ABSTRACTThe anaerobic sporeformerClostridium difficileis the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent morphotype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact ofC. difficilespores with the host and for spore persistence during recurrent episodes of infection. Although some studies on the biology of the exosporium have been conducted (J. Barra-Carrasco et al., J Bacteriol 195:3863–3875, 2013,http://dx.doi.org/10.1128/JB.00369-13; J. Phetcharaburanin et al., Mol Microbiol 92:1025–1038, 2014,http://dx.doi.org/10.1111/mmi.12611), there is a lack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electron micrographs, we analyzed the variability of the spore's outermost layers in various strains and found distinctive variability in the ultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, we observed that although this layer was stable during spore purification, it was partially lost after 6 months of storage at room temperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease in the levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that the presence of the exosporium marker CdeC on spores obtained fromC. difficilebiofilms depended on the biofilm culture conditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of the exosporium surface ofC. difficilespores. These findings have direct implications and should be considered in the development of novel methods to diagnose and/or removeC. difficilespores by using exosporium proteins as targets.
Complete Genome Sequences and Transmission Electron Micrographs of Listeria Phages of the Genus Homburgvirus
