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Andrea Wilcks

Publications and source records attributed to Andrea Wilcks.

16 recordsLinked to original sources
PubMed2006

Persistence of Bacillus thuringiensis bioinsecticides in the gut of human-flora-associated rats.

The capability of two bioinsecticide strains of Bacillus thuringiensis (ssp. israelensis and ssp. kurstaki) to germinate and persist in vivo in the gastrointestinal tract of human-flora-associated rats was studied. Rats were dosed either with vegetative cells or spores of the bacteria for 4 consecutive days. In animals fed spores, B. thuringiensis cells were detected in faecal and intestinal samples of all animals, whereas vegetative cells only poorly survived the gastric passage. Heat-treatment of intestinal samples, which kills vegetative cells, revealed that B. thuringiensis spores were capable of germination in the gastrointestinal tract. In one animal fed spores of B. thuringiensis ssp. kurstaki, these bacteria were detected at high density (10(3)-10(4) CFU g(-1) faecal and intestinal samples) even 2 weeks after the last dosage. In the same animal, passage of B. thuringiensis ssp. kurstaki to the spleen was observed; however, no other adverse effects were observed. Denaturing gradient gel electrophoresis of PCR-amplified bacterial 16S rRNA genes in faecal samples revealed no major effect of B. thuringiensis on the composition of the indigenous gut bacteria. Additionally, no cytotoxic effect was detectable in gut samples by Vero cell assay.

Animal Feed↗
PubMed2006

Horizontal transfer of tet(M) and erm(B) resistance plasmids from food strains of Lactobacillus plantarum to Enterococcus faecalis JH2-2 in the gastrointestinal tract of gnotobiotic rats.

Two wild-type strains of Lactobacillus plantarum previously isolated from fermented dry sausages were analysed for their ability to transfer antibiotic resistance plasmids in the gastrointestinal tract. For this purpose, we used gnotobiotic rats as an in vivo model. Rats were initially inoculated with the recipient Enterococcus faecalis JH2-2 at a concentration of 10(10) CFU mL(-1). After a week, either of the two donors L. plantarum DG 522 (harbouring a tet(M)-containing plasmid of c. 40 kb) or L. plantarum DG 507 [harbouring a tet(M)-containing plasmid of c. 10 kb and an erm(B)-containing plasmid of c. 8.5 kb] was introduced at concentrations in the range of 10(8)-10(10) CFU mL(-1). Two days after donor introduction, the first transconjugants (TCs) were detected in faecal samples. The detected numbers of tet(M)-TCs were comparable for the two donors. In both cases, this number increased to c. 5 x 10(2) CFU g(-1) faeces towards the end of the experiment. For erm(B)-TCs, the number was significantly higher and increased to c. 10(3) CFU g(-1) faeces. To our knowledge, this is the first study showing in vivo transfer of wild-type antibiotic resistance plasmids from L. plantarum to E. faecalis.

Animals↗
PubMed2006

A 90-day safety study in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA).

Genetically modified plants expressing insecticidal traits offer a new strategy for crop protection, but at the same time present a challenge in terms of food safety assessment. The present 90-day feeding study was designed to assess the safety of a rice variety expressing the snowdrop Galanthus nivalis lectin (GNA lectin), and forms part of a EU-funded project where the objective has been to develop and validate sensitive and specific methods to assess the safety of genetically modified foods. Male and female Wistar rats were given a purified diet containing either 60% genetically modified or parental rice for 90 days. This corresponds to a mean daily GNA lectin intake of approximately 58 and 67mg/kg body weight for males and females, respectively. Prior to the animal study comprehensive analytical characterization of both rice materials was performed. The chemical analyses showed a number of statistically significant differences, with the majority being within the ranges reported in the literature. In the animal study a range of clinical, biological, immunological, microbiological and pathological parameters were examined. A number of significant differences were seen between groups fed the two diets, but none of them were considered to be adverse. In conclusion, the design of the present animal study did not enable us to conclude on the safety of the GM food. Additional group(s) where the expressed gene products have been spiked to the diet should be included in order to be able to distinguish whether the observed effects were due to the GNA lectin per se or to secondary changes in the GM rice.

