The Bacterial and Fungal Diversity of an Aged PAH- and Heavy Metal-Contaminated Soil is Affected by Plant Cover and Edaphic Parameters.

Industrial wasteland soils with aged PAH and heavy metal contaminations are environments where pollutant toxicity has been maintained for decades. Although the communities may be well adapted to the presence of stressors, knowledge about microbial diversity in such soils is scarce. Soil microbial community dynamics can be driven by the presence of plants, but the impact of plant development on selection or diversification of microorganisms in these soils has not been established yet. To test these hypotheses, aged-contaminated soil samples from a field trial were collected. Plots planted with alfalfa were compared to bare soil plots, and bacterial and fungal diversity and abundance were assessed after 2 and 6 years. Using pyrosequencing of 16S rRNA gene and ITS amplicons, we showed that the bacterial community was dominated by Proteobacteria, Actinobacteria, and Bacteroidetes and was characterized by low Acidobacteria abundance, while the fungal community was mainly represented by members of the Ascomycota. The short-term toxic impact of pollutants usually reduces the microbial diversity, yet in our samples bacterial and fungal species richness and diversity was high suggesting that the community structure and diversity adapted to the contaminated soil over decades. The presence of plants induced higher bacterial and fungal diversity than in bare soil. It also increased the relative abundance of bacterial members of the Actinomycetales, Rhizobiales, and Xanthomonadales orders and of most fungal orders. Multivariate analysis showed correlations between microbial community structure and heavy metal and PAH concentrations over time, but also with edaphic parameters (C/N, pH, phosphorus, and nitrogen concentrations).

Insight into the primary mode of action of TiO2 nanoparticles on Escherichia coli in the dark.

Large-scale production and incorporation of titanium dioxide nanoparticles (NP-TiO2 ) in consumer products leads to their potential release into the environment and raises the question of their toxicity. The bactericidal mechanism of NP-TiO2 under UV light is known to involve oxidative stress due to the generation of reactive oxygen species. In the dark, several studies revealed that NP-TiO2 can exert toxicological effects. However, the mode of action of these nanoparticles is still controversial. In the present study, we used a combination of fluorescent probes to show that NP-TiO2 causes Escherichia coli membrane depolarization and loss of integrity, leading to higher cell permeability. Using both transcriptomic and proteomic global approaches we showed that this phenomenon translates into a cellular response to osmotic stress, metabolism of cell envelope components and uptake/metabolism of endogenous and exogenous compounds. This primary mechanism of bacterial NP-TiO2 toxicity is supported by the observed massive cell leakage of K(+) /Mg(2+) concomitant with the entrance of extracellular Na(+), and by the depletion of intracellular ATP level.

Ecotoxicological assessment of organic wastes spread on land: Towards a proposal of a suitable test battery.

The land spreading of organic wastes in agriculture is a common practice in Europe, under the regulation of the Directive 86/278/EEC. One of the objectives of this Directive is to prevent harmful effects of organic wastes on soil, plants and animals. Despite this regulatory framework, there is still a lack of harmonized ecotoxicological test strategy to assess the environmental hazard of such wastes. The aim of this study was to provide a first step towards the a priori ecotoxicological assessment of organic wastes before their land use. For that purpose, nine different organic wastes were assessed using direct (i.e. terrestrial tests) and indirect (i.e. tests on water eluates) approaches, for a total of thirteen endpoints. Then, multivariate analyzes were used to discriminate the most relevant test strategy, among the application rates and bioassays used. From our results, a draft of test strategy was proposed, using terrestrial bioassays (i.e. earthworms and plants) and a concentration range between one and ten times the recommended application rates of organic wastes.

The use of soil mites in ecotoxicology: a review.

Mites, and especially soil-inhabiting ones, have been less studied than the other invertebrates used in bio-assays for the assessment of soil quality and the hazards of chemicals, although these organisms are included in the regulatory assessment scheme of pesticides. The recent advances in the development of test methods for soil mites groups have provided more information on their sensitivities towards chemicals, which needs to be presented for a more robust assessment of the current trends in soil mite ecotoxicology. Moreover, interestingly mite is the only taxa for which test methods were developed and standardized on predatory organisms. This review summarizes the different protocols for the assessment of chemicals using soil-inhabiting mites, including laboratory, semi-field and field studies. Among the data found in the literature, most of the chemicals assessed with mites were pesticides, while a few environmental samples were assessed with these organisms. Their sensitivities towards chemicals were then compared and discussed regarding other soil invertebrates. Finally, we conclude on the usefulness of soil mites in ecotoxicology, and provide future research trail in this area.

