Comparative metagenomics reveals poultry and swine farming are hotspots for multidrug and tetracycline resistance.

Antibiotic misuse in livestock is a major threat to human health, as bacteria are quickly developing resistance to them. We performed a comparative analysis of 25 faecal metagenomes from swine, poultry, cattle, and humans to investigate their resistance profiles. Our analysis revealed that all genes conferring resistance to antibiotic classes assessed except tetracyclines were more prevalent in poultry manure than in the remaining species. We detected clinically relevant antibiotic resistance genes, such as mcr-1 which confers resistance to polymyxins. Among them, extended-spectrum ß-lactamase blaCTX-M genes were particularly abundant in all species. Poultry manure was identified as a hotspot for multidrug resistance, which may compromise medical treatment options. Urgent actions in the livestock industry are imperative to hamper the emergence and spread of antibiotic resistance.

Hypersaline mining effluents affect the structure and function of stream biofilm.

The salinisation of freshwater ecosystems is a global environmental problem that threatens biodiversity, ecosystem functioning and human welfare. The aim of this study was to investigate the potential impact of a realistic salinity gradient on the structure and functioning of freshwater biofilms. The salinity gradient was based on the real ion concentration of a mining effluent from an abandoned mine in Germany. We exposed biofilm from a pristine stream to 5 increasing salinities (3 to 100 g L-1) under controlled conditions in artificial streams for 21 days. We evaluated its functional (photosynthetic efficiency, nutrient uptake, and microbial respiration) and structural responses (community composition, algal biomass and diatom, cyanobacteria and green algae metrics) over time. Then we compared their responses with an unexposed biofilm used as control. The functionality and structure of the biofilm exposed to the different salinities significantly decreased after short-term and long-term exposure, respectively. The community composition shifted to a new stable state where the most tolerant species increased their abundances. At the same time, we observed an increase in the community tolerance (measured as Pollution-Induced Community Tolerance) along the salinity gradient. This study provides relevant information on the salt threshold concentrations that can substantially damage algal cells (i.e., between 15 and 30 g L-1). The results provide new insights regarding the response and adaptation of stream biofilm to salinity and its potential implications at the ecosystem level.

Nitrosation and nitration of diclofenac and structurally related nonsteroidal anti-inflammatory drugs (NSAIDs) in nitrifying activated sludge.

Diclofenac (DCF) is a highly consumed non-steroidal anti-inflammatory drug that is excreted partially metabolized and is poorly removed during wastewater treatment. Previous findings demonstrated that DCF in wastewater treatment plants (WWTP) is partially removed to nitro/nitroso compounds. The reactive nitrogen species, that are microbially produced during nitrification in the activated sludge of WWTP, were suspected to be involved in the transformation of DCF. Therefore, here, we investigated the molecular features governing such biotransformation and the role played by nitrifying bacteria by biodegradation experiments at lab scale in enriched nitrifying sludge bioreactors spiked with DCF and other structurally related non-steroidal anti-inflammatory drugs (NSAIDs). We provided evidence of the incorporation of NO/NO2 groups into DCF originated from ammonia by isotopically labelled biodegradation experiments. Nitroso and nitro-derivatives were tentatively identified for all NSAIDs studied and biotransformation mechanisms were proposed. Our findings from biodegradation experiments performed under different incubation conditions suggested that biotransformation of DCF and its related NSAIDs might not only be microbially mediated by ammonia oxidizing bacteria, but other nitrifiers co-occurring in the activated sludge as ammonia oxidizing archaea and nitrite oxidizing bacteria. Follow-up studies should be conducted to disentangle such complex behaviour in order to improve removal of these contaminants in WWTPs.

Surfactin Stimulated by Pectin Molecular Patterns and Root Exudates Acts as a Key Driver of the Bacillus-Plant Mutualistic Interaction.

