Diagnostic Real-Time PCR Assays for the Detection of Emetic Bacillus cereus Strains in Foods and Recent Food-Borne Outbreaks

Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5′ nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.     

Submarine foraging behavior of alcids in an artificial environment

We used an artificial environment at Sea World, Inc, San Diego, to study underwater foraging behavior of alcids. Larger birds dove longer and had greater wingbeat frequencies. The pigeon guillemot Cepphus columba was the only species to use both feet and wings for propulsion; all others used just wings. Aggressive interactions underwater were common. Competition among alcids in the wild may occur primarily underwater, and artificial environments may be the best means to study such interactions.     

Cg2091 encodes a polyphosphate/ATP-dependent glucokinase of Corynebacterium glutamicum

The Corynebacterium glutamicum gene cg2091 is encoding a polyphosphate (PolyP)/ATP-dependent glucokinase (PPGK). Previous work demonstrated the association of PPGK to PolyP granules. The deduced amino acid sequence of PPGK shows 45% sequence identity to PolyP/ATP glucomannokinase of Arthrobacter sp. strain KM and 50% sequence identity to PolyP glucokinase of Mycobacterium tuberculosis H37Rv. PPGK from C. glutamicum was purified from recombinant Escherichia coli. PolyP was highly preferred over ATP and other NTPs as substrate and with respect to the tested PolyPs differing in chain length; the protein was most active with PolyP₇₅. Gel filtration analysis revealed that PolyP supported the formation of homodimers of PPGK and that PPGK was active as a homodimer. A ppgK deletion mutant (ΔppgK) showed slowed growth in minimal medium with maltose as sole carbon source. Moreover, in minimal medium containing 2 to 4% (w/v) glucose as carbon source, ΔppgK grew to lower final biomass concentrations than the wild type. Under phosphate starvation conditions, growth of ΔppgK was reduced, and growth of a ppgK overexpressing strain was increased as compared to wild type and empty vector control, respectively. Thus, under conditions of glucose excess, the presence of PPGK entailed a growth advantage.     

A Pediocin-Producing Lactobacillus plantarum Strain Inhibits Listeria monocytogenes in a Multispecies Cheese Surface Microbial Ripening Consortium

The growth of Listeria monocytogenes WSLC 1364, originating from a cheese-borne outbreak, was examined in the presence and in the absence of a pediocin AcH-producing Lactobacillus plantarum strain on red smear cheese. Nearly complete inhibition was observed at 10² CFU of L. monocytogenes per ml of salt brine solution, while contamination with Listeria mutants resistant to pediocin resulted in high cell counts of the pathogen on the cheese surface. The inhibition was due to pediocin AcH added together with the L. plantarum culture to the brine solution but not to bacteriocin production in situ on cheese. Pediocin resistance developed in vitro at different but high frequencies in all 12 L. monocytogenes strains investigated, and a resistant mutant remained stable in a microbial surface ripening consortium over a 4-month production process in the absence of selection pressure. In conclusion, the addition of a L. plantarum culture is a potent measure for combating Listeria in a contaminated production line, but because of the potential development of resistance, it should not be used continuously over a long time in a production line.     

Trabecular bone strains around a dental implant and associated micromotions—A micro-CT-based three-dimensional finite element study

The first objective of this computational study was to assess the strain magnitude and distribution within the three-dimensional (3D) trabecular bone structure around an osseointegrated dental implant loaded axially. The second objective was to investigate the relative micromotions between the implant and the surrounding bone. The work hypothesis adopted was that these virtual measurements would be a useful indicator of bone adaptation (resorption, homeostasis, formation).In order to reach these objectives, a μCT-based finite element model of an oral implant implanted into a Berkshire pig mandible was developed along with a robust software methodology. The finite element mesh of the 3D trabecular bone architecture was generated from the segmentation of μCT scans. The implant was meshed independently from its CAD file obtained from the manufacturer. The meshes of the implant and the bone sample were registered together in an integrated software environment. A series of non-linear contact finite element (FE) analyses considering an axial load applied to the top of the implant in combination with three sets of mechanical properties for the trabecular bone tissue was devised. Complex strain distribution patterns are reported and discussed. It was found that considering the Young’s modulus of the trabecular bone tissue to be 5, 10 and 15 GPa resulted in maximum peri-implant bone microstrains of about 3000, 2100 and 1400. These results indicate that, for the three sets of mechanical properties considered, the magnitude of maximum strain lies within an homeostatic range known to be sufficient to maintain/form bone. The corresponding micro-motions of the implant with respect to the bone microstructure were shown to be sufficiently low to prevent fibrous tissue formation and to favour long-term osseointegration.     

