Guinea fowl eggshell structural analysis at different scales reveals how organic matrix induces microstructural shifts that enhance its mechanical properties.

Guinea fowl eggshells have an unusual structural arrangement that is different from that of most birds, consisting of two distinct layers with different microstructures. This bilayered organization, and distinct microstructural characteristics, provides it with exceptional mechanical properties. The inner layer, constituting about one third of the eggshell thickness, contains columnar calcite crystal units arranged vertically as in most bird shells. However, the thicker outer layer has a more complex microstructural arrangement formed by a switch to smaller calcite domains with diffuse/interlocking boundaries, partly resembling the interfaces seen in mollusk shell nacre. The switching process that leads to this remarkable second-layer microstructure is unknown. Our results indicate that the microstructural switching is triggered by changes in the inter- and intracrystalline organic matrix. During production of the outer microcrystalline layer in the later stages of eggshell formation, the interactions of organic matter with mineral induce an accumulation of defects that increase crystal mosaicity, instill anisotropic lattice distortions in the calcite structure, interrupt epitaxial growth, reduce crystallite size, and induce nucleation events which increase crystal misorientation. These structural changes, together with the transition between the layers and each layer having different microstructures, enhance the overall mechanical strength of the Guinea fowl eggshell. Additionally, our findings provide new insights into how biogenic calcite growth may be regulated to impart unique functional properties. STATEMENT OF SIGNIFICANCE: Avian eggshells are mineralized to protect the embryo and to provide calcium for embryonic chick skeletal development. Their thickness, structure and mechanical properties have evolved to resist external forces throughout brooding, yet ultimately allow them to crack open during chick hatching. One particular eggshell, that of the Guinea fowl, has structural features very different from other galliform birds - it is bilayered, with an inner columnar mineral structure (like in most birds), but it also has an outer layer with a complex microstructure which contributes to its superior mechanical properties. This work provides novel and new fundamental information about the processes and mechanisms that control and change crystal growth during the switch to microcrystalline domains when the second outer layer forms.

Purification, characterization, and lytic activity against Naegleria fowleri of two amoebicins produced by Bacillus licheniformis A12.

Bacillus licheniformis A12 produces two amoebolytic substances (amoebicins A12-A and A12-B) in liquid media during sporulation. Both substances have been purified and characterized. They are heat- and protease-resistant peptides containing aspartic acid, glutamic acid, serine, proline, and tyrosine in a molar ratio of 5:2:2:2:2. No fatty acids or carbohydrates have been detected. Their molecular weight is 1,430. Purified amoebicins A12-A and A12-B exhibit amoebolytic action against Naegleria fowleri. They also exhibit antibiotic action against yeasts (Saccharomyces heterogenicus and Cryptococcus neoformans) and several fungal species (Aspergillus niger, Microsporum canis, Mucor plumbeus, and Trychophyton mentagrophytes). Their antibacterial spectrum appears to be restricted to Bacillus megaterium, Corynebacterium glutamicum, and Sarcina sp.

Antagonistic action of the bacterium Bacillus licheniformis M-4 toward the amoeba Naegleria fowleri.

Free-living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis). In a broad bacterial screening from soil and water we have isolated three strains (M-4, D-13 and A-12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp. and also against different Gram-positive and Gram-negative bacteria. Physical-chemical characteristics, partial purification and biological activities of a substance produced by the M-4 strain have been investigated. This substance (m-4) is stable at high temperature (up to 100 degrees C) and extremes of pH (2.5-9.5) and also at -20 degrees C for months. Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium. Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m-4.