Bacillus thuringiensis - Interest as agent for biocontrol of pests with possible human repercussions: Quantitative culture; Molecular identification (PCR and sequencing)

Information 12-30-2018.

Bacillus thuringiensis is a gram-positive bacterium, facultative anaerobic, bacillus-shaped, sporulated. This species is characterized by producing parasporal crystalline inclusions with toxic proteins for larvae of different orders of insects and other invertebrates. The biological control of insect pests by these insecticidal crystalline proteins represents one of the most fruitful uses of biological control agents. The main targets of bioinsecticides based on B. thuringiensis are larvae of herbivorous lepidoptera such as cabbage worm or others, some coleoptera, nematodes, mites and protozoa. For this purpose, strain B. thuringiensis serotype kurstaki HD1 is generally used.

This species of Bacillus, is included in the Bacillus cereus.group. The Bacillus cereus group includes at least four species: B. cereus, B. thuringiensis, Bacillus anthracis, Bacillus mycoides and two other proposed species Bacillus weihenstephanensis and Bacillus pseudomycoides. The taxonomy of the group is controversial, and some consider that the species Bacillus cereus is a subspecies of B. cereus sensu lato. The identification and differentiation of the bacteria of the B. cereus group is difficult due to the genetic and phenotypic similarities between them.

Some of the species included in the Bacillus cereus group are potentially enterotoxic for humans and animals, which requires the rapid detection of the B. cereus group. B. cereus is frequently found in the soil and can be isolated from raw milk and dairy products. B. cereus also causes foodborne illnesses associated with various toxins, causing diarrhea in higher animals.

B. thuringiensis is difficult to differentiate due to its culture and metabolic characteristics of the other species of the B. cereus group when it loses a plasmid in which the cry gene is found, which gives it some properties.

To differentiate the members of the B. cereus group, several tests have been proposed, including whole-genome DNA hybridization, sequence analysis of 16S-23S operons, intergenic spacer region gyrB-gyrA, enzyme electrophoresis, Pulsed field gel electrophoresis analysis, amplified fragment length polymorphisms, virulence factors, random PCR and restriction fragment length polymorphisms of PCR products.

In adverse conditions, Bacillus thuringiensis forms endospores and, at the same time, crystalline parasporal inclusions that contain one or more insecticidal proteins such as Cry and Cyt proteins. Cry proteins are toxic by ingestion and their specificity is determined by their binding to the receptors in the epithelial cells of the insect´s midgut. Commercial biopesticides based on B. thuringiensis have been used for more than 30 years and transgenic plants that express Cry proteins have been used since 1996 for the control of insect pests. Some strains of B. thuringiensis also synthesize other insecticidal molecules that are secreted. Several types of secreted toxins have been described with very different structures and modes of action. For example, Cry1I is a lepidopteran-specific protein toxin secreted during the early stationary phase. The beta exotoxin is another extracellular insecticidal toxin expressed during vegetative growth. This toxin is an adenine nucleotide analog and probably acts as an inhibitor of DNA-dependent RNA polymerases. Due to the toxicity to vertebrates, their public use is prohibited in many countries. Several strains of B. thuringiensis and Bacillus cereus secrete some toxic proteins during their vegetative growth that do not form crystals and that have no homology with the Cry proteins. These vegetative insecticidal proteins, called VIP proteins, are the most recently discovered insecticidal proteins of B. thuringiensis and a system for their nomenclature has been proposed. Vip1 and Vip2 are proteins of 100 and 52 kDa, respectively, that form a binary toxin with activity against the larvae of some species of coleoptera. Based on its homology to other binary toxins, it has been proposed that Vip1 proteins would bind to specific receptors in midgut cells, associating as a multimer, which would form a channel. Vip2 would pass through this pore and, based on its homology with the ADP-ribosyltransferases, would modify the monomeric actin and inhibit its polymerization. Vip3 does not share homology with any other known protein and has been shown to have a broad insecticidal spectrum. This 88-kDa toxin is processed in the insect´s midgut to an active form that is toxic to several lepidopteran species after binding to specific receptors in the midgut cells. Some species of insects exposed to a certain toxin have developed cross resistance to other related toxins, so the identification of new toxins could be useful for control programs in cases of resistance. In this way, new toxins with different modes of action could be used in the formulations of B. thuringiensis and in transgenic plants to avoid the phenomenon of cross-resistance. Vip proteins appear to bind to different target sites to which Cry proteins bind in epithelial cells of the midgut of the tested species, so these proteins are candidates for use in conjunction with Cry proteins in insect pest management . The use of this combination of toxins could be applied to extend the toxicity spectrum and minimize the risk of cross-resistance. The detection of the vip2 and vip3 genes in the B. thuringiensis strains was carried out using methods based on the polymerase chain reaction (PCR). The amplification products of the PCR reactions confirm the presence of vip genes, but this approach only allows the assignment of the gene to a vip family (vip1, vip2 or vip3) and does not allow the identification of specific genes within a family.

Due to the genetic similarities between B. thuringiensis and B. cereus, which could cause gastrointestinal diseases and somatic infections, the safety of B. thuringiensis as a bioinsecticide has been questioned, although there is only some information on clinical cases related to the use of B. thuringiensis. However, neither the medical practice nor the methods used for the detection of food pathogens discriminate between B. thuringiensis and B. cereus as causal agents in relation to contamination of human food or diseases, so the actual proportion of the disease is unknown. participation of B. thuringiensis in these diseases, which are generally attributed to B. cereus. There is a study in which it has been shown that B. thuringiensis is involved in an outbreak of gastroenteritis in four people. In this study, the strain was initially identified as B. cereus, but was later found to be B. thuringiensis.

Only a few attempts have been made to isolate B. thuringiensis from different types of food, and none of them has included quantification. B. thuringiensis has recovered from a wide variety of foods, such as pasta, rice, spices, grains, bread, legumes and milk. The isolates obtained belonged to the serotype kurstaki or neoleonensis. B. thuringiensis kurstaki was isolated from grapes for human consumption and it was hypothesized that the bacteria could have originated from biopesticide residues.

 

Tests carried out in IVAMI:

 

  • Cultivation in semi-selective media for bacteria of the Bacillus cereus group, among which is Bacillus thuringiensis.
  • Molecular detection (PCR) of cry genes, specific for Bacillus thuringiensis.

 

Recommended sample:

 

  • Vegetable product.
  • Products derived from vegetables.
  • Products of animal origin that could contain this bacterium.

Conservation and sending of the sample:

 

  • Refrigerated (preferred) for less than 2 days.
  • Frozen: more than 2 days.

 

Delivery of results:

 

  • Culture and molecular identification: 5 to 10 days.

Cost of the test:

 

  • Cultivation in semi-selective media (semi-selective): Consult ivami@ivami.com