The aim of this study was to determine the effect of the Salmonella phage cocktail SalmoFree® in the composition of the microbiota of the cecum of broilers. The study was performed supplying the phage cocktail to chickens reared with commercial purposes at a farm under production scale conditions. Our study also included the characterization of the dominant bacterial microbiota present at the grower stage of broilers in production conditions. Our results suggest a process of normal microbiota maturation characterized by a transition towards a higher diverse community. This observation was independent of the treatment applied, further demonstrating that phages do not affect the normal development of the microbiota.
Surprisingly, our results suggest that the behaviour of the microbiota in the period of the experiments (age 17–36 days), corresponding to the grower phase of the chickens, is similar to what have been previously reported in experimental chickens reared in a controlled environment [48, 49]. For instance, our observations support the stabilization dynamic of the microbiota at this developmental stage. Likewise, the observation of a microbial community similar in older chickens to the one observed at slaughter age was consolidated (Figs. 2 and 3). Additionally, the age of the animal was the variable that had the highest influence over the microbiota variation, in agreement with previous studies [48, 50]. This is a key evidence suggesting that microbiota approximations done under controlled environments do not differ largely from farming conditions.
Analyses of similarities among communities over time allowed us to identify two main moments of microbiota development at the last stage of the production cycle (PERMANOVA, P < 0,05) (Fig. 3). The 3rd week, representing the first week of the experiment and the week just after the change to the grower diet, is where the community seems to be more uniform and with a significantly higher abundance of bacteria such as Ruminococcaceae, Bacillales, Coprobacter, Hydrogenoanaerobacterium and Barnesiellaceae (Fig. 5) compared to Parasuterella and Flavobacteriaceae. The other two weeks, the 4rd and 5th weeks, exhibit a higher variation in abundance and diversity, which could be attributed to the change in diet. During the weeks 4th and 5th the microbiota becomes populated by Phascolarctobacterium, Desulfovibrionaceae, Megamonas, Odoribacter, Rikenellaceae and Alistipes. These bacteria could represent biomarkers of microbiota maturation at these rearing conditions (altitude: 1230 m.s.n.m.; litter composition: ground; average of no. of chickens/m2: 13.86; average of area house in m2: 645.61). Nevertheless, further studies are necessary to confirm the generalization of our current results to other farms and conditions.
Analysis of the dynamics of the core microbiome identified members that were reported previously in the literature as being part of the most abundant genera in the microbiome of chicken cecum . In one previous study, authors performed a comprehensive day-to-day microbiome analysis of the chicken cecum from day 3 to 35 using experimental chickens in a controlled environment. Authors identified the most abundant genera, also identified in our core analysis, as: Escherichia.Shigella, Eisenbergiella, Ruminiclostridium, Flavonifractor, Anaerotruncus, Faecalibacterium, Lachnoclostridium, Megamonas, Intestinimonas, Shuttleworthia, Subdoligranulum, Tyzzerella, Lactobacillus, Blautia and Erysipelotrichaceae, among others (Table 3).
The core composition of the microbiota identified key members at the last stage of the production cycle (Fig. 3) (Table 3). Those microorganisms may be responsible for important metabolic processes in the microbiota of broiler chickens. Among these, there are some important degraders of complex polysaccharides and producers of short chain fatty acids (SCFA). For instance, Eisenbergiella, Lachnoclostridium of the family Lachnospiraceae play an important role in the production of butyrate which is the preferred energy source for the gut epithelial cells . Another butyrate producer found in the core was Intestinimonas . Megamonas and Bacteroides were detected as well; these bacteria are known to produce propionate as the main end product of the degradation of complex plant polysaccharides. Although propionate is a less preferred energy source than butyrate, its production might represent an efficient balance between energy acquisition from available nutrients and sustained growth . Other bacteria present in the core microbiome involved in producing SCFA were Subdoligranulum, Faecalibacterium Alistipes, Coprobacter, Blautia and Butyricimonas .
On the other hand, Campylobacter, Helicobacter and Megamonas are bacteria carrying hydrogenases, which have the potential to serve as hydrogen sinks that facilitate succinate production . Succinate is an important metabolite in both host and microbial processes. Meanwhile, the presence of Oscillibacter, a Clostridium cluster IV member, has been identified as an anaerobe producer of valerate and associated with diet-induced obesity . Surprisingly, Bifidobacterium, a butyrate producer, was not detected while it has been reported consistently as a dominant member of the chicken microbiota [48–50].
