Pre- and Postharvest Strategies for Pleurotus ostreatus Mushroom in a Circular Economy Approach.

Mushroom cultivation presents a viable solution for utilizing agro-industrial byproducts as substrates for growth. This process enables the transformation of low-economic-value waste into nutritional foods. Enhancing the yield and quality of preharvest edible mushrooms, along with effectively preserving postharvest mushrooms, stands as a significant challenge in advancing the industry. Implementing pre- and postharvest strategies for Pleurotus ostreatus (Jacq.) P. Kumm (oyster mushroom) within a circular economy framework involves optimizing resource use, minimizing waste, and creating a sustainable and environmentally friendly production system. This review aimed to analyze the development and innovation of the different themes and trends by bibliometric analysis with a critical literature review. Furthermore, this review outlines the cultivation techniques for Pleurotus ostreatus, encompassing preharvest steps such as spawn production, substrate preparation, and the entire mushroom growth process, which includes substrate colonization, fruiting, harvesting, and, finally, the postharvest. While novel methodologies are being explored for maintaining quality and extending shelf-life, the evaluation of the environmental impact of the entire mushroom production to identify areas for improvement is needed. By integrating this knowledge, strategies can be developed for a more sustainable and circular approach to Pleurotus ostreatus mushroom cultivation, promoting environmental stewardship and long-term viability in this industry.

Effects of Different Irrigation Regimes, Nitrogen Levels and Storage Conditions on Volatiles of 'Gala' Apple.

With a characteristic flavour and aroma, "Maçã de Alcobaça" are apples produced in the western region of the mainland of Portugal. Given the known influence of pre-harvest cultural techniques and post-harvest conservation methods on fruit quality, this work evaluated the effect of cultural factors and conservation methods on the volatile profile of 'Gala' apples. Tests were carried out during four seasons (2018 to 2021) in two 'Gala' apple orchards (F and S) maintained with different irrigation rates and nitrogen fertilisation [normal irrigation and normal nitrogen (Control, NINN), normal irrigation and excess nitrogen (NIEN), excess irrigation and normal nitrogen (EINN), excess irrigation and excess nitrogen (EIEN)], and under three storage conditions [Controlled Atmosphere + 1-methylcyclopropene (CA+1-MCP), Dynamic Controlled Atmosphere (DCA) and DCA+1-MCP]. The intact fruit volatiles were isolated by headspace solid-phase microextraction (HS-SPME) and analysed by Gas Chromatography with Flame Ionisation Detection and Gas Chromatography-Mass Spectrometry at harvest (T0) and after 8 months of storage (T8). HS-SPME volatiles from 'Gala' apples, obtained at T0 in control conditions, were characterised by trans,trans-α-farnesene dominance (36-69%), followed by hexyl acetate (5-23%) and hexyl hexanoate (3-9%). The four irrigation and nitrogen treatments did not evidence main changes in the apple volatile profile. Instead, storage conditions changed the ratio between compounds; previously undetected compounds attained high percentages and decreased the intensity of the dominant compounds in the control conditions. Although all storage conditions tested changed the volatile profile and emanation intensity, the effect was more accentuated in storage for 8 months with DCA+1-MCP.

Valorisation of Wasted Immature Tomato to Innovative Fermented Functional Foods.