Animals↗
PubMed2006

Safety testing of GM-rice expressing PHA-E lectin using a new animal test design.

The 90-day animal study is the core study for the safety assessment of genetically modified foods in the SAFOTEST project. The model compound tested in the 90-day study was a rice variety expressing the kidney bean Phaseolus vulgaris lectin agglutinin E-form (PHA-E lectin). Female Wistar rats were given a nutritionally balanced purified diet with 60% parental rice, 60% PHA-E rice or 60% PHA-E rice spiked with 0.1% recombinant PHA-E lectin for 90 days. This corresponded to a mean daily PHA-E lectin intake of approximately 0, 30 and 100mg/kg body weight for each group, respectively. The spiking was used to increase the specificity and to demonstrate the sensitivity of the study. A range of biological, biochemical, microbiological and pathological parameters were examined and significant differences in weight of small intestine, stomach and pancreas and plasma biochemistry were seen between groups. Included in this paper are also data from the molecular characterisation and chemical analysis of the PHA-E rice, from the construction and production of the PHA-E lectin, and from the preceding 28-day in vivo study where the toxicity of the pure PHA-E lectin was determined. In conclusion, the combined use of information from the compositional analysis, the 28-day study and the characterisation of the PHA-E rice and the PHA-E lectin has improved the design of the 90-day study. The spiking procedure has facilitated the interpretation of the results of the study and transferred it into a valuable tool for the future safety testing of genetically modified foods.

Animals↗
PubMed2006

A 90-day safety study of genetically modified rice expressing Cry1Ab protein (Bacillus thuringiensis toxin) in Wistar rats.

An animal model for safety assessment of genetically modified foods was tested as part of the SAFOTEST project. In a 90-day feeding study on Wistar rats, the transgenic KMD1 rice expressing Cry1Ab protein was compared to its non-transgenic parental wild type, Xiushui 11. The KMD1 rice contained 15mg Bt toxin/kg and based on the average feed consumption the daily intake was 0.54mg Bt toxin/kg body weight. No adverse effects on animal behaviour or weight gain were observed during the study. Blood samples collected one week prior to sacrifice were analyzed and compared for standard haematological and biochemical parameters. A few parameters were significantly different, but all within the normal reference intervals for rats of this breed and age and not in relation to any other findings, thus not considered treatment related. Upon sacrifice a large number of organs were weighed, macroscopic and histopathological examinations were performed with only minor changes to report. The aim of the study was to use a known animal model in performance of safety assessment of a GM crop, in this case KMD1 rice. The results show no adverse or toxic effects of KMD1 rice when tested in the design used in this 90-day study. Nevertheless the experiences from this study lead to the overall conclusion that safety assessment for unintended effects of a GM crop cannot be done without additional test group(s).

Animals↗
PubMed2006

Fate and effect of ingested Bacillus cereus spores and vegetative cells in the intestinal tract of human-flora-associated rats.

The fate and effect of Bacillus cereus F4433/73R in the intestine of human-flora-associated rats was studied using bacteriological culturing techniques and PCR-denaturing gradient gel electrophoresis in combination with cell assays and immunoassays for detection of enterotoxins. In faecal samples from animals receiving vegetative cells, only few B. cereus cells were detected. Spores survived the gastric barrier well, and were in some cases detected up to 2 weeks after ingestion. Selective growing revealed no major changes in the intestinal flora during passage of B. cereus. However, denaturing gradient gel electrophoresis analysis with universal 16S rRNA gene primers revealed significant changes in the intestinal microbiota of animals dosed with spores. Vero cell assays and a commercial kit (BCET-RPLA) did not reveal any enterotoxin production from B. cereus F4433/73R in the intestinal tract.