Bacillus velezensis is considered as a model species belonging to the so-called Bacillus subtilis complex that evolved typically to dwell in the soil rhizosphere niche and establish an intimate association with plant roots. This bacterium provides protection to its natural host against diseases and represents one of the most promising biocontrol agents. However, the molecular basis of the cross talk that this bacterium establishes with its natural host has been poorly investigated. We show here that these plant-associated bacteria have evolved a polymer-sensing system to perceive their host and that, in response, they increase the production of the surfactin-type lipopeptide. Furthermore, we demonstrate that surfactin synthesis is favored upon growth on root exudates and that this lipopeptide is a key component used by the bacterium to optimize biofilm formation, motility, and early root colonization. In this specific nutritional context, the bacterium also modulates qualitatively the pattern of surfactin homologues coproduced in planta and forms mainly variants that are the most active at triggering plant immunity. Surfactin represents a shared good as it reinforces the defensive capacity of the host. IMPORTANCE Within the plant-associated microbiome, some bacterial species are of particular interest due to the disease protective effect they provide via direct pathogen suppression and/or stimulation of host immunity. While these biocontrol mechanisms are quite well characterized, we still poorly understand the molecular basis of the cross talk these beneficial bacteria initiate with their host. Here, we show that the model species Bacillus velezensis stimulates the production of the surfactin lipopeptide upon sensing pectin as a cell surface molecular pattern and upon feeding on root exudates. Surfactin favors bacterial rhizosphere fitness on one hand and primes the plant immune system on the other hand. Our data therefore illustrate how both partners use this multifunctional compound as a unique shared good to sustain a mutualistic interaction.

Adverse cutaneous effects of mitogen-activated protein kinase inhibitors in children.

BACKGROUND: Mitogen-activated protein kinase inhibitors (MEKi) are currently used for the treatment of central nervous system tumors in children and have shown promising results. Cutaneous adverse effects are among the most common toxicities described in adults, but few studies exist in pediatric patients. OBJECTIVE: We aimed to describe the cutaneous adverse effects associated with MEKi in pediatric patients. METHODS: A retrospective study was carried out at our pediatric hospital in Barcelona, Spain, in patients undergoing treatment with MEKi. RESULTS: Sixty-one children between 1 and 18 years of age were included. All patients developed cutaneous toxicity. Eczema, hair abnormalities, oral aphthae, and paronychia were among the most common cutaneous side effects. CONCLUSIONS: Recognizing skin toxicity in pediatric patients under treatment with MEKi is essential to establishing appropriate education and therapy, thereby improving treatment tolerability and minimizing avoidable interruptions in treatment.

Propranolol-resistant infantile hemangioma successfully treated with sirolimus.

Infantile hemangiomas are the most common benign vascular tumors in childhood. Propranolol is the first-line treatment for infantile hemangiomas, but failures may occur. Sirolimus, an mTOR inhibitor, is a promising drug for the treatment of vascular malformations and vascular tumors. We present the case of a child with multiple infantile hemangiomasthat was successfully treated with sirolimus and propranolol after failure of combined propranolol and prednisolone treatment.

S100 negative granular cell tumor of the oral cavity: dermoscopy and surgical approach.

We report the case of a 47-year-old male patient with S100 negative granular cell tumor of the oral cavity, focusing on dermoscopic features as well as surgical approach, not previously reported in the literature. The study contributes to the literature on dermoscopy and surgical treatment for this tumor and provides a practical approach to differentiating non-neural granular cell tumors and granular cell tumors.

S100 negative granular cell tumor of the oral cavity: dermoscopy and surgical approach

Abstract: We report the case of a 47-year-old male patient with S100 negative granular cell tumor of the oral cavity, focusing on dermoscopic features as well as surgical approach, not previously reported in the literature. The study contributes to the literature on dermoscopy and surgical treatment for this tumor and provides a practical approach to differentiating non-neural granular cell tumors and granular cell tumors.

[Metabolic syndrome in children aged 6 to 12 years with obesity in public schools of seven municipalities in the State of Mexico]. / Síndrome metabólico en niños de 6 a 12 años con obesidad, en escuelas públicas de siete municipios del Estado de México.

OBJECTIVE: To estimate the prevalence of Metabolic Syndrome (MS), its components and its association with obesity and cardiovascular risk in school age children. MATERIALS AND METHODS: A cross-sectional study of 1,017 children from 6 to 12 years of age. Anthropometric information, blood pressure, glucose, insulin, cholesterol, HDLc, LDLc and triglycerides were collected. The presence of MS and its components were determined by criteria of Cook et. al. (2003), modified for children and adolescents. In the analysis, the children were grouped by aged group of 6 to 9 and 10 to 12 years. The association between MS, obesity and cardiovascular risk was estimated using Student's t-test and Mann-Whitney U test, Chi-square test and logistic regression. RESULTS: The MS was present with 54.6% in obese children. Obesity was the characteristic of greater association to suffer from MS with an OR=8.62 p<0.001. CONCLUSIONS: MS is high among children with obesity, and is mostly associated with insulin resistance and atherogenic risk.