In this study, the lactic fermentation of immature tomatoes as a tool for food ingredient production was evaluated as a circular economy-oriented alternative for valorising industrial tomatoes that are unsuitable for processing and which have wasted away in large quantities in the field. Two lactic acid bacteria (LAB) were assessed as starter cultures in an immature tomato pulp fermentation to produce functional food ingredients with probiotic potential. The first trial evaluated the probiotic character of Lactiplantibacillus plantarum (LAB97, isolated from immature tomato microbiota) and Weissella paramesenteroides (C1090, from the INIAV collection) through in vitro gastrointestinal digestion simulation. The results showed that LAB97 and C1090 met the probiotic potential viability criterion by maintaining 6 log10 CFU/mL counts after in vitro simulation. The second trial assessed the LAB starters' fermentative ability. Partially decontaminated (110 °C/2 min) immature tomato pulp was used to prepare the individually inoculated samples (Id: LAB97 and C1090). Non-inoculated samples, both with and without thermal treatment (Id: CTR-TT and CTR-NTT, respectively), were prepared as the controls. Fermentation was undertaken (25 °C, 100 rpm) for 14 days. Throughout storage (0, 24, 48, 72 h, 7, and 14 days), all the samples were tested for LAB and Y&M counts, titratable acidity (TA), solid soluble content (SSC), total phenolic content (TPC), antioxidant capacity (AOx), as well as for organic acids and phenolic profiles, and CIELab colour and sensory evaluation (14th day). The LAB growth reached ca. 9 log10 CFU/mL for all samples after 72 h. The LAB97 samples had an earlier and higher acidification rate than the remaining ones, and they were highly correlated to lactic acid increments. The inoculated samples showed a faster and higher decrease rate in their SSC levels when compared to the controls. A nearly two-fold increase (p < 0.05) during the fermentation, over time, was observed in all samples' AOx and TPC (p < 0.05, r = 0.93; similar pattern). The LAB97 samples obtained the best sensory acceptance for flavour and overall appreciation scores when compared to the others. In conclusion, the L. plantarum LAB97 starter culture was selected as a novel probiotic candidate to obtain a potential probiotic ingredient from immature tomato fruits.

Influence of Air-Drying Conditions on Quality, Bioactive Composition and Sensorial Attributes of Sweet Potato Chips.

The drying process is an essential thermal process for preserving vegetables and can be used in developing dried products as healthy alternative snacks. The effects of air-drying conditions using a convection dryer with hot air at different temperatures (60°, 65°, 70°, 75°, and 80 °C, in the range 5-200 min, at a fixed air speed of 2.3 m/s) were tested on the quality of slices (2.0 ± 0.1 mm) of dried sweet potato (Bellevue PBR). For each time and temperature, drying condition, physicochemical parameters (moisture content, CIELab color, texture parameters, total phenolic and carotenoid contents) and a sensory evaluation by a panel at the last drying period (200 min) were assessed. Drying time was shown to have a more significant effect than temperature on the quality of dried sweet potato as a snack, except for carotenoid content. Given the raw tuber content, thermal degradation (p < 0.05) of total phenolic compounds (about 70%), regardless of tested conditions, contrasted with the higher stability of total carotenoids (<30%). The dried product, under optimal conditions (≥75 °C for 200 min), achieved a moisture content (≤10%) suitable for preservation, providing a crispy texture with favourable sensory acceptance and providing a carotenoid content similar to the raw product.

In vitro activity of imipenem/relebactam against Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal (SUPERIOR and STEP studies).

OBJECTIVES: To study the in vitro activity of imipenem/relebactam and comparators and the imipenem/relebactam resistance mechanisms in a Pseudomonas aeruginosa collection from Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17) surveillance studies. METHODS: P. aeruginosa isolates (n = 474) were prospectively recovered from complicated urinary tract (cUTI), complicated intra-abdominal (cIAI) and lower respiratory tract (LRTI) infections in 11 Portuguese and 8 Spanish ICUs. MICs were determined (ISO broth microdilution). All imipenem/relebactam-resistant P. aeruginosa isolates (n = 30) and a subset of imipenem/relebactam-susceptible strains (n = 32) were characterized by WGS. RESULTS: Imipenem/relebactam (93.7% susceptible), ceftazidime/avibactam (93.5% susceptible) and ceftolozane/tazobactam (93.2% susceptible) displayed comparable activity. The imipenem/relebactam resistance rate was 6.3% (Portugal 5.8%; Spain 8.9%). Relebactam restored imipenem susceptibility to 76.9% (103/134) of imipenem-resistant isolates, including MDR (82.1%; 32/39), XDR (68.8%; 53/77) and difficult-to-treat (DTR) isolates (67.2%; 45/67). Among sequenced strains, differences in population structure were detected depending on the country: clonal complex (CC)175 and CC309 in Spain and CC235, CC244, CC348 and CC253 in Portugal. Different carbapenemase gene distributions were also found: VIM-20 (n = 3), VIM-1 (n = 2), VIM-2 (n = 1) and VIM-36 (n = 1) in Spain and GES-13 (n = 13), VIM-2 (n = 3) and KPC-3 (n = 2) in Portugal. GES-13-CC235 (n = 13) and VIM type-CC175 (n = 5) associations were predominant in Portugal and Spain, respectively. Imipenem/relebactam showed activity against KPC-3 strains (2/2), but was inactive against all GES-13 producers and most of the VIM producers (8/10). Mutations in genes affecting porin inactivation, efflux pump overexpression and LPS modification might also be involved in imipenem/relebactam resistance. CONCLUSIONS: Microbiological results reinforce imipenem/relebactam as a potential option to treat cUTI, cIAI and LRTI caused by MDR/XDR P. aeruginosa isolates, except for GES-13 and VIM producers.

