Anaerobic Transformation and Detoxification of Sulfamethoxazole by Sulfate-Reducing Enrichments and Desulfovibrio vulgaris.

Sulfamethoxazole (SMX) is a veterinary antibiotic that is not efficiently removed from wastewater by routine treatment and therefore can be detected widely in the environment. Here, we investigated whether microbial anaerobic transformation can contribute to the removal of SMX in constructed systems. We enriched SMX-transforming mixed cultures from sediment of a constructed wetland and from digester sludge of a wastewater treatment plant. Transformation of SMX was observed in both sulfate-reducing and methanogenic cultures, whereas nitrate-reducing cultures showed no SMX transformation. In sulfate-reducing cultures, up to 90% of an initial SMX concentration of 100-250 µM was removed within 6 weeks of incubation, and the experiments demonstrated that the transformation was microbially catalyzed. The transformation products in sulfate-reducing cultures were identified as the reduced and isomerized forms of the isoxazole SMX moiety. The transformation products did not spontaneously reoxidize to SMX after oxygen exposure, and their antibacterial activity was significantly decreased compared to SMX. Population analyses in sequential transfers of the sulfate-reducing cultures revealed a community shift toward the genus Desulfovibrio. We therefore tested a deposited strain of Desulfovibrio vulgaris Hildenborough for its capacity to transform SMX and observed the same transformation products and similar transformation rates as in the enrichment cultures. Our work suggests that an initial anaerobic step in wastewater treatment can reduce the concentration of SMX in effluents and could contribute to decreased SMX concentrations in the environment.

Increased levels of antibiotic resistance in urban stream of Jiulongjiang River, China.

The rapid global urbanization and other extensive anthropogenic activities exacerbated the worldwide human health risks induced by antibiotic resistance genes (ARGs). Knowledge of the origins and dissemination of ARGs is essential for understanding modern resistome, while little information is known regarding the overall resistance levels in urban river. In this study, the abundance of multi-resistant bacteria (MRB) and ARGs was investigated using culture-based method and high-throughput qPCR in water samples collected from urban stream and source of Jiulongjiang River, China, respectively. The abundance of MRB (conferring resistance to three combinations of antibiotics and vancomycin) was significantly higher in urban samples. A total of 212 ARGs were detected among all the water samples, which encoded resistance to almost all major classes of antibiotics and encompassed major resistant mechanisms. The total abundance of ARGs in urban samples (ranging from 9.72 × 10(10) to 1.03 × 10(11) copies L(-1)) was over two orders of magnitude higher than that in pristine samples (7.18 × 10(8) copies L(-1)), accompanied with distinct ARGs structures, significantly higher diversity, and enrichment of ARGs. Significant correlations between the abundance of ARGs and mobile genetic elements (MGEs) were observed, implicating the potential of horizontal transfer of ARGs. High abundance and enrichment of diverse ARGs and MGEs detected in urban river provide evidence that anthropogenic activities are responsible for the emergence and dissemination of ARGs to the urban river and management options should be taken into account for minimizing the spread of ARGs.

Carbon and hydrogen stable isotope fractionation of sulfamethoxazole during anaerobic transformation catalyzed by Desulfovibrio vulgaris Hildenborough.

The fate of antibiotics in aquatic environments is of high concern and approaches are needed to assess the transformation of antibiotics in wastewater treatment plants. Here we used the model organism Desulfovibrio vulgaris Hildenborough to analyze compound specific isotope fractionation associated with anaerobic transformation of the antibiotic sulfamethoxazole (SMX). The results show that the rearrangement of the isoxazole ring in SMX is leading to significant carbon and hydrogen isotopic fractionation (εC = -5.8 ± 0.7‰, εH = -34 ± 9‰) during anaerobic transformation. The observed carbon isotopic fractionation is significantly higher than the values reported for aerobic degradation (εC = -0.6 ± 0.1‰) or abiotic reactions (εC = -0.8 to -4.8‰ for photolysis, εC = -0.8 to -2.2‰ for advanced oxidation). This indicates that carbon isotope fractionation can be used as a parameter to differentiate reaction mechanisms of SMX transformation. The corresponding apparent kinetic isotope effect (AKIEC) for anaerobic transformation of SMX was 1.029 ± 0.003, suggesting that the mechanism for anaerobic transformation is distinct from the mechanism reported for microbial aerobic degradation (AKIEC = 1.006 ± 0.001). In addition, dual-element (C-H) isotope analysis of SMX was performed in the present study, which was achieved by utilizing gas chromatography (GC) as the separation method instead of routine liquid chromatography. This dual-element isotope analysis resulted in a Λ value of 4.5 ± 2.2. Overall, compound specific isotope analysis can be a feasible tool to monitor the mitigation of SMX in wastewater treatment plants.

