Rise of toxic cyanobacterial blooms is promoted by agricultural intensification in the basin of a large subtropical river of South America.

Toxic cyanobacterial blooms are globally increasing with negative effects on aquatic ecosystems, water use and human health. Blooms' main driving forces are eutrophication, dam construction, urban waste, replacement of natural vegetation with croplands and climate change and variability. The relative effects of each driver have not still been properly addressed, particularly in large river basins. Here, we performed a historical analysis of cyanobacterial abundance in a large and important ecosystem of South America (Uruguay river, ca 1900 km long, 365,000 km2 basin). We evaluated the interannual relationships between cyanobacterial abundance and land use change, river flow, urban sewage, temperature and precipitation from 1963 to the present. Our results indicated an exponential increase in cyanobacterial abundance during the last two decades, congruent with an increase in phosphorus concentration. A sharp shift in the cyanobacterial abundance rate of increase after the year 2000 was identified, resulting in abundance levels above public health alert since 2010. Path analyses showed a strong positive correlation between cyanobacteria and cropland area at the entire catchment level, while precipitation, temperature and water flow effects were negligible. Present results help to identify high nutrient input agricultural practices and nutrient enrichment as the main factors driving toxic bloom formation. These practices are already exerting severe effects on both aquatic ecosystems and human health and projections suggest these trends will be intensified in the future. To avoid further water degradation and health risk for future generations, a large-scale (transboundary) change in agricultural management towards agroecological practices will be required.

Las floraciones de cianobacterias tóxicas vienen aumentando drásticamente a nivel mundial con efectos negativos en los ecosistemas acuáticos, los usos del agua y la salud humana. Los principales mecanismos promotores de las floraciones son la eutrofización, la construcción de represas, la contaminación con residuos urbanos, la pérdida de vegetación natural y el cambio y la variabilidad climáticos. Los efectos relativos de cada determinante aún no se han abordado adecuadamente, particularmente en las grandes cuencas fluviales de América del Sur. En este trabajo, realizamos un análisis histórico de la abundancia de cianobacterias en un gran e importante ecosistema de América del Sur (el Río Uruguay, c.a. 1.900 km de largo, cuenca de 365.000 km2). Evaluamos las relaciones entre la abundancia de cianobacterias y el cambio en los usos del suelo, el caudal de los ríos, la contaminación urbana, la temperatura y la precipitación desde 1963 hasta el presente. Nuestros resultados evidencian un aumento exponencial en la abundancia de cianobacterias durante las últimas dos décadas, de forma congruente con el aumento en la concentración de fósforo en agua. Fue identificado además, un cambio brusco en la tasa de aumento de la abundancia de cianobacterias después del año 2000, lo que resultó en niveles de alerta por encima de riesgo para la salud pública desde 2010. Los análisis estadísticos indicaron una fuerte y positiva correlación entre las cianobacterias y el área de cultivo en la cuenca, mientras que la precipitación, la temperatura y el flujo de agua fueron insignificantes. Estos resultados contribuyen a identificar que las prácticas agrícolas con alto aporte de nutrientes y el enriquecimiento de nutrientes son los principales impulsores de la formación de floraciones tóxicas. Estas prácticas ya están teniendo graves efectos en los ecosistemas acuáticos y la salud humana y las proyecciones sugieren que se intensificarán en el futuro. Para evitar una mayor degradación de la calidad del agua y el incremento de los riesgos para la salud de las generaciones futuras, se requerirá un cambio a gran escala (transfronterizo) en la gestión agrícola hacia prácticas agroecológicas.

Genotyping and Multivariate Regression Trees Reveal Ecological Diversification within the Microcystis aeruginosa Complex along a Wide Environmental Gradient.

