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Whole Genome Sequencing Reveals Biopesticidal Origin of Bacillus thuringiensis in Foods.

ABSTRACT: Bacillus thuringiensis is a microbial insecticide widely used to control agricultural pests. Although generally regarded as safe, B. thuringiensis is phylogenetically intermingled with the foodborne pathogen B. cereus sensu stricto and has been linked to foodborne outbreaks. Limited data on the pathogenicity potential of B. thuringiensis and the occurrence of biopesticide residues in food compromise a robust consumer risk assessment. In this study, we analyzed whole-genome sequences of 33 B. thuringiensis isolates from biopesticides, food, and human fecal samples linked to outbreaks. All food and outbreak-associated isolates genomically matched (≤ 6 wgSNPs; ≤ 2 cgSNPs) with one of six biopesticide strains, suggesting biopesticide products as their source. Long-read sequencing revealed a more diverse virulence gene profile than previously assumed, including a transposase-mediated disruption of the promoter region of the non-hemolytic enterotoxin gene nhe and a bacteriophage-mediated disruption of the sphingomyelinase gene sph in some biopesticide strains. Furthermore, we provide high-quality genome assemblies of seven widely used B. thuringiensis biopesticide strains, which will facilitate improved microbial source tracking and risk assessment of B. thuringiensis-based biopesticides in the future.

SUBMITTER: Biggel M

PROVIDER: S-EPMC8790155 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBacillus thuringiensis (Bt) is a gram-positive ubiquitous saprophytic bacterium that produces proteins (Crystal protein, Vegetative insecticidal protein, and Secreted insecticidal protein) toxic to insects during its growth cycle. In the present study, the whole genome of a locally isolated B. thuringiensis strain BA04 was sequenced to explore the genetic makeup and to identify the genes responsible to produce insecticidal proteins including the virulence factors. The strain was isolated from the soil sample of the Kaziranga National Park, Assam, North-Eastern part of India (Latitude: 26°34'39.11''N and Longitude: 93°10'16.04''E).ResultsThe whole genome sequencing (WGS) of the BA04 strain revealed that it has a circular genome of size 6,113,005 bp with four numbers of plasmids. A total of 6,111 genes including two novel crystal protein-encoding genes (MH753362.1 and MH753363.1) were identified. The BLASTn analysis of MH753362.1 showed 84% similarities (maximum identity) with Cry1Ia (KJ710646.1) gene, whereas MH753363.1 exhibited 66% identity with Insecticidal Crystal Protein (ICP)-6 gene (KM053257.1). At the protein level, MH753362.1 and MH753363.1 shared 79% identity with Cry1Ia (AIW52613.1) and 40% identity with Insecticidal Crystal Protein (ICP)-6 (AJW76687.1) respectively. Three-dimensional structures of these two novel protein sequences revealed that MH753362.1 have 48% structural similarity with Cry8ea1 protein, whereas MH753363.1 showed only 20% structural similarity with Cry4Aa protein. Apart from these insecticidal genes, the strain was also found to contain virulence and virulence-associated factors including the antibiotic resistance genes and Clustered regularly interspaced short palindromic repeat (CRISPR) sequences.ConclusionThis is the first report on the whole genome sequence of Bt strain BA04 isolated from Assam, a North-Eastern state of India. The WGS of strain BA04 unveils the presence of two novel types of insecticidal crystal protein-encoding genes which can be used for the development of insect-resistant transgenic crops. Additionally, the strain could be used for the formulations of effective biopesticides. The WGS provides the fastest and cheapest platform for a better understanding of the genetic makeup of a strain and helps to explore the role of virulence genes in pathogenicity against the insect host.

Project description:Bacillus thuringiensis (Bt), used as a biological control agent (BCA), can persist on plants, and from there can be introduced into the final food product. In routine food safety diagnostics, these Bt residues cannot be distinguished from natural populations of Bacillus cereus present in plants and all are enumerated as "presumptive B. cereus." In this study, information on eventual use of Bt biopesticides, brand, application times and intervals provided by three food processing companies in Belgium, were integrated with quantitative data on presumptive B. cereus measured from fresh to frozen food products. This information together with data on genomic similarity obtained via whole genome sequencing (WGS) and cry gene profiling using a quantitative real-time PCR (qPCR) assay, confirmed that six out of 11 Bt isolates originated from the applied Bt biocontrol products. These identified Bt strains were shown to carry enterotoxin genes (nhe, hbl, cytK-2) and express Hbl enterotoxin in vitro. It was also noted that these Bt biopesticide strains showed no growth at standard refrigeration temperatures and a low or moderate biofilm-forming ability and cytotoxic activity. Our results also showed that the use of Bt as a BCA on spinach plants in the field led to higher residual counts of Bt in spinach (fresh or frozen) in the food supply chain, but the residual counts exceeding at present commonly assumed safety limit of 105 CFU/g was only found in one fresh spinach sample. It is therefore recommended to establish a pre-harvest interval for Bt biopesticide application in the field to lower the likelihood of noncompliance to the generic B. cereus safety limit. Furthermore, WGS was found to be the best way to identify Bt biopesticide isolates at the strain level for foodborne outbreaks and clinical surveillance. The developed qPCR assay for screening on the presence of cry genes in presumptive B. cereus can be applied as a rapid routine test as an amendment to the already existing test on Bt crystal proteins determined via phase-contrast microscopy.

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Whole Genome Sequencing Reveals Biopesticidal Origin of Bacillus thuringiensis in Foods.

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