Global deletome profile of Saccharomyces cerevisiae exposed to lithium.

The increasing use of lithium (Li) in new technologies raises the question of its impact on living microorganisms. In the present study, we aimed to identify putative Li targets and resistance mechanisms in the yeast model Saccharomyces cerevisiae using a deletomic approach based on the screening of a collection of 4733 knockout mutants under Li exposure. This screening highlighted 60 mutants resistant to Li and 124 mutants sensitive to Li. Through functional enrichment analyses, transport systems were identified as playing a central role in cell resistance to toxic concentrations of Li. In contrast, the AKT/protein kinase B family, signal transduction or cell communication were identified as potential toxic targets of Li. The majority of the mutants with a Li-sensitive phenotype were also sensitive to other alkali and alkaline earth metals, whereas the Li-resistance phenotype was mostly resistant to Na but poorly resistant to other metals. A comparison with the results of deletomics studies carried out in the presence of other metals highlighted Li-specific phenotypes. Three genes (NAM7, NMD2, UPF3) of the nonsense-mediated decay pathway were specifically involved in resistance to Li. In contrast, mutants with the NCA2, SPT20, GCN5, YOR376W, YPK3, and DCW1 genes deleted were specifically resistant to Li. These genes encode various functions from putative mannosidase to constitution of the Spt-Ada-Gcn5 acetyltransferase complex. This work provides a better understanding of potential specific resistance mechanisms and cellular targets of Li in yeast.

Multiple factors affecting Ixodes ricinus ticks and associated pathogens in European temperate ecosystems (northeastern France).

In Europe, the main vector of tick-borne zoonoses is Ixodes ricinus, which has three life stages. During their development cycle, ticks take three separate blood meals from a wide variety of vertebrate hosts, during which they can acquire and transmit human pathogens such as Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. In this study conducted in Northeastern France, we studied the importance of soil type, land use, forest stand type, and temporal dynamics on the abundance of ticks and their associated pathogens. Negative binomial regression modeling of the results indicated that limestone-based soils were more favorable to ticks than sandstone-based soils. The highest tick abundance was observed in forests, particularly among coniferous and mixed stands. We identified an effect of habitat time dynamics in forests and in wetlands: recent forests and current wetlands supported more ticks than stable forests and former wetlands, respectively. We observed a close association between tick abundance and the abundance of Cervidae, Leporidae, and birds. The tick-borne pathogens responsible for Lyme borreliosis, anaplasmosis, and hard tick relapsing fever showed specific habitat preferences and associations with specific animal families. Machine learning algorithms identified soil related variables as the best predictors of tick and pathogen abundance.

Characterization of bacterial resistance in treated hospital wastewater.

This article seeks to characterize the bacterial profile of pediatric hospital wastewater samples collected at the outlet of a wastewater treatment plant, and to estimate their relative susceptibility to antimicrobial agents. A total of 64 strains were isolated in the wastewater samples, of which 49 were identified as belonging to different families: Enterobacteriaceae (e.g. Escherichia coli, Klebsiella sp., Citrobacter sp.) comprised 57.2% of the identified bacteria, non-Enterobacteriaceae (e.g. Aeromonas sp., Pseudomonas sp.) comprised 30.6%, and Streptococcaceae (e.g. Enterococcus sp.) comprised 12.2%. The tests of the susceptibility of the bacteria to the antimicrobial agents used in the hospital showed that 100% of the bacterial species found discharged in the hospital wastewater treatment system were resistant to one or more of the antimicrobial agents according to the criteria of the U.S. Clinical Laboratory Standards Institute/National Committee for Clinical Laboratory Standards. The antimicrobial agent tests showed that meropenem, norfloxacin, ciprofloxacin, levofloxacin, and cefepime were the most effective antimicrobials against bacteria of the Enterobacteriaceae family. For bacteria of the non-Enterobacteriaceae family, norfloxacin, ciprofloxacin, levofloxacin, and cefepime presented the most effective antimicrobial action, whereas for bacteria of the Streptococcaceae family, ampicillin, vancomycin, and gentamicin were the most effective antimicrobials. Hospital wastewater treatment plants could be considered as places of selection pressure for bacterial resistance because of the presence of antibiotic-resistant bacteria coming from sewers or created at the treatment plant.

