J Clin Pathol 1991;44:455-458

Toxin production by Bacillus pumilus

B Hoult, A F Tuxford

AbstractTwo strains of Bacillus pumilus (Mlland M38) and one strain each of Bacilluscereus (M27), Bacillus subtilis (M67),and Enterobacter agglomerans (M14)were identified from the air ofLancashire cotton mills. These strainswere tested for cytopathic effects in Verocells; B pumilus and B cereus strainswere also examined for haemolyticactivity, lecithinase production, andproteolytic action on casein. Roundingand clumping of the Vero cells occurredafter the addition of supernatantsprepared from B pumilus and B cereusstrains; finger-like projections developedin the cells treated with B pumilussupernatants. Minimal effects occurredwith B subtilis and E agglomerans.After two hours of exposure B pumilus(Mll) produced the greatest effect, buttreatment with trypan blue showed thatmost cells exposed to the Ml1 strainwere still alive after 96 hours ofexposure; those exposed to the super-natant prepared from the M38 strain ofB pumilus were. dead. Sheep erythro-cytes were lysed more rapidly byB cereus than by B pumilus, B cereus(strongly positive) had a greater effect onlecithin than either of the B pumilusstrains (M38 weakly positive, Ml1negative). All hydrolised casein but theeffect was more rapid with one of theB pumilus (Ml 1) strains.

It is concluded that not only do thetoxins of B pumilus differ from those ofB cereus, but there are also differencesbetween the toxins produced by the twostrains ofB pumilus (M1 and M38).

Tuffnell,5 however he did not find Entagglomerans (previously known as Erwiniaherbicola), although his culture media wouldhave supported the growth of these bacteria.He reported that the predominant specieswere B pumilus and B subtilis in the cottonmills where byssinosis occurred and Bmegaterium in the jute mills, where the diseasewas absent. Twenty five years later Tuxfordand Moogan isolated these three species ofBacillus and also Ent agglomerans among 21different airborne bacterial species in theLancashire cotton mills (the jute mills hadclosed).6 It seemed logical, therefore, toexamine airborne bacteria, including Bpumilus and Ent agglomerans isolated from theLancashire cotton mills, for toxin production.There are various techniques for detecting

toxins produced by bacteria includingobservation of the cytopathic effects on livingcells. Monolayers of Vero cells (derived fromgreen monkey kidney) have been used for thedetection of Clostridium perfringens toxin,7 Bcereus toxin,8 and enterotoxin of enteropathic.Escherichia coli.9 As this cell line has beenused to detect toxins from both Gramnegative and Gram positive bacteria, includ-ing B cereus, it seemed to be suitable fortesting both Bacillus species and Entagglomerans.

Other manifestations of toxin production bythe Bacillus species are the ability to lyseerythrocytes of different animal species(haemolysins), to attack proteins (proteolyticactivity), and to break down lethicin by theaction of lecithinase. Some of these propertieshave been used by Turnbull and Kramerl' intheir scheme for the identification of B cereus,and it was decided to modify their techniquesto detect similar properties in strains of Bpumilus and some of the other species.

Department ofPathological Sciences,Division ofBacteriology,University ofManchester, OxfordRoad, ManchesterM13 9PT.B HoultA F TuxfordCorrespondence to:Dr B Hoult

Accepted for publication6 December 1990

In recent years attention has focused on therole of bacterial toxins in causing disease.Flindt has shown that preparations fromBacillus subtilis were responsible for causingrespiratory symptoms in workers producingwashing powders containing enzymes.'B pumilus, which produces toxin, has beenisolated from guinea-pigs with experimentallyinduced enterocolitis associated with clinda-mycin' and toxin from B cereus has beenshown to cause food poisoning.3

Current opinion is that endotoxin fromEnterobacter agglomerans (a Gram negativebacterium found on plants) has an importantrole in producing the symptoms of byssinosis,the cotton and flax workers' respiratorydisease.4 When the bacterial contents of the airin Lancashire cotton mills were studied by

MethodsBACTERIAThe bacteria used in this series of experimentswere from a collection isolated in 1986 from theair of 18 Lancashire cotton spinning mills, arepresentative number of which were stored onnutrient agar slopes for future use. Included inthe collection were 14 strains ofB pumilus, fourof B cereus, two of Ent agglomerans and fivestrains of B subtilis. Fifty six strains from 21bacterial species were screened initially fortoxic effects on Vero cells; 12 out of the 14strains of B pumilus and three out of four of Bcereus were cytopathic; all the B subtilis and Entagglomerans strains proved to be non-cytopathic. Two strains ofB pumilus (M 1 andM38) and one of B cereus (M27) were selected

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for further examination. Strains of B subtilis(M67) and Ent agglomerans (M14) were alsoincluded as non-cytopathic controls in some ofthe experiments.The identities of the previously isolated

strains were reconfirmed by Gram staining,cultural appearances, and API galleries forbiochemical characterisation (20B for Bacillusspecies and 20E for Ent agglomerans). Cultureswere also tested for their ability to growanaerobically, and spore stains were performedon older cultures of Bacillus species.

