APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

Oct. 1999, p. 4637–4645 Vol. 65, No. 10

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Toxigenic Strains of Bacillus licheniformis Related to Food PoisoningM. S. SALKINOJA-SALONEN,1* R. VUORIO,1 M. A. ANDERSSON,1 P. KAMPFER,2 M. C. ANDERSSON,3

T. HONKANEN-BUZALSKI,4 AND A. C. SCOGING5

Department of Applied Chemistry and Microbiology1 and Animal Reproduction, Department of Clinical Sciences,Saarentaus,3 FIN-00014 University of Helsinki, and Department of Food Microbiology, National Veterinary

and Food Research Institute (EELA), 00231 Helsinki,4 Finland; Institut fur Angewandte Mikrobiologie,Justus-Liebig Universitat, D-35390 Giessen, Germany2; and Food Hygiene Laboratory, Central Public

Health Laboratory, Public Health Laboratory Service, London NW9 5HT, United Kingdom5

Received 18 November 1998/Accepted 5 May 1999

Toxin-producing isolates of Bacillus licheniformis were obtained from foods involved in food poisoning inci-dents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the in-hibition of boar spermatozoan motility, has been shown previously to be a sensitive assay for the emetic toxinof Bacillus cereus, cereulide. Cell extracts of the toxigenic B. licheniformis isolates inhibited sperm motility, dam-aged cell membrane integrity, depleted cellular ATP, and swelled the acrosome, but no mitochondrial damagewas observed. The responsible agent from the B. licheniformis isolates was partially purified. It showed physico-chemical properties similar to those of cereulide, despite having very different biological activity. The toxic agentwas nonproteinaceous; soluble in 50 and 100% methanol; and insensitive to heat, protease, and acid or alkaliand of a molecular mass smaller than 10,000 g mol21. The toxic B. licheniformis isolates inhibited growth ofCorynebacterium renale DSM 20688T, but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55°C but not at 10°C, and were nondistinguish-able from the type strain of B. licheniformis, DSM 13T, by a broad spectrum of biochemical tests. Ribotypingrevealed more diversity; the toxin producers were divided among four ribotypes when cut with PvuII and amongsix when cut with EcoRI, but many of the ribotypes also contained nontoxigenic isolates. When ribotyped withPvuII, most toxin-producing isolates shared bands at 2.8 6 0.2, 4.9 6 0.3, and 11.7 6 0.5 or 13.1 6 0.8 kb.

Bacillus licheniformis, Bacillus subtilis, and Bacillus pumiluscomprise the subtilis group, which has been associated with arange of clinical conditions, food spoilage such as ropy bread,and incidents of food-borne gastroenteritis (27). B. lichenifor-mis has also been associated with septicemia, peritonitis, oph-thalmitis, and food poisoning in humans, as well as with bovinetoxemia and abortions (14, 28). B. licheniformis is a commoncontaminant of dairy products (7).

Most food poisoning incidents attributed to Bacillus speciesare associated with Bacillus cereus, but the relevance of thesubtilis group as food poisoning organisms is being increasinglyrecognized. B. cereus toxins have been well documented (12),but involvement of toxins produced by B. licheniformis has notyet been demonstrated. Food-borne B. licheniformis outbreaksare predominantly associated with cooked meats and vegeta-bles (20, 24, 26). We report here on toxin-producing isolates ofB. licheniformis obtained from foods involved in food poison-ing incidents, from raw milk, and from industrially producedbaby food.

(A preliminary report on this work has been presented pre-viously [29].)

MATERIALS AND METHODS

Bacterial cultures. Corynebacterium renale DSM 20688T, B. licheniformis DSM13T, Bacillus amyloliquefaciens DSM 7T, B. pumilus DSM 27T, and B. cereus DSM31T were obtained from the German Collection of Microorganisms and CellCultures, Braunschweig, Germany, and B. subtilis ATCC 6051T was obtainedfrom the American Type Culture Collection, Manassas, Va. The emetic-toxin-producing strains of B. cereus, 4810/72, NC7401, and F-5881, are described

elsewhere (3). The bacteria were cultured for toxicity assays on blood or brainheart infusion (Difco Laboratories, Detroit, Mich.) or in Trypticase soy agar(LAB M, Bury, England) plates or Trypticase soy broth. Temperature tolerancewas tested at 10°C (with B. cereus DSM 31T as a positive control) and at 55°C(60.5°C) in liquid medium on a shaking incubator (150 rpm) and at 28, 37, and55°C (60.5°C) on agar plates.

Biological analyses. The following phenotypic traits were assayed as describedby Smibert and Krieg (25): hydrolysis of lecithin, of starch, and of casein andlysozyme resistance (7 days, 25°C); hemolysis (bovine blood agar plates, readafter 24 and 72 h at 37°C); and anaerobic growth (read after 1 and 7 days, 37°C).The brain heart infusion agar plates were preincubated in anaerobic chambersbefore use. Lipase activity was assayed with modified Sierra lipolysis agar con-taining peptone (25) (10 g), NaCl (5 g), CaCl2 (0.1 g), beef extract (3 g), ferrouscitrate (0.2 g), and agar (15 g) per liter. After autoclaving, 0.5 ml of Victoria BlueB (stock, 0.1 g/150 ml) and 0.1 ml of Tween 80 were added per 10 ml of themedium. Microconcentrator membranes were obtained from Amicon Ltd.,Stonehouse, United Kingdom.

