is an opportunistic pathogen that can cause, and then take advantage of, intestinal inflammation to disrupt the commensal microbiota, compromise gut barriers, and colonize host tissues (1–3
). Differences in pre-existing microbial populations may dictate the course and severity of a Salmonella
), and successful Salmonella
colonization of the host is linked to an inflammation-associated disturbance of the gut microbiota (1–3
). Thus, it may be important to support beneficial microbial populations to mitigate Salmonella
colonization in pigs.
Prebiotics are dietary compounds that can facilitate the growth and metabolic activities of beneficial microbes, usually resulting in an increase in short chain fatty acids (SCFAs) concentrations in the distal intestinal tract, a major site of Salmonella
). Increased SCFAs can have beneficial impacts on host tissues, bolstering barrier functions, enhancing tolerance to commensal organisms (and therefore reduced inflammation), and encouraging oxidative metabolism in gut tissues (10
). The combination of these effects diminishes oxygen potential at the mucosa and limits inflammation-associated reactive molecules, thereby restricting terminal electron acceptor availability required for bacterial respiration (11
). In gut ecosystems with limited resources to support respiration, anaerobic fermentative microorganisms outcompete bacteria that use respiration, such as Salmonella
). Previous research has detailed the beneficial effects of dietary resistant potato starch (RPS) in the gut environment, and how these effects may shift ecological conditions to be less hospitable and exploitable by Salmonella
To build on our previous research (14
), we investigated the ability of various in-feed additives to support beneficial microbes as well as reduce the colonization and shedding of multidrug-resistant (MDR) Salmonella enterica
serovar I 4,,12:i:- (Salmonella
I 4,,12:i:-) in post-nursery swine. Recently, certain lineages of Salmonella
I 4,,12:i:- have become a significant concern in pig production due to their acquisition of antibiotic-, metal-, and biocide-resistance genes (16
). In the current study, we evaluated standard commercial swine diets containing additions of an RPS, a fatty acid-natural flavoring feed additive (FAM), or resistant corn starch (RCS) for their abilities to modify the swine intestinal microbiota, promote production of SCFAs known to benefit gut health, and limit Salmonella
colonization and shedding.
The RPS response-phenotype observed in this study is in line with previous descriptions of animals fed resistant starch. A reduction in alpha diversity was observed in RPS-fed animals that parallels similar responses noted for pigs administered type 2 resistant starches in feed (23
). The increases in butyrate, valerate, and caproate observed in the RPS-fed animals align with our previous research of the impacts of RPS intake on the swine gut ecosystem (14
). In addition, many of the OTUs associated with the phenotype of interest in this study, belong to the same taxonomic groups enriched in the RPS-fed pigs in our previous study. For example, OTUs belonging to the genera Megasphaera
, and Syntrophococcus
were identified in the RPS response-phenotype in both studies (14
), despite the pigs being sourced from different herds. Other studies that administered resistant starch to various animal species detected similar increases in these taxa as well. Olsenella
was previously associated with resistant starch intake in broilers (20
) as well as swine (25
). A recent clinical trial observed increases in Bifidobacterium
, and Prevotella
in humans fed resistant starch (19
), and another study detected enrichments of Bifidobacterium
in swine fed resistant starch (18
While RPS-fed pigs exhibited significant shifts in bacterial communities and cecal SCFAs, feeding pigs RCS did not result in the same phenotype. In line with our results, a recent study in humans showed that dietary RPS stimulated SCFA production while a high amylose corn starch failed to do so (26
). Additionally, within the RPS group of the human study, not all individuals responded uniformly, and the presence of key primary starch degraders in the microbiota was important for the increased SCFA phenotype. In humans it was suggested that bacteria belonging to the genus Ruminococcus
were important primary degraders of RPS that developed butyrogenic cross-feeding interactions with other members of the microbiota. In addition, gut ecosystems with Bifidobacteria
as major primary degraders did not exhibit the increased butyrate as seen in ecosystems with Ruminococcus
as major primary degraders. The findings from our present study are somewhat different as we did not detect an association of Ruminococcus
OTUs with the increased SCFA phenotype, but rather OTUs from the genera Bifidobacterium
held central positions in our phenotype-response networks and were associated with both increased SCFAs as well as reduced Salmonella
fecal shedding. However, in addition to differing host species between the studies, it is important to note that diversity of ecological roles exist within these genera and not all species of a genera will behave identically. Furthermore, our study was conducted in the context of a Salmonella
disturbance which further complicates comparisons between the studies and interpretation of these interactions.