Addressing the ecological and evolutionary processes underlying biodiversity patterns is essential to identify the mechanisms shaping community structure and function. In bacteria, the formation of new ecologically distinct populations (ecotypes) is proposed as one of the main drivers of diversification. New ecotypes arise when mutations in key functional genes or acquisition of new metabolic pathways by horizontal gene transfer allow the population to exploit new resources, permitting their coexistence with the parental population. We previously reported the presence of microcystin-producing organisms of the Microcystis aeruginosa complex (toxic MAC) through an 800-km environmental gradient ranging from freshwater to estuarine-marine waters in South America. We hypothesize that the success of toxic MAC in such a gradient is due to the existence of very closely related populations that are ecologically distinct (ecotypes), each specialized to a specific arrangement of environmental variables. Here, we analyzed toxic MAC genetic diversity through quantitative PCR (qPCR) and high-resolution melting analysis (HRMA) of a functional gene (mcyJ, microcystin synthetase cluster). We explored the variability of the mcyJ gene along the environmental gradient by multivariate classification and regression trees (mCART). Six groups of mcyJ genotypes were distinguished and associated with different combinations of water temperature, conductivity, and turbidity. We propose that each mcyJ variant associated with a defined environmental condition is an ecotype (or species) whose relative abundances vary according to their fitness in the local environment. This mechanism would explain the success of toxic MAC in such a wide array of environmental conditions. IMPORTANCE Organisms of the Microcystis aeruginosa complex form harmful algal blooms (HABs) in nutrient-rich water bodies worldwide. MAC HABs are difficult to manage owing to the production of potent toxins (microcystins) that resist water treatment. In addition, the role of microcystins in the ecology of MAC organisms is still elusive, meaning that the environmental conditions driving the toxicity of the bloom are not clear. Furthermore, the lack of coherence between morphology-based and genomic-based species classification makes it difficult to draw sound conclusions about when and where each member species of the MAC will dominate the bloom. Here, we propose that the diversification process and success of toxic MAC in a wide range of water bodies involves the generation of ecotypes, each specialized in a particular niche, whose relative abundance varies according to its fitness in the local environment. This knowledge can improve the generation of accurate prediction models of MAC growth and toxicity, helping to prevent human and animal intoxication.

Spatial and temporal dynamics and potential pathogenicity of fecal coliforms in coastal shallow groundwater wells.

Access to water through shallow groundwater wells is a common practice in coastal settlements. This, coupled with a lack of planning for wastewater disposal promotes fecal contamination of groundwater and poses a threat to human health. Here, the spatial and temporal dynamics of groundwater fecal contamination was evaluated during summer and winter (2013 and 2014) in a coastal protected area having a high touristic relevance (Cabo Polonio, Uruguay). Fecal coliforms (FC) abundance in groundwater was significantly higher during summer, related to an influx of ~ 1000 tourists per day. A significant spatial autocorrelation was found in 2014, when the abundance of FC in a well was influenced by its three nearest wells (Moran and Geary tests). The applied statistical models (mixed models) indicated that total phosphorus and organic matter were the variables significantly explaining FC abundance. The risk for human health was estimated using groundwater-extracted DNA and qPCR of genes encoding for E. coli virulence factors (stx1, stx2, and eae). Potential Shiga toxin-producing enteropathogenic and enterohemorrhagic pathotypes were detected, even at FC abundances ≤ 1 CFU (100 mL-1). Moreover, we found that contaminated groundwater reached the beach, being the presence of FC in sand detected even in winter and showing its highest frequency nearby groundwater wells consistently having high FC abundance (hot spots). Altogether, the results show that fecal contamination of shallow groundwater in Cabo Polonio involves a risk for human health that intensifies during summer (associated to a significant increase of tourists). This contamination also impacts the beach, where FC can remain through the whole year.

Biogeography of the cyanobacterium Raphidiopsis (Cylindrospermopsis) raciborskii: Integrating genomics, phylogenetic and toxicity data.