Animals↗
PubMed2006

Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensis-based insecticides on fresh fruits and vegetables.

A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic.

Bacillus thuringiensis↗
PubMed2005

Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food.

Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain. A new observation was that 31 out of the 40 randomly selected B. cereus-like strains could be classified as Bacillus thuringiensis due to crystal production and/or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis.

Bacillus cereus↗
PubMed2005

Characterization of transferable tetracycline resistance genes in Enterococcus faecalis isolated from raw food.

The prevalence of tetracycline resistance, and of specific genetic determinants for this resistance was investigated in 1003 strains of Enterococcus faecalis isolated from various raw food products originating from five categories including chicken meat, other poultry meat, beef, pork, and 'other'. For the 238 resistant isolates identified, the ability to transfer the resistant phenotype to a given recipient in vitro was investigated. New and interesting observations were that the tet(L) resistance determinant was more readily transferred than tet(M), and that the presence of Tn916-like elements known to encode tet(M) did not correlate with increased transferability of the resistant phenotype.

Animals↗
PubMed2004

Persistence of DNA studied in different ex vivo and in vivo rat models simulating the human gut situation.

This study aimed to evaluate the possibility of DNA sequences from genetically modified plants to persist in the gastrointestinal (GI) tract. PCR analysis and transformation assays were used to study DNA persistence and integrity in various ex vivo and in vivo systems using gnotobiotic rats. DNA studied was either plasmid DNA, naked plant DNA or plant DNA embedded in maize flour. Ex vivo experiments performed by incubating plant DNA in intestinal samples, showed that DNA is rapidly degraded in the upper part of the GI tract whereas degradation is less severe in the lower part. In contrast, plasmid DNA could be recovered throughout the GI tract when intestinal samples were taken up to 5 h after feeding rats with plasmid. Furthermore, DNA isolated from these intestinal samples was able to transform electro-competent Escherichia coli, showing that the plasmid was still biologically active. The results indicate that ingested DNA may persist in the GI tract and consequently may be present for uptake by intestinal bacteria.

Animals↗
PubMed2003

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes.

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.

Amino Acid Sequence↗
PubMed2002

Isolation and characterization of Bacillus cereus-like bacteria from faecal samples from greenhouse workers who are using Bacillus thuringiensis-based insecticides.

OBJECTIVES: Since the discovery of the insecticidal activity of Bacillus thuringiensis at the beginning of the twentieth century, this bacterium has been used increasingly against various insect pests. In spite of the extensive use of B. thuringiensis, only sporadic clinical case reports have been published. In recent years, the close relationship between B. thuringiensis and the human pathogen Bacillus cereus has been confirmed. In practice, only the insecticidal activity of B. thuringiensis distinguishes the two species. However, both species are composed of thousands of isolates with varying potential for causing adverse effects in humans. The aim of this study was to employ molecular biology methods for assessment of occupational exposure to B. thuringiensis-based biopesticides by determination of specific genetic information including plasmid profiles and random amplified polymorphic DNA (RAPD). METHODS: Faecal samples from 12 persons, working in Danish greenhouses, were collected for microbial analysis. Seven persons were using B. thuringiensis-based insecticides, whereas five persons were employed at greenhouses that did not use B. thuringiensis. The bacteria were isolated on B. cereus-specific solid substrate, and colonies were further identified using the polymerase chain reaction (PCR). The PCR method was used for the identification of the enterotoxin genes HblA and BceT. The expression of enterotoxins was detected with two commercial serological kits. Primers specific for 16S-23S spacer region were used to identify the bacteria as members of the B. cereus group. Several primers towards insecticidal genes have been used in order to further characterize the isolates as subspecies of B. thuringiensis. RESULTS: Two faecal samples from the B. thuringiensis-exposed greenhouse workers were positive for B. cereus-like bacteria. One isolate displayed intracellular crystalline inclusions characteristic of B. thuringiensis, production of and genes for B. cereus enterotoxins and it was PCR-positive for an insecticidal toxin primer set. RAPD profiles of the faecal isolate were identical to that of strains isolated from a commercial product. CONCLUSIONS: The methods applied have verified that the faecal isolate was identical to the B. thuringiensis isolate found in the biopesticide used. This is the first reported case of isolation of a bacterial biopesticide from human faeces. The biopesticide was shown to harbour and express enterotoxin genes. However, there is no evidence that this caused any adverse effects to the person from whom these bacteria were isolated.

