Bacillus thuringiensis (Bt)

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Development of methods to assess the fate and effects of Bacillus thuringiensis (Bt) Cry1F and Cry3Bb1 proteins

Development of methods to assess the fate and effects of Bacillus thuringiensis (Bt) Cry1F and Cry3Bb1 proteins

In 1996, crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins were introduced to the agricultural market. The acreage on which these crops are grown has increased by 18 million ha in the last 10 years. The terrestrial and aquatic fate of transgenic Bt proteins are key parameters governing exposure of non-target organisms in the environment. Because of potential non-target effects on terrestrial and aquatic organisms, it is important to accurately quantify Bt protein exposure with environmental fate studies. However, conflicting results have been found for terrestrial fate studies, and there is almost no information on the aquatic fate of transgenic Bt proteins. The various results seen in the terrestrial fate studies are likely in part due to differences in the analytical methods used. Currently, there is no reliable and accurate analytical technique for quantifying transgenic Bt proteins from environmental matrices with high recovery efficiency. The goals of this study were to improve the extraction of Bt proteins from environmental matrices. A series of spike-and-recovery experiments using the lepidopteran-active Bt Cry1F and the coleopteran- active Bt Cry3Bb1 were conducted to determine the best extraction method for these two proteins. A differential extraction was found for these two classes of proteins, with the best- available buffer for Cry1F being a biomimetic buffer and the best-available buffer for Cry3Bb1 being a high-salt, high-pH buffer. Despite a good extraction efficiency of Cry1F from soil, the biomimetic buffer was not able to extract any Cry1F from the soil of field plots containing Herculex 1 corn. The results from this study indicate a need for extraction buffers that can overcome the adsorption of Bt proteins to surface-active particles in soil. Several solid-phase extraction (SPE) methods were attempted for cleanup and concentration of Bt proteins from environmental matrices. Although good retention to the SPE tubes was achieved using carbon-18 and strong-anion-exchange cartridges, an efficient method for eluting Bt proteins from the tubes was not achieved.
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LARVICIDAL ACTIVITY OF BACILLUS THURINGIENSIS ISOLATED FROM Bt COTTON RHIZOSPHERE SOIL AGAINST ANOPHELES MOSQUITO LARVAE (CULICIDAE)

LARVICIDAL ACTIVITY OF BACILLUS THURINGIENSIS ISOLATED FROM Bt COTTON RHIZOSPHERE SOIL AGAINST ANOPHELES MOSQUITO LARVAE (CULICIDAE)

Mosquitoes are the important vectors of several human diseases such as malaria, filariasis, dengue fever, and yellow fever causing serious health problems to world human populations. At present, mosquito-borne diseases are a greater threat in tropical and subtropical countries [1]. The resurgence of these diseases is due to the increase in mosquito breeding places. These disease-causing insects are generally controlled by conventional insecticides [2]. Further, the indiscriminate use of neurotoxic insecticides caused various environmental toxic problems to non-target organisms and insecticidal resistance [3]. Globally, there are conscientious efforts to overcome these problems, and great emphasis is placed recently on eco-friendly and economically viable methodologies for insect control. An alternative approach for mosquito control is the use of natural products such as plant and microorganisms. Bacillus thuringiensis (Bt) is a soil-dwelling, Gram-positive, rod-shaped, and spore-forming bacterium that produces crystal (Cry) proteins that are toxic to insect species among the orders Lepidoptera , Diptera, and Coleoptera [4-7]. The lethality of Bt to the insects is mainly attributed by the Cry and cytolytic protein (Cyt) proteins, which was produced during the growth cycle [4,8]. These Bt strains are found in two different states as either vegetative and can be found as spores. These endospores are resistant to any environmental stress. They are metabolically inactive and a resting form of the bacterium which is a completely different structural form, chemical composition and enzymatic constitution from its vegetative state. The insecticidal activities of the various toxins are differing, but they are considered to be harmless to higher organisms including humans [9]. The Cry proteins are protoxins which can be converted to active toxins on ingestion by a susceptible insect [10]. The microbial insecticides have significant importance in the pest management. Further, when compared with chemical pesticides, these
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Genetic Basis for Variability of Cry1Ac Expression Among Commercial Transgenic <em>Bacillus thuringiensis</em> (Bt) Cotton Cultivars in the United States

