Isolation and characterization of Bacillus cereus-like bacteria from faecal samples from greenhouse workers who are using Bacillus thuringiensis-based insecticides.
OBJECTIVE
Since the discovery of the insecticidal activity of Bacillus thuringiensis at the beginning of the twentieth century, this bacterium has been used increasingly against various insect pests. In spite of the extensive use of B. thuringiensis, only sporadic clinical case reports have been published. In recent years, the close relationship between B. thuringiensis and the human pathogen Bacillus cereus has been confirmed. In practice, only the insecticidal activity of B. thuringiensis distinguishes the two species. However, both species are composed of thousands of isolates with varying potential for causing adverse effects in humans. The aim of this study was to employ molecular biology methods for assessment of occupational exposure to B. thuringiensis-based biopesticides by determination of specific genetic information including plasmid profiles and random amplified polymorphic DNA (RAPD).


METHODS
Faecal samples from 12 persons, working in Danish greenhouses, were collected for microbial analysis. Seven persons were using B. thuringiensis-based insecticides, whereas five persons were employed at greenhouses that did not use B. thuringiensis. The bacteria were isolated on B. cereus-specific solid substrate, and colonies were further identified using the polymerase chain reaction (PCR). The PCR method was used for the identification of the enterotoxin genes HblA and BceT. The expression of enterotoxins was detected with two commercial serological kits. Primers specific for 16S-23S spacer region were used to identify the bacteria as members of the B. cereus group. Several primers towards insecticidal genes have been used in order to further characterize the isolates as subspecies of B. thuringiensis.


RESULTS
Two faecal samples from the B. thuringiensis-exposed greenhouse workers were positive for B. cereus-like bacteria. One isolate displayed intracellular crystalline inclusions characteristic of B. thuringiensis, production of and genes for B. cereus enterotoxins and it was PCR-positive for an insecticidal toxin primer set. RAPD profiles of the faecal isolate were identical to that of strains isolated from a commercial product.


CONCLUSIONS
The methods applied have verified that the faecal isolate was identical to the B. thuringiensis isolate found in the biopesticide used. This is the first reported case of isolation of a bacterial biopesticide from human faeces. The biopesticide was shown to harbour and express enterotoxin genes. However, there is no evidence that this caused any adverse effects to the person from whom these bacteria were isolated.
Jensen
Gert B
GB
Department of Chemical Working Environment, National Institute of Occupational Health, Copenhagen, Denmark. gbj@ami.dk
Larsen
Preben
P
Jacobsen
Bodil L
BL
Madsen
Bodil
B
Wilcks
Andrea
A
Smidt
Lasse
L
Andrup
Lars
L
D001409:Bacillus cereus / Q000235:genetics / Q000302:isolation & purification*; D001413:Bacillus thuringiensis / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D003718:Denmark; D005243:Feces / Q000382:microbiology*; D006801:Humans; D016273:Occupational Exposure / Q000032:analysis*; D010572:Pest Control, Biological; D019105:Random Amplified Polymorphic DNA Technique
D001427:Bacterial Toxins; D017931:DNA Primers; D004269:DNA, Bacterial