Comparison analyses between treated and control farmhouses must be done with caution, because of the conditions and characteristics of the current trials (variation of temperature, humidity, feed composition; the antibiotic intervention, and phage’s cross-contamination). Altogether, it seems that the treatment patterns visualized in trial 1 are not evident in trial 2. For example, in trial 1 the diversity seems to be higher in the control group, also in trial 1 the relative abundance of Campylobacter sp. is lower in the treatment group while in trial 2 this abundance is low in both experimental groups. We proposed that these dissimilarities might be an effect of the presence of phages due to cross-contamination starting at the beginning of the trial 2 in all treatments. This event could generate a bias in trial 2.
In this line, our data suggest a low trend of alpha diversity in treated farmhouses. Although an increase in microbial diversity in the gut has been linked to improved health in the elderly , a reduced alpha diversity in samples subjected to phages has been reported previously as well as in trials with probiotics in chickens, where treated groups exhibit a lower diversity compared to non-treated groups . An alternative explanation for the observed reduction of diversity could lie in the presence of opportunistic pathogens . In some cases, the microbiota associated with disease is more diverse due to the presence of opportunistic pathogens that have the capacity to colonize this niche. Thus, the alpha diversity reduction could be attributed to a lower presence of opportunistic pathogens.
Differential analyses by treatment group determined that few taxa are significantly associated with the addition of phages, supporting the statement that phages are not affecting the structure of the microbiome. Particularly interesting is the evidence of the reduction of Campylobacter in treated farmhouses. Campylobacter is also considered an important food-borne pathogen associated to the consumption of poultry products and its importance in public health is substantial . In addition, when this opportunistic pathogen is highly abundant in chickens, it has been demonstrated to cause damage to the gut . The observed reduction of Campylobacter might suggest a beneficial modulation of phages while the reduction in Salmonella and pathogenic Enterobacteriaceae such as Escherichia coli represents a great added value to the application of SalmoFree® in broilers. Additionally, the presence of Campylobacter jejuni has been associated with a higher abundance of E coli not only in broilers  but also in humans . It is important to highlight that the reduction of Campylobacter is likely occurring as a secondary effect of the phage lysis on Salmonella, since is not expected that phages infect strains of this genera given the specificity of the cocktail towards Salmonella sp. strains. Furthermore, the correlated increase in Helicobacter abundance with the decrease in Campylobacter supports the proposal of a competitive dynamic between these two genera .
The increase of Butyricimonas and Rikenellaceae following the phage treatment is noteworthy. These two genera are reported as beneficial bacteria in chickens due to their enrichment in samples treated with probiotics .
Since the relative abundance of Salmonella could not be obtained, the analysis of Enterobacteriaceae family is highly important to elucidate the performance of the phage cocktail. We consider this analysis useful since members of this family are phylogenetically closely related. Enterobacteria such as E. coli and Salmonella enterica are facultative anaerobic pathogens that occupy the same niche [64, 65], hence knowing the behaviour of the family will help us to understand what happened with Salmonella. Furthermore, the Enterobactereaceae family has been reported as one consistent biomarker of poor gut health in poultry , as it has been negatively correlated with performance of production variables .
Regarding the Enterobateriaceae analysis, our data confirmed that in the presence of the phages the abundance of this taxon is reduced. Although the reduction of this group was observed for both experimental groups in trial 1 and trial 2, it might be a consequence of the cross contamination of phages that occurred in control houses (Table 2). Apparently, the higher effect of phages is around the 4th week of the production cycle; this time point also corresponds to the moment where more microbial changes are occurring. This finding identified the importance of the second dose and suggests it as a relevant dose in the regime proposed for SalmoFree®. The result that the reduction on the second trial was even better, let us to hypothesize that the reduction of the target could be higher with the application of phages by consecutive cycles.
Equally important is the maintenance of low abundance at slaughter. Hence, the application of phages is achieving its main objective that is to reduce the presence of Salmonella (an Enterobacteriaceae member) when chickens arrive in the slaughterhouse in order to impact directly the risk of food poisoning and reduce infection cases in humans.