thermoru-ber), which were removed from the Bacil-

lus genus. There are currently 11 recog-nized species of Brevibacillus (Table 5).

Brevibacillus laterosporus(formerly Bacil-lus laterosporus) strains possess larvicidalactivity, albeit at low levels. Three specieshave been associated with human infec-tions, Brevibacillus agri, Brevibacillusbrevis, and Brevibacillus laterosporus.Fourteen distinct chromosomal restrictionfragment patterns were observed among29 strains of Brevibacillus laterosporustested (Zahner and others 1999), suggest-ing a range of diversity among Brevibacil-lus laterosporusstrains.

Ecology ofBacillus,

Sporolactobacillus, andBrevibacillus

Bacillus.Bacillusspecies are commonlyassociated with soil, and as such are iso-lated almost ubiquitously from soil, water,dust, and air. They are associated withcommercial production of antibiotics, in-dustrial chemicals, and enzymes. Theyalso play a role in food spoilage, and heatresistance of thermophilic Bacillussporesare especially problematic to the dried-milk industry. The use of Bacillus subtilisin fermentation of some food is traditionalin Eastern countries: Bacillus subtilisstrainnatto is used in the production of the tradi-

tional fermented Japanese legume food,natto (Hosoi and Kiuchi 2003).

Bacillusspecies are normally allochtho-nous microbes to the human intestinal

tract and are found as a result of inadvert-ent ingestion of contaminated foods or in-gestion of fermented foods such as natto.They are not normal colonizing inhabit-ants of the human intestinal tract. The tran-sient nature of the Bacillus species hasbeen established in feeding studies show-ing that 1 wk after cessation of feeding, Ba-cillusis no longer isolated from subjects.

Sporolactobacillus.The habitats of themembers of the genus Sporolactobacillus,apart from the original isolation fromchicken feed, are believed to be the soil,milk products, and pickle (as contami-nants). The incidence of these sporeform-

ers in the environment is low. Doores andWesthoff (1983), using a selective methodspecific for sporolactobacilli, examinedsamples of food, beverages, plant, andanimal material. Only 2 out of 699 sam-ples examined were positive for Sporolac-tobacillus, documenting the rarity of thisspecies in these environments. Strains ofSporolactobacilluswere found to surviveexposure to low pH (Hyronimus and oth-ers 2000), although the procedure usedto assay this resistance did not allow dis-crimination between spores and vegeta-

Table 3Errors in nomenclature of sporeformers contained in commercial probi-otic products

Product Indications on label Identified as Reference

Enterogermina (Italy)1 B. subtilis B. clausii Green and others 1999,Senesi and others 2001

Lactipan plus (Italy) Lactobacillus sporogenesB. subtilis Hoa and others 2000Domuvar (Italy) B. subtilis B. clausii Hoa and others 2000Bactisubtil (France) B. subtilis B. cereus Hoa and others 2000Subtyl (Vietnam) B. subtilis Bacillus spp.2 Hoa and others 2000Biosubtil Dalat (Vietnam) B. subtilis B. cereus Hoa and others 2000Biosubtil Nha Trang B. subtilis B. pumilus Green and others 1999

1The label of this product was recently amended, and it now correctly states B. clausii.2Authors suggested that strain of this product could belong to a new species, named B. vietnami.

Table 5Validly named Brevibacillusspecies (Shida and others 1996; Loganand others 2002)

Species

Brevibacillus agriBrevibacillus borstelensisBrevibacillus brevisBrevibacillus centrosporus

Brevibacillus choshinensisBrevibacillus formosusBrevibacillus invocatusBrevibacillus laterosporusBrevibacillus parabrevisBrevibacillus reuszeriBrevibacillus thermoruber

Table 4Validly named Sporolacto-bacillus species. Five species and 2 sub-species are at the moment validly citedin the List of Bacterial Names withStanding in Nomenclature, while the lat-est edition of Manual of Systematic Bac-teriology enlisted only the type species

Sporolactobacillus inulinus(Kitahara andSuzuki 1963; Kitahara and Lai 1967).

Species

Sporolactobacillus inulinusSporolactobacillus kofuensisSporolactobacillus lactosusSporolactobacillus nakayamae subspnakayamaeSporolactobacillus nakayamae subspracemicusSporolactobacillus terrae

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tive cells. Rychen and Simoes Nunes(1993, 1995) have evaluated Sporolacto-bacillusstrain P44 for enhancing feed ef-ficiency in pigs.

