ANtibioticS and mobile resistance elements in WastEwater Reuse applications: risks and innovative solutions

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Heterogeneous photocatalysis using UVA-LEDs for the removal of antibiotics and antibiotic resistant bacteria from urban wastewater treatment plant effluents

Publication . Biancullo, Francesco; Moreira, Nuno F. F.; Ribeiro, Ana R.; Manaia, Célia M.; Faria, Joaquim L.; Nunes, Olga C.; Castro-Silva, Sergio M.; Silva, Adrian M. R.

Secondary urban wastewater samples were spiked with azithromycin (AZT), trimethoprim (TMP), ofloxacin (OFL) and sulfamethoxazole (SMX) at 100 mu g L-1 to investigate the efficiency of a TiO2-photocatalytic treatment using UVA-LEDs. Different operating parameters were studied, such as the irradiation conditions, catalyst load and the use of methanol as carrier solvent and radical scavenger. The most efficient conditions to treat spiked urban wastewater (4 LEDs symmetrically distributed and 1.00 g L-1 of catalyst) were also assessed on the removal of the antibiotics at real concentrations, as well as on the inactivation and regrowth of bacteria after 3-day storage (total and resistant heterotrophs, Escherichia coli and enterococci). Clindamycin (CLI) was targeted when SMX was not detected. One-hour treatment was enough to reduce the analysed antibiotics to values below the detection limits and to decrease the bacterial load by 2 log-units. Bacterial regrowth was observed for total heterotrophs, after the storage of photocatalytic treated wastewater, to values close to pre-treatment. However, the antibiotic resistance percentage of such stored wastewater was always similar or lower than that of secondary urban wastewater. Thus, the potential of this process as part of the tertiary treatment is demonstrated, but conditions must be adjusted to minimize microbial regrowth.

2019Journal article

Restricted access

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

Publication . Fortunato, Gianuario; Vaz-Moreira, Ivone; Becerra-Castro, Cristina; Nunes, Olga C.; Manaia, Célia M.

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.

2018Journal article

Open access

Inter-laboratory calibration of quantitative analyses of antibiotic resistance genes

Publication . Rocha, Jaqueline; Cacace, Damiano; Kampouris, Ioannis; Guilloteau, Hélène; Jäger, Thomas; Marano, Roberto B.M.; Karaolia, Popi; Manaia, Célia M.; Merlin, Christophe; Fatta-Kassinos, Despo; Cytryn, Eddie; Berendonk, Thomas U.; Schwartz, Thomas

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are widely distributed in the environment where they represent potential public health threats. Quantitative PCR (qPCR) is a suitable approach to detect and quantify ARGs in environmental samples. However, the comparison of gene quantification data between different laboratories is challenging since the data are predominantly obtained under non-harmonized conditions, using different qPCR protocols. This study aimed at carrying out an inter-laboratory calibration in order to assess the variability inherent to the qPCR procedures for quantification of ARGs. With this aim, samples of treated wastewater collected in three different countries were analysed based on common DNA extract pools and identical protocols as well as distinct equipment, reagents batches, and operators. The genes analysed were the 16S rRNA, vanA, blaTEM, qnrS, sul1, blaCTXM-32 and intI1 and the artificial pNORM1 plasmid containing fragments from the seven targeted genes was used as a reference. The 16S rRNA gene was the most abundant, in all the analysed samples, followed by intI1, sul1, qnrS, and blaTEM, while blaCTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1, intI1, blaTEM and qnrS the inter-laboratory variation was below 28% (3–8%, 6–18%, 8–21%, 10–24%, 15–28%, respectively). While it may be difficult to fully harmonize qPCR protocols due to equipment, reagents and operator variations, the inter-laboratory calibration is an adequate and necessary step to increase the reliability of comparative data on ARGs abundance in different environmental compartments and/or geographic regions.

2020Journal article

Restricted access

Assessing the risk of antibiotic resistance transmission from the environment to humans: non-direct proportionality between abundance and risk

Publication . Manaia, Célia M.

The past decade has witnessed a burst of study regarding antibiotic resistance in the environment, mainly in areas under anthropogenic influence. Therefore, impacts of the contaminant resistome, that is, those related to human activities, are now recognized. However, a key issue refers to the risk of transmission of resistance to humans, for which a quantitative model is urgently needed. This opinion paper makes an overview of some risk-determinant variables and raises questions regarding research needs. A major conclusion is that the risks of transmission of antibiotic resistance from the environment to humans must be managed under the precautionary principle, because it may be too late to act if we wait until we have concrete risk values.

2017Journal article

Restricted access

Continuous ozonation of urban wastewater: removal of antibiotics, antibiotic-resistant Escherichia coli and antibiotic resistance genes and phytotoxicity

Publication . Iakovides, I. C.; Michael-Kordatou, I.; Moreira, N. F. F.; Ribeiro, A. R.; Fernandes, T.; Pereira, M. F. R.; Nunes, O. C.; Manaia, C. M.; Silva, A. M. T.; Fatta-Kassinos, D.

This work evaluated the removal of a mixture of eight antibiotics (i.e. ampicillin (AMP), azithromycin (AZM), erythromycin (ERY), clarithromycin (CIA), ofloxacin (OFL), sulfamethoxazole (SMX), trimethoprim (TMP) and tetracycline (TC)) from urban wastewater, by ozonation operated in continuous mode at different hydraulic retention times (HRTs) (i.e. 10, 20, 40 and 60 min) and specific ozone doses (i.e. 0.125, 0.25, 0.50 and 0.75 gO(3) gDOC(-1)). As expected, the efficiency of ozonation was highly ozone dose-and contact time-dependent. The removal of the parent compounds of the selected antibiotics to levels below their detection limits was achieved with HRT of 40 min and specific ozone dose of 0.125 gO(3) gDOC(-1). The effect of ozonation was also investigated at a microbiological and genomic level, by studying the efficiency of the process with respect to the inactivation of Escherichia coli and antibiotic-resistant E. coli, as well as to the reduction of the abundance of selected antibiotic resistance genes (ARG5). The inactivation of total cultivable E. coli was achieved under the experimental conditions of HRT 40 min and 0.25 gO(3) gDOC(-1), at which all antibiotic compounds were already degraded. The regrowth examinations revealed that higher ozone concentrations were required for the permanent inactivation of E. coli below the Limit of Quantification (

2019Journal article

Open access