Similar to previous studies indicating that appropriate primary degraders are key in phenotypic responses to resistant starches, in our study many of the OTUs associated with increased butrayte, valerate, and caproate do not belong to taxa known to produce these compounds. Production of these three SCFAs from dietary fiber generally requires a primary degrader to perform the initial breakdown of the polymers. Secondary fermenters can then cross-feed using the released simple sugars and the metabolic outputs of primary degraders (such as acetate, lactate, and succinate) to produce the final fermentation products (27
). Therefore, observations of OTUs associating with SCFAs that are not typically produced by organisms represented in the OTUs may signify important primary degraders that provide simpler carbon sources to secondary fermenters that cross-feed to produce butyrate, valerate, and caproate. Our study identified OTUs belonging to the genera Prevotella_7
, and Olsenella
, as candidate organisms that could fill this primary degrader niche. Furthermore, our results suggest that the interindividual variations observed in the RPS response-phenotype could be linked to differences in abundance of the important primary starch degraders, in line with previous work (29
). Specifically, the pigs with the highest concentrations of SCFAs and lowest levels of Salmonella
fecal shedding having the most robust populations of these potential primary starch degraders.
While the SCFA butyrate is known to be a microbial metabolite of central importance, our data suggest the reduction in Salmonella
fecal shedding in RPS-fed animals was more strongly associated with the longer SCFAs valerate and caproate. These two longer SCFAs may be indicators of conditions that favor bacteria which are fermentative specialists. It has been proposed that longer SCFAs can be produced using propionate or butyrate as substrates, with the same metabolic machinery as used in butyrate production in a reverse beta-oxidation process (31
). It follows that valerate and caproate may only be produced once conditions that favor fermentative specialists are established and shorter SCFAs such as acetate, propionate, and butyrate are abundant. Additionally, while butyrate is a high priority metabolic input for intestinal epithelial cells, valerate and caproate may be higher priority substrates than butyrate for oxidation in the mucosa (32
). Knowledge of the health benefits of longer SCFAs is more limited than that of butyrate, but the longer SCFAs also provide benefits to intestinal host-microbiota systems. Valerate may provide some protection from auto-immune disorders such as eczema (33
), as well as a protective effect against Clostridium difficile
). These longer, less well-characterized, SCFAs deserve further investigation into their roles in the reduction in Salmonella
colonization and shedding.
Our phenotype association networks suggest that increased SCFA production was closely related to reduced Salmonella
fecal shedding. Robust SCFA production plays key roles in health and barrier function in gut-associated mucosal tissues (12–14
); additionally, increased concentrations of SCFAs are known to reduce the luminal pH which can have bactericidal effects on pathogens such as Salmonella
). Furthermore, SCFA production by commensals and oxidation of these SCFAs by gut tissues can play a central role in driving environmental conditions in the gut toward those that favor fermentative specialists to the detriment of those microbes that prefer to use respiratory metabolisms, such as Salmonella
relies on generating an inflammation-linked oxidative disturbance to disrupt healthy commensal ecosystems, thereby aiding in host colonization and environmental dissemination (1–3
). Only the bacterial communities in RPS-fed pigs exhibited consistent differences from those in the control pigs throughout the experiment which suggests the RPS communities reacted differently to Salmonella
inoculation compared with all other treatments.
Differences in microbial community composition within the RPS-fed pigs helps explain the variation in Salmonella
shedding and within this group. One hypothesis is that different gut communities process RPS in distinct ways with different metabolic end products, and certain microbial functions are required for a response that most benefits the host (30
). The activities of some communities may be more beneficial for host tissues, providing a gut environment that is more resilient to incursion by opportunistic pathogens. Previous work has identified many ecosystem services that are associated with increased pathogen resistance (11
). Although our study design did not allow the interrogation of why certain RPS-fed pigs came to harbor communities that provided increased resistance, we observed that the RPS-fed communities that produced the most SCFAs butyrate, valerate, and caproate provided the most resistance to Salmonella
. Not all primary degraders of resistant starch have the same capacities to share the simple sugars that result from primary starch degradation (29
), and the presence of other cross-feeding members can also be a major determinant of resistant starch response phenotypes (37
). This study as well as previous work suggests that these primary degraders or other crossfeeding members may play a large role in determining the RPS response phenotype and therefore the Salmonella
shedding phenotype as well.