Genetic Basis for Variability of Cry1Ac Expression Among Commercial Transgenic <em>Bacillus thuringiensis</em> (Bt) Cotton Cultivars in the United States

The amount of Cry1Ac δ -endotoxin in transgenic Bacillus thuringiensis Berliner (Bt) or Bollgard cotton varies among commercial cultivars. These differences in expression have been correlated with survival levels in Lepidop- tera, indicating that all Bollgard cultivars do not provide the same level of control. The objective of this study was to determine if differences in overall expression among commercial cultivars of Bollgard cotton were under simple genetic control. These findings could influence the way breeders select cultivars by evaluating for efficacy in insect control in addition to agronomic traits. Two sets of crosses were made in the greenhouse with cultivars that express the endotoxin at high and low levels. The parents and F 1 and F 2 gen-
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Pathogenicity of indigenous soil isolate of Bacillus thuringiensis to Helicoverpa armigera Hübner 1809 (Lepidoptera: Noctuidae)

Pathogenicity of indigenous soil isolate of Bacillus thuringiensis to Helicoverpa armigera Hübner 1809 (Lepidoptera: Noctuidae)

Microbial bio-pesticides are eco-friendly and target spe- cific alternates to hazardous synthetic pesticides (Kumar and Singh 2015; Majeed et al. 2017). Worldwide, an an- nual increase of 10% in the use of bio-pesticides has been estimated and among them, approximately 90% formula- tions are derived from Bacillus thuringiensis (Bt) (Kumar and Singh 2015; Osman et al. 2015). However, there is a great potential to look for the indigenous strains and iso- lates of Bt and characterize their pathogenicity and tox- icity against insect pest species (Sree and Varma 2015).

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Bacillus thuringiensis (Bt) Toxin Susceptibility and Isolation of Resistance Mutants in the Nematode Caenorhabditis elegans

Bacillus thuringiensis (Bt) Toxin Susceptibility and Isolation of Resistance Mutants in the Nematode Caenorhabditis elegans

The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin’s effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.
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Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection

Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection

ABSTRACT Bacillus thuringiensis (Bt) is armed to complete a full cycle in its insect host. During infection, virulence factors are expressed under the control of the quorum sensor PlcR to kill the host. After the host’s death, the quorum sensor NprR controls a necrotrophic lifestyle, allowing the vegetative cells to use the insect cadaver as a bioincubator and to survive. Only a part of the Bt population sporulates in the insect cadaver, and the precise composition of the whole population and its evolution over time are unknown. Using fluorescent reporters to record gene expression at the single-cell level, we have determined the differentia- tion course of a Bt population and explored the lineage existing among virulent, necrotrophic, and sporulating cells. The dynamics of cell differentiation were monitored during growth in homogenized medium, biofilm formation, and colonization of insect larvae. We demonstrated that in the insect host and in planktonic culture in rich medium, the virulence, necrotrophism, and sporulation regulators are successively activated in the same cell. In contrast, in biofilms, activation of PlcR is dispensable for NprR activation and we observed a greater heterogeneity than under the other two growth conditions. We also showed that sporulating cells arise almost exclusively from necrotrophic cells. In biofilm and in the insect cadaver, we identified an as-yet- uncharacterized category of cells that do not express any of the reporters used. Overall, we showed that PlcR, NprR, and Spo0A act as interconnected integrators to allow finely tuned adaptation of the pathogen to its environment.
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Evaluation of The commercial Bacillus thuringiensis (BT) products

