Toxicity assessment of pharmaceutical compounds on mixed culture from activated sludge using respirometric technique: The role of microbial community structure.

Micropollutants of emerging concern such as pharmaceuticals can significantly affect the performance of secondary biological processes in wastewater treatment plants. The present study is aimed to evaluate the toxicity and inhibition of three pharmaceutical compounds (caffeine, sulfamethoxazole and carbamazepine) on two cultures of microbial consortia enriched from wastewater aerobic activated sludge. One of them was acclimated to pharmaceuticals and the other was non-acclimated as control bioassay. The toxic and inhibitory effects on these cultures were assessed by respirometric tests through the oxygen uptake rate as an indicator of their capacity to degrade a readily available carbon source. Higher values of toxicity and inhibition of pharmaceutical compounds were observed for the control culture as compared to the acclimated one. Sulfamethoxazole and carbamazepine exhibited higher toxicity and inhibition effects than caffeine in both acclimated and control cultures. The microbial diversity of the two cultures was also studied. The composition of microbial community of acclimated and control cultures, was determined by targeting the 16S ribosomal RNA gene. It was observed that Proteobacteria was the most abundant phylum, with Gammaproteobacteria dominating both cultures. Control culture was dominated by Gammaproteobacteria and mostly by the genera Pseudomonas and Sodalis, which belong to common families present in wastewater. Results suggested that the acclimated culture to the three pharmaceuticals was mostly comprised of the extremely multiresistant genera Escherichia-Shigella (38%) of Gammaproteobacteria, resulting to higher resistance as compared to the control culture (Escherichia-Shigella, 7%). Finally, the microbial structure of the microorganisms present in a real bioreactor, which was initially seeded with the acclimated culture and fed in a continuous mode with the selected pharmaceuticals, was also analyzed. The continuous loading of pharmaceuticals in the bioreactor affected its microbial diversity, leading to the dominance of Betaproteobacteria and to the resistant genus Rhizobium of Alphaproteobacteria.

Assessment of the impact of three pesticides on microbial dynamics and functions in a lab-to-field experimental approach.

The toxicity of pesticides on soil microorganisms is as an emerging area of concern. Novel and well-standardized tools could be now used to provide a robust assessment of the ecotoxicity of pesticides on soil microorganisms. We followed a tiered lab-to-field approach to assess the toxicity of three pesticides, widely used at EU level, (chlorpyrifos (CHL), isoproturon (IPU) and tebuconazole (TBZ)) on (i) the abundance of 11 microbial taxa and 8 functional microbial groups via q-PCR and (ii) the activity of enzymes involved in biogeochemical cycles via fluorometric analysis. Correlation of microbial measurements with the concentration of pesticides, and their transformation products (TPs) in soil enabled the identification of the compounds driving the effects observed. At lab tests (×1, ×2 and ×10 the recommended dose), CHL and TBZ significantly reduced the relative abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) which recovered by the end of the study, while all pesticides induced a persistent reduction in the relative abundance of sulfur-oxidizing bacteria (SOB). The two demethylated metabolites of IPU (MD-IPU and DD-IPU) adversely affected P-cycling enzymes and leucine aminopeptidase (Leu). At field tests (×1, ×2 and ×5 the recommended dose), a persistent reduction on the relative abundance of AOA was induced by all pesticides, but only CHL and its hydrolysis product 3,5,6 trichloro-2-pyridynol (TCP) soil levels were negatively correlated with AOA relative abundance. Our findings suggest that ammonia-oxidizing microorganisms constitute the most responsive microbial group to pesticides and could be potential candidates for inclusion in pesticide risk assessment.

Genome-wide population structure and evolutionary history of the Frizarta dairy sheep.

In the present study, we used genomic data, generated with a medium density single nucleotide polymorphisms (SNP) array, to acquire more information on the population structure and evolutionary history of the synthetic Frizarta dairy sheep. First, two typical measures of linkage disequilibrium (LD) were estimated at various physical distances that were then used to make inferences on the effective population size at key past time points. Population structure was also assessed by both multidimensional scaling analysis and k-means clustering on the distance matrix obtained from the animals' genomic relationships. The Wright's fixation F ST index was also employed to assess herds' genetic homogeneity and to indirectly estimate past migration rates. The Wright's fixation F IS index and genomic inbreeding coefficients based on the genomic relationship matrix as well as on runs of homozygosity were also estimated. The Frizarta breed displays relatively low LD levels with r 2 and |D'| equal to 0.18 and 0.50, respectively, at an average inter-marker distance of 31 kb. Linkage disequilibrium decayed rapidly by distance and persisted over just a few thousand base pairs. Rate of LD decay (ß) varied widely among the 26 autosomes with larger values estimated for shorter chromosomes (e.g. ß=0.057, for OAR6) and smaller values for longer ones (e.g. ß=0.022, for OAR2). The inferred effective population size at the beginning of the breed's formation was as high as 549, was then reduced to 463 in 1981 (end of the breed's formation) and further declined to 187, one generation ago. Multidimensional scaling analysis and k-means clustering suggested a genetically homogenous population, F ST estimates indicated relatively low genetic differentiation between herds, whereas a heat map of the animals' genomic kinship relationships revealed a stratified population, at a herd level. Estimates of genomic inbreeding coefficients suggested that most recent parental relatedness may have been a major determinant of the current effective population size. A denser than the 50k SNP panel may be more beneficial when performing genome wide association studies in the breed.

Challenging the Wigglesworthia, Sodalis, Wolbachia symbiosis dogma in tsetse flies: Spiroplasma is present in both laboratory and natural populations.

