Bacillus thuringiensis serotype H34 isolated from human andinsecticidal strains serotypes 3a3b and H14 can lead to death of

immunocompetent mice after pulmonary infection

Eric Hernandez a, Franc°oise Ramisse b;*, Thierry Cruel c, Robert le Vagueresse a,Jean-Didier Cavallo d

a Laboratoire de Biologie, HIA Percy, 92141 Clamart, Franceb Centre d'Etudes du Bouchet Laboratoire de Microbiologie, BP 3, 91710 Vert-le-Petit, France

c Laboratoire d'Anatomo Pathologie, H.I.A du Val de Graêce, 75014 Paris, Franced Laboratoire de Biologie, HIA Begin, Saint-Mandeè, France

Received 24 September 1998; received in revised form 8 January 1999; accepted 12 January 1999

Abstract

In 1995, we isolated a strain of Bacillus thuringiensis serotype H34 from severe human tissue necrosis. This bacterium wasable to induce myonecrosis in immunosuppressed mice after cutaneous infection. Its potential pathogenicity forimmunocompetent hosts was investigated in a mouse model of pulmonary infection. Mice infected intranasally by asuspension containing 108 spores died within 8 h in a clinical toxic-shock syndrome. In the same conditions, infection with amutant without crystalline toxin, with the supernatant from a culture containing 108 bacteria ml31 and by the insecticidal strainserotypes 3a3b or H14 led to identical results. Lower inocula simply induced a local inflammatory reaction with bacterialpersistence observed during the course of 10 days. z 1999 Federation of European Microbiological Societies. Published byElsevier Science B.V. All rights reserved.

Keywords: Bacillus thuringiensis ; Pulmonary infection; Hemolysin

1. Introduction

The ubiquitous soil bacterium Bacillus thuringien-sis encodes a diverse array of pesticidal proteinswidely used around the world for insect pest control.Recently, DNA sequences encoding the delta-endo-toxin have been included in plants to promote resist-ance to insects.

Infection in humans is unusual, and apart fromgastrointestinal tract infections or those followinglaboratory contamination, there are only two clinicalreports of B. thuringiensis infection [1,2]. In 1995, weisolated a strain of B. thuringiensis var. konkukian(serotype H34) from soft tissue necrosis followingsevere war wounds caused by a land mine explosion.The ability of this strain to induce myonecrosis inimmunosuppressed mice after cutaneous infectionhas been previously described [1]. Since B. thuringien-sis spores and its parasporal body are commonly

0928-8244 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 9 2 8 - 8 2 4 4 ( 9 9 ) 0 0 0 0 5 - X

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* Corresponding author. Tel. : +33 (1) 6990-8381;Fax: +33 (1) 6493-5266; E-mail: f.ramisse@wanadoo.fr

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used as aerosolised biopesticides [3], the aim of thisstudy was to evaluate the potential pathogenicity ofthis strain and of the insecticidal serotypes 3a3b andH14 for immunocompetent mice after pulmonary ex-perimental infection.

2. Materials and methods

2.1. Mice

Mice used were 5-week-old female BALB/c(Charles River, Saint-Aubin-les-Elbeuf, France)kept in a biosafety containment facility in groupsof ¢ve, with sterile water and food.

2.2. Bacteria

B. thuringiensis serotype H34-konkukian was iso-lated from a severe war wound infection [1]. B. thur-ingiensis H34 without crystalline toxin was a sponta-neous mutant isolated from the same patient. Bothstrains were identi¢ed with biochemical tests and H-serotyping performed by the WHO collaboratingcentre for Entomopathogens (Dr. Lecadet, Uniteèdes Bacteèries Entomopathogeénes, Institut Pasteur,Paris, France). B. thuringiensis serotype H12 was ob-tained from a clinical specimen and was consideredas clinically irrelevant. B. thuringiensis serotypes3a3b and H14 were obtained from Abbot laborato-ries.

2.3. Cultures

Spore suspensions were prepared from a 10-day-old culture on poor agar medium (yeast extract, 10 gl31 ; NaCl, 5 g l31 ; Agar, 20 g l31) suspended insterile water and incubated for 1 h at 65³C in orderto kill vegetative forms. Dilutions were made in ster-ile water to obtain inocula of 105^108 spores permouse. Supernatants were obtained from a 24-h sta-tionary-phase cultures containing 108 CFU ml31

centrifuged 15 min at 7000Ug and ¢ltered on 0.22Wm.

2.4. Infection

For each strain, four groups of ¢ve mice were

infected intranasally, under light ether anaesthesia,by suspensions containing from 105^108 spores in50 Wl. Five mice were infected with the supernatant.Each experiment was repeated three times.

2.5. Bacterial counts

Lungs were dissected from the main bronchi, di-luted in sterile PBS (Sigma), and plated on trypti-case-soy agar medium. Bacterial counts from lunghomogenates were expressed in log10 CFU ml31 asmean þ S.E.

