In vitro interactions of Alternaria mycotoxins, an emerging class of food contaminants, with the gut microbiota: a bidirectional relationship

Abstract

The human gut microbiota plays an important role in the maintenance of human health. Factors able to modify its composition might predispose the host to the development of pathologies. Among the various xenobiotics introduced through the diet, Alternaria mycotoxins are speculated to represent a threat for human health. However, limited data are currently available about the bidirectional relation between gut microbiota and Alternaria mycotoxins. In the present work, we investigated the in vitro effects of different concentrations of a complex extract of Alternaria mycotoxins (CE; containing eleven mycotoxins; e.g. 0.153 µM alternariol and 2.3 µM altersetin, at the maximum CE concentration tested) on human gut bacterial strains, as well as the ability of the latter to metabolize or adsorb these compounds. Results from the minimum inhibitory concentration assay showed the scarce ability of CE to inhibit the growth of the tested strains. However, the growth kinetics of most of the strains were negatively affected by exposure to the various CE concentrations, mainly at the highest dose (50 µg/mL). The CE was also found to antagonize the formation of biofilms, already at concentrations of 0.5 µg/mL. LC-MS/MS data analysis of the mycotoxin concentrations found in bacterial pellets and supernatants after 24 h incubation showed the ability of bacterial strains to adsorb some Alternaria mycotoxins, especially the key toxins alternariol, alternariol monomethyl ether, and altersetin. The tendency of these mycotoxins to accumulate within bacterial pellets, especially in those of Gram-negative strains, was found to be directly related to their lipophilicity.

Keywords: Adsorption; Biofilm; Chemical mixture; Lipophilicity; Microbiome.

Fig. 1

Modifications of the area under the curve (AUC) of growth curves induced by 24 h incubation of bacterial strains with different concentrations of the Alternaria extract. Reported numbers refer to mean ± SD of increase or reduction of AUC compared to the respective control (strain + DMSO). Percentages of DMSO varied from 0.1% to 0.001% (for treatments with 50 µg/mL or 0.5 µg/mL of CE, respectively). Each condition was tested in triplicate. Significant differences to the DMSO control were evaluated by Student’s t test (*p < 0.05)

Fig. 2

Representative growth curves expressed as optical density measured at 600 nm of different bacterial strains during treatment with the maximum (50 µg/mL, left column) and minimum (0.5 µg/mL, right column) concentration of the Alternaria extract (CE). Bacterial growth curves colored in black or red refer to the DMSO-treated or CE-treated strains, respectively

Fig. 3

Effects on biofilm formation induced by 24 h and 48 h incubation of strains with different concentrations of the Alternaria extract. Differences between treated and control samples were evaluated by Student’s t test (*p < 0.05; **p < 0.01). Cut-off values were calculated as follow: OD_cut-off = mean ODs of uninoculated medium + 3 times standard deviation. ^#Indicates no growth

Fig. 4

Bar charts showing the amount of the most affected mycotoxins recovered in pellets and supernatants of the tested strains after 24 h incubation with 25 µg/mL of the Alternaria extract. Data are reported as mean ± SD and differences between the total mycotoxin recovery in samples and media controls (media + CE) were evaluated by Student’s t test (*p < 0.05). AF A. finegoldii, AT A. timonensis, AM A. muciniphila, BC B. caccae, BE B. eggerthii, BT B. thetaiotaomicron, BV B. vulgatus, PD P. distasonis, EC E. coli, LH L. hominis, BL B. longum, B. sp. Bifidobacterium sp., CI C. innocuum, RB R. bicirculans

Fig. 5

Recoveries of mycotoxins from bacterial pellets. a Heatmap showing the average amount (in % compared to the total amount recovered) of mycotoxins found after 24 h incubation with 25 µg/mL of CE in pellets of all strains tested. b Double-axis plot showing mean ± SD (in % compared to the total amount recovered) of mycotoxin concentrations found in pellets of Gram-negative and positive strains (blue and light blue columns, respectively; left axis), and the mean value of theoretical lipophilicity (solid line; right axis) of mycotoxins. Significant differences between Gram-negative and -positive strains were evaluated by Student’s t test (*p < 0.05; **p < 0.01)

Fig. 6

Score-loading plot of the first two principal components from PCA highlighting strain distribution in the bidimensional space and the contribution of mycotoxin concentrations found in pellets and supernatants. AF A. finegoldii, AT A. timonensis, AM A. muciniphila, BC B. caccae, BE B. eggerthii, BT B. thetaiotaomicron, BV B. vulgatus, PD P. distasonis, EC E. coli, LH L. hominis, BL B. longum, B. sp. Bifidobacterium sp., CI C. innocuum, RB R. bicirculans