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- blaTEM and vanA as indicator genes of antibiotic resistance contamination in a hospital–urban wastewater treatment plant systemPublication . Narciso-da-Rocha, Carlos; Varela, Ana R.; Schwartz, Thomas; Nunes, Olga C.; Manaia, Celia M.Four indicator genes were monitored by quantitative PCR in hospital effluent (HE) and in the raw and treated wastewater of the municipal wastewater treatment plant receiving the hospital discharge. The indicator genes were the class 1 integrase gene intI1, to assess the capacity of bacteria to be involved in horizontal gene transfer processes; blaTEM, one of the most widespread antibiotic resistance genes in the environment, associated with Enterobacteriaceae; vanA, an antibiotic resistance gene uncommon in the environment and frequent in clinical isolates; and marA, part of a locus related to the stress response in Enterobacteriaceae. Variation in the abundance of these genes was analysed as a function of the type of water, and possible correlations with cultivable bacteria, antimicrobial residue concentrations, and bacterial community composition and structure were analysed. HE was confirmed as an important source of blaTEM and vanA genes, and wastewater treatment showed a limited capacity to remove these resistance genes. The genes blaTEM and vanA presented the strongest correlations with culturable bacteria, antimicrobial residues and some bacterial populations, representing interesting candidates as indicator genes to monitor resistance in environmental samples. The intI1 gene was the most abundant in all samples, demonstrating that wastewater bacterial populations hold a high potential for gene acquisition
- Genotypic diversity and antibiotic resistance in Sphingomonadaceae isolated from hospital tap waterPublication . Narciso-da-Rocha, Carlos; Vaz-Moreira, Ivone; Manaia, Célia M.The aim of this study was to infer about the modes and extent of dispersion of Sphingomonadaceae via tap water. Sphingomonadaceae isolated from tap water samples in different places of a hospital were compared, based on intra-species genetic variability and antibiotic resistance phenotypes. These isolates were also compared with others isolated before from houses and dental chairs, served by the same municipal water supply system. Sphingomonadaceae from hospital tap water comprised members of the genera Sphingomonas, Sphingobium, Novosphingobium and Blastomonas. In general, distinct genotypes of Sphingomonadaceae were detected in different hospital areas and in tap water outside the hospital, suggesting these bacteria are not persistent or widespread in the urban water distribution system. Possible intrinsic antibiotic resistance, observed in most or all members of the family or of a genus, was observed for colistin in Sphingomonadaceae, aminoglycosides in the genus Blastomonas and beta-lactams in the genus Sphingobium. Possible acquired resistance phenotypes, not common to all members of a given species, comprised fluoroquinolones, cephalosporins and sulphonamides. Although the potential of Sphingomonadaceae as opportunistic pathogens may be low, the capacity of these bacteria to thrive in water supply systems, combined with the intrinsic or acquired antibiotic resistance, may raise the risk associated with their occurrence in hospital tap water.
- The influence of the autochthonous wastewater microbiota and gene host on the fate of invasive antibiotic resistance genesPublication . Narciso-da-Rocha, Carlos; Manaia, Célia M.The aim of this study was to assess the fate of invasive antibiotic resistance genes (ARG) discharged in wastewater. With this objective, antibiotic resistant bacteria (ARB) known to harbor specific ARG were inoculated in wastewater (hospital effluent, or municipal raw and treated wastewater) and in ultra-pure sterile water microcosms. Two sets of wastewater ARB isolates were used - set 1, Enterococcus faecalis, Acinetobacter johnsonii, Klebsiella pneumoniae and set 2, Enterococcus faecium, Acinetobacter johnsonii, Escherichia coli. Non-inoculated controls were run in parallel. Samples were collected at the beginning and at the end (15 days) of the incubation period and the abundance of the genes 16S rRNA, intI1, blaTEM and vanA and the bacterial community composition were analyzed. In general, the genes blaTEM and vanA had lower persistence in wastewater and in ultra-pure water than the genes 16S rRNA or the class 1 integron integrase intI1. This effect was more pronounced in wastewater than in ultra-pure water, evidencing the importance of the autochthonous microbiota on the elimination of invasive ARG. Wastewater autochthonous bacterial groups most correlated with variations of the genes intI1, blaTEM and vanA were members of the classes Gammaproteobacteria, Bacilli or Bacteroidia. For blaTEM, but not for vanA, the species of the ARB host was important to determine its fate. These are novel findings on the ecology of ARB in wastewater environments.
