Elisabet Marti

Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river.

Antibiotic resistance has become a major health concern; thus, there is a growing interest in exploring the occurrence of antibiotic resistance genes (ARGs) in the environment as well as the factors that contribute to their emergence. Aquatic ecosystems provide an ideal setting for the acquisition and spread of ARGs due to the continuous pollution by antimicrobial compounds derived from anthropogenic activities. We investigated, therefore, the pollution level of a broad range of antibiotics and ARGs released from hospital and urban wastewaters, their removal through a wastewater treatment plant (WWTP) and their presence in the receiving river. Several antimicrobial compounds were detected in all water samples collected. Among antibiotic families, fluoroquinolones were detected at the highest concentration, especially in hospital effluent samples. Although good removal efficiency by treatment processes was observed for several antimicrobial compounds, most antibiotics were still present in WWTP effluents. The results also revealed that copy numbers of ARGs, such as blaTEM (resistance to β-lactams), qnrS (reduced susceptibility to fluoroquinolones), ermB (resistance to macrolides), sulI (resistance to sulfonamides) and tetW (resistance to tetracyclines), were detected at the highest concentrations in hospital effluent and WWTP influent samples. Although there was a significant reduction in copy numbers of these ARGs in WWTP effluent samples, this reduction was not uniform across analyzed ARGs. Relative concentration of ermB and tetW genes decreased as a result of wastewater treatment, whereas increased in the case of blaTEM, sulI and qnrS genes. The incomplete removal of antibiotics and ARGs in WWTP severely affected the receiving river, where both types of emerging pollutants were found at higher concentration in downstream waters than in samples collected upstream from the discharge point. Taken together, our findings demonstrate a widespread occurrence of antibiotics and ARGs in urban and hospital wastewater and how these effluents, even after treatment, contribute to the spread of these emerging pollutants in the aquatic environment.

The role of aquatic ecosystems as reservoirs of antibiotic resistance

Although antibiotic resistance has become a major threat to human health worldwide, this phenomenon has been largely overlooked in studies in environmental settings. Aquatic environments may provide an ideal setting for the acquisition and dissemination of antibiotic resistance, because they are frequently impacted by anthropogenic activities. This review focuses primarily on the emergence and dissemination of antibiotic resistance in the aquatic environment, with a special emphasis on the role of antibiotic resistance genes.

Exploring the links between antibiotic occurrence, antibiotic resistance, and bacterial communities in water supply reservoirs

Antibiotic resistance represents a growing global health concern due to the overuse and misuse of antibiotics. There is, however, little information about how the selective pressure of clinical antibiotic usage can affect environmental communities in aquatic ecosystems and which bacterial groups might be responsible for dissemination of antibiotic resistance genes (ARGs) into the environment. In this study, chemical and biological characterization of water and sediments from three water supply reservoirs subjected to a wide pollution gradient allowed to draw an accurate picture of the concentration of antibiotics and prevalence of ARGs, in order to evaluate the potential role of ARGs in shaping bacterial communities, and to identify the bacterial groups most probably carrying and disseminating ARGs. Results showed significant correlation between the presence of ARG conferring resistance to macrolides and the composition of bacterial communities, suggesting that antibiotic pollution and the spreading of ARG might play a role in the conformation of bacterial communities in reservoirs. Results also pointed out the bacterial groups Actinobacteria and Firmicutes as the ones probably carrying and disseminating ARGs. The potential effect of antibiotic pollution and the presence of ARGs on the composition of bacterial communities in lacustrine ecosystems prompt the fundamental question about potential effects on bacterial-related ecosystem services supplied by lakes and reservoirs.

Prevalence of antibiotic resistance genes and bacterial community composition in a river influenced by a wastewater treatment plant.

Antibiotic resistance represents a global health problem, requiring better understanding of the ecology of antibiotic resistance genes (ARGs), their selection and their spread in the environment. Antibiotics are constantly released to the environment through wastewater treatment plant (WWTP) effluents. We investigated, therefore, the effect of these discharges on the prevalence of ARGs and bacterial community composition in biofilm and sediment samples of a receiving river. We used culture-independent approaches such as quantitative PCR to determine the prevalence of eleven ARGs and 16S rRNA gene-based pyrosequencing to examine the composition of bacterial communities. Concentration of antibiotics in WWTP influent and effluent were also determined. ARGs such as qnrS, bla TEM, bla CTX-M, bla SHV, erm(B), sul(I), sul(II), tet(O) and tet(W) were detected in all biofilm and sediment samples analyzed. Moreover, we observed a significant increase in the relative abundance of ARGs in biofilm samples collected downstream of the WWTP discharge. We also found significant differences with respect to community structure and composition between upstream and downstream samples. Therefore, our results indicate that WWTP discharges may contribute to the spread of ARGs into the environment and may also impact on the bacterial communities of the receiving river.

Removal of microbial indicators from municipal wastewater by a membrane bioreactor (MBR).

The impact of removable and irremovable fouling on the retention of viral and bacterial indicators by the submerged microfiltration membrane in an MBR pilot plant was evaluated. Escherichia coli, sulphite-reducing Clostridium spores, somatic coliphages and F-specific RNA bacteriophages were used as indicators. The membrane demonstrated almost complete removal of E. coli and sulphite-reducing Clostridium spores. However, there was no correlation with membrane fouling. The phage removal varied in accordance with the irremovable fouling, rising from 2.6 to 5.6 log(10) units as the irremovable fouling increased (measured by the change in the transmembrane pressure). In contrast, removable fouling did not have any effect on the retention of viruses by the membrane. These results indicate that irremovable membrane fouling may affect the removal efficiency of MBRs and, therefore, their capacity to ensure the required microbiological standards for the permeate achieved.

