Efficient bioaccumulation of tungsten by Escherichia coli cells expressing the Sulfitobacter dubius TupBCA system.

Tungsten (W) is a valuable element with considerable industrial and economic importance that belongs to the European Union list of critical metals with a high supply risk. Therefore, the development of effective and new methods for W recovery is essential to ensure a sustainable supply. In the present study, the Sulfitobacter dubius W transport system TupABC was explored in order to demonstrate both its functionality in Escherichia coli cells and to construct a bioaccumulator (EcotupW). The complete gene cluster tupBCA or partial gene cluster tupBC were cloned in an expression vector and transformed into E. coli. Metal accumulation was evaluated when each construct strain was grown with three separate metal oxyanions (tungstate, molybdate or chromate). The specificity of the bioaccumulator was determined by competition assays using cells grown with mixed solutions of metal oxyanions (W/Mo and W/Cr). The results showed the relevance of the TupA protein in the TupABC transporter system to W-uptake and also allowed Mo and Cr accumulations, although with amounts 1.7 and 2.9-fold lower than W, respectively. To identify the importance of the valine residue in the accumulation efficiency of the VTTS motif, site-directed mutagenesis of tupA was performed. A mutant with a threonine residue, instead of the respective valine, confirmed that W was internalized by nearly double the amount compared to the native form. The findings indicated that cells carrying the native S. dubius TupABC system were great W-bioaccumulators and could be promising tools for W recovery.

Toxicity of the bionematicide 1,4-naphthoquinone on non-target soil organisms.

The main goal of the present study was to evaluate the ecotoxicological effects of 1,4-naphthoquinone (1,4-NTQ), a natural-origin compound presenting nematicidal activity, that can be obtained from walnut husk, in plants and soil invertebrates, including non-target soil nematode communities. This research was part of an ongoing project that aims to develop environmentally-friendly nematicides obtained from agricultural residues. The battery of ISO tests included emergence and growth of corn (Zea mays) and rape (Brassica napus); avoidance with the earthworm Eisenia andrei and the collembolan Folsomia candida; and reproduction with the previous species plus the enchytraeid Enchytraeus crypticus. A novel soil nematode community assay was also performed. ISO tests and nematode assays were conducted using a natural uncontaminated soil that was spiked with a range of 1,4-NTQ concentrations. Toxicity of 1,4-NTQ was found for all test-species and the most sensitive were F. candida and E. andrei. After 7 days of exposure to 1,4-NTQ, nematode abundance decreased along the concentration gradient, and a partial recovery was observed after 14 days (1,4-NTQ <48 mg kg-1 soil). The number of nematode families consistently decreased in both periods. Overall, results indicate that a 1,4-NTQ concentration of <20 mg kg-1 could be environmentally safe but preliminary data suggest that it might be ineffective for the target-nematodes, root-knot nematodes, Meloidogyne spp., and root-lesion nematodes, Pratylenchus spp. In addition, if higher dosages of 1,4-NTQ bionematicide are necessary, the potential recovery of non-target organisms under real field scenarios also needs to be assessed.

Virulence factors and infection ability of Pseudomonas aeruginosa isolates from a hydropathic facility and respiratory infections.

AIMS: To compare the virulence pool and acute infection ability of Pseudomonas aeruginosa isolates from a hydropathic facility, used to treat respiratory conditions by inhalation of untreated natural mineral water, with clinical isolates from respiratory infections. METHODS AND RESULTS: Pseudomonas aeruginosa isolates from a hydropathic facility and from respiratory infections were typed by pulsed-field gel electrophoresis. Nonclonal representatives of each population were selected. 18 virulence-encoding genes were screened by polymerase chain reaction and statistically compared by multiple correspondence analysis. Homogeneous distribution of genes between populations but higher genetic association in aquatic isolates was observed, as well as distinct virulence pool according to location in the water system. Acute infection ability of selected isolates from each population, in Galleria mellonella model, showed lower LD50 of the majority of the hydropathic isolates and significant variations in LD50 of biofilm isolates from different equipments. CONCLUSIONS: Hydrotherapy Ps. aeruginosa isolates present similar virulence to isolates from respiratory infections. Hydrotherapy users may be exposed to different microbiological risks when using different treatment equipments. SIGNIFICANCE AND IMPACT OF THE STUDY: Twenty-one million people use hydropathic facilities in Europe, and the majority present risk factors to pneumonia. This study demonstrates the health risk associated with this practice. Revision of European regulations should be considered.