Evaluation of The commercial Bacillus thuringiensis (BT) products

Abstract: Bacillus thuringiensis (BT) is the most widely used biological insecticide. This bacterium makes protein crystals during sporulate. In this study, effectiveness commercial formulations of Bacillus thuringiensis were tested against 3 moth larvae insect. Concentrations 0.125, 0.062, 0.031, 0.015, 0.25, 0.5, 1, 2 gram and 1.25, 0.625 and 0.315 gram were provided from Bacillus thuringiensis and were effected on Triboliom. The results showed that the ratio of 250 to 500 gram in 100 liters for every Petri of treatment and the amount of 5 gram flour is caused death of the insect .
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Study of toxicity of bacillus thuringiensis cry protein isolated from 1953 strain of bacillus thuringiensis on the rat hepatic and renal tissues in  a 90 day feed design

Study of toxicity of bacillus thuringiensis cry protein isolated from 1953 strain of bacillus thuringiensis on the rat hepatic and renal tissues in a 90 day feed design

Bacillus thuringiensis is a spore forming gram positive bacteria that have a number of different chemical compounds which are used for different types of insecticidal purposes. Though other various species of Bacillus are used for insecticidal purpose the effect on mammalian system has not been elaborated till now. In our study we observed some adverse effects of the cry toxins on the mammalian tissues i.e. specifically in the renal and hepatic tissues. The renal tissues and the h tissues are the markers of toxin excretion and accumulation in physiological systems. Some results show no significant differences at all while some showed potent differences. Our study in brief reflects the different biochemical and histological changes that occurred by Bt Cry proteins in daily diet to the group of animals for observing the toxicological effects o
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A strain of Bacillus thuringiensis containing a novel

A strain of Bacillus thuringiensis containing a novel

by p19 and orf1-orf2 genes on Cyt1aa protein expression in acrystalliferous strain of Bacillus thuringiensis.. Screening by Polymerase Chain Reaction of Bacillus thuringiensis[r]

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Comparative Genomics of Bacillus thuringiensis Reveals a Path to Specialized Exploitation of Multiple Invertebrate Hosts

Comparative Genomics of Bacillus thuringiensis Reveals a Path to Specialized Exploitation of Multiple Invertebrate Hosts

ABSTRACT Understanding the genetic basis of host shifts is a key genomic ques- tion for pathogen and parasite biology. The Bacillus cereus group, which encom- passes Bacillus thuringiensis and Bacillus anthracis, contains pathogens that can infect insects, nematodes, and vertebrates. Since the target range of the essential virulence factors (Cry toxins) and many isolates is well known, this group presents a powerful system for investigating how pathogens can diversify and adapt to phylogenetically distant hosts. Specialization to exploit insects occurs at the level of the major clade and is associated with substantial changes in the core genome, and host switching between insect orders has occurred repeatedly within subclades. The transfer of plasmids with linked cry genes may account for much of the adaptation to particular insect orders, and network analysis implies that host specialization has produced strong associations between key toxin genes with similar targets. Analysis of the dis- tribution of plasmid minireplicons shows that plasmids with orf156 and orf157, which carry genes encoding toxins against Lepidoptera or Diptera, were contained only by B. thuringiensis in the specialized insect clade (clade 2), indicating that tight genome/plasmid associations have been important in adaptation to invertebrate hosts. Moreover, the accumulation of multiple virulence factors on transposable ele- ments suggests that cotransfer of diverse virulence factors is advantageous in terms of expanding the insecticidal spectrum, overcoming insect resistance, or through gains in pathogenicity via synergistic interactions between toxins.
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Bacillus thuringiensis  Grundlagen und Einsatz im biologischen und integrierten Pflanzenschutz

Bacillus thuringiensis Grundlagen und Einsatz im biologischen und integrierten Pflanzenschutz

Im Rahmen ihrer langjährigen Tätigkeit als wissenschaftliche Mitarbeiterin des Instituts für Biologischen Pflanzenschutz des Julius Kühn-Instituts, Bundesforschungsinstitut für Kulturpflan- zen hielt die Autorin anlässlich der gemeinsamen Lehrveranstal- tung „Biologischer und Integrierter Pflanzenschutz“ mit dem Fachbereich Biologie der Technischen Universität Darmstadt in den Jahren 2008 und 2009 je eine Vorlesung über „Bacillus thuringiensis“. Die Inhalte dieser Vorlesungen wurden in Form der vorliegenden Broschüre aufgearbeitet.