Effects on activated sludge bacterial community exposed to sulfamethoxazole.

The bacterial community shift on a lab scale Sequencing Batch Reactor (SBR) fed with synthetic wastewater and exposed to 50μgL(-1) of sulfamethoxazole (SFX) for 2months was investigated in this study. The impact on biological nutrient removal performance and SFX removal efficiencies were also studied. Satisfactory biological nutrient removal was observed as regards to COD and Nitrogen. SFX removal efficiencies ranged between 20% and 50% throughout the experimental period, enhanced within the aerobic phases of the SBR cycle, with no evident signs of biomass acclimation. Nevertheless, denaturing gradient gel electrophoresis (DGGE) analysis showed significant variance leading to not only the fading, but also the emergence of new species in the bioreactor bacterial community after SFX dosage. According to the phylogenetic analysis, bacteria belonging to Betaproteobacteria and Gammaproteobacteria classes were the dominant species, among them, the Thiotrix spp. (Gammaproteobacteria) cell number increased due to its tolerance to the antibiotic. On the other hand, the classes Sphingobacteria, Actinobacteria, Chloroflexi and Chlorobi were found to be more vulnerable to the antibiotic load and disappeared. The sulphonamide resistance gene sulI was also quantified and discussed, as there are very few studies on bacterial resistance in lab-scale treatment reactors.

Bacteriophages as a reservoir of extended-spectrum β -lactamase and fluoroquinolone resistance genes in the environment

Six antibiotic resistance genes (blaCTX-M , blaSHV , blaTEM , qnrA, qnrB and qnrS) were quantified by qPCR in both phage and bacterial DNA fractions of environmental water samples in order to determine the contribution of phages to the dissemination of antibiotic resistance genes (ARGs) in the environment. Although the highest copy numbers (p <0.05) of ARGs were detected in the bacterial DNA fraction, qnrS and blaSHV genes were found in the phage DNA from all samples analysed, reaching up to 4 log10 copy numbers/mL in hospital samples. These results indicate that bacteriophages are a potential reservoir of resistance genes and may act as efficient vehicles for horizontal gene transfer.

Real-Time PCR assays for quantification of qnr genes in environmental water samples and chicken feces.

ABSTRACT Real-time PCR assays were developed for the enumeration of plasmid-mediated quinolone resistance (PMQR) determinants, such as the qnrA, qnrB, and qnrS genes, in different water samples and chicken feces. The results indicate that the developed assays are specific and sensitive for the quantification of qnr genes in complex samples.

Prevalence of antibiotic-resistant fecal bacteria in a river impacted by both an antibiotic production plant and urban treated discharges

In this study, the abundance and spatial dynamics of antibiotic-resistant fecal bacteria (Escherichia coli, total coliforms and Enterococcus spp.) were determined in water and sediment samples from a river impacted by both antibiotic production plant (APP) and urban wastewater treatment plant (WWTP) discharges. Agar dilution and disk diffusion methods were also used for antimicrobial susceptibility testing. Two antimicrobial agents, cephalexin (25 μg/ml) and amoxicillin (50 μg/ml), were evaluated using the agar dilution method for E. coli, total coliforms (TC) and Enterococcus spp., whereas the degree of sensitivity or resistance of E. coli isolates to penicillin (10 U), ampicillin (10 μg), doxycycline (30 μg), tetracycline (30 μg), erythromycin (15 μg), azithromycin (15 μg) and streptomycin (10 μg) was performed using the disk diffusion method. Real-time PCR assays were used to determine the prevalence of three antibiotic-resistance genes (ARGs). The agar dilution method showed that most E. coli isolates and TC were resistant to amoxicillin, especially after receiving the APP discharges. Antibiotic resistances to amoxicillin and cephalexin were higher after the APP discharge point than after the WWTP effluent. The disk diffusion method revealed that 100% of bacterial isolates were resistant to penicillin and erythromycin. Multidrug-resistant bacteria were detected and showed a higher proportion at the WWTP discharge point than those in the APP. Highly multidrug-resistant bacteria (resistance to more than 4 antibiotics) were also detected, reaching mean values of 41.6% in water samples and 50.1% in sediments. The relative abundance of the blaTEM, blaCTX-M and blaSHV genes was higher in samples from the treatment plants than in those collected upstream from the discharges, especially for water samples collected at the APP discharge point. These results clearly demonstrate that both the APP and the WWTP contribute to the emergence and spread of antibiotic resistance in the environment.

Use of pyrosequencing to explore the benthic bacterial community structure in a river impacted by wastewater treatment plant discharges.

In this study, we determined the diversity and composition of benthic bacterial communities collected in river sediments upstream and downstream from a wastewater treatment plant (WWTP). Pyrosequencing of bacterial 16S rRNA genes revealed notable differences between the communities from upstream and downstream sites. In particular, a higher relative abundance of Acidobacteria, Chloroflexi, Deltaproteobacteria and Firmicutes and a lower proportion of Gammaproteobacteria and Verrucomicrobia sequences were detected at the downstream site compared to the upstream site. These findings represent a first approximation of the impact of WWTP discharges on environmental microbial communities.