Identification of dominant sulfamethoxazole-degraders in pig farm-impacted soil by DNA and protein stable isotope probing.

Increasing introduction of antibiotic residues from humans and animal farming into the environment impacts the functioning of natural ecosystems and significantly contributes to the propagation of antibiotic resistance. Microbial degradation is the major sink of antibiotics in soil but the identification of in situ degrading populations is challenging. Here, we investigated sulfamethoxazole-degrading bacteria in soil microcosms by culture-independent DNA and protein stable isotope probing. 0.5% of the carbon from 13C6-labeled sulfamethoxazole amended to soil microcosms was transformed to 13CO2 demonstrating partial mineralization of the antibiotic. DNA stable isotope probing revealed incorporation of 13C from 13C6-labeled sulfamethoxazole into Actinobacteria and among them into the families Intrasporangiaceae, Nocardioidaceae, and Gaiellaceae and the order Solirubrobacterales. Protein stable isotope probing demonstrated the incorporation of 13C from 13C6-labeled sulfamethoxazole into proteins of bacteria of the families Intrasporangiaceae, Nocardioidaceae and the order Solirubrobacterales, which is consistent with the results of DNA stable isotope probing. The 13C abundance of 60 to 80% in several taxonomically relevant proteins indicated that Intrasporangiaceae directly acquired carbon from 13C6-labeled sulfamethoxazole. The results highlight the crucial role of yet-uncultivated indigenous bacteria for antibiotics degradation, and the potential of cultivation-independent stable isotope based molecular approaches to elucidate the structure of antibiotic-degrading populations in complex microbial communities under natural conditions.

Tracking antibiotic resistome during wastewater treatment using high throughput quantitative PCR.

Wastewater treatment plants (WWTPs) contain diverse antibiotic resistance genes (ARGs), and thus are considered as a major pathway for the dissemination of these genes into the environments. However, comprehensive evaluations of ARGs dynamic during wastewater treatment process lack extensive investigations on a broad spectrum of ARGs. Here, we investigated the dynamics of ARGs and bacterial community structures in 114 samples from eleven Chinese WWTPs using high-throughput quantitative PCR and 16S rRNA-based Illumina sequencing analysis. Significant shift of ARGs profiles was observed and wastewater treatment process could significantly reduce the abundance and diversity of ARGs, with the removal of ARGs concentration by 1-2 orders of magnitude. Whereas, a considerable number of ARGs were detected and enriched in effluents compared with influents. In particular, seven ARGs mainly conferring resistance to beta-lactams and aminoglycosides and three mobile genetic elements persisted in all WWTPs samples after wastewater treatment. ARGs profiles varied with wastewater treatment processes, seasons and regions. This study tracked the footprint of ARGs during wastewater treatment process, which would support the assessment on the spread of ARGs from WWTPs and provide data for identifying management options to improve ARG mitigation in WWTPs.

[Comparative Investigation of Antibotic Resistance Genes Between Wastewater and Landfill Leachate].

The increasing output of wastewater and landfill leachate during the rapid urbanization exacerbated the human health risks induced by antibiotic resistance genes(ARGs). For further insight into antibiotic resistance genes(ARGs) of wastewater (adding leachate before treatment) and landfill leachate, high-throughput qPCR technique was used to investigate their abundance and diversities. A total of 187 unique ARGs were detected in wastewater, which was significantly higher than that in leachate (39 unique ARGs were detected)(P<0.05). A total of 119 antibiotic resistance genes were significantly enriched in wastewater compared with the landfill leachate (P<0.05), especially the tnpA-04 and blaVEB, which belongs to mobile gene elements(MGEs) and ß-Lactamase, increased by 3338-fold and 1061-fold, respectively. Our results showed that the profile of ARGs was significantly different between the wastewater and leachate, and the wastewater was the important reservoir of ARGs.

[Long-term manure application induced shift of diversity and abundance of antibiotic resistance genes in paddy soil].

To investigate the effect of long-term manure application on the profiles of antibiotic resistance genes (ARGs) in paddy soil, the abundance of ARGs targeted by 295 primers sets were measured by using high-throughput quantitative PCR technique. A total of 107 types of ARGs were detected in long-term manured paddy soil, which is significantly higher than that in control paddy soil (P < 0.05). PCA analysis and UPGMA cluster demonstrated that long-term manure application significantly changed the ARGs diversity and abundance in paddy soils. There were 49 types antibiotic resistance genes were enriched significantly in paddy soil applied with manure (P < 0.05), especially the mexF, which belongs to quinolones-chloramphenicol resistance genes, increased 1791-fold compared with paddy soil of no manure application. Our results showed that long-term application of manure in paddy soil resulted in the shift of abundance and diversity of ARGs, high-throughput quantitative PCR could be a powerful tool concerning the environmental ARGs dimensions.