Raphidiopsis (Cylindrospermopsis) raciborskii, a globally distributed bloom-forming cyanobacterium, produces either the cytotoxin cylindrospermopsin (CYL) in Oceania, Asia and Europe or the neurotoxin saxitoxin (STX) and analogues (paralytic shellfish poison, PSP) in South America (encoded by sxt genetic cluster) and none of them in Africa. Nevertheless, this particular geographic pattern is usually overlooked in current hypotheses about the species dispersal routes. Here, we combined genomics, phylogenetic analyses, toxicity data and a literature survey to unveil the evolutionary history and spread of the species. Phylogenies based on 354 orthologous genes from all the available genomes and ribosomal ITS sequences of the taxon showed two well-defined clades: the American, having the PSP producers; and the Oceania/Europe/Asia, including the CYL producers. We propose central Africa as the original dispersion center (non-toxic populations), reaching North Africa and North America (in former Laurasia continent). The ability to produce CYL probably took place in populations that advanced to sub-Saharan Africa and then to Oceania and South America. According to the genomic context of the sxt cluster found in PSP-producer strains, this trait was acquired once by horizontal transfer in South America, where the ability to produce CYL was lost.

Identifying Invaders: The Case of Ceratium furcoides (Gonyaulacales, Dinophyceae) in South America.

Ceratium furcoides is an invasive freshwater dinoflagellate that in the last three decades has expanded its geographic distribution in South America, being recently found in Paraná River floodplain (Argentina). Despite growing concern about the presence and impacts of this invader, information regarding genetic diversity in the Southern Hemisphere is missing. This work constitutes the first phylogenetic characterization of Ceratium populations of South America, particularly, from the Paraná system. After taxonomic identification as C. furcoides based on morphological traits, two sequencing-based approaches were applied using the ribosomal 18S gene: Sanger sequencing to isolated individuals and high-throughput amplicon sequencing (HTS) to environmental DNA. The sequence of C. furcoides obtained shared 100% identity to Asian sequences, and formed a highly supported clade in the constructed reference phylogenetic tree. HTS helped to recover low-frequency genetic variants suggesting the presence of different population of C. furcoides, and to alert potential invasion in its early stages.

Application of ancient DNA to the reconstruction of past microbial assemblages and for the detection of toxic cyanobacteria in subtropical freshwater ecosystems.

Ancient DNA (aDNA) analysis of lake sediments is a promising tool for detecting shifts in past microbial assemblages in response to changing environmental conditions. We examined sediment core samples from subtropical, freshwater Laguna Blanca (Uruguay), which has been severely affected by cultural eutrophication since 1960 and where cyanobacterial blooms, particularly those of the saxitoxin-producer Cylindrospermopsis raciborskii, have been reported since the 1990s. Samples corresponding to ~1846, 1852, 2000 and 2007 AD were selected to perform denaturing gradient gel electrophoresis (DGGE) analysis of the 16S-23S rRNA intergenic transcribed spacer (ribosomal ITS) to compare their prokaryotic assemblage composition. Each stratum showed different ITS patterns, but the composition of 21st century samples was clearly different than those of mid-19th century. This compositional change was correlated with shifts in sediment organic matter and chlorophyll a content, which were significantly higher in recent samples. The presence of saxitoxin-producing cyanobacteria was addressed by quantitative real-time PCR of the sxtU gene involved in toxin biosynthesis. This gene was present only in recent samples, for which clone libraries and ITS sequencing indicated the presence of Cyanobacteria. Phylogenetic analyses identified C. raciborskii only in the 2000 sample, shortly after several years when blooms were recorded in the lake. These data suggest the utility of aDNA for the reconstruction of microbial assemblage shifts in subtropical lakes, at least on centennial scales. The application of aDNA analysis to genes involved in cyanotoxin synthesis extends the applicability of molecular techniques in palaeolimnological studies to include key microbial community characteristics of great scientific and social interest.

The Microcystis-microbiome interactions: origins of the colonial lifestyle.