Zinc Biofortification in Vitis vinifera: Implications for Quality and Wine Production.

Nowadays, there is a growing concern about micronutrient deficits in food products, with agronomic biofortification being considered a mitigation strategy. In this context, as Zn is essential for growth and maintenance of human health, a workflow for the biofortification of grapes from the Vitis vinifera variety Fernão Pires, which contains this nutrient, was carried out considering the soil properties of the vineyard. Additionally, Zn accumulation in the tissues of the grapes and the implications for some quality parameters and on winemaking were assessed. Vines were sprayed three times with ZnO and ZnSO4 at concentrations of 150, 450, and 900 g ha-1 during the production cycle. Physiological data were obtained through chlorophyll a fluorescence data, to access the potential symptoms of toxicity. At harvest, treated grapes revealed significant increases of Zn concentration relative to the control, being more pronounced for ZnO and ZnSO4 in the skin and seeds, respectively. After winemaking, an increase was also found regarding the control (i.e., 1.59-fold with ZnSO4-450 g ha-1). The contents of the sugars and fatty acids, as well as the colorimetric analyses, were also assessed, but significant variations were not found among treatments. In general, Zn biofortification increased with ZnO and ZnSO4, without significantly affecting the physicochemical characteristics of grapes.

Imipenem-Relebactam Susceptibility in Enterobacterales Isolates Recovered from ICU Patients from Spain and Portugal (SUPERIOR and STEP Studies).

Imipenem-relebactam is a novel ß-lactam-ß-lactamase inhibitor combination. We evaluated the in vitro activity of imipenem-relebactam and comparators against Enterobacterales clinical isolates recovered in 8 Spanish and 11 Portuguese intensive care units (ICUs) (SUPERIOR, 2016-2017; STEP, 2017-2018). Overall, 747 Enterobacterales isolates (378 Escherichia coli, 252 Klebsiella spp., 64 Enterobacter spp., and 53 other species) were prospectively collected from ICU patients with complicated intraabdominal (cIAI), complicated urinary tract (cUTI), and lower respiratory tract (LRTI) infections. MICs were determined (ISO-broth microdilution), and whole-genome sequencing (WGS) was performed in a subset of isolates displaying susceptible and resistant imipenem-relebactam MICs. Imipenem-relebactam (98.7% susceptible) showed similar activity to ceftazidime-avibactam (99.5% susceptible) and higher than ceftolozane-tazobactam (86.9% susceptible). Imipenem-relebactam was inactive against 1.3% (10/747) isolates, all of them due to carbapenemase production (9 K. pneumoniae and 1 E. cloacae). Imipenem-relebactam was active against 100% of extended-spectrum ß-lactamase (ESBL)-E. coli and ESBL-Klebsiella spp. isolates and 80.4% of carbapenemase-Klebsiella spp. producers. Carbapenemase genes were confirmed by WGS in 41 Klebsiella spp.: OXA-48 (20/41), KPC-3 (14/41), OXA-181 (4/41), NDM-1 (1/41), OXA-48 + VIM-2 (1/41), and KPC-3 + VIM-2 (1/41). In Klebsiella spp. isolates, relebactam restored imipenem susceptibility in all KPC-3 producers, and resistant isolates (7/41) were mostly OXA-48 + CTX-M-15-K. pneumoniae high-risk clones (7/9). Intercountry differences were detected as follows: OXA-48 (17/21) was dominant in Spain, unlike KPC-3 (14/15) in Portugal. Imipenem-relebactam was 100% active against CTX-M-15-ST131-H30Rx-E. coli high-risk clone, predominant in both countries. Our results depict the potential role of imipenem-relebactam in ICU patients with cIAIs, cUTIs, and LRTIs due to wild-type ESBL- and carbapenemase-producing Enterobacterales, particularly KPC producers. IMPORTANCE We comparatively evaluate the in vitro activity of a drug combination consisting of a carbapenem (imipenem) and a novel inhibitor of beta-lactamases (relebactam), a mechanism that destroys beta-lactam antibiotics. We assess the activity against a collection of Enterobacterales clinical isolates recovered from difficult-to-treat infections in patients admitted to different intensive care units in Portugal and Spain. Imipenem-relebactam shows excellent activity in avoiding common resistance mechanisms in this setting, such as extended-spectrum beta-lactamases and carbapenemases widely distributed, including KPCs. We show few resistant isolates (<2%). Molecular characterization by whole-genome sequencing shows that most of the resistant isolates produced specific carbapenemase, such as OXA-48 or metalo-betalactamases. Our study updates the activity of imipenem-relebactam in light of current epidemiology in a hospital setting in which the use of this combination is needed due to the presence of infections due to multidrug-resistant isolates.