Brevibacillus. The habitat of Brevibacil-lusoverlaps with that of Bacillus. This ge-nus is associated with the soil, isolateshave been found in the dairy environment,and some are used in industrial microbiol-

ogy applications. There is little informationabout their use as probiotic agents, andmost reference to probiotic Brevibacillusspecies is as former Bacillusspecies. Theiroriginal habitat is not the intestinal tract. Astrain of Brevibacillusbreviswas studied forits use for biocontrol ofplant pathogens due totheir antimicrobial pro-duction (Edwards andSeddon 2001). Anoth-er example of the useof Brevibacillusis witha strain (HPD31) of thespecies choshinensisused as an efficientproducer of recombi-nant human epidermalgrowth factor (Miyau-chi and others 1999).This strain also servesto convert biologicallyinactive multimers ofepidermal growth fac-tor into biologically ac-tive monomers.

Efficacy ofBacillus,

Sporolactobacillus, andBrevibacillusRecently, the term probiotic was de-fined as live microorganisms adminis-tered in adequate amounts which confera health effect on the host (FAO/WHO2001). Implicit in this definition is that, forthe term probiotic to be used, a healtheffect must be demonstrated. Althoughthe published literature substantiatinghealth effects of sporeforming probiot-ics in humans is sparse, a variety ofproducts for human consumption whichcontain sporeformers are available com-mercially (Table 1). In some countries, Ba-cillus-based probiotics have been suc-

cessfully introduced by pharmaceuticalcompanies. For example, Hanmi Pharma-ceutical Co., Ltd. in China has a Bacillusprobiotic marketed as a therapeutic drugwith clinical evidence and full regulationby the federal authorities. On the otherend of the spectrum are undefined mix-tures of soil organisms sold as dietarysupplements by a variety of companies,which presumably contain sporeformers.The following discussion will highlightsome publications documenting physio-logical effects in humans of sporeforming

bacteria. A review summarizes the use ofa commercial spore-containing probioticproduct, Enterogermina, as an antidiar-rheal probiotic (Mazza 1994). Studiesaimed at animal feed applications are notthe focus for this discussion.

Initial efforts to document a physiologi-cal impact of probiotic bacteria often fo-cus on the following three criteria: (1) in-

herent characteristics of strains that wouldenable intestinal tract survival, (2) the fateof the fed bacterium, and (3) the impact ofconsumption of the live bacterium on in-testinal flora. (It should be noted, howev-

er, that effects beyondan impact on intesti-nal flora, and at ex-traintestinal sites, havebeen documented formany probiotic strains[Reid and others2003]). A few suchstudies have beendone with sporeform-ers. Hyronimus andothers (2000) evaluat-ed the acid and biletolerance of vegetativecells of 13 strains ofSporolactobaci l lus,Bacillus laevolacticus,Bacillus racemilacti-cus, and Bacillus co-agulansin the vegeta-tive state. They found

that only Bacillus racemilacticus andBa-cillus coagulans tolerated oxgall above0.3%. Strains of Bacillus laevolacticus

and Sporolactobacillustolerated pH 3 for3 h, but none of the 13 strains evaluatedsurvived pH 2.0 for 3 hr. Although resultssuggest that none ofthe strains evaluatedhave the necessaryability to survive un-der conditions presentin the human gas-trointestinal tract,these studies wereconducted on vegeta-tive cells. This studydid not test sporeswhich would presum-

ably tolerate suchconditions well. How-ever, the 3-h incuba-tion time used in thisstudy is longer thanwould be likely to beexperienced duringgastric transit. Incubation times of 30 minto 1 hr are more realistic. Spinosa andothers (2000a) studied the fate of Bacillussubtilis and Bacillus clausii spores afterintragastric inoculation into mice. Theydetermined that, although spores sur-

vived gastrointestinal transit, their levelsdeclined exponentially to negligible num-bers in less than 1 wk and no significantlevel of vegetative cells could be recov-ered from the ileum, colon, or feces (veg-etative cells were recovered up to 72 h).The authors concluded that the sporeform of the Bacillus must mediate anyprobiotic effect. However, Casula and

Cutting (2002) tested the ability of Bacil-lus subtilisspores to germinate in the in-testinal tract of mice. Using a chimericgene expressed only in vegetative cells ofBacillus subtilisas the basis for their as-say, they documented the presence ofvegetative cells after feeding of spores.This supports the conclusion that germi-nation of Bacillus subtilisin the intestinedoes occur, and may in fact mediate aprobiotic effect. In a different approach,Hoa and others (2001) studied the behav-ior of Bacillus subtilis spores adminis-tered by intragastric gavage to mice. Theydid not differentially count spores andvegetative forms and checked the pres-ence of spores only up to 96 hr after ad-ministration. They found that in some ex-periments the number of spores was larg-er than expected from the size of the in-oculum. Their results suggest that limitedgermination of spores into vegetativeforms could occur. Jadamus and others(2001) demonstrated germination of Ba-cilluscereusvar. toyoispores in intestinalsamples from both broiler chickens andsuckling piglets, and concluded that ger-mination of spores was a necessary pre-requisite for its possible probiotic effects.

