Florfenicol exposure increases susceptibility to S. Enteritidis infection
We established a study design (Fig. 1a) in which SPF chicks were divided into four groups: orally administered florfenicol (FT); S. Enteritidis-infected by oral gavage (ST); simultaneously of FFC-treated and S. Enteritidis infected (FST); and an untreated control group (NT). Under the SPF environment, all chicks cultured negative for Salmonella spp. until experimental infection with S. Enteritidis. The control group remained culture negative for Salmonella spp. throughout the study. The colonization and translocation of S. Enteritidis in the intestinal tract of chicks directly determines its survival and pathogenicity. Therefore, we quantified S. Enteritidis levels in the caecum, spleen, and liver. The number of S. Enteritidis (log10 CFU/g tissue) significantly increased by 25.49% (cecal contents, P < 0.01), 23.04% (spleen, P < 0.01) and 21.33% (liver, P < 0.01), respectively, in the FST group relative to that of in the ST group at 3 days post-infection (dpi). Similar results were observed at days 18 (10 dpi) and 25 (17 dpi), although the Salmonella loads were less than those observed at 3 dpi (Fig. 1b).
Florfenicol administration aggravates S. Enteritidis-induced intestinal morphology and barrier injury
As FFC intervention made the chicks more susceptible to Salmonella infection, it is possible that antibiotics disrupted the immature intestinal barrier homeostasis of chicks, altered intestinal permeability, and facilitated greater translocation of Salmonella to their internal organs. Therefore, we investigated the effects of FFC administration on S. Enteritidis-induced changes in intestinal morphology. Hematoxylin and eosin (H & E) staining showed that the chicks in the NT group exhibited an intact ileal mucosa, neat intestinal villi, deep crypts, and a clear and complete gland structure, similar to that observed for the FT group (Fig. S1a and b). The ST group displayed an incomplete structure of the ileal mucosa, villi had a shorter length and sparse distribution, and crypts were shallow (Fig. S1c). However, the FST group morphology included loss of mucosal structures, atrophic crypts, and lamina propria bowel edema (Fig. S1d). Histological injury (Fig. S1e) was scored based on H & E-stained images; the scores indicated tissue damage. The score for the ST group (7.13 ± 0.44) was significantly higher than control (0.63 ± 0.18). Relative to the ST group, FFC pre-administration significantly increased the ileum injury score (10.75 ± 0.45). Scanning electron microscopy (SEM) showed that the NT group had complete ileal villi, forming full and closely arranged structures (Fig. 2e), whereas the FT group also had intact ileal villi, but their arrangement was relatively loose (Fig. 2f). As expected, the ileal villi of the ST group were damaged (Fig. 2g) and those of the FST group showed more severe damage (Fig. 2h). These results suggested that although FFC has less effect on intestinal morphology, intestinal injury was aggravated by Salmonella invasion.
An effect of FFC on intestinal barrier function in the ileum after S. Enteritidis infection was also observed exacerbating. The FFC pre-treatment can exacerbate the S. Enteritidis-induced increase in permeability of the Ileum (Fig. 3a-d). The serum diamine oxidase (DAO) and lipopolysaccharide (LPS) levels of the FT group were significantly higher (P < 0.001 and 0.05 respectively) than those of the NT group (Fig. 3c and d). In the case of Salmonella infection, serum D-lactate, DAO, and LPS levels in both the ST and FST groups were significantly increased (P < 0.001) relative to those of the NT group. However, FFC treatment significantly increased (P < 0.001) serum D-lactate, DAO, and LPS levels in chicks exposed to Salmonella (Fig. 3b-d). Alcian blue staining indicated that FFC significantly decreased (P < 0.05) the acidic mucin of ileum relative to the ileum of chicks in the NT group, as evident by the quantitative evaluation of positive Alcian blue staining (Fig. 2a and b) using integral optical density measurement (Fig. 3a). Similarly, FFC treatment also significantly decreased (P < 0.01) the mean density of acidic mucin (Fig. 2d and Fig. 3A) after exposure to Salmonella. Transcriptional analysis of a range of relevant intestinal barrier genes (Fig. 5) showed that FFC treatment significantly altered the transcription of (Claudin 1, IL-17A, and IFN-α) in the FT group. However, in the presence of Salmonella, FFC significantly reduced the expression of ZO-1, Occludin, Claudin 1, MUC2, and TFF2, and significantly increased the expression of IL-17A, IL-22, and IFN-α. Furthermore, treatment with FFC significantly decreased (P < 0.01) secretory immune globulin A (SIgA) secretion, but had no effect on serum IgG (Fig. 3e and f). Nevertheless, FFC treatment reduced SIgA secretion to a higher degree (P < 0.001) after Salmonella infection (Fig. 3f). Thus, FFC intervention, to some extent, increased the permeability of the intestinal mucosal of chicks, reduced mucosal immunity, and substantially increased the extent of damage to the intestinal mucosal barrier after Salmonella infection.