Multi-Target Alternative Approaches to Promoting Fresh-Cut Carrots' Bioactive and Fresh-like Quality.

Fresh-cut fruits and vegetables, as near-fresh foods, are a quick and easy solution to a healthy and balanced diet. The rapid degradation of nutritional and sensory quality during the processing and storage of a product is critical and plant-type-dependent. The introduction of disruptive technological solutions in fresh-cut processing, which could maintain fresh-like quality with less environmental impact, is an emerging research concept. The application of abiotic stress treatments (heat shock and UV-C) induces metabolic responses and microbial effects in plant tissues, potentially slowing down several quality senescence pathways. The previously selected combined and single effects of heat shock (100 °C/45 s; in the whole root) and UV-C (2.5 kJ/m2) treatments and two packaging conditions (oriented polypropylene (OPP) vs. micro-perforated OPP films) on controlling critical degradation pathways of fresh-cut carrots and on promoting bioactive and sensory quality during storage (5 °C, 14 days) were studied. Among the tested combinations, synergistic effects on the quality retention of fresh-cut carrots were only attained for applying heat shock associated with micro-perforated OPP film packaging. Its effects on reducing (3.3 Log10 CFU/g) the initial contamination and controlling microbiological spoilage (counts below the threshold limit of 7.5 Log10 CFU/g), increasing the bioactive content (38% and 72% in total phenolic content and chlorogenic acid, respectively), and preserving fresh quality attributes prove to be a viable alternative technology for shredded carrot processing.

Efficient Assessment and Large-Scale Conservation of Intra-Varietal Diversity of Ancient Grapevine Varieties: Case Study Portugal.

There are thousands of ancient grapevine varieties in Europe, each one having a high level of intra-varietal diversity with regard to important economic traits (yield, soluble solids content, acidity, anthocyanins, and others). However, this potential has become exposed to a process of genetic erosion since the middle of the last century. The main objective of this work is to present experimental strategies for conservation and utilization of intra-varietal diversity. A concrete example is given about the actions performed in Portugal since 1978. Two main approaches for the conservation of intra-varietal diversity were performed: (1) strict conservation (in pots and in the field without experimental design) for future generations; and (2) conservation and, simultaneously, evaluation of the intra-varietal variability for selection to fulfil the immediate needs of the grape and wine sector (in the field with experimental design). More than 30,000 accessions of Portuguese autochthonous varieties are conserved. Using the theory of mixed models, intra-varietal diversity of the yield was found for the 59 varieties studied. The conservation and the evaluation of the intra-varietal diversity for quantitative traits will allow to extract high economic value, as well as to ensure its utilization to meet the objectives of the vine and wine sector.

Influence of a heat-shock pre-treatment on wound-induced phenolic biosynthesis as an alternative strategy towards fresh-cut carrot processing.