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CHARACTERIZATION OF A NATIVE BACILLUS THURINGIENSIS ISOLATES FROM MALAYSIA THAT PRODUCES EXOSPORIUM-ENCLOSED PARASPORAL INCLUSION

CHARACTERIZATION OF A NATIVE BACILLUS THURINGIENSIS ISOLATES FROM MALAYSIA THAT PRODUCES EXOSPORIUM-ENCLOSED PARASPORAL INCLUSION

to granular surface textures. De Respinis et al. (2006) mentioned that the colony morphology of B. thuringiensis and B. cereus is indistinguishable and both species show the typical morphology of circular or irregular colonies in white or grey; granular, milky or mat aspect and minimum 5 mm of diameter. In addition to smooth colonies with irregular shape, Lima et al. (2002) also characterized a B. thuringiensis isolate that produced dark yellowish colonies. Apart from the characteristics stated, many also reported that B. thuringiensis strains formed “fried-egg” appearance on agar plate (Travers et al., 1987; El-kersh et al., 2012; Mahalakshmi et al., 2012). The descriptions for colony morphology are very subjective. Bt-S84-13a showed different colony morphology when compared with Bti, however, it possessed the common morphological appearance as described in the literature, and most importantly, it also displayed the “fried-egg” morphology, which is in agreement with the earlier reports.
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Yield analysis and adaptation for Bacillus thuringiensis (Bt) and non-Bacillus thuringiensis (Bt) Cotton varieties in the kingdom of Eswatini

Yield analysis and adaptation for Bacillus thuringiensis (Bt) and non-Bacillus thuringiensis (Bt) Cotton varieties in the kingdom of Eswatini

Abstract— Cotton in Eswatini contributes 2.1 % of the country’s Gross Domestic Product owing to low cotton yield due to high pest pressure. Eswatini farmers grow Alba QM 301 a conventional non Bt variety which is affected by bollworm. Cotton is no longer profitable and farmers are quitting the industry, yet it is the only source of livelihood in drought prone areas of Eswatini . Countries lik e India and South Africa have replaced conventional cotton with high yielding Bt or genetically modified cotton.The study analyses yield and adaptation of Bt cotton under rain fed condition.Bt cotton hybrid was evaluated under field condition for adaptation and yield performance in 2016 and 2017 season. Two Bt cotton varieties JKCH 1947 Bt and JKCH 1050 Bt were tested against the local variety Alba Plus QM 301 and JKC 724 both Non Bt (NBt).JKCH 1947 recorded significantly higher seed cotton yield per ha of 3070 k g/ha on the first year. It was closely followed by JKCH 1050 with a yields of 2955 k g/ha.The number of boll per plant was also significant higher compared the control. Alba Plus QM 301 and JKC 724 both Non Bt (NBt) recorded the lower yields of 2066 and 821 k g/ha respectively, under the same condition with less number of bolls per plant. Similar observations were recorded on the second year, JKCH1947 and JKCH 1050 recording 1765k g/ha and 1865k g/ha respectively. A similar trend was observed onthe number of bolls per plant,higher number of bolls were recorded in JKCH 1050 Bt followed by JKCH 1947 Bt. Alba Plus QM 301 NBt and JKC 724 NBt recorded fewer boll in both years. All varieties showed good adaptability to local environment with good plant stand. Keywords— Bt cotton, rain fed conditions, seed cotton.
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Article -   Encoding of BT (Bacillus thuringiensis) pro tein by fu sion gene

