Florfenicol exposure increases susceptibility to S. enteritidis infection
We established a study design (Fig. 1a) in which SPF chicks were fed either the FFC-treated (FT), the S. Enteritidis-infected (ST) or simultaneous treatment of S. Enteritidis and FFC (FST). Under the SPF environment, all chicks were cultured negative for Salmonella spp. until experimental infection with S. enteritidis and 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 the survival and pathogenicity of S. enteritidis. Therefore, we examined S. enteritidis levels in the caecum, spleen and liver and found that the antibiotic (FFC) could promote S. enteritidis colonization and translocation in the intestines of chicks. 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 compared with those in the ST group at 3 days post-infection (dpi). Similar results were observed at day 18 (10 dpi) and day 25 (17 dpi), although their Salmonella loads are less than the 3 dpi (Fig. 1b). The above results indicate a robust influence of antibiotic upon susceptibility to oral S. enteritidis infection in neonatal chicks.
Florfenicol administration aggravates S. enteritidis-induced morphology and intestinal barrier function injury
The FFC intervention made the chicks more susceptible to Salmonella infection. It is possible that antibiotics disrupted the immature intestinal barrier homeostasis of the chicks, thus changing the intestinal permeability and making the chicks carrying more Salmonella in their internal organs. Therefore, we investigated the effects of FFC administration on S. enteritidis-induced intestinal morphology injury. H&E staining showed that the NT group exhibited an intact structure of the ileal mucosa, neat intestinal villi, deep crypts, and a clear and complete gland structure, as also observed in the FT group (Additional file 1: Figure S1a, b). The ST group showed that the structure of the ileal mucosa was incomplete, villi had a shorter length and sparse distribution, and the crypts were shallow (Additional file 1: Figure S1c). However, the FST group increased loss of mucosal structures, and atrophic crypts as well as lamina propria bowel edema can be observed (Additional file 1: Figure S1d). The histological injury score (Additional file 1: Figure S1e) was assessed based on the H&E staining images, and the score showed quantifiable results of tissue damage. The score of the chicks in the ST group (7.13 ± 0.44) was significantly higher than normal chicks (0.63 ± 0.18). Compared with the ST group, the FFC pre-administration (10.75 ± 0.45) significantly increased the injury score of the ileum. We also utilized SEM to examine the intestinal structure in different groups. The results showed that the NT group had complete ileal villi, which formed full and closely arranged structures (Fig. 2e), the FT group also had intact ileal villi, but the arrangement structure was relatively loose (Fig. 2f). As expected, the ileal villi in the ST group were damaged (Fig. 2g), whereas those in FST group showed more severely (Fig. 2h). These results suggest that, although FFC has less effect on intestinal morphology, it can aggravate the intestinal morphological damage in the presence of Salmonella invasion.
The effect of FFC on intestinal barrier function changes in ileum after S. enteritidis infection were also examined. We found that FFC can exacerbate the S. enteritidis-induced Ileum permeability increase (Fig. 3a-d). The serum DAO and LPS levels in the FT group were significantly (P < 0.001 and 0.05 respectively) higher than those in the NT group (Fig. 3c, d). In the case of Salmonella infection, serum D-lactate, DAO and LPS levels in both ST and FST group were significantly (P < 0.001) increased compared with that in NT group. However, FFC treatment significantly (P < 0.001) increased the serum D-lactate, DAO and LPS contents exposed to Salmonella infection (Fig. 3b-d). Alcian blue staining indicated that FFC significantly (P < 0.05) decreased the acidic mucin of ileum compared with NT group, as evident by the quantitative evaluation of positive Alcian blue staining (Fig. 2a, b) using the integral optical density measurement (Fig. 3a). Similarly, FFC treatment also significantly decreased (P < 0.01) mean density of acidic mucin (Fig. 2d; Fig. 3a) exposed to Salmonella infection. Transcriptional analysis of a range of relevant intestinal barrier genes was used to determine changes between these groups (Fig. 5). FFC treated significantly altered the gene transcription (Caludin1, IL-17A, IFN-α) in FT group. However, in the case of Salmonella infection, the FFC significantly reduced the expression of ZO-1, Occludin, Caludin1, MUC2 and TFF2, and significantly increased the expression of IL-17A, IL-22 and IFN-α. Furthermore, treatment with FFC significantly (P < 0.01) decreased SIgA secretion, but had no effect on serum IgG (Fig. 3e, f). Nevertheless, FFC treated reduced the SIgA secretion more seriously (P < 0.001) after Salmonella infection (Fig. 3f). These results indicate that FFC intervention, to some extent, increased the intestinal mucosal permeability of chicks, reduced mucosal immunity and significantly increased the degree of damage to the intestinal mucosal barrier after Salmonella infection.