In fresh-cut vegetables, plant tissues are often challenged by (a)biotic stresses that act in combination, and the response to combinatorial stresses differs from that triggered by each individually. Phenolic induction by wounding is a known response contributing to increase products phenolic content. Heat application is a promising treatment in minimal processing, and its interference on the wound-induced response is produce-dependent. In carrot, two-combined stress effects were evaluated: peel removal vs. shredding, and heat application (100 °C/45 s) vs. shredding, on changes in total phenolic content (TPC) during 10 days (5 °C). By applying the first stress combination, a decrease in TPC was verified on day 0 (∼50%), ascribed to the high phenolic content of peels. Recovery of initial fresh carrot levels was achieved after 7 days owing to phenolic biosynthesis induced by shredding. For the second combination, changes in TPC, phenylalanine-ammonia-lyase (PAL), and peroxidase (POD) activity of untreated (Ctr) and heat-treated (HS) peeled shredded carrot samples were evaluated during 10 days. The heat-shock did not suppress phenolic biosynthesis promoted by PAL, although there was a two-day delay in TPC increments. Notwithstanding, phenolic accumulation after 10 days exceeded raw material TPC content. Also, the decrease in POD activity (30%) could influence quality degradation during storage.

Selection of Autochthonous LAB Strains of Unripe Green Tomato towards the Production of Highly Nutritious Lacto-Fermented Ingredients.

Lactic fermentation of unripe green tomatoes as a tool to produce food ingredients is a viable alternative for adding value to industrial tomatoes unsuitable for processing and left in large quantities in the fields. Fermentation using starter cultures isolated from the fruit (plant-matrix adapted) can have advantages over allochthonous strains in obtaining fermented products with sensory acceptability and potentially probiotic characteristics. This paper details the characterisation of the unripe green tomato lactic microbiota to screen LAB strains for use as starter cultures in fermentation processes, along with LAB strains available from INIAV's collection. Morphological, biochemical (API system), and genomic (16S rDNA gene sequencing) identification showed that the dominant LAB genera in unripe green tomato are Lactiplantibacillus, Leuconostoc, and Weissella. Among nine tested strains, autochthonous Lactiplantibacillus plantarum and allochthonous Weissella paramesenteroides showed tolerance to added solanine (200 ppm) and the best in vitro probiotic potential. The results indicate that the two LAB strains are promising candidates for manufacturing probiotic fermented foods from unripe green tomatoes.

Potential Phenotyping Methodologies to Assess Inter- and Intravarietal Variability and to Select Grapevine Genotypes Tolerant to Abiotic Stress.

Plant phenotyping is an emerging science that combines multiple methodologies and protocols to measure plant traits (e.g., growth, morphology, architecture, function, and composition) at multiple scales of organization. Manual phenotyping remains as a major bottleneck to the advance of plant and crop breeding. Such constraint fostered the development of high throughput plant phenotyping (HTPP), which is largely based on imaging approaches and automatized data retrieval and processing. Field phenotyping still poses major challenges and the progress of HTPP for field conditions can be relevant to support selection and breeding of grapevine. The aim of this review is to discuss potential and current methods to improve field phenotyping of grapevine to support characterization of inter- and intravarietal diversity. Vitis vinifera has a large genetic diversity that needs characterization, and the availability of methods to support selection of plant material (polyclonal or clonal) able to withstand abiotic stress is paramount. Besides being time consuming, complex and expensive, field experiments are also affected by heterogeneous and uncontrolled climate and soil conditions, mostly due to the large areas of the trials and to the high number of traits to be observed in a number of individuals ranging from hundreds to thousands. Therefore, adequate field experimental design and data gathering methodologies are crucial to obtain reliable data. Some of the major challenges posed to grapevine selection programs for tolerance to water and heat stress are described herein. Useful traits for selection and related field phenotyping methodologies are described and their adequacy for large scale screening is discussed.

Pervasive hybridization with local wild relatives in Western European grapevine varieties.

Grapevine (Vitis vinifera L.) diversity richness results from a complex domestication history over multiple historical periods. Here, we used whole-genome resequencing to elucidate different aspects of its recent evolutionary history. Our results support a model in which a central domestication event in grapevine was followed by postdomestication hybridization with local wild genotypes, leading to the presence of an introgression signature in modern wine varieties across Western Europe. The strongest signal was associated with a subset of Iberian grapevine varieties showing large introgression tracts. We targeted this study group for further analysis, demonstrating how regions under selection in wild populations from the Iberian Peninsula were preferentially passed on to the cultivated varieties by gene flow. Examination of underlying genes suggests that environmental adaptation played a fundamental role in both the evolution of wild genotypes and the outcome of hybridization with cultivated varieties, supporting a case of adaptive introgression in grapevine.