Taken together, these studies suggest thatsome germination may occur in vivo, butstill unresolved is the role of these differ-

ent cell states in medi-ating physiological in-teractions in vivo.

Impact on fecal mi-croflora. Regardingthe impact of spore-formers on fecal mi-croflora, Adami andCavazzoni (1999)studied the effect ofBacillus coagulansCNCM I-1061 on the

profile of bacteria infeces in a piglet mod-el. They found that Ba-cillus coagulans con-sumption (~1011 cfu/kg feed, 50% spores)increased aerobic and

anaerobic sporeformers, decreased lacto-cocci, enterococci, anaerobic cocci, andfecal coliforms over time. Hosoi and oth-ers (1999) tested the impact of Bacillussubtilis(natto) on the fecal flora of mice.Spores did not affect fecal Enterobacteri-

Studies suggest thatsome germination may

occur in vivo, but stillunresolved is the role

of these different cell

states in mediatingphysiological interac-

tions in vivo

Though published lit-erature substantiating

health effects of spore-

forming probiotics is

sparse, a variety ofproducts which con-

tain sporeformersare available

commercially

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aceae nor Enterococcus spp., regardlessof diet groups, but differences in Bacteroi-daceaeand Lactobacilluswere observed.No changes were observed when auto-claved spores were administered. Studieson the impact of spores on the fecal floraof humans have not been reported.

Production of antimicrobials.The pro-duction of antimicrobials is considered to

be a pathogen-inhibiting mechanism ex-hibited by probioticbacteria, and suchcompounds havebeen shown to be pro-duced by some spore-formers. Dozens of dif-ferent peptide antibiot-ics exhibiting antago-nism against a broadspectrum of microbeshave been identifiedfrom the Bacillus ge-nus. Most are not use-ful clinically, however,although polymyxin produced by Bacilluspolymxya is a notable exception to thistrend. Lee and others (2001) documentedthat Bacillus polyfermenticusproduced abacteriocin, polyfermenticin SCD. This heatlabile, proteinase K-sensitive compoundprimarily inhibited other Bacillusspecies.Pinchuk and others (2001) identified twodistinct heat-stable, protease-resistant anti-Helicobacter pyloriactivities produced byBacillus subtilis3 in vitro. One activity wasidentified as amicoumacin A. However, itis not clear how these antimicrobials couldbe produced in the stomach where Heli-

cobacter is found and where evidencesuggests that the vegetative cell would bedestroyed. Therefore, their in vivo rele-vance remains to be demonstrated. Hyron-imus and others (1998) identified an inhib-itory substance, coagulin, produced byBacillus coagulansI4. This bacteriocin-likecompound was plasmid-linked, heat-sta-ble, protease-sensitive, and exhibited bothbacteriocidal and bacteriolytic actionagainst related and unrelated bacteria(Leuconostoc, Oenococcus, Listeria, Pedi-ococcus, andEnterococcus). This discov-ery was followed up by Le Marrec andothers (2000), who characterized coagulin

by N-terminal sequencing of the purified44-amino acid peptide and found it quitesimilar to pediocins produced by Pedio-coccus acidilactici, differing by only a sin-gle C-terminus amino acid. Furthermore,high homology was identified between theplasmid-encoded coagulin gene in Bacil-lus coagulansand the genes encoding thepediocin peptide from Pediococcus. Somegood basic experimentation has been con-ducted on Bacillus bacteriocins, but nostudies documenting the effectiveness ofthese bacteriocins produced in vivo on

pathogen inhibition have been conducted.Animal models.Demonstrating a physi-

ological benefit in animal models is anoth-er important step in establishing probioticefficacy. Sorokulova and others (1997)tested several commercial products (Bio-sporin from Ukraine, Subalin fromUkraine, Bactisubtil from Yugoslavia, andCereobiogen from China) containing Bacil-

lusspecies for Campylobacter inhibition.In vitro inhibition ofseveral C. jejuniand C.coli strains was ob-served by Biosporinproducts containingBacillus subtilisor Ba-cillus licheniformisand by the Bacillussubt i l i s -conta in ingSubalin product. Nei-ther the Bactisubtil northe Cereobiogen prod-ucts demonstrated invitro inhibition of the

