Trace Metal Exposure from Dietary Supplements Differentially Inhibits Gram-Positive and Gram-Negative Bacterial Growth

Implications for Gut Microbial Homeostasis

Abstract

Dietary supplements can carry trace metal contamination, yet this hazard is rarely screened. Copper, zinc, and iron enter multivitamin, herbal, and algal products through raw material residues or deliberate fortification. Inductively coupled plasma mass spectrometry (ICP-MS) reports metal content but not biological toxicity, which depends on bioavailability and matrix chemistry. We built a low-cost dual-species bacterial biosensor that reads out metal toxicity as a growth-inhibition signature. Escherichia coli C600 (Gram-negative) and Bacillus megaterium (Gram-positive) were incubated with three reference metals (CuSO₄·5H₂O, ZnSO₄·7H₂O, FeSO₄·6H₂O) at 0, 1, 10, and 50 ppm, and with three commercial supplements (Ashwagandha, Multivitamin, Spirulina) as 10% v/v aqueous extracts. Growth was tracked over 6 hours by OD₆₀₀ in liquid cuvettes at 90-minute intervals and by endpoint colony counts on metal-loaded LB agar. One-way ANOVA with Tukey HSD and two-way ANOVA tested concentration and species × concentration effects. All six species × metal ANOVAs were significant (p < 0.001), and B. megaterium was 4–5× more sensitive than E. coli to Cu and Zn. Species × concentration interactions were also significant by two-way ANOVA (p < 0.01). Multivitamin extract inhibited growth to a level comparable with ~10 ppm Cu exposure; Spirulina suppressed apparent OD without a matching drop in colony count, which identified that signal as pigment interference rather than toxicity. The Gram-type-specific inhibition fingerprints support the dual-species, dual-readout platform as a rapid biological screen for trace metal contamination in dietary supplements.