Antimicrobial and Heavy Metal Resistance in Escherichia coli from Pig Fecal Samples

Abstract

Antimicrobial resistance (AMR) has been driven mainly by antibiotic use, but other environmental pressures, such as heavy metals and biocides, can also drive resistance through a process known as co-selection. Some of these metals, historically or indirectly introduced into agricultural settings and still present in soil and water (including residual mercury), may help maintain resistant bacteria even when antibiotic exposure is reduced. Although this potential link has been suggested, it still remains underexplored in livestock-associated bacteria. This study investigated the relationship between antibiotic and heavy metal resistance in Escherichia coli (a standard indicator organism for AMR monitoring) isolated from pig fecal samples. Given that frozen storage is often used in research and surveillance, we compared freshly collected and long-term frozen samples to test whether storage conditions influenced bacterial culturability. We specifically asked whether archived/biobanked fecal material, collected for genetic work and not preserved for culture, could still be used to assess AMR and possible co-selection retrospectively. The work followed two complementary lines of investigation: first, culture-based phenotypic testing of E. coli isolates for resistance to antibiotics (oxytetracycline and streptomycin) and metals (copper and mercury); and second, metagenomic analysis of fecal datasets to assess the broader resistome genetically (AMR potential). The metagenomic approach not only provided an overview of resistance gene diversity but also allowed us to explore whether antibiotic and metal resistance genes occurred within the same samples and how these patterns compared with the phenotypes observed in cultured bacteria. The results showed that freezing at −80 °C reduced bacterial culturability in water samples to almost zero within the first two months, but fecal samples remained much more resilient. Even after extended storage, fecal material retained a substantial fraction of culturable bacteria, making it a reliable source for downstream resistance testing. Within the freshly collected fecal samples, several E. coli strains displayed resistance to antibiotics and metals, and the metagenomic analysis of fecal material confirmed the presence of multiple resistance genes related to antibiotic, metal, and biocide tolerance, although clear genetic co-occurrence between them could not be demonstrated. These findings suggest that metals in livestock environments may play a ‘’hidden role’’ in sustaining AMR. This highlights the importance of considering antibiotics, metals, and biocides as concurrent pressures. Moreover, co-resistant E. coli can be readily isolated from pig feces under normal production conditions. By combining field sampling, culture-based testing, and metagenomic analysis, this study contributes to a more comprehensive understanding of AMR’s environmental drivers and supports the development of improved surveillance strategies in agriculture.