Camplylobacter strains. Only Biosporinand Subalin were tested in the mousemodel of Camplylobacter infection. Theauthors concluded that a single preventa-tive oral dose of either of these productscaused a protective effect; however, theendpoints for protective effect and the sta-tistical significance of their results were notclear in the paper. In a model of Vibrioin-fection in rabbits, Hattori and others(1965) demonstrated that Bacillus coagu-lansP-22 inhibited Vibrio-induced chang-es (histological appearance and exudatesaccumulation) in intestinal tissue.

Animal feed supplements. Most otherpapers on the role of sporeformers in ani-mals are focused ontheir use as animalfeed supplements. Al-though this is not thefocus of this paper,their effectiveness assuch is noteworthy. Forexample, Cavazzoniand others (1998) test-ed the effect of Bacilluscoagulans as facilita-tors of growth and effi-cient food conversion

in chickens in 2 inde-pendent studies. Thestudies compared Ba-cillus coagulansor virginiamycin supple-ments to the diet to a control group withno supplements. In the first study, the Ba-cillus coagulans-treated group outper-formed the control group and was equiva-lent to or better than the antibiotic-fedgroup with mean body weight and dailyweight gain from day 7 through the end ofthe study at day 49. Feed intake was un-changed. In the second study, the same re-

sults were observed, except statistically sig-nificant changes did not occur until day33 of the study. Similar results are the basisfor several commercial animal supplementproducts. Their effectiveness resulting incessation or reduction of use of antibioticsin animal feeds would be important.

Impact on immune function. Modula-tion of immune function is a postulated

mechanism of action of probiotic bacteria,including sporeformers. Heat- or formalde-hyde-inactivated Bacillusspecies exhibit-ed immunopotentiating activity in mono-nuclear leukocytes isolated from humans(Prokeov and others 1994). But a fewstudies have been published on immuneenhancement by orally consumed spore-forming bacteria. Muscettola and others(1992) demonstrated that the Enterogermi-na product containing different strains ofBacillus clausii spores administered to iceincreased interferon production ex vivobystimulated peritoneal and spleen cells.Ciprandi and others (1986) evaluated theeffects of Bacillus clausii(formerly desig-nated Bacillus subtilis) spores and vegeta-tive cells on mitogenic T-cell proliferationand mitogen-induced lymphokine pro-duction by mononuclear cells isolatedfrom healthy volunteers. Only vegetativecells stimulated mitogen-induced mono-nuclear cell proliferation. Neither sporesnor vegetative cells modified interleukin-2or -interferon production. Vacca and oth-ers (1983) tested immunomodulating ef-fects of Bacillus clausiispores (Enteroger-mina) in an open label study of 11 multi-ple myeloma patients serving as their own

controls. Spores were administered 3 /d(6 109spores/d) for 21-d periods, alter-nated with a 21-d restperiod following 7-dcytostatic therapy. Im-proved cell-mediatedimmunity parameters,including response ofE-rosettes and mono-cyte chemotaxis, wasobserved after sporetreatment. Further-more, 4 patients expe-rienced a decrease ofrecurrent respiratory

infections occurringbefore immunomodu-lating treatment. No

side effects were reported to the sporetreatment. Kozuka and others (2000) andGoto and others (2000) have used engi-neered strains of Brevibacillusas deliveryagents for mucosal adjuvants.

Human studies.Published human clini-cal trials with oral consumption of spore-formers are few. One study on the impactof sporeformers on reduction of blood lip-ids was reported. This study was conduct-

Physiological differ-

ences between spores

and vegetative cellssuggest that probiotic

effects differ based onphysiological state

Studies on the impactof sporeformers on

promoting human

health are intriguingbut scanty

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ed with Bacillus coagulans (marketed asLactobacillus sporogenes). Althoughthere are 2 retrievable references, these pa-pers report the same data (Mohan and oth-ers 1990a, b). The data were derived froman open-label, nonrandomized study of17 hyperlipidemic patients. Patients re-ceived 2 tablets 3 x/dfor a total daily admin-

istration of 3.6 108spores for 12 wk. Re-ductions in total cho-lesterol (330 to 226mg %) and LDL (267to 173 mg %) were re-corded, but no dietarycontrol was conduct-ed. No adverse inci-dents were reported.These results suggestinteresting pilot studyresults, but cannot beconsidered conclusive for a role of Bacil-lus coagulansin reducing serum lipids inhyperlipidemic patients. This is the only re-trievable study on Lactobacillus sporo-genes in humans.