Species of the Microcystis genus are the most common bloom-forming toxic cyanobacteria worldwide. They belong to a clade of unicellular cyanobacteria whose ability to reach high biomasses during blooms is linked to the formation of colonies. Colonial lifestyle provides several advantages under stressing conditions of light intensity, ultraviolet light, toxic substances and grazing. The progression from a single-celled organism to multicellularity in Microcystis has usually been interpreted as individual phenotypic responses of the cyanobacterial cells to the environment. Here, we synthesize current knowledge about Microcystis colonial lifestyle and its role in the organism ecology. We then briefly review the available information on Microcystis microbiome and propose that changes leading from single cells to colonies are the consequence of specific and tightly regulated signals between the cyanobacterium and its microbiome through a biofilm-like mechanism. The resulting colony is a multi-specific community of interdependent microorganisms.

Effect of hydrological modification on the potential toxicity of Microcystis aeruginosa complex in Salto Grande reservoir, Uruguay.

It is widely known that the environmental conditions caused by the construction of reservoirs favor the proliferation of toxic cyanobacteria and the formation of blooms due to the high residence time of the water, low turbidity, temperature regimes, among others. Microcystin-producing cyanobacteria such as those from the Microcystis aeruginosa complex (MAC) are the most frequently found organisms in reservoirs worldwide, being the role of the environment on microcystin production poorly understood. Here, we addressed the community dynamics and potential toxicity of MAC cyanobacteria in a subtropical reservoir (Salto Grande) located in the low Uruguay river. Samples were taken from five different sites (upstream, inside the reservoir and downstream) during contrasting seasons (summer and winter) to analyze: (i) the MAC community structure by amplicon sequencing of the phycocyanin gene spacer, (ii) the genotype diversity of microcystin-producing MAC by high resolution melting analysis of the mcyJ gene, and (iii) the abundance and mcy transcription activity of the microcystin-producing (toxic) fraction. We found that MAC diversity decreased from summer to winter but, despite the observed changes in MAC community structure, the abundance of toxic organisms and the transcription of mcy genes were always higher inside the reservoir, regardless of the season. Two different genotypes of toxic MAC were detected inside the reservoir, one associated with low water temperature (15 °C) and one thriving at high water temperature (31 °C). These findings indicate that the environmental conditions inside the reservoir reduce community diversity while promoting the proliferation of toxic genotypes that actively transcribe mcy genes, whose relative abundance will depend on the water temperature.

A georeferenced rRNA amplicon database of aquatic microbiomes from South America.

The biogeography of bacterial communities is a key topic in Microbial Ecology. Regarding continental water, most studies are carried out in the northern hemisphere, leaving a gap on microorganism's diversity patterns on a global scale. South America harbours approximately one third of the world's total freshwater resources, and is one of these understudied regions. To fill this gap, we compiled 16S rRNA amplicon sequencing data of microbial communities across South America continental water ecosystems, presenting the first database µSudAqua[db]. The database contains over 866 georeferenced samples from 9 different ecoregions with contextual environmental information. For its integration and validation we constructed a curated database (µSudAqua[db.sp]) using samples sequenced by Illumina MiSeq platform with commonly used prokaryote universal primers. This comprised ~60% of the total georeferenced samples of the µSudAqua[db]. This compilation was carried out in the scope of the µSudAqua collaborative network and represents one of the most complete databases of continental water microbial communities from South America.

Coastal bacterioplankton community diversity along a latitudinal gradient in Latin America by means of V6 tag pyrosequencing.

The bacterioplankton diversity of coastal waters along a latitudinal gradient between Puerto Rico and Argentina was analyzed using a total of 134,197 high-quality sequences from the V6 hypervariable region of the small-subunit ribosomal RNA gene (16S rRNA) (mean length of 60 nt). Most of the OTUs were identified into Proteobacteria, Bacteriodetes, Cyanobacteria, and Actinobacteria, corresponding to approx. 80% of the total number of sequences. The number of OTUs corresponding to species varied between 937 and 1946 in the seven locations. Proteobacteria appeared at high frequency in the seven locations. An enrichment of Cyanobacteria was observed in Puerto Rico, whereas an enrichment of Bacteroidetes was detected in the Argentinian shelf and Uruguayan coastal lagoons. The highest number of sequences of Actinobacteria and Acidobacteria were obtained in the Amazon estuary mouth. The rarefaction curves and Good coverage estimator for species diversity suggested a significant coverage, with values ranging between 92 and 97% for Good coverage. Conserved taxa corresponded to aprox. 52% of all sequences. This study suggests that human-contaminated environments may influence bacterioplankton diversity.