OBJETIVO: Estimar la prevalencia de síndrome metabólico(SM), sus componentes y su asociación con la obesidad y el riesgo cardiovascular en niños en edad escolar. MATERIAL Y MÉTODOS: Estudio transversal de 1 017 niños de 6 a 12 años de edad. Se recabó información antropométrica, presión sanguínea, glucosa, insulina, colesterol, colesterol de las lipoproteínas de alta densidad (HDLc, por sus siglas en inglés), colesterol de las lipoproteínas de baja densidad (LDLc, por sus siglas en inglés) y triglicéridos. La presencia de SM y sus componentes se determinaron a partir de los criterios de Cook y colaboradores (2003), modificados para niños y adolescentes. Para el análisis se establecieron dos grupos de edad, con niños de 6 a 9, por un lado, y de 10 a 12 años de edad, por el otro. La asociación entre SM, obesidad y riesgo cardiovascular fue estimada mediante las pruebas t de Student y U de Mann-Whitney, ji cuadrada y regresión logística. RESULTADOS: El SM estuvo presente en 54.6% de los niños obesos. La obesidad fue la característica de mayor asociación para padecer SM, con una razón de momios (RM) de 8.62 (p<0.001). CONCLUSIONES: El SM es alto en niños con obesidad, y su asociación con la resistencia a la insulina y el riesgo aterogénico es considerable.

Síndrome metabólico en niños de 6 a 12 años con obesidad, en escuelas públicas de siete municipios del Estado de México / Metabolic syndrome in children aged 6 to 12 years with obesity in public schools of seven municipalities in the State of Mexico

Resumen Objetivo Estimar la prevalencia de síndrome metabólico (SM), sus componentes y su asociación con la obesidad y el riesgo cardiovascular en niños en edad escolar. Material y métodos Estudio transversal de 1 017 niños de 6 a 12 años de edad. Se recabó información antropométrica, presión sanguínea, glucosa, insulina, colesterol, colesterol de las lipoproteínas de alta densidad (HDLc, por sus siglas en inglés), colesterol de las lipoproteínas de baja densidad (LDLc, por sus siglas en inglés) y triglicéridos. La presencia de SM y sus componentes se determinaron a partir de los criterios de Cook y colaboradores (2003), modificados para niños y adolescentes. Para el análisis se establecieron dos grupos de edad, con niños de 6 a 9, por un lado, y de 10 a 12 años de edad, por el otro. La asociación entre SM, obesidad y riesgo cardiovascular fue estimada mediante las pruebas t de Student y U de Mann-Whitney, ji cuadrada y regresión logística. Resultados: El SM estuvo presente en 54.6% de los niños obesos. La obesidad fue la característica de mayor asociación para padecer SM, con una razón de momios (RM) de 8.62 (p<0.001). Conclusiones El SM es alto en niños con obesidad, y su asociación con la resistencia a la insulina y el riesgo aterogénico es considerable.

Abstract Objective To estimate the prevalence of Metabolic Syndrome (MS), its components and its association with obesity and cardiovascular risk in school age children. Materials and methods A cross-sectional study of 1,017 children from 6 to 12 years of age. Anthropometric information, blood pressure, glucose, insulin, cholesterol, HDLc, LDLc and triglycerides were collected. The presence of MS and its components were determined by criteria of Cook et al. (2003), modified for children and adolescents. In the analysis, the children were grouped by aged group of 6 to 9 and 10 to 12 years. The association between MS, obesity and cardiovascular risk was estimated using Student's t-test and Mann-Whitney U test, Chi-square test and logistic regression. Results The MS was present with 54.6% in obese children. Obesity was the characteristic of greater association to suffer from MS with an OR=8.62 p<0.001. Conclusions MS is high among children with obesity, and is mostly associated with insulin resistance and atherogenic risk.

A novel sample preparation procedure for effect-directed analysis of micro-contaminants of emerging concern in surface waters.

