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Genetic variation in the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain

Publication . Ferreira, Catarina; Bogas, Diana; Bikarolla, Santosh K.; Varela, Ana Rita; Frykholm, Karolin; Linheiro, Raquel; Nunes, Olga C.; Westerlund, Fredrik; Manaia, Célia M.

Bacteria harboring conjugative plasmids have the potential for spreading antibiotic resistance through horizontal gene transfer. It is described that the selection and dissemination of antibiotic resistance is enhanced by stressors, like metals or antibiotics, which can occur as environmental contaminants. This study aimed at unveiling the composition of the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain (H1FC54) under different mating conditions. To meet this objective, plasmid pulsed field gel electrophoresis, optical mapping analyses and DNA sequencing were used in combination with phenotype analysis. Strain H1FC54 was observed to harbor five plasmids, three of which were conjugative and two of these, pH1FC54_330 and pH1FC54_140, contained metal and antibiotic resistance genes. Transconjugants obtained in the absence or presence of tellurite (0.5 μM or 5 μM), arsenite (0.5 μM, 5 μM or 15 μM) or ceftazidime (10 mg/L) and selected in the presence of sodium azide (100 mg/L) and tetracycline (16 mg/L) presented distinct phenotypes, associated with the acquisition of different plasmid combinations, including two co-integrate plasmids, of 310 kbp and 517 kbp. The variable composition of the conjugative plasmidome, the formation of co-integrates during conjugation, as well as the transfer of non-transferable plasmids via co-integration, and the possible association between antibiotic, arsenite and tellurite tolerance was demonstrated. These evidences bring interesting insights into the comprehension of the molecular and physiological mechanisms that underlie antibiotic resistance propagation in the environment.

2019Journal article

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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

Insights on sulfamethoxazole bio-transformation by environmental proteobacteria isolates

Publication . Reis, Patrícia J. M.; Homem, Vera; Alves, Arminda; Vilar, Vítor J.P.; Manaia, Celia M.; Nunes, Olga C.

Although sulfonamide residues are frequently reported as freshwaters contaminants, information on the ability of native bacteria to modify these synthetic antibiotics is scarce. Our purpose was to investigate the potential of bacteria from different aquatic environments to cleave or transform sulfamethoxazole (SMX) and infer on their ability to reduce the toxicity of this antibiotic. From a collection of about 100 Proteobacteria, 47 strains previously isolated from drinking water, surface water, and wastewater grew in the presence of 200 μMSMX, and were further studied. Out of these, 14 strains, mostly from mineral drinking water, transformed SMX into equimolar amounts of the lesser toxic derivative N4-acetyl-sulfamethoxazole. The highest percentage of SMX transformation was recorded for two strains affiliated to Pseudomonas mandelii. For P. mandelii McBPA4 higher SMX transformation rate and extent were observed in fed-batch (∼8 μMSMX/h, 81%) than in batch conditions (∼5 μMSMX/h, 25%), but similar specific transformation rates were found in both cultivation modes (∼20 μmolSMX/gcell dry weight/h), indicating the dependence of the process on the microbial load. These results evidence that the capacity to transform synthetic antibiotics may be common among bacteria and highlight the potential of environmental bacteria in attenuating the potential adverse effects of pollution with sulfonamides.

2018Journal article

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Solar treatment (H2O2, TiO2-P25 and GO-TiO2 photocatalysis, photo-Fenton) of organic micropollutants, human pathogen indicators, antibiotic resistant bacteria and related genes in urban wastewater

Publication . Moreira, Nuno F. F.; Narciso-da-Rocha, Carlos; Pólo-Lopez, M. Immaculada; Pastrana-Martínez, Luisa M.; Faria, Joaquim L.; Manaia, Célia M.; Fernandez-Ibáñez, Pilar; Nunes, Olga C.; Silva, Adrian M. T.

Solar-driven advanced oxidation processes were studied in a pilot-scale photoreactor, as tertiary treatments of effluents from an urban wastewater treatment plant. Solar-H2O2, heterogeneous photocatalysis (with and/or without the addition of H2O2 and employing three different photocatalysts) and the photo-Fenton process were investigated. Chemical sulfamethoxazole, carbamazepine, and diclofenac) and biological contaminants (faecal contamination indicators, their antibiotic resistant counterparts, 16S rRNA and antibiotic resistance genes), as well as the whole bacterial community, were characterized. Heterogeneous photocatalysis using TiO2-P25 and assisted with H2O2 (P25/H2O2) was the most efficient process on the degradation of the chemical organic micropollutants, attaining levels below the limits of quantification in less than 4 h of treatment (corresponding to QUV < 40 kJ L 1). This performance was followed by the same process without H2O2, using TiO2-P25 or a composite material based on graphene oxide and TiO2. Regarding the biological indicators, total faecal coliforms and enterococci and their antibiotic resistant (tetracycline and ciprofloxacin) counterparts were reduced to values close, or beneath, the detection limit (1 CFU 100mL 1) for all treatments employing H2O2, even upon storage of the treated wastewater for 3-days. Moreover, P25/H2O2 and solar-H2O2 were the most efficient processes in the reduction of the lysed genes. However, this reduction was transient for 16S rRNA, intI1 and sul1 genes, since after 3-days storage of the treated wastewater their abundance increased to values close to pre-treatment levels. Similar behaviour was observed for the genes qnrS (using TiO2-P25), blaCTX-M and blaTEM (using TiO2-P25 and TiO2-P25/H2O2). Interestingly, higher proportions of sequence reads affiliated to the phylum Proteobacteria (Beta- and Gammaproteobacteria) were found after 3-days storage of treated wastewater than before its treatment. Members of the genera Pseudomonas, Rheinheimera and Methylotenera were among those with overgrowth.

2018Journal article

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Proteobacteria become predominant during regrowth after water disinfection

Publication . Becerra-Castro, Cristina; Macedo, Gonçalo; Silva, Adrian M.T.; Manaia, C. M.; Nunes, Olga C.

Disinfection processes aimat reducing the number of viable cells through the generation of damages in different cellular structures and molecules. Since disinfection involves unspecific mechanisms, some microbial populationsmay be selected due to resilience to treatment and/or to high post-treatment fitness. In this study, the bacterial community composition of secondarily treated urban wastewater and of surface water collected in the intake area of a drinking water treatment plant was compared before and 3-days after disinfection with ultraviolet radiation, ozonation or photocatalytic ozonation. The aim was to assess the dynamics of the bacterial communities during regrowth after disinfection. In all the freshly collected samples, Proteobacteria and Bacteroidetes were the predominant phyla (40–50% and 20–30% of the reads, respectively). Surface water differed from wastewater mainly in the relative abundance of Actinobacteria (17% and b5% of the reads, respectively). After 3-days storage at light and room temperature, disinfected samples presented a shift of Gammaproteobacteria (from 8 to 10% to 33–65% of the reads) and Betaproteobacteria (from 14 to 20% to 31–37% of the reads), irrespective of the type of water and disinfection process used. Genera such as Pseudomonas, Acinetobacter or Rheinheimera presented a selective advantage after water disinfection. These variations were not observed in the non-disinfected controls. Given the ubiquity and genome plasticity of these bacteria, the results obtained suggest that disinfection processes may have implications on the microbiological quality of the disinfected water.

2016Journal article

Restricted access