Metagenomic profiling of raw wastewater in Portugal highlights microbiota and resistome signatures of public health interest beyond the usual suspects.

In response to the rapid emergence and dissemination of antimicrobial resistant bacteria (ARB) and genes (ARGs), integrated surveillance systems are needed to address antimicrobial resistance (AMR) within the One Health Era. Wastewater analyses enable biomarker monitoring at the sewershed level, offering timely insights into pathogen circulation and ARB/ARGs trends originating from different compartments. During two consecutive epidemic waves of the COVID-19 pandemic in Portugal, taxonomic and functional composition of raw urban wastewater from two wastewater treatment plants (WWTPs) representing one million in equivalent population, located in the main urban areas of the country, were profiled by shotgun metagenomics. Hospital wastewater from two central hospitals located in the WWTPs catchment areas were also sequenced. The resistome and virulome were profiled using metagenomic assemblies without taxonomic constraint, and then specifically characterized for ESKAPE pathogens. Urban and hospital wastewater exhibited specific microbiota signatures, Pseudomonadota dominated in the first and Bacteroidota in the latter. Correlation network analyses highlighted 85 (out of top 100) genera co-occurring across samples. The most frequent ARGs were classified in the multidrug, tetracyclines, and Macrolides, Lincosamides, Streptogramins (MLS) classes. Links established between AMR determinants and bacterial hosts evidenced that the diversity and abundance of ARGs is not restricted to ESKAPE, being also highly predominant among emergent enteropathogens, like Aeromonas and Aliarcobacter, or in the iron (II) oxidizer Acidovorax. The Aliarcobacter genus accumulated high abundance of sulphonamides and polymyxins ARGs, while Acinetobacter and Aeromonas hosted the highest abundance of ARGs against beta-lactams. Other bacteria (e.g. Clostridioides, Francisella, Vibrio cholerae) and genes (e.g. vanA-type vancomycin resistance) of public health interest were detected, with targeted monitoring efforts being needed to establish informative baseline data. Altogether, results highlight that wastewater monitoring is a valuable component of pathogen and AMR surveillance in healthy populations, providing a community-representative snapshot of public health trends beyond priority pathogens.

A wastewater-based epidemiology tool for COVID-19 surveillance in Portugal.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater produced interest in its use for sentinel surveillance at a community level and as a complementary approach to syndromic surveillance. With this work, we set the foundations for wastewater-based epidemiology (WBE) in Portugal by monitoring the trends of SARS-CoV-2 RNA circulation in the community, on a nationwide perspective during different epidemiological phases of the pandemic. The Charité assays (E_Sarbecco, RdRP, and N_Sarbecco) were applied to monitor, over 32-weeks (April to December 2020), the dynamics of SARS-CoV-2 RNA at the inlet of five wastewater treatment plants (WWTP), which together serve more than two million people in Portugal. Raw wastewater from three Coronavirus disease 2019 (COVID-19) reference hospitals was also analyzed during this period. In total, more than 600 samples were tested. For the first weeks, detection of SARS-CoV-2 RNA was sporadic, with concentrations varying from 103 to 105 genome copies per liter (GC/L). Prevalence of SARS-CoV-2 RNA increased steeply by the end of May into late June, mainly in Lisboa e Vale do Tejo region (LVT), during the reopening phase. After the summer, with the reopening of schools in mid-September and return to partial face-to-face work, a pronounced increase of SARS-CoV-2 RNA in wastewater was detected. In the LVT area, SARS-CoV-2 RNA load agreed with reported trends in hotspots of infection. Synchrony between trends of SARS-CoV-2 RNA in raw wastewater and daily new COVID-19 cases highlights the value of WBE as a surveillance tool, particularly after the phasing out of the epidemiological curve and when hotspots of disease re-emerge in the population which might be difficult to spot based solely on syndromic surveillance and contact tracing. This is the first study crossing several epidemiological stages highlighting the long-term use of WBE for SARS-CoV-2.

The Mitochondrial Prohibitin (PHB) Complex in C. elegans Metabolism and Ageing Regulation.

The mitochondrial prohibitin (PHB) complex, composed of PHB-1 and PHB-2, is an evolutionarily conserved context-dependent modulator of longevity. This extremely intriguing phenotype has been linked to alterations in mitochondrial function and lipid metabolism. The true biochemical function of the mitochondrial PHB complex remains elusive, but it has been shown to affect membrane lipid composition. Recent work, using large-scale biochemical approaches, has highlighted a broad effect of PHB on the C. elegans metabolic network. Collectively, the biochemical data support the notion that PHB modulates, at least partially, worm longevity through the moderation of fat utilisation and energy production via the mitochondrial respiratory chain. Herein, we review, in a systematic manner, recent biochemical insights into the impact of PHB on the C. elegans metabolome.

