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- Antibiotic resistance in European wastewater treatment plants mirrors the pattern of clinical antibiotic resistance prevalencePublication . Pärnänen, Katariina M. M.; Narciso-da-Rocha, Carlos; Kneis, David; Berendonk, Thomas U.; Cacace, Damiano; Do, Thi Thuy; Elpers, Christian; Fatta-Kassinos, Despo; Henriques, Isabel; Jaeger, Thomas; Karkman, Antti; Martinez, Jose Luis; Michael, Stella G.; Michael-Kordatou, Irene; O’Sullivan, Kristin; Rodriguez-Mozaz, Sara; Schwartz, Thomas; Sheng, Hongjie; Sørum, Henning; Stedtfeld, Robert D.; Tiedje, James M.; Giustina, Saulo Varela Della; Walsh, Fiona; Vaz-Moreira, Ivone; Virta, Marko; Manaia, Célia M.Integrated antibiotic resistance (AR) surveillance is one of the objectives of the World Health Organization global action plan on antimicrobial resistance. Urban wastewater treatment plants (UWTPs) are among the most important receptors and sources of environmental AR. On the basis of the consistent observation of an increasing north-to-south clinical AR prevalence in Europe, this study compared the influent and final effluent of 12 UWTPs located in seven countries (Portugal, Spain, Ireland, Cyprus, Germany, Finland, and Norway). Using highly parallel quantitative polymerase chain reaction, we analyzed 229 resistance genes and 25 mobile genetic elements. This first trans-Europe surveillance showed that UWTP AR profiles mirror the AR gradient observed in clinics. Antibiotic use, environmental temperature, and UWTP size were important factors related with resistance persistence and spread in the environment. These results highlight the need to implement regular surveillance and control measures, which may need to be appropriate for the geographic regions.
- Metal(loid)-contaminated soils as a source of culturable heterotrophic aerobic bacteria for remediation applicationsPublication . Pires, Carlos; Franco, Albina R.; Pereira, Sofia I. A.; Henriques, Isabel; Correia, António; Magan, Naresh; Castro, Paula M. L.Heavy metal-contaminated soils are a serious environmental problem. Herein, the culturable heterotrophic bacterial community present on two metal(loid)-contaminated sites in the Northern Portugal was investigated. The bacterial counts ranged from 5.96 to 7.69 and 7.04 to 7.51 (log CFUg(-1) soil) in Sites 1 and 3, respectively. The bacterial population was predominantly composed of Firmicutes, Proteobacteria, and Actinobacteria on both sites. The most represented genera in Site 1 were Bacillus (41%) and Pseudomonas (27%), whereas Arthrobacter (21%) and Pseudomonas (13%) were the most represented genera in Site 3. Several bacterial isolates showed tolerance to high concentrations of metal(loid)s, suggesting that both contaminated sites are a valuable source of metal(loid)-tolerant bacteria, which may be further used in bioremediation and/or phytoremediation processes.
