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Culture-dependent and culture-independent diversity surveys target different bacteria: a case study in a freshwater sample

Publication . Vaz-Moreira, Ivone; Egas, Conceição; Nunes, Olga C.; Manaia, Celia

Compared 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.

2011Journal article

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Diversity and antibiotic resistance of aeromonas spp. in drinking and waste water treatment plants

Publication . Figueira, Vania; Vaz-Moreira, Ivone; Silva, Márcia; Manaia, Célia M.

The taxonomic diversity and antibiotic resistance phenotypes of aeromonads were examined in samples from drinking and waste water treatment plants (surface, ground and disinfected water in a drinking water treatment plant, and raw and treated waste water) and tap water. Bacteria identification and intra-species variation were determined based on the analysis of the 16S rRNA, gyrB and cpn60 gene sequences. Resistance phenotypes were determined using the disc diffusion method. Aeromonas veronii prevailed in raw surface water, Aeromonas hydrophyla in ozonated water, and Aeromonas media and Aeromonas puntacta in waste water. No aeromonads were detected in ground water, after the chlorination tank or in tap water. Resistance to ceftazidime or meropenem was detected in isolates from the drinking water treatment plant and waste water isolates were intrinsically resistant to nalidixic acid. Most of the times, quinolone resistance was associated with the gyrA mutation in serine 83. The gene qnrS, but not the genes qnrA, B, C, D or qepA, was detected in both surface and waste water isolates. The gene aac(6’)-ib-cr was detected in different waste water strains isolated in the presence of ciprofloxacin. Both quinolone resistance genes were detected only in the species A. media. This is the first study tracking antimicrobial resistance in aeromonads in drinking, tap and waste water and the importance of these bacteria as vectors of resistance in aquatic environments is discussed.

2011Journal article

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Bottled mineral water as a potential source of antibiotic resistant bacteria

Publication . Falcone-Dias, Maria Fernanda; Vaz-Moreira, Ivone; Manaia, Célia M.

The antibiotic resistance phenotypes of the cultivable bacteria present in nine batches of two Portuguese and one French brands of commercially available mineral waters were examined. Most of the 238 isolates recovered on R2A, Pseudomonas Isolation agar or on these culture media supplemented with amoxicillin or ciprofloxacin, were identified (based on 16S rRNA gene sequence analysis) as Proteobacteria of the divisions Beta, Gamma and Alpha. Bacteria resistant to more than three distinct classes of antibiotics were detected in all the batches of the three water brands in counts up to 102 CFU/ml. In the whole set of isolates, it was observed resistance against all the 22 antimicrobials tested (ATB, bio- Me´rieux and disc diffusion), with most of the bacteria showing resistance to three or more classes of antibiotics. Bacteria with the highest multi-resistance indices were members of the genera Variovorax, Bosea, Ralstonia, Curvibacter, Afipia and Pedobacter. Some of these bacteria are related with confirmed or suspected nosocomial agents. Presumable acquired resistance may be suggested by the observation of bacteria taxonomically related but isolated from different brands, exhibiting distinct antibiotic resistance profiles. Bottled mineral water was confirmed as a possible source of antibiotic resistant bacteria, with the potential to be transmitted to humans.

2012Journal article

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Diversity and antibiotic resistance in Pseudomonas spp. from drinking water

Publication . Vaz-Moreira, Ivone; Nunes, Olga C.; Manaia, Célia M.

Pseudomonas spp. are common inhabitants of aquatic environments, including drinking water. Multiantibiotic resistance in clinical isolates of P. aeruginosa is widely reported and deeply characterized. However, the information regarding other species and environmental isolates of this genus is scant. This study was designed based on the hypothesis that members of the genus Pseudomonas given their high prevalence, wide distribution in waters and genetic plasticity can be important reservoirs of antibiotic resistance in drinking water. With this aim, the diversity and antibiotic resistance phenotypes of Pseudomonas isolated from different drinking water sources were evaluated. The genotypic diversity analyses were based on six housekeeping genes (16S rRNA, rpoD, rpoB, gyrB, recA and ITS) and on pulsed field gel electrophoresis. Susceptibility to 21 antibiotics of eight classes was tested using the ATB PSE EU (08) and disk diffusion methods. Pseudomonas spp. were isolated from 14 of the 32 sampled sites. A total of 55 non-repetitive isolates were affiliated to twenty species. Although the same species were isolated from different sampling sites, identical genotypes were never observed in distinct types of water (water treatment plant/distribution system, tap water, cup fillers, biofilm, and mineral water). In general, the prevalence of antibiotic resistance was low and often the resistance patterns were related with the species and/or the strain genotype. Resistance to ticarcillin, ticarcillin with clavulanic acid, fosfomycin and cotrimoxazol were the most prevalent (69–84%). No resistance to piperacillin, levofloxacin, ciprofloxacin, tetracycline, gentamicin, tobramycin, amikacin, imipenem or meropenem was observed. This study demonstrates that Pseudomonas spp. are not so widespread in drinking water as commonly assumed. Nevertheless, it suggests that water Pseudomonas can spread acquired antibiotic resistance, preferentially via vertical transmission.

2012Journal article

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Bacillus purgationiresistans sp. nov., isolated from a drinking-water treatment plant

Publication . Vaz-Moreira, Ivone; Figueira, Vânia; Lopes, Ana R.; Lobo-da-Cunha, Alexandre; Sproer, Cathrin; Schumann, Peter; Nunes, Olga C.; Manaia, Célia M.

A Gram-positive, aerobic, non-motile, endospore-forming rod, designated DS22T, was isolated from a drinking-water treatment plant. Cells were catalase- and oxidase-positive. Growth occurred at 15–37 6C, at pH 7–10 and with ,8% (w/v) NaCl (optimum growth: 30 6C, pH 7–8 and 1–3% NaCl). The major respiratory quinone was menaquinone 7, the G+C content of the genomic DNA was 36.5 mol% and the cell wall contained meso-diaminopimelic acid. On the basis of 16S rRNA gene sequence analysis, strain DS22T was a member of the genus Bacillus. Its closest phylogenetic neighbours were Bacillus horneckiae NRRL B-59162T (98.5% 16S rRNA gene sequence similarity), Bacillus oceanisediminis H2T (97.9 %), Bacillus infantis SMC 4352-1T (97.4 %), Bacillus firmus IAM 12464T (96.8 %) and Bacillus muralis LMG 20238T (96.8 %). DNA–DNA hybridization, and biochemical and physiological characterization allowed the differentiation of strain DS22T from its closest phylogenetic neighbours. The data supports the proposal of a novel species, Bacillus purgationiresistans sp. nov.; the type strain is DS22T (5DSM 23494T5NRRL B-59432T5LMG 25783T).

2012Journal article

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