- Diversity and antibiotic resistance of Acinetobacter spp. in water from the source to the tapPublication . Narciso-da-Rocha, Carlos; Vaz-Moreira, Ivone; Svensson-Stadler, Liselott; Moore, Edward R. B.; Manaia, Célia M.Acinetobacterspp. are ubiquitous bacteria in the environment.Acinetobacterspp. isolated from a municipal drinking water treatment plant and from connected tap waterwere identified to the species level on the basis ofrpoB genepartial sequence analysis. Intraspecies variation wasassessed based on the analysis of partial sequences of house-keeping genes (rpoB,gyrB, andrecA). Antibiotic resistancewas characterized using the disk diffusion method and iso-lates were classified as wild or non-wild type (non-WT),according to the observed phenotype. The strains ofAcinetobacterspp. were related to 11 different validly pub-lished species, although three groups of isolates, presentinglowrpoB sequence similarities with previously describedspecies, may represent new species. Most of the isolateswere related to the speciesA. johnsoniiandA. lwoffii.These two groups, as well as others related to the speciesA. parvusandA. tjernbergiae, were detected in the watertreatment plant and in tap water. Other strains, related to thespeciesA. pittiiandA. beijerinckii, were isolated only fromtap water. Most of the isolates (80 %) demonstrated wild type (WT) to all of the 12 antibiotics tested. Non-WT fortetracycline, meropenem, and ceftazidime, among others,were observed in water treatment plant or in tap watersamples. Although, in general, this study suggests a lowprevalence of acquired antibiotic resistance in waterAcinetobacterspp., the potential of some species to acquireand disseminate resistance via drinking water is suggested.
- 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.
- 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.
- Hydromonas duriensis gen. nov., sp. nov., isolated from freshwaterPublication . Vaz-Moreira, Ivone; Narciso-da-Rocha, Carlos; De Brandt, Evie; Vandamme, Peter; Ferreira, A. C. Silva; Lobo-da-Cunha, Alexandre; Nunes, Olga C.; Manaia, Célia M.An aerobic, Gram-stain-negative rod, designated strain A2P5T , was isolated from the Douro river, in Porto, Portugal. Cells were catalase- and oxidase-positive. Growth occurred at15–30 8C, at pH 6–8 and in the presence of 1 % (w/v) NaCl. The major respiratory quinone was Q8, the genomic DNA had a G+C content of 47¡1 mol%, and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol were amongst the major polar lipids. On the basis of 16S rRNA gene sequence analysis, strain A2P5T was observed to be a member of the family Burkholderiaceae, but could not be identified as a member of any validly named genus. The low levels of 16S rRNA gene sequence similarity to other recognized taxa (,91 %), together with the comparative analysis of phenotypic and chemotaxonomic characteristics, supported the proposal of a novel species of a new genus within the family Burkholderiaceae. The name Hydromonas duriensis gen. nov., sp. nov. is proposed. The type strain of Hydromonas duriensis is A2P5T (5LMG 28428T 5CCUG 66137T).
