Biofilm-forming capacity of soil nitrogen-fixing bacteria and related gene expression under florfenicol combined with copper environmental stress

Objective  The ecotoxic effects of florfenicol and copper (Cu) residues on soil nitrogen-fixing bacteria were studied to provide a basis for evaluating the environmental risks of veterinary drugs.

Method  A dominant nitrogen-fixing bacterium RpEC2071 was isolated from peanut root enclosure and treated under florfenicol and Cu stress. We set the blank group (0 μg/mL), florfenicol group (40 μg/mL), Cu group (200 μg/mL) and mixed group (flufenicol 40 μg/mL, Cu 200 μg/mL), and collected samples at multiple time points after dosing. The phenol-sulfuric acid method and 96-well microplate method were used to study the effects of florfenicol and Cu alone or in combination on the production of extracellular polysaccharides and biofilm formation of nitrogen-fixing bacterium. RT-qPCR was used to determine nitrogen-fixing bacterium biofilm formation and the mRNA expression levels of nitrogen fixation-related genes.

Result  Florfenicol and Cu alone promoted the formation of biofilms, and the biofilm formation capacity of both was about twice that of the blank group. Under mixed stress, the biofilm formation was inhibited, and the biofilm formation capacity of the blank group was 3.1 times that of it. The results of extracellular polysaccharide secretion were basically consistent with the results of the determination of biofilm formation ability. Biofilm-related genes were significantly positively correlated with nitrogen metabolism regulatory genes, and the addition of florfenicol and Cu changed the expression levels of genes such as fliQ,ntrX andnnrR, and it would produce synergistic or antagonistic effects after the combination treatment of florfenicol and Cu.

Conclusion  The individual or combined stress of florfenicol and Cu affects the expression of biofilm-related genes in nitrogen-fixing bacteria RpEC2071, which in turn affects its ability to form biofilm. This research reveals the ecotoxic potential of veterinary drugs and heavy metal residue stresses on soil nitrogen-fixing bacteria, using florfenicol and Cu as examples. As a result, this can lead to the impairment of the nitrogen-fixing ecosystem in soil.