PREPARATION OF SUPERNATANTS FORINOCULATION INTO TISSUE CULTUREThe bacteria were cultured overnight innutrient broth at 370C, nutrient agar (OxoidBroth No 2 with 1% agar) bijoux slopes wereinoculated from these cultures and incubatedfor 48 hours at 37°C. The cultures wereharvested into 0 5 ml of Gibco's minimalessential medium (MEM), supplemented with1% glutamine and 1% fetal calf serum. Thesuspensions were mixed for 30 seconds on avortex mixer, left at room temperature for 60minutes, then centrifuged for 15 minutes at1800 x g. Before use the supernatants werefiltered through a sterile millipore filter of0-2 im pore size. Uninoculated nutrient agarslopes were treated in the same way to providebacteria-free supernatants to act as negativecontrols.

TISSUE CULTUREWells of tissue culture microtitre plates(Falcon) were inoculated with 200 ,l of Verocells grown in MEM supplemented with 10%fetal calfserum, and incubated at 37°C for 24 to48 hours in a carbon dioxide incubator until thecell sheet was confluent. The growth mediumwas then replaced with 200 pul of bacterialsupernatant and duplicate plates wereincubated at 37°C in 5% carbon dioxide andexamined at intervals (30 and 60 minutes, two,four, 24, and 48 hours) using an invertedmicroscope to detect cytopathic effects.Supernatants were tested in duplicate andsupernatants from non-cytopathic species,similarly treated, were included as negativecontrols. To establish whether the effect wascytotonic (more than 50% of cells showingrounding) or cytotoxic (death of more than50% of cells),"' trypan blue was added toseveral wells each day to determine the numberof dead cells. In a separate experiment theregenerative capacity of the tissue culture wastested by removing supernatants after 30 and60 minutes of exposure and replacing with 200pl of either growth or maintenance medium.The plates were reincubated at 37°C in carbondioxide and the Vero cells were examined daily.

OTHER TOXIC EFFECTSTwo strains of B pumilus (M 1I and M38) andthe M27 strain ofB cereus (as a positive control)were examined for haemolytic, phos-pholipolytic, and proteolytic effects using themethods ofTurnbull and Kramer,'0 modified as

follows: Supernatants were prepared byinoculating one drop of an 18 hour nutrientbroth culture (Oxoid) into 50 ml of brain heartinfusion broth (Oxoid) and incubating in a

shaking water bath at 37°C for 48 hours.Bacteria were removed by centrifugation andfiltration. Removal was confirmed by inoculat-ing the filtrates onto nutrient agar plates,incubating overnight at 37°C and examiningfor absence of bacterial growth.

Haemolytic effectTwofold dilutions of the supernatants inphosphate buffered saline (PBS) (Dulbecco Atablets; Oxoid) were made in WHO plates andequal volumes of 0 5% washed sheep erythro-cytes suspended in PBS were added to give finalconcentrations in the wells from 1 in 10 to 1 in1240. The plates were incubated at 37°C andinspected for haemolysis after four and 24hours.

Phospholipolytic activityThis was determined by placing 50 MI ofsupernatant in 5 mm diameter wells cut inlecithin agar (10% egg yolk emulsion agar,Oxoid). The plates were incubated at 37°C andthe level of activity determined by measuringthe diameter of the zones of opacity aroundthe wells after four, eight, and 24 hours'incubation.

Proteolytic activityWells of 5 mm diameter were cut in casein(50% skimmed milk) agar plates; these werefilled with 50 MuI ofsupernatant and incubated at37°C. Zone diameters around the wells weremeasured after four, eight, and 24 hours.

All experiments were performed in duplicateand were repeated.

ResultsTypical toxic effects on the Vero cell sheets areshown in the figs 1-4. In the wells containingsupernatant with cytopathic properties thecells showed rounding and clumping with theformation of holes in the cell sheet; longprojections developed in cells treated with Bpumilus supernatants (fig 1). B cereus caused acytopathic effect but without the finger-likeprojections (fig 2); B subtilis supernatant hadlittle or no effect on the Vero cells, and minimalcytopathic effects were also observed in thosewells treated with Ent agglomerans (fig 3).

