1. Introduction
Poultry meat production is expanding rapidly, as global meat consumption is constantly increasing [
1]. As antibiotic growth promoters (AGPs) are still used in many countries to increase performance in poultry production, their usage increased simultaneously. However, AGPs contribute to the development and increase of antibiotic-resistant bacteria such as extended-spectrum beta-lactamase (ESBL) producing bacteria in poultry [
2]. Many countries have, therefore, banned AGPs for environmental and public health concerns [
3,
4]. AGPs in poultry production are well known to increase animal performance and infection resistance, and thus, alternatives are demanded to compensate for this loss [
5,
6]. Among the commercially existing alternatives, probiotics and phytobiotics have been reported to impact on the performance and health in poultry [
7].
Regarding probiotics, many publications have shown health promoting effects that are associated with the modifications on gut microbiota [
8,
9].
Bacillus spp., yeasts, and lactic acid bacteria are commonly used as probiotics in animal nutrition. Among the lactic acid bacteria, the lactobacilli have the advantage that they are “generally regarded as safe” (GRAS-status) and are indigenous to the intestinal tract. Certain
Lactobacillus agilis strains are able to modify the presence of pathogenic bacteria in vitro [
10] and ex vivo [
11] or regulate the gut microbiota in broiler chickens in vivo [
12,
13]. In some studies, several
L. salivarius strains have also been shown to promote animal health [
14,
15]. Proper supplementation of certain probiotic strains may also lead to the immunomodulation of the host [
16], and consequently, resilience against pathogenic bacteria may be increased [
17,
18]. However, those effects primarily depend on the specific strain, and it is still unclear if immunomodulation is a direct effect of the probiotics or a response to a modified microbiota. Nevertheless, the health-promoting effect of probiotics has often been observed, and this effect may sometimes lead to an improvement in performance. Finally, host-specific probiotics were reported to show better survival and colonization of the strain in the intestinal tract of the host [
19,
20].
Phytobiotics have also been studied as an alternative to AGPs due to their strong antibacterial activity in vitro and their beneficial influence on animal health and performance in broiler production systems [
21,
22,
23]. Phytobiotics have the potential to inhibit pathogenic bacteria [
24] and show a range of host-related responses such as improvement in antioxidative status [
25], intestinal barrier functions [
26] as well as a beneficial modification the digestive or immune function of the host [
27]. These modes of action may contribute to an increase in growth performance [
28,
29]. However, like probiotics, due to a lack of studies, it is still unclear if these effects are directly induced by the presence of the phytobiotics or are an indirect effect of changes within the intestinal microbiota.
Synergistic feed additives are thought to act by combining their individual effects that lead to a superior effect than their sole application [
30]. This principle has been applied for combinations of probiotics and prebiotics with the intent to promote beneficial bacteria and at the same time, supply suitable substrates for the probiotic [
31]. There are also reports on other combinations of feed additives like organic acids combined with phytases [
32], probiotics [
33], or phytobiotics [
34]. To our best knowledge, only one study evaluated the use of a probiotic
E. faecium strain with a commercial phytobiotic product as a combination in broiler chickens. This study showed beneficial effects on animal health, but no effect on animal performance [
35]. In summary, the use of such possibly synergistic combinations of feed additives has not received attention in the field of poultry nutrition. Therefore, a concept was designed and applied in this study to combine host-specific probiotic
Lactobacillus strains with specific phytobiotics to invoke beneficial synergistic effects for the animal.
Both probiotics and phytobiotics are known to modulate the intestinal microbiota in poultry [
36]. Therefore, studies on possible synergistic effects must include an in-depth analysis of the bacterial composition and activity. Furthermore, in light of the evolution of ESBL-producing
Enterobacteriaceae in poultry [
2], the impact of the feed additives on the colonization of antibiotic-resistant bacteria is of high interest.
As young animals are still in the process of developing a mature microbiota [
37], this progress may be modified more easily via feed additives [
9]. Thus, young broiler chickens were used to investigate the effect of these feed additives on the intestinal microbiota.
Thus, the aim of this study was to compare two different Lactobacillus strains and two different commercial phytobiotics as well as their combinations on the possible synbiotic activity in young broiler chickens.
4. Discussion
Accumulating numbers of studies show that novel additives such as probiotics or phytobiotics may be used as alternatives to in-feed AGPs. However, the efficiency of the alternative additives depends on many factors like uptake concentration, overall diet, supplementation method, or the rearing environment [
50]. To maximize the efficiency of those alternatives, the combination in accordance with a synergistic concept is a favorable solution that may act beyond their single applications. The present study investigated the synergistic effects of probiotics and phytobiotic feed additives on the intestinal microbiota in young broiler chickens. The gut microbiome is a key to understand animal health and nutrition better [
51], and thus, this study focused on the bacterial composition and –activity in crop and caecum of young broiler chickens that has not yet developed a stable microbiota.
