FEMS Immunology and Medical Microbiology 24 (1999) 43^47

Bacillus thuringiensis serotype H34 isolated from human and insecticidal 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 b

a Laboratoire de Biologie, HIA Percy, 92141 Clamart, France 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, France d 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 was able to induce myonecrosis in immunosuppressed mice after cutaneous infection. Its potential pathogenicity for immunocompetent hosts was investigated in a mouse model of pulmonary infection. Mice infected intranasally by a suspension containing 108 spores died within 8 h in a clinical toxic-shock syndrome. In the same conditions, infection with a mutant without crystalline toxin, with the supernatant from a culture containing 108 bacteria ml31 and by the insecticidal strain serotypes 3a3b or H14 led to identical results. Lower inocula simply induced a local inflammatory reaction with bacterial persistence observed during the course of 10 days. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bacillus thuringiensis; Pulmonary infection ; Hemolysin

1. Introduction The ubiquitous soil bacterium Bacillus thuringiensis encodes a diverse array of pesticidal proteins widely used around the world for insect pest control. Recently, DNA sequences encoding the delta-endotoxin have been included in plants to promote resistance to insects. * Corresponding author. Tel.: +33 (1) 6990-8381; Fax: +33 (1) 6493-5266; E-mail: [email protected]

Infection in humans is unusual, and apart from gastrointestinal tract infections or those following laboratory contamination, there are only two clinical reports of B. thuringiensis infection [1,2]. In 1995, we isolated a strain of B. thuringiensis var. konkukian (serotype H34) from soft tissue necrosis following severe war wounds caused by a land mine explosion. The ability of this strain to induce myonecrosis in immunosuppressed mice after cutaneous infection has been previously described [1]. Since B. thuringiensis 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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used as aerosolised biopesticides [3], the aim of this study was to evaluate the potential pathogenicity of this strain and of the insecticidal serotypes 3a3b and H14 for immunocompetent mice after pulmonary experimental infection.

infected intranasally, under light ether anaesthesia, by suspensions containing from 105 ^108 spores in 50 Wl. Five mice were infected with the supernatant. Each experiment was repeated three times.

2. Materials and methods

Lungs were dissected from the main bronchi, diluted in sterile PBS (Sigma), and plated on trypticase-soy agar medium. Bacterial counts from lung homogenates were expressed in log10 CFU ml31 as mean þ S.E.

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 groups of ¢ve, with sterile water and food. 2.2. Bacteria B. thuringiensis serotype H34-konkukian was isolated from a severe war wound infection [1]. B. thuringiensis H34 without crystalline toxin was a spontaneous mutant isolated from the same patient. Both strains were identi¢ed with biochemical tests and Hserotyping performed by the WHO collaborating centre for Entomopathogens (Dr. Lecadet, Uniteè des Bacteèries Entomopathogeénes, Institut Pasteur, Paris, France). B. thuringiensis serotype H12 was obtained from a clinical specimen and was considered as clinically irrelevant. B. thuringiensis serotypes 3a3b and H14 were obtained from Abbot laboratories. 2.3. Cultures Spore suspensions were prepared from a 10-dayold culture on poor agar medium (yeast extract, 10 g l31 ; NaCl, 5 g l31 ; Agar, 20 g l31 ) suspended in sterile water and incubated for 1 h at 65³C in order to kill vegetative forms. Dilutions were made in sterile water to obtain inocula of 105 ^108 spores per mouse. Supernatants were obtained from a 24-h stationary-phase cultures containing 108 CFU ml31 centrifuged 15 min at 7000Ug and ¢ltered on 0.22 Wm. 2.4. Infection For each strain, four groups of ¢ve mice were

2.5. Bacterial counts

2.6. Histological examinations Histological examinations were performed immediately after death or 48 h after infection. Mice were killed by injection of a pentothal overdose. Lungs were perfused with 500 Wl of 10% formaldehyde by intratracheal injection, taken out and ¢xed in 10% formalin. The tissues were embedded in para¤n blocks and sectioned at 4^5 Wm thickness. The sections were mounted onto regular slides for staining with haematoxylin-eosin-sa¡ron or Gram stain. 2.7. Haemolytic activity Supernatants were centrifuged on Centriprep 30 (Amicon, Epernon, France) in order to concentrate and separate the proteins by molecular weight. Haemolytic activities were quanti¢ed by incubating 50 Wl of serial 2-fold dilutions of supernatants with 50 Wl of 0.5% packed and washed rabbit erythrocytes. The mixtures were incubated 60 min at 37³C and left to sediment for 60 min at room temperature [4]. The titre was given by the last dilution giving 100% haemolysis. Erythrocytes with sterile broth were used as negative control and erythrocytes with distilled water as positive control.