Impact of environmental conditions on the ripening of Queijo de Évora PDO cheese.

"Queijo de Évora" is a traditional Portuguese cheese from raw ewe's milk and granted with PDO label. It is ripened traditionally in rooms with empirical control of temperature and humidity. Nowadays, almost all cheese factories use rooms with temperature and humidity control, but still a significant heterogeneity among cheeses is acknowledged due to unequal distribution of environmental conditions. This paper discusses the influence of the environmental conditions on the ripening of Queijo de Évora, including the application of computational fluid dynamics in steady state conditions. Experimental data was obtained in cheeses ripened along the traditional ripening cycle, in different locations. A significant influence of environmental conditions was observed, especially air velocity and humidity, affecting physical-chemical, microbiological and sensory characteristics. Locations with higher air velocity, presented cheeses with lower moisture content, higher mesophilic bacteria count, darker appearance and higher number of holes. Locations with higher humidity presented cheeses with lower scores on some sensorial parameters like appearance, firmness and intensity of odor. The results of computational fluid dynamics made possible the identification of areas in and around the cheese stacks were the air distribution is less than adequate or uneven, which may influence the evolution of cheese during ripening.

Staphylococcus saprophyticus From Clinical and Environmental Origins Have Distinct Biofilm Composition.

Biofilm formation has been shown to be critical to the success of uropathogens. Although Staphylococcus saprophyticus is a common cause of urinary tract infections, its biofilm production capacity, composition, genetic basis, and origin are poorly understood. We investigated biofilm formation in a large and diverse collection of S. saprophyticus (n = 422). Biofilm matrix composition was assessed in representative strains (n = 63) belonging to two main S. saprophyticus lineages (G and S) recovered from human infection, colonization, and food-related environment using biofilm detachment approach. To identify factors that could be associated with biofilm formation and structure variation, we used a pangenome-wide association study approach. Almost all the isolates (91%; n = 384/422) produced biofilm. Among the 63 representative strains, we identified eight biofilm matrix phenotypes, but the most common were composed of protein or protein-extracellular DNA (eDNA)-polysaccharides (38%, 24/63 each). Biofilms containing protein-eDNA-polysaccharides were linked to lineage G and environmental isolates, whereas protein-based biofilms were produced by lineage S and infection isolates (p < 0.05). Putative biofilm-associated genes, namely, aas, atl, ebpS, uafA, sasF, sasD, sdrH, splE, sdrE, sdrC, sraP, and ica genes, were found with different frequencies (3-100%), but there was no correlation between their presence and biofilm production or matrix types. Notably, icaC_1 was ubiquitous in the collection, while icaR was lineage G-associated, and only four strains carried a complete ica gene cluster (icaADBCR) except one that was without icaR. We provided evidence, using a comparative genomic approach, that the complete icaADBCR cluster was acquired multiple times by S. saprophyticus and originated from other coagulase-negative staphylococci. Overall, the composition of S. saprophyticus biofilms was distinct in environmental and clinical isolates, suggesting that modulation of biofilm structure could be a key step in the pathogenicity of these bacteria. Moreover, biofilm production in S. saprophyticus is ica-independent, and the complete icaADBCR was acquired from other staphylococci.

Staphylococcus saprophyticus Causing Infections in Humans Is Associated with High Resistance to Heavy Metals.

