Niels Bohse Hendriksen

Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis.

ABSTRACT Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereusand B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceTgene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of thebceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes inB. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

Characterization of Emetic Bacillus weihenstephanensis, a New Cereulide-Producing Bacterium†

ABSTRACT Cereulide production has until now been restricted to the species Bacillus cereus. Here we report on two psychrotolerant Bacillus weihenstephanensis strains, MC67 and MC118, that produce cereulide. The strains are atypical with regard to pheno- and genotypic characteristics normally used for identification of emetic B. cereus strains. MC67 and MC118 produced cereulide at temperatures of as low as 8°C.

Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9

Aims: To identify and characterize new bacteriocins from a collection of 41 strains belonging to 27 subspecies of Bacillus thuringiensis, and to evaluate the safety of the producers.

Biolog Substrate Utilisation Assay for Metabolic Fingerprints of Soil Bacteria: Incubation Effects

Bacterial communities can be described by their enzymatic potentials using the Biolog substrate utilisation assay. We have investigated tetrazolium reduction and cell growth during incubation of Pseudomonas fluorescens MM6 and soil bacteria in Biolog plates. Increasing the inoculum size shortened the lag phase before formazan formation. For the soil bacteria, increasing the inoculum also resulted in a higher rate constant of formazan formation, and the final number of wells with formazan formation increased. Both MM6 and soil bacteria proliferated in the wells both with and without specific carbon sources after inoculation. With soil bacteria, the presence of clay, humic substances, and dissolved organic matter increased the background coloration and may have resulted in cell growth. The growth led to increased culturability (CFU/AODC) and rate of colony-appearance and decreased the diversity of the bacterial communities within each well. Metabolic fingerprinting of bacterial communities using Biolog plates thus depends on aerobic growth of a fraction of the community.

Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains

The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.

The Bacillus cereus bceT enterotoxin sequence reappraised.

Bacillus cereus is a known opportunistic human pathogen belonging to the B. cereus group. Establishment of the pathogenesis most likely involves several gene products. One of these gene products, a single gene component named bceT, has been cloned and described from B. cereus B-4ac [Agata et al., Microbiology 141 (1995) 983-988]. However, our sequences of the bceT region from 16 B. cereus group strains showed inconsistency with the published bceT sequence. Only part of the bceT sequence had homology to our sequences. This initiated a more thorough investigation of the bceT sequence. Restriction site search and database searches intimated that the cloned bceT was created by an incidental joining of four DNA fragments during ligation. One of these fragments had 93% homology to an open reading frame (ORF 101) located within the pathogenic island of the Bacillus anthracis pXO1 virulence plasmid. We suggest that the reported enterotoxic activity of the original cloned bceT construct could be due to either the fusion gene or the fragment with homology to ORF 101 in pXO1.

Estimating the effects of Cry1F Bt‐maize pollen on non‐target Lepidoptera using a mathematical model of exposure