Physiological tests. The food poisoning isolates (coded F) of B. licheniformiswere identified by 25 phenotypic and biochemical tests as described in reference10. Good anaerobic growth and utilization of propionate were used to distinguishthe strains from B. subtilis. All B. licheniformis isolates were characterized byusing API 50 CH cassettes (bioMerieux, Marcy l’Etoile, France), read after 24and 48 h at 37°C with Bacillus identification profile database API Lab1 (version2.1) and with a battery of 87 physiological tests, as described previously (17). Thereaction profiles of these tests were compared with a database (16).

Toxicity tests. Bacteria were grown on tryptic soy agar plates for 10 days at28°C to obtain mainly sporulated and lysed cells, verified by phase-contrastmicroscopy. Colonies were scraped from the agar and suspended in aqua des-tillata to 100 mg ml21. The suspension was treated by repeated freeze-thawcycles and filtered (0.2-mm pore size). The permeate was diluted in dimethylsulfoxide and tested for toxicity by using the same concentration of dimethylsulfoxide as the blank.

The motility inhibition of boar spermatozoa by the cell extracts was tested asdescribed for the emetic toxin of B. cereus (3). Of each bacterial strain, two to fiveindependently prepared extracts were tested. The sperm motility inhibition bythe extracts was given as the concentration required to block motility of 50% ofthe cells (see Table 1) or by indicating the percentage of motile cells (see Table3). Three microscopic fields of 102 spermatozoa (magnification, 3200) wereanalyzed for motility with a Hamilton-Thorne sperm analyzer (HTM-S, version7.2; Hamilton-Thorne Research, Danvers, Mass.) as described in reference 3.The results were confirmed by subjective estimation of motility by phase-contrastmicroscopy (5 to 10 fields; magnification, 3200).

* Corresponding author. Mailing address: Department of AppliedChemistry and Microbiology, P.O. Box 56 (Biocenter), 00014 Univer-sity of Helsinki, Finland. Phone: 358-9-70859300. Fax: 358-9-70859301.E-mail: mirja.salkinoja-salonen@helsinki.fi.

4637

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.

The cell membrane-damaging capacity of the bacterial extracts was measuredby selective staining with ethidium homodimer and calcein AM, carried out asdescribed in reference 15. Damage to acrosomes was recorded by light micros-copy as described elsewhere (30). Mitochondrial damage was documented bytransmission electron microscopy of spermatozoan thin sections as describedelsewhere (3). ATP loss from the spermatozoan was assayed as described else-where (3).

C. renale DSM 20688T growth inhibition (4) was read after 2 days at 28°C fromtwo to three replicate Trypticase soy agar plates with wells each holding 150 to200 ml of the cell extract of the isolate to be tested.

Ribotyping. Ribotyping was performed with a robotized instrument as de-scribed in reference 31. The B. licheniformis strains were prepared and analyzedsimilarly to the B. cereus strains (23). In short, the DNA was restriction endo-nuclease cut with EcoRI or PvuII and hybridized to phosphorescently labeledEscherichia coli whole ribosomal operon. Fragment sizes were determined withthe GelCompar program (version 4.0; Applied Maths BVBA, Kortrijk, Belgium)from the ribotypes produced by the RiboPrinter (Qualicon, Wilmington, Del.) withDNA molecular size markers (1.1, 2.2, 3.2, 6.5, 9.6, and 48 kb) in every third lane.

RESULTS

Detection of toxin-producing B. licheniformis isolates. In to-tal, 210 B. licheniformis isolates involved in food poisoning orsuspected food poisoning and 29 strains and isolates originat-ing from veterinary samples, food packaging material, air, andindustrial contaminants were studied for toxicity by two tests,the spermatozoan motility test and the C. renale DSM 20688T

growth inhibition test. Thirteen strains were found to be pos-itive in one or both toxicity tests. Isolates toxic by both tests(n 5 10) originated from incident-associated food or clinicalspecimens from Finland and the United Kingdom over a pe-riod of .10 years, mainly from cases where B. licheniformis wasisolated from food in high numbers (104 to 105 CFU g21). Inaddition, toxic isolates were obtained from the udder of a cowthat had clinically recovered from severe mastitis. Table 1 is acompilation of the properties of the 13 positive and 9 nontoxicstrains.

Table 1 shows that crude cell extracts (filtered through 0.2-mm-pore-size filters) prepared from 10 B. licheniformis isolatesinhibited boar spermatozoan motility when spermatozoa wereexposed to the extracts. This protocol has been shown to be asensitive indicator for the presence (in nanograms per millili-ter) of the emetic toxin of B. cereus (3). The 10 toxic isolatesincluded two isolates (553/1 and 553/2) cultured from infantfeed formula following an infant fatality (Table 1). Cell ex-tracts prepared from these 10 isolates also inhibited growthof C. renale DSM 20688T. The type strain of B. licheniformis,DSM 13T, was not toxic by either test. The type strains ofB. subtilis (ATCC 6051T) and B. pumilus (DSM 27T) were alsotested and found to be nontoxic to sperm cells.

Two B. licheniformis isolates (575U/5 and 575E/P) of eighttested from unused infant feed packages of the same brand asthat connected to the fatal case blocked sperm motility andinhibited C. renale (Table 1; the six nontoxic isolates are notshown). Toxic strains were also isolated from a fecal specimenof a hospital patient with acute-phase food poisoning symp-toms (F287/91) and from food poisoning cases connected withcurry rice and fast foods (F2943/92, F5520/96, and F231/97).Two of three isolates from milk (each from a separate quarterof the udder) of a postmastitic cow were inhibitory to C. renaleDSM 20688T (Table 1; the nontoxic isolate is not shown) andblocked boar spermatozoan motility. Even though all sperm-toxic isolates inhibited growth of C. renale, the reverse was nottrue. However, the sperm cells were exposed to extracts cor-responding to 2 to 4 mg (wet weight) of B. licheniformis cellsml21, whereas the amount used in the C. renale test corre-sponded to 20 to 40 mg (wet weight) of B. licheniformis cellsper well in the agar plates. The C. renale test, using a higheramount of the agent, may thus have detected weak toxin pro-

ducers which were undetectable in the sperm test. High dosescould not be tested with sperm cells because of nonspecificinterference by the crude bacterial extracts. The results thus donot exclude the possibility that both effects may have beencaused by the same toxic agent. Extracts prepared from thetype strain of B. cereus (DSM 31T) strongly inhibited growth ofC. renale at amounts equivalent to 20 to 40 mg of B. cereus cellsper well.