Influence of microorganisms on uranium release from mining-impacted lake sediments under various oxygenation conditions.

Microbial processes can be involved in the remobilization of uranium (U) from reduced sediments under O2 reoxidation events such as water table fluctuations. Such reactions could be typically encountered after U-bearing sediment dredging operations. Solid U(IV) species may thus reoxidize into U(VI) that can be released in pore waters in the form of aqueous complexes with organic and inorganic ligands. Non-uraninite U(IV) species may be especially sensitive to reoxidation and remobilization processes. Nevertheless, little is known regarding the effect of microbially mediated processes on the behaviour of U under these conditions.

Unsuspected diversity of arsenite-oxidizing bacteria as revealed by widespread distribution of the aoxB gene in prokaryotes.

In this study, new strains were isolated from an environment with elevated arsenic levels, Sainte-Marie-aux-Mines (France), and the diversity of aoxB genes encoding the arsenite oxidase large subunit was investigated. The distribution of bacterial aoxB genes is wider than what was previously thought. AoxB subfamilies characterized by specific signatures were identified. An exhaustive analysis of AoxB sequences from this study and from public databases shows that horizontal gene transfer has likely played a role in the spreading of aoxB in prokaryotic communities.

Characterization of the ars gene cluster from extremely arsenic-resistant Microbacterium sp. strain A33.

The arsenic resistance gene cluster of Microbacterium sp. A33 contains a novel pair of genes (arsTX) encoding a thioredoxin system that are cotranscribed with an unusual arsRC2 fusion gene, ACR3, and arsC1 in an operon divergent from arsC3. The whole ars gene cluster is required to complement an Escherichia coli ars mutant. ArsRC2 negatively regulates the expression of the pentacistronic operon. ArsC1 and ArsC3 are related to thioredoxin-dependent arsenate reductases; however, ArsC3 lacks the two distal catalytic cysteine residues of this class of enzymes.

Simple or complex organic substrates inhibit arsenite oxidation and aioA gene expression in two ß-Proteobacteria strains.

Microbial transformation of arsenic species and their interaction with the carbon cycle play a major role in the mobility of this toxic metalloid in the environment. The influence of simple or complex organic substrates on arsenic bio-oxidation was studied using two bacterial strains: one - the arsenivorans strain of Thiomonas delicata - is able to use AsIII as sole energy source; the other, Herminiimonas arsenicoxydans, is not. Experiments were performed at two AsIII concentrations (75 and 2 mg/L). At 75 mg/L As, for both strains, expression of aioA gene decreased when yeast extract concentration was raised from 0.2 to 1 g/L. At 2 mg/L As, the presence of either yeast extract or simple (succinate or acetate) organic substrates in the medium during bacterial growth decreased the AsIII-oxidation rate by both strains. When added specifically during oxidation test, yeast extract but not simple organic substrates seems to have a negative effect on AsIII oxidation. Taken together, results confirm the negative influence of simple or complex organic substrates on the kinetics of microbial AsIII oxidation and suggest that this effect results from different mechanisms depending on the type of organic substrate. Further, for the first time, the influence of a complex organic substrate, yeast extract, on aioA gene expression has been evidenced.

Inhibition of transcription in Staphylococcus aureus by a primary sigma factor-binding polypeptide from phage G1.

The primary sigma factor of Staphylococcus aureus, sigma(SA), regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with sigma(SA) both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of sigma(SA) that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-sigma factor may occlude the -35 element recognition domain of sigma(SA). As would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of sigma(SA) to DNA and sigma(SA)-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.

Toxicity mechanisms of ZnO UV-filters used in sunscreens toward the model cyanobacteria Synechococcus elongatus PCC 7942.