The effect of Bacillus subtilison patientswith slow or static urinary flow (as a bio-marker of urinary tract infection risk) wasstudied (Meroni and others 1983). Thestudy was a randomized, placebo-con-trolled trial of 80 elderly adults (mean age75.5 y) treated daily for 6 mo with 2 vials(number of spores per vial was not indicat-ed) of Bacillus subtilisATCC 9799 sporesgiven orally. During the 5th and 6thmonths, a statistically significant reduction

in the number of patients with at least 1positive urine culture during each monthand consecutive altered sediment leuko-cyte counts (leukocytes >106/24 h) wasobserved.

The studies published on the impact ofsporeformers on promoting human healthare intriguing but scanty. It can be con-cluded that some strains of sporeformers(1) produce antipathogenic compounds,(2) production of these antipathogeniccompounds may result in an antipatho-genic response in some animal models, (3)the resistance of spores to environmentalstress ensures that these bacteria can pass

through the gastric barrier alive and canremain viable for long periods in commer-cial preparations without refrigeration, (4)vegetative cell or cell components react toimmune cells enhancing immune re-sponse, but the extent of this response inhumans has not been adequately substan-tiated, (5) no adverse incidents have beenreported in published human trials, and (6)retrievable references on controlled hu-man trials reporting the role of sporeform-ers in human health are few and are limit-ed to Bacillusspecies.

Mechanisms of action.The fundamen-tal physiological differences betweenspores and vegetative cells suggest thatprobiotic effects may be mediated bymechanisms which differ based on physi-ological state. Most research on mecha-nisms of efficacy of probiotics in general

has been conductedon nonsporeformers.

Preliminary evidenceindicates that sporesof Bacillus subtilisdonot associate closelywith intestinal cells,nor do they elicit im-mune response(Tompkins, unpub-lished data, 2002).Hosoi and others(1999) have shownthat autoclaved sporesdo not influence the

intestinal microflora in mice. Such studieshave led to the hypothesis that probioticefficacy of spores may be mediatedthrough metabolites or enzymatic activi-ties. The Bacillus species produce pro-teases (for example, subtilisin), which aiddigestion and reduce allergenicity. Theyare also said to produce vitamin K2(Hosoi and Kiuchi 2003). The catalaseand subtilisin produced by Bacillusspe-cies have been shown in vitroto promoteLactobacillus growth (Hosoi and others2000). However, some evidence of sporegermination in vivohas been publishedin animal models (Casula and Cutting2002; Hoa and others 2001; Jadamus

and others 2001). It isnot possible at thistime to concludeabout the role of thespore or vegetativestates in mediatingprobiotic function. Instudies conducted inchickens fed dietary B.subtilisnatto for 28 d(Samanya and Yamau-chi 2002), intestinalhistologies (such asvillus height, cell area,and cell mitosis) were

altered relative to thecontrols. Additionally, it was observedthat blood ammonia was depressed. Itwas hypothesized that that intestinal func-tion was activated by the depressedblood ammonia concentration. Other evi-dence for physiological activity camefrom a study focused on antigenotoxicityactivity of 16 Bacillusstrains (Caldini andothers 2002). Deactivation of 4-nitroquin-oline-1-oxide in a short-term bacterial as-say was demonstrated. Mechanisms ofaction of sporeformers as probiotics re-

main to be defined, but may include met-abolic activities of the microbe, secretionof antimicrobials, and immunomodula-tion. Certainly more research is neededon the mechanisms of probiotic action ofsporeformers.

