Here, we made use of molecular tools to describe egg-related microbial communities. We first examined bacterial communities using two sampling techniques, egg swabs and crushed eggshells. Using the most adequate methodology (crushed egg shells), we compared the structure, abundance, and composition of both bacterial and fungal communities on early and late incubated eggs.
Egg swabs versus eggshell pieces techniques to assess microbial communities in eggshells
The structure of bacterial communities based on eggshell pieces differed from those found using swabs, supporting similar findings from poultry sciences. In chicken eggs, it has been shown that depending on the method used to recover bacteria associated with the eggshell, different subsets of bacterial communities can be observed (Musgrove et al. 2005; Kawasaki et al. 2008; Chousalkar and Roberts 2012). The natural porosity of eggshells is likely the reason for these differences. For instance, aerobic bacteria recovered from “the crush method” exceeded the number of cells obtained using a “shell rinse method” (Kawasaki et al. 2008). In an experiment where eggshell surfaces were inoculated with Salmonella cells, the number of cells obtained from crushed eggshells previously washed was about half the number of cells recovered from their own egg rinsates (Kawasaki et al. 2008). In line with these studies, we detected more than five times the amount of microbial DNA when using the whole eggshell, enabling a proper quantification of the DNA concentration in each sample through fluorescence. The DNA concentration was sufficiently large to perform a range of molecular analyses to determine abundance and diversity of bacterial and fungal communities associated with eggshell in the same sample.
Importantly, our results showed that different extraction methods uncover different bacterial communities, as the overall bacterial structure based on eggshell swabs exhibited low levels of similarity when compared to the eggshell pieces coming from the same egg. Moreover, when comparing different eggs, bacterial communities obtained by the swab method shared higher similarity than did those obtained by crush method (eggshell pieces). In addition, for comparative purposes, we had to set the template DNA used for PCR-DGGE at the same concentration for both methods. Because the DNA concentration obtained with the swab method was low and suboptimal when compared to other studies dealing with environmental samples (Pereira e Silva et al. 2012), this limited our bacterial analyses for comparison. The use of the eggshell pieces allowed us to overcome the limitations of low-template concentration and therefore provided a more complete description of community structure and diversity of microorganisms associated with the eggshells.
Despite the advantages that using eggshell pieces provide for the molecular analyses of microbial communities, egg destruction might not be desirable in every study. In particular, the use of eggshell swabs may remain unavoidable in studies dealing with embryo survival (Martín-Gálvez et al. 2010). Our results raise awareness that swabbing does not capture the entire microbiome, and therefore, the findings obtained based on this method should be interpreted keeping this limitation in mind. For studies that focus on egg contents (and require egg destruction), we advocate that working with eggshells is a better alternative.
Shifts in microbial communities through incubation
It has been hypothesized that incubation reduces the water on the egg surface to prevent bacterial growth (Cook et al. 2003, 2005a,b; D'Alba et al. 2010; but see Wang et al. 2011), when compared to exposed (unincubated) eggs. This bacteriostatic effect of incubation has been attributed as an important mechanism controlling the hatching success (Cook et al. 2003, 2005a; Beissinger et al. 2005). It is less clear, however, if microbial communities change during incubation, and if this can be correlated with embryo survival. Even though the presence of bacteria is often seen as a potential risk for embryo development, due to the presence of pathogenic species, it is important to realize that bacterial communities are highly diverse and that only a small subset of the total comprise pathogens. Additionally, the concept of pathogenicity cannot be generalized to a certain genus or species, as it depends on an intricate interaction between bacterial strain, host species, and host health. Alternatively, the microbial communities associated with eggshells can be seen as a protective barrier against pathogenic species. It has been hypothesized that incubation should prevent the growth of pathogenic strains while enhancing the growth of beneficial ones (Cook et al. 2005b; Shawkey et al. 2009).
Considering the bacterial communities, our results showed an overall decrease in diversity during incubation, whereas a few bacterial OTUs that preferentially grew to the detriment of others, remained after 3 weeks of incubation. The higher number of specific bacterial OTUs observed at late incubation might explain the overall increase in bacterial abundance. One possible explanation is that bacteria that are selected during incubation probably grow in the empty places left by dead cells as is the case in bacterial soil invasion (Eisenhauer et al. 2013). Our results contrast with the only molecular study addressing this issue, on Pearly-eyed Thrashers (Shawkey et al. 2009), where bacterial abundance and structure did not change during incubation. However, this disparity could be explained by methodological issues. First, Shawkey et al. (2009) used the swab method for DNA extraction, which does not comprise the bacterial communities found inside the eggshell pores. Second, Shawkey et al. (2009) quantified bacterial abundance based on the overall DNA concentration obtained per sample, which includes other sources of DNA (fungal, animal) in addition to that of bacterial origin. In our case, we used more specific methods, such as qPCR, which is commonly used to quantify microbial DNA in a range of environmental samples (Bach et al. 2002; Pereira e Silva et al. 2012). Third, the contrasting results might be a reflection of the environment where the samples were collected and bird species. Moreover, our study was performed on captive birds in semi-natural conditions, on unfertilized eggs, and may thus not be entirely representative of what happens in the wild. Further molecular studies are needed to unravel how bacterial communities vary through the incubation period.