The use of Collembola avoidance tests to characterize sewage sludges as soil amendments.

The ecotoxicological characterization of sewage sludge takes into account the additive, antagonistic and synergistic effects that occur as a result of multi-chemical interactions. Such an evaluation therefore is essential to complement the chemical analysis that, although required by law, is clearly insufficient. Using a tiered approach in the toxic evaluation of sewage sludge allows for characterization of toxicity in a timely manner. According to the literature, reproduction tests with Folsomia candida are suitable tools for the toxic assessment of organic sludges. Therefore, the inclusion of Collembola avoidance tests at a screening level (low tier), and acting as a trigger for longer-period tests (high tier; e.g. reproduction test), may provide a successful strategy, and may complement the currently proposed test battery. To evaluate the use of both avoidance and reproduction tests with collembolans in such a tiered approach, three sewage sludges (urban, olive and electroplating industries) were mixed in with a field-collected soil at different concentrations. Avoidance and reproduction tests were performed with the soil-sludge mixtures after 0, 4 and 12 weeks of incubation. The tests detected no toxicity in soil-sludge mixtures of urban and olive sludges at any incubation period. Mixtures with sludge from the electroplating industry induced toxicity only in the avoidance tests with freshly prepared and 4-week incubated samples. These results demonstrate the ability of Collembola avoidance tests to assess sewage sludge toxicity over time and its potential for hazardous sludge characterization at low tier levels.

Alternaria infectoria brain abscess in a child with chronic granulomatous disease.

In the present report, we describe the first case of a phaeohyphomycotic brain abscess in a 5-year-old boy with chronic granulomatous disease (CGD) admitted to hospital with seizures. A computed tomography (CT) scan revealed a cerebral abscess and the microbiology study showed a dark, melanin-pigmented fungus, exhibiting only sterile hyphae. This fungus was identified by the amplification and sequencing of the 5.8S RNA gene and of the adjacent internal transcriber spacer domains, ITS1 and ITS2, as Alternaria infectoria. Due to the impossibility of a surgical excision, and although several therapeutic strategies were attempted, the patient died. Limitations in the routine identification procedures and therapeutic options of this emerging opportunistic agent are highlighted in this report.

Diversity of chromium-resistant and -reducing bacteria in a chromium-contaminated activated sludge.

AIMS: This study attempts to establish a relationship between the Cr(VI) resistance of the culturable microbial community and the Cr(VI) resistance and Cr(VI)-reducing ability of representative strains of each population, in order to assess whether these are exclusive characteristics of one microbial group or abilities shared among many groups. METHODS AND RESULTS: A group of 48 Cr(VI)-resistant isolates, with different colony types, was isolated from chromium-contaminated activated sludge. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis protein patterns and fatty acid methyl ester analysis identified six populations, representing 54% of the isolated bacteria, as belonging to the genera Acinetobacter and Ochrobactrum. The remaining populations included strains identified as species of the beta-Proteobacteria and high G + C Gram-positive bacteria. The Cr(VI) resistance and reduction ability of the strains were tested. All but two isolates grew in the presence of 1 mmol l(-1) Cr(VI). During enrichment, all isolates were able to survive to 2 mmol l(-1) Cr(VI) and complete Cr(VI) reduction was achieved. Representative strains of each population were able to partially reduce (5.4-39.1%) the Cr(VI) present in the growth medium. CONCLUSIONS: Most of the identified isolates have never been reported to be Cr(VI)-resistant and/or Cr(VI)-reducing strains. The mechanisms of Cr(VI) resistance and reduction may differ from group to group; therefore, it is evident that both Cr(VI) resistance and reduction are shared abilities and not an exclusive characteristic of a single group, possibly reflecting horizontal genetic transfer resulting from selective pressure in this contaminated environment. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study of a microbial community under chronic chromate stress and, as the success of microbial-based metal remediation technologies requires a better understanding of the microbial community and the population response to metal stress, it may contribute to the implementation of a strategy of bioremediation of chromate-contaminated environments.

Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant.

Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant. These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system. The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains. One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P. fluorescens. The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System. Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile. This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P. aeruginosa or to any of the other type strains tested. Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P. alcaligenes. This study shows that characteristics similar to P. aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.

Computer-aided comparison of protein electrophoretic patterns for grouping and identification of heterotrophic bacteria from mineral water.

The microflora of a natural mineral water was studied immediately after bottling (T0) and after 7 d storage (T7) during 6 months, and isolates were clustered by SDS-PAGE of whole-cell protein profiles. Isolates from each cluster were further characterized by API 20NE, fatty acid composition and quinone profiles. The numerical analysis of the electrophoregrams of all bacteria isolated from the mineral water formed 15 clusters and five unclustered strains. Except for five minor clusters, all clusters were composed of strains isolated over several months. The numerical analysis of the electrophoregrams of bacteria isolated immediately after bottling formed 15 clusters while after 7 d storage only four of these populations could be isolated, indicating the populations present in the mineral water were stable and that changes occurring after bottling probably resulted from a selection process. Only one unclustered strain was identified simultaneously by all the systems, as Sphingomonas paucimobilis. The monitoring of the aquifer and the bottling system, and the construction of a large database with bacteria of the autochthonous flora allows the detection of alterations in the aquifer by changes in the microflora.

Characterization of Legionella species by numerical analysis of whole-cell protein electrophoresis.

The results of a computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of 291 isolates and 74 reference strains belonging to all known species of the genus Legionella revealed that the majority of the species of this genus can be adequately identified by this method. The type strain of Legionella bozemanii did not cluster with the other strains of this species, and the only strain of Legionella geestiana available clustered with the strains of Legionella feeleii. When we performed a numerical analysis by omitting certain portions of the pattern containing dense bands, all of the species could be distinguished. Our results also show that the type strains of Legionella nautarum and Legionella londiniensis deposited in the National Collection of Type Cultures do not correspond to the type strains deposited in the American Type Culture Collection. We used the results of a fatty acid and ubiquinone composition analysis to complement the SDS-PAGE results for several strains whose identities as determined by indirect immunofluorescence were doubtful. Computer-assisted SDS-PAGE of whole-cell proteins can be used in the classification of Legionella species and to identify and screen large numbers of isolates for further, in-depth taxonomic studies of smaller numbers of strains.

Survival of allochthonous bacteria in still mineral water bottled in polyvinyl chloride (PVC) and glass.

The mortality of Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa, based on the culturability of these bacteria, was assessed in non-carbonated mineral water, bottled in polyvinyl chloride (PVC) containing the indigenous flora, sterile mineral water bottled in PVC, sterile mineral water in glass containers, and sterile tap water in glass containers. There was a general decrease in the culturability of these organisms in the four test waters, except that Ps. aeruginosa grew in sterile tap water. Escherichia coli and Kl. pneumoniae had the highest mortality rates under the conditions tested, while Ent. cloacae had a very low and constant mortality rate that would have resulted in the persistence of this organism in mineral water for a long period of time. After a sharp initial decrease in culturability, Ps. aeruginosa also had a very low mortality rate in mineral water bottled in PVC.

Heterotrophic plate counts and the isolation of bacteria from mineral waters on selective and enrichment media.

The heterotrophic plate counts of 15 brands of bottled non-carbonated mineral waters were examined and found to be generally high and variable. Four selective or enrichment media for the enumeration of coliforms (m-Endo LES and m-lauryl sulphate agar) and Pseudomonas aeruginosa (cetrimide-nalidixic acid agar and malachite green broth) were used to isolate several species of Gram-negative bacteria. Strains identified as CDC gr IVc-2 and Comamonas (Ps.) acidovorans were the two most commonly isolated. Considerable variation in populations was seen between the brands, as well as between two batches of the same mineral water.

Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water.

The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9-12 months. The plate counts in R2A medium incubated at 22 degrees and 37 degrees C were low initially, increasing to 10(4)-10(5) cfu/ml within a few days of bottling. The number of bacteria recovered at 22 degrees C from PVC bottles was fairly constant during the storage period, but the population isolated at 37 degrees C decreased markedly after storage for 1 year. The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis. Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.