- Quinolone-resistant Escherichia coli isolated from birds of prey in Portugal are genetically distinct from those isolated from water environments and gulls in Portugal, Spain and SwedenPublication . Vredenburg, Jana; Varela, Ana Rita; Hasan, Badrul; Bertilsson, Stefan; Olsen, Björn; Narciso-da-Rocha, Carlos; Bonnedahl, Jonas; Stedt, Johan; Costa, Paulo Martins Da; Manaia, Célia M.The influence of geographic distribution and type of habitat on the molecular epidemiology of ciprofloxacin resistant Escherichia coli was investigated. Ciprofloxacin resistant E. coli from wastewater, urban water with faecal contamination and faeces of gulls, pigeons and birds of prey, from Portugal, Spain and Sweden were compared based on multilocus sequence typing (MLST) and quinolone resistance genetic determinants. Multi-locus sequence typing allowed the differentiation of E. coli lineages associated with birds of prey from those inhabiting gulls and waters. E. coli lineages of clinical relevance, such as the complex ST131, were detected in wastewater, streams and gulls in Portugal, Spain and Sweden. Quinolone resistance was due to gyrA and parC mutations, although distinct mutations were detected in birds of prey and in wastewater, streams and gulls isolates. These differences were correlated with specific MLST lineages, suggesting resistance inheritance. Among the plasmid-mediated quinolone resistance genes, only aac(6′)-ib-cr and qnrS were detected in wastewater, streams and gulls isolates, but not in birds of prey. The horizontal transfer of the gene aac(6′)-ib-cr could be inferred from its occurrence in different MLST lineages
- Oryzisolibacter propanilivorax gen. nov., sp nov., a propanil-degrading bacteriumPublication . Vaz-Moreira, Ivone; Narciso-da-Rocha, Carlos; Lopes, Ana Rita; Carvalho, Gilda; Lobo-da-Cunha, Alexandre; Whitman, William B.; Snauwaert, Cindy; Vandamme, Peter; Manaia, Célia M.; Nunes, Olga C.Strain EPL6T, a Gram-negative, motile, short rod was isolated from a propanil and 3,4-dichloroaniline enrichment culture produced from rice paddy soil. Based on the analyses of the 16S rRNA gene sequence, strain EPL6T was observed to be a member of the family Comamonadaceae , sharing the highest pairwise identity with type strains of the species Alicycliphilus denitrificans K601T (96.8 %) and Melaminivora alkalimesophila CY1T (96.8 %). Strain EPL6T was able to grow in a temperature range of 15–37 °C, pH 6–9 and in the presence of up to 4 % (w/v) NaCl and tested positive for catalase and oxidase reactions. The major respiratory quinone was Q8. The genomic DNA had a G+C content of 69.4±0.9 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, and the major fatty acid methyl esters comprised C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH). Comparison of the genome sequence of strain EPL6T and of its closest neighbours, Melaminivora alkalimesophila CY1T and Alicycliphilus denitrificans K601T, yielded values of ANI ≤84.1 % and of AAI ≤80.3 %. Therefore, the genetic, phylogenetic, phenotypic and chemotaxonomic characteristics support the classification of this organism into a new taxon. Considering the genetic divergence of strain EPL6T from the type strains of the closest species, which belong to distinct genera, we propose a new genus within the family Comamonadaceae , named Oryzisolibacter propanilivorax gen. nov., sp. nov., represented by the isolate EPL6T as the type strain of the species (=LMG 28427T=CECT 8927T).
- Multidrug resistance phenotypes are widespread over different bacterial taxonomic groups thriving in surface waterPublication . Narciso-da-Rocha, Carlos; Manaia, Célia M.The environment is the original and most ancient source of the antibiotic resistance determinants that threat the human health nowadays. In the environment, water is a privileged habitat and mode of dissemination of bacteria of different origins. Freshwater bodies that cross urban areas are supposed to hold a complex mixture of both human/animal origin and strictly environmental bacteria. In this study,wewere interested in unveiling the bacterial diversity in urban river transects and, simultaneously, investigate the occurrence of antibiotic resistant bacteria, in particular themultidrug resistant (MDR).With this aim, water and sediments of two rivers were sampled from an urban transect and the bacterial diversitywas assessed based on 16S rRNA gene-based community analysis and, simultaneously, total heterotrophic bacteria were isolated in the presence and in the absence of antibiotics. The three predominant phyla were Proteobacteria, Bacteroidetes and Actinobacteria, in water, or Acidobacteria, in sediments. MDR bacteria were observed to belong to the predominant phyla observed in water, mostly of the classes Gamma- and Betaproteobacteria (Proteobacteria) and Sphingobacteriia and Flavobacteriia (Bacteroidetes) and belonged to genera of ubiquitous (Pseudomonas, Acinetobacter, Stenotrophomonas) ormainly environmental (Chitinophaga, Chryseobacterium) bacteria. The observation that MDR bacteria are widespread in the environment and over distinct phylogenetic lineages has two relevant implications: i) the potential of environmental bacteria as source or facilitators for antibiotic resistance acquisition; ii) the need to complement cultureindependent methods with culture-based approaches in order to identify major sources of MDR profiles.