Here, we reported that late incubation favoured mostly species belonging to the Gammaproteobacteria class, which comprised 57% of the total number of OTUs found, whereas this bacterial class accounted for 22% on early incubated eggs. These changes were mostly due to the presence of OTUs affiliated to Salmonella enterica (range: 98–99% similarity, when the closest hit was assigned at the genus level, Appendix A5), which belongs to the family Enterobacteriaceae. Culture-dependent and molecular methods have detected this bacterial family at both early and late incubation stages (Shawkey et al. 2009; Ruiz-de-Castañeda et al. 2011b), indicating that this family is an important component of the egg microbiome. Based on molecular methods, Shawkey et al. (2009) have shown that the number of bacterial taxa belonging to the Enterobacteriaceae family tended to increase from early to late incubation, even though a significant increase was observed only when comparing unincubated and incubated eggs (Shawkey et al. 2009). We also observed a twofold increase in Staphylococcus-like OTUs throughout incubation (order Bacillales). Thus, although incubation might limit the growth of potential pathogenic bacterial species, our study shows that some of these species tend to increase from early to late incubation. However, their actual role remains unclear due to the lack of information on their pathogenicity. Testing for bacterial pathogenicity would require in vivo experiments (when the specific isolates are available) or the use of more specific molecular tools targeting type III and/or IV secretion systems (see Deane et al. 2010) for instance. To our knowledge, only one study established a real effect of bacteria on bird fitness: Soler et al. (2012) have shown that Enterococcus and Enterobacteriaceae on eggshell were negatively associated with hatching success. Moreover, while pathogenicity is often argued when Enterococcaceae, Enterobacteriaceae, or Staphylococcaceae are described, none of the studies discussed their potential beneficial or commensal role. For instance, in humans, some of these family-related species were often found in the gut without being consistently harmful (i.e., Enterococci described in Byappanahalli et al. 2012). Considering that pathogenicity is most likely strain- and host-dependent, additional in-depth studies are certainly required for more conclusive explanations.
Fungi are undoubtedly part of the microbiome associated with the egg environment. They have been described in nest materials and might colonize eggshells (Baggott and Graeme-Cook 2002). It has been postulated that fungi might be able to break down the cuticle to facilitate bacterial trans-shell penetration, by increasing the number of pores accessible (Board et al. 1964, 1979; Board and Halls 1973). The probability of bacterial infection of egg contents was shown to be positively associated with fungal growth on eggshells (Cook et al. 2003). Moreover, fungi have been described in poultry industries (see Szablewski et al. 2010; Nowaczewski et al. 2011). Nevertheless, comparing our results with previous studies proved to be challenging because of the lack of data on fungi associated with eggshells. So far, they have been detected in egg contents after exposure to their natural environmental conditions (Cook et al. 2003, 2005a). Using cultivation methods, Godard et al. (2007) showed that eggshells were free from fungi most of the time at the laying day, but their number increased over time on exposed eggs, possibly due to the water on the egg surface. Conversely, using molecular tools, we showed that fungi are a constant constituent of the egg microbiome at early incubation, although their importance reduced during incubation, as only about half of the eggshells still harbored fungal DNA at the late incubation period. This reduction could be due to the humidity control of the egg caused by incubation behavior, because fungi abundance can be correlated with the water content on the eggshell (Godard et al. 2007). Additionally, we observed a strong decrease in their diversity, indicating that only a few selected species were able to cope with the lower level of humidity. However, as we did not measure water content on the eggshell, we cannot discriminate between resistance to low humidity or differences in humidity between eggs.
Interestingly, some of the fungi identified in our study have been reported in the avian gut microbiota, and their prevalence may depend on the avian species (Cafarchia et al. 2006). This includes Cryptococcus laurentii, Cryptococcus uniguttulatus, Debaryomyces hansenii, and Rhodotorula rubra, which have been identified in feral pigeon cloaca (Littman and Walter 1967; Mattsson et al. 1999). Up to fourteen fungal species have been also described in the lower intestinal tract of these birds (Ramirez et al. 1976). The presence of fungi on the eggshells of early incubated eggs might be explained under the hypothesis of vertical transmission. Additionally, some fungi may grow through incubation while others die out. The presence of new OTUs after incubation could be explained by other sources as fungi have been for instance found in feathers of pigeons (Deshmukh 2004) and other avian species (Mandeel et al. 2011) and often mentioned in avian ecological studies (i.e., Bisson et al. 2007; Brilhante et al. 2012). More molecular-based studies focusing on other bird species are needed to determine whether these results are consistent or not between bird species and environmental conditions, and the possible role of fungal species on embryo survival.