Risk of chronic kidney disease in 260 patients with lupus nephritis-analysis of a nationwide multicentre cohort with up to 35 years of follow-up.

OBJECTIVES: To compare proliferative (PLN) and membranous (MLN) lupus nephritis (LN) regarding clinical and laboratory presentation and long-term outcomes; To investigate predictors of progression to chronic kidney disease (CKD). METHODS: Multicentre observational study, with retrospective analysis of a prospective cohort, using data from the Rheumatic Diseases Portuguese Registry-Reuma.pt. Patients with biopsy-proven PLN, MLN and mixed LN were included. Cox regression survival analysis was used to investigate predictors of CKD. RESULTS: 260 patients were included. Median follow-up was 8 years (IQR 11; minimum 1, maximum 35 years). MLN patients presented with significantly lower serum creatinine (0.70 (IQR 0.20; minimum 0.50, maximum 1.30) mg/dl vs 0.80 (IQR 0.31; minimum 0.26, maximum 2.60) in PLN, p= 0.003). Proteinuria levels did not differ between groups (p= 0.641). Levels of complement were reduced in PLN but nearly normal in MLN patients, and there were fewer patients with positive anti-dsDNA antibodies in the MLN group (p< 0.001). One year after the beginning of treatment, 62% of the patients achieved EULAR/ERA-EDTA complete response, with further 5% achieving partial response. Patients with lower proteinuria at diagnosis were more likely to achieve a complete renal response at one year, however, proteinuria at diagnosis or at one year did not predict long term CKD. Estimated glomerular filtration rate (eGFR) ≤75 mL/min/1.73 m2 at one year was the strongest predictor of progression to CKD (HR 23 [95% CI 8-62], p< 0.001). Other possible predictors included the use of azathioprine for induction of remission, older age at diagnosis and male sex. CONCLUSION: Proteinuria levels did not predict LN histologic class in our cohort. eGFR cutoff of 75 mL/min/1.73 m2 after one year of treatment was strongly predictive of progression to CKD.

Blastocystis occurrence and subtype diversity in European wild boar (Sus scrofa) from the Iberian Peninsula.

The ongoing increase in wild boar populations across Europe has fostered human-wildlife conflicts, including the transmission of emerging pathogens with zoonotic importance. Blastocystis is a ubiquitous, faecal-oral transmitted protist that can cause gastrointestinal illnesses and is observed in humans and animals worldwide. The role of wildlife in the epidemiology of Blastocystis is insufficiently understood. Thus, we investigated the occurrence and subtype diversity of Blastocystis in free-ranging wild boars from the Iberian Peninsula using conventional PCR and next-generation amplicon sequencing of a fragment of the ssu RNA gene. A total of 459 wild boar faecal samples were collected across Spain (n = 360) and Portugal (n = 99) between 2014 and 2021. Blastocystis was present in 15.3% (70/459; 95% CI 12.1-18.9) of the wild boars analysed, and its occurrence was significantly higher in Portugal (34.3%, 34/99; 95% CI 25.1-44.6) than in Spain (10.0%, 36/360; 95% CI 7.1-13.6). Seven Blastocystis subtypes (ST5, ST10b, ST13-ST15, ST24b, and ST43) were detected among the surveyed wild boar populations, with greater variability detected in Portuguese samples. ST5 was identified in all the Blastocystis-positive animals, whereas 14.3% of them harboured ST mixed colonisations. Our results demonstrate that Blastocystis ST5 is particularly adapted to infect wild boars. The additional identification of zoonotic STs reinforces the role of wild boars as spreaders of zoonotic infections with public health significance.

Occurrence and molecular characterization of Enterocytozoon bieneusi in wild and domestic animal species in Portugal.

The phylum Microsporidia encompasses a diverse group of obligate, intracellular, and spore-forming organisms able to infect a wide range of animal hosts. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and animals. Little is known about the presence and epidemiology of E. bieneusi in wildlife. We investigated E. bieneusi occurrence and genetic diversity in wild and domestic mammals, through molecular-detection methods, from different regions across Portugal. A total of 756 samples were collected from 288, 242, and 226 wild carnivores, wild ungulates, and domestic animals, respectively. Overall, eight specimens were E. bieneusi-positive (1.1%, 8/756) obtained from five wild (Iberian lynx, Iberian wolf, red fox, stone marten, and wild boar) and one domestic (sheep) host. Nucleotide sequence analysis identified four genotypes of E. bieneusi, Type IV, Wildboar3, BEB6, and PtEbIX. Three of those genotypes belong to Groups 1 (Type IV and Wildboar3) and 2 (BEB6), which are known to contain genotypes capable of infecting a variety of hosts, including humans, highlighting their public health importance. PtEbIX belongs to the dog-specific Group 11. This study represents the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal in wild and domestic animals to date and the first worldwide identification of E. bieneusi in wolf species. Our study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals.

The identification of human-pathogenic genotypes of fungi-related Enterocytozoon bieneusi in wild carnivores and ungulates in Portugal suggests cross-species infection events and overlapping of the sylvatic and domestic transmission cycles, demonstrating a potential transmission risk to humans.

Endoparasites of the Iberian wolf (Canis lupus signatus) and mesocarnivores in Central Portugal.

At the end of the nineteenth century, massive population declines were observed in carnivores due to the emergence of infectious diseases. This study aims to investigate, by means of coprological analysis, the prevalence and intensity of the parasites that infect the endangered Iberian wolf Canis lupus signatus and two mesocarnivores (the red fox Vulpes vulpes and the stone marten Martes foina) in Central Portugal. In total, 67.2% of the samples screened were infected; Toxascaris leonina (40.6%) was the parasite with the highest prevalence, followed by Ancylostomatidae and Eimeria spp. (28.1%). Eimeria spp. was found in stone marten with the highest infection rate (37,800 OPG), followed by T. leonina (10,100 EPG) in a red fox sample. Moderate to high levels of parasitic infections were identified in 73.3% of red foxes from the western area. Our results highlight the possibility of cross-infection among these carnivore species and cross-contamination in the wildlife-livestock-human interface.