2.6. Histological examinations

Histological examinations were performed imme-diately after death or 48 h after infection. Mice werekilled by injection of a pentothal overdose. Lungswere perfused with 500 Wl of 10% formaldehyde byintratracheal injection, taken out and ¢xed in 10%formalin. The tissues were embedded in para¤nblocks and sectioned at 4^5 Wm thickness. The sec-tions were mounted onto regular slides for stainingwith haematoxylin-eosin-sa¡ron or Gram stain.

2.7. Haemolytic activity

Supernatants were centrifuged on Centriprep 30(Amicon, Epernon, France) in order to concentrateand separate the proteins by molecular weight. Hae-molytic activities were quanti¢ed by incubating 50 Wlof serial 2-fold dilutions of supernatants with 50 Wlof 0.5% packed and washed rabbit erythrocytes. Themixtures were incubated 60 min at 37³C and left tosediment for 60 min at room temperature [4]. Thetitre was given by the last dilution giving 100% hae-molysis. Erythrocytes with sterile broth were used asnegative control and erythrocytes with distilled wateras positive control.

3. Results

3.1. Infections with spore suspensions

All the mice instilled with 108 spores of B. thur-ingiensis var. konkukian died within 8 h with a clin-ical toxic-shock syndrome. Autopsy showed large

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haemorrhagic lique¢ed lesions of the lungs and theliver. Histological examinations of the lung revealedlesions of acute bronchitis associated with ulceration,injuries of the mucociliary apparatus, oedema andalveolar damage. These lesions were associated witha neutrophilic in¢ltrate. Blood cultures obtainedafter intracardiac puncture were positive and re-vealed a pure culture of B. thuringiensis. In thesame conditions, inocula from 105 to 107 sporesper mouse led only to a local in£ammatory reaction,with a bacterial persistence observed for at least 10days (Table 1). Bacterial counts gave the same resultswhen the lung homogenates were heated or not at65³C in order to kill vegetative forms.

Infection by the mutant without parasporal bodyinclusion led to the same results, including for thepulmonary bacterial counts.

Instillation of 108 spores of B. thuringiensis sero-type 3a3b led to a lethality of 80%. The lethality wasonly 4O% with B. thuringiensis serotype H14 whenusing the same inoculum (Table 2). Histological ex-aminations revealed identical lesions to those ob-served with B. thuringiensis serotype H34.

Infection by 108 spores of B. thuringiensis H12,simply induced a lung in£ammatory reaction.

3.2. Nasal instillation of supernatants

Since the obvious lesions were lung haemorrhagicsu¡usions, we supposed, as previously described inthe mice cutaneous infectious model [1], that the se-creted haemolysins of B. thuringiensis were the toxinsinvolved in the pathogenicity. Two di¡erent haemo-lysins, closely related to those of Bacillus cereus, havebeen described [5^7]. While one, thiol-dependent, isinhibited by cholesterol, the other is not. Instillationwith the supernatant of a stationary phase culture(24 h at 37³C) containing 108 CFU of B. thuringien-sis H34 led to an 80% lethality rate. This ¢guredropped to 10% when cholesterol was added to thesupernatant at the concentration of 60 Wg ml31. Incontrast, the supernatant of B. thuringiensis H12 wasnot able to kill the mice.

After ¢ltration of the supernatants on Centriprep30, the haemolytic activity corresponding to the frac-tion higher than 30 kDa was: 1/256 for B. thurin-giensis H34 crystal� ; 1/128 for B. thuringiensis sero-type 3a3b; 1/32 for serotype H14; and 1/2 for B.thuringiensis serotype H12. The concentrated frac-tion was able to kill mice in 30 min for B. thurin-giensis H34 and in less than 5 h for B. thuringiensis3a3b.

The fractions corresponding to the proteins of a

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Table 1Pulmonary bacterial counts of Bacillus thuringiensis H34

Time 108 spores per mouse 107 spores per mouse 105 spores per mouse 103 spores per mousepostinfection (log10 8) (log10 7) (log10 5) (log10 3)

4 h 7.30 þ 0.12 ND ND ND8 h dead ND ND ND

24 h 6.87 þ 0.17 4.33 þ 0.14 2.81 þ 0.2648 h 7.09 þ 0.25 4.79 þ 0.24 3.17 þ 0.0210 days 5.20 þ 0.16 3.16 þ 0.13 1.10 þ 0.08

Values are mean þ S.E. of mice per point. ND, not determined.

Table 2Titration of the haemolytic activity in the supernatants (24 h after ¢ltration on Centriprep 30), and lethality rates induced by 108 sporesml31

Strain Haemolysin titre Lethality at 24 h postinfection due to 108 spores

B. thuringiensis H34 cry� 1/256 100%B. thuringiensis 3a3b 1/128 80%B. thuringiensis H14 1/32 40%B. thuringiensis H12 1/2 0%

Titres are the last dilution giving 100% haemolysis.

