TY - JOUR
T1 - A rationale for the high limits of quantification of antibiotic resistance genes in soil
AU - Fortunato, Gianuario
AU - Vaz-Moreira, Ivone
AU - Becerra-Castro, Cristina
AU - Nunes, Olga C.
AU - Manaia, Célia M.
N1 - Funding Information:
This work is part of a project that has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 675530 .
Funding Information:
This work was supported by National Funds from FCT – Fundação para a Ciência e a Tecnologia, Portugal, through projects UID/Multi/50016/2013, WaterJPI/0001/2013 STARE – “Stopping Antibiotic Resistance Evolution”, by project POCI-01-0145-FEDER-006939 (LEPABE– UID/EQU/00511/2013), funded by European Regional Development Fund through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI), and by national funds through FCT – Fundação para a Ciência e a Tecnologia ; project “LEPABE-2-ECO-INNOVATION” – NORTE-01-0145-FEDER-000005, funded by Norte Portugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund .
Funding Information:
IVM and CBC were supported by the FCT grants SFRH/BPD/87360/2012 and SFRH/BPD/87152/2012 , respectively.
Funding Information:
IVM and CBC were supported by the FCT grants SFRH/BPD/87360/2012 and SFRH/BPD/87152/2012, respectively.
Publisher Copyright:
© 2018 Elsevier Ltd
PY - 2018/12
Y1 - 2018/12
N2 - 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, blaTEM, blaOXA, blaIMP, blaVIM) 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. The results suggest that the limits of quantification and detection of antibiotic resistance genes may be too high to infer about potential risks.
AB - 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, blaTEM, blaOXA, blaIMP, blaVIM) 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. The results suggest that the limits of quantification and detection of antibiotic resistance genes may be too high to infer about potential risks.
KW - LOD - Limit of detection
KW - LOQ - Limit of quantification
KW - Quantitative PCR
KW - Risk assessment
KW - Wastewater
UR - http://www.scopus.com/inward/record.url?scp=85054416046&partnerID=8YFLogxK
U2 - 10.1016/j.envpol.2018.09.128
DO - 10.1016/j.envpol.2018.09.128
M3 - Article
C2 - 30300875
AN - SCOPUS:85054416046
SN - 0269-7491
VL - 243
SP - 1696
EP - 1703
JO - Environmental Pollution
JF - Environmental Pollution
ER -