No abstract

27Wastewater treatment plants (WWTPs) provide optimal conditions for the maintenance and 28 spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In this 29 work we describe the occurrence of antibiotic resistant faecal coliforms and their mechanisms 30 of antibiotic resistance in the effluent of two urban WWTPs in Ireland. Effluent samples were 31 collected from two WWTPs in Spring and Autumn of 2015 and 2016. The bacterial 32 susceptibility patterns to 13 antibiotics were determined. The phenotypic tests were carried 33 out to identify AmpC or extended-spectrum β-lactamase (ESBL) producers. The presence of 34 ESBL genes were detected by PCR. Plasmids carrying ESBL genes were transformed into 35Escherichia coli DH5α recipient and underwent plasmid replicon typing to identify 36 incompatibility groups. More than 90% of isolated faecal coliforms were resistant to 37 amoxicillin and ampicillin, followed by tetracycline (up to 39.82%), ciprofloxacin (up to 38 31.42%) and trimethoprim (up to 37.61%). Faecal coliforms resistant to colistin and 39 imipenem were detected in all effluent samples. Up to 53.98% of isolated faecal coliforms 40 expressed a multi-drug resistance (MRD) phenotype. AmpC production was confirmed in 41 5.22% of isolates. The ESBL genes were confirmed for 11.84% of isolates (9.2% of isolates 42 carried bla TEM , 1.4% bla , 0.2% bla CTX-M-1 and 1% bla CTX-M-15 ). Plasmids extracted from 43 52 ESBL isolates were successfully transformed into recipient E. coli. The detected plasmid 44 incompatibility groups included the IncF group, IncI1, IncHI1/2 and IncA/C. These results 45 provide evidence that treated wastewater is polluted with ARB and MDR faecal coliforms 46 and are sources of ESBL-producing, carbapenem and colistin resistant Enterobacteriaceae. 47 48 49 50 Keywords 51 Antibiotic resistance, multidrug resistance, AmpC, extended-spectrum β-lactamase (ESBL), 52 plasmids 53 54 55 56 3 57 Importance 58 Antibiotic resistant bacteria (ARB) are an emerging environmental concern with a potential 59 impact on human health. The results provide the evidence that treated wastewater is polluted 60 with antibiotic resistant bacteria containing mobile resistance mechanisms of importance to 61 clinical treatment of pathogens and multi-drug resistant (MDR) faecal coliforms. They are 62 sources of relatively high proportions of ESBL-producing Enterobacteriaceae, and include 63 carbapenem and colistin resistant Enterobacteriaceae. The significance of this study is the 64 identification of the role of WWTPs as a potential control point to reduce or stop the 65 movement of ESBL, MDR and colistin resistant bacteria into the environment from further 66 upstream sources, such as human or animal waste.

Extended-spectrum β-lactamase (ESBL)/AmpC producing bacteria are one of the critical priority resistant bacteria that contributes to treatment failure and increased death rates. In this work we aimed to study the role of wastewater treatment plants (WWTPs) as reservoirs of ESBL/AmpC producing faecal coliforms. The effluent samples were collected from two WWTPs and faecal coliforms were isolated from all samples using the membrane filtration method. Bacterial isolates were subjected to antimicrobial susceptibility testing toward cefotaxime and ceftazidime. The isolates that showed a resistance phenotype to these antibiotics were considered as putative ESBL/AmpC producing bacteria. These bacteria were subjected to the AmpC test using a protocol with phenylboronic acid. The AmpC negative strains in the AmpC test served as samples for multiplex PCR containing primers specific for blaTEM, blaSHV and blaCTX-M. In total, 498 faecal coliforms were isolated from WWTP effluent samples. For the antibiotic susceptibility testing 99 isolates were considered as ESBL/AmpC producing bacteria. Among them, 26 isolates were found to be positive in the AmpC test. The PCR results revealed that 49 isolates carried blaTEM, 6 bla SHV12, 1 blaCTX-M1 and 5 blaCTX-M15. The ESBL/AmpC producing faecal coliforms in WWTP effluent are discharged to the receiving water environment. These data need to be considered when analysing the risk of WWTP effluent to the environment and to human health, as many of the bacteria identified are not analysed in assessment of risk of pollution from WWTPs globally.

The Advanced Biotechnology for Intensive-Freshwater Aquaculture Wastewater Reuse (ABAWARE) project, which is part of the European Commission’s Water Joint Programming Initiative 2016 Joint Call, aims to increase the efficiency and resilience of water use in aquaculture and minimise its negative impact on the environment and human health. This research, which forms one part of the total ABAWARE project, aimed to ascertain the impact of using microbiota and certain plant species, in conjuncture with a more traditional Recirculated Aquaculture System (RAS), as a filtering system had on the arb and G abundance in various samples taken from an aquaculture facility. Sediment and water samples were taken from the inflow, the main fish basin, after the bioactive ponds before filtration, and after filtration. The resistance genes present in these samples were detected using the Wafergen smartchip real-time qPCR system. This system allows for the simultaneous quantification of 348 distinct Antibiotic Resistance Genes for each sample. The samples also underwent microbiome analysis via 16S rRNA metagenomic sequencing. Mothur was used to analyse the sequencing data. This data informs us of the changes in the microbial population changes that are enacted by the various stages within the aquaculture facility.

26Wastewater treatment plants (WWTPs) provide optimal conditions for the maintenance and 27 spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In this 28 work we describe the occurrence of antibiotic resistant faecal coliforms and their mechanisms 29 of antibiotic resistance in the effluent of two urban WWTPs in Ireland. Effluent samples were 30 collected from two WWTPs in Spring and Autumn of 2015 and 2016. The bacterial 31 susceptibility patterns to 13 antibiotics were determined. The phenotypic tests were carried out 32 to identify AmpC or extended-spectrum β-lactamase (ESBL) producers. The presence of ESBL 33 genes were detected by PCR. Plasmids carrying ESBL genes were transformed into 34Escherichia coli DH5α recipient and underwent plasmid replicon typing to identify 35 incompatibility groups. More than 90% of isolated faecal coliforms were resistant to 36 amoxicillin and ampicillin, followed by tetracycline (up to 39.82%), ciprofloxacin (up to 37 31.42%) and trimethoprim (up to 37.61%). Faecal coliforms resistant to colistin and imipenem 38 were detected in all effluent samples. Up to 53.98% of isolated faecal coliforms expressed a 39 multi-drug resistance (MRD) phenotype. AmpC production was confirmed in 5.22% of 40 isolates. The ESBL genes were confirmed for 11.84% of isolates (9.2% of isolates carried 41 blaTEM, 1.4% blaSHV-12, 0.2% blaCTX-M-1 and 1% blaCTX-M-15). Plasmids extracted from 52 ESBL 42 isolates were successfully transformed into recipient E. coli. The detected plasmid 43 incompatibility groups included the IncF group, IncI1, IncHI1/2 and IncA/C. These results 44 provide evidence that treated wastewater is polluted with ARB and MDR faecal coliforms and 45 are sources of ESBL-producing, carbapenem and colistin resistant Enterobacteriaceae. 46 47 48 49 Keywords 50 Antibiotic resistance, multidrug resistance, AmpC, extended-spectrum β-lactamase (ESBL), 51 plasmids 52 53 54 55 56 3