Metagenomic analysis of Raphidiopsisraciborskii microbiome: beyond the individual.

Raphidiopsisraciborskii is a toxic, invasive bacteria with a defined biogeographic pattern attributed to the generation of ecotypes subjected to local environmental filters and to phenotypic plasticity. The interactions taking place between the cyanobacterium and the other bacteria inhabiting the external polysaccharide-rich matrix surrounding the cells, or phycosphere, may be ecotype-specific and would have different influence on the carbon and nutrient cycling in the ecosystem. Here, we describe the bacterial community or microbiome (assessed by 16S rRNA metagenomics) associated to two R.raciborskii strains that have been described as different ecotypes: the saxitoxin-producer MVCC19 and the non-toxic LB2897. Our results showed that both ecotypes share 50% of their microbiomes and differ in their dominant taxa. The taxon having the highest abundance in the microbiome of MVCC19 was Neorhizobium (22.5% relative abundance), while the dominant taxon in LB2897 was the Planctomycetes SM1A02 (26.2% relative abundance). These groups exhibit different metabolic capabilities regarding nitrogen acquisition (symbiotic nitrogen-fixing in Neorhizobium vs. anammox in SM1A02), suggesting the existence of ecotype-specific microbiomes that play a relevant role in cyanobacterial niche-adaptation. In addition, as saxitoxin and analogues are nitrogen-rich (7 atoms per molecule), we hypothesise that saxitoxin-producing R.raciborskii benefits from external sources of nitrogen provided by the microbiome bacteria. Based on these findings, we propose that the mechanisms involved in the assembly of the cyanobacterial microbiome community are ecotype-dependent.

Teaching during the COVID-19 Pandemic: Sharing Results and Data Obtained from the Ames Test.

We present a resource for instructors that contains results and data sets from the Ames test. Our aim is to share the results we have collected in previous semesters with other instructors, so they will be able to "conduct" the Ames test without the need to set foot in a laboratory classroom. Instructors will be able to use our online resource to perform the test remotely, as a supplement to their laboratory classroom, or even under hybrid circumstances. The coronavirus disease 2019 (COVID-19) pandemic brought many changes, including the way we, as instructors, were able to carry out our educational curricula, since access to laboratory classrooms was not always possible. While COVID-19 restrictions are still in place, and thus access to laboratory classrooms is limited or null, instructors can use our online resource, without the need to set foot in a laboratory classroom. When COVID-19 restrictions are lifted and access to laboratory classrooms is permitted, instructors can follow the procedures we describe and compare their results with ours, which appear in Results and Discussion, or use our data sets as take-home assignments for their students. In addition to its use in detecting the potential mutagenicity of different samples, we have found the Ames test to be extremely useful for developing problem-solving skills by means of exercises like the ones included in this resource. Furthermore, the potential of this test as a starting point for problem-based learning is remarkable. Some suggestions for its use in active learning settings are provided.

Machine learning methods for imbalanced data set for prediction of faecal contamination in beach waters.

Predicting water contamination by statistical models is a useful tool to manage health risk in recreational beaches. Extreme contamination events, i.e. those exceeding normative are generally rare with respect to bathing conditions and thus the data is said to be imbalanced. Modeling and predicting those rare events present unique challenges. Here we introduce and evaluate several machine learning techniques and metrics to model imbalanced data and evaluate model performance. We do so by using a) simulated data-sets and b) a real data base with records of faecal coliform abundance monitored for 10 years in 21 recreational beaches in Uruguay (N ≈ 19000) using in situ and meteorological variables. We discuss advantages and disadvantages of the methods and provide a simple guide to perform models for a general audience. We also provide R codes to reproduce model fitting and testing. We found that most Machine Learning techniques are sensitive to imbalance and require specific data pre-treatment (e.g. upsampling) to improve performance. Accuracy (i.e. correctly classified cases over total cases) is not adequate to evaluate model performance on imbalanced data set. Instead, true positive rates (TPR) and false positive rates (FPR) are recommended. Among the 52 possible candidate algorithms tested, the stratified Random forest presented the better performance improving TPR in 50% with respect to baseline (0.4) and outperformed baseline in the evaluated metrics. Support vector machines combined with upsampling method or synthetic minority oversampling technique (SMOTE) performed well, similar to Adaboost with SMOTE. These results suggests that combining modeling strategies is necessary to improve our capacity to anticipate water contamination and avoid health risk.

