Genotoxicity of 4-nonylphenol and nonylphenol ethoxylate mixtures by the use of Saccharomyces cerevisiae D7 mutation assay and use of this text to evaluate the efficiency of biodegradation treatments.

Nonylphenol ethoxylates (NPnEOs, where n is the number of ethoxylic units in the molecule) are non-ionic surfactants widely used for domestic and industrial purposes. 4-Nonylphenol (4-NP), the main product of NPnEO biodegradation, is a toxic xenobiotic compound classified as endocrine disrupter. While numerous studies reported the toxicity and oestrogenic activity of nonylphenols, little is known about the mutagenicity of these compounds. In this paper, the genotoxicity of 4-NP and NPnEO mixtures was evaluated by using the D7 strain of Saccharomyces cerevisiae as experimental model. The same genotoxicity tests were applied to effluents deriving from experimental packed-bed bioreactors, developed for the treatment of NPnEO contaminated wastewater, in order to evaluate the residual genotoxic potential with respect to the influent waste. The target compounds fed to the bioreactors were 4-NP and NPnEO mixtures possessing an average of 5 or 1.5 ethoxylic units (Igepal CO-520 and Igepal CO-210, respectively). The results showed that 4-NP induced significant cytotoxic effect on S. cerevisiae cells at 50 mg/L, as well as mutagenic effects at the lowest tested concentrations (12 and 25 mg/L). 4-NP was the most genotoxic compound among those assayed, followed by Igepal CO-210, whereas Igepal CO-520 did not induce genotoxicity at any of the assayed concentrations. The genotoxic effects of 4-NP on yeast cells disappeared after the treatment of 4-NP artificially contaminated water in the bioreactor. This indicates that the biological treatment is capable of removing not only the pollutant, but also the toxicity associated to the compound and its degradation metabolites. This study represents, to the best of our knowledge, the first report that evaluates the genotoxicity of both 4-NP, NPnEOs and their potential aerobic degradation products on an eukaryotic organism. The obtained results suggest that the S. cerevisiae D7 strain is a very effective model microorganism to study the induction of genotoxic damage by the compounds under study. Moreover, this yeast assay has been proved effective to evaluate the detoxification effect deriving from biotreatment processes.

Nonylphenol polyethoxylate degradation in aqueous waste by the use of batch and continuous biofilm bioreactors.

An aerobic bacterial consortium (Consortium A) was recently obtained from textile wastewater and was capable of degrading 4-nonylphenol and nonylphenol polyethoxylates (NPnEOs). In the perspective of developing a biotechnological process for the treatment of effluents from activated sludge plants fed with NPnEO contaminated wastewater, the capability of Consortium A of biodegrading an industrial mixture of NPnEOs in the physiological condition of immobilized cells was investigated. Two identically configured packed bed reactors were developed by immobilizing the consortium on silica beads or granular activated carbon. Both reactors were tested in batch and continuous mode by feeding them with water supplemented with NPnEOs. The two reactors were monitored through chemical, microbiological and molecular integrated methodology. Active biofilms were generated on both immobilization supports. Both reactors displayed comparable NPnEO mineralization under batch and continuous conditions. FISH analyses evidenced that the biofilms evolved with time by changing the reactor operation mode and the organic load. Taken together, the data collected in this study provide a preliminary strong indication on the feasibility of Consortium A-based biofilm technology for the decontamination of NPnEO containing effluents.

Characterization of 4-nonylphenol-degrading bacterial consortium obtained from a textile wastewater pretreatment plant.

4-Nonylphenol (4-NP) isomers are toxic and recalcitrant compounds often resulting, together with short-chain ethoxylated nonylphenol (NPnEO, where n is the number of ethylene oxide units), from NPnEO biodegradation in conventional activated sludge plants. In this work, a microbial consortium, defined as Consortium A, capable of removing 100 mg/L of 4-NP with no accumulation of metabolites with aromatic moiety was isolated from textile wastewaters after enrichment with 4-NP. The consortium showed remarkable degradation activities toward several short-chain NPnEO congeners. Culture-dependent techniques were used to isolate from the consortium twenty-six strains assigned to seven different amplified ribosomal DNA restriction analysis groups. Two- and three-member cocultures were prepared with the strains showing highest 4-NP-degrading capabilities, but neither the single strains nor the cocultures were as efficient in 4-NP degradation as Consortium A. FISH was used to characterize the microbial composition of Consortium A: it evidenced a strong occurrence of Proteobacteria and, in particular, of Gammaproteobacteria along with a relevant stability of the culture. Therefore, the isolated consortium has the potential of being used in the development of a biotechnological process for the tertiary treatment of effluents of activated sludge plants fed with NPnEO-contaminated wastewaters.