Zinc oxide (ZnO) nanoparticles are commonly used in sunscreens for their UV-filtering properties. Their growing use can lead to their release into ecosystems, raising question about their toxicity. Effects of these engineered nanomaterials (ENMs) on cyanobacteria, which are important primary producers involved in many biogeochemical cycles, are unknown. In this study, we investigated by several complementary approaches the toxicological effects of two marketed ZnO-ENMs (coated and uncoated) on the model cyanobacteria Synechococcus elongatus PCC 7942. It was shown that despite the rapid adsorption of ENMs on cell surface, toxicity is mainly due to labile Zn released by ENMs. Zn dissipates cell membrane potential necessary for both photosynthesis and respiration, and induces oxidative stress leading to lipid peroxidation and DNA damages. It leads to global downregulation of photosystems, oxidative phosphorylation, and transcription/translation machineries. This also translates into significant decrease of intracellular ATP content and cell growth inhibition. However, there is no major loss of pigments and even rather an increase in exposed cells compared to controls. A proposed way to reduce the environmental impact of Zn would be the improvement of the coating stability to prevent solubility of ZnO-ENMs.

Pleiotropic effects of rfa-gene mutations on Escherichia coli envelope properties.

Mutations in the rfa operon leading to severely truncated lipopolysaccharide (LPS) structures are associated with pleiotropic effects on bacterial cells, which in turn generates a complex phenotype termed deep-rough. Literature reports distinct behavior of these mutants in terms of susceptibility to bacteriophages and to several antibacterial substances. There is so far a critical lack of understanding of such peculiar structure-reactivity relationships mainly due to a paucity of thorough biophysical and biochemical characterizations of the surfaces of these mutants. In the current study, the biophysicochemical features of the envelopes of Escherichia coli deep-rough mutants are identified from the molecular to the single cell and population levels using a suite of complementary techniques, namely microelectrophoresis, Atomic Force Microscopy (AFM) and Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) for quantitative proteomics. Electrokinetic, nanomechanical and proteomic analyses evidence enhanced mutant membrane destabilization/permeability, and differentiated abundances of outer membrane proteins involved in the susceptibility phenotypes of LPS-truncated mutants towards bacteriophages, antimicrobial peptides and hydrophobic antibiotics. In particular, inner-core LPS altered mutants exhibit the most pronounced heterogeneity in the spatial distribution of their Young modulus and stiffness, which is symptomatic of deep damages on cell envelope likely to mediate phage infection process and antibiotic action.

Diversity of arsenite transporter genes from arsenic-resistant soil bacteria.

A PCR approach was developed to assess the occurrence and diversity of arsenite transporters in arsenic-resistant bacteria. For this purpose, three sets of degenerate primers were designed for the specific amplification of approximately 750bp fragments from arsB and two subsets of ACR3 (designated ACR3(1) and ACR3(2)) arsenite carrier gene families. These primers were used to screen a collection of 41 arsenic-resistant strains isolated from two soil samples with contrasting amounts of arsenic. PCR results showed that 70.7% of the isolates contained a gene related to arsB or ACR3, with three of them carrying both arsB and ACR3-like genes. Phylogenetic analysis of the protein sequences deduced from the amplicons indicated a prevalence of arsB in Firmicutes and Gammaproteobacteria, while ACR3(1) and ACR3(2) were mostly present in Actinobacteria and Alphaproteobacteria, respectively. In addition to validating the use of degenerate primers for the identification of arsenite transporter genes in a taxonomically wide range of bacteria, the study describes a novel collection of strains displaying interesting features of resistance to arsenate, arsenite and antimonite, and the ability to oxidize arsenite.

Antimicrobial drug discovery through bacteriophage genomics.

Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases.

Impact of manufactured TiO2 nanoparticles on planktonic and sessile bacterial communities.

In the present study, we conducted a 2 week microcosm experiment with a natural freshwater bacterial community to assess the effects of titanium dioxide nanoparticles (TiO2-NPs) at various concentrations (0, 1, 10 and 100 mg/L) on planktonic and sessile bacteria under dark conditions. Results showed an increase of planktonic bacterial abundance at the highest TiO2-NP concentration, concomitant with a decrease from that of sessile bacteria. Bacterial assemblages were most affected by the 100 mg/L TiO2-NP exposure and overall diversity was found to be lower for planktonic bacteria and higher for sessile bacteria at this concentration. In both compartments, a 100 mg/L TiO2-NPs exposure induced a decrease in the ratio between the Betaproteobacteria and Bacteroidetes. For planktonic communities, a decrease of Comamonadaceae was observed concomitant with an increase of Oxalobacteraceae and Cytophagaceae (especially Emticicia). For sessile communities, results showed a strong decrease of Betaproteobacteria and particularly of Comamonadaceae.