Effect of Cry1F maize on the behavior of susceptible and resistant Spodoptera frugiperda and Ostrinia nubilalis

Understanding the behavior of pests targeted with Bacillus thuringiensis Berliner (Bt) crops is important to define resistance management strategies. Particularly the study of larval movement between plants is important to determine the feasibility of refuge configurations. Exposure to Bt maize, Zea mays L. (Poaceae), has been suggested to increase larval movement in lepidopteran species but few studies have examined the potential for resistance to interact with behavioral responses to Bt toxins. Choice and no‐choice experiments were conducted with Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) and Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) to determine whether Cry1F resistance influences neonate movement. Leaf discs of Cry1F maize and the corresponding isoline were used to characterize behavioral responses. In both experiments, the location (on or off of plant tissues) and mortality of susceptible and Cry1F resistant neonates was recorded for 5 days, but the analysis of larvae location was performed until 7 h. Our results indicated no strong difference between resistant and susceptible phenotypes in S. frugiperda and O. nubilalis, although a small percentage of susceptible neonates in both species abandoned maize tissue expressing Cry1F. However, significant behavioral differences were observed between species. Ostrinia nubilalis exhibited increased movement between leaf discs, whereas S. frugiperda selected plant tissue within the first 30 min and remained on the chosen plant regardless of the presence of Cry1F. Spodoptera frugiperda reduced larval movement may have implications to refuge configuration. This study represents the first step toward understanding the effects of Cry1F resistance on Lepidoptera larval behavior. Information regarding behavioral differences between species could aid in developing better and more flexible resistance management strategies.     

Effect of Circulating Forms of Soy Isoflavones on the Oxidation of Low Density Lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.     

Hepatocyte growth factor and other fibroblast secretions modulate the phenotype of human bronchial epithelial cells

The luminal airway surface is lined with epithelial cells that provide a protective barrier from the external environment and clear inhaled pathogens from the lung. To accomplish this important function, human bronchial epithelial (HBE) cells must be able to rapidly regenerate a mucociliary layer of cells following epithelial injury. Whereas epithelial-fibroblast interactions are known to modulate the airway architecture during lung development and repair, little is known about how these two cells interact. Using a primary HBE and lung fibroblast coculture system, we demonstrate that 1) subepithelial fibroblasts provide a suitable environment for differentiation of HBE cells into a polarized ciliated phenotype despite being cultured in media that induces terminal squamous differentiation and growth arrest in the absence of fibroblasts, 2) HBE cells cocultured with subepithelial fibroblasts exhibit augmented ciliogenesis, accelerated wound repair, and diminished polarized ion transport compared with cells grown in control conditions, and 3) hepatocyte growth factor (HGF) is important for subepithelial fibroblast modulation of HBE cell differentiation. These results provide a model to study fibroblast modulation of epithelial phenotype and indicate that HGF secreted by subepithelial fibroblasts contributes to HBE cell differentiation.     

Does secondary chemistry enable lichens to grow on iron-rich substrates?

Lichen substances are shown to increase or to inhibit the adsorption of Fe at cation exchange sites. The influence on the adsorption strongly differs between individual lichen substances and is different for Fe²⁺ and Fe³⁺. These results add a new biological role to the known functions of lichen secondary metabolites. In an experiment with cellulose filters, which were soaked with acetone solutions of lichen substances and were then incubated with micromolar solutions of FeCl₂ or FeCl₃, many lichen substances were found to increase Fe³⁺ adsorption, whereas others had no effect. Most lichen substances had no effect on Fe²⁺ adsorption, but two were found to reduce and one to increase the level of adsorption. Lichens of Fe-poor and -rich sites contain lichen substances with different adsorption behavior towards Fe²⁺ and Fe³⁺. All the studied lichen substances, which only occur in lichens of Fe-poor sites, turned out to be effective Fe³⁺ adsorbents. Lichens of Fe-bearing rock and slag, however, were found to lack lichen substances, or to contain substances that did not adsorb Fe³⁺ and had no effect on Fe²⁺ adsorption, or thirdly, to contain substances that increased Fe³⁺ adsorption, but decreased Fe²⁺ adsorption. These results suggest that lichen substances do play a significant role in Fe adsorption in lichens and determine their tolerance to excess concentrations of Fe. Notwithstanding the strong correlation between the secondary chemistry of lichen species and their preference for Fe-rich or Fe-poor substrates, the postulated mechanism of temporary Fe adsorption by lichen substances has to be subject of future biochemical research.