- A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plantsPublication . Marano, Roberto B.M.; Fernandes, Telma; Manaia, Célia M.; Nunes, Olga; Morrison, Donald; Berendonk, Thomas U.; Kreuzinger, Norbert; Tenson, Tanel; Corno, Gianluca; Fatta-Kassinos, Despo; Merlin, Christophe; Topp, Edward; Henn, Leonie; Scott, Andrew; Heß, Stefanie; Slipko, Katarzyna; Laht, Mailis; Kisand, Veljo; Cesare, Andrea Di; Karaolia, Popi; Michael, Stella G.; Petre, Alice L.; Rosal, Roberto; Pruden, Amy; Riquelme, Virginia; Agüera, Ana; Esteban, Belen; Luczkiewicz, Aneta; Kalinowska, Agnieszka; Leonard, Anne; Gaze, William H.; Adegoke, Anthony A.; Stenstrom, Thor A.; Pollice, Alfieri; Salerno, Carlo; Schwermer, Carsten U.; Krzeminski, Pawel; Guilloteau, Hélène; Donner, Erica; Drigo, Barbara; Libralato, Giovanni; Guida, Marco; Bürgmann, Helmut; Beck, Karin; Garelick, Hemda; Tacão, Marta; Henriques, Isabel; Martínez-Alcalá, Isabel; Guillén-Navarro, Jose M.; Popowska, Magdalena; Piotrowska, Marta; Quintela-Baluja, Marcos; Bunce, Joshua T.; Polo-López, Maria I.; Nahim–Granados, Samira; Pons, Marie-Noëlle; Milakovic, Milena; Udikovic-Kolic, Nikolina; Ory, Jérôme; Ousmane, Traore; Caballero, Pilar; Oliver, Antoni; Rodriguez-Mozaz, Sara; Balcazar, Jose L.; Jäger, Thomas; Schwartz, Thomas; Yang, Ying; Zou, Shichun; Lee, Yunho; Yoon, Younggun; Herzog, Bastian; Mayrhofer, Heidrun; Prakash, Om; Nimonkar, Yogesh; Heath, Ester; Baraniak, Anna; Abreu-Silva, Joana; Choudhury, Manika; P. Munoz, Leonardo; Krizanovic, Stela; Brunetti, Gianluca; Maile-Moskowitz, Ayella; Brown, Connor; Cytryn, EddieThe World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum β-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.
- A molecular and multivariate approach to the microbial community of a commercial shallow raceway marine recirculation system operating with a moving bed biofilterPublication . Matos, Ana; Borges, Maria-Teresa; Peixe, Carla; Henriques, Isabel; Pereira, Carlos M.; Castro, Paula M. L.Information on commercial aquaculture systems bacterial communities is scarce. The present study was performed in a turbot shallow raceway recirculating aquaculture system throughout 221 days after initial fish stocking. The dynamics of bacteria attached to a Moving Bed Biofilm Reactor and the distribution of the system culturable planktonic heterotrophic bacteria were assessed. Different levels of free heterotrophic bacteria were found over time: <= 2.6 x 10(4) CFU mL(-1) in incoming and ozonated water and >= 1.5 x 10(6) CFU mL(-1) in raceway and biofilter water. 16S rRNA gene sequencing of nine strains persistently isolated from the system affiliated with Bacteroidetes, alpha-Proteobacteria or gamma-Proteobacteria. Polymerase chain reaction-denaturating gradient gel electrophoresis (PCR-DGGE) data showed temporal and spatial segregation of attached biofilter community. The first 32 days were the most important for bacterial establishment with higher culturable bacteria biomass loadings and higher community diversity. Multivariate canonical correspondence analysis of PCR-DGGE showed that the most common aquaculture water quality descriptors explained 70% of the DGGE pattern variability.
- Bacterial community dynamics within an aerobic granular sludge reactor treating wastewater loaded with pharmaceuticalsPublication . Amorim, Catarina L.; Alves, Marta; Castro, Paula M.L.; Henriques, IsabelPharmaceuticals are micropollutants often present in wastewater treatment systems. In this study, the potential impact of such micropollutants on the bacterial population within aerobic granular sludge (AGS) bioreactor was investigated. The AGS bacterial community structure and composition were accessed combining DGGE fingerprinting and barcoded pyrosequencing analysis. Both revealed the existence of a dynamic bacterial community, independently of the pharmaceuticals presence. The AGS microbiome at both phylum and class levels varied over time and, after stopping pharmaceuticals feeding, the bacterial community did not return to its initial composition. Nevertheless, most of the assigned OTUs were present throughout the different operational phases. This core microbiome, represented by over 72% of the total sequences in each phase, probably played an important role in biological removal processes, avoiding their failure during the disturbance period. Quantitative-PCR revealed that pharmaceuticals load led to gradual changes on the abundance of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and polyphosphate-accumulating organisms (PAO) but their persistence during that phase demonstrated the resilience of such bacterial groups. AGS microbiome changed over time but a core community was maintained, probably ensuring the accomplishment of the main biological removal processes.
