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- Evolution of gentamicin and arsenite resistance acquisition in Ralstonia pickettii water isolatesPublication . Ferro, Pompeyo; Vaz-Moreira, Ivone; Manaia, Célia M.Ralstonia pickettii are ubiquitous in water environments. Members of this species are frequently, but not always, resistant to both gentamicin and arsenite. Gentamicin and arsenite co-resistance and the putative molecular mechanisms were investigated. A group of 37 R. pickettii strains isolated from drinking water and hospital wastewater were characterized for gentamicin and arsenite resistance phenotypes, the number and size of plasmids, and screened for genetic elements associated with arsenite tolerance, Integrative and Conjugative Elements (ICEs), among other. The genomes of three representative strains were compared. Most gentamicin resistant (GR) isolates (32/33) were resistant to arsenite, and harbored ICE- and ars operon-related genes. These genetic elements were not detected in any of the five arsenite susceptible strains, regardless of the GR (n = 1) or gentamicin susceptibility (GS) (n = 4) phenotype. The comparison of the genomes of two GR (one resistant and one susceptible to arsenite) and one GS strains suggested that these phenotypes correspond to three phylogroups, distinguished by presence of some genes only in GR isolates, in addition to point mutations in functional genes. The presence of ICEs and ars operon-related genes suggest that arsenite resistance might have been acquired by GR lineages.
- 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).
- The role of environmental stressors and genetic information on the antimicrobial tolerance of Ralstonia pickettiiPublication . Ferro, Pompeyo; Vaz-Moreira, Ivone; Tamames, Javier; Martínez, José L.; Manaia, C. M.
- 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).
- Neighbor urban wastewater treatment plants display distinct profiles of bacterial community and antibiotic resistance genesPublication . Fernandes, Telma; Vaz-Moreira, Ivone; Manaia, Célia M.Urban wastewater treatment plants (UWTPs) are among the major recipients of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues in urban environments. Although during treatment, bacteria of human and animal origin are removed, some are able to survive, persisting in the final effluent. The occurrence of these bacteria, especially those harboring ARGs, may have a direct impact on the quality of the treated wastewater that is returned to the environment. In this study, we aimed to assess if the final effluent bacterial communities of three UWTPs (PT1, PT2, and PT3) located next to each other were distinct and if such differences were related with the antibiotic resistance profiles. It was observed that the bacterial community (16S rRNA gene Illumina sequencing) and load of selected ARGs of final effluent differed among the three UWTPs, irrespective of sampling time. Members of the families Aeromonadaceae, Campylobacteraceae, Veillonellaceae, [Weeksellaceae], and Porphyromonadaceae were observed to be positively correlated with some ARGs (bla(CTX-M), bla(OXA-A), bla(SHV)) and intI1 (p < 0.05), while Intrasporangiaceae were observed to be negatively correlated. While Aeromonadaceae are recognized relevant ARG harbors, the other bacterial families may represent bacteria that co-exist with the ARG hosts, which may belong to minor bacterial groups omitted in the analyses. These findings suggest the importance of bacterial dynamics during treatment to the ARB&ARGs removal, a rationale that may contribute to design new strategies to apply in the UWTPs to prevent the spread of antibiotic resistance.
- 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.
- Microbacterium invictum sp. nov., isolated from homemade compostPublication . Vaz-Moreira, Ivone; Lopes, Ana R.; Faria, Cátia; Sproer, Cathrin; Schumann, Peter; Nunes, Olga C; Manaia, Célia M.Strain DC-200T was isolated from homemade compost produced from kitchen refuse and characterized using a polyphasic approach. The isolate was a Gram-positive motile short rod, facultatively aerobic, catalase-positive and oxidase-negative, and was able to grow at 10–37 6C, pH 6.0–9.5 and with up to 5% of NaCl. The peptidoglycan was of the type B1 alpha and the muramic acid residues were glycolylated. The major fatty acids were anteiso-C15 : 0 and anteiso- C17 : 0. The predominant respiratory menaquinones were MK-11 and MK-12. The G+C content of the genomic DNA was 70 mol%. Based on the analysis of the 16S rRNA gene sequence, the closest phylogenetic neighbours of strain DC-200T were Microbacterium lacus A5E-52T (98.7 %) and Microbacterium aoyamense KV-492T (98.2 %). The phenetic characterization of the isolate supports its inclusion within the genus Microbacterium; however, its distinctive phenotypic features and the results from the 16S rRNA gene sequence analysis and the DNA–DNA hybridization study suggest that the isolate represents a novel species. The name Microbacterium invictum sp. nov. is proposed. The type strain is DC-200T (5DSM 19600T5LMG 24557T).