Description of the toxigenic B. licheniformis isolates. All tox-ic B. licheniformis isolates grew anaerobically; were lecithinasenegative, lipolytic (i.e., they hydrolyzed Tween 80; isolates cod-ed F and 123/3 were not tested), and lysozyme sensitive; andhydrolyzed starch and casein (123/3 was not tested). The 10sperm-toxic strains grew at 55 but not at 10°C in Trypticase soybroth. Twelve of 17 strains inhibitory to C. renale (Table 1) werebeta-hemolytic, and 9 of the 10 sperm-toxic isolates were (theexception was F231/97) also beta-hemolytic. Four of the 15beta-hemolytic isolates were not inhibitory to C. renale and notsperm toxic. The type strain of B. licheniformis (DSM 13T) wasnonhemolytic and nontoxic. The growth inhibition of C. renaleand/or toxicity to boar spermatozoa was thus not due to theproduction of beta-hemolysins by the B. licheniformis isolates.

All isolates listed in Table 1 could use propionate as the solecarbon source, the characteristic which distinguishes B. licheni-formis from B. subtilis and B. pumilus. The sperm-toxic andnontoxic strains of B. licheniformis were compared by using 87biochemical traits. The 21 B. licheniformis isolates presented inTable 1 were characterized as follows. The results presentedfor the 21 isolates are identical to those obtained for the typestrain DSM 13T. Type strain DSM 13T and all isolates assim-ilated N-acetyl-D-glucosamine, L-arabinose, p-arbutin, D-cello-biose, D-fructose, D-galactose, gluconate, D-glucose, D-man-nose, D-maltose, a-D-melibiose, L-rhamnose, D-ribose, sucrose,salicin, D-trehalose, D-xylose, i-inositol, maltitol, D-mannitol,D-sorbitol, acetate, propionate, cis-aconitate, trans-aconitate,4-aminobutyrate, citrate, fumarate, DL-3-hydroxybutyrate, DL-lactate, oxoglutarate, pyruvate, L-alanine, L-aspartate, L-orni-thine, and L-proline. Type strain DSM 13T and all isolates didnot assimilate adonitol, putrescine, adipate, azelate, glutarate,itaconate, L-malate, mesaconate, suberate, b-alanine, L-histi-dine, L-leucine, L-phenylalanine, L-serine, L-tryptophan, 3-hy-droxybenzoate, 4-hydroxybenzoate, and phenylacetate. Allisolates (including type strain DSM 13T) did not hydrolyzeesculin, para-nitrophenyl-b-D-galactopyranoside, para-nitro-phenyl-a-D-glucopyranoside, para-nitrophenyl-b-D-glucopy-ranoside, bis-para-nitrophenyl-phosphate, and L-glutamyl-g-3-carboxy-para-nitroanilide. Type strain DSM 13T but noneof the isolated hydrolyzed para-nitrophenyl-b-D-glucuronide,para-nitrophenyl-phenyl-phosphonate, para-nitrophenyl-phos-phorylcholine, 2-deoxythymidine-59-para-nitrophenyl-phos-phate, L-alanine-para-nitroanilide, and L-proline-para-nitroani-lide. The results confirmed their identity as B. licheniformis,with a Willcox probability of P . 0.99 (17). No biochemicaldifference was detected between the isolates that were toxicand those that were nontoxic to sperm cells or C. renale DSM20688T: all biochemical reactions were the same as those of thetype strain B. licheniformis DSM 13T.

Ribotype patterns of toxic and nontoxic B. licheniformisstrains. The 21 isolates and the type strain of B. licheniformisin Table 1 were ribotyped by using EcoRI and PvuII, and themultiband patterns obtained are shown in Fig. 1. Ten distinctribotype patterns were obtained with EcoRI, and 11 were ob-tained with PvuII. In the ribotype patterns obtained with PvuII(Fig. 1A), the sperm-toxic isolates clustered in four groups, inthree (Fig. 1, lanes A, B, and E) of which the fragment patternswere closely similar. Two of these ribotypes (A and B) also

4638 SALKINOJA-SALONEN ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.

TA

BL

E1.

Origins

andtoxicities

ofB

.licheniformis

isolatesa

Isolatecode

Sourceof

isolateC

ountry

Description

ofillness

CF

Ug

offood

21

Toxic

propertiesof

theB

.licheniformis

isolates

Onset

(h)or

phaseSym

ptom(s)

No.ill/no.risk

Hem

olysisT

oxicityto

boarsperm

dInhibition

zone(m

m)

ofC

.renale

F3648/90

Icecream

UK

Acute

SC,V

,D2/2

Beta

2,

2F

287/91F

ecesof

foodpoisoning

patientU

KA

cuteN

,V,D

,AP

1/1B

eta1

1.

20F

2943/92C

urriedchicken

andm

ayonnaisesandw

ichU

K5

N,SC

,D1/1?

33

106

Beta

1.