Summary 1. In farmland biodiversity, a potential risk to the larvae of non‐target Lepidoptera from genetically modified (GM) Bt‐maize expressing insecticidal Cry1 proteins is the ingestion of harmful amounts of pollen deposited on their host plants. A previous mathematical model of exposure quantified this risk for Cry1Ab protein. We extend this model to quantify the risk for sensitive species exposed to pollen containing Cry1F protein from maize event 1507 and to provide recommendations for management to mitigate this risk. 2. A 14‐parameter mathematical model integrating small‐ and large‐scale exposure was used to estimate the larval mortality of hypothetical species with a range of sensitivities, and under a range of simulated mitigation measures consisting of non‐Bt maize strips of different widths placed around the field edge. 3. The greatest source of variability in estimated mortality was species sensitivity. Before allowance for effects of large‐scale exposure, with moderate within‐crop host‐plant density and with no mitigation, estimated mortality locally was <10% for species of average sensitivity. For the worst‐case extreme sensitivity considered, estimated mortality locally was 99·6% with no mitigation, although this estimate was reduced to below 40% with mitigation of 24‐m‐wide strips of non‐Bt maize. For highly sensitive species, a 12‐m‐wide strip reduced estimated local mortality under 1·5%, when within‐crop host‐plant density was zero. Allowance for large‐scale exposure effects would reduce these estimates of local mortality by a highly variable amount, but typically of the order of 50‐fold. 4. Mitigation efficacy depended critically on assumed within‐crop host‐plant density; if this could be assumed negligible, then the estimated effect of mitigation would reduce local mortality below 1% even for very highly sensitive species. 5.  Synthesis and applications. Mitigation measures of risks of Bt‐maize to sensitive larvae of non‐target lepidopteran species can be effective, but depend on host‐plant densities which are in turn affected by weed‐management regimes. We discuss the relevance for management of maize events where cry1F is combined (stacked) with a herbicide‐tolerance trait. This exemplifies how interactions between biota may occur when different traits are stacked irrespective of interactions between the proteins themselves and highlights the importance of accounting for crop management in the assessment of the ecological impact of GM plants.

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats

Aims:  To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats.

Characterization of polyvalent and safe Bacillus thuringiensis strains with potential use for biocontrol.

Sixteen Bacillus thuringiensis (Bt) strains were screened for their anti‐insect, antibacterial and antifungal determinants by phenotypic tests and PCR targeting major insecticidal proteins and complements, chitinases, lactonases, β‐1,3‐glucanases and zwittermicinA. Six strains had genes of at least two major insecticidal toxins and of insecticidal complements. With regard to fungal biocontrol, all the strains inhibited Fusarium oxysporum and Aspergillus flavus growth and four strains had all or most of the antifungal determinants examined, with strain Bt HD932 showing the widest antifungal activity spectrum. Autolysins, bacteriocin and AHL‐lactonases were produced by all or most of the tested strains with different activity spectra including pathogens like Listeria monocytogenes. Safety evaluation was carried out via PCR by screening the B. cereus psychrotolerance‐related genes, toxin genes and the virulence pleiotropic regulator plcR. Diarrheal enterotoxins and other toxin genes were widespread among the collection with strains Bt HD9 and H45 lacking psychrotolerance‐related genes, while five strains were positive. Only three strains (BMG1.7, H172, H156) resulted positive with primer sets targeting partial or complete plcR gene. By Vero Cell Assays, Bt HD868 followed by Bt HD9 were shown to be the safest strains. These polyvalent and safe Bt strains could be very promising in field application. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Widespread Occurrence of Bacterial Human Virulence Determinants in Soil and Freshwater Environments

ABSTRACT The occurrence of 22 bacterial human virulence genes (encoding toxins, adhesins, secretion systems, regulators of virulence, inflammatory mediators, and bacterial resistance) in beech wood soil, roadside soil, organic agricultural soil, and freshwater biofilm was investigated by nested PCR. The presence of clinically relevant bacterial groups known to possess virulence genes was tested by PCR of 16S and 23S rRNA genes. For each of the virulence genes detected in the environments, sequencing and NCBI BLAST analysis confirmed the identity of the PCR products. The virulence genes showed widespread environmental occurrence, as 17 different genes were observed. Sixteen genes were detected in beech wood soil, and 14 were detected in roadside and organic agricultural soils, while 11 were detected in the freshwater biofilm. All types of virulence traits were represented in all environments; however, the frequency at which they were detected was variable. A principal-component analysis suggested that several factors influenced the presence of the virulence genes; however, their distribution was most likely related to the level of contamination by polycyclic aromatic hydrocarbons and pH. The occurrence of the virulence genes in the environments generally did not appear to be the result of the presence of clinically relevant bacteria, indicating an environmental origin of the virulence genes. The widespread occurrence of the virulence traits and the high degree of sequence conservation between the environmental and clinical sequences suggest that soil and freshwater environments may constitute reservoirs of virulence determinants normally associated with human disease.