Bacterial ectosymbionts and virulence silencing in a Fusarium oxysporum strain.

In the present article we have ascertained the presence of a consortium of ectosymbiotic bacteria belonging to Serratia, Achromobacter, Bacillus and Stenotrophomonas genera associated to the mycelium of the antagonistic Fusarium oxysporum MSA 35 [wild-type (WT) strain]. Morphological characterization carried out on the WT strain, on the F. oxysporum MSA 35 without ectosymbionts [cured (CU) strain] and on the pathogenic F. oxysporum f.sp. lactucae (Fuslat 10) showed that the ectosymbionts, present only in the WT strain, caused a depleted production of micro conidia and aerial hyphae, and a change in shape and dimension of the latter. Virulence tests showed that the cured Fusarium was a pathogenic strain and, as shown by polymerase chain reaction and microscope analysis, pathogenicity was correlated with the capability of the cured hyphae of penetrating lettuce roots. Accordingly, the hyphae of the WT strain were impaired in entering the plant roots. Typing experiments provided evidence that both CU and WT strains belong to F. oxysporum f.sp. lactucae. This implies that the antagonistic effect of WT Fusarium is not a fungal trait, but it is due to the interaction with the ectosymbiotic bacteria. Expression analysis showed that fmk1, chsV and pl1 genes involved in F. oxysporum pathogenicity are not expressed in the WT strain whereas they are expressed in the cured fungus. These results, together with the hyphal characteristics, suggest that the inability of WT strain to penetrate the plant roots could be due to alterations in the expression profile of cell wall-degrading enzymes. In conclusion, we demonstrated a modulation of F. oxysporum gene expression in response to the interaction with the ectosymbiotic bacteria. Preliminary researches indicated that the presence of bacteria attached to the hyphae of antagonistic F. oxysporum is not an isolated phenomenon. Further investigations are necessary to better understand the rule and the diffusion of ectosymbiotic bacteria among antagonistic Fusarium.

Induction and characterization of morphologic mutants in a natural Saccharomyces cerevisiae strain.

Saccharomyces cerevisiae is a good model with which to study the effects of morphologic differentiation on the ecological behaviour of fungi. In this work, 33 morphologic mutants of a natural strain of S. cerevisiae, obtained with UV mutagenesis, were selected for their streak shape and cell shape on rich medium. Two of them, showing both high sporulation proficiency and constitutive pseudohyphal growth, were analysed from a genetic and physiologic point of view. Each mutant carries a recessive monogenic mutation, and the two mutations reside in unlinked genes. Flocculation ability and responsiveness to different stimuli distinguished the two mutants. Growth at 37 degrees C affected the cell but not the colony morphology, suggesting that these two phenotypes are regulated differently. The effect of ethidium bromide, which affects mitochondrial DNA replication, suggested a possible "retrograde action" of mitochondria in pseudohyphal growth.

Degradation of low-ethoxylated nonylphenols by a Stenotrophomonas strain and development of new phylogenetic probes for Stenotrophomonas spp. detection.

An aerobic bacterium (BCc6), isolated from nonylphenol polyethoxylates (NPEOs)-contaminated sludge, was shown to be capable of degrading low-ethoxylated NPEO mixtures. Sequencing of 16S rRNA gene (rDNA) showed that it clustered with Stenotrophomonas nitritireducens. Fluorescent in situ hybridization (FISH), performed on BCc6 strain and on the previously isolated Stenotrophomonas BCaL2, also involved in NPEO degradation but clustering with S. maltophilia, showed that strain BCc6 did not hybridize with the S. maltophilia-specific probe, and neither of the two strains hybridized with probes targeted to the Gammaproteobacteria site, rDNA analyses performed on the two strains evidenced two new polymorphisms, the first one at the 23S rRNA Gammaproteobacteria site, characterizing the known members of the Stenotrophomonas genus, and the other one at the 16S rRNA level, characteristic of S. nitritireducens. Two new FISH probes were designed accordingly, tested on control bacterial cultures, and employed for in situ monitoring of Stenotrophomonas representatives.

Characterization of Saccharomyces cerevisiae natural populations for pseudohyphal growth and colony morphology.

In this work we have analyzed the colony and cellular morphologies of natural populations of Saccharomyces cerevisiae strains in response to different environmental stimuli. Among one thousand strains grown on YPD medium, 2.5% exhibited a rough (R) colony phenotype versus a smooth (S) phenotype. When grown on the ammonium-deficient medium SLAD, 56% of the strains showed a filamentous phenotype, often associated (43.8%) with an invasive phenotype, while 4.7% of the strains exhibited only an invasive phenotype. The rough phenotype on YPD was always associated with the filamentous phenotype on SLAD. A subset of 52 strains was further characterized for the growth phenotype under different stimuli (nitrogen deprivation, addition of alcohols, growth on proline as sole nitrogen source). On 27 strains, genetic analysis of the spore products was also performed. The entire set of data showed a wide distribution of dimorphism in the yeast population and great variability with respect to the dimorphic switch capability. Some strains grew with peculiar colony morphologies under different environmental stimuli and some showed colony morphology variations. Ecological implications of the wide spreading of dimorphic behavior and the occurrence of peculiar colony morphologies in natural yeasts are discussed.

