Phenotypic Copper Resistance in Aerobic Intestinal Bacteria from Children with Different Levels of Copper-Exposure

Copper is an environmental element, essential for life, to which humans are exposed by inhalation, hand-to-mouth contamination, or ingestion of food and water. In this work, bacterial copper susceptibility (amount of copper able to inhibit bacterial growth) of aerobic bacteria from the intestinal microbiota of healthy children in Spain was explored. To establish the possible effect of children's exposure to copper in the selection of copper-resistant organisms, the prevalence of copper-resistance among bacteria isolated in stool samples of 233 children belonging to the INMA cohort of the Spanish Project for Environment and Childhood Research was studied. Stool samples were seeded into Szybalsky-type agar plates containing specific culture media and a gradient of copper sulphate (< 0.68 - 2.05 mM). Culture media was suitable for growth of Gamma-Proteobacteria (mostly Enterobacteriaceae), Enterococcus and Staphylococcus. Colonies growing at intermediate (IR: < 0.68 - 1.36 mM) and/or higher copper concentrations (HR: 1.36 - 2.05 mM) were characterized by MaldiTOF assays. Sixty different species of copper-resistant organisms were detected. For Enterobacteriaceae, HR colonies were detected in 64.5% of seeded fecal samples, mostly corresponding to genus Escherichia (77% of positive samples); Enterobacter (15%), Citrobacter (13%), and Klebsiella (4.7%). However, Escherichia coli populations have a significantly lower proportion of high copper resistant colonies (49%) than the ensemble of Klebsiella-Enterobacter- Citrobacter colonies (77%). In HR-colonies of Firmicutes, Enterococcus genus was found in 97.3% of fecal samples, predominantly E. faecium (86%, of the positive Enterococcus samples), E. faecalis (37%), and E. hirae (6.9%). No significant correlation was found between counts of HR-colonies and the copper concentrations found in dry hair of the children studied (10 - 30 mcg/g). Copper-resistant populations in the children's intestine might have evolved in the copper-rich external environment.