Safety of sporeforming probiotics

The commercial uses of Bacillus spe-

cies are numerous and include the pro-duction of antibiotics, amino acids, en-zymes, and fermented beans. This facthelps establish a cornerstone for safetyevaluation of these bacteria. However, theuse of Bacillusas a probiotic involves di-rect consumption of high concentrationsof viable microbes. It is rare for any sub-stance to be considered safe for any use.The differences between consumption ofcertain Bacillus species as part of a fer-mented food and consumption of otherspecies of Bacillusat concentrated dosesas a biotherapeutic must be consideredfundamentally different uses with safetyevaluation ramifications. With the excep-tion of Bacillus anthracis and Bacilluscereus, Bacillusspecies have not general-ly been considered pathogenic. Indeed,spores of Bacillusare regularly consumedby animals and man inadvertentlythrough the food and feed supply, and insome fermented foods. The occurrence ofBacillus species (subtilis, licheniformis,cereus, circulans, thuringiensis, sphaeri-cus, badius, firmus, megaterium, my-coides, andsphaericus) and Brevibacilluslaterosporusas components of fermentedsoy or locust beans was demonstrated

with random amplifiedpolymorphic DNAanalysis (Sarkar andothers 2002), docu-menting the associa-tion of these spore-formers with ferment-ed foods. However,there have been manyrecent reports of infec-tions associated withBacillus species, andthese cases are note-worthy when consid-ering the safety of Ba-cillus as a probiotic.

While food poisoning is perhaps thegreatest risk of Bacillusspecies, there arereports of serious local and opportunisticsystemic infections and abortions bythese microorganisms (SCAN 2000a).There is sufficient evidence to suggest thatprobiotic products based on sporeform-ing bacteria undergo more rigorous eval-uation before marketing. Many of theseproducts may prove not to be a threat tothe health and well-being of the consum-er, but insufficient characterization makes

There have been no

toxicity data published

on the various Bacillusspecies with respect to

their use as human

probiotics

To date the FDA hasnot granted GRAS sta-

tus for any sporeform-

er probiotic applica-tion

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this determination difficult. To date, theonly commercially available Bacillusthathas been investigated in safety and toxici-ty studies and shown to be safe is Bacillussubtilis and even in this case the datahave not been widely published.

A misconception perpetuated by somemanufacturers and retailers is that Bacillusspecies are Generally Recognized as Safe

(GRAS) by the Federal Drug Administration(FDA) in the United States of America.While it is true that some Bacillusspeciesare recognized as GRAS for specific appli-cations such as enzyme production, todate the FDA has not granted GRAS statusfor any sporeformer probiotic application.While probiotic bacteria of the genera Lac-tobacillusand Bifidobacteriumhave beensubjected to rigorous testing for acute andchronic toxicity (Donohue and others1993) and panels of experts have revei-wed relevant data and concluded theirsafety for use as probiotics (Adams andMarteau 1995), there have been no toxici-ty data published on the various Bacillusspecies with respect to their use as humanprobiotics. The production of emetic andenterotoxins in clinically relevant strains ofBacillushas been more extensively stud-ied. Significant numbers of toxigenicstrains of Bacillus subtilis, Brevibacillus lat-erosporous, and others have been report-ed. Effective toxin detection assays, whichshould be applied to sporeformers used inprobiotic products (Beattie and Williams1999), have been developed. Althoughadherence to intestinal epithelial cells isfrequently touted as a valuable characteris-

tic of probiotic strains, including spore-formers, studies have suggested that Bacil-lus adhesion is a virulence mechanism(Andersson and others 1998; Rowan and

others 2001).The safety of Bacillussubtilisand Bacil-

lus amyloliquefacienshas been reviewed(de Boer and Diderichsen 1991). This re-view specifically examined published inci-dences of Bacillusinfections. These infec-tions were not due to direct ingestion ofBacillus, but from other sources. The find-ings showed that infections most frequent-

ly appeared in people with a history of en-docarditis, who were immunosuppressedor had recently undergone surgery. Whilethe paper also admits that reported casesof food poisoning byBacillus subtilisarevery low, it points out that exact and reli-able figures are hard to obtain. This is be-cause hospitals do not necessarily differ-entiate between Bacillus cereusand otherspecies of Bacillusas agents of food poi-soning. Bacillusspecies have been associ-ated with nosocomial bacteremia (Richardand others 1988).

Cases of infection resulting from Bacil-lusprobiotic consumption have been re-ported. Oggioni and others (1998) report-ed septicaemia caused by Bacillus subtilisstrains from a probiotic preparation usedby an immunocompromised patient. Spi-nosa and others (2000b) examined 2published examples of Bacillusinfectionswhich may have been linked to a com-mercial probiotic preparation. The infec-tious agent found in one case of cholang-itis in France in 1996 and one case of re-current septicemia in Italy in 1998, wereindistinguishable from a Bacillus strainfound in an Italian probiotic product (Ba-cillus clausii). However, the authors could

not confirm a causal role of the Italianprobiotic in the infections. Both infectionsoccurred in immunosuppressed patients.The French patient had undergone a kid-

ney transplant and the Italian patient wasundergoing chemotherapy. The authorsrecommended the development of a pub-lic database for all alimentary and probi-otic strains where characteristics relevantfor identification and typing could be de-posited.