Muscle dysfunction in axial spondylarthritis: the MyoSpA study.

OBJECTIVES: We aimed to investigate muscle physical properties, strength, mass, physical performance, and the prevalence of sarcopenia in patients with axial spondylarthritis (axSpA) compared to the healthy controls (HC). METHODS: We performed a cross-sectional study on 54 participants: 27 patients with axSpA and 27 HC, matched by age, gender, and level of physical activity. Muscle physical properties (stiffness, tone and elasticity), muscle strength (five-times sit-to-stand [5STS] test), muscle mass, physical performance (measured through gait speed) and sarcopenia were compared between the groups. Linear regression models were conducted allowing adjustment for relevant variables. RESULTS: Patients with axSpA (mean age 36.5 (SD 7.5) years, 67% males, mean disease duration 6.5 (3.2) years) had no significant difference in segmental muscle stiffness, tone or elasticity, compared with the HC, despite showing a slight numerically higher lower lumbar (L3-L4) stiffness [median 246.5 (IQR 230.5-286.5) vs. 232.5 (211.0-293.5), p=0.38]. No participants presented sarcopenia. Patients with axSpA, compared to the HC, had lower total strength [B=1.88 (95% CI 0.43;3.33)], as well as lower strength in the upper (B=-17.02 (-27.33;-6.70)] and lower limbs [B=-11.14 (-18.25;-4.04)], independently of muscle physical properties. Patients had also significantly lower gait speed than the HC [B=-0.11 (-0.21;-0.01)], adjusted for muscle mass, strength and muscle physical properties. CONCLUSIONS: Young axSpA patients with a relatively short disease duration presented similar segmental muscle physical properties as the HC and had no sarcopenia. Patients with axSpA had reduced physical performance and lower strength compared to the HC, despite normal muscle mass, suggesting a possible muscle dysfunction. Gait characteristics may be a potential biomarker of interest in axSpA.

Drug-Cannabinoid Interactions in Selected Therapeutics for Symptoms Associated with Epilepsy, Autism Spectrum Disorder, Cancer, Multiple Sclerosis, and Pain.

Clinical practice entails a translation of research that assists in the use of scientific data and therapeutic evidence for the benefit of the patient. This review critically summarizes the potential impact of cannabinoids in conjunction with other drugs when associated with treatments for epilepsy, autism spectrum disorder, cancer, multiple sclerosis, and chronic pain. In these associations, potential drug interactions may occur and alter the predicted clinical results. Therefore, the potential for drug interactions must always be assessed to avoid therapeutic failures and/or increased side effects. Some effects may be additive, synergistic, or antagonistic, but changes in absorption, distribution, metabolism, particularly through cytochrome P450 (CYP) isoenzymes (e.g., CYP2C9 and CYP3A4), and excretion may also occur. For example, the combination of cannabis-derived compounds and the antifungal drug ketoconazole, a CYP3A4 inhibitor, increases the plasma concentration of Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). In contrast, rifampicin, a CYP3A4 inducer, stands out for reducing plasma THC levels by approximately 20-40% and 50% to 60% for CBD. Other CYP3A4 inhibitors and inducers are likely to have a similar effect on plasma concentrations if co-administered. Pharmacokinetic interactions with anticonvulsant medications have also been reported, as have pharmacodynamic interactions between cannabinoids and medications with sympathomimetic effects (e.g., tachycardia, hypertension), central nervous system depressants (e.g., drowsiness, ataxia), and anticholinergics (e.g., tachycardia and somnolence). Although further studies are still pending, there is currently clinical evidence supporting drug interactions with cannabinoids, requiring doctors to evaluate the risk of drug combinations with cannabinoids and vice versa. The tables provided here were designed to facilitate the identification of biorelevant interactions that may compromise therapeutic efficacy and toxicity.

Molecular detection and characterization of Blastocystis in herbivore livestock species in Portugal.

Blastocystis is a ubiquitous intestinal protist in humans and animals worldwide. The traditional livestock free-roaming raising system in rural communities increases the risk of infection with contact with a wider range of pathogens transmitted via the faecal-oral route associated with that wildlife-livestock-human interface. However, no studies have been conducted to determine the occurrence and subtype distribution of Blastocystis in livestock in Portugal. Here, we collected 180 faecal samples from herbivore livestock (cattle, goats, horses, and sheep) in different regions of the country to investigate Blastocystis prevalence and subtype diversity using PCR and next-generation amplicon sequencing. Blastocystis was present in 40.6% (73/180; 95% CI: 33.31-48.11) of the samples (goats, 81.0%; sheep, 60.9%; cattle, 32.2%). None of the horse samples were Blastocystis-positive. Eighteen subtypes were detected (ST1-ST3, ST5-ST7, ST10, ST13, ST14, ST21, ST23-ST26, ST30, ST42-ST44). Mixed infections were detected in 97.3% of the Blastocystis-positive samples. Potentially zoonotic subtypes were identified in 75.0%, 96.4%, and 100% of the Blastocystis-positive specimens collected from cattle, sheep, and goats, respectively. These results demonstrate that cattle, sheep, and goats harbour a high diversity of Blastocystis subtypes in the study regions. Importantly, our data provide novel molecular evidence strongly suggesting that some Blastocystis STs/ST subgroups