- Assessment of rhizospheric culturable bacteria of Phragmites australis and Juncus effusus from polluted sitesPublication . Pereira, Sofia I. A.; Pires, Carlos; Henriques, Isabel; Correia, António; Magan, Naresh; Castro, Paula M. L.This study aimed at the isolation and characterization of metal(loid)-tolerant bacteria from the rhizosphere of Phragmites australis and Juncus effusus plants growing in two long-term contaminated sites in Northern Portugal. Site 1 had higher contamination than Site 3. Bacteria were isolated using metal(loid)-supplemented (Cd, Zn, and As) media. Isolates were grouped by random amplified polymorphic DNA and identified by 16S rRNA gene sequencing. Strains were also examined for their metal(loid) tolerance. The counts of metal(loid)-tolerant bacteria were higher in Site 1 and ranged between log 7.17 CFU g(-1) soil in As-containing medium and log 7.57 CFU g(-1) soil in Zn-containing medium, while counts at Site 3 varied between log 5.33 CFU g(-1) soil in Cd-containing medium and log 6.97 CFUg(-1) soil in As-containing medium. The composition of bacterial populations varied between locations. In Site 1, the classes Actinobacteria (36%) and Bacilli (24%) were well represented, while in Site 3 strains were mainly affiliated to classes Actinobacteria (35%), 'y-Proteobacteria (35%), and 13-Proteobacteria (12%). The order of metal(loid) toxicity for the isolated strains was Cd > As > Zn. Overall, 10 strains grew at 500 mg Cd L-1, 1000 mg Zn L-1, and 500 mg As L-1, being considered the most metal(loid)-tolerant bacteria. These strains belonged to genera Cupriavidus, Burkholderia, Novosphingobium, Sphingo bacterium, Castellaniella, Mesorhizobium, Chryseobacterium, and Rhodococcus and were mainly retrieved from Site 1. The multiple metal(loid)-tolerant strains isolated in this study have potential to be used in bioremediation/phytoremediation.
- Bacterial lineages putatively associated with the dissemination of antibiotic resistance genes in a full-scale urban wastewater treatment plantPublication . Narciso-da-Rocha, Carlos; Rocha, Jaqueline; Vaz-Moreira, Ivone; Lira, Felipe; Tamames, Javier; Henriques, Isabel; Martinez, Jose Luis; Manaia, Célia M.Urban wastewater treatment plants (UWTPs) are reservoirs of antibiotic resistance. Wastewater treatment changes the bacterial community and inevitably impacts the fate of antibiotic resistant bacteria and antibiotic resistance genes (ARGs). Some bacterial groups are major carriers of ARGs and hence, their elimination during wastewater treatment may contribute to increasing resistance removal efficiency. This study, conducted at a full-scale UWTP, evaluated variations in the bacterial community and ARGs loads and explored possible associations among them. With that aim, the bacterial community composition (16S rRNA gene Illumina sequencing) and ARGs abundance (real-time PCR) were characterized in samples of raw wastewater (RWW), secondary effluent (sTWW), after UV disinfection (tTWW), and after a period of 3 days storage to monitoring possible bacterial regrowth (tTWW-RE). Culturable enterobacteria were also enumerated. Secondary treatment was associated with the most dramatic bacterial community variations and coincided with reductions of ~2 log-units in the ARGs abundance. In contrast, no significant changes in the bacterial community composition and ARGs abundance were observed after UV disinfection of sTWW. Nevertheless, after UV treatment, viability losses were indicated ~2 log-units reductions of culturable enterobacteria. The analysed ARGs (qnrS, blaCTX-M, blaOXA-A, blaTEM, blaSHV, sul1, sul2, and intI1) were strongly correlated with taxa more abundant in RWW than in the other types of water, and which associated with humans and animals, such as members of the families Campylobacteraceae, Comamonadaceae, Aeromonadaceae, Moraxellaceae and Bacteroidaceae. Further knowledge of the dynamics of the bacterial community during wastewater treatment and its relationship with ARGs variations may contribute with information useful for wastewater treatment optimization, aiming at a more effective resistance control.
