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- Acinetobacter rudis sp. nov., isolated from raw milk and raw wastewaterPublication . Vaz-Moreira, Ivone; Novo, Ana; Hantsis-Zacharov, Elionora; Lopes, Ana Rita; Gomila, Margarita; Nunes, Olga C.; Manaia, Célia M.; Halpern, MalkaTwo bacterial strains, G30T and A1PC16, isolated respectively from raw milk and raw wastewater, were characterized using a polyphasic approach. Chemotaxonomic characterization supported the inclusion of these strains in the genus Acinetobacter, with Q-8 and Q-9 as the major respiratory quinones, genomic DNA G+C contents within the range observed for this genus(38–47 mol%) and C16 : 0, C18 : 1v9c and C16 : 1v7c/iso-C15 : 0 2-OH as the predominant fatty acids. The observation of 16S rRNA gene sequence similarity lower than 97% with other Acinetobacter species with validly published names led to the hypothesis that these isolates could represent a novel species. This hypothesis was supported by comparative analysis of partial sequences of the genes rpoB and gyrB, which showed that strains G30T and A1PC16 did not cluster with any species with validly published names, forming a distinct lineage. DNA–DNA hybridizations confirmed that the two strains were members of the same species, which could be distinguished from their congeners by several phenotypic characteristics. On the basis of these arguments, it is proposed that strains G30T and A1PC16 represent a novel species, for which the name Acinetobacter rudis sp. nov. is proposed. The type strain is strain G30T (5LMG 26107T 5CCUG 57889T 5DSM 24031T 5CECT 7818T).
- 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
- Antibiotic resistance, antimicrobial residues and bacterial community composition in urban wastewaterPublication . Novo, Ana; André, Sandra; Viana, Paula; Nunes, Olga C.; Manaia, Célia M.This study was based on the hypothesis that the occurrence of antimicrobial residues and antibiotic resistant bacteria in the sewage could be correlated with the structure and composition of the bacterial community and the antibiotic resistance loads of the final effluent. Raw and treated wastewater composite samples were collected from an urban treatment plant over 14 sampling dates. Samples were characterized for the i) occurrence of tetracyclines, penicillins, sulfonamides, quinolones, triclosan, arsenic, cadmium, lead, chromium and mercury; ii) antibiotic resistance percentages for tetracycline, sulfamethoxazole, ciprofloxacin and amoxicillin and iii) 16S rRNA gene-DGGE patterns. The data of corresponding samples, taking into account the hydraulic residence time, was analyzed using multivariate analysis. Variations on the bacterial community structure of the final effluent were significantly correlated with the occurrence of tetracyclines, penicillins, sulfonamides, quinolones and triclosan in the raw inflow. Members of the class Epsilonproteobacteria presented positive correlations with those antimicrobials, whereas negative correlations were observed with Beta and Gammaproteobacteria and Firmicutes. Antibiotic resistance percentages presented different trends of variation in heterotrophs/enterobacteria and in enterococci, varied over time and after wastewater treatment. Antibiotic resistance was positively correlated with the occurrence of tetracyclines residues and high temperature. A relationship between antibiotic residues, bacterial community structure and composition and antibiotic resistance is demonstrated. Further studies, involving more wastewater treatment plants may help to elucidate this complex relationship.
- A pilot study combining ultrafiltration with ozonation for the treatment of secondary urban wastewater: organic micropollutants, microbial load and biological effectsPublication . Graça, Cátia A. L.; Ribeirinho-Soares, Sara; Abreu-Silva, Joana; Ramos, Inês I.; Ribeiro, Ana R.; Castro-Silva, Sérgio M.; Segundo, Marcela A.; Manaia, Célia M.; Nunes, Olga C.; Silva, Adrián M. T.Ozonation followed by ultrafiltration (O3 + UF) was employed at pilot scale for the treatment of secondary urban wastewater, envisaging its safe reuse for crop irrigation. Chemical contaminants of emerging concern (CECs) and priority substances (PSs), microbial load, estrogenic activity, cell viability and cellular metabolic activity were measured before and immediately after O3 + UF treatment. The microbial load was also evaluated after one-week storage of the treated water to assess potential bacteria regrowth. Among the organic micropollutants detected, only citalopram and isoproturon were not removed below the limit of quantification. The treatment was also effective in the reduction in the bacterial loads considering current legislation in water quality for irrigation (i.e., in terms of enterobacteria and nematode eggs). However, after seven days of storage, total heterotrophs regrew to levels close to the initial, with the concomitant increase in the genes 16S rRNA and intI1. The assessment of biological effects revealed similar water quality before and after treatment, meaning that O3 + UF did not produce detectable toxic by-products. Thus, the findings of this study indicate that the wastewater treated with this technology comply with the water quality standards for irrigation, even when stored up to one week, although improvements must be made to minimise microbial overgrowth.