The Mitochondrial PHB Complex Determines Lipid Composition and Interacts With the Endoplasmic Reticulum to Regulate Ageing.

Metabolic disorders are frequently associated with physiological changes that occur during ageing. The mitochondrial prohibitin complex (PHB) is an evolutionary conserved context-dependent modulator of longevity, which has been linked to alterations in lipid metabolism but which biochemical function remains elusive. In this work we aimed at elucidating the molecular mechanism by which depletion of mitochondrial PHB shortens the lifespan of wild type animals while it extends that of insulin signaling receptor (daf-2) mutants. A liquid chromatography coupled with mass spectrometry approach was used to characterize the worm lipidome of wild type and insulin deficient animals upon PHB depletion. Toward a mechanistic interpretation of the insights coming from this analysis, we used a combination of biochemical, microscopic, and lifespan analyses. We show that PHB depletion perturbed glycerophospholipids and glycerolipids pools differently in short- versus long-lived animals. Interestingly, PHB depletion in otherwise wild type animals induced the endoplasmic reticulum (ER) unfolded protein response (UPR), which was mitigated in daf-2 mutants. Moreover, depletion of DNJ-21, which functionally interacts with PHB in mitochondria, mimicked the effect of PHB deficiency on the UPRER and on the lifespan of wild type and insulin signaling deficient mutants. Our work shows that PHB differentially modulates lipid metabolism depending on the worm's metabolic status and provides evidences for a new link between PHB and ER homeostasis in ageing regulation.

1H-NMR-Based Endometabolome Profiles of Burkholderia cenocepacia Clonal Variants Retrieved from a Cystic Fibrosis Patient during Chronic Infection.

During cystic fibrosis (CF) chronic lung infections, bacteria of the Burkholderia cepacia complex (Bcc) are exposed for several years to a stressful and changing environment. These environmental challenges results in genetic changes of the initial infecting strain with the consequent diversification of genotypes and phenotypes. The exploitation of functional and comparative genomic approaches has suggested that such diversification is associated with massive metabolic remodeling but these alterations are poorly understood. In the present work, we have explored a high resolution 1H-NMR-based metabolomic approach coupled to multivariate analysis to compare the endometabolome of three B. cenocepacia clonal variants retrieved from a CF patient from the onset of infection (IST439) until death with cepacia syndrome after 3.5 years (IST4113 and IST4134), to complement former proteomic and transcriptomic analyses. A fourth clonal variant (IST4129) retrieved from the same CF patient when the clinical condition worsened during the last months of life, was also examined since it was found to lack the third replicon. The metabolomic profiles obtained, based on the complete 1H-NMR spectra, highlight the separation of the four clonal variants examined, the most distinct profile corresponding to IST4129. Results indicate a variable content of several amino acids in the different isolates examined and suggest that glycolysis and the glyoxylate shunt are favored in late variants. Moreover, the concentration of two metabolites with demonstrated cellular protective functions against stress, glycine-betaine and trehalose, is different in the different isolates examined. However, no clear correlation could be established between their content and stress tolerance. For example, IST4113, previously found to be the most resistant variant to antimicrobials of different classes, exhibits low levels of trehalose and glycine-betaine but the highest resistance to heat and oxidative stress. Also, IST4129, with a high level of glycine-betaine but lacking the third replicon, previously associated with stress resistance and virulence, exhibits the highest susceptibility to all the stresses tested. Taken together, results from this study provide insights into the metabolic diversification of B. cenocepacia clonal variants during long-term infection of the CF airways.

Analysis of the effect of the mitochondrial prohibitin complex, a context-dependent modulator of longevity, on the C. elegans metabolome.

The mitochondrial prohibitin complex, composed of two proteins, PHB-1 and PHB-2, is a context-dependent modulator of longevity. Specifically, prohibitin deficiency shortens the lifespan of otherwise wild type worms, while it dramatically extends the lifespan under compromised metabolic conditions. This extremely intriguingly phenotype has been linked to alterations in mitochondrial function and in fat metabolism. However, the true function of the mitochondrial prohibitin complex remains elusive. Here, we used gas chromatography coupled to a flame ionization detector (GC/FID) and ¹H NMR spectroscopy to gain molecular insights into the effect of prohibitin depletion on the Caenorhabditis elegans metabolome. We analysed the effect of prohibitin deficiency in two different developmental stages and under two different conditions, which result in opposing longevity phenotypes, namely wild type worms and daf-2(e1370) insulin signalling deficient mutants. Prohibitin depletion was shown to alter the fatty acid (GC/FID) and ¹H NMR metabolic profiles of wild type animals both at the fourth larval stage of development (L4) and at the young adult (YA) stage, while being more pronounced at the later stage. Furthermore, wild type and the diapause mutant daf-2(e1370), either expressing or not prohibitin, were clearly distinguishable based on their metabolic profiles, revealing changes in fatty acid composition, as well as in carbohydrate and amino acid metabolism. Moreover, the metabolic data indicate that daf-2(e1370) mutants are more robust than the wild type animals to changes induced by prohibitin depletion. The impact of prohibitin depletion on the C. elegans metabolome will be discussed herein in the scope of its effect on longevity. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging. Guest Editor: Aleksandra Trifunovic.

