Research on Drivers of Antibiotic Resistance in the Neuse River Basin

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Maya doing heavy metal resistance tests on cultured samples

Antimicrobial compounds are life-saving tools to treat infections caused by bacteria, viruses, or fungi. As far back as 2600 B.C.E., ancient Egyptians used heavy metals like copper as antimicrobial agents; Greeks, Romans, Aztecs, and other major civilizations also used heavy metals as antimicrobials to treat illness and disinfect water.

Intentional use of heavy metals as antimicrobials has diminished since the discovery and commercial-use of synthetic antibiotics in the mid-20th century; however, heavy metals from mining and smelting, agriculture, and domestic and industrial wastewater can enter the environment and place selective pressures on microorganisms. As organisms are exposed to heavy metals and/or other antimicrobial compounds, they evolve and adapt. These adaptations help organisms survive in the presence of widely available synthetic antibiotics, contributing to urgent and emerging health hazards associated with antimicrobial resistance. Life-threatening bacteria, fungi, and viruses can survive and grow in the presence of the very medicines designed to inactivate them.

As antimicrobial resistance proliferates in microorganisms across environmental compartments, people are at higher risk of exposure through a range of activities. In fact, at the current rate of spread, one estimate has suggested that deaths linked to antimicrobial resistance (10 million) could exceed cancer-related deaths (8.2 million) by 2050. Among those affected, immunocompromised groups (e.g., people who undergo chemotherapies, burn-wound victims, and people with diabetes) are most at risk of dying from life-threatening infections.

Additionally, antimicrobial resistance and its impacts on animal health are a potential risk to economic stability. According to the 2016 Review on Antimicrobial Resistance (AMR), global GDP could fall by an estimated 2% to 3.5% due to AMR, costing the world up to $100 trillion.

In surface water environments, the concentration of heavy metals is several orders of magnitude higher than antibiotics. Past research shows heavy metal concentrations have an influence on antibiotic resistance, typically involving a process called “co-occurrence,” in which heavy metal resistance and antibiotic resistance occur in the same microorganism. Heavy metals can be toxic to bacteria at high concentrations, which, in turn, may lead bacteria to acquire and be genetically selected for traits that allow them to resist these metals. These traits also help to fight against antibiotics.

Among the most ubiquitous metals in surface waters are zinc and copper. Copper and zinc are additives in agricultural animal feeds and often wash from farms into surface waters through runoff. Furthermore, metals-based pesticides are another notable source of heavy metals in surface waters. Copper sulfate, for instance, is commonly used as an algicide in North Carolina, especially in wastewater treatment plants.  

In order to better understand the occurrence and co-occurrence of resistance to metals and synthetic antibiotics in environmental compartments, I undertook a study of AMR organisms in the Neuse River watershed. This work characterized the occurrence of antibiotic resistance and metals resistance in enterobacteriaceae (a group of Gram-negative bacteria that includes E. coli) in the Neuse River basin. I also sought to measure the co-occurrence of metals resistance and resistance to common synthetic antibiotics, and to determine which environmental factors, if any, are associated with antibiotic resistance in Enterobacteriaceae in surface waters. As part of this work, I took water samples and water quality measurements from four surface water sites in the Neuse River Basin: the Eno River, Morgan Creek, the Neuse River in Raleigh, and the Neuse River Estuary.

Samples were transported to the Water Institute’s environmental microbiology lab at UNC, where I cultured enterobacteriacea from each sample by membrane filtration, selecting isolates for further characterization. Gram-negative bacteria such as ennterobacteriaceae include species responsible for many diseases relevant to human and animal health, and some of these have become increasingly resistant to multiple first-line antibiotics over the past decade, making their occurrence a topic of particular interest.

By regrowing cells isolated from cultured bacterial colonies, I was able to test which bacteria are resistant to copper, zinc, or both, and which bacteria are resistant to certain antibiotics,using standard methods.

Analysis of these results showed associations between antibiotic resistance and occurrence of metal resistance, particularly resistance to zinc, among enterobacteraceae in the Neuse River watershed. These results did not indicate whether the association is causal. However, it is common for resistance to multiple different compounds to be transferred together from one organism to another when a common factor or a common genetic sequence contributes to multiple categories of resistance, whether through co-selection, horizontal gene transfer, or other means. If such mechanisms are important in the Neuse River watershed, this could suggest that increasing occurrence of metals such as zinc in the surface waters and sediments of the watershed might contribute to increased occurrence of antimicrobial resistance to drugs of public health importance among enterobacteriacea in the watershed as well. Many human activities in the watershed can contribute to increasing levels of zinc in other metals, including storm water runoff, runoff from animal feeding operations using zinc and other metals, discharge of metal-containing wastewater treatment plant effluent, and others. Further work may help understand whether such sources are important contributors of metals in surface water in the Neuse River watershed, whether these inputs are potentially associated with increased levels of metal resistance and/or antibiotic resistance among bacteria in surface waters in the watershed and, if so, whether there are potential public health implications and opportunities for prevention and management of AMR hazards in the watershed associated with these potential factors.


This work also found that water temperature was associated with antibiotic resistance and metal resistance among Enterobacteriaceae in collected samples. In other studies, higher temperatures appeared to enhance antibiotic resistance mechanisms in Gram-negative bacteria, and the significant association between antibiotic resistance and metal resistance at higher temperatures that we found suggests the potential for greater occurrence and/or proliferation of antimicrobial resistance in warmer waters.

In addition, high salinity appeared to have an association with resistance to at least three antibiotics in one species of Gram-negative bacteria studied. The significant associations of high temperature and salinity with antibiotic resistance is of interest because climate change continues to exacerbate these factors. As temperatures increase, sea levels rise and salinity tends to increase in estuarine systems like the Neuse. Our team also found associations between pH and mutidrug-resistance in some samples. A variety of natural and anthropogenic factors can influence surface water pH.

Looking ahead, understanding how metal concentrations in surfaces waters, other natural environmental factors, and human activities influence antimicrobial resistance in enterobacteriaceae in the Neuse River watershed and other North Carolina surface waters can help inform efforts to monitor and manage associated hazards in our state. Ultimately, this could help reduce the spread of antimicrobial resistance, protecting public health and our state’s economy.

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