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Investigating the impact of UV-C/H2O2 and sunlight/H2O2 on the removal of antibiotics, antibiotic resistance determinants and toxicity present in urban wastewater

Publication . G. Michael, Stella; Michael-Kordatou, Irene; Nahim-Granados, Samira; Polo-López, Maria Inmaculada; Rocha, Jaqueline; Martínez-Piernas, Ana B.; Fatta-Kassinos, Pilar; Manaia, Célia M.

This work aimed at exploring the impact of UV-C/H2O2 and sunlight/H2O2 processes, applied at pilot scale, on removing: (i) ciprofloxacin and sulfamethoxazole, (ii) cultivable Escherichia coli and Pseudomonas aeruginosa grown in the presence and absence of sub-minimal inhibitory concentrations of ciprofloxacin and sulfamethoxazole and (iii) the genes 16S rRNA and selected antibiotic resistance genes (ARGs) (i.e., sul1, blaCTX-M, qnrS, tetM, etc.) from urban wastewater. The major antibiotic transformation products (TPs) formed, were elucidated and the chronic toxicity of the whole effluent mixture against Vibrio fischeri was evaluated. The capability of the processes, in terms of the elimination of the antibiotics present in urban wastewater, varied among the two light sources used: both antibiotics were fully removed during UV-C/Η2Ο2, whereas only ciprofloxacin was removed during the sunlight/H2O2. The photo-transformation of the antibiotics led to the identification of 21 and 18 TPs of ciprofloxacin and sulfamethoxazole, respectively, while all of them retained their core moiety, responsible for the antibacterial activity. All the UV-C/H2O2-treated samples were found to be toxic, whereas the luminescence of V. fischeri was not inhibited when tested in the sunlight/H2O2-treated samples. During both processes, E. coli, P. aeruginosa and the colonies of these species still viable in the presence of antibiotics, were successfully inactivated to values below the detection limit. However, sunlight/H2O2 has not achieved complete disinfection, as regrowth of E. coli and P. aeruginosa colonies was observed after 48 h of storage of the treated effluent. Finally, none of the technologies tested was able to completely remove the target ARGs, confirming their inability to prevent the spread of resistance determinants to the environment.

2020Journal article

Restricted access

Neighbor urban wastewater treatment plants display distinct profiles of bacterial community and antibiotic resistance genes

Publication . Fernandes, Telma; Vaz-Moreira, Ivone; Manaia, Célia M.

Urban wastewater treatment plants (UWTPs) are among the major recipients of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues in urban environments. Although during treatment, bacteria of human and animal origin are removed, some are able to survive, persisting in the final effluent. The occurrence of these bacteria, especially those harboring ARGs, may have a direct impact on the quality of the treated wastewater that is returned to the environment. In this study, we aimed to assess if the final effluent bacterial communities of three UWTPs (PT1, PT2, and PT3) located next to each other were distinct and if such differences were related with the antibiotic resistance profiles. It was observed that the bacterial community (16S rRNA gene Illumina sequencing) and load of selected ARGs of final effluent differed among the three UWTPs, irrespective of sampling time. Members of the families Aeromonadaceae, Campylobacteraceae, Veillonellaceae, [Weeksellaceae], and Porphyromonadaceae were observed to be positively correlated with some ARGs (bla(CTX-M), bla(OXA-A), bla(SHV)) and intI1 (p < 0.05), while Intrasporangiaceae were observed to be negatively correlated. While Aeromonadaceae are recognized relevant ARG harbors, the other bacterial families may represent bacteria that co-exist with the ARG hosts, which may belong to minor bacterial groups omitted in the analyses. These findings suggest the importance of bacterial dynamics during treatment to the ARB&ARGs removal, a rationale that may contribute to design new strategies to apply in the UWTPs to prevent the spread of antibiotic resistance.

