Medical-grade honey enriched with antimicrobial peptides has enhanced activity against antibiotic-resistant pathogens.

Honey has potent activity against both antibiotic-sensitive and -resistant bacteria, and is an interesting agent for topical antimicrobial application to wounds. As honey is diluted by wound exudate, rapid bactericidal activity up to high dilution is a prerequisite for its successful application. We investigated the kinetics of the killing of antibiotic-resistant bacteria by RS honey, the source for the production of Revamil® medical-grade honey, and we aimed to enhance the rapid bactericidal activity of RS honey by enrichment with its endogenous compounds or the addition of antimicrobial peptides (AMPs). RS honey killed antibiotic-resistant isolates of Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus faecium, and Burkholderia cepacia within 2 h, but lacked such rapid activity against methicillin-resistant S. aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. It was not feasible to enhance the rapid activity of RS honey by enrichment with endogenous compounds, but RS honey enriched with 75 µM of the synthetic peptide Bactericidal Peptide 2 (BP2) showed rapid bactericidal activity against all species tested, including MRSA and ESBL E. coli, at up to 10-20-fold dilution. RS honey enriched with BP2 rapidly killed all bacteria tested and had a broader spectrum of bactericidal activity than either BP2 or honey alone.

Nucleotide sequence of the chicken HMGI-C cDNA and expression of the HMGI-C and IGF1 genes in autosomal dwarf chicken embryos.

Mutations in the genes for high mobility group protein I-C (HMGI-C) and insulin-like growth factor 1 (IGF1) are known to be responsible for dwarf phenotypes in the mouse. Because the locus for autosomal dwarfism (adw) in the chicken maps to a region which is syntenic to a region in the human and mouse in which the HMGI-C and IGF1 genes are located, HMGI-C and IGF1 are likely candidate genes for adw in the chicken. In this study their possible role in the establishment of this phenotype has been investigated. We have cloned and sequenced the complete coding region of the chicken HMGI-C cDNA. Comparison with its human counterpart revealed a nucleotide sequence conservation of 84%. Only nine amino acids are present principally in the N-terminal segment before the first DNA-binding domain. Northern blot analysis showed no difference in the expression of the HMGI-C gene between adw and wild-type chicken embryos. Also no mutations in either the HMGI-C or the IGF1 RNA nucleotide sequence were detected in adw chicken embryos.

Bulked segregant analysis using microsatellites: mapping of the dominant white locus in the chicken.

In order to perform a linkage study, the genotypes of a large number of individuals from a segregating population need to be determined. In case the phenotype to be mapped is influenced by a single locus or a major gene, sampling of the DNA from individual animals with the same phenotype into a single pool (bulked segregant) can reduce the number of typings. In this study we used bulked segregant analysis in order to map the Dominant White locus in the chicken. In a pilot experiment, we showed that allele frequencies can be accurately estimated from pooled samples using fluorescently labeled microsatellite markers. A segregating population for the Dominant White locus was obtained by performing a cross between a white male chicken (Genotype li for Dominant White) and a black female chicken (ii). The resulting progeny of 21 white and 18 black chickens were divided in two pools. Genotypes for both the parents and the pools were determined using 168 fluorescently labeled microsatellite markers, of which 68 were informative. The relative allele frequencies between the pools were estimated for these 68 informative markers. One marker (MCW188) was found to segregate with the Dominant White locus. Subsequent typing of all individuals from this cross and an additional 148 animals from five different families showed only two recombinants between the marker and the Dominant White locus, resulting in a LODlinkage score (log10 of odds) of 36. Using the pooled DNA approach, the Dominant White locus was successfully mapped on linkage group 22 of the East Lansing reference family at a distance of 2 cM from MCW188.

Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees.

Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii x N. sanderae were transformed with the gene for hEGF, equipped with a nectary-targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml⁻¹. There is a significant linear relationship (P<0.001) between hEGF concentration and induction of hEGF-receptor phosphorylation. Since the flower morphology of these plants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF-receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.

Development and mapping of polymorphic microsatellite markers derived from a chicken brain cDNA library.

Until now the genetic linkage map in chicken has ben based mainly on random genomic markers. The addition of expressed sequence tags (ESTs) to the genetic linkage maps is becoming more important because ESTs can form the basis for comparative mapping studies. This may be helpful for the detection of candidate genes for quantitative trait loci (QTLs). In our study we used a (TG)13 repeat as probe for the detection of microsatellites in a chicken brain cDNA library. After hybridization 0.15% of the cDNA clones gave a positive signal. The cDNA complexity of the library was high; of the 90 cDNA clones that were sequenced 60 occurred only once. For 29 clones primer sets for the polymerase chain reaction could be developed. Twenty-one microsatellites were polymorphic on one or more of the test panels and 15 markers could be mapped on either or both of the international reference families. Because sequence homology between chicken and mammalian cDNAs is sometimes low it was difficult to assess the level of sequence homology that indicated a true homologous transcript. In our study seven cDNA cones, of which three could be mapped, showed a relatively high percentage of sequence homology with sequences found in other species. Because sequencing and mapping of expressed sequence tags in human and mouse is progressing very rapidly, it is predicted that further information will soon be readily available. Therefore, increasing the number of expressed sequences on the chicken genetic linkage map will be of value for comparative mapping studies in the near future.

The HMGI-C gene is a likely candidate for the autosomal dwarf locus in the chicken.

In order to map the autosomal dwarf (adw) locus in the chicken, 11 segregating families were created. Initially five of these families were used for a linkage experiment in which the genome was scanned with microsatellites using a technique called bulked segregant analysis. Subsequently animals from 11 families were typed individually for microsatellites that appeared to be linked. We were able to detect genetic linkage of the adw locus to five different microsatellite markers on chromosome 1, the closest showing a recombination fraction of only 0.03 (LOD score 32.12). In mice the phenotype pygmy shows a striking similarity to the autosomal dwarf phenotype in chickens, both having a disproportionately large head. The pygmy locus has been mapped on mouse chromosome 10 and found to represent a mutation in the gene coding for high-mobility group protein I-C (HMGI-C). Considering the synteny between regions of chicken chromosome 1, mouse chromosome 10 and human chromosome 12, and taking into account both the phenotypic characteristics and the mode of inheritance of the chicken adw and the mouse pygmy loci, the HMGI-C gene is a major candidate gene for the adw locus in the chicken. Fluorescence in situ hybridization of metaphase chromosomes with the chicken HMGI-C gene as a probe, showed that the chicken HMGI-C gene is indeed closely linked to marker LEI146 on chromosome 1.

Developing microsatellite markers from cDNA: a tool for adding expressed sequence tags to the genetic linkage map of the chicken.

A chicken embryonic cDNA library was screened with a (TG)13 probe in order to develop polymorphic microsatellite markers. The redundancy of the embryonic cDNA library with a chicken brain cDNA library, which was used for microsatellite development in a previous study, was extremely high. Of the 300 (TG)13 positive clones, only 80 were unique for the embryonic cDNA library. Still, nine expressed sequences derived from the embryonic cDNA library were mapped in the Wageningen (WAU) resource population. In addition seven microsatellite markers from the chicken brain cDNA library, which were monomorphic or unlinked in the two international reference families in the previous study, were also mapped in the WAU population. Three of the 16 mapped chicken expressed sequence tags (ESTs) showed relatively high percentages of sequence similarity to sequences found in other species. As two of these genes, RAB6 and ZFX/ZFY, have been mapped in humans, they contribute to the comparative map of the chicken.