Rapid freshwater discharge on the coastal ocean as a mean of long distance spreading of an unprecedented toxic cyanobacteria bloom.

Cyanobacterial toxic blooms are a worldwide problem. The Río de la Plata (RdlP) basin makes up about one fourth of South America areal surface, second only to the Amazonian. Intensive agro-industrial land use and the construction of dams have led to generalized eutrophication of main tributaries and increased the intensity and duration of cyanobacteria blooms. Here we analyse the evolution of an exceptional bloom at the low RdlP basin and Atlantic coast during the summer of 2019. A large array of biological, genetic, meteorological, oceanographic and satellite data is combined to discuss the driving mechanisms. The bloom covered the whole stripe of the RdlP estuary and the Uruguayan Atlantic coasts (around 500 km) for approximately 4 months. It was caused by the Microcystis aeruginosa complex (MAC), which produces hepatotoxins (microcystin). Extreme precipitation in the upstream regions of Uruguay and Negro rivers' basins caused high water flows and discharges. The evolution of meteorological and oceanographic conditions as well as the similarity of organisms' traits in the affected area suggest that the bloom originated in eutrophic reservoirs at the lower RdlP basin, Salto Grande in the Uruguay river, and Negro river reservoirs. High temperatures and weak Eastern winds prompted the rapid dispersion of the bloom over the freshwater plume along the RdlP northern and Atlantic coasts. The long-distance rapid drift allowed active MAC organisms to inoculate freshwater bodies from the Atlantic basin, impacting environments relevant for biodiversity conservation. Climate projections for the RdlP basin suggest an increase in precipitation and river water flux, which, in conjunction with agriculture intensification and dams' construction, might turn this extraordinary event into an ordinary situation.

Morphology captures toxicity in Microcystis aeruginosa complex: Evidence from a wide environmental gradient✰.

Blooms of the Microcystis aeruginosa complex (MAC) consist of mixtures of toxin-producing and non-toxin-producing populations, but the environmental conditions that determine their relative abundance and shift are not clear. Morphological traits reflect the responses of MAC organisms to environmental changes, thus they could be useful to improve the predictability of the abundance of both toxic and nontoxic populations. In this work, the response of MAC toxic populations to environmental conditions and their relationship with morphology (size of organisms) were investigated in different water bodies (reservoir, river, and estuary) covering wide salinity (0-33) and temperature (10-36 °C) gradients. Sub-surface water samples were collected and divided into 4 size classes (mesh size 〈20 µm, 20-60 µm, 60-150 µm and〉 150 µm) and three toxicity proxies were assessed (mcyE gene and transcripts copy numbers and microcystin concentration) for each size-class. For all the size-classes, the logarithm of the number of mcyE gene copies per sample was proportional to the logarithm of the corresponding biovolume fraction, showing that MAC biovolume is a good indicator of toxicity potential. When toxicity was analyzed through mcyE transcript abundance and microcystin concentration, the largest size fraction (>150 µm) showed the highest toxicity values of both proxies. Nevertheless, mcyE transcription and toxin production per cell were higher in the colonies retained in the 60 to 150 µm size fractions, followed by single cells (<20 µm). At the reservoir, where environmental variability is low, the total abundance of mcyE gene copies was significantly explained by MAC biovolume, regardless of the environmental conditions. However, when data from the reservoir to the estuary were modeled, biovolume and temperature (with a minor contribution of salinity and wind intensity) were selected in the best models. According to these results, the size distribution of MAC biovolume appears as a good predictor of active toxin production, being the colonies in the 60-150 µm size fraction good indicators of higher toxicity. These results can be used to predict MAC toxicity based on the size structure of the community.