2019Journal article

Open access

Removal of microorganisms and antibiotic resistance genes from treated urban wastewater: a comparison between aluminium sulphate and tannin coagulants

Publication . Grehs, Bárbara W. N.; Lopes, Ana Rita; Moreira, Nuno F. F.; Fernandes, Telma; Linton, Maria A. O.; Silva, Adrián M. T.; Manaia, Célia M.; Carissimi, Elvis; Nunes, Olga C.

The presence of antibiotic resistant-bacteria (ARB) and antibiotic resistance genes (ARG) in treated effluents of urban wastewater treatment plants (WWTP) may represent a threat to the environment and public health. Therefore, cost-effective technologies contributing to minimize loads of these contaminants in the final effluents of WWTP are required. This study aimed at assessing the capacity of coagulation to reduce the ARB&ARG load in secondary treated urban wastewater (STWW), as well as the impact of the process on the structure and diversity of the bacterial community. Coagulation performance using aluminium sulphate, a synthetic substance, and tannins, a biowaste, was compared. Samples were analysed immediately before (STWW) and after the coagulation treatment (Alu, Tan), as well as after 3-days storage in the dark at room temperature (RSTWW, RAlu, RTan), to assess possible reactivation events. Both coagulants decreased the turbidity and colour and reduced the bacterial load (16S rRNA gene copy number, total heterotrophs (HET), and ARB (faecal coliforms resistant to amoxicillin (FC/AMX) or ciprofloxacin (FC/CIP) up to 1–2 log immediately after the treatment. Both coagulants reduced the load of intl1, but in average, aluminium sulphate was able to decrease the content of the analysed ARGs (blaTEM and qnrS) to lower levels than tannin. Reactivation after storage was observed mainly in RTan. In these samples the load of the culturable populations and qnrS gene prevalence increased, sometimes to values higher than those found in the initial wastewater. Reactivation was also characterized by an increment in Gammaproteobacteria relative abundance in the bacterial community, although with distinct patterns for RTan and RAlu. Curvibacter, Undibacterium and Aquaspirillum were among the most abundant genera in RAlu and Aeromonas, Pseudomonas and Stenotrophomonas in RTan. These bacterial community shifts were in agreement with the variations in the culturable bacterial counts of HET for RTan and FC/CIP for RAlu. In summary, the overall performance of aluminium sulphate was better than that of tannins in the treatment of treated urban wastewater.

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

The influence of the autochthonous wastewater microbiota and gene host on the fate of invasive antibiotic resistance genes

Publication . Narciso-da-Rocha, Carlos; Manaia, Célia M.

The aim of this study was to assess the fate of invasive antibiotic resistance genes (ARG) discharged in wastewater. With this objective, antibiotic resistant bacteria (ARB) known to harbor specific ARG were inoculated in wastewater (hospital effluent, or municipal raw and treated wastewater) and in ultra-pure sterile water microcosms. Two sets of wastewater ARB isolates were used - set 1, Enterococcus faecalis, Acinetobacter johnsonii, Klebsiella pneumoniae and set 2, Enterococcus faecium, Acinetobacter johnsonii, Escherichia coli. Non-inoculated controls were run in parallel. Samples were collected at the beginning and at the end (15 days) of the incubation period and the abundance of the genes 16S rRNA, intI1, blaTEM and vanA and the bacterial community composition were analyzed. In general, the genes blaTEM and vanA had lower persistence in wastewater and in ultra-pure water than the genes 16S rRNA or the class 1 integron integrase intI1. This effect was more pronounced in wastewater than in ultra-pure water, evidencing the importance of the autochthonous microbiota on the elimination of invasive ARG. Wastewater autochthonous bacterial groups most correlated with variations of the genes intI1, blaTEM and vanA were members of the classes Gammaproteobacteria, Bacilli or Bacteroidia. For blaTEM, but not for vanA, the species of the ARB host was important to determine its fate. These are novel findings on the ecology of ARB in wastewater environments.

2017Journal article

Restricted access