Prolonged zinc exposure modulates biofilm metabolic activity and conjugation in Enterococcus faecalis

Zinc oxide (ZnO), including its nanoparticulate form (ZnONPs), is widely used in agriculture and accumulates in the environment, where it may impose sustained selective pressure on microbial communities. However, the impact of prolonged zinc exposure on horizontal gene transfer and conjugation dynamics in Enterococcus faecalis remains poorly understood.

We exposed Enterococcus faecalis OG1RF:pCF10 (donor) and OG1SSp (recipient) to prolonged zinc exposure (20 serial passages) and analyzed phenotypic and transcriptional changes associated with conjugation and virulence-related traits. Chronic exposure to ZnO and ZnONPs was associated with pronounced aggregation in the plasmid-carrying donor strain, reduced optical density values, and significantly lower recoverable CFU/mL at 24 h, although extensive clumping likely affected CFU recovery. Zinc exposure was also associated with increased metabolic activity within established biofilms, while gelatinase production and antibiotic susceptibility remained unchanged. ZnONP-adapted recipient cells showed a significant increase in conjugation frequency, whereas ZnO-adapted recipients and zinc-adapted donors showed non-significant upward trends. Notably, transcription of genes within the plasmid-encoded prgQ conjugation operon was increased even in the absence of exogenous pheromone stimulation. In contrast, short-term zinc exposure did not enhance plasmid transfer, indicating that increased conjugation required long-term adaptation rather than acute stress.

These findings indicate that prolonged zinc exposure is associated with altered aggregation, biofilm-associated metabolic activity, and conjugation dynamics in E. faecalis. However, the underlying mechanisms remain unresolved and may involve a combination of physiological, regulatory, and genetic adaptations arising from long-term exposure.