Article - Encoding of BT (Bacillus thuringiensis) pro tein by fu sion gene

Cry1Ac belongs to Cry1 d- endotoxin family and is active against Helicoverpa armigera and Plutella xylostella but it shows no mortality on Spodoptera litura. On the other hand recently discoveredVip3A is highly toxic to Spodoptera litura, but not to Helicoverpa armigera. Since Helicoverpa armigera, Plutella xylostella S.litura both are being notorious pest on some economically important crops like cotton, tomato, cabbage, cauliflower and insecticidal genes having toxicity on these pests is desirable. In the present study we describe cloning of two different Bt gene, cry1Ac and Vip3A fusion plasmid, its successful expression in E.coli cells and toxicity analysis was P.xylostella
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Effect of Chlorine and Temperature on Larvicidal Activity of Cuban Bacillus thuringiensis Isolates

Effect of Chlorine and Temperature on Larvicidal Activity of Cuban Bacillus thuringiensis Isolates

ter but there are not visible reduction of Cry and Cyt toxins by treatment with chlorine in SDS-PAGE analysis. Chlorine is a non-selec- tive oxidant with a number of effects over the living systems: reacts with a variety of cellu- lar components (30), deactivates enzymatic ac- tive sites, decreases the biological functions of proteins and produces deleterious effects on DNA (31). In addition chlorinated waters af- fect the permeability of the cytoplasmic mem- brane (32), which could lead to cell death and inhibit the growth of B. thuringiensis (33).

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Suppressing a plant-parasitic nematode with fungivorous behavior by fungal transformation of a Bt cry gene

Suppressing a plant-parasitic nematode with fungivorous behavior by fungal transformation of a Bt cry gene

Background: Pine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (PWN), is an impor- tant destructive disease of pine forests worldwide. In addition to behaving as a plant-parasitic nematode that feeds on epithelial cells of pines, this pest relies on fungal associates for completing its life cycle inside pine trees. Manipulating microbial symbionts to block pest transmission has exhibited an exciting prospect in recent years; however, trans- forming the fungal mutualists to toxin delivery agents for suppressing PWN growth has received little attention. Results: In the present study, a nematicidal gene cry5Ba3, originally from a soil Bacillus thuringiensis (Bt) strain, was codon-preferred as cry5Ba3Φ and integrated into the genome of a fungus eaten by PWN, Botrytis cinerea, using Agrobacterium tumefaciens-mediated transformation. Supplementing wild-type B. cinerea extract with that from the cry5Ba3Φ transformant significantly suppressed PWN growth; moreover, the nematodes lost fitness significantly when feeding on the mycelia of the cry5Ba3Φ transformant. N-terminal deletion of Cry5Ba3Φ protein weakened the nematicidal activity more dramatically than did the C-terminal deletion, indicating that domain I (endotoxin-N) plays a more important role in its nematicidal function than domain III (endotoxin-C), which is similar to certain insecticidal Cry proteins.
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A new ecotoxicological test method for genetically modified plants and other stressors in soil with the black fungus gnat Bradysia impatiens (Diptera): current status of test development and dietary effects of azadirachtin on larval development and emerge

A new ecotoxicological test method for genetically modified plants and other stressors in soil with the black fungus gnat Bradysia impatiens (Diptera): current status of test development and dietary effects of azadirachtin on larval development and emergence rate