3. Results 3.1. Infections with spore suspensions All the mice instilled with 108 spores of B. thuringiensis var. konkukian died within 8 h with a clinical toxic-shock syndrome. Autopsy showed large

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Table 1 Pulmonary bacterial counts of Bacillus thuringiensis H34 Time postinfection

108 spores per mouse (log10 8)

107 spores per mouse (log10 7)

105 spores per mouse (log10 5)

103 spores per mouse (log10 3)

4 8 24 48 10

7.30 þ 0.12 dead

ND ND 6.87 þ 0.17 7.09 þ 0.25 5.20 þ 0.16

ND ND 4.33 þ 0.14 4.79 þ 0.24 3.16 þ 0.13

ND ND 2.81 þ 0.26 3.17 þ 0.02 1.10 þ 0.08

h h h h days

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

haemorrhagic lique¢ed lesions of the lungs and the liver. Histological examinations of the lung revealed lesions of acute bronchitis associated with ulceration, injuries of the mucociliary apparatus, oedema and alveolar damage. These lesions were associated with a neutrophilic in¢ltrate. Blood cultures obtained after intracardiac puncture were positive and revealed a pure culture of B. thuringiensis. In the same conditions, inocula from 105 to 107 spores per mouse led only to a local in£ammatory reaction, with a bacterial persistence observed for at least 10 days (Table 1). Bacterial counts gave the same results when the lung homogenates were heated or not at 65³C in order to kill vegetative forms. Infection by the mutant without parasporal body inclusion led to the same results, including for the pulmonary bacterial counts. Instillation of 108 spores of B. thuringiensis serotype 3a3b led to a lethality of 80%. The lethality was only 4O% with B. thuringiensis serotype H14 when using the same inoculum (Table 2). Histological examinations revealed identical lesions to those observed 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 haemorrhagic su¡usions, we supposed, as previously described in the mice cutaneous infectious model [1], that the secreted haemolysins of B. thuringiensis were the toxins involved in the pathogenicity. Two di¡erent haemolysins, closely related to those of Bacillus cereus, have been described [5^7]. While one, thiol-dependent, is inhibited by cholesterol, the other is not. Instillation with the supernatant of a stationary phase culture (24 h at 37³C) containing 108 CFU of B. thuringiensis H34 led to an 80% lethality rate. This ¢gure dropped to 10% when cholesterol was added to the supernatant at the concentration of 60 Wg ml31 . In contrast, the supernatant of B. thuringiensis H12 was not able to kill the mice. After ¢ltration of the supernatants on Centriprep 30, the haemolytic activity corresponding to the fraction higher than 30 kDa was: 1/256 for B. thuringiensis H34 crystal‡ ; 1/128 for B. thuringiensis serotype 3a3b; 1/32 for serotype H14; and 1/2 for B. thuringiensis serotype H12. The concentrated fraction was able to kill mice in 30 min for B. thuringiensis H34 and in less than 5 h for B. thuringiensis 3a3b. The fractions corresponding to the proteins of a

Table 2 Titration of the haemolytic activity in the supernatants (24 h after ¢ltration on Centriprep 30), and lethality rates induced by 108 spores ml31 Strain B. B. B. B.

thuringiensis thuringiensis thuringiensis thuringiensis

H34 cry‡ 3a3b H14 H12

Haemolysin titre

Lethality at 24 h postinfection due to 108 spores

1/256 1/128 1/32 1/2

100% 80% 40% 0%

Titres are the last dilution giving 100% haemolysis.

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molecular weight under 30 kDa were not haemolytic and were not able to kill mice after experimental infection. 3.3. Inactivated supernatant The lethal fraction of the supernatant of B. thuringiensis H34 heated at 70³C for 10 min did not express haemolytic activity. When instilled into mice, the fraction was unable to kill them.