The level of Salmonella colonization is strongly associated with the triggering of intestinal inflammation [53]. We therefore hypothesized that increased colonization may be linked to increased mucosal inflammation. Indeed, FFC-treated chicks exhibited higher levels of gut inflammation after Salmonella infection (Fig. 4). This was verified by quantification of pro- and anti-inflammatory cytokines in ileal tissues. Salmonella infection after FFC-pretreatment significantly increased levels of cytokines IL-1β (Fig. 4a), IL-6 (Fig. 4b), IL-8 (Fig. 4c), TNF-α (Fig. 4e), and IFN-γ (Fig. 4f), whereas the level of IL-10 (Fig. 4d) was significantly decreased. Moreover, levels of inflammatory cytokines (IL-1β, IL-6, TNF-α and IFN-γ) were significantly increased in the FT group. These results indicated that FFC-pretreatment exacerbated the Salmonella-induced inflammatory response.
Florfenicol administration alters the gut microbiota
The composition and density of the gut microbiota play an important role in combating Salmonella invasion. Oral pretreatment with antibiotics decreases colonization resistance and leads to an post-treatment expansion of Salmonella loading in the gut [53]. Thus, we hypothesized that the higher Salmonella population observed in the chicks of the FFC-treated group may be linked to the disruption of microbiota composition and density. To this end, the microbiota compositions of the cecal content of chicks at 3, 10, and 17 dpi were determined by 16S rRNA gene sequencing. Fig. S2 illustrates microbiota diversity, represented as boxplots of measures of α-diversity, Shannon index, Observed Species and Pielou index. The α-diversity at 3 dpi was neither affected by FFC treatment nor by S. Enteritidis infection (Fig. S2a). However, a significant decrease in α-diversity was observed in the FST group at 10 dpi (Fig. S2b). Fig. S2c shows that the Shannon and Pielou index of the cecal microbial communities were significantly increased in the FST group at 17 dpi. These results indicated that the α-diversity of gut microbiota from chicks is not significant affected by a single FFC treatment or Salmonella challenge. However, the combination of both treatments significantly disturbed cecal α-diversity.
Phylum and genus distributions of microbial compositions are shown in Fig. S3. Firmicutes (67.85 – 99.62%) dominated the microbiota in all four groups at three different stages of infection (Fig. S3a). At 3, 10, and 17 dpi, the FST chicks had the highest relative abundance of Proteobacteria (2.68%, 1.30%, and 1.75%, respectively) relative to the other three groups (Fig. S3a). At 17 dpi, the FFC (9.23%) chicks had significantly reduced relative abundance of Bacteroidetes relative to the NT group (31.26%). Salmonella infection (23.87%) had negligible effect on Bacteroidetes abundance. However, infection with Salmonella after pretreatment with FFC almost eliminated the growth of Bacteroidetes (0.01%) (Fig. S3). We applied the LEfSe (linear discriminant analysis effect size) method to identify abundant bacterial taxa among these groups; only those taxa that obtained a log LDA (linear discriminant analysis) score > 3 were ultimately considered. A cladogram from phylum to genus level abundance is shown in Fig. 6. In total, 21, 21, and 28 differentially abundant taxa were identified at 3, 10, and 17 dpi, respectively (Fig. 6). In the untreated control chicks, LEfSe highlighted the greater differential abundance of Lactobacillus at 3 and 10 dpi, and Bacteroides at 17 dpi. Notably, the relative abundance of Enterobacteriaceae was significantly higher in the FST group than in the other three groups at all three time points. However, the other taxa were altered irregularly at different times in different groups. The relative abundance of these biomarkers is shown in Fig. S4, wherein consistent results were obtained. We also established taxonomic cladograms at 11 day (3 dpi), with the relative abundance of the taxa node of each group shown as a pie chart; only those taxa with relative abundance > 0.1% were considered (Fig. 7a). Similarly, the abundance ratio of Lactobacillus was considerably higher in the control group than in the other three groups. Additionally, the abundance ratio of Enterobacteriaceae in the FST group dominated among all four groups. Furthermore, Salmonella was only found in the challenged groups (ST and FST) at the genus level, with the abundance ratio of Salmonella in the FFC pretreatment group significantly higher than those of the untreated groups (Fig. 7a). We measured the cecal loads of these biomarkers and two intestinal protective bacteria by quantitative real-time PCR (qPCR) (Fig. 7b). At day 11 (3 dpi), FFC pre-treatment significantly reduced the densities of total bacteria, Lactobacillus, Clostridium butyricum and Faecalibacterium prausnitzii. Although Salmonella infection had no effect on cecal bacterial densities, chicks with Salmonella infection after pretreatment with FFC harbored much higher densities of Enterobacteriaceae, and lower densities of Lactobacillus, Bacteroides, C. butyricum and F. prausnitzii relative to the control group. At day 25 (17 dpi), C. butyricum and F. prausnitzii were present at equivalent densities in the cecal content of all four groups. However, significant differences in the bacterial densities of total bacteria, Lactobacillus, Bacteroides, and Enterobacteriaceae were still apparent between the NT and FST groups or between the ST and FST groups (Fig. 7b). Lactobacillus and Bacteroides are generally considered as beneficial bacteria that provide protection for the gut, whereas Enterobacteriaceae are potential pathogens in poultry and humans. These observations suggested that FFC exposure significantly decreased the abundance of Lactobacillus in chicks, and this inhibitory effect may provide a growth advantage for Enterobacteriaceae, especially Salmonella.