20F

2667/94M

incedbeef

pieU

K12

AP,D

1/11

310

82

,2

F2896/95

Tandooriking

prawn

bU

KB

eta2

,2

F9229/95

Blue

cheese,saladdressing

bU

K12

N,A

P,D6/9

Beta

2,

2F

4647/96Pancake

UK

N,V

1/?1.1

310

8B

eta2

.10

F5520/96

Curry

riceb

UK

V1/1

1.13

106

Beta

1.

10F

231/97Profiteroles

bU

K7

3/?3.1

310

51

1.

10F

5734/93V

anillasauce

Norw

ayN

,V,B

.2

2,

2123/3

Vanilla

pudding(reconstituted)

bF

inland6–8

SC,V

,D111/124

(6hospitalized,1

fatal)N

D2

.20

553/1Infant

feed(form

ula)b

Finland

1/1(fatal)

cA

lpha,beta1

1.

10553/2

Infantfeed

(formula)

bF

inland1/1

(fatal)c

Alpha,beta

11

.10

575U/5

Infantfeed

formula,unused

packageF

inlandB

eta1

1.

10575E

/PInfant

feedform

ula,unusedpackage

Finland

Beta

1.

10H

ulta52/97

Milk

frompostm

astiticcow

,1stquarter

Finland

Beta

11

.10

Hulta

53/97M

ilkfrom

postmastitic

cow,2nd

quarterF

inlandB

eta2

.10

Hulta

54/97M

ilkfrom

postmastitic

cow,3rd

quarterF

inlandB

eta1

1.

10T

SP19U

nusedfood

packagingpaperboard

Finland

2,

2T

SP29aU

nusedfood

packagingpaperboard

Finland

2.

1048/87

Air

(garbagedum

p)F

inlandB

eta2

,2

DSM

13T

Type

strain,culturecollection

2,

2

aA

bbreviations:UK

,United

Kingdom

;SC,stom

achcram

ps;V,vom

iting;D,diarrhea;N

,nausea;AP,abdom

inalpain;B,oralburning;N

D,not

determined.

bM

orethan

oneB

acillussp.present

inthe

associatedfood.

cIsolates553/1

and553/2

arefrom

thesam

eincident.

dSperm

cellmotility

inhibitionis

indicatedas

follows:

11

,inhibitionat

,2

mg

ofB

.licheniformis

cells;1

,inhibitionat

4m

g(w

etw

eight)of

cellsper

mlof

extendedboar

semen;

2,no

inhibitionat

4m

gm

l 21.

VOL. 65, 1999 TOXIGENIC BACILLUS LICHENIFORMIS FROM FOOD 4639

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.

contained nontoxic isolates. The three related ribotype pat-terns shared bands at 2.7 6 0.2, 5.0 6 0.2, and 11.6 6 0.1 kb(Table 2). The type strain B. licheniformis DSM 13T (nontoxic)has ribotype A together with another nontoxic strain and twotoxic strains (lane A, Fig. 1). The fourth toxic strain ribotype(lane H in Fig. 1) shared the bands at 2.7 and 5.0 kb but had,in addition, four larger fragments (13 to 35 kb). When cut withEcoRI, one ribotype pattern contained the type strain DSM13T and seven other isolates, of which five were toxic (Fig. 1B,lane A). The other sperm-toxic isolates were scattered amongsix EcoRI ribotype patterns. The results thus show that amongthe toxic strains there was considerable diversity in ribotypes,indicating that the toxic isolates were not a clone. However, thePvuII ribotypes containing toxic isolates shared bands of sim-ilar sizes (2.5, 5.0, 7 to 8, 11.6, or 13.6 kb [Table 2]). Thefragment sizes obtained by the unweighted pair group methodwith averages algorithm and a commercial computer programfor 22 isolates analyzed with an automated ribotyper over aperiod of 1 year showed standard deviations of approximately5% for fragments of ,10 kb and 5 to 10% for fragments of 10to 36 kb. Considering this reproducibility, an automated searchfor fragments of specific sizes may be useful as a preliminaryscreening method for toxic strains.

Effects of toxic B. licheniformis extracts on boar spermato-zoa. The responses, shown as four different viability parame-ters, of boar spermatozoa to cell extracts prepared from ninesperm-toxic isolates of B. licheniformis (Table 1) are compiledin Table 3. None of the extracts was cytolytic toward sperma-tozoa. All nine isolates inhibited motility of the exposed sper-matozoa and damaged cell membrane integrity (Fig. 2), de-pleted the cellular ATP content (Table 3), and swelled theacrosome (Fig. 3). None of these extracts swelled the mito-chondria as observed by transmission electron microscopy(Fig. 4). Extracts prepared from the type strains of B. licheni-formis, B. subtilis, B. pumilus, Bacillus mycoides, and B. cereuscaused none of the effects exhibited by the strains of B. licheni-

formis (Table 3). Extracts prepared from three emetic-toxin-producing strains of B. cereus (4810/72, NC7401, and F-5881)were tested in the same assay; all inhibited spermatozoan mo-tility at extremely low concentrations (corresponding to ,0.002mg [wet weight] of cells ml21) and swelled the mitochondria inthe sperm tail but had no effect on the membrane integrity, cellATP content, or the acrosome (Table 3). The extracts of eachof the three emetic B. cereus strains had no effect on the growthof C. renale at doses corresponding to 20 to 40 mg (wet weight)of cells per well in the agar plate.