- Sphingobium vermicomposti sp. nov., isolated from vermicompostPublication . Vaz-Moreira, Ivone; Faria, Cátia; Lopes, Ana R.; Svensson, Liselott; Falsen, Enevold; Moore, Edward R. B.; Ferreira, António C. Silva; Nunes, Olga C.; Manaia, Célia M.Strain VC-230T was isolated from homemade vermicompost produced from kitchen waste. The isolate was a Gram-negative-staining, catalase- and oxidase-positive, motile rod-shaped bacterium able to grow at 15–37 6C and pH 6–8. On the basis of 16S rRNA gene sequence analysis, strain VC-230T was determined to belong to the family Sphingomonadaceae by its clustering with type strains of the genus Sphingobium, with Sphingobium chlorophenolicum ATCC 33790T (97.7 %) and Sphingobium herbicidovorans DSM 11019T (97.4 %) as its closest neighbours. The polar lipid pattern, the presence of spermidine and ubiquinone 10, the predominance of the cellular fatty acids C18 : 1v7c/9t/12t, C16 : 1v7c and C16 : 0 and the G+C content of the genomic DNA supported the affiliation of this organism to the genus Sphingobium. The phylogenetic, chemotaxonomic, phenotypic and DNA–DNA hybridization analyses verify that strain VC-230T represents a novel species, for which the name Sphingobium vermicomposti sp. nov. is proposed. The type strain is VC-230T (5CCUG 55809T 5DSM 21299T).
- Multidrug resistance in quinolone-resistant gram-negative bacteria isolated from hospital effluent and the municipal wastewater treatment plantPublication . Vaz-Moreira, Ivone; Varela, Ana Rita; Pereira, Thamiris V.; Fochat, Romário C.; Manaia, C. M.This study is aimed to assess if hospital effluents represent an important supplier of multidrug-resistant (MDR) Gram-negative bacteria that, being discharged in the municipal collector, may be disseminated in the environment and bypassed in water quality control systems. From a set of 101 non-Escherichia coli Gram-negative bacteria with reduced susceptibility to quinolones, was selected a group of isolates comprised by those with the highest indices of MDR (defined as nonsusceptibility to at least one agent in six or more antimicrobial categories, MDR ‡6) or resistance to meropenem or ceftazidime (n = 25). The isolates were identified and characterized for antibiotic resistance phenotype, plasmid-mediated quinolone resistance (PMQR) genes, and other genetic elements and conjugative capacity. The isolates with highest MDR indices were mainly from hospital effluent and comprised ubiquitous bacterial groups of the class Gammaproteobacteria, of the genera Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Klebsiella, and Pseudomonas, and of the class Flavobacteriia, of the genera Chryseobacterium and Myroides. In this group of 25 strains, 19 identified as Gammaproteobacteria harbored at least one PMQR gene (aac(6¢)-Ib-cr, qnrB, qnrS, or oqxAB) or a class 1 integron gene cassette encoding aminoglycoside, sulfonamide, or carbapenem resistance. Most of the E. coli J53 transconjugants with acquired antibiotic resistance resulted from conjugation with Enterobacteriaceae. These transconjugants demonstrated acquired resistance to a maximum of five classes of antibiotics, one or more PMQR genes and/or a class 1 integron gene cassette. This study shows that ubiquitous bacteria, other than those monitored in water quality controls, are important vectors of antibiotic resistance and can be disseminated from hospital effluent to aquatic environments. This information is relevant to support management options aiming at the control of this public health problem.
- 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.