A rationale for the high limits of quantification of antibiotic resistance genes in soil

Abstract

The determination of values of abundance of antibiotic resistance genes (ARGs) per mass of soil is extremely useful to assess the potential impacts of relevant sources of antibiotic resistance, such as irrigation with treated wastewater or manure application. Culture-independent methods and, in particular, quantitative PCR (qPCR), have been regarded as suitable approaches for such a purpose. However, it is arguable if these methods are sensitive enough to measure ARGs abundance at levels that may represent a risk for environmental and human health. This study aimed at demonstrating the range of values of ARGs quantification that can be expected based on currently used procedures of DNA extraction and qPCR analyses. The demonstration was based on the use of soil samples spiked with known amounts of wastewater antibiotic resistant bacteria (ARB) (Enterococcus faecalis, Escherichia coli, Acinetobacter johnsonii, or Pseudomonas aeruginosa), harbouring known ARGs, and also on the calculation of expected values determined based on qPCR.The limits of quantification (LOQ) of the ARGs (vanA, qnrS, bla TEM , bla OXA , bla IMP , bla VIM ) were observed to be approximately 4 log-units per gram of soil dry weight, irrespective of the type of soil tested. These values were close to the theoretical LOQ values calculated based on currently used DNA extraction methods and qPCR procedures. The observed LOQ values can be considered extremely high to perform an accurate assessment of the impacts of ARGs discharges in soils. A key message is that ARGs accumulation will be noticeable only at very high doses. The assessment of the impacts of ARGs discharges in soils, of associated risks of propagation and potential transmission to humans, must take into consideration this type of evidence, and avoid the simplistic assumption that no detection corresponds to risk absence.

Performance of polycarbonate, cellulose nitrate and polyethersulfone filtering membranes for culture-independent microbiota analysis of clean waters

“…Quantitative PCR is considered a sensitive method, capable of detecting low gene copy numbers, however the limits of quantification are limited by the amount of DNA that can be recovered from the samples to analyze [4,17]. Clean waters are a good example of samples that contain low loads of biomass and low yields of DNA extraction, always requiring a concentration step, normally using membrane filtration [18,19].…”

Comparison of Culture- and Quantitative PCR-Based Indicators of Antibiotic Resistance in Wastewater, Recycled Water, and Tap Water

“…clinical samples, groundwater, wastewater, manure, and soil [11,12,13,14,15,16,17,18,19,20]. Nevertheless, qPCR is not exempt from biases, which include the inability to ascertain gene expression (when DNA based), the potential for qPCR inhibition, and the inability to directly discriminate between extracellular and intracellular DNA [13,21,22,23,24].…”

Development and validation of novel PCR primers for identification of plasmid-mediated colistin resistance (mcr) genes in various environmental settings

“…For some genes in a given sample it is impossible to perform their quantification (since the copy number of the gene is below the limit of quantification [LOQ]) or detection (since the gene occurs in a quantity below the limit of detection [LOD]). Such situation was, for example, observed for ARGs of clinical relevance is soils (Fortunato et al, 2018).…”