Staphylococcus saprophyticus is a common pathogen of the urinary tract, a heavy metal-rich environment, but information regarding its heavy metal resistance is unknown. We investigated 422 S. saprophyticus isolates from human infection and colonization/contamination, animals, and environmental sources for resistance to copper, zinc, arsenic, and cadmium using the agar dilution method. To identify the genes associated with metal resistance and assess possible links to pathogenicity, we accessed the whole-genome sequence of all isolates and used in silico and pangenome-wide association approaches. The MIC values for copper and zinc were uniformly high (1,600 mg/liter). Genes encoding copper efflux pumps (copA, copB, copZ, mco, and csoR) and zinc transporters (zinT, czrAB, znuBC, and zur) were abundant in the population (20 to 100%). Arsenic and cadmium showed various susceptibility levels. Genes encoding the ars operon (arsRDABC), an ABC transporter and a two-component permease, were linked to resistance to arsenic (MICs ≥ 1,600 mg/liter; 14% [58/422]; P < 0.05). At least three cad genes (cadA or cadC and cadD-cadX or czrC) and genes encoding multidrug efflux pumps and hyperosmoregulation in acidified conditions were associated with resistance to cadmium (MICs ≥ 200 mg/liter; 20% [85/422]; P < 0.05). These resistance genes were frequently carried by mobile genetic elements. Resistance to arsenic and cadmium were linked to human infection and a clonal lineage originating in animals (P < 0.05). Altogether, S. saprophyticus was highly resistant to heavy metals and accumulated multiple metal resistance determinants. The highest arsenic and cadmium resistance levels were associated with infection, suggesting resistance to these metals is relevant for S. saprophyticus pathogenicity.

Foodborne Origin and Local and Global Spread of Staphylococcus saprophyticus Causing Human Urinary Tract Infections.

Staphylococcus saprophyticus is a primary cause of community-acquired urinary tract infections (UTIs) in young women. S. saprophyticus colonizes humans and animals but basic features of its molecular epidemiology are undetermined. We conducted a phylogenomic analysis of 321 S. saprophyticus isolates collected from human UTIs worldwide during 1997-2017 and 232 isolates from human UTIs and the pig-processing chain in a confined region during 2016-2017. We found epidemiologic and genomic evidence that the meat-production chain is a major source of S. saprophyticus causing human UTIs; human microbiota is another possible origin. Pathogenic S. saprophyticus belonged to 2 lineages with distinctive genetic features that are globally and locally disseminated. Pangenome-wide approaches identified a strong association between pathogenicity and antimicrobial resistance, phages, platelet binding proteins, and an increased recombination rate. Our study provides insight into the origin, transmission, and population structure of pathogenic S. saprophyticus and identifies putative new virulence factors.

Confronting Ceftolozane-Tazobactam Susceptibility in Multidrug-Resistant Enterobacterales Isolates and Whole-Genome Sequencing Results (STEP Study).

Ceftolozane-tazobactam (C/T) is frequently used for infections caused by multidrug-resistant (MDR)-Enterobacterales isolates. Whole-genome sequencing (WGS, Illumina-Hiseq 4000/NovaSeq 6000, OGC, UK) was used to study the population structure, the resistome and the virulome of C/T-susceptible and -resistant MDR Escherichia spp. (n=30) and Klebsiella spp. (n=78) isolates, recovered from lower respiratory, intra-abdominal and urinary tract infections of ICU patients from 11 Portuguese Hospitals (STEP study, 2017-2018). Minimum inhibitory concentrations (MICs) were determined (ISO-broth microdilution, breakpoints EUCAST-2020). In Escherichia spp., a weak concordance between the phenotypic and the WGS method (P=0.051) was observed in the carbapenemase detection (3/30) [blaVIM-2 (2/3), blaKPC-3 (1/3)]; VIM-2-Escherichia coli isolates were C/T-susceptible and only the KPC-3-Escherichia marmotae producer showed C/T-resistance. Overall, CTX-M-15-E. coli-ST131-O25:H4-H30-Rx (11/30) was the most frequent subclone, followed by CTX-M-27-E. coli-ST131-O25:H4-H30 (4/4). Moreover, a wide resistome and virulome were detected in all E. coli isolates. Among Klebsiella spp. isolates [K. pneumoniae (67/78), K. aerogenes (7/78), K. oxytoca (2/78), K. variicola (2/78)], concordance (P<0.001) was observed between the phenotypic and the genomic carbapenemase detection (21/78) [blaKPC-3 (14/21), blaOXA-48 (3/21), blaOXA-181 (3/21)]. A high correlation between C/T-resistance and carbapenemase detection was established (P<0.05). Overall, a high clonal diversity was observed, mainly in KPC-3-producing K. pneumoniae isolates. An extensive resistome was detected in Klebsiella spp. isolates, whereas virulence determinants were mostly identified in carbapenemase producers (P<0.001). WGS is a powerful tool for typing characterization and microbiological study of MDR-Enterobacterales pathogens. Furthermore, carbapenemase genes are associated with C/T-resistance in Klebsiella spp., but other mechanisms might also be involved.