Hilbeck et al. [21] described a structured, stepwise selec- tion procedure for the identification of organism groups potentially suitable as test species for the assessment of GMP. Applying this procedure for the receiving environ- ments of Germany, larvae of the dipteran family Sciari- dae were identified as a potential test taxon because of their ecological relevance in Central European agricul- tural soils [8, 33]. Similar to other soil dwelling dipteran larvae, Sciaridae can reach high numbers in species and individuals in agricultural fields in the temperate regions of Central Europe [29] and participate in organic matter decomposition. A comprehensive appraisal of the ecolog- ical importance and ecosystem function of soil dwelling Diptera, including Sciaridae, is given by Frouz [16]. Of the worldwide 1700 species [30], approximately 340 spe- cies occur in Germany [29]. Whereas Lycoriella castane- scens (Diptera: Sciaridae) has been listed as a promising candidate species [21], no culture of L. castanescens was available at the start of the study. However, for another sciarid species, Bradysia impatiens, a culture existed at the Julius-Kühn-Institute (JKI) in Kleinmachnow (Ger- many). Büchs [6, 7] performed tests with B. impatiens and Bacillus thuringiensis (Bt) maize and confirmed the exposure of the larval stage to the Bt toxin from MON810 maize. Bradysia spp. are found in agricultural fields at abundances similar to those of L. castanescens [8, 33], but no published records of the species B. impatiens from agricultural fields exist [19]. For the test development, B. impatiens was used as there is no reason to believe that both sciarid species differ in terms of susceptibility to GMP material. Both are destruents of soil organic matter, feed on fungi and have similar life history parameters [4, 9, 24, 29].
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Field efficacy of genetically modified FK 95 Bollgard II cotton for control of bollworms, Lepidoptera, in Ghana

Field efficacy of genetically modified FK 95 Bollgard II cotton for control of bollworms, Lepidoptera, in Ghana

Conventional breeding for resistance against these pests has not yielded the desired results. Consequently, they are managed mainly with insecticides in Ghana and many other cotton-growing areas in West Africa [2–4]. Globally, cotton is responsible for about 16–25% of all chemical insecticides used in agriculture, which is more than what is used for any other single crop [6, 7, 10]. Although most of these insecticides are effective for control, they pose health hazards to farmers who use them and also contaminate the environment. Moreover, they are expensive and their indiscriminate use can cause emergence of resistant biotypes in insect populations resulting in control failures such as those reported for pyrethroid insecticide use in parts of West Africa [5, 8, 9]. In an effort to scale down on insecticide use, amidst fears for environmental contamination and insect resistance build-up, genetic modification of plants for resistance to insect pests has been found to be a better and environ- mentally friendlier alternative [10]. Genetic modification with the soil-borne bacterium Bacillus thuringiensis (Bt) Berliner has been used to control insect pests in several crops [11–13]. Genetically modified cotton contains the Bt gene(s) that produce(s) toxins or bio-pesticides inside the plant to offer protection against insects. It has spe- cific activity against lepidopteran insects such as the boll- worm complex due to specific receptors and conditions in the caterpillar’s gut that allow activation of the Bt crys- tal proteins [14, 15]. The objective of the present study was to evaluate the field efficacy of the genetically modi- fied Bollgard II (BG II) cotton, FK 95 BG II for control of cotton bollworms in Ghana.
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Genetic Structure of Population of Bacillus cereus and B  thuringiensis Isolates Associated with Periodontitis  and Other Human Infections

Genetic Structure of Population of Bacillus cereus and B thuringiensis Isolates Associated with Periodontitis and Other Human Infections

The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.
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Whole genome phylogeny of Bacillus by feature frequency profiles (FFP)

Whole genome phylogeny of Bacillus by feature frequency profiles (FFP)

The findings of FFP analyses were fully supported by SNP phylogenies construed by kSNP (alignment – free sequence analysis method) and Parsnp (core genome alignment method). By comparing the NJ trees inferred from FFP analysis (Fig. 1) and kSNP analysis (Fig. 2), we found a high level of similarity between two phylogenies. The clades of I - b and I - d clades in FFP tree are consistent with the Bacillus anthracis and Bacillus thuringiensis clades in kSNP tree, whilst the cluster I in FFP tree is corresponding to the clade of Bacillus cereus sensu lato in kSNP tree. While the core genome SNP tree constructed by Parsnp failed to cover all the species studied, the exclusion of other Bacillus species from the major clus- ter was actually a support for the monophyly of Bacillus cereus sensu lato. This is because Parsnp is lim- ited in intraspecific alignment and can only tolerate genomes with high similarity ( >=97%). Genomes from other species will be automatically excluded from the full alignment 22 .
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