Introduction
Recently, strains of Bacillus thuringiensis were discovered that had toxic activity against two-winged and rigid-winged insect species(Ben-Dov, 2014; Zghal et al., 2018; Domínguez-Arrizabalaga et al., 2020; Cao et al., 2022). In this regard, the larvicidal activities of these strains may be broader than previously thought, as it was assumed that they were effective only against lepidopterans. During the last three decades, an intensive search for natural isolates that could be of economic importance for the control of insect pests has been carried out (Xiao & Wu, 2019). Screening procedures have yielded tens of thousands of isolates that are in both publicly and privately owned collections (Pinto et al., 2012). These isolates in collections, therefore, could be reservoirs that would act as toxins in pest control.
B. thuringiensis produces insecticidal proteins, the primary type of crystalline (Cry) proteins (Bravo et al., 2007). Actively growing vegetative cells are non-toxic since they synthesize proteins that do not produce crystals. At the same time, some strains synthesize Cry proteins that are endotoxic to some species of Coleoptera. Thus, Δ-endotoxins mainly contain cytolytic (Cyt) and Cry proteins (Pardo-López et al., 2019). However, Cyt and Cry proteins have different sequence homology, although they assume similar action modes relative to cell lysis, resulting in irreversible damages in the mid-gut of insects (Bravo et al., 2007).
There are several reasons for the increased interest in B. thuringiensis . In particular, many insects are becoming resistant to insecticides. At the same time, the presence of resistance to strains ofB. thuringiensis may also be increasing. Classification by pathotype of B. thuringiensis strains is difficult because not only the great diversity of δ endotoxic genes is known (Aronson, 2002), but also their multiplicity even within one strain (Palma et al., 2014). Therefore, it is necessary to search for reliable methods to classifyB. thuringiensis strains. Serotyping and classification according to biochemical features proved to be the most effective methods. Serotyping of B. thuringiensis was developed by examining flagellar antigens (De Barjac & Frachon, 1990). Two issues remain relevant: a) whether serotyping reflects the full diversity of B. thuringiensis ; b) to what extent serotyping may be relevant to genetic type exchanges between species and subspecies related to Bacillus cereus .
The two above bacteria are very similar in biochemical traits and genetic properties (Wei et al., 2019). Comparative analysis of conserved genes in the core genomes and pangenomes of related Bacillusspecies revealed numerous overlapping loci in these strains. Poornima et al. (2012) also reported that these bacteria’s genetic and phenotypic properties are almost indistinguishable. They synthesize parasporins, a new functional category of inclusion proteins capable of destroying cancer cells first identified in the B. thuringiensis isolate. Although this strain possesses parasporal inclusions typical of B. thuringiensis species, an in-depth study is required on its morphological and molecular differences relative to other related species.
Furthermore, it has been shown that B. cereus species cannot be reliably identified via standard biotyping (Yusuf et al., 2018). Hence, biochemical experiments may not be sufficient for differentiation. Instead, an insecticidal crystalline protein has been used as a distinguishing feature to differentiate these bacteria (Pardo-López et al., 2013).
Some of the factors that distinguish these two bacteria are the pathogenicity of samples B. cereus causing gastrointestinal disorders, while B. thuringiensis is also involved in diarrhoeal epidemics. Insecticidal crystalline proteins (δ-endotoxin) encoded bycry genes have been reported as one of the distinctive featuresof B. thuringiensis (Bravo et al., 2007). Since using a biomarker to differentiate the B. cereus is quite complicated and time-consuming, a highly effective identifier is urgently required to replace previous ones with less efficient performance, sometimes providing false results. Based on transcription regulator genes and Cry protein genes for B. thuringiensis (Pardo-López et al., 2013), a study was conducted to test and compare the effectiveness of the developed biomarker to the existing Cry protein marker (cry 2) in distinguishing B. thuringiensis from B. cereus strains. Cry2 was found to be the most abundant crystalline protein in B. thuringiensis (Bravo et al., 2007).
It has been established that B. thuringiensis is quite commonly associated with the feces of many animals, particularly herbivorous mammals (Rahman et al., 2022). Therefore, this work aimed to investigate fecal animal isolates to determine flagellar (H) antigenic serotypes, as well as the insect pathogenic activity of B. thuringiensisstrains necessary for their differentiation. The data indicated daily plant material consumed with food, in which high concentrations of these bacteria were present. In practical applications, obtained results may assist in studying the toxicity of B. thuringiensis serotypes against several types of human cancer cells that would help treat tumors.