The levels of Salmonella colonization are tightly interconnected with the trigger of intestinal inflammation . This suggested that the increased colonization levels might be linked to increased mucosal inflammation. Indeed, FFC treated chicks featured higher levels of gut inflammation after Salmonella infection (Fig. 4). This was verified by quantitative analysis of proinflammatory cytokines and anti-inflammatory cytokines in ileum tissue. Salmonella infection after FFC-pretreated significantly increased the level of the cytokines IL-1β (Fig. 4a), IL-6 (Fig. 4b), IL-8 (Fig. 4c), TNF-α (Fig. 4e), and IFN-γ (Fig. 4f), whereas IL-10 (Fig. 4d) was significantly decreased. Moreover, the inflammatory cytokines (IL-1β, IL-6, TNF-α and IFN-γ) were also significantly increased in the FT group. These results indicate that the FFC exacerbated the Salmonella-induced inflammatory response. It is possible that antibiotic-treated causes Gram-negative bacterium releases LPS, which promotes intestinal inflammation, and this can be confirmed by serum LPS levels. Moreover, FFC may also shifts the gut microbiota and metabolic profiling of neonatal chicks, causing microbiotic and metabolic disorders and support Salmonella colonization.
Florfenicol administration alters the gut microbiota
The composition and density of the gut microbiota play an important role in combating Salmonella invasion, and oral pretreatment with antibiotics decreases colonization resistance and leads to an obviously post-antibiotic expansion of the Salmonella loading in the gut . Thus, we hypothesized that the more Salmonella population observed in FFC-treated group might be linked to a disorder of the microbiota composition and density. To this end, comparative microbiota analysis of the cecal content of different groups at three different stages of infection (Day 3, 10 and 17 post-infection) was performed by 16S rRNA gene sequencing. Additional file 2: Figure S2 shows estimates of the diversity of the microbiota, presented as plots of the Shannon index, Observed Species and Pielou index measure of α-diversity. The α-diversity of the cecal microbiotas from chicks at 3 dpi was not neither affected by FFC treatment nor S. Enteritidis infection (Additional file 2: Figure S2a). However, a significantly decrease in alpha diversity was observed in FST group at 10 dpi (Additional file 2: Figure S2b). And Additional file 2: Figure S2c shows that the Shannon and Pielou index of the cecal microbial communities are significantly increase in the FST group at 17 dpi. These results indicated that the α-diversity of gut microbiota from chicks are not significant affected by a single FFC treatment or Salmonella challenge. However, the infection of Salmonella after pretreatment with antibiotics significantly disturbed the alpha diversity of chicks.
The results of phylum and genus distributions of microbial composition are shown in Additional file 3: Figure S3 and Additional file 4: Figure S4, respectively. Firmicutes (71.40 – 99.62%) dominated the chicks gut microbiota in the four groups at three different stages of infection (Additional file 3: Figure S3). At 3-, 10- and 17-days post infection, the FST group had the highest relative abundance of Proteobacteria (2.68%, 1.30%, and 1.32%, respectively) compared with other three groups (Additional file 3: Figure S3). And at 17-days post infection, compared with the NT group (27.40%), the FFC (7.76%) significantly reduced the relative abundance of Bacteroidetes, and Salmonella infection (22.27%) has less effect on Bacteroidetes. However, the infection of Salmonella after pretreatment with FFC almost limits the growth of Bacteroidetes (0.01%) (Additional file 4: Figure S4). We further applied the LEfSe method to identify specifically abundant bacterial taxa among these groups (only those taxa that obtained a log linear discriminant analysis [LDA] scores > 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 bacterial taxa were identified at three different stages of infection, respectively (Fig. 6). In the non-treated chicks, LEfSe highlights 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 compared with other three groups at all three different points. However, the other taxa were changed irregularly at different times in different groups. Moreover, the relative abundance of these biomarkers was showed in Additional file 5: Figure S5, and consistent results are obtained. We also established taxonomic cladogram at 11 day (3 dpi), and the relative abundance of taxa node in each group was showed in the form of a pie chart (only those taxa that the relative abundance > 0.1% were ultimately considered) (Fig. 7a). Similarly, the abundance ratio of Lactobacillus in the control group was significantly higher than the other three groups. Additionally, the abundance ratio of Enterobacteriaceae in the FST group was dominated among these four groups. Furthermore, at the genus level, Salmonella was only found in the challenged groups, and the abundance ratio of Salmonella in the FFC pretreatment group was significantly higher than that in the unpretreated group (Fig. 7a). Additionally, we determined the cecal loads of these biomarkers and two intestinal protective bacteria by quantitative PCR (qPCR) (Fig. 7b). At 11 day (3 dpi), the FFC pre-treatment significantly reduced the densities of total bacteria, Lactobacillus, clostridium butyricum and faecalibacterium prausnitzii. Although single Salmonella infection had no effect on densities in the cecal contents, Salmonella infection after pretreatment with FFC group harbored much higher densities of Enterobacteriaceae, and lower densities of Lactobacillus, Bacteroides, clostridium butyricum and faecalibacterium prausnitzii than the control group. At 25 day (17 dpi), the clostridium butyricum and faecalibacterium prausnitzii were present at equivalent densities in the cecal contents of four groups. However, the significant differences in the bacterial densities of total bacteria, Lactobacillus, Bacteroides and Enterobacteriaceae were still apparent between NT and FST group or ST and FST group (Fig. 7b). The Lactobacillus and Bacteroides are generally considered as the beneficial bacteria that provide protection for the gut, whereas the Enterobacteriaceae was known to be the potential pathogens of poultry and/or humans. These observations suggest that FFC exposure significantly decreased the abundance of Lactobacillus in chicks, and this inhibitory effect may provide a growth advantage for Enterobacteriaceae, especially Salmonella, in the gut of chicks.