Probiotics generally do not reduce the total amount or activity of bacteria in the gut, but can sometimes increase bacterial metabolite concentrations in broiler chicken [
52,
53]. On the other hand, phytobiotics are often used due to their strength in vitro antibacterial activity [
54]. The active ingredients in the phytobiotic products were carvacrol and cinnamaldehyde as well as additionally eugenol in formulation L. All three substances have been shown to inhibit a range of bacteria in vitro and show diverse effects on performance, immunology, and reduction of pathogenic bacteria in broiler chicken [
55,
56,
57]. The probiotic
Lactobacillus strains in this study were previously characterized in vitro regarding their resilience against both phytobiotic products (data not shown). Both strains showed a high tolerance in vitro, which could be confirmed in vivo, especially for the
L. salivarius strain. In fact, a strong synergistic effect was observed for the species
L. salivarius with formulation L, which may indicate that the functionality of LS1 increased accordingly when applied as a synergistic product. However, the species
L. agilis was strongly inhibited by the effects of the formulation C in vivo, but in combination with LA73 an increase of this species was observed in the crop. This also points to a synergistic effect for increased LA73 colonization in combination with formulation C. Taken together; the data suggest that indigenous
L. agilis strains may be much more sensitive to phytobiotic pressure compared to the supplemented
L. agilis strain. This reflects strain-specific differences in lactobacilli in general. Therefore, synergistic effects seem to be in effect regarding certain combinations of probiotic and phytobiotics. Unfortunately, to the best knowledge of the authors, there are not many reports on phytobiotic modifications of the intestinal microbiota in broiler chicken, studies on combined usage with probiotics are even rare [
35]. However, studies in humans, pigs, and rats show that the absorption of the mentioned essential oils occurs in the upper small intestine [
58,
59]. It is therefore probable that pancreatic enzymes in poultry attack these substances, and resorption of their metabolites could be expected before they reach the caecum. Consequently, it is unlikely that relevant concentrations of the phytobiotics reached the hindgut. This implies that results on crop and caecum microbiota should be viewed separately from different angles and that modifications of the caecal microbiota are largely due to bacterial- or host-related changes in the upper intestinal tract.
The crop plays an essential role in the transient storage and moisturization of feed [
60]. It is also viewed as a pre-gastric fermentation chamber that defines the input of bacteria into the gut [
61]. Generally, the crop of broiler chicken is heavily dominated by certain dominant
Lactobacillus species [
62,
63,
64], which was also observed in this study. Of the few studies on the subject, one report with a probiotic
L. salivarius strain showed no effect on crop lactobacilli after administration [
65]. This was also observed for the number of lactobacilli in this study, but the single addition of probiotic strains significantly enhanced their quantity compared to the control group. Thus, both strains were able to colonize the crop. This was not unexpected, as both probiotic strains were originally isolated from the broiler intestine and already demonstrated great potential for in vitro survival under-stimulated gastric stress and epithelial adherence in our previous study [
38].
Significant positive synergistic effects on relative Lactobacillus spp. abundance were only observed for L. salivarius in combination with formulation L as well as for L. agilis with both phytobiotics. In general, the supplementation of the probiotic strains seemed to be the overriding effect on Lactobacillus spp. abundance, while the additional phytobiotic supplementation showed only minor effects. Similarly, a slight non-significant decrease of species richness was observed for combination groups, but significant differences for microbiota diversity (Shannon-index) did not show clear synergistic effects.
The impact of the probiotics and phytobiotics on the crop microbiota also extended to non-dominant bacteria. For instance, the
Clostridium sensu stricto 1 genus exhibited the highest abundance apart from the lactobacilli. This
Clostridium genus has been shown to be associated with necrotic enteritis and
Clostridium perfringens infection models [
66,
67]. However, the
Clostridium sensu stricto 1 cluster also contains species such as
C. butyricum, which has also been used as a probiotic in poultry [
68]. It is, therefore, difficult to assign a positive, indifferent or negative role to this genus. Nevertheless, the comparison of
Clostridium sensu stricto 1 sequencing data to the much more sensitive
C. perfringens qPCR data did not show any correlation (data not shown). We can, therefore, conclude that this genus probably did not include
C. perfringens. The abundance of
Clostridium sensu stricto 1 was high in single probiotic and formulation L supplemented feed groups but was dramatically reduced in combinations of LS1 with both phytobiotic products and especially in LA73 with formulation L. Thus, synergistic effects in the significant reduction of this
Clostridium spp. were visible only for certain combinations. Although the additional eugenol in formulation L may have played a role in enhancing the
Clostridium sensu stricto 1 abundance compared to formulation C in single supplementation; this does not account for its total inhibition in combination with both probiotic strains. These results signify again that the synergistic mode of action on certain bacteria are not additive but rely on the impact of the feed additives on other bacteria. In this case, the concomitant responses of
Faecalibacterium spp.,
Blautia spp., and an unidentified
Clostridiales may have played a role in the significant modification of
Clostridium sensu stricto 1.