TABLE 2. Fragment sizes observed in the B. licheniformis PvuIIribotypes containing toxic isolates

Fragment sizerange (kb)

Size of fragment for PvuII ribotypea:

A B E H

35.1–36.0 35.5 (63.7)19.1–35.0 19.3 (60.9)16.1–19.0 16.8 (61.0)13.1–16.0 13.8 13.1 (60.8)10.1–12.0 11.7 (60.5) 11.8 (60.1) 11.39.1–10.0 9.9 (60.5) 10.0 (60.2)8.1–9.0 8.4 (60.3) 8.5 (60.1) 8.3 (60.4)7.1–8.0 7.1 (60.2) 7.2 (60.2) 7.76.6–7.0 6.8 (60.2) 7.0 (60.1) 7.0 (60.3)6.1–6.5 6.2 (60.2) 6.3 (60.1)4.1–6.0 5.2 (60.1) 4.8 (60.1) 5.7 4.9 (60.2)2.0–4.0 2.6 (60.1) 2.6 (60.1) 2.8 2.8 (60.1)

a Sizes are shown in kilobases 6 standard deviations calculated for the isolateswith identical ribotypes. For the patterns of the isolates in ribotypes A, B, E, andH (similarity value of 0.95), see the ribotype images in lanes A, B, E, and H,respectively, in Fig. 1. The sizes are means of all strains sharing the ribotype.Ribotype A included two toxic and two nontoxic isolates, ribotype B includedfour toxic isolates and one nontoxic isolate, ribotype E included one toxic isolateand no nontoxic isolates, and ribotype H included three toxic and two nontoxicisolates.

FIG. 1. Ribotyping of 25 isolates and strains of B. licheniformis of different origins, with PvuII (A) or EcoRI (B) and hybridization with labeled whole ribosomaloperon of E. coli. The patterns obtained from B. amyloliquefaciens DSM 7T, B. cereus DSM 31T, and B. subtilis ATCC 6051T are also shown. Strains indicated as beingof the same ribotype exhibited patterns with a similarity value of .0.95. (A) Lane A, DSM 13T, 553/2 (toxic), 575E/P (toxic), and TSP29a; lane B, 553/1 (toxic), F287/91(toxic), F231/97 (toxic), Hulta 53/97, and Hulta 54/97 (toxic); lane E, F2943/92 (toxic); lane H, 575U/5 (toxic), F2667/94, F5520/96 (toxic), Hulta 52/97 (toxic), andTSP19; lanes C, D, F, G, I, J, and K, no toxic isolates; lanes L, M, and N, reference strains B. amyloliquefaciens DSM 7T, B. subtilis ATCC 6051T, and B. cereus DSM31T, respectively. (B) Lane A, DSM 13T, 553/1 (toxic), 575U/5 (toxic), F281/91 (toxic), F5734/93, TSP29a, and Hulta 52/97 (toxic); lane C, F9229/95, Hulta 53/97, andHulta 54/97 (toxic); lane D, F2943/92 (toxic), F2896/95, and F231/97 (toxic); lane E, 575E/P (toxic); lane F, 553/2 (toxic); lane I, F5520/96 (toxic); lanes B, G, H, andJ, no toxic isolates; lanes K, L, and M, reference strains B. amyloliquefaciens DSM 7T, B. subtilis ATCC 6051T, and B. cereus DSM 31T, respectively.

4640 SALKINOJA-SALONEN ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.

These results show that the sperm-toxic agent(s) producedby the toxic strains of B. licheniformis was uniform in action.The exerted toxic effect differed in biological activity from thatof the cereulide produced by the emetic strains of B. cereus. Itis interesting that the extracts prepared from the two sperm-toxic B. licheniformis isolates (Hulta 52/97 and Hulta 54/97)originating from raw milk from a postmastitic cow exhibitedtoxic responses in spermatozoan cells quantitatively and qual-itatively similar to those seen after exposure to the toxic foodpoisoning isolates, including those from an unused package ofbranded infant feed (Tables 1 and 3). The effects on the sper-matozoan plasma membrane and on the acrosomal responseto B. licheniformis extracts were dose dependent. The toxicthreshold of these effects was equivalent to 2 to 4 mg (wetweight) of bacterial cells per ml of extended boar semen for alltoxic B. licheniformis strains (Fig. 2 and Table 1).

Toxic activities of extracts prepared from the B. licheniformisisolates 553/1, 553/2, 575U/5, Hulta 52/97, and Hulta 54/97were insensitive to heat (100°C for 20 min), inactivation bypronase (200 mg ml21, 3 h), acid (pH 2 with HCl for 30 min),and alkali (pH 12 with NaOH for 30 min). The observed heatstability suggests that the toxin(s) was not an enzyme. The toxicagent was more soluble in 50 or 100% methanol than in water.As a solution in 50% (vol/vol) methanol, the toxic agent wasfilterable through microconcentrator membranes with a nom-inal cutoff of 10,000 g mol21 but not as a water extract, indi-cating a tendency to hydrophobic interactions. The 10,000-gmol21 filtrates of the toxic extracts exhibited unaltered toxiceffects on the spermatozoa. These results show that the B. li-cheniformis sperm-toxic agent was nonproteinaceous, heat sta-ble, and nonpolar and of an apparent mass smaller than 10,000g mol21. The agent inhibiting the growth of C. renale also

survived the treatments listed above, indicating that it was thesame or a similar type of compound as the agent blockingspermatozoan motility.

DISCUSSION

This paper is to our knowledge the first demonstration oftoxins produced by B. licheniformis isolates associated withhuman disease. Toxins produced by B. licheniformis were de-tected by the boar spermatozoan motility inhibition assay,which has been reported as a sensitive and specific test fordetecting emetic-toxin-producing B. cereus strains (3).