Effects of Bacillus supernatants

Sheep cell haemolysin Lecithinase Protease

Bacterial species Hours Titre Hours Zone Hours Zone

B cereus M27 4 1/80 24 20 mm 24 20 mmB pumilus MI1 24 1/40 24 0mm 8 20mmB pumilus M38 24 1/40 24 7mm 24 20mm

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Toxin production by Bacillus pumilus

Figure 1 B pumilus(Ml I)): cytopathic effecton Vero cells after 18hours' incubation.

Figure 2 B cereus:cytopathic effect on Verocells after 18 hours'incubation.

Figure 3 E agglomerans:minimal cytopathic effecton Vero cells after 48hours' incubation.

Figure 4 Vero cellsincubated withoutbacterial supernatant for48 hours.

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Negative control supernatants had no effect onthe tissue culture (fig 4). B pumilus (MI 1) and Bcereus produced maximum effects on the cellsheet within two hours of inoculation; Bpumilus (M38) had little effect in the first 24hours, but by 48 hours showed a typical Bpumilus type effect on the cell sheet (fig 1).Treatment with trypan blue showed that at 48hours very few cells treated with B pumilus(M38) had died, but by 96 hours most were

dead; this was considered to be a cytotoxiceffect." Examination of the cells exposed to theMll strain of B pumilus showed a cytotoniceffect" in that most cells were still alive after 96hours. Immediately after the development of

the first holes in the cell sheet the supernatantwas replaced with either maintenance mediumor growth medium and the plates were re-incubated for 48 hours. Cell sheets treatedwith maintenance medium were unchangedbut regeneration of the cell sheet had occurredin those wells containing growth medium andthe cytopathic effect was no longer visible.

Results of the other effects of the super-natants ofB cereus and B pumilus are shown inthe table. Sheep erythrocytes were lysed in fourhours by B cereus supernatant at a titre of 1/80;the B pumilus supernatants at a titre of1/40 produced haemolysis in 24 hours. Zonesites of 20 mm in the lecithin plates wereobserved after 24 hours' incubation withB cereus. Different effects were observed withthe two strains of B pumilus, M38 producedvery small zones (7 mm diameter) after 24hours' incubation, whereas M 1I had no effecton the lecithin even after prolonged incubation.Zones of 20 mm in the casein plates wereproduced by all three strains, but the time toproduce the zones varied; after eight hours azone had occurred around Mll; it took 24hours' incubation to develop around M38 andthe strain of B cereus.

DiscussionThese experiments have confirmed that super-natants prepared from B pumilus have toxicproperties. Whether all the effects were causedby one substance has not been determined. Thefiltrable toxins seemed to be produced late in thebacterial growth cycle or possibly were releasedfollowing cell injury or death. Bropy andKnoop2 found that filtrates prepared frombrain-heart infusion cultures of six B pumilusisolates (from guinea pigs with clindamycininduced enterocolitis) produced aggregation ofY1 adrenal cells within four hours and cell lysisafter 18 hours. Our results cannot be compareddirectly with those of Brophy and Knoop,however, as the methods used for detectionwere not the same.

In our experiments the effects of the Bpumilus supernatants were different from thosepreviously described for B cereus.'0 The varia-tions in the effects of the supernatants of Bpumilus were unexpected; in the originalscreening M38 had so little effect on the cellsheets after 24 hours it was chosen as the non-toxigenic strain for further experiments; theseshowed that M38 supernatant was slow to actbut lethal after incubation at 37°C for 96 hours.The cells treated with the B pumilus M 1Isupernatant showed a pronounced cytotonicresponse before 48 hours but were still alive at96 hours.The reasons for the choice of Vero cells have

already been given, but it is interesting to notethat no cytopathic effect occurred with super-natants of Ent agglomerans or B subtilis.Thompson et al only examined the disruptiveeffect ofB cereus in Vero cells for 12 hours andrecorded cytotoxicity as the reciprocal of thatdilution which resulted in complete loss ofcytopathic activity.'