The similarity of microbial communities (β-diversity) between groups was visualized using principal-coordinate analysis (PCoA) of Bray-Curtis distances. PCoA plots for 3 dpi showed that microbial communities from Salmonella or FFC treated chicks are clearly different from those of the untreated chicks. The first axis of the PCoA plot shows 19.0% of variation in bacterial diversity while the second axis shows 13.0% (Fig. 7c). The first axis roughly distinguishes the antibiotic pre-treated chicks and non-pretreated chicks, and the second axis roughly distinguishes the infected and non-infected chicks. The PCoA at 10 dpi shows that the microbiota composition was very similar between the NT and FT groups, whereas the ST and FST groups are still obviously distinguish from the NT group (Fig. S5a). Intriguingly, at day 25 (17 dpi), the PCoA plot demonstrated that both the microbiota composition of ST and FT groups tends toward the NT group, whereas composition of the FST group is still strikingly divergent from the NT group (Fig. S5b). These findings suggested that a single FFC or Salmonella treatment alters the microbiota composition, with recovery two weeks after infection. Whereas FFC pretreatment hindered the recovery of microbiota composition of chicks after Salmonella infection.
Florfenicol administration alters the metabolic profiling
We hypothesized that differences in key metabolites may be crucial to the effect of Salmonella colonization on chicks. Therefore, we analyzed metabolomes by LC-MS to determine differential levels of metabolites on day 11 (3 dpi) in cecal contents. The PCA score plot shows that the metabolome of NT group and ST group was significantly separated among the four groups, whereas there was no clear distinction in cecal metabolites between the FT and FST groups (Fig. 8a). Orthogonal projections to latent structures discriminant analysis (OPLS-DA) and permutation test plot of OPLS-DA were performed. As shown in Fig. 8, cecal metabolites of the NT group were clearly distinguished from those of the FT group (Fig. 8b), ST group (Fig. 8d), and FST group (Fig. 8f). In addition, there was a clear separation between the FST group and ST group in cecal metabolites (Fig. 8h).
From the OPLS-DA models, we identified 72 differential metabolites between the NT and FT groups, 42 differential metabolites between the NT and ST groups, 69 differential metabolites between the NT and FST groups, and 57 differential metabolites between the FST and ST groups, using to the threshold VIP > 1 and p < 0.05 (Welch’s t test). The differential metabolites are listed in Supplementary Table S2. Next, we performed pathway enrichment analysis based on these differential metabolites to better understand the effect of FFC on metabolism (Fig. 9). Linoleic acid metabolism, aminoacyl-tRNA biosynthesis, lysine biosynthesis, phenylalanine metabolism and lysine degradation were enriched after FFC treatment (Fig. 9a). Arginine and proline metabolism, lysine biosynthesis, lysine degradation and D-glutamine and D-glutamate metabolism were enriched by Salmonella infection (Fig. 9b). Linoleic acid metabolism, aminoacyl-tRNA biosynthesis, lysine biosynthesis, butanoate metabolism and phenylalanine metabolism were enriched in FFC pre-treated, Salmonella infected chicks (Fig. 9c). Linoleic acid metabolism was enriched between the FST and ST groups (Fig. 9d). These data indicated that linoleic acid metabolism is the most noteworthy metabolic pathway in the FFC-treated groups with or without Salmonella challenge. We mapped the metabolic pathway of linoleic acid based on the identified differential metabolites, as well as the relative amounts (means ± SD) of these metabolites in the four groups (Fig. 9e). The metabolites that affect the metabolic pathways of linoleic acid are primarily linoleic acid, 12,13-EpOME, and 12,13-diHOME; the relative amounts of these metabolites in the FT and FST groups were significantly higher than in the NT and ST groups. Notably, the relative levels of 12,13-EpOME and 12,13-diHOME were significantly higher in the FFC-pretreated group, but were negligible in the non-pretreated group (Fig. 9e).
