Cefotaxime Exposure-Caused Oxidative Stress, Intestinal Damage and Gut Microbial Disruption in Artemia sinica

Abstract

Cefotaxime (CTX) is an easily detectable antibiotic pollutant in the water environment, but little is known about its toxic effects on aquatic invertebrates, especially on the intestine. Here, we determined the oxidative stress conditions of A. sinica under CTX exposure with five concentrations (0, 0.001, 0.01, 0.1 and 1 mg/L) for 14 days. After that, we focused on changes in intestinal tissue morphology and gut microbiota in A. sinica caused by CTX exposure at 0.01 mg/L. We found malondialdehyde (MDA) was elevated in CTX treatment groups, suggesting the obvious antibiotic-induced oxidative stress. We also found CTX exposure at 0.01 mg/L decreased the villus height and muscularis thickness in gut tissue. The 16S rRNA gene analysis indicated that CTX exposure reshaped the gut microbiota diversity and community composition. Proteobacteria, Actinobacteriota and Bacteroidota were the most widely represented phyla in A. sinica gut. The exposure to CTX led to the absence of Verrucomicrobia in dominant phyla and an increase in Bacteroidota abundance. At the genus level, eleven genera with an abundance greater than 0.1% exhibited statistically significant differences among groups. Furthermore, changes in gut microbiota composition were accompanied by modifications in gut microbiota functions, with an up-regulation in amino acid and drug metabolism functions and a down-regulation in xenobiotic biodegradation and lipid metabolism-related functions under CTX exposure. Overall, our study enhances our understanding of the intestinal damage and microbiota disorder caused by the cefotaxime pollutant in aquatic invertebrates, which would provide guidance for healthy aquaculture.

Keywords: Artemia sinica; cefotaxime; gut microbiota; intestinal tissue; oxidative stress.

Figure 1

The effects of CTX exposure at five different concentrations for 14 days on (a) T-AOC and (b) MDA in A. sinica. * and ** represent significant differences between the control group and the CTX treatment group. * p-value < 0.05, and ** p-value < 0.01. T-AOC = total antioxidant capacity, MDA = malondialdehyde.

Figure 2

Morphological changes in the appearance (a) and intestinal tissues (b) of A. sinica under 0.01 mg/L CTX exposure. CK = the control group, CTX = the 0.01 mg/L CTX treatment group.

Figure 3

Effects of 0.01 mg/L CTX on intestinal structure of A. sinica in (a) villus height; and (b) muscularis thickness. **** represents significant differences (p-value < 0.001) between the CK group and the CTX group. CK = the control group, CTX = the 0.01 mg/L CTX treatment group.

Figure 4

Relative abundance of (a) top 10 bacterial at phylum level; and (b) top 20 bacterial at genus level in A. sinica gut microbiota of the CK and CTX groups. CK = the control group, CTX = the 0.01 mg/L CTX treatment group.

Figure 5

Genera with significant differences in abundance of gut microbiota in A. sinica after 0.01 mg/L CTX exposure. * and ** represent significant differences between the CK group and the CTX group (* p-value < 0.05, ** p-value < 0.01). The values shown on heatmap were z-scores generated by z-normalization of relative species abundance. CK1, CK2 and CK3 were the three samples from the CK group, while CTX1, CTX2, CTX3 were the three samples from the CTX group. CK = the control group, CTX = the 0.01 mg/L CTX treatment group.

Figure 6

The cladogram to depict the key and most differentially abundant taxa associated with CTX exposure in A. sinica gut microbiota. Logarithmic LDA score = 3, and p-value = 0.05. CK = the control group, CTX = the 0.01 mg/L CTX treatment group.

Figure 7

Mean relative abundance of predicted functions at KEGG class 2 level in A. sinica gut microbiota of the CK and CTX group. CK = the control group, CTX = the 0.01 mg/L CTX treatment group.

Figure 8

Functional differences at KEGG class 3 level between the CK and CTX groups. CK = the control group, CTX = the 0.01 mg/L CTX treatment group.