Characterization of strain-specific Bacillus cereus swimming motility and flagella by means of specific antibodies.

One of the multiple factors determining the onset of the diarrhoeal disease caused by enteropathogenic Bacillus cereus is the ability of the bacteria to actively move towards the site of infection. This ability depends on flagella, but it also varies widely between different strains. To gain more insights into these strain-specific variations, polyclonal rabbit antisera as well as monoclonal antibodies (mAbs) were generated in this study, which detected recombinant and natural B. cereus flagellin proteins in Western blots as well as in enzyme immunoassays (EIAs). Based on mAb 1A11 and HRP-labelled rabbit serum, a highly specific sandwich EIA was developed. Overall, it could be shown that strain-specific swimming motility correlates with the presence of flagella/flagellin titres obtained in EIAs. Interestingly, mAb 1A11, recognizing an epitope in the N-terminal region of the flagellin protein, proved to inhibit bacterial swimming motility, while the rabbit serum rather decreased growth of selected B. cereus strains. Altogether, powerful tools enabling the in-depth characterization of the strain-specific variations in B. cereus swimming motility were developed.

Enteropathogenic Potential of Bacillus thuringiensis Isolates from Soil, Animals, Food and Biopesticides.

Despite its benefits as biological insecticide, Bacillus thuringiensis bears enterotoxins, which can be responsible for a diarrhoeal type of food poisoning. Thus, all 24 isolates from foodstuffs, animals, soil and commercially used biopesticides tested in this study showed the genetic prerequisites necessary to provoke the disease. Moreover, though highly strain-specific, various isolates were able to germinate and also to actively move, which are further requirements for the onset of the disease. Most importantly, all isolates could grow under simulated intestinal conditions and produce significant amounts of enterotoxins. Cytotoxicity assays classified 14 isolates as highly, eight as medium and only two as low toxic. Additionally, growth inhibition by essential oils (EOs) was investigated as preventive measure against putatively enteropathogenic B. thuringiensis. Cinnamon Chinese cassia showed the highest antimicrobial activity, followed by citral, oregano and winter savory. In all tests, high strain-specific variations appeared and must be taken into account when evaluating the hazardous potential of B. thuringiensis and using EOs as antimicrobials. Altogether, the present study shows a non-negligible pathogenic potential of B. thuringiensis, independently from the origin of isolation. Generally, biopesticide strains were indistinguishable from other isolates. Thus, the use of these pesticides might indeed increase the risk for consumers' health. Until complete information about the safety of the applied strains and formulations is available, consumers or manufacturers might benefit from the antimicrobial activity of EOs to reduce the level of contamination.

Assessing the toxic potential of enteropathogenic Bacillus cereus.

The diarrheal type of food poisoning caused by enteropathogenic Bacillus cereus has been linked to various exotoxins. Best described are the non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), and cytotoxin K (CytK). Due to the ubiquitous prevalence of B. cereus in soil and crops and its ability to form highly resistant endospores, contaminations during food production and processing cannot be completely avoided. Although phylogenetically closely related, enteropathogenic B. cereus strains show a high versatility of their toxic potential. Thus, functional tools for evaluating the pathogenic potential are urgently needed in order to predict hazardous food contaminations. As the diarrheal syndrome is the result of a toxico-infection with enterotoxin production in the intestine, the entire passage of the bacteria within the host, from spore survival in the stomach, spore germination, host cell adherence, and motility, to enterotoxin production under simulated intestinal conditions was compared in a panel of 20 strains, including high pathogenic as well as apathogenic ones. This approach resulted in an overarching virulence analysis scheme. In parallel, we searched for potential toxico-specific secreted markers to discriminate low and high pathogenic strains. To this end, we targeted known exotoxins using an easy to implement immunoblotting approach as well as a caseinolytic exoprotease activity assay. Overall, Nhe component B, sphingomyelinase, and exoproteases showed good correlation with the complex virulence analysis scheme and can serve as a template for future fast and easy risk assessment tools to be implemented in routine diagnostic procedures and HACCP studies.

Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus.

A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related.

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

Clarification and 3-D visualization of immunolabeled human placenta villi.

We report here the successful 3D visualization of human placenta villous structures on the order of ∼1 mm3 by a combination of immunolabeling, rapid tissue clarification and laser scanning confocal microscopy. The resultant image sets exhibit a complex arrangement of villi and their contained vasculature that mirrors their arrangement in situ.