A novel sample preparation procedure relying on Solid Phase Extraction (SPE) combining different sorbent materials on a sequential-based cartridge was optimized and validated for the enrichment of 117 widely diverse contaminants of emerging concern (CECs) from surface waters (SW) and further combined chemical and biological analysis on subsequent extracts. A liquid chromatography coupled to high resolution tandem mass spectrometry LC-(HR)MS/MS protocol was optimized and validated for the quantitative analysis of organic CECs in SW extracts. A battery of in vitro CALUX bioassays for the assessment of endocrine, metabolic and genotoxic interference and oxidative stress were performed on the same SW extracts. Satisfactory recoveries ([70-130]%) and precision (< 30%) were obtained for the majority of compounds tested. Internal standard calibration curves used for quantification of CECs, achieved the linearity criteria (r2 > 0.99) over three orders of magnitude. Instrumental limits of detection and method limits of quantification were of [1-96] pg injected and [0.1-58] ng/L, respectively; while corresponding intra-day and inter-day precision did not exceed 11% and 20%. The developed procedure was successfully applied for the combined chemical and toxicological assessment of SW intended for drinking water supply. Levels of compounds varied from < 10 ng/L to < 500 ng/L. Endocrine (i.e. estrogenic and anti-androgenic) and metabolic interference responses were observed. Given the demonstrated reliability of the validated sample preparation method, the authors propose its integration in an effect-directed analysis procedure for a proper evaluation of SW quality and hazard assessment of CECs.

Pesticide Mixture Toxicity in Surface Water Extracts in Snails (Lymnaea stagnalis) by an in Vitro Acetylcholinesterase Inhibition Assay and Metabolomics.

Many chemicals in use end up in the aquatic environment. The toxicity of water samples can be tested with bioassays, but a metabolomic approach has the advantage that multiple end points can be measured simultaneously and the affected metabolic pathways can be revealed. A current challenge in metabolomics is the study of mixture effects. This study aims at investigating the toxicity of an environmental extract and its most abundant chemicals identified by target chemical analysis of >100 organic micropollutants and effect-directed analysis (EDA) using the acetylcholinesterase (AChE) bioassay and metabolomics. Surface water from an agricultural area was sampled with a large volume solid phase extraction (LVSPE) device using three cartridges containing neutral, anionic, and cationic sorbents able to trap several pollutants classes like pharmaceuticals, pesticides, PAHs, PCBs, and perfluorinated surfactants. Targeted chemical analysis and AChE bioassay were performed on the cartridge extracts. The extract of the neutral sorbent cartridge contained most of the targeted chemicals, mainly imidacloprid, thiacloprid, and pirimicarb, and was the most potent AChE inhibitor. Using an EDA approach, other AChE inhibiting candidates were identified in the neutral extract, such as carbendazim and esprocarb. Additionally, a metabolomics experiment on the central nervous system (CNS) of the freshwater snail Lymnaea stagnalis was conducted. The snails were exposed to the extract, the three most abundant chemicals individually, and a mixture of these. The extract disturbed more metabolic pathways than the three most abundant chemicals individually, indicating the contribution of other chemicals. Most pathways perturbed by the extract exposure overlapped with those related to exposure to neonicotinoids, like the polyamine metabolism involved in CNS injuries. Metabolomics for the straightforward comparison between a complex mixture and single compound toxicity is still challenging but, compared to traditional biotesting, is a promising tool due to its increased sensitivity.

Investigating the formation and toxicity of nitrogen transformation products of diclofenac and sulfamethoxazole in wastewater treatment plants.

Diclofenac (DCF) and sulfamethoxazole (SMX) are highly consumed pharmaceuticals and concentrated in effluents from conventional wastewater treatment plants (WWTPs) since they are not completely eliminated. Under microbial mediated nitrification/denitrification processes occurring in nitrifying activated sludge DCF biotransformed into its nitroso and nitro derivatives (NO-DCF and NO2-DCF, respectively). SMX was biotransformed under denitrification conditions in water/sediment batch reactors into its nitro and desamino derivatives (NO2-SMX and Des-SMX, respectively). Four transformation products (TPs) from DCF and SMX were analized in wastewaters (WW) and receiving surface waters (SW). Nitrifying/denitrifying-derivatives of DCF and SMX were detected for the first time in WW and SW at one order of magnitude lower than their parent compounds. Relationships observed among levels of NO-DCF, NO2-DCF and nitrogen-species tentatively suggested that nitrification/denitrification processes are involved in nitration and nitrosation of DCF during biological WW treatment. Acute toxicity of analytes to Daphnia magna and Vibrio fischeri was assessed individually and in mixtures with other relevant micropollutants. Individual effects showed these compounds as not harmful and not toxic. However, synergism effects observed in mixtures evidenced that contribution of these compounds to overall toxicity of complex environmental samples, should not be dismissed.

