Antibiotic residues and resistance in the environment

Here you find guidance, methods and tools to measure antibiotic residues and resistance in the environment.

Data from the environment is important for understanding the full magnitude of the antibiotic resistance problem and for planning targeted actions from a One Health perspective. Data from the environment can help to:

(i) quantify concentrations of antibiotic residues

(ii) assess the spread of resistant bacteria and resistance genes

(iii) identify hotspots of resistance, and

(iv) detect potential health risks.

To learn more about drivers and consequences of antibiotic resistance in the environment, visit  Understand –  Antibiotics in the Environment.

What and how to measure?

Antibiotic resistant bacteria, antibiotic resistance genes, and antibiotic residues in the ecosystem can be monitored using different analytical methods. Water, sediment, and soil samples are commonly used to identify antibiotic concentrations and resistance elements.

Antibiotic resistant bacteria

Cultured-based methods

Culture-based methods are well-standardized for detecting specific bacteria, determining resistance levels and providing information on potential risks to human health. These techniques primarily apply to bacteria that can be isolated and cultured. It is important to highlight that the majority of environmental bacteria cannot easily be cultured. Therefore culture methods alone cannot comprehensively characterize antibiotic resistance in a specific environment.

Antibiotic resistance genes

Most environmental microbes cannot be cultured in the laboratory due to challenges in simulating environmental conditions. Over the years, this has caused the larger reservoir of resistance to be overlooked. The development and harnessing of molecular methods on environmental samples has allowed identification of antibiotic resistance genes in non-cultivable bacteria.

Molecular methods

Polymerase chain reaction (PCR) and metagenomic analysis have been used for detecting genes resistant to antibiotics in environmental samples. With next-generation sequencing (NGS) it is possible  to obtain millions of reads representing the bacterial community and to characterise the resistomes.

Antibiotic residues

By analyzing antibiotic concentrations in wastewater treatment plants, soils, surface waters, and groundwaters, we can gain a better understanding of how antibiotics persist once they reach the environment. This can also provide a snapshot of the classes and quantity of antibiotics used in humans and in food animal production.

• Susceptibility testing: These tests utilize bacteria that are sensitive to antibiotics to determine the presence of specific antibiotics in a sample. Disc diffusion involves placing discs coated with the sample of interest on plates with bacteria; a halo of cleared growth around the disc indicates that an antibiotic killed the bacteria.
• Physical and chemical assays: Antibiotics can be identified in a complex sample using physical and chemical assays that focus on the characteristics of the molecules such as their size, charge, binding characteristics, or reactive properties. Antibiotic concentrations in milk, serum, meat, soil, and water samples have been quantified using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS).
• Immunoassays: Antibiotics can be detected in highly complex liquid or processed solid samples using immunoassays. This is based on the specificity of interactions between antibodies and the antibiotic of interest.

Pharmaceutical pollution of the world’s rivers

Description: This study from the University of York analyzed pollution from 61 active pharmaceutical ingredients (API) in 258 rivers around the world in 2021. 13 of these were antibiotics.

Place/settings: Water samples were collected from 1,052 locations in 104 countries.

Sampling: Standardized water-sampling kits were used. Videos and a step-by-step guide on the sample collection procedure were provided to all collaborators. A sampling campaign with 5-10-sampling sites along the rivers were designed by the different collaborators. Typically sites would be upstream, within, and downstream a populated area plus at additional points of interest like wastewater treatment discharges Two samples were collected at each site and all sites within one river campaign were sampled on the same day. After sample collection, samples were frozen and sent to a single centre for analysis.

Analysis method: High-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Findings: Concentrations of 9 of the 13 antimicrobials were higher than the proposed safe limits in regards to resistance development  for at least one sampling site. More specifically, ciprofloxacin exceeded the safe limit at 64 sites and metronidazole was 300 times higher than the safe target at one sampling site. The samples most polluted with active pharmaceutical ingredients were from low- to middle-income countries predominantly in sub-Saharan Africa, south Asia, and South America.

Selected Resources