Ecophysiological differences of betaproteobacterial populations in two hydrochemically distinct compartments of a subtropical lagoon.

We studied the population sizes and substrate incorporation patterns of three phylogenetic groups of Betaproteobacteria in a coastal subtropical lagoon that is characterized by a sharp transition from humic freshwater to turbid brackish water. Various cellular processes were addressed by short-term incubations with four radiolabelled compounds and microautoradiographic assessment of substrate incorporation. Group-specific differences in the abundances and the respective physiological state of the three populations were observed upon transfer from the humic-rich compartment to the main body of the lagoon (estimated at 1-2 days). Members of the clade B of Polynucleobacter (PnecB) experienced only an insignificant change in cell numbers, but displayed a general metabolic downshift, carbon metabolism (glucose incorporation) being most affected. By contrast, bacteria from the closely related Polynucleobacter C clade (PnecC) clearly differed in total abundances and in the numbers of DNA-synthesizing or glucose incorporating cells. At the same time, PnecC bacteria maintained comparable levels of protein synthesis (leucine uptake) in both lagoon compartments, and the proportion of cells incorporating N-acetylglucosamine was even higher in the main body of the lagoon. Members of the R-BT lineage showed little changes in cell numbers, DNA synthesis and carbon metabolism. Altogether, the observed patterns of substrate metabolism suggest that different bacterial populations in the lagoon undergo specific physiological adjustments in response to changing environmental conditions.

Alterations in the Gut Microbiota of Rats Chronically Exposed to Volatilized Cocaine and Its Active Adulterants Caffeine and Phenacetin.

A role of the gut microbiota in influencing brain function and emotional disorders has been suggested. However, only a few studies have investigated the gut microbiota in the context of drug addiction.Cocaine can be smoked (i.e., crack or coca paste) and its consumption is associated with a very high abuse liability and toxicity. We have recently reported that cocaine base seized samples contained caffeine and phenacetin as main active adulterants, which may potentiate its motivational, reinforcing, and toxic effects. However, the effect of volatilized cocaine and adulterants on the gut microbiota remained unknown. In the present study, we evaluated the effect of volatilized cocaine and two adulterants on the structure, diversity, and functionality of the gut microbiota in rats. Animals were chronically exposed to the fume of cocaine, caffeine, and phenacetin during 14 days. At the end of the treatment, feces were collected and the structure, composition, and functional predictions of the gut microbiota were analyzed. Cocaine significantly decreased the community richness and diversity of the gut microbiota while both cocaine and phenacetin drastically changed its composition. Phenacetin significantly increased the Firmicutes-Bacteroidetes ratio compared to the control group. When the predicted metagenome functional content of the bacterial communities was analyzed, all the treatments induced a dramatic decrease of the aromatic amino acid decarboxylase gene. Our findings suggest that repeated exposure to volatilized cocaine, as well as to the adulterants caffeine and phenacetin, leads to changes in the gut microbiota. Future studies are needed to understand the mechanisms underlying these changes and how this information may support the development of novel treatments in drug addiction.

Microbial Source Tracking Analysis Using Viral Indicators in Santa Lucía and Uruguay Rivers, Uruguay.