4. Discussion Infection by B. thuringiensis is very uncommon in humans, but this bacterium is not identi¢ed in routine culture. In this particular case, B. thuringiensis has been identi¢ed in our medical laboratory by the Vitek System (Vitek, St. Louis, MO) which was using, at the time, the industrial software. When using the medical software, this bacterium was identi¢ed as B. cereus. Compared to B. thuringiensis which is resistant to penicillin G and ampicillin, B. cereus expresses a higher resistance pattern and is resistant to penicillin G, ampicillin, ticarcillin and cefalotin, but remains sensitive to piperacillin [1^8]. Except for B. thuringiensis H12, commonly recovered in soils and used as control, all the strains tested in this protocol were, to di¡erent degrees, pathogenic for mice. The commercial strains of B. thuringiensis are commonly used in agriculture and in forestry with no case of pathogenicity described for over 30 years, but infectious concentrations used for infection in this protocol are very high compared to those used in agriculture. However, it has never been described that any serotype of B. thuringiensis was able to kill mice when applied at a high concentration by the pulmonary route. B. thuringiensis H34 isolated from our patient is not used as a biopesticide, and is perhaps a rare and unusual isolate expressing a high level of a B. cereus-like haemolytic toxin. However, this argument is not available for B. thuringiensis 3a3b and H14 which were obtained from a commercial source. In the case of B. thuringiensis H34, pathogenicity in mice does not seem to be correlated with the production of the delta-endotoxin. A decrease in lethality rate observed when cholesterol was added to the

supernatant indicates that the thiol-dependent haemolysin is probably implicated in the pathogenicity of B. thuringiensis serotypes H34, 3a3b, and H14. Surprisingly, the concentration of 107 organisms per mouse did not produce enough toxin to kill the mice. This observation suggests that a high concentration of haemolysin is required for lethality. This hypothesis was con¢rmed by the instillation of concentrated supernatants which were able to kill mice in less than 30 min for B. thuringiensis H34 and 5 h for serotype 3a3b. However, haemolysin is probably not involved alone in pathogenicity: a lower inoculum induced pulmonary lesions with bacterial persistence observed during a 10-day period. This persistence was not associated with bacterial multiplication (Table 1). The observation that bacterial counts in lung homogenates, after heating at 65³C or not, were the same, suggests that the inoculum is maintained as sporulated forms. The exact localisation of spores in lungs is unknown, but we suppose that the spores are ingested, but not destroyed, by the alveolar phagocytic cells. Ongoing experiments will be performed to con¢rm this hypothesis and to investigate the correlation between the serotypes, the amount of thiol-dependent haemolysin, the phospholipase production, and the pulmonary pathogenicity in mice.

Acknowledgments We gratefully acknowledge Karine David, Agneés Labarre and Rosy Smith for their technical assistance.

References [1] Hernandez, E., Ramisse, F., Cruel, T., Ducoureau, J.P., Alonso, J.M. and Cavallo, J.D. (1998) Bacillus thuringiensis serovar H34-konkukian superinfection: report of one case and experimental evidence of pathogenicity in immunosuppressed mice. J. Clin. Microbiol. 36 (7), 2138^2139. [2] Damgaard, P.H., Granum, P.E., Bresciani, J., Torregrossa, M.V., Eilenberg, J. and Valentino, L. (1997) Characterization of Bacillus thuringiensis isolated from infections in burn wounds. FEMS Immunol. Med. Microbiol. 18, 47^53. [3] Aronson, A.I., Beckman, W. and Dunn, P. (1986) Bacillus thuringiensis and related insect pathogens. Microbiol. Rev. 50, 1^24.

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E. Hernandez et al. / FEMS Immunology and Medical Microbiology 24 (1999) 43^47 [4] Coolbaugh, J.C., Wende, R.D. and Williams, R.P. (1972) Microtitration of Bacillus cereus hemolysin. Appl. Microbiol. 24, 997^998. [5] Coolbaugh, J.C. and Williams, R.P. (1978) Production and characterization 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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Bacillus thuringiensis and Bacillus cereus. FEMS Microbiol. Lett. 63, 205^209. [7] Matsuyama, J., Yamamoto, K., Miwatani, T. and Honda, T. (1995) Monoclonal antibody developed against a hemolysin of Bacillus thuringiensis. Microbiol. Immunol. 39, 619^622. [8] Cavallo, J.D., Hernandez, E. and Dubrous, J.P. (1997) Bacillus cereus agent d'infection des plaies de guerre. Meèd. Armeèes 25 (5), 373^378.

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