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Inhibitory effect of incubation on microbial growth has extensively been studied in wild bird populations using culture-based methods and conflicting results exist on whether incubation selectively affects the growth of microbes on the egg surface. In this study, we employed culture-independent methods, quantitative PCR and 16S rRNA gene pyrosequencing, to elucidate the effect of incubation on the bacterial abundance and bacterial community composition on the eggshells of the Eurasian Magpie ( Pica pica ). We found that total bacterial abundance increased and diversity decreased on incubated eggs while there were no changes on non-incubated eggs. Interestingly, Gram-positive  Bacillus , which include mostly harmless species, became dominant and genus  Pseudomonas , which include opportunistic avian egg pathogens, were significantly reduced after incubation. These results suggest that avian incubation in temperate regions may promote the growth of harmless (or benevolent) bacteria and suppress the growth of pathogenic bacterial taxa and consequently reduce the diversity of microbes on the egg surface. We hypothesize that this may occur due to difference in sensitivity to dehydration on the egg surface among microbes, combined with the introduction of  Bacillus  from bird feathers and due to the presence of antibiotics that certain bacteria produce.

Effect of Incubation on Bacterial Communities of Eggshells in a Temperate Bird, the Eurasian Magpie (Pica pica)