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the "Wigglesworthia-Sodalis-Wolbachia dogma" operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G. tachinoides, members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density in G. f. fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G. f. fuscipes.

"LOVE TO HATE" pesticides: felicity or curse for the soil microbial community? An FP7 IAPP Marie Curie project aiming to establish tools for the assessment of the mechanisms controlling the interactions of pesticides with soil microorganisms.

Pesticides end up in soil where they interact with soil microorganisms in various ways. On the Yin Side of the interaction, pesticides could exert toxicity on soil microorganisms, while on the Yang side of interaction, pesticides could be used as energy source by a fraction of the soil microbial community. The LOVE TO HATE project is an IAPP Marie Curie project which aims to study these complex interactions of pesticides with soil microorganisms and provide novel tools which will be useful both for pesticide regulatory purposes and agricultural use. On the Yin side of the interactions, a new regulatory scheme for assessing the soil microbial toxicity of pesticides will be proposed based on the use of advanced standardized tools and a well-defined experimental tiered scheme. On the Yang side of the interactions, advanced molecular tools like amplicon sequencing and functional metagenomics will be applied to define microbes that are involved in the rapid transformation of pesticides in soils and isolate novel pesticide biocatalysts. In addition, a functional microarray has been designed to estimate the biodegradation genetic potential of the microbial community of agricultural soils for a range of pesticide groups.

Sequence variations in alleles of the avirulence gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola lead to loss of recognition of the AvrPphE protein within bean cells and a gain in cultivar-specific virulence.

The bean halo blight pathogen, Pseudomonas syringae pv. phaseolicola (Psph), is differentiated into nine races based on the presence or absence of five avirulence (avr) genes in the bacterium, which interact with corresponding resistance genes. R1-R5, in Phaseolus vulgaris. The resistance gene R2 is matched by avrPphE, which is located adjacent to the cluster of hrp genes that are required for pathogenicity of Psph. Although only races 2, 4, 5 and 7 are avirulent on cultivars with R2 (inducing the hypersensitive response; HR), homologues of avrPphE are present in all races of Psph. DNA sequencing of avrPphE alleles from races of Psph has demonstrated two routes to virulence: via single basepair changes conferring amino acid substitutions in races 1, 3, 6 and 9 and an insertion of 104bp in the allele in race 8. We have demonstrated that these base changes are responsible for the difference between virulence and avirulence by generating transconjugants of a virulent race harbouring plasmids expressing the various alleles of avrPphE. Agrobacterium tumefaciens-directed expression of avrPphE from race 4 in bean leaves induced the HR in a resistance gene-specific manner, suggesting that the AvrPphE protein is alone required for HR induction and is recognized within the plant cell. The allele from race 6, which is inactive if expressed in Psph, elicited a weak HR if expressed in planta, whereas the allele from race 1 did not. Our results suggest that the affinity of interaction between AvrPphE homologues and an unknown plant receptor mediates the severity of the plant's response. Mutation of avrPphE alleles did not affect the ability to colonize bean from a low level of inoculum. The avirulence gene avrPphB, which matches the R3 resistance gene, also caused a gene-specific HR following expression in the plant after delivery by A. tumefaciens.

Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola.

The 154-kb plasmid was cured from race 7 strain 1449B of the phytopathogen Pseudomonas syringae pv. phaseolicola (Pph). Cured strains lost virulence toward bean, causing the hypersensitive reaction in previously susceptible cultivars. Restoration of virulence was achieved by complementation with cosmid clones spanning a 30-kb region of the plasmid that contained previously identified avirulence (avr) genes avrD, avrPphC, and avrPphF. Single transposon insertions at multiple sites (including one located in avrPphF) abolished restoration of virulence by genomic clones. Sequencing 11 kb of the complementing region identified three potential virulence (vir) genes that were predicted to encode hydrophilic proteins and shared the hrp-box promoter motif indicating regulation by HrpL. One gene achieved partial restoration of virulence when cloned on its own and therefore was designated virPphA as the first (A) gene from Pph to be identified for virulence function. In soybean, virPphA acted as an avr gene controlling expression of a rapid cultivar-specific hypersensitive reaction. Sequencing also revealed the presence of homologs of the insertion sequence IS100 from Yersinia and transposase Tn501 from P. aeruginosa. The proximity of several avr and vir genes together with mobile elements, as well as G+C content significantly lower than that expected for P. syringae, indicates that we have located a plasmid-borne pathogenicity island equivalent to those found in mammalian pathogens.

HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro.

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ(Psph) was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ(Psph) to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ(Psph)-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ(Psph). The HrpZ(Psph)-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl-. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants.

Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease.

The avrPphF gene was cloned from Pseudomonas syringae pathovar phaseolicola (PPH:) races 5 and 7, based on its ability to confer avirulence towards bean cultivars carrying the R1 gene for halo-blight resistance, such as Red Mexican. avrPphF comprised two open reading frames, which were both required for function, and was located on a 154 kb plasmid (pAV511) in PPH: Strain RW60 of PPH:, lacking pAV511, displayed a loss in virulence to a range of previously susceptible cultivars such as Tendergreen and Canadian Wonder. In Tendergreen virulence was restored to RW60 by avrPphF alone, whereas subcloned avrPphF in the absence of pAV511 greatly accelerated the hypersensitive resistance reaction caused by RW60 in Canadian Wonder. A second gene from pAV511, avrPphC, which controls avirulence to soybean, was found to block the activity of avrPphF in Canadian Wonder, but not in Red Mexican. avrPphF also conferred virulence in soybean. The multiple functions of avrPphF illustrate how effector proteins from plant pathogens have evolved to be recognized by R gene products and, therefore, be classified as encoded by avirulence genes.