- Genotypic and phenotypic traits of blaCTX-M-carrying Escherichia coli strains from an UV-C-treated wastewater effluentPublication . Tavares, Rafael D. S.; Tacão, Marta; Figueiredo, Ana S.; Duarte, Ana S.; Esposito, Fernanda; Lincopan, Nilton; Manaia, Célia M.; Henriques, IsabelWastewater treatment plants (WWTPs) are relevant sources of antibiotic resistance into aquatic environments. Disinfection of WWTPs’ effluents (e.g. by UV-C irradiation) may attenuate this problem, though some clinically relevant bacteria have been shown to survive disinfection. In this study we characterized 25 CTX-M-producing Escherichia coli strains isolated from a WWTP’s UV-C-irradiated effluent, aiming to identify putative human health hazards associated with such effluents. Molecular typing indicated that the strains belong to the phylogroups A, B2 and C and clustered into 9 multilocus sequence types (STs), namely B2:ST131 (n ¼ 7), A:ST58 (n ¼ 1), A:ST155 (n ¼ 4), C:ST410 (n ¼ 2), A:ST453 (n ¼ 2), A:ST617 (n ¼ 2), A:ST744 (n ¼ 1), A:ST1284 (n ¼ 3) and a putative novel ST (n ¼ 3). PCR-screening identified 9 of the 20 antibiotic resistance genes investigated [i.e. sul1, sul2, sul3, tet(A), tet(B), blaOXA-1-like, aacA4, aacA4-cr and qnrS1]. The more prevalent were sul1, sul2 (n ¼ 15 isolates) and tet(A) (n ¼ 14 isolates). Plasmid restriction analysis indicated diverse plasmid content among strains (14 distinct profiles) and mating assays yielded cefotaxime-resistant transconjugants for 8 strains. Two of the transconjugants displayed a multi-drug resistance (MDR) phenotype. All strains were classified as cytotoxic to Vero cells (9 significantly more cytotoxic than the positive control) and 10 of 21 strains were invasive towards this cell line (including all B2:ST131 strains). The 10 strains tested against G. mellonella larvae exhibited a virulent behaviour. Twenty-four and 7 of the 25 strains produced siderophores and haemolysins, respectively. Approximately 66% of the strains formed biofilms. Genome analysis of 6 selected strains identified several virulence genes encoding toxins, siderophores, and colonizing, adhesion and invasion factors. Freshwater microcosms assays showed that after 28 days of incubation 3 out of 6 strains were still detected by cultivation and 4 strains by qPCR. Resistance phenotypes of these strains remained unaltered. Overall, we confirmed WWTP’s UV-C-treated outflow as a source of MDR and/or virulent E. coli strains, some probably capable of persisting in freshwater, and that carry conjugative antibiotic resistance plasmids. Hence, disinfected wastewater may still represent a risk for human health. More detailed evaluation of strains isolated from wastewater effluents is urgent, to design treatments that can mitigate the release of such bacteria.