The similarity of microbial communities (β-diversity) was visualized through PCoA of Bray-Curtis distances. At 3 dpi. The PCoA plots showed that microbial communities from Salmonella or FFC treated chicks clearly separate from those of the non-treated chicks. The first axis of the PCoA explained 19.0% of the variation in bacterial diversity while the second axis explained 13.0% (Fig. 7C). The first axis can roughly distinguish the antibiotic pre-treated chicks and non-pretreated chicks, and second axis can roughly distinguish the Salmonella infected birds and non-infected birds. The PCoA at 10 dpi showed that the microbiota composition was very similar between the NT and FT chicks, whereas the ST and FST group are still obviously distinguish from the NT group (Additional file 6: Figure S6a). Intriguingly, at 25 day (17 dpi), the PCoA demonstrated that both the microbiota composition of ST and FT group is tended to the NT group, whereas microbiota composition of FST group is still a striking divergence from the NT group (Additional file 6: Figure S6b). These findings suggest that a single FFC or Salmonella treatment can cause some changes in microbiota composition of chicks, and they would generally recover after two weeks. Whereas FFC pretreatment hindered the recovery from microbiota composition of chicks for 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 conducted metabolomic analysis by LC-MS to determine the differential levels of metabolites on day 11 (3 dpi) in cecal contents of different groups. The principal-coordinate analysis (PCA) score plot showed that the metabolome of NT group and ST group was significantly separated among four groups, whereas there was no clear distinction in cecal metabolites between the FT and FST group (Fig. 8). For further analysis, orthogonal projections to latent structures-discriminate analysis (OPLS-DA) and permutation test plot of OPLS-DA was carried out to explore the differences between these groups. As shown in Additional file 7: Figure S7, the OPLS-DA showed that the cecal metabolites of the NT group were clearly distinguished from those of the FT group (Additional file 7: Figure S7a), ST group (Additional file 7: Figure S7c), and FST group (Additional file 7: Figure S7e). In addition, there was also a clear separation between the FST group and ST group in cecal metabolites (Additional file 7: Figure S7g).
From the OPLS-DA models, we identified 72 differential metabolites between NT and FT group, 42 differential metabolites between NT and ST group, 69 differential metabolites between NT and FST group, and 57 differential metabolites between FST and ST group according to the threshold (VIP > 1, and p < 0.05; Welch’s t test). The significantly differential metabolites of these groups are shown in Supplementary Table 3. We next performed the pathway enrichment analysis based on these differential metabolites to comprehensively understand the effect of FFC on metabolism of chicks (Fig. 9). Linoleic acid metabolism, aminoacyl-tRNA biosynthesis, lysine biosynthesis, phenylalanine metabolism and lysine degradation were enriched after FFC treated (Fig. 9a). Arginine and proline metabolism, lysine biosynthesis, lysine degradation and D-glutamine and D-glutamate metabolism were enriched after Salmonella infected (Fig. 9b). Linoleic acid metabolism, aminoacyl-tRNA biosynthesis, lysine biosynthesis, butanoate metabolism and phenylalanine metabolism were enriched in Salmonella infection after FFC pretreatment group (Fig. 9c). Linoleic acid metabolism was enriched between the FST and ST group (Fig. 9d). The above results indicate that the linoleic acid metabolism is the most remarkable metabolism pathway in FFC treated group with or without Salmonella challenge. We next mapped the metabolic pathway of linoleic acid based on identified differential metabolites, and the relative amount (mean ± SD) of these metabolites in four groups was also showed (Fig. 9e). The metabolites that affect the metabolic pathways of linoleic acid mainly include linoleic acid, 12,13-EpOME and 12,13-diHOME, and the relative amount of these metabolites in the FT and FST group was significantly higher than the NT and ST group. Notably, the relative levels of 12,13-EpOME and 12,13-diHOME were significantly high in the FFC-pretreated group, but almost none in the non-pretreated group (Fig. 9e).