Concentration and risk of pharmaceuticals in freshwater systems are related to the population density and the livestock units in Iberian Rivers.

Considerable amounts of pharmaceuticals are used in human and veterinary medicine, which are not efficiently removed during wastewater and slurries treatment and subsequently entering continuously into freshwater systems. The intrinsic biological activity of these non-regulated pollutants turns their presence in the aquatic environment into an ecological matter of concern. We present the first quantitative study relating the presence of pharmaceuticals and their predicted ecotoxicological effects with human population and livestock units. Four representative Iberian River basins (Spain) were studied: Llobregat, Ebro, Júcar and Guadalquivir. The levels of pharmaceuticals were determined in surface water and sediment samples collected from 77 locations along their stream networks. Predicted total toxic units to algae, Daphnia and fish were estimated for pharmaceuticals detected in surface waters. The use of chemometrics enabled the study of pharmaceuticals for: their spatial distribution along the rivers in two consecutive years; their potential ecotoxicological risk to aquatic organisms; and the relationships among their occurrence and predicted ecotoxicity with human population and animal farming pressure. The Llobregat and the Ebro River basins were characterized as the most polluted and at highest ecotoxicological risk, followed by Júcar and Guadalquivir. No significant acute risks of pharmaceuticals to aquatic organisms were observed. However potential chronic ecotoxicological effects on algae could be expected at two hot spots of pharmaceuticals pollution identified in the Llobregat and Ebro basins. Analgesics/antiinflammatories, antibiotics and diuretics were the most relevant therapeutic groups across the four river basins. Among them, hydrochlorothiazide and gemfibrozil, as well as azithromycin and ibuprofen were widely spread and concentrated pharmaceuticals in surface waters and sediments, respectively. Regarding their predicted ecotoxicity, sertraline, gemfibrozil and loratidine were identified as the more concerning compounds. Significantly positive relationships were found among levels of pharmaceuticals and toxic units and population density and livestock units in both surface water and sediment matrices.

Transcriptomic, biochemical and individual markers in transplanted Daphnia magna to characterize impacts in the field.

Daphnia magna individuals were transplanted across 12 sites from three Spanish river basins (Llobregat, Ebro, Jucar) showing different sources of pollution. Gene transcription, feeding and biochemical responses in the field were assessed and compared with those obtained in re-constituted water treatments spiked with organic eluates obtained from water samples collected at the same locations and sampling periods. Up to 166 trace contaminants were detected in water and classified by their mode of action into 45 groups that included metals, pharmaceuticals, pesticides, illicit drugs, and other industrial compounds. Physicochemical water parameters differentiated the three river basins with Llobregat having the highest levels of conductivity, metals and pharmaceuticals, followed by Ebro, whereas the Jucar river had the greatest levels of illicit drugs. D. magna grazing rates and cholinesterase activity responded similarly than the diversity of riparian benthic communities. Transcription patterns of 13 different genes encoding for general stress, metabolism and energy processes, molting and xenobiotic transporters corroborate phenotypic responses differentiated sites within and across river basins. Principal Component Analysis and Partial Least Square Projections to Latent Structures regression analyses indicated that measured in situ responses of most genes and biomarkers and that of benthic macroinvertebrate diversity indexes were affected by distinct environmental factors. Conductivity, suspended solids and fungicides were negatively related with the diversity of macroinvertebrates cholinesterase, and feeding responses. Gene transcripts of heat shock protein and metallothionein were positively related with 11 classes of organic contaminants and 6 metals. Gene transcripts related with signaling paths of molting and reproduction, sugar, protein and xenobiotic metabolism responded similarly in field and lab exposures and were related with high residue concentrations of analgesics, diuretics, psychiatric drugs, ß blockers, illicit drugs, trizoles, bisphenol A, caffeine and pesticides. These results indicate that application of omic technologies in the field is a promising subject in water management.

