Discussion
The intestinal microbiota acts as a physical barrier for incoming pathogens and plays an important role in the host resistance against infections by both direct interactions with pathogenic bacteria via competitive exclusion, such as occupation of attachment sites or consumption of nutrient sources, and indirectly by influencing the immune system via production of antimicrobial substances (Sekirov et al., 2010). Development of the gut microbiota in chickens occurs immediately after hatching and by getting older, this microbiome becomes very diverse until it reaches a relatively stable dynamic state (Pan and Yu, 2014).
Interactions of the intestinal microbiome with the host and certain microorganisms have profound effects on bird health, and are therefore of great importance for poultry production. Consequently, in the present study, the composition of the gut microbiota of chickens in a longitudinal study from day 1 to day 28 of age was analyzed and the differences between content and mucosa-associated gut microbiota were investigated. In order to extend the range of analyses comparisons were performed between control chickens and chickens infected at 14 days of age with C. jejuni.
In this study, a high diversity of phyla (15 in the jejunal and 4 in the cecal mucosal samples) was found at day 1 of life, indicating a rapid intake of environmental organisms after birth. In addition, the composition of the gut microbiota differed substantially between young and older birds, with Proteobacteria being significantly more present at the first day of life and decreasing thereafter, whereas the Firmicutes were the predominant phylum in older birds. This is in agreement with Lu et al. (2003) who found that the gut is firstly colonized by the phylum Proteobacteria, particularly by the family Enterobacteriaceae. In older birds, the phylum Firmicutes mainly represented by Lachnospiraceae, Ruminococcaceae, Clostridiaceae, and Lactobacillaceae dominated. As a consequence, the chicken gut is firstly colonized by facultative aerobes which are substituted later on by anaerobes. Obviously, oxygen consumption by the aerobic bacteria alters the gut ecosystem toward more reducing conditions, which facilitates subsequent growth and colonization of the obligate anaerobes (Wise and Siragusa, 2007).
Besides Proteobacteria and Firmicutes, also lower abundant phyla (e.g., Actinobacteria and Tenericutes) changed significantly with time, indicating high dynamics in the re-organization of the whole microbiome through time. Taken together, the present study revealed that the chicken gut is largely dominated by the phyla Proteobacteria and Firmicutes, with lower proportions of Actinobacteria, Bacteroidetes, and Tenericutes. Similarly, previous studies have also shown that Firmicutes, Bacterioidetes, and Proteobacteria are the most common phyla in the chicken ceca (Wei et al., 2013; Oakley et al., 2014; Sergeant et al., 2014).
Interestingly, jejunal, and cecal microbiota were found to be distinct and certain acid-tolerant bacteria, mostly Acidobacteria, were present in the jejunum only. Altogether, the results demonstrated that the abundance of bacteria varied between the jejunum and the cecum, with some species more present in the jejunum (e.g., Acinetobacter and Acidobacteria) and others (e.g., Bacteroides and Clostridium) being predominant in the cecum of chickens. This and other variations can be explained by the fact that feed passes quickly through the foregut and is retained for hours in the hindgut. In addition, the small intestine is mainly responsible for food digestion and absorption, while the large intestine, especially the cecum, is responsible for microbial fermentation, further nutrient absorption and detoxification of substances that are harmful to the host (Gong et al., 2002).
Chickens investigated in the current study had a high abundance of E. coli and E. faecalis (best type strain hits) in the first week of life which might potentially increase their resistance to other bacterial infections. E. coli, a facultative anaerobe bacterium, was the dominant species in the early life of chickens. Thus, a depletion of E. coli during the second week of life could potentially affect the host susceptibility to enteric pathogen infections, representing a key role for these gut microbiota in host resistance. This decrease in E. coli abundance has been attributed with a beginning dominance of anaerobes (Zhu and Joerger, 2003). It may be possible that such disturbances in the community structure allow a pathogen to colonize and proliferate. Anyhow, it remains hypothetical whether these diversity changes influence the susceptibility to pathogens and the outcome of infection.
The current results revealed that E. coli, E. faecalis, C. paraputrificum, and C. sartagoforme (best type strain hits) were more predominant in the mucosa than in the lumen, suggesting significant implications for birds' health, considering that the mucosa-associated bacteria are of great importance in the host mucosal responses with consequences for the mucosal barrier (Ott et al., 2004).
Despite the high prevalence of Campylobacter in chickens the mechanism of colonization in the gut is still poorly understood. The high bacterial load in the gut and the establishment of a latent infection characterized by continuous shedding indicates that Campylobacter in chickens can modify the microbiota composition. In the current study it could be shown that Campylobacter colonization shifted the two major phyla towards an enrichment of Firmicutes with concomitant reduction of Proteobacteria. Interestingly, a reverse correlation between Firmicutes and Proteobacteria was observed, suggesting a possible antagonistic interaction between these two phyla. According to Pan and Yu (2014) alterations in one phyla or species may not only affect the host directly, but can also disrupt the entire microbial community. Notably, bacterial taxa belonging to the phyla Firmicutes are known to be involved in the degradation of complex carbohydrates (not absorbed by the host) and in the production of SCFAs (Thibodeau et al., 2015). Thus, the SCFAs production by Firmicutes might, at least partially, explain their dominance in the infected birds, which have a high SCFAs requirement as a source of energy for C. jejuni to colonize the chicken gut. Furthermore, Brown et al. (2012) reported that members of the phylum Firmicutes can inhibit the growth of opportunistic pathogens, such as E. coli, which has also been shown in the present study.