Correlation between the differential gut microbiota and metabolites
After observing marked differences in metabolite content as well as the microbial composition after FFC pre-treatment, we tested for specific correlations between the microbial taxa and key metabolites. Spearman correlation analysis revealed an association between four bacterial genera and nine discriminant metabolites in FFC-pretreated chicks (Fig. 10a). Enterobacteriaceae was a taxon with strong correlation, particularly with linoleic acid, 12,13-EpOME, 12,13-diHOME and L-tyrosine (positive correlations), whereas only L-ascorbic acid was negatively correlated. Furthermore, Clostridium positively correlated with L-palmitoyl carnitine, linoleic acid, 12,13-diHOME and L-tyrosine, while the taxon negatively correlated with L-ascorbic acid, anandamide, and 4-pyridoxic acid. The genus Lactobacillus negatively correlated with L-palmitoyl carnitine, linoleic acid, 12,13-EpOME, 12,13-diHOME, and L-tyrosine, and positively correlated with L-ascorbic acid. Lastly, a weaker positive correlation was detected between Ruminococcus and metabolites 4-pyridoxic acid and gamma-aminobutyric acid. Canonical correspondence analysis (CCA) showed that Enterobacteriaceae was the most important bacterial taxon influencing linoleic acid metabolism (including linoleic acid, 12,13-EpOME, and 12,13-diHOME) after FFC pre-treatment (Fig. 10b). The correlation network between differential bacterial taxa and metabolites consisted of 13 nodes and 22 edges. We found that the metabolic pathway of linoleic acid has a strong positive correlation with Enterobacteriaceae, whereas Lactobacillus has a negative correlation with it (Fig. 10c).
As linoleic acid can be converted into conjugated linoleic acid (CLA) by Lactobacillus [54], and a significantly negative correlation between linoleic acid and Lactobacillus was observed in our study, we hypothesized that the non-FFC pretreated chicks (more abundance of Lactobacillus) may have higher CLA levels. However, CLA is an isomer of linoleic acid, so the use of untargeted metabolomics cannot distinguish between these compounds. Therefore, we employed targeted LC-MS to detect compounds including linoleic acid, 9c,11t-CLA, 10t,11c-CLA, 12,13-EpOME, and 12,13-diHOME (Fig. 10d). In line with the results of metabolic profiling, linoleic acid, 12,13-EpOME, and 12,13-diHOME levels were higher in the FFC-pretreated groups. Moreover, we observed a higher more CLAs concentrations in the cecal contents of non-FFC pretreated chicks, with the levels of 9c,11t-CLA significantly higher than that of10t,11c-CLA (Fig. 10d). Spearman correlation analysis showed a strong association between the abundance of Lactobacillus and CLA concentrations (Fig. S6). Collectively, these findings suggested that 12,13-EpOME and 12,13-diHOME may be the key metabolites for the propagation of gut colonization of Salmonella, whereas CLA may limit the Salmonella growth during infection.
Contrasting effects of conjugated linoleic acid and 12,13-diHOME on S. Enteritidis colonization
We pre-administered CLA and 12,13-diHOME to newly-hatched chicks before infecting them with S. Enteritidis (Fig. 11a). By 3 dpi, Salmonella loads in the caecum, spleen, and liver were significantly reduced in the chicks pretreated with CLA, whereas they were significantly increased by pre-treatment with 12,13-diHOME (Fig. 11b). Consistent with the fecal Salmonella loads, pre-treatment with CLA significantly reduced enteropathy at 3 dpi, whereas 12,13-diHOME significantly increased it (Fig. S8). Furthermore, CLA-pretreated chicks exhibited a decrease in intestinal permeability (serum D-lactate, DAO and LPS levels), and pro-inflammatory factors (IL-1β, IL-6, IL-8, TNF-α and IFN-γ), as well as a significant increase in IL-10 levels. Notably, 12,13-diHOME-pretreated chicks exhibited contrasting results (Fig. 12). We also compared the effect of these two metabolites on the expression of genes related to intestinal barrier function after Salmonella infection (Fig. 13). We found that CLA significantly increased the expression of ZO-1 and Occludin, whereas the 12,13-diHOME significantly reduced the expression ZO-1, Occludin, Claudin1, and MUC2. CLA also significantly increased the expression of IL-17A (Fig. 13). To evaluate whether orally administered CLA and 12,13-diHOME reach the gut lumen, we quantified their concentrations in the cecal contents, and observed a significant increase in CLA and 12,13-diHOME relative to non-treated chicks (data not shown). Together, these results demonstrated that pretreatment with CLA attenuates Salmonella colonization, whereas 12,13-diHOME promotes it (Fig. 14).