Aggregation-based cooperation during bacterial aerobic degradation of polyethoxylated nonylphenols.

Three bacterial strains were isolated from activated sludge samples of two treatment plants receiving domestic and industrial wastewaters containing polyethoxylated nonylphenols. One strain (VA160) was isolated on rich medium, and the other two (BCaL1 and BCaL2) on mineral medium containing two industrial mixtures of nonylphenol ethoxylates as the sole carbon source. Strain VA160 was a Gram-positive, spore forming, filamentous bacterium, producing aggregates during growth in liquid medium. On the basis of phylogenetic analysis the strains were assigned to the Bacillus (VA160), Acinetobacter (BCaL1) and Stenothrophomonas (BCaL2) genera. High performance liquid chromatography analysis showed that only the Acinetobacter and Stenothrophomonas strains were involved in the degradation of polyethoxylated nonylphenols. Bacillus VA160, however, when co-cultured with the two degrading strains, induced the formation of cell aggregates and facilitated NPEO degradation. Fluorescent in situ hybridisation on the activated sludge sample from which Bacillus VA160 was isolated, using probes for Gram-positive bacteria with low G + C content, showed that bacteria belonging to this group specifically occurred inside the examined flocs. These observations suggest that the enhanced biodegradation of polyethoxylated nonylphenols in the three-membered co-culture is favoured by VA160-induced aggregation of BcaL1 and BcaL2 cells involved in the process.

Characterization of four olive-mill-wastewater indigenous bacterial strains capable of aerobically degrading hydroxylated and methoxylated monocyclic aromatic compounds.

Seven aerobic bacterial strains capable of degrading several of the monocyclic aromatic compounds occurring in the phenolic fraction of olive-mill wastewaters (OMWs) were isolated from an Italian OMW. The results of the 16S rDNA restriction analysis evidenced that these strains are distributed among four different groups. One strain of each group was taxonomically characterized by sequencing the amplified 16S rDNA, and the four strains were assigned to the genera Comamonas (strain AV1A), Ralstonia (strain AV5BG), Pseudomonas (strain AV2A) and Sphingomonas (strain AV6C). The four strains, when checked for the ability to degrade nine monocyclic aromatic compounds abundant in OMWs, were found to significantly metabolize five to eight of them, both as resting cells and growing cells. Specific enzyme analyses of the same selected strains showed: (1) the occurrence of O-demethylating activities towards four methoxylated mono-aromatic acids in three of the four studied strains (strains AV1A, AV5BG and AV6C), (2) ring-cleavage activity towards protocatechuic acid in all of the strains, and (3) a ring-cleavage activity towards catechol in strain AV6C. The isolates described here exhibit a biodegradation potential towards monocyclic aromatic compounds of OMWs that is markedly broader and higher than that displayed by other aerobic bacteria described previously. These features make them excellent candidates for removing the low-molecular-weight phenolic compounds persisting in the effluent following anaerobic digestion of OMWs.

Crosses between Saccharomyces cerevisiae and Saccharomyces bayanus generate fertile hybrids.

Crossings between strains of Saccharomyces cerevisiae and Saccharomyces bayanus were carried out. Genetic, molecular and electrophoretic karyotyping data indicated that interspecific hybrids were obtained. The hybrid cells segregated "grande" and "petite" colonies, and the latter ranged between 20 and 50%; unlike "grande" colonies, "petite" colonies did not sporulate and did not ferment maltose. In the hybrids, the extent of sporulation varied between 10 and 20%; only very rare asci (around 10(-4)) held viable ascospores. Clones from the viable ascospores sporulated and produced asci with viable ascospores able to give mating with spores from both hybrid derivatives and parental species. Fertile asci could derive from allotetraploid cells generated by endomitotic events in allodiploid cells, a mechanism that enables overcoming the species barrier between S. cerevisiae and S. bayanus.

Sarcoma do estroma da mama: relato de caso / Breast stromal sarcoma: case report

Sarcoma de estroma da mama é uma entidade rara, correspondente a menos de 1 por cento de todas as neoplasias da mama. Os autores relatam um caso de sarcoma mamário diagnosticado no exame anatomopatológico da peça cirúrgica, fazendo consideraçöes acerca do diagnóstico clínico, radiológico e aspectos histológicos do tumor