Quantitative 1H-NMR-metabolomics reveals extensive metabolic reprogramming and the effect of the aquaglyceroporin FPS1 in ethanol-stressed yeast cells.

A metabolomic analysis using high resolution 1H NMR spectroscopy coupled with multivariate statistical analysis was used to characterize the alterations in the endo- and exo-metabolome of S. cerevisiae BY4741 during the exponential phase of growth in minimal medium supplemented with different ethanol concentrations (0, 2, 4 and 6% v/v). This study provides evidence that supports the notion that ethanol stress induces reductive stress in yeast cells, which, in turn, appears to be counteracted by the increase in the rate of NAD+ regenerating bioreactions. Metabolomics data also shows increased intra- and extra-cellular accumulation of most amino acids and TCA cycle intermediates in yeast cells growing under ethanol stress suggesting a state of overflow metabolism in turn of the pyruvate branch-point. Given its previous implication in ethanol stress resistance in yeast, this study also focused on the effect of the expression of the aquaglyceroporin encoded by FPS1 in the yeast metabolome, in the absence or presence of ethanol stress. The metabolomics data collected herein shows that the deletion of the FPS1 gene in the absence of ethanol stress partially mimics the effect of ethanol stress in the parental strain. Moreover, the results obtained suggest that the reported action of Fps1 in mediating the passive diffusion of glycerol is a key factor in the maintenance of redox balance, an important feature for ethanol stress resistance, and may interfere with the ability of the yeast cell to accumulate trehalose. Overall, the obtained results corroborate the idea that metabolomic approaches may be crucial tools to understand the function and/or the effect of membrane transporters/porins, such as Fps1, and may be an important tool for the clear-cut design of improved process conditions and more robust yeast strains aiming to optimize industrial fermentation performance.

Genome-wide identification of Saccharomyces cerevisiae genes required for maximal tolerance to ethanol.

The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H(+)-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme beta-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPS1 expression contributes to decreased [(3)H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production.

The RIM101 pathway has a role in Saccharomyces cerevisiae adaptive response and resistance to propionic acid and other weak acids.

The physiological function of the Saccharomyces cerevisiae RIM101 signaling pathway is extended in this study beyond alkaline pH-induced responses. The transcription factor Rim101p is demonstrated to be required for maximal tolerance to weak acid-induced stress, at pH 4.0, but does not exert protection against low pH itself (range 4.5-2.5), when a strong acid is used as the acidulant. The Rim101p-dependent alterations of the yeast transcriptome following exposure to propionic acid stress (at pH 4.0) include genes of the previously described Rim101p regulon but also new target genes, in particular KNH1, involved in cell wall beta-1,6-glucan synthesis and the uncharacterized ORF YIL029c, both required for maximal propionic acid resistance. Clustering of the genes that provide resistance to propionic acid reveals the enrichment of those involved in protein catabolism through the multivesicular body pathway and in the homeostasis of internal pH and vacuolar function. The analysis of the network of interactions established among all the identified propionic acid resistance determinants shows an enrichment of interactions around the RIM101 gene and highlights the role of proteins involved in Rim101p proteolytic processing. RIM101 expression is shown to be required to counteract propionic acid-induced cytosolic acidification and for proper vacuolar acidification and cell wall structure, these having positive implications for a robust adaptive response and resistance to stress promoted by this food preservative.

YEASTRACT-DISCOVERER: new tools to improve the analysis of transcriptional regulatory associations in Saccharomyces cerevisiae.

The Yeast search for transcriptional regulators and consensus tracking (YEASTRACT) information system (www.yeastract.com) was developed to support the analysis of transcription regulatory associations in Saccharomyces cerevisiae. Last updated in September 2007, this database contains over 30 990 regulatory associations between Transcription Factors (TFs) and target genes and includes 284 specific DNA binding sites for 108 characterized TFs. Computational tools are also provided to facilitate the exploitation of the gathered data when solving a number of biological questions, in particular the ones that involve the analysis of global gene expression results. In this new release, YEASTRACT includes DISCOVERER, a set of computational tools that can be used to identify complex motifs over-represented in the promoter regions of co-regulated genes. The motifs identified are then clustered in families, represented by a position weight matrix and are automatically compared with the known transcription factor binding sites described in YEASTRACT. Additionally, in this new release, it is possible to generate graphic depictions of transcriptional regulatory networks for documented or potential regulatory associations between TFs and target genes. The visual display of these networks of interactions is instrumental in functional studies. Tutorials are available on the system to exemplify the use of all the available tools.