The toxins of B. licheniformis inhibited sperm motility (Table1) by interfering with the cellular energy metabolism in amanner different from that shown with the emetic toxin ofB. cereus, a toxin that causes swelling of mitochondria (3, 21).The toxic threshold of the B. licheniformis extracts was $100times higher than those observed for emetic-toxin-producingB. cereus strains (3). The occurrence of B. cereus in sensitivefoods is regulated in many countries (in Finland, the limit is#103 CFU g21). There is no restriction on B. licheniformis,which often occurs in high numbers in foods (we observed 104

to 108 CFU g21 [Table 1]). The toxigenicity observed in thepresent work may thus be of food poisoning significance. Thetoxic B. licheniformis extracts induced the acrosome reaction(Table 3 and Fig. 3), a very likely novel trait among bacterialtoxins, and possibly indicating an impact on the cellular signal-ling system.

Despite differences in biological activity, the sperm-toxicagents from the isolates of B. licheniformis studied (Table 3)were similar in many physicochemical properties to cereulide,the emetic toxin of B. cereus (1, 3). Cereulide is a dodecadep-

TABLE 3. Effects of cell extracts from food- and food poisoning-related isolates of B. licheniformis andfrom reference strains on boar spermatozoaa

Extract prepared from eachof the strains or isolates

Observed response

Damage to energy metabolismof sperm cells

% of sperm cells in which morphologicaldamage observed

% of spermcells motilec

ATP (mg ml21) ofextended sperm

Membraneintegrityd

Acrosomeswellingb

Mitochondrialswellinge

B. licheniformis isolates from foodF287/91, F2943/92, F5220/96, F231/97, 553/1, 553/2,

and 575U/5,1 ,0.1 .70 .50 ,1

Raw milk isolates (postmastitic cow) Hulta 52/97and Hulta 54/97

,1 ND f .70 ND ,1

Emetic-toxin-producing strains of B. cereus 4810/72,NC7401, and F-5881

,1 .3 ,20 ,10 .50

Type strains of related speciesB. licheniformis DSM 13T .60 .3 ,20 ,10 ,1B. subtilis ATCC 6051T .60 .3 ,20 ND ,1B. pumilus DSM 27T .60 .3 ,20 ND ,1B. mycoides ATCC 6462T .60 .3 ,20 ,10 ,1B. cereus DSM 31T .60 .3 ,20 ,10 ,1

Control exposuresReagents only .60 .3 ,20 ,10 ,1Freeze-thaw ,1 ,0.1 .70 ND ND

a Extended boar semen (30 3 106 to 60 3 106 sperm cells ml21) was exposed for 1 to 3 days to 10 ml of cell extracts (0.2-mm-pore-size filter filtrate) ml21 preparedfrom plate-grown cultures. Two to three extracts of each strain or isolate were tested with sperm from three individual boars. The average difference between the resultswas ,20% in all cases.

b Degenerated acrosomes were detected after Giemsa staining by light microscopy (Fig. 3).c Percentage of sperm cells expressing high motility as shown by the Hamilton-Thorne sperm motility analyzer (3).d As observed by vitality staining and fluorescence microscopy (15) (Fig. 2).e Swollen and disrupted mitochondria as observed by transmission electron microscopy (Fig. 4D).f ND, not determined.

VOL. 65, 1999 TOXIGENIC BACILLUS LICHENIFORMIS FROM FOOD 4641

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.

FIG. 2. Fluorescence micrographs of boar spermatozoa stained for determination of viability after being exposed to cell extracts of different B. licheniformis strains.(A) Sperm cells (5 3 106) in 1 ml of extended (with commercial extender) boar semen were exposed to cell extract from 4 mg (wet weight) of B. licheniformis DSM13T cells. Over 80% of the spermatozoa showed intact cell membranes. Similar results were obtained with spermatozoa exposed to the negative control (staining green).(B) Effect of extract prepared from 2 mg (wet weight) of cells of B. licheniformis 553/2. Fifty percent of the sperm cells lost integrity in the cell membrane (stainingorange). (C) Sperm cells were exposed to extract prepared from 4 mg (wet weight) of the same isolate as in panel B. Seventy percent of sperm cells lost cell membraneintegrity (red). Magnification in all panels, 32,000 (i.e., sperm head dimensions are 2 to 3 by 3 to 5 mm).

4642 SALKINOJA-SALONEN ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.

FIG. 3. Light micrographs of Giemsa stained boar spermatozoa exposed to cell extracts of B. cereus and toxic and nontoxic strains of B. licheniformis. (A and B)Extended boar semen (1 ml) exposed to extract from 4 mg (wet weight) of cells of the emetic B. cereus strain 4810/72 (A) or the (nontoxic) B. licheniformis type strainDSM 13T (B). Over 90% of the exposed sperm cells showed heavily staining dark intact acrosomes, similar to spermatozoa exposed to negative control extract (datanot shown). (C) Extended boar semen (2 ml) exposed to B. licheniformis 553/2 (extract from 4 mg [wet weight] of cells). Over 50% of the cells showed lightly stainingfused acrosomes or swollen and disrupted acrosomes. Magnification in all panels, 32,000 (i.e., sperm head dimensions are 2 to 3 by 3 to 5 mm).

VOL. 65, 1999 TOXIGENIC BACILLUS LICHENIFORMIS FROM FOOD 4643

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.

sipeptide structurally resembling valinomycin (1). Peptide tox-ins are nonribosomally produced by a wide range of microor-ganisms (18). Also, some strains of B. licheniformis are knownto produce peptide antibiotics (13), such as bacitracin (11, 18)and amoebicins (8, 9, 19), some of which have been used asantimicrobial agents, but none have so far been shown to beassociated with food poisoning. The B. licheniformis toxins

reported in this paper also possessed antimicrobial activity,demonstrated by the induction of large inhibition zones by cellextracts of many of the B. licheniformis isolates when intro-duced onto plate cultures of the actinomycete C. renale DSM20688T (Table 1). C. renale has been shown elsewhere to besusceptible toward human pathogenic Staphylococcus aureusstrains producing staphylococcin BacR1 (4) and to be associ-

