Methods
Methods Used to Quantify AMR in Wastewater
Methods
Wastewater AMR surveillance uses multiple complementary approaches.
Modern surveillance combines genomics, culture-based detection, chemical analysis, environmental metadata, and clinical datasets to provide a holistic view of AMR dynamics.
| Type of method | Methodology | Description | Pros. | Cons. | Refs. |
|---|---|---|---|---|---|
| Mass spectrometry-based | LC-MS | Liquid chromatography coupled with mass spectrometry (LC-MS) to elucidate antimicrobial compound concentrations in wastewater | By combining HPLC and MS, the strengths of both techniques can be used | Initial costs, requires skilled personnel to set up | [139] |
| Sequencing-based | Sequencing using Illumina | Next-generation sequencing of microbial genetic material in a wastewater sample using the Illumina platform | Technology used widely, lowest cost, wide range of instruments, lowest error rates | Long sequence runs, shortest read lengths, no real-time data access | [140,141] |
| Sequencing-based | Sequencing using Oxford Nanopore technology | Next-generation sequencing of microbial genetic material in a wastewater sample using the Oxford Nanopore platform | Fast sequencing, longest confirmed reads, small instrument footprint, lowest instrument and consumable cost, real-time data access | Highest error rate of all the platforms | [142] |
| Sequencing-based | Sequencing using Ion Torrent technology | Next-generation sequencing of microbial genetic material in a wastewater sample using the Ion Torrent platform | Fast run time, comparatively cheap, long reads possible | High error rate, lower overall data output | [143] |
| Sequencing-based | Sequencing using Pacific Biosciences technology | Next-generation sequencing of microbial genetic material in a wastewater sample using the Pacific Biosciences platform | Fast sequencing runs, long reads, real-time measurement of base incorporation | Largest instrument footprint, lower output per run, higher error rates | [144,145] |
| Sequencing-based | Metagenomic sequencing | Can be defined as the sequencing of all genomes in a sample | Gathers information on all genomes in a sample, discovery of novel organisms, no a priori data needed | DNA of environmental microorganisms cannot be extracted completely, the sequencing may miss low-abundance microorganisms, there is no “gold standard” for bioinformatic software | [146,147] |
| Sequencing-based | Sequencing using 16S region of microbial DNA | Sequencing of the microbial 16S rRNA region of genetic material using a sequencing technology | Targets and reads a region of the 16S rRNA gene which is found in all Bacteria and Archaea, relatively cheap, lots of computational pipelines available | Can only identify organisms that have a 16S rRNA gene, multicopy variation of the 16S rRNA gene, 16S rRNA gene variable regions cannot typically resolve species | [148,149] |
| Sequencing-based | Whole-genome sequencing | Sequencing of the genetic material of a single organism using sequencing technology. Can identify all the genes in a genome including ARGs, and contribute to high-resolution genome assembly and identification of bacterial species/strains | Provides a high-resolution, base-by-base view of the genome, lots of computational pipelines available | The processing involves a few extra steps compared to 16S rRNA sequencing, is more expensive, computationally intense | [46,150] |
| PCR-based | Measuring resistance genes using qPCR | Using quantitative polymerase chain reaction (qPCR) to quantify the quantity of AMR genes | Comparatively cheap (compared to most sequencing technologies), fast method of measuring resistance genes | Need prior sequence data of the specific target gene of interest, needs standard curve analysis, susceptible to impurities present in the sample | [151,152] |
| PCR-based | Using droplet digital PCR | Using droplet digital PCR (ddPCR) to quantify the quantity of AMR genes | Accurate absolute quantification of pathogens, less contamination, no need for standard curves, more resilient to inhibitory substances | Clinical application of ddPCR is still not popular, there are fewer references available | [144,153,154] |
| Culture-based | Culture-based methods | With or without antibiotic or selective media, allows for the identification of specific taxa | Relatively quick, cheap | Lacks resolution (number of taxa studied), ignores "unculturable" bacteria, low sensitivity (compared to molecular methods), works best for bacteria that replicate efficiently in rich media within 24 h, slow growing and viable but not culturable (VBNC) bacteria are not detected. | [54] |
Reference – Clarke et al., 2024