Interestingly, similar changes in relative abundance were observed for the putatively pathogenic genera Aeromonas spp. and Acinetobacter spp. Furthermore, C. perfringens positive samples in the crop were generally lower in combination groups, which points to their potential to a synergistic potential to reduce detrimental bacteria in the intestinal tract. Nevertheless, synergistic effects for these bacteria were not visible for all combinations. Therefore, the response of the intestinal microbiota to different phytobiotics seems to be quite diverse. However, as beneficial synergistic effects are clearly visible regarding putatively pathogenic bacteria, the combination of certain probiotic and phytobiotic products may be advantageous for animal health.
Overall, bacterial activity in terms of bacterial metabolites was lower in the crop of feed groups with single phytobiotic addition, although only the reduction in lactate was significant. Carvacrol, cinnamaldehyde, and eugenol are all known to inhibit bacterial growth in vitro, and consequently, their activity [
69,
70]. Our results indicate that both phytobiotic formulations indeed inhibited bacterial metabolism, although no significant changes in the absolute bacterial counts were observed. Consequently, at the employed in-feed concentrations, the phytogenic products may not inhibit total bacterial growth
per se, but significantly reduce their activity in the crop. However, the production of lactate or acetate in the intestinal tract is usually considered beneficial, as it may inhibit pathogenic or other bacteria detrimental to the host [
71,
72]. The increased lactate concentration in certain combinations of phyto- and probiotics points to a beneficial synergistic effect. Still, the synergism seems to depend on specific combinations and cannot be classified as an additive effect of individual supplementation.
We also monitored the ex vivo survival of an ESBL producing, but non-pathogenic
E. coli strain in the intestinal contents because ESBL producing enterobacteria have become a worldwide concern in poultry production [
73]. The results of our study show synergistic effects on reducing the ex vivo survival of the
E. coli model strain in crop contents compared to the control. The in vivo results on the quantification of the
Escherichia group show a similar reducing synergism except for LA73/formulation C. Lactobacilli are known for their antagonistic activity against enterobacteria [
74,
75] and both probiotic strains showed exceptional inhibitory activity against the
E. coli strain in vitro and ex vivo [
38]. Contrary to these results, data from both the ex vivo assay and the
Escherichia group quantification showed that the single addition of the probiotic strains had only slight effects on
E. coli survival. Thus, the inhibitory activity of the phytobiotic products was probably necessary to enhance the impact of the probiotic strains. However, the survival of the
E. coli strain was also affected in the caecum, where active phytobiotic concentrations are considered low. This indicates that different modes of action may be in play.
In the context of ESBL producing enterobacteria and transfer of their resistance genes, the presence of the enterobacterial class 1 integron integrase 1 gene (
int1) was also monitored. This enzyme is a key protein in the incorporation of foreign DNA in enterobacteria [
76]. Its copy numbers correlated highly to the count of the
Escherichia group (
p < 0.0001; 0.551 coefficient) as well as to the count of enterobacteria (
p < 0.0001; 0.423 coefficient). However, only the combination of LA73 and formulation L showed a reducing effect on
int1 concentration. The
int1 gene is widely distributed in enterobacteria, and it is likely that certain enterobacterial species or strains responded differently to the supplementation of the feed additives. Nevertheless, the results clearly show that synergistic effects of probiotics and phytobiotics may be superior to a single addition to combat the spread of enterobacterial antibiotic resistance.
In the caecum, fermentation of undigested nutrients occurs [
77], and the bacterial composition and activity is largely determined by incoming nutrients as well as bacteria from the small intestine [
78]. Their metabolites (SCFA) can be used as an energy source by the host and may contribute to meet the energy requirements of the animal. Furthermore, the caecum also determines the output of the potentially detrimental bacteria into the environment and thus has an important impact on stable hygiene.
In this study, the impact of the feed additives on the caecal bacterial composition was much less pronounced compared to changes observed in the crop. This may point to the fact that the active compounds in the phytobiotic products (carvacrol, cinnamaldehyde, and eugenol) are metabolized in the small intestine. Consequently, their active concentration may be drastically reduced. Nevertheless, an antagonistic relationship between the abundance of a dominating unidentified Clostridiales genus and Faecalibacterium spp. was observed, as the significant reduction of Faecalibacterium spp. was always offset by a trend for an increase of the unidentified Clostridiales genus. As noticed for other parameters, a trend for synergistic effects was again visible for the combination LA73 and formulation L.
The dominating bacteria in the caecum are most likely to be the most prominent producers of SCFA from undigested nutrients, and indeed, an increase in the relative abundance of the unidentified
Clostridiales genus always corresponded with increased acetate concentrations. The increased metabolite production points to an enhanced capacity to ferment undigested nutrients and indicates a more mature microbiota. As a mature microbiota is viewed as beneficial [
79], the caecal microbiota, especially in animals, fed the combination LA73 and formulation L, could have developed faster than the microbiota in other feed groups.
There were two noteworthy exceptions to the generally low response of the caecal microbiota: a reduced colonization of C. perfringens and reduced ex vivo survival of the ESBL-producing E. coli in all treatment groups. Apparently, adverse conditions for these two detrimental species existed due to the addition of the feed additives. However, as the phytobiotic concentration is believed to be low in the caecum, these adverse conditions may have mainly originated from interbacterial competition or host-related responses that were induced in the crop or small intestine.