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Measure  –  Antibiotic resistance

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.

Box image showing the antibiotic resistance parameters you can measure in the environment and overview of techniques
Antibiotic resistance parameters you can measure in the environment and overview of techniques.

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.

Escherichia coli and Enterococcus spp. are examples of fecal indicators bacteria that shuold be monitored in water and wastewater. In 2021, the WHO and the Advisory Group on Integrated Surveillance on AMR (AGISAR) created a standardised protocol to monitor extended spectrum B-lactamase (ESBL) producing E. coli as a key indicator across the One Health spectrum in low resource settings.

Pseudomonas aeruginosa and Aeromonas spp. are also sewage-dwelling bacteria that could be considered given their relevance as informative antibiotic resistance indicators.

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.

Figure 2. Examples of bacterial genes that confer resistance to different antibiotic classes. Information source: Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance.

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.

Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance

Description:  This study aimed to compare resistomes from various countries to improve understanding of the global transmission patterns of individual resistance genes.

Place/Setting: 243 cities in 101 countries.

Sampling: 757 samples of raw, untreated, urban sewage prior to any processing were collected and frozen by project partners worldwide. Sampling was conducted twice a year (June and November) from 2016 to 2019. All samples were shipped to a single centre for analysis.

Analysis method: Metagenomic sequencing analysis.

Findings: Streptococcus, Acinetobacter, Klebsiella, Pseudomonas, and Acidovorax were among the most common bacterial genera found in the samples. There was evidence of 557 distinct resistance genes. Abundances differed between sites and continents. There were regional  patterns in resistomes that varied by drug class. The analysis also revealed patterns of global transmission as well as geographic limitation in the spread of certain common resistance genes.

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 assaysAntibiotics 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.

Sampling sites for analysed API pollution in 258 of the world’s rivers.

Selected Resources

Tools and methodology

Resources Description
EMBRACE-WATERS statement: Recommendations for reporting of studies on antimicrobial resistance in wastewater and related aquatic environments Article providing guidance on how to standardize antimicrobial resistance studies in the wastewater sector.
WHO integrated global surveillance on ESBL-producing E. coli using a “One Health” approach Protocol that includes standard methodologies on ESBL-producing E.coli for the human, food chain, and environmental sectors to be implemented in LMICs to facilitate the establishment of an integrated multisectoral surveillance on AMR.
Antimicrobial Resistance Monitoring of Water Environments: A Framework for Standardized Methods and Quality Control Article/framework for standardizing AMR monitoring of wastewater, recycled water, and surface water, leveraging the expertise of multiple stakeholders (from academia to governments).
Pharmaceutical pollution of the world’s rivers Article and methodology. Global-scale study that describes pollution of active pharmaceutical ingredients (API) in the rivers of >50% of the world’s countries using a sensitive and internationally validated sampling and analytical method.

A previous study outlines the method further. Instructional videos are also available online 1) Global Monitoring of Pharmaceuticals Project: Sample Collection Video 2) Global Monitoring of Pharmaceuticals Project_Collection of water in the sampling bucket.

A framework for standardized qPCR-targets and protocols for quantifying antibiotic resistance in surface water, recycled water and wastewater Review that describes which gene targets are most commonly measured by qPCR to quantify antibiotic resistance in surface water, recycled water, and wastewater.
Trends of pharmaceutical residues in rivers, suspended particular matter and fish – New insights by new analytical methods for active substances, their metabolites and transformation products Report from German Environment Agency highlighting innovative analytical methods for quantifying high-priority pharmaceutical residues in water, sediment, suspended matter, and biota.
A conceptual framework for the environmental surveillance of antibiotics and antibiotic resistance Journal article/Framework for how to use environmental samples for surveillance of antibiotic consumption and antibiotic resistance. The framework contains five objectives of surveillance together with proposed markers and sampling sites.
Methods for field measurement of antibiotic concentrations: limitations and outlook Review that describes current methods (microbiological assays, physical and chemical assays, immunoassays, aptasensors and biosensors) to measure antibiotic concentrations and their applicability for on-site use.

Data and reports

Resources Description
Bracing for Superbugs: Strengthening environmental action in the One Health response to antimicrobial resistance Report from UNEP that synthesizes current evidence and knowledge gaps on the environmental dimension of AMR. It analyzes three economic sectors and their value chains: pharmaceuticals and other chemicals, agriculture and food, and healthcare.
The development of an integrated environment–human risk approach for the prioritisation of antibiotics for policy decisions Article that proposes a ranking of antibiotics based on an integrated environment–human risk approach, and using data from China. Antibiotics are ranked based on different risk scores such as overall risk, antibiotic resistance risk to environment, ecotoxicity risk and antibiotic resistance risk to human health.
Global Analysis and Assessment of Sanitation and Drinking-Water (GLAAS) Reports. GLAAS is a UN-Water initiative implemented by WHO. The objective is to provide policy- and decision-makers at all levels with a reliable, easily accessible and comprehensive reports of WASH systems analysis around the world in order to support making informed decisions for sanitation, drinking-water and hygiene.
Database “Pharmaceuticals in the environment” Database. A downloadable and searchable database of worldwide Measured Environmental Concentrations (MEC) of pharmaceuticals published in peer-reviewed journals from 2017 to 2020. Allows you to search by substance or region to find reports.
Industrial Wastewater Treatment Technology Database (IWTT) Database of wastewater treatment technologies. Search for pilot and full-scale technologies by industry (such as hospital), treatment technology (such as UV), or pollutant (such as different antibiotics). Curated by the US EPA.
Pharmaceuticals in the environment initiatives Database of initiatives and technology pilots in EU Member States to address pharmaceuticals in the environment and pharmaceutical waste (including in wastewater). Searchable by areas of interest (for example AMR), geographic area, country etc. Curated by Health Care Without Harm Europe (Safer Pharma campaign).
Pharmaceuticals in drinking water Report from WHO provides guidance and recommendations for managing the emerging concern about pharmaceuticals (including antibiotics) in drinking water.
European Nucleotide Archive Database. Open-access platform for the management, sharing, archiving, and dissemination of sequence data. Global surveillance of antibiotic resistance can be found in the following: – Sample rounds can be found under PRJEB40798, PRJEB40816, PRJEB40815 and PRJEB27621. -Sequencing data from sampling sites are deposited under PRJEB51229.
The Comprehensive Antibiotic Resistance Database Database with reference sequences to support analysis of resistance genes, their products, and associated phenotypes in for example environmental samples.


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