Distinct epidemiology and resistance mechanisms affecting ceftolozane/tazobactam in Pseudomonas aeruginosa isolates recovered from ICU patients in Spain and Portugal depicted by WGS.

OBJECTIVES: To analyse the epidemiology, the resistome and the virulome of ceftolozane/tazobactam-susceptible or -resistant Pseudomonas aeruginosa clinical isolates recovered from surveillance studies in Portugal (STEP, 2017-18) and Spain (SUPERIOR, 2016-17). METHODS: P. aeruginosa isolates were recovered from intra-abdominal, urinary tract and lower respiratory tract infections in ICU patients admitted to 11 Portuguese and 8 Spanish hospitals. MICs were determined (ISO-standard broth microdilution, EUCAST 2020 breakpoints). A subset of 28 ceftolozane/tazobactam-resistant P. aeruginosa isolates were analysed and compared with 28 ceftolozane/tazobactam-susceptible P. aeruginosa strains by WGS. RESULTS: Clonal complex (CC) 235 (27%) and CC175 (18%) were the most frequent, followed by CC244 (13%), CC348 (9%), CC253 (5%) and CC309 (5%). Inter-hospital clonal dissemination was observed, limited to a geographical region (CC235, CC244, CC348 and CC253 in Portugal and CC175 and CC309 in Spain). Carbapenemases were detected in 25 isolates (45%): GES-13 (13/25); VIM type (10/25) [VIM-2 (4/10), VIM-20 (3/10), VIM-1 (2/10) and VIM-36 (1/10)]; and KPC-3 (2/25). GES-13-CC235 (13/15) and VIM type-CC175 (5/10) associations were observed. Interestingly, KPC-3 and VIM-36 producers showed ceftolozane/tazobactam-susceptible phenotypes. However, ceftolozane/tazobactam resistance was significantly associated with GES-13 and VIM-type carbapenemase production. Six non-carbapenemase producers also displayed ceftolozane/tazobactam resistance, three of them showing known ceftolozane/tazobactam resistance-associated mutations in the PBP3 gene, ftsI (R504C and F533L). Overall, an extensive virulome was identified in all P. aeruginosa isolates, particularly in carbapenemase-producing strains. CONCLUSIONS: GES-13-CC235 and VIM type-CC175 were the most frequent MDR/XDR P. aeruginosa clones causing infections in Portuguese and Spanish ICU patients, respectively. Ceftolozane/tazobactam resistance was mainly due to carbapenemase production, although mutations in PBP-encoding genes may additionally be involved.

Response of Mycorrhizal 'Touriga Nacional' Variety Grapevines to High Temperatures Measured by Calorespirometry and Near-Infrared Spectroscopy.

Heat stress negatively affects several physiological and biochemical processes in grapevine plants. In this work, two new methods, calorespirometry, which has been used to determine temperature adaptation in plants, and near-infrared (NIR) spectroscopy, which has been used to determine several grapevine-related traits and to discriminate among varieties, were tested to evaluate grapevine response to high temperatures. 'Touriga Nacional' variety grapevines, inoculated or not with Rhizoglomus irregulare or Funneliformis mosseae, were used in this study. Calorespirometric parameters and NIR spectra, as well as other parameters commonly used to assess heat injury in plants, were measured before and after high temperature exposure. Growth rate and substrate carbon conversion efficiency, calculated from calorespirometric measurements, and stomatal conductance, were the most sensitive parameters for discriminating among high temperature responses of control and inoculated grapevines. The results revealed that, although this vine variety can adapt its physiology to temperatures up to 40 °C, inoculation with R. irregulare could additionally help to sustain its growth, especially after heat shocks. Therefore, the combination of calorespirometry together with gas exchange measurements is a promising strategy for screening grapevine heat tolerance under controlled conditions and has high potential to be implemented in initial phases of plant breeding programs.