FIG. 4. Thin cross sections of the middle segments of boar spermatozoa exposed for 4 days to cell extracts of B. licheniformis 553/2 and F287/91, the B. licheniformistype strain DSM 13T, and an emetic toxin producer strain, B. cereus 4810/72. (A and B) Sperm cells exposed to cell extracts prepared from 4 mg (wet weight) of isolates553/2 and F287/91. These sperm cells had lost motility and ATP and showed damaged cell plasma membrane, but the mitochondria were intact. (C) Spermatozoonexposed to extract of the type strain B. licheniformis DSM 13T (nontoxic). These cells displayed normal motility and cellular ATP content after exposure, and the figureshows an intact plasma membrane. (D) Sperm cell exposed to cell extract from 2 mg (wet weight) of the emetic strain B. cereus 4810/72 (12) ml21. These cells havea morphologically intact plasma membrane and normal ATP content, but over 90% of the cells exposed showed no motility, and their mitochondria were swollen witha disrupted outer membrane. Bars, 200 nm.

4644 SALKINOJA-SALONEN ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.

ated with the production of the exfoliative toxin B (22). Theanticorynebacterial activity of B. licheniformis isolates wascaused by an agent(s) physicochemically similar to that toxic tospermatozoa (both are resistant to heat, acid, and alkali andsoluble in methanol). Sperm toxin-producing B. licheniformisstrains were not readily differentiated from nontoxigenicstrains by only biochemical and physiological criteria (Table 1;see also above). Ribotyping revealed great genetic diversity;the toxigenic strains thus formed no clone. However, the toxicribotypes were related (shared bands [Table 2]); only 4 of the11 PvuII ribotypes contained toxigenic strains. Automated ri-botyping may be useful as a preliminary screening test forputative toxin producers.

It is interesting that the ribotype of a toxic isolate obtainedfrom the baby food associated with a fatal food poisoning(553/1) was identical to that of the isolate obtained from anunopened package of the same brand (575U/5) and to that ofa toxic isolate cultured from the raw milk of a cow that had ap-parently recovered from mastitis (Hulta 52/97). As B. licheni-formis is a sporeformer and likely to survive all industrial pro-cessing of milk, such as the manufacture of milk powder andwhey concentrate, such a finding may indicate a possible routeof infection.

These results also indicate genetic diversity among the toxicB. licheniformis isolates: two different toxigenic ribotypes wereisolated from two different quarters of the same udder of acow, two different toxigenic ribotypes were isolated from thesame batch of baby food, and two toxigenic strains with differ-ent ribotypes were recovered from an unused package of com-mercial baby food. It is possible that genotypically distinguish-able different toxigenic isolates might have been detected alsoin the other food poisoning cases listed in Table 1, had morethan one isolate been available for study.

Recombinant strains of B. licheniformis are used to produceindustrial enzymes on a large scale, e.g., carbohydrase andprotease used in food processing (5, 6). The species is consid-ered safe and has generally recognized-as-safe status with theU.S. Food and Drug Administration (6). In the light of ourfindings, the generally recognized-as-safe status of the speciesB. licheniformis may require reassessment.

ACKNOWLEDGMENTS

This work was financially supported by the Academy of Finland(M.S.S.) and the Centre of Excellence Fund of the University ofHelsinki (M.S.S.). We thank the Institute of Biotechnology of theUniversity of Helsinki for the use of the electron microscope.

We are grateful to Jyrki Juhanoja for expert technical support inelectron microscopy, to Paula Hyvonen (EELA, Kuopio) for donatingstrain 123/3, to Irina Tsitko for help with the GelCompar program, andto Irmgard Suominen and Camelia Apetroaie for their contributions insperm toxicity testing.

REFERENCES

1. Agata, N., M. Ohta, M. Masashi, and M. Isobe. 1995. A novel dodecadep-sipeptide, cereulide, is an emetic toxin of Bacillus cereus. FEMS Microbiol.Lett. 129:17–20.

2. Andersson, M., M. Laukkanen, E.-L. Nurmiaho-Lassila, F. A. Rainey, S. I.Niemela, and M. S. Salkinoja-Salonen. 1995. Bacillus thermosphaericus, sp.nov., a new thermophilic ureolytic Bacillus isolated from air. Syst. Appl.Microbiol. 18:203–220.

3. Andersson, M. A., R. Mikkola, J. Helin, M. C. Andersson, and M. S. Salki-noja-Salonen. 1998. A novel sensitive bioassay for detection of Bacilluscereus emetic toxin and related depsipeptide ionophores. Appl. Environ.Microbiol. 64:1338–1343.

4. Crupper, S. S., A. J. Gies, and J. J. Iandolo. 1997. Purification and charac-terization of staphylococcin BacR1, a broad-spectrum bacteriocin. Appl.Environ. Microbiol. 63:4185–4190.

5. de Boer, S. A., F. G. Priest, and B. Diedrichsen. 1994. On the industrial use

of Bacillus licheniformis: a review. Appl. Microbiol. Biotechnol. 40:595–598.6. Food and Drug Administration. 1983. 21 CFR 184:1027. Fed. Regist. 48:

329–340.7. Frank, J. F. 1997. Milk and dairy products, p. 100–116. In M. P. Doyle, L. R.

Beuchat, and T. J. Montville (ed.), Food microbiology: fundamentals andfrontiers. American Society for Microbiology, Washington, D.C.

8. Galvez, A., E. Valdivia, A. Gonzalez-Segura, M. Lebbadi, M. Martinez-Bueno, and M. Maqueda. 1993. Purification, characterization and lytic ac-tivity against Naegleria fowleri of two amoebocins produced by Bacillus li-cheniformis A12. Appl. Environ. Microbiol. 59:1480–1486.