Simultaneous determination of diclofenac, its human metabolites and microbial nitration/nitrosation transformation products in wastewaters by liquid chromatography/quadrupole-linear ion trap mass spectrometry.

An analytical method was developed and validated for the first determination of five major human metabolites of the non-steroidal anti-inflammatory drug diclofenac as well as two microbial transformation products in wastewater. The method was based on the extraction of diclofenac and the chemically synthetized compounds by solid-phase extraction (SPE), using a hydrophilic-lipophilic balanced polymer followed by liquid chromatography (LC) coupled to hybrid quadrupole-linear ion trap mass spectrometry (QqLIT-MS). Quantitation was performed by the internal standard approach, to correct for matrix effects. The accuracy of the method was generally higher than 40% for raw and treated wastewater with a precision below 12%. In wastewater influent and effluent samples the detection limits for the majority of target compounds were 0.3-2.5ngL(-1) and 0.1-3.1ngL(-1), respectively. The method was applied to the analysis of influent and effluent wastewater samples from urban wastewater treatment plants. Moreover, to obtain an extra tool for confirmation and identification of the studied diclofenac-derived compounds, Information-Dependent Acquisition (IDA) experiments were performed, with selected reaction monitoring (SRM) as the survey scan and an enhanced product ion (EPI) scan as the dependent scan. Diclofenac and its major human metabolite, 4'-hydroxydiclofenac were detected in all samples at concentrations of 331-1150ngL(-1) and 585-6000ngL(-1), respectively. Neither microbial transformation product of diclofenac was detected in any of the influent samples analyzed, but in effluents, their concentrations ranged from 4 to 105ngL(-1).

Hydrological variation modulates pharmaceutical levels and biofilm responses in a Mediterranean river.

The Llobregat is a Mediterranean river that is severely impacted by anthropogenic pressures. It is characterized by high flow variability which modulates its chemical and biological status. The present work evaluates the effects of flow changes on the concentration of pharmaceutically active compounds (PhACs) and their relationship to cellular parameters of river biofilms. To this end, at two selected sampling sites at the lower course of the Llobregat river, surface water samples were collected twice a week over two hydrologically different periods exhibiting low and high river flows. Higher levels of PhACs were detected at the downstream sampling site. Irrespective of the flow regime, analgesics, anti-inflammatories and lipid regulators were the most abundant substances at both sampling sites with total concentrations of up to 1,000 ng/L and 550 ng/L at the upstream and downstream sites, respectively. Antibiotics (fluoroquinolones) and psychiatric treatment drugs were also detected at high levels in the second campaign achieving concentrations of up to 500 ng/L. The principal component analysis (PCA) performed with the PhACs concentrations of the two campaigns revealed differences in the various therapeutic groups depending on sampling site and period. After a flash flood event during the second sampling period, dilution of PhACs occurred, but their average concentrations measured before the flood were restored within two weeks. For the majority of compounds, PhAC concentrations displayed an inverse relationship with river discharge The effects of water containing different concentrations of PhACs on biofilm communities were evaluated and related to flow regime variations. Translocation of biofilm communities from a less to a more polluted site of the river demonstrated an increase in bacteria mortality in the translocated biofilms. After the flood, extracellular peptidase activity and chlorophyll-a concentration were significantly reduced, and biofilm growth rate was significantly lower.

Modelling the emerging pollutant diclofenac with the GREAT-ER model: application to the Llobregat River Basin.

The present research aims at giving an insight into the increasingly important issue of water pollution due to emerging contaminants. In particular, the source and fate of the non-steroidal anti-inflammatory drug diclofenac have been analyzed at catchment scale for the Llobregat River in Catalonia (Spain). In fact, water from the Llobregat River is used to supply a significant part of the Metropolitan Area of Barcelona. At the same time, 59 wastewater treatment plants discharge into this basin. GREAT-ER model has been implemented in this basin in order to reproduce a static balance for this pollutant for two field campaigns data set. The results highlighted the ability of GREAT-ER to simulate the diclofenac concentrations in the Llobregat Catchment; however, this study also pointed out the urgent need for longer time series of observed data and a better knowledge of wastewater plants outputs and their parameterization in order to obtain more reliable results.