Some efforts to characterize the antibiot-ic resistance of Bacillus strains used as

probiotic products have been made. BothCiffo (1984) and Mazza and others (1992)evaluated 4 strains of Bacillus clausiiwhich comprise the commercial productEnterogermina for their resistance to thera-peutic antibiotics. Mazza and others(1992) went on to test the stability andtransferability of these resistance traits. Sta-ble resistance to cephalosporins, mac-rolides, and quinolones was observed, buttransfer of these resistance phenotypes toother bacteria in vitroor in vivowas notdetected.

An important aspect of establishing safe-ty of a probiotic is proper taxonomic char-acterization of the bacteria in the product.Green and others (1999) tested 2 commer-cial products (Enterogermina and Biosub-tyl) and showed that neither was com-posed of Bacillus subtilisas claimed by themanufacturers. This was based on severalkey phenotypic characteristics (amylaseactivity and alkaline growth) and total 16SrDNA sequences. The Enterogermina strainmost closely aligned with the Sporolacto-bacillusgroup (Bacillus alcalophilussub-group) and the Biosubtyl strain was mostclosely related to Bacillus pumilus.

It is imperative that sporeforming probi-

otic product be accurately represented tothe consumers. One approach is for indus-try to develop and use objective and sci-ence-based guidelines for commercial

Figure 2Randomly amplified polymorphic DNA-polymerase chain reaction profiling [with dendrogram] is an example ofone technique that can be used to establish strain identity, which is a crucial early step in the manufacturing of a well-characterized probiotic. It is also an essential tool to ensure that the microbe has not changed during storage or produc-tion. Note that there are 2 profiles shown for R0179: the first lane represents the strain before production (mother culture) andthe second lane represents the strain after industrial production (commercial product). Image courtesy of Dr. Denis Roy,

Agriculture & Agri-Food Canada, St. Hyacinthe, QC, Canada.

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products. Alternatively, it is likely that gov-ernments will recognize the need to im-pose more stringent regulations regardingthese particular microorganisms. The fol-lowing list of recommendations is basedpartially on the current guidelines pro-posed researchers (Donohue and Salmin-en 1996; Przyrembel 2001) and world or-ganizations (SCAN

2000a, b; FAO/WHO2001). It is suggestedthat they be consid-ered the minimal safe-ty information forcompanies to bringsporeforming bacteriato market as probiot-ics:

1. Each strain ofbacterium in the prod-uct must be isolated,named, and taxonomi-cally identified. Speci-ation must be un-equivocally estab-lished using the mostcurrent, valid method-ology. Generally, acombination of gene-based and phenotypic techniques are nec-essary. 16s rRNA gene sequencing withcomparison to type strains from a recog-nized culture collection such as ATCC, theEuropean culture collections (DSMZ,LMG, CIP, NCIMB) or Japanese collection(IAM) is the best available approach formost microbes. A strain-specific patternderived from total chromosomal (for exam-

ple, pulsed field gel electrophoresis, ran-domly amplified polymorphic DNA-poly-merase chain reaction), or rDNA must beestablished (Figure 2). If the identity of thestrain or strains of bacteria in a product isin question, no conclusions can be madeon its safety.

2. Nomenclature of the bacteria mustadhere to the current, scientifically recog-nized names. Protracted use of older ormisleading nomenclature is not acceptableon product labels. Current nomenclaturecan be ascertained as indicated in the ear-lier section of this paper on nomenclature.

Announcement on a Validation List,

which validates bacterial names publishedelsewhere. The Validation List is publishedin the Intl J of Syst and Evolutionary Micro-biol.

3. Sufficient in vitrocharacterization ofeach strain of bacteria should be conduct-ed, including: antibiotic resistance profile,production of emetic or enterotoxins, gas-tric acid resistance, and bile resistance.Bacterial strains that demonstrate transfer-able antibiotic resistance should not beemployed. A detailed scheme for testingtoxin production has been already recom-

mended by the Scientific Committee onAnimal Nutrition (SCAN 2000b). Anystrains capable of producing toxins mustnot be used as a probiotic.

Bacteria should be characterized for thepresence of plasmids or transferable DNAvectors. Bacillus plasmids have beenshown to mediate interspecies transfer of

antibiotic-resistance

(Koehler and Thorne1987), and this riskmust be avoided.

