Antibiotic resistance detection in wastewater breakthrough

Researchers at the University of Illinois Urbana-Champaign, USA, have developed a novel method for detecting antibiotic resistance genes in wastewater.

Growing concern over antibiotic resistance

Antibiotic resistance is a growing global concern, one that threatens our ability to prevent and treat bacterial infections in humans and animals. While antibiotics are powerful modern tools for combatting infections, bacteria change and adapt over time in response to antibiotic exposure, therefore decreasing effectiveness. Widespread overuse and misuse of antibiotics in the healthcare and food industries further accelerate this problem.

Beyond direct exposure to antibiotics, resistance is also passed between different bacteria through the transfer of small pieces of bacterial DNA called antibiotic resistance genes. There are over 5,000 identified genes with this ability, and they can be found in clinical samples, as well as bodies of water, originating from hospitals, farms, and sewage systems.

Developing a better method of detection

Now, civil and environmental engineering graduate student Yuqing Mao and Professor Helen Nguyen based at the university's Carl R. Woese Institute for Genomic Biology have developed a 'CRISPR-enriched metagenomics' method for the enhanced surveillance of antibiotic resistance genes in wastewater.

The method enhances our ability to track and monitor the spread of antibiotic resistance genes, lowering the detection limit by an order of magnitude, from 10-4 to 10-5, compared to standard metagenomics and during testing found 1,189 more antibiotic resistance genes and 61 more gene families which are low in abundance in wastewater samples.

Profession Nguyne told media: "Antibiotic resistance genes can reduce the life-saving power of drugs used to treat bacterial infections. Wastewater detection with clinical significance allows public health authorities and physicians to anticipate what is circulating in communities."

Replacing time consuming detective work

Wastewater contains numerous different antibiotic resistance genes mixed together with genetic material from various sources including humans, viruses, and bacteria. However, antibiotic resistance genes only make up a tiny percentage of the total DNA content, which means uncovering them in wastewater samples requires sensitive detection methods.

The most common technique is quantitative polymerase chain reaction, qPCR. This method uses RNA guides called primers to identify the specific DNA sequences of known antibiotic resistance genes, which are then amplified for detection.

Mao told media: "qPCR is a sensitive method that many people in public health are well trained to do, but it requires primer design and validation which is very time consuming. Since qPCR is used to pull out targeted gene sequences, all the other genetic material in the sample remains completely unknown."

The second method, metagenomics, is not as sensitive as qPCR, but captures a more complete story of the genetic information contained in a sample. This method involves breaking all the sample DNA into millions of smaller fragments which are then simultaneously sequenced. Computational algorithms piece together the full DNA sequences for comparison against databases to determine their identities.

Mao added: "Antibiotic resistance genes make up less than one percent of DNA in the sample. Using standard metagenomics methods, 99.9 per cent of the DNA detected is not associated with antibiotic resistance genes."

Enriching the samples

To enrich the volume of antibiotic resistance genes associated fragments in the samples, the researchers collaborated with, Joanna Shisler, from the university's Department of Microbiology. Together, they leveraged the CRISPR-Cas9 system, a gene editing tool.

Unlike with standard metagenomics method, CRIPSR-Cas9 allows for targeted fragmentation within antibiotic resistance genes. By designing a pool of 6,010 different guide RNAs that could specifically bind to DNA at different sites found in antibiotic resistance genes, the Cas9 protein could be directed to cut at these locations.

Mao concluded: "Our new CRISPR method increases the abundance of antibiotic resistance gene fragments in the sample, which increases their chances to be read and detected. CRISPR also has better potential for multiplexed assays than something like PCR because the molecular interaction is simple and straightforward for CRISPR."

The research was published by Water Research, under the title' Enhanced detection for antibiotic resistance genes in wastewater samples using a CRISPR-enriched metagenomic method'.