- The impact of antibiotic exposure in water and zebrafish gut microbiomes: a 16S rRNA gene-based metagenomic analysisPublication . Almeida, Ana Rita; Alves, Marta; Domingues, Inês; Henriques, IsabelIn order to supply human demand for food, the aquaculture industry has been growing fast in the last years, being fish usually cultivated in overcrowded conditions. Hence, to prevent the rapidly disease spreading, antibiotics may be applied to both sick and healthy animals. Due to its broad spectrum, oxytetracycline (OTC) is one of the most used antibiotics in food-production. Yet, although useful to prevent infections, antibiotics may reshape aquatic animals' microbiome, disturbing hosts’ welfare. However, the impact of this exposure to the organism microbiome and its surrounding environment is poorly understood. Then, the objective of this study was to analyze in detail the long-term effect of OTC in both zebrafish gut and water microbiomes. Zebrafish adults were exposed, via water, for two months to three concentrations of OTC (0, 10 and 10000 μg/L). Total DNA was extracted from gut and water samples and the V3–V4 region of the bacterial 16 S rRNA gene was sequenced using Illumina technology. Results of alpha and beta-diversity analyses revealed that long-term exposure to OTC impacted both zebrafish gut and water microbiomes. In water samples, effects were observed even at the lowest (10 μg/L) OTC concentration tested resulting in an increase in Deltaproteobacteria, namely the Myxococcales and Bdellovibrionales orders. On the other hand, effects on zebrafish gut were only observed at the highest concentration with the selection of Alphaproteobacteria and Actinobacteria classes. Although these classes are common in fish gut, the increase of Actinobacteria may represent a health problem since some genera like Gordonia are linked to some human infection disease. Nevertheless, in both gut and water, it was observed a decrease in Gamaproteobacteria, probably due to OTC mode of action. In silico functional metagenomic analysis revealed that OTC exposure selected general detoxification mechanisms. In addition, the abundance of functional genes involved in Quorum Sensing (QS) increased under OTC exposure suggesting that QS may help bacteria to survive OTC stress. Thus, future studies should consider post-exposure scenarios for a deeper analysis of the water and zebrafish gut resistome, since bacteria may react differently after exposure ceased.
- Fate of cefotaxime-resistant enterobacteriaceae and ESBL-producers over a full-scale wastewater treatment process with UV disinfectionPublication . Silva, Isabel; Tacão, Marta; Tavares, Rafael D.S.; Miranda, Rita; Araújo, Susana; Manaia, Célia M.; Henriques, IsabelDisinfection by UV radiation is one of themost promising solutions to reduce the bacterial load and antibiotic resistance in the final effluents of urban wastewater treatment plants (UWTP). Our aim was to evaluate the fate of cefotaxime-resistant Enterobacteriaceae and Extended Spectrum Beta-Lactamase (ESBL) producers in a full-scale system that includes UV-C disinfection. Over treatment, the abundance of cefotaxime-resistant Enterobacteriaceaewas reduced,with reductions of 1.9 log units after secondary treatment (STWsamples) and 1.8 log following UV disinfection (UTW samples). These reductions, did not reflect the variations in the prevalence of cefotaximeresistant Enterobacteriaceae, estimated to be of 3% in rawwastewater (RW), 18% in STWand 3% in UTW. A significant increase of cefotaxime-resistant bacterial counts (0.5 log; p b 0.05)was observed after 3 days of storage. In a total of 1799 cefotaxime-resistant Enterobacteriaceae isolates, 15% harboured blaCTX-M (n = 274), 11% blaTEM (n = 194) and 4% blaSHV (n = 72). While the ESBL gene prevalence decreased over treatment, the prevalence of the intI1 gene decreased after ST but slightly increased in UTW samples. The blaCTX-M-carriers were identified as Escherichia coli and Klebsiella pneumoniae, mostly multi-drug resistant (90.5%) and carrying integrase genes (82.8%). The blaCTX-M gene variants (48 blaCTX-M-15, 9 blaCTX-M-32, 8 blaCTX-M-1, 5 blaCTX-M-27, and 2 blaCTX-M-14) were flanked by ISEcp1, ISEcp1/IS26, IS903 and ORF477 in 8 different arrangements. The IncF plasmid replicon type was highly prevalent among blaCTX-M-carrying Escherichia coli (74.5%) while IncR predominated among K. pneumoniae (54.5%). Our results confirmed the potential of UV-C disinfection to remove antibiotic resistant bacteria. Still, resistant Enterobacteriaceae (about 30 × 106 cells per m3 of water), presenting traits that might potentiate antibiotic resistance spread, are released in the final effluent. In addition, a significant regrowthwas observed after storage. These results suggest that improvements ofwastewater disinfection are still required tominimize the risks associated with UWTP discharges