*4.3. The Effect of Single and Combined Stressors on the Metabolomic Diversity of Gut Microbiota*

The results also confirmed our second hypothesis, since NPs resulted in an increase and enrofloxacin resulted in the decrease in the overall carbon respiration rate of the *Daphnia* gut microbiota. This may be due to a changing bacteria abundance, their metabolic condition, and their ability of utilising different carbon sources. The decrease in the metabolic rate in the presence of enrofloxacin is consistent with the existing data on the negative effect of antibiotics on the bacteria metabolism, since there is a link between antibiotic-induced cellular respiration and bactericidal lethality. Antibiotics disturb the metabolic state of bacteria, which impacts the antibiotic efficacy [89,90]. The positive effect of NPs on the respiration of the bacteria in the *Daphnia* gastrointestinal tract may be due to the provision of an additional sorption surface, which not only facilitates the formation of bacterial biofilms, but may also affect the ability to degrade organic compounds that absorb on such surfaces. For example, the sorption of proteins on surfaces can change their conformation, making them more accessible to proteolytic enzymes and subject to faster hydrolysis [91]. Our results are the first to demonstrate the effect of NPs on the metabolic profile of the gut microbiota. Moreover, the results are also the first to demonstrate the interaction between the stressors on the metabolic profile of the gut microbiota, which was apparent in the increased inhibitory effect of enrofloxacin in the presence of NPs, and in the reduced positive effect of NPs in the presence of enrofloxacin.

The results also confirmed our third hypothesis, as the presence of NPs and enrofloxacin affected the metabolic fingerprints measured as the relative use of different carbon sources as compared to the control. The functional structure of the bacteria community had changed. The presence of each of the stressors resulted in a relative increase in the usage of carboxylic acids, amino acids, and carbohydrates and in the relative decrease in the usage of phosphorylated carbons and complex carbon sources with the control, which suggests that both the enrofloxacin and NPs forced the bacteria to exploit the easily digestible carbon sources [92]. In general, these results are in accordance with the existing literature. For instance, it has been shown that the presence of polystyrene NPs altered the carbohydrate metabolism of marine medaka fish gut microbiota [23]. However, Zhang et al. [51] did not show any significant effect of NPs on the predicted metabolic pathways of the gut microbiota in the same fish species. A picture of the negative impact of NPs on the metabolic fingerprint emerges from the research on the freshwater biofilm done with the Biolog EcoPlate method, in which the NPs' presence (1, 5 and 10 mg × <sup>L</sup>−1) reduced the microbial metabolic functional diversity. The total carbon metabolism remained constant with growing NP concentrations, but the utilisation of some specific carbon sources (e.g., esters) had changed [93]. It was revealed that the presence of sulfamethazine affected the function of the gut microbiota of medaka fish, so the carbohydrate metabolism was significantly decreased [23]. Zhang et al. [51] presented similar results, showing a decrease in the carbohydrate metabolism in marine medaka females who had been exposed to low concentrations of sulfamethazine (0.5 mg × <sup>g</sup>−<sup>1</sup> supplied in fish food); thus, also the lipid

and amino acid metabolism was enhanced under these conditions. In the murine model, other antibiotics, like amoxicillin, have been shown to elevate the expression of genes responsible for starch utilisation by *Bacteroides thetaiotaomicron* [94].

Moreover, the NPs and enrofloxacin which were acting together had a different effect on the metabolic fingerprints than the NPs and enrofloxacin which were acting separately, suggesting the existence of the interaction between the two stressors. The simultaneous presence of sulfamethazine and NPs also significantly altered the carbohydrate metabolism, as He et al. [23] discovered. However, it should be pointed out that the results from He et al. [23] and Zhang et al. [51] concerning the effects of antibiotics and NPs on changes in the gut microbial metabolism fingerprint were obtained by the bioinformatics prediction. Our results stem from the direct measurement of the microbial metabolic fingerprint as the function of different respiration intensities of various carbon sources by the *Daphnia* gut microbial community.