In 1930 Krontowski produced evidence ofcellular regeneration following removal of

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bacterial supernatant.'2 Working with Cory-nebacterium diphtheriae toxin and chick embryocultures, he showed reduction in the area oftissue growth when diluted toxin was added tothe growth medium; reversion to normalgrowth medium induced regeneration(equivalent in size to that ofthe control culture)within three subsequent passages. He alsodescribed the development of projections fromsome of the cells treated with diphtheria toxin.In our experiments regeneration occurredwhen the cytopathic B pumilus supernatant wasreplaced in the wells by growth medium, butdid not occur when it was replaced bymaintenance medium. Ifregeneration occurredat all, however, it was more likely to happen inresponse to the substitution with growthmedium as the higher concentration of fetal calfserum in this medium is intended to supportactive growth rather than mere survival of thetissue culture.These experiments do not provide direct

evidence that the Bacillus species have a role inthe aetiology of byssinosis, but the detection ofthese toxic effects justify further work. Further-more, serum antibodies to these strains havebeen shown in cotton mill workers. 3 Onehypothesis is that toxin is produced at bodytemperature after inhalation of the bacteria.This would account for symptoms increasingduring the shift'4; the disappearance ofsymptoms when away from the mill might beexplained by the regeneration of damagedtissue,"' if the inhaled toxin-producing bacteriahad been overcome by host defences. Thishypothesis does not explain the Monday onset(first working day) but there might be aquantitative factor or some host factor.

If this hypothesis is correct it would beinteresting to investigate the role of iron intoxin production because the concentration iscritical for the production of C diphtheriaetoxin." Strippers and grinders who areresponsible for the maintenance of the cardingmachines,'5 and who might therefore have

greater exposure to iron particles and todamaged bacteria, are those most commonlyaffected by byssinosis. Obviously there is stillmuch work to be done to test these hypotheses.

This work was supported by grants from the British CottonGrowing Association Ltd Work People's Collections Fund andthe Cotton Industry War Memorial Trust.

We thank Mrs J Crosby for preparation of the photographicprints.

1 Flindt MLH. Pulmonary disease due to inhalation ofderivatives of Bacillus subtilis containing proteolyticenzyme. Lancet 1969;ii: 1 177-81.

2 Brophy PF, Knoop FC. Bacillus pumilus in the induction ofclindamycin-associated enterocolitis in guinea pigs. InfectImmun 1982;35:289-95.

3 Turnbull PCB, J0rgensen K, Kramer JM, et al. Severeclinical conditions associated with Bacillus cereus and theapparent involvement of exotoxins. J Clin Pathol1979;32:289-93.

4 Rylander R, Hagland P, Lindholm M. Endotoxin in cottondust and respiratory function decrement amongst cottonworkers in an experimental card room. Am Rev Resp Dis1985;131:209-13.

5 Tuffnell P. The relationship of byssinosis to the bacteria andfungi in the air of textile mills. Br J Ind Med 1960;17:304-6.

6 Tuxford AF, Moogan IJ. Bacteriological studies inLancashire cotton mills. Proceedings of the 10th CottonDust Research Conference, Las Vegas, 1986. Memphis:National Cotton Council, 1986:72-6.

7 Borriello SP, Larson HE, Welch AR, et al. EnterotoxigenicClostridium perfringens: a possible cause of antibiotic-associated diarrhoea. Lancet 1984;i:305-7.

8 Thompson NE, Ketterhagen MJ, Bergdoll MS, et al.Isolation and some properties of an enterotoxin producedby Bacillus cereus. Infect Immun 1984;43:887-94.

9 Speirs JI, Stravric S, Konowalchuk J. Assay of Escherichiacoli heat-labile enterotoxin with vero cells. Infect Immun1977;16:617-22.

10 Tumbull PCB, Kramer JM. Non gastrointestinal Bacilluscereus infections: an analysis ofexotoxin production over atwo year period. J Clin Pathol 1983;36:1091-6.

11 Potomski J, Burke V, Robinson J, et al. Aeromonas cytotoxicenterotoxin cross reactive with cholera toxin. J MedMicrobiol 1987;23:179-86.

12 Krontowski AA. Quantitative untersuchengen uber diewirkung des diphtherietoxins auf das wachstum und denstoffwechel der gewebskulturen. Zentr Bakt Parasit Abt Iorig 1930;117:328-48.

13 Tuxford AF, Hoult B, Sigsgaard T. In: Jacobs RR,Wakelyn PJ, eds. Antibodies to Bacillus species in sera ofDanish cotton workers. Proceedings ofthe 13th Cotton DustResearch Conference, Nashville, 1989: Memphis: NationalCotton Council, 1989:147-8.

14 Schilling RSF. Byssinosis in the British cotton textileindustry. Br Med Bull 1950;7:52-6.

15 Prausnitz C. Investigations on respiratory dust disease inoperatives in the cotton industry. Medical Research Councilspecial report series 212. London: HMSO, 1936:5-73.

16 Barkdale L. Corynebacterium diphtheriae and its relatives.Bacteriol Rev 1970;34:378-422.

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