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Little is known about secretion of outer membrane vesicles (OMVs) by  Cronobacter . In this study, OMVs isolated from  Cronobacter sakazakii, Cronobacter turicensis , and  Cronobacter malonaticus  were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven  Cronobacter  species while a few genes (OMATP gene,  groEL, ompC, mipA, ctp , and  ompX ) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of  Cronobacter , and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport.

Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.