- Paenibacillus residui sp. nov., isolated from urban waste compostPublication . Vaz-Moreira, Ivone; Figueira, Vânia; Lopes, Ana Rita; Pukall, Rüdiger; Spröer, Cathrin; Schumann, Peter; Nunes, Olga C.; Manaia, Célia M.Two bacterial strains, MC-246T and MC-247, were isolated from municipal urban waste compost and characterized by a polyphasic approach. Both isolates were Gram-stain-variable, endosporeforming rods that were catalase-, oxidase- and b-galactosidase-positive, and able to grow at 25– 50 6C and pH 7.0–9.0, with optimum growth at 37 6C and pH 7. The predominant cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0 and iso-C17 : 0; the major respiratory quinone was menaquinone MK-7; the cell wall peptidoglycan was of type A1c; and the DNA G+C content was 49 mol%. These characteristics, as well as data from 16S RNA gene sequence analysis, showed that these strains were affiliated with the genus Paenibacillus; the type strains of Paenibacillus ginsengarvi and Paenibacillus hodogayensis were among their closest neighbours (,94.2 % sequence similarity). Nevertheless, the hypothesis that strains MC246T and MC-247 could represent a novel species was supported by the low 16S rRNA gene sequence similarity values shared with other members of the genus Paenibacillus and by the observation of distinct biochemical and physiological traits. Strains MC-246T and MC-247 shared 99.6 % 16S rRNA gene sequence similarity and showed almost identical MALDI-TOF mass spectra, but could be distinguished at the phenotypic and genotypic level. However, DNA–DNA hybridization between strains MC-246T and MC-247 resulted in values above 70 % indicating that both organisms represent a single species, for which the name Paenibacillus residui sp. nov. is proposed; the type strain is MC-246T (5DSM 22072T 5CCUG 57263T).
- In memoriam. Farewell MiltonPublication . Manaia, Célia M.; Albuquerque, Luciana; Veríssimo, António; Morais, Paula V.; Nunes, Olga C.
- Ultrafiltration after ozonation of urban wastewater: tackling bacterial regrowthPublication . Soares, S. R.; Graça, C. A. L.; Abreu-Silva, J.; Manaia, C. M.; Ribeiro, A. R.; Silva, A. M. T.; Nunes, O. C.
- Environmental factors influencing molinate biodegradation by a two-member mixed culture in rice paddy field floodwaterPublication . Barreiros, Luisa; Peres, Joana; Azevedo, Nuno F.; Manaia, Célia M.; Nunes, Olga C.Bioaugmentation is reported as a feasible approach for the treatment of pesticide contaminated environments. Aiming the development and future implementation of a bioremediation process to treat natural waters polluted with molinate, a molinate-mineralizing culture, composed of Pseudomonas chlororaphis ON1 and Gulosibacter molinativorax ON4T (designated DC1), was assayed in paddy field floodwater microcosms. The influence of abiotic (temperature, presence of other herbicides) and biotic (floodwater autochthonous microbiota) factors on molinate mineralization by culture DC1 was assessed. In parallel, the proportion of the introduced strains in filter-sterilized floodwaters was monitored by fluorescent in situ hybridization (FISH). Molinate mineralization and growth of culture DC1 were observed under all tested conditions, although the kinetic parameters (maximum specific growth and degradation rates) were significantly affected by the environmental conditions and culture media used. Additionally, these two factors were observed to have a statistically significant interaction. The lowest values of both kinetic parameters were observed at 15 °C. The presence of the herbicides propanil, bentazone and MCPA, frequently applied in rice culture protection, did not affect the degrading capacity of culture DC1. Furthermore, it was possible to infer that the autochthonous microbiota does not retard or limit molinate biodegradation, given the growth and degradation rates by culture DC1 were higher in non-sterile microcosm assays. Although G. molinativorax ON4T is known to promote the initial breakdown of molinate, P. chlororaphis ON1 appeared to be responsible to pull up the process, since higher proportions of this organism were found at the exponential growth and molinate degradation phase. Culture DC1 is efficient, versatile and presents a promising potential to be applied as a bioaugmentation tool for the remediation of environmental waters contaminated with molinate.
- Distinct behavior between multi-antibiotic resistant escherichia coli strains towards reactive oxygen speciesPublication . Sousa, Vera M.; Mendes, Adélio; Manaia, C. M.; Nunes, Olga C.
- Culture-dependent and culture-independent diversity surveys target different bacteria: a case study in a freshwater samplePublication . Vaz-Moreira, Ivone; Egas, Conceição; Nunes, Olga C.; Manaia, CeliaCompared with culture-independent approaches, traditionally used culture-dependent methods have a limited capacity to characterizewatermicrobiota. Nevertheless, for almost a century the latter have been optimized to detect and quantify relevant bacteria. A pertinent question is if culture-independent diversity surveys give merely an extended perspective of the bacterial diversity or if, even with a higher coverage, focus on a different set of organisms. We compared the diversity and phylogeny of bacteria in a freshwater sample recovered by currently used culture-dependent and culture-independent methods (DGGE and 454 pyrosequencing). The culture-dependent diversity survey presented lower coverage than the other methods. However, it allowed bacterial identifications to the species level, in contrast with the other procedures that rarely produced identifications below the order. Although the predominant bacterial phyla detected by both approaches were the same (Proteobacteria, Actinobacteria, Bacteroidetes), sequence similarity analysis showed that, in general, different operational taxonomical units were targeted by each method. The observation that culture-dependent and independent approaches target different organisms has implications for the use of the latter for studies in which taxonomic identification has a predictive value. In comparison to DGGE, 454 pyrosequencing method had a higher capacity to explore the bacterial richness and to detect cultured organisms, being also less laborious.