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molecular weight under 30 kDa were not haemolyticand were not able to kill mice after experimentalinfection.

3.3. Inactivated supernatant

The lethal fraction of the supernatant of B. thur-ingiensis H34 heated at 70³C for 10 min did notexpress haemolytic activity. When instilled intomice, the fraction was unable to kill them.

4. Discussion

Infection by B. thuringiensis is very uncommon inhumans, but this bacterium is not identi¢ed in rou-tine culture. In this particular case, B. thuringiensishas been identi¢ed in our medical laboratory by theVitek System (Vitek, St. Louis, MO) which was us-ing, at the time, the industrial software. When usingthe medical software, this bacterium was identi¢ed asB. cereus. Compared to B. thuringiensis which is re-sistant to penicillin G and ampicillin, B. cereus ex-presses a higher resistance pattern and is resistant topenicillin G, ampicillin, ticarcillin and cefalotin, butremains sensitive to piperacillin [1^8].

Except for B. thuringiensis H12, commonly recov-ered in soils and used as control, all the strains testedin this protocol were, to di¡erent degrees, pathogenicfor mice. The commercial strains of B. thuringiensisare commonly used in agriculture and in forestrywith no case of pathogenicity described for over 30years, but infectious concentrations used for infec-tion in this protocol are very high compared to thoseused in agriculture. However, it has never been de-scribed that any serotype of B. thuringiensis was ableto kill mice when applied at a high concentration bythe pulmonary route. B. thuringiensis H34 isolatedfrom our patient is not used as a biopesticide, andis perhaps a rare and unusual isolate expressing ahigh level of a B. cereus-like haemolytic toxin. How-ever, this argument is not available for B. thuringien-sis 3a3b and H14 which were obtained from a com-mercial source.

In the case of B. thuringiensis H34, pathogenicityin mice does not seem to be correlated with the pro-duction of the delta-endotoxin. A decrease in lethal-ity rate observed when cholesterol was added to the

supernatant indicates that the thiol-dependent hae-molysin is probably implicated in the pathogenicityof B. thuringiensis serotypes H34, 3a3b, and H14.Surprisingly, the concentration of 107 organismsper mouse did not produce enough toxin to kill themice. This observation suggests that a high concen-tration of haemolysin is required for lethality. Thishypothesis was con¢rmed by the instillation of con-centrated supernatants which were able to kill micein less than 30 min for B. thuringiensis H34 and 5 hfor serotype 3a3b. However, haemolysin is probablynot involved alone in pathogenicity: a lower inocu-lum induced pulmonary lesions with bacterial persis-tence observed during a 10-day period. This persis-tence was not associated with bacterial multiplication(Table 1). The observation that bacterial counts inlung homogenates, after heating at 65³C or not, werethe same, suggests that the inoculum is maintainedas sporulated forms. The exact localisation of sporesin lungs is unknown, but we suppose that the sporesare ingested, but not destroyed, by the alveolar phag-ocytic cells.

Ongoing experiments will be performed to con¢rmthis hypothesis and to investigate the correlation be-tween the serotypes, the amount of thiol-dependenthaemolysin, the phospholipase production, and thepulmonary pathogenicity in mice.

Acknowledgments

We gratefully acknowledge Karine David, AgneésLabarre and Rosy Smith for their technical assis-tance.

References

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[2] Damgaard, P.H., Granum, P.E., Bresciani, J., Torregrossa,M.V., Eilenberg, J. and Valentino, L. (1997) Characterizationof Bacillus thuringiensis isolated from infections in burnwounds. FEMS Immunol. Med. Microbiol. 18, 47^53.

[3] Aronson, A.I., Beckman, W. and Dunn, P. (1986) Bacillusthuringiensis and related insect pathogens. Microbiol. Rev.50, 1^24.

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[4] Coolbaugh, J.C., Wende, R.D. and Williams, R.P. (1972) Mi-crotitration of Bacillus cereus hemolysin. Appl. Microbiol. 24,997^998.

[5] Coolbaugh, J.C. and Williams, R.P. (1978) Production andcharacterization of two hemolysins of Bacillus cereus. Can.J. Microbiol. 24, 1289^1295.

[6] Honda, T., Shiba, A., Seo, S., Yamamoto, J., Matsuyama, J.and Miwatani, T. (1991) Identity of hemolysins produced by

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[7] Matsuyama, J., Yamamoto, K., Miwatani, T. and Honda, T.(1995) Monoclonal antibody developed against a hemolysin ofBacillus thuringiensis. Microbiol. Immunol. 39, 619^622.

[8] Cavallo, J.D., Hernandez, E. and Dubrous, J.P. (1997) Bacil-lus cereus agent d'infection des plaies de guerre. Meèd. Armeèes25 (5), 373^378.

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