4. A review of safetyshould be conductedby an independent3rd party panel of ex-perts qualified in thefield. Depending onthe genus and speciesused, the intendeddose, and the targetconsumers, safetycharacterization maybe conducted as fol-lows. The ability to ad-here to, invade, andmodulate the immunesystem of appropriatehuman cell lines

should be evaluated. Strongly adherent orinvasive strains of bacteria in vegetativeor spore form should not be used as pro-biotics. Acute toxicity and embryo toxicitystudies would contribute to understand-ing of safety. Testing of each strain in con-centrated form (in both vegetative andspore state), and the final product, on atleast one mammalian species may be

needed. Repeated dosage chronic toxicitystudies, for a minimum of 9 mo, shouldbe performed on each strain in concen-trated form (in both vegetative and sporestate) and the final product on at least 2mammals, preferably a rodent and onelarger species (for example, pig, rabbit, orcat).

5. Label and marketing literature mustlist contraindications for Bacillus speciesor sporeforming bacteria, including specif-ic references to patients or consumers whoare immunosuppressed as a result of HIVinfection, chemotherapy, or allograft thera-py.

6. Marketing literature or product labelshould provide:

(a) Indications for use supported by clin-ical evidence.

(b) Clear definition of the genus, species,strain, and concentration of each bacterialcomponent of the product.

(c) An adverse reaction reporting tele-phone number must be clearly indicatedon the product label.

Conclusions

The probiotic industry is in a growthphase, with enhanced activity in tradition-al functional food use and also expandedinterest in use for specific therapeutic in-dications. In general, current governmentlegislation does not provide definitionsregarding the suitability of various bacte-ria for food and supplement use. In mostcountries, products marketed as pharma-

ceuticals must meet premarket criteria forefficacy and safety. This approach pro-vides consumers with assurances onproduct quality. In many cases, use ofprobiotics in dietary supplements (in theU.S.) or foods does not require premarketapproval, but this does not constitute per-mission to market uncharacterized prod-ucts with unsubstantiated safety and effi-cacy. The responsible manufacturer mustconsider the standard for safety as essen-tially the same, regardless of the market-ing niche for the product. In fact, safetyfor a food or supplement could be con-sidered more stringent, in that risk/benefitassessment is acceptable for pharmaceuti-cal products but not for foods or supple-ments. Many companies are promotingthe therapeutic use of Bacillusand othersporeforming bacteria in concentrateddosages, but these companies should notcompromise on safety assessments fortheir products. Companies are leveragingthe temperature resistance of the sporesin order to make shelf-stable label andmarketing claims. Some of these compa-nies are deliberately misusing nomencla-ture, such as referring to their Bacillus co-agulans strains as Lactobacillus sporo-

genes, to take advantage of the more pre-dominant scientific literature that existsfor the lactobacilli. In the end, the con-sumers concern is that safe, efficaciousproducts are available to them. In thecase of many sporeforming bacteria soldas probiotics, their concerns have notbeen adequately addressed.

In contrast, probiotic research with thelactobacilli and bifidobacteria has pro-gressed quite steadily. Numerous articlesreviewing the safety (Borriello and others2003) and efficacy (Guarner and Malage-lada 2003) of this type of probiotic havebeen published in recent years. A Medline

search of articles using the keyword pro-biotic and limited to clinical studies overthe past 10 years resulted in 123 referenc-es. Not one included the terms Bacillus,Sporolactobacillus, or Brevibacillus.Much remains to be done to substantiatesporeformers as probiotics.

References:Adami A, Cavazzoni V. 1999. Occurrence of selected

bacterial groups in the feces of piglets fed with Ba-cillus coagulans as probiotic. J Basic Microbiol39:3-9.

In many cases, use of

probiotics does not re-quire premarket ap-

proval, but this does

not constitute permis-sion to market unchar-

acterized products

with unsubstantiatedsafety and efficacy

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MS 20020667

Author Sanders is with Dairy and Food CultureTechnologies, 7119 S. Glencoe Ct., Centennial,CO 80122 U.S.A. Author Tompkins is Dir. of Re-search, Institut Rosell Inc., Montreal, QC, Canada,and author Morelli is Prof. of Food Biotechnology,Istituto di Microbiologia U.C.S.C., Piacenza, Italy.Direct inquires to author Sanders (E-mail:[email protected]).
mailto:[email protected]:[email protected]

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