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.