Correlation between the differential gut microbiota and metabolites
After finding marked differences in the content of metabolites as well as the microbial composition after FFC-pretreated, we analyzed whether there were any specific correlations between the microbial taxa and key metabolites. The Spearman correlation analysis revealed an association between four bacterial genera with nine discriminant metabolites in FFC-pretreated chicks (Fig. 10a). Enterobacteriaceae is a taxon with strong correlation, particularly with linoleic acid, 12,13-EpOME, 12,13-diHOME and L-tyrosine (positive correlations), while only L-ascorbic acid negatively correlated. Furthermore, the Clostridium positively correlated with L-palmitoylcarnitine, linoleic acid, 12,13-diHOME and L-tyrosine, while the taxon negatively correlated with L-ascorbic acid, anandamide and 4-pyridoxic acid. The Lactobacillus genus negatively correlated with L-palmitoylcarnitine, linoleic acid, 12,13-EpOME, 12,13-diHOME and L-tyrosine, and positively correlated with L-ascorbic acid. Lastly, a less strong positive correlation was detected between the Ruminococcus and 4-pyridoxic acid and gamma-aminobutyric acid. The CCA test showed that the Enterobacteriaceae was the most important bacterial factor influencing the linoleic acid metabolism (including linoleic acid, 12,13-EpOME, 12,13-diHOME) after FFC-pretreated (Fig. 10b). Additionally, the correlation network between differential bacterial taxa and metabolites is consists of 13 nodes and 22 edges. And the results also showed that the metabolic pathway of linoleic acid has a strong positive correlation with Enterobacteriaceae, while Lactobacillus negatively correlated (Fig. 10c).
Since linoleic acid can be produced by Lactobacillus into CLA , and in our study, there is a significant negative correlation between the linoleic acid and Lactobacillus. Therefore, we hypothesized that the non-FFC pretreated chicks (more abundance of Lactobacillus) may have more CLA contents. However, CLA is an isomer of linoleic acid, and the use of untargeted metabolomics detection cannot distinguish these substances, so we using the targeted LC-MS to detect these substances including linoleic acid, 9c,11t-CLA, 10t,11c-CLA, 12,13-EpOME and 12,13-diHOME (Fig. 10d). In line with results of the metabolic profiling, the contents of linoleic acid, 12,13-EpOME and 12,13-diHOME were higher in the FFC-pretreated groups. Moreover, we confirmed more CLAs concentrations in the cecal contents of non-FFC pretreated chicks, and the 9c,11t-CLA level is significantly higher than the10t,11c-CLA (Fig. 10d). Furthermore, the Spearman correlation analysis showed a strong association between the abundance of lactobacillus and CLA concentrations (Additional file 8: Figure S8). Collectively, these findings hinted that 12,13-EpOME and 12,13-diHOME may be the key metabolites for prolonging gut colonization of Salmonella, whereas the CLA may limit the Salmonella growth during infection.
Conjugated linoleic acid Attenuates, yet 12,13-diHOME promotes, the S. enteritidis Colonization
To address if CLA and 12,13-diHOME affect the Salmonella colonization more directly, we pre-administered these compounds to newly hatched chicks before infected with S. Enteritidis (Fig. 11a). By day 3 post-infection, the Salmonella loads in the caecum, spleen and liver were significantly reduced in the chicks pretreated with CLA, whereas those were significantly increased in the chicks pretreated with 12,13-diHOME (Fig. 11b). Consistent with the fecal Salmonella loads, the pre-treatment of CLA significantly reduced, whereas 12,13-diHOME significantly increased the enteropathy of chicks by 3 dpi (Additional file 10: Figure S10). Furthermore, CLA-pretreated chicks also exhibited decreases intestinal permeability (serum D-lactate, DAO and LPS levels), and decreases pro-inflammatory levels (IL-1β, IL-6, IL-8, TNF-α and IFN-γ), as well as a significant increase in IL-10 levels. Similarly, the 12,13-diHOME-pretreated chicks gets the opposite results (Fig. 12). We also compared the effect of these two metabolites on the expression of intestinal barrier function genes after Salmonella infection (Fig. 13). The results showed that the CLA significantly increased the expression of ZO-1 and Occludin, whereas the 12,13-diHOME significantly reduced the expression ZO-1, Occludin, Caludin1 and MUC2, and significantly increased the expression of IL-17A (Fig. 13). To evaluate whether orally administration CLA and 12,13-diHOME reach to the gut lumen, we quantified the concentrations of these substances in the cecal contents, and observed a significant increase in cecal contents levels compared with non-treated chicks (Additional file 11: Figure S11). Together, these results demonstrate that pretreated CLA can attenuates, yet 12,13-diHOME promotes the Salmonella colonization in the gut of neonatal chicks.