9. Galvez, A., M. Maqueda, P. Cordovilla, M. Martinez-Bueno, M. Lebbadi,and E. Valdivia. 1994. Characterization and biological activity againstNaegleria fowleri of amoebocins produced by Bacillus licheniformis D-13.Antimicrob. Agents Chemother. 38:1314–1319.

10. Gordon, R. E., W. C. Haynes, and C. Hor-Nay Pang. 1973. The genusBacillus, p. 3–14. In Agriculture handbook no. 427. United States Depart-ment of Agriculture, Washington, D.C.

11. Grafe, U. 1992. Biochemie der Antibiotika: Struktur-Biosynthese-Wirk-mechanismus, p. 164–170. Spektrum Akademischer Verlag GmbH, Heidel-berg, Germany.

12. Granum, P. E. 1997. Bacillus cereus, p. 327–336. In M. P. Doyle, L. R.Beuchat, and T. J. Montville (ed.), Food microbiology: fundamentals andfrontiers. American Society for Microbiology, Washington, D.C.

13. Horowitz, S., and W. M. Griffin. 1991. Structural analysis of Bacillus licheni-formis 86 surfactant. J. Ind. Microbiol. 7:45–52.

14. Johnson, C. T., G. R. Lupson, and K. E. Lawrence. 1994. The bovine pla-centome in bacterial and mycotic abortions. Vet. Rec. 134:263–266.

15. Juonala, T., S. Lintukangas, T. Nurttila, and M. Andersson. 1998. Relation-ship between semen quality and fertility in 106 A1-boars. Reprod. Domest.Anim. 33:155–158.

16. Kampfer, P. 1991. Application of miniaturized physiological and biochemicaltests in numerical classification and identification of some bacilli. J. Gen.Appl. Microbiol. 37:225–247.

17. Kampfer, P., E. B. M. Denner, S. Meyer, E. R. B. Moore, and H.-J. Busse.1997. Classification of “Pseudomonas azotocolligans” Anderson 1955, 132, inthe genus Sphingomonas as Sphingomonas trueperi sp. nov. Int. J. Syst. Bac-teriol. 47:577–583.

18. Kleinkauf, H., and H. von Dohren. 1996. A nonribosomal system of peptidesynthesis. Eur. J. Biochem. 236:335–351.

19. Lebbadi, M., A. Galvez, E. Valdivia, M. Martinez-Bueno, and M. Maqueda.1994. Purification of amoebicin m4-A from Bacillus licheniformis M-4. Arch.Microbiol. 162:98–102.

20. Lund, B. M. 1990. Foodborne diseases due to Bacillus and Clostridiumspecies. Lancet 336:982–986.

21. Mahler, H., A. Pasi, J. M. Kramer, P. Schulte, A. C. Scoging, W. Baer, andS. Kraehenbuehl. 1997. Fulminant liver failure in association with the emetictoxin of Bacillus cereus. N. Engl. J. Med. 336:1143–1148.

22. Melish, M. E., and L. A. Glasgow. 1971. Staphylococcal scalded skin syn-drome: the expanded clinical syndrome. J. Pediatr. 78:958–967.

23. Pirttijarvi, T. S., M. A. Andersson, A. Scoging, and M. S. Salkinoja-Salonen.1999. Evaluation of methods for recognizing strains of Bacillus cereus withfood poisoning potential among industrial contaminants. Syst. Appl. Micro-biol. 22:133–144.

24. Rosenkvist, H., and Å. Hansen. 1995. Contamination profiles and character-ization of Bacillus species in wheat bread and raw materials for bread pro-duction. Int. J. Food Microbiol. 26:353–363.

25. Smibert, R. M., and N. R. Krieg. 1994. Phenotypic characterization, p.607–654. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg(ed.), Methods for general and molecular bacteriology. American Society forMicrobiology, Washington, D.C.

26. Tatzel, R., W. Ludwig, K.-H. Schleifer, and P. R. Wallnofer. 1994. Identifi-cation of Bacillus strains isolated from milk and cream with classical andnucleic acid hybridization methods. J. Dairy Res. 61:529–535.

27. Turnbull, P. 1997. The role of Bacillus genus in infection. Culture 18:5–8.28. Turnbull, P. C. B., and J. M. Kramer. 1995. Bacillus, p. 349–356. In P. R.

Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.),Manual of clinical microbiology, 6th ed. American Society for Microbiology,Washington, D.C.

29. Vuorio, R., M. A. Andersson, T. Johansson, T. Honkanen-Buzalski, andM. S. Salkinoja-Salonen. 1998. Toxin producing Bacillus licheniformis strainsin infant food. Poster presented at International Conference on FoodbornePathogens, Detection and Typing, The Hague, The Netherlands, 20 to 21April 1998.

30. Watson, P. F. 1975. The use of Giemsa stain to detect changes in acrosomesof frozen ram spermatozoa. Vet. Rec. 97:12–13.

31. Zaitsev, G. M., I. V. Tsitko, F. A. Rainey, N. I. Govorukhina, Y. A. Trotsenko,J. S. Uotila, E. Stackebrandt, and M. S. Salkinoja-Salonen. 1998. Newaerobic ammonium-dependent and obligately oxalotrophic bacteria: descrip-tion of Ammoniphilus oxalaticus, gen. nov., sp. nov., and Ammoniphilus ox-alivorans gen. nov., sp. nov. Int. J. Syst. Bacteriol. 48:151–161.

VOL. 65, 1999 TOXIGENIC BACILLUS LICHENIFORMIS FROM FOOD 4645

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 23

Oct

ober

202

1 by

210

.195

.103

.243

.