Another possibility is that RPS-fed communities could help limit Salmonella
numbers if they could withstand or buffer the oxidative stress Salmonella
induces in the intestine. In line with this idea, we noted that many of the central OTUs in our RPS phenotype association network belonged to taxa able to cope with microaerobic conditions or perform some form of respiration. For example, species within both the Bifidobacterium
genera are microaerophiles (39
); similarly, members of Prevotella_7
produce respiratory menaquinones (41
), suggesting they may utilize respiration when conditions allow. The ability to withstand the conditions induced by a Salmonella
infection may have allowed the important primary starch degraders to maintain their keystone roles during the Salmonella
-induced oxidative disturbance. The communities in higher shedding RPS-fed pigs may not have had primary degraders that could cope with the Salmonella
disturbance. If these keystone members of the communities could not cope with oxidative stress, the foodwebs they feed would collapse which could result in reduced SCFA production and further increased oxidative conditions in which Salmonella
can thrive. Future efforts into characterizing communities associated with an improved RPS response phenotype should include culturing efforts to better characterize the ecological niche of important members. These results highlight taxa that should be investigated for their abilities to enhance the beneficial effects of RPS and improve colonization resistance.
The presence of endogenous competitor organisms provides yet another alternative hypothesis for the within-RPS treatment variations in the Salmonella
fecal shedding phenotype. For example, endogenous Enterobacteriaceae
can have a major influence on the Salmonella
colonization phenotype in mice (43
). Mice already colonized by endogenous Enterobacteriaceae
organisms prior to a Salmonella
challenge were resistant to colonization. The hypothesized mechanism for this effect was that these pre-existing microbes had a similar respiration-based metabolism and occupied the same niche in the gut ecosystem that Salmonella
colonizes; this nutritional competition excluded Salmonella
. These protective microbes, such as other Enterobacteriaceae
, competing with Salmonella
were often of low abundance in the microbiota and generally below the limit of detection of many culture-independent approaches like 16S rRNA amplicon sequencing. Other work has suggested similar roles for Proteobacteria
helping to maintain anaerobic gut environments in other species (44
). Unfortunately, the design of our study did not allow us to speculate on the presence of such organisms in the animals shedding low levels of Salmonella
. Future studies that seek to characterize microbial features that impact Salmonella
colonization and shedding should consider employing culture-based or other approaches that allow the interrogation of more rare members of the microbiota.
This study detailed alterations in the swine gut microbiota and SCFA production through the course of a Salmonella challenge in the context of different in-feed additives. Only the RPS-fed group had consistent, meaningful differences when compared with the control group. The RPS-fed animals had a different Salmonella-induced change in their gut microbial communities, an enrichment of RPS- and health-associated microbial taxa, increased concentrations of health-associated SCFAs, and lower measurements of Salmonella fecal shedding relative to the control animals. Within the RPS-fed animals, interindividual variation in response phenotypes was evident. In RPS-fed pigs, those shedding the lowest levels of Salmonella had higher cecal concentrations of butyrate, valerate, caproate, and succinate and higher abundances of many OTUs from genera such as Bifidobacteria, Olsenella, Prevotella_7, and others. Collectively, the data suggest that RPS may be an effective option for limiting Salmonella colonization and shedding (including MDR Salmonella serovar I 4,,12:i:-) in swine, provided the appropriate bacterial communities that can utilize RPS as a substrate are present in the gut.
We thank the following for their outstanding contributions to this work: Trey Faaborg and the Iowa State University Swine Nutrition Farm for animal care; USDA-ARS Animal caretakers Brian Conrad, Dalene Whitney, Jeremy Spieker; and technicians Margaret Walker, Eli Whalen, Zahra Bond, Briony Atkinson, and Kellie Winter, for assistance with sample collection and processing; David Alt for MiSeq expertise.
This research used resources provided by the SCINet project of the USDA Agricultural Research Service, ARS project number 0500-00093-001-00-D. This research was supported by appropriated funds from USDA-ARS CRIS project 5030-3200-113-00D, 5030-31320-004-00D, and 5030-31000-006-00D and an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the authors’ and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE. Conflict of Interest Statement Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendations or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer. We declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Study conception: S.M.D.B., B.L.B., B.J.K., and C.L.L.; study planning and design: S.M.D.B., B.L.B., B.J.K., C.L.L., J.M.T., and K.A.B.; diet formulations: B.J.K.; sample collection and processing: S.M.D.B., B.L.B., J.M.T., D.C.S., B.J.K., K.A.B., and C.L.L.; bioinformatics & statistical analysis: J.M.T.; discussion and interpretation of results: S.M.D.B., B.L.B., J.M.T., D.C.S., B.J.K., K.A.B., and C.L.L.; manuscript writing: J.M.T.; manuscript review: S.M.D.B., B.L.B., J.M.T., D.C.S., B.J.K., K.A.B., and C.L.L.