The aim of this study was to determine the origin (human, bovine or porcine) and the concentration of the fecal sources of contamination in waters from Santa Lucía basin and Uruguay River in Uruguay by using host-specific viral markers (adenoviruses and polyomaviruses) as microbial source tracking (MST). Between June 2015 and May 2016, monthly collections of surface water samples were performed in six sites in Santa Lucía basin and four sites in Uruguay River (n = 120 samples). Viral concentration was carried out using an absorption-elution method. Detection and quantification of human and porcine adenovirus (HAdV and PAdV, respectively) and human and bovine polyomavirus (HPyV and BoPyV, respectively) were performed by quantitative PCR (qPCR). To evaluate the infectivity of circulating HAdV, an integrated cell culture-qPCR (ICC-qPCR) was used. A logistic regression analysis was carried out to estimate the influence of environmental variables on the virus presence in surface waters. Overall, HAdV was the prevalent (18%; 21/120) followed by BoPyV (11%; 13/120) and HPyV (3%; 3/120), whereas PAdV was not detected in this study. The mean concentration ranged from 1.5 × 104 genomic copies/L (gc/L) for HAdV to 1.8 × 102 gc/L for HPyV. Infective HAdVs were observed in two out of ten analyzed samples. A significant effect of environmental temperature (p = 0.001) and river (p = 0.012) on the presence of human viruses was found. These results suggest that fecal contamination could affect the water quality of these rivers, showing deficiencies in the procedure of sewage discharge from regional cities, livestock and dairy farms.

Colonization of overlaying water by bacteria from dry river sediments.

We studied the diversity, community composition and activity of the primary microbial colonizers of the water above freshly re-wetted sediments from a temporary river. Dried sediments, collected from Mulargia River (Sardinia, Italy), were covered with sterile freshwater in triplicate microcosms, and changes of the planktonic microbial assemblage were monitored over a 48 h period. During the first 9 h bacterial abundance was low (1.5 x 10(4) cells ml(-1)); it increased to 3.4 x 10(6) cells ml(-1) after 28 h and did not change thereafter. Approximately 20% of bacteria exhibited DNA de novo synthesis already after 9 h of incubation. Changes of the ratios of (3)H-leucine to (3)H-thymidine incorporation rates indicated a shift of growth patterns during the experiment. Extracellular enzyme activity showed a maximum at 48 h with aminopeptidase activity (430.8 +/- 22.6 nmol MCA l(-1) h(-1)) significantly higher than alkaline phosphatase (98.6 +/- 4.3 nmol MUF l(-1) h(-1)). The primary microbial colonizers of the overlaying water - as determined by 16S rRNA gene sequence analysis - were related to at least six different phylogenetic lineages of Bacilli and to Alphaproteobacteria (Brevundimonas spp. and Caulobacter spp.). Large bacterial cells affiliated to one clade of Bacillus sp. were rare in the dried sediments, but constituted the majority of the planktonic microbial assemblage and of cells with detectable DNA-synthesis until 28 h after re-wetting. Their community contribution decreased in parallel with a rise of flagellated and ciliated protists. Estimates based on cell production rates suggested that the rapidly enriched Bacillus sp. suffered disproportionally high loss rates from selective predation, thus favouring the establishment of a more heterogenic assemblage of microbes (consisting of Proteobacteria, Actinobacteria and Cytophaga-Flavobacteria). Our results suggest that the primary microbial colonizers of the water above dried sediments are passively released into the plankton and that their high growth potential is counteracted by the activity of bacterivorous protists.

An iron-regulated outer-membrane protein of Proteus mirabilis is a haem receptor that plays an important role in urinary tract infection and in in vivo growth.

Proteus mirabilis, a common cause of urinary tract infections, expresses iron-regulated outer-membrane proteins (OMPs) in response to iron restriction. It has been suggested that a 64 kDa OMP is involved in haemoprotein uptake and that this might have a role in pathogenesis. In order to confirm this hypothesis, this study generated a P. mirabilis mutant strain (P7) that did not express the 64 kDa OMP, by insertion of the TnphoA transposon. The nucleotide sequence of the interrupted gene revealed that it corresponded to a haemin receptor precursor. Moreover, in vitro growth assays showed that the mutant was unable to grow using haemoglobin and haemin as unique iron sources. The authors also carried out in vivo growth and infectivity assays and demonstrated that P7 was not able to survive in an in vivo model and was less efficient than wild-type strain Pr 6515 in colonizing the urinary tract. These results confirmed that the P. mirabilis 64 kDa iron-regulated OMP is a haem receptor that has an important role for survival and multiplication of these bacteria in the mammalian host and in the development of urinary tract infection.