Bacillus cereus↗
PubMed1999

Functional insights into pGI2, a cryptic rolling-circle replicating plasmid from Bacillus thuringiensis.

Detailed functional analysis revealed the modular organization of pGI2, a 9672 bp plasmid from Bacillus thuringiensis H1.1 that harbours the 4149 bp transposon Tn4430. Whereas the pGI2 leading-strand replicon was identified through deletion experiments, sequence comparisons indicated the presence of an ssot-like single-strand origin commonly found among Bacillus plasmids. Southern hybridization confirmed the existence of ssDNA intermediates, but only in the case of plasmid derivatives lacking the ssot site. Moreover, the pGI2 replication protein Rep displayed significant similarity with that of pTX14-3, a 7-6 kb plasmid from B. thuringiensis serovar israelensis, suggesting that both elements are representatives of a new family of rolling-circle replicating (RCR) plasmids. In addition, both plasmids share a conserved 320 bp region downstream of their rep genes which, in the case of pGI2, appeared indispensable for replication. This region is therefore likely to correspond to, or to be part of, the actual double-strand origin of both plasmids. Another interesting feature of pGI2 is the presence of a mobilization (Mob) protein, as demonstrated by its ability to be mobilized by the conjugative plasmid pAW63 from B. thuringiensis serovar kurstaki HD73. The same transfer system was also used to unambiguously demonstrate similar properties of the related Mob-like protein from pTX14-3. A closer analysis of this family of related Mob proteins suggested a subdomanial organization among its members. Finally, the 270 residue pGI2 ORF2 was shown to be related to ORF43 of pMRC01, a 60 kb conjugative plasmid from Lactococcus lactis subsp. lactis. Although no function has been assigned to the putative ORF43 protein, it is located downstream of a bacteriocin operon, next to an IS946 element. pGI2 appears thus far as an assemblage of functional modules with no obvious metabolic function, presumably acting as a reservoir of carrier (rep and sso), rearrangement (Tn4430) or recruiting (Mob) entities for its bacterial host.

Amino Acid Sequence↗
PubMed1998

Characterization of plasmid pAW63, a second self-transmissible plasmid in Bacillus thuringiensis subsp. kurstaki HD73.

Bacillus thuringiensis subspecies kurstaki HD73, toxic for lepidopteran larvae, contains two large self-transmissible plasmids of approximately 75 kb, pHT73 and pAW63. The conjugative plasmid pHT73 has been studied extensively and has been shown to harbour the toxin gene cry1Ac, the transposon Tn4430 and several insertion sequences. In this study it was demonstrated that the minor plasmid pAW63 is also self-transmissible and about 10-30 times more efficient in mobilizing plasmid pBC16. To facilitate direct selection for pAW63 transfer, the plasmid was tagged with the tetracycline resistance transposon Tn5401 and in intraspecies matings it was found that after 2 h, all recipients had acquired a copy of the plasmid. Mating experiments demonstrated that pAW63 could be transferred to Bacillus thuringiensis subsp. israelensis, Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus sphaericus, and that the conjugative functions were expressed in these hosts. Hybridization studies showed that the replicons of pAW63 and pHT73 were distinct from one another. Sequences homologous to transposon Tn4430 and several insertion sequences were, however, shown to reside on both plasmids.

Bacillus↗