Improvement of the Caenorhabditis elegans growth and reproduction test to assess the ecotoxicity of soils and complex matrices.

A growth and reproduction test using the nematode Caenorhabditis elegans was recently standardized by the International Organization for Standardization (ISO). Performing the ISO 10872 protocol (2010) revealed some drawbacks when applied to soil or soil mixed with complex matrices. The authors propose some modifications to the current protocol to normalize the test conditions. An appropriate range of moisture conditions was determined as a percentage of the water-holding capacity (WHC) of the soil. According to the authors' results, C. elegans tests can be performed in the range of 60% to 100% WHC. To ensure that the modifications of the protocol did not affect the organisms' recovery, extraction ratios for the juveniles were subsequently estimated. The modified protocol was found to be as reliable as the standard one concerning recovery of juveniles (over 80%). The protocol was also applied to several chemicals to investigate their potential as reference chemicals for soil toxicity tests. Boric acid, copper chloride, and nickel sulfate showed deleterious effects in a concentration-dependent manner for the growth and reproduction of C. elegans. Finally, the modified protocol was used to assess the growth and reproduction of C. elegans in soil amended with a limed sewage sludge. The authors conclude that the C. elegans modified protocol is a promising tool for the assessment of soil toxicity as well as the toxicity of mixtures with complex matrices.

Modifications of the bacterial reverse mutation test reveals mutagenicity of TiO(2) nanoparticles and byproducts from a sunscreen TiO(2)-based nanocomposite.

The bacterial reverse mutation test, recommended by the Organization for Economic Co-operation and Development (OECD) to determine genotoxicity of chemical compounds, has been recently used by several authors to investigate nanoparticles. Surprisingly, test results have been negative, whereas in vitro mammalian cell tests often give positive genotoxic responses. In the present study, we used the fluctuation test procedure with the Salmonella typhimurium strains TA97a, TA98, TA100 and TA102 to determine the mutagenic potential of TiO(2) nanoparticles (NP-TiO(2)) and showed that, when it is used conventionally, this test is not suitable for nanoparticle genotoxicity assessment. Indeed, the medium used during exposure prevents electrostatic interactions between bacterial cells and nanoparticles, leading to false-negative responses. We showed that a simple pre-exposure of bacteria to NP-TiO(2) in a low ionic strength solution (NaCl 10mM) at a pH below the nanoparticle isoelectric points (pH 5.5) can strongly improve the accuracy of the test. Thus, based on these improvements, we have demonstrated the genotoxicity of the engineered NP-TiO(2) tested and a NP-TiO(2) byproduct from a sunscreen nanocomposite. It was also shown that strain TA102 is more sensitive than the other strains, suggesting an oxidative stress-mediated mechanism of genotoxicity.

Role of electrostatic interactions in the toxicity of titanium dioxide nanoparticles toward Escherichia coli.

The increasing production and use of titanium dioxide nanoparticles (NP-TiO(2)) has led to concerns about their possible impact on the environment. Bacteria play crucial roles in ecosystem processes and may be subject to the toxicity of these nanoparticles. In this study, we showed that at low ionic strength, the cell viability of Escherichia coli was more severely affected at pH 5.5 than at pH 7.0 and pH 9.5. At pH 5.5, nanoparticles (positively charged) strongly interacted with the bacterial cells (negatively charged) and accumulated on their surfaces. This phenomenon was observed in a much lower degree at pH 7.0 (NP-TiO(2) neutrally charged and cells negatively charged) and pH 9.5 (both NP-TiO(2) and cells negatively charged). It was also shown that the addition of electrolytes (NaCl, CaCl(2), Na(2)SO(4)) resulted in a gradual reduction of the NP-TiO(2) toxicity at pH 5.5 and an increase in this toxicity at pH 9.5, which was closely related to the reduction of the NP-TiO(2) and bacterial cell electrostatic charges.