Besides these major shifts, also low abundant phyla (e.g., Actinobacteria and Tenericutes) were affected by the Campylobacter infection, which could also disequilibrate the microbiome composition. Similarly, Johansen et al. (2006) found in a denaturing gradient gel electrophoresis (DGGE) based experiment that C. jejuni colonization affected the development and complexity of the microbial communities of the ceca over 17 days of age. Furthermore, Qu et al. (2008) noted that the community structure of the cecal microbiome from the C. jejuni challenged chicken has greater diversity and evenness with a higher abundance of Firmicutes at the expense of the Bacteroidetes and other taxa. Sofka et al. (2015) also reported that Campylobacter carriage, assessed in samples from slaughter houses, was associated with moderate modulations of the cecal microbiome as revealed by an increase in Streptococcus and Blautia relative abundance in birds of 56 days of age, originating from different farms and production types. Recently, Thibodeau et al. (2015) found also that C. jejuni colonization induced a moderate alteration of the chicken cecal microbiome beta-diversity at 35 days of age.
This study's results strongly suggest that the Campylobacter associated alterations of the gut microbiota were a direct effect due to the interaction of C. jejuni with the microbiota or a consequence of the host responses or even a combination of both (Barman et al., 2008; Mon et al., 2015). The obtained results indicate that the influence of a Campylobacter infection on microbial communities was more pronounced at 14 dpi than at 7 dpi. This could be explained by an increased load of Camplyobacter at the later time point as demonstrated in recent studies using the same C. jejuni strain (Awad et al., 2014, 2015a,b, 2016).
We also found significant differences in the abundance of certain bacterial species in the infected birds compared with the controls. C. jejuni caused a significant decrease in E. coli (best type strain hit) in the microbiota of infected birds in both jejunum and cecum. This is in agreement with our previous study which showed that Campylobacter colonization decreased E. coli loads in the jejunum and cecum at 7 dpi and at 14 dpi, but increased E. coli translocation to the liver and spleen of the infected birds as determined by conventional bacteriology (Awad et al., 2016). Thus, the current results pointed out that the relative abundance of E. coli could be an important determinant of susceptibility for a Campylobacter infection in particular and Gram-negative pathogens in general.
In contrast to the Campylobacter -E. coli interaction, it was found that the relative abundance of Clostridium spp. was higher in the infected birds compared with the negative controls, indicating a link between C. jejuni and Clostridium. This confirms data from an earlier study in which a positive correlation between high levels of Clostridium perfringens (>6 log) and the colonization of C. jejuni were found by real-time quantitative PCR (Skånseng et al., 2006; Thibodeau et al., 2015). This might be due to the fact that C. jejuni acts as a hydrogen sink leading to improved growth conditions for some Clostridia through increased fermentation (Kaakoush et al., 2014). This link can also be explained by the fact that the Clostridium organic acid production could be used by C. jejuni as an energy source. Furthermore, it was found that a Campylobacter infection induces excess mucous production in the intestine (Molnár et al., 2015) which consequently may enhance Clostridium proliferation due to the fact that an increase in mucin secretion in the gut provides an opportunity for Clostridium spp. to proliferate (M'Sadeq et al., 2015). Overall, the higher abundance of Campylobacter and Clostridium spp. might result in a higher endotoxin production with subsequent increase in intestinal permeability that facilitates the colonization and enhances bacterial translocation from the intestine to the internal organs, which is well in agreement with our pervious results (Awad et al., 2015a, 2016).
Finally, the strong shifts in the bacterial microbiome in the current study might help to explain why a Campylobacter infection is age dependent and chickens in the field become mainly colonized at an age of two to 4 weeks (Newell and Fearnley, 2003; Conlan et al., 2007). In agreement with this, Bereswill et al. (2011) demonstrated that a shift of intestinal microbiota in humans was linked with an increased susceptibility for C. jejuni. Finally, Haag et al. (2012) demonstrated that C. jejuni colonization in mice depends on the microbiota of the host and vice versa and Campylobacter colonization induces a shift of the intestinal microbiota. This was also observed in the present study as community structures were more dissimilar at the OTUs level in the infected birds compared with the controls. Moreover, in the infected birds, the population of beneficial microbes, such as E. coli and E. desmolans were comparatively lower than the potentially pathogenic bacteria, such as Clostridium spp., rendering the need for modulation of the gut microbiota to improve the gut health of the infected birds.