The complex intestinal microbial ecosystem is characterized by a close microbe-microbe-host relationship, which reaches a climax status. However, this homeostasis can be shifted into a state of dysbiosis by e.g. antimicrobial agents, diet, immune deficiency or infection, and thereby possibly facilitate the expansion of the resistome . Despite the well-studied and highly diverse microbial ecosystem in older individuals, the colonization of the neonatal gut in both humans and pigs is a de novo establishment of a microbial ecosystem undergoing several consecutive steps under the influence of genetic, maternal, and environmental (dietary and medical) factors [13, 14, 15]. Moreover, the early neonatal phase seems to determine the microbial profile and intestinal health later in life. In pigs, the very early colonizers between birth and 2 days of age are mainly enterobacteria, streptococci and clostridia of maternal and environmental origin [13, 14, 16]. This is supported by our findings here, where Escherichia, Streptococcus, Shigella, Enterococcus, Collinsella, Fusobacterium and Ruminococcus were the dominating genera in FP and FP-CD piglets. Under normal conditions, these genera become later outcompeted by others such as Alistipes, Bacteroides, Fusobacterium and Lactobacillus [13, 16]. Because FP and FP-CD piglets were transferred into the artificial rearing units within 6 h after birth and had no further contact to the mothers surroundings, it seems likely that these piglets habored an ‘immature-like’ microbiome as compared with SP and WP piglets, accompanied by a further expansion of these early colonizers through the formula diet. For example, formula with high lactose content promoted the proliferation of enterobacteria in neonatal piglets . We have previously shown that formula feeding also promotes CDI in neonatal piglets and the expansion of naturally colonizing CD in FP piglets . This is in line with studies in formula-fed infants showing a higher intestinal colonization with CD and E. coli as compared to breast-fed infants [18, 19]. Breastmilk is considered the gold standard for infant nutrition and this same is true for neonatal piglets. It seems likely that maternal antibodies in the porcine milk may provide protection against CDI in the suckling offspring. For example, we have recently shown that sow colostrum and serum contain antibodies against CD toxin A . In addition, sow colostrum exerted a protective effect against CD toxin-induced effects in porcine intestinal cell model IPEC-J2 . A formula-induced altered or delayed intestinal colonization and low abundance of lactobacilli or clostridia may decrease colonization resistance thereby promoting CD dissemination [16, 22, 23]. The expansion of Escherichia, Streptococcus, Shigella, Enterococcus phylotypes through the formula diet is of particular relevance because they represent a presumed source of persisting multi-drug resistant pathogens through further forward selection under medical treatment or other stress factors in the gut environment later in life. Interestingly, strong positive correlations between CD counts and abundance of Staphylococcus spp., Enterococcus spp. and Peptostreptococcus spp. Pasteurella spp., and Globicatella spp. taxa have been found in healthy suckling piglets, which suggests that CD and other potential pathogens could contribute to a gut inflammation and AR spread . It is known that certain groups of bacteria such as Proteobacteria and enterococci carry a high abundance of AR genes . Under normal conditions, their cell concentration is not high enough for efficient AR gene transfer. However, in a state of dysbiosis where colonization resistance is disrupted, an expansion of Proteobacteria and enterococci may lead to an increased abundance of AR genes, as we have found in artificially reared piglets. Moreover, it has been shown that gut inflammation boosts AR gene transfer between pathogenic and commensal Enterobacteriaceae in mice . Similarly, non-virulent CD may acquire virulence from virulent CD types via conjugative transposons which are known to encode toxin and AR genes, therefore increasing the AR load in the gut environment [25, 26].
In pigs, for example, reports have linked the use of supra-nutritional levels of trace elements such as Cu and Zn in swine diets with an increased prevalence of multi-resistant E. coli strains . A co-selection for AR genes within the intestinal microbial communities during or after an initial state of dysbiosis could even further promote an expansion of the bacterial resistome.
In the present study, a total of 83 AR genes were identified, conferring resistance to members of 11 classes of antimicrobial agents. A higher abundance of these genes was determined in FP as well as FP-CD piglets as compared to the other groups. However, the abundance of certain AR genes such as tet(Q), tet(W), lnu(C), lsa(E), or ant(6)-Ib in S, SP and WP groups was apparently linked to Megasphaera, Bifidobacterium, Lactobacillus, or Prevotella thereby confirming these genera as common carriers of these AR genes . Moreover, the observed co-occurrence of some AR genes among all groups could be attributed to microbial community composition and co-occurrence of certain bacterial taxa in general but also to gene co-localization in bacterial genomes. Plasmid co-location of AR genes such as blaCTX−M genes and mph(A) was previously reported in extended-spectrum β-lactamase-producing E. coli isolates from farm animals (including pigs) in Germany .A recent study showed clusters of co-occurring AR genes in pig environments including such as tet(W), tet(Q), tet(B), mefA, blaTEM . The present study shows that such co-occurrences are rather dynamic and that the abundance of AR genes largely depends on the microbiome composition. As shown, the latter is influenced by age (e.g. SP vs. WP vs. S), diet and rearing environment (e.g. SP vs. FP), and CDI (FP vs. FP-CD). However, our study also supports the hypothesis that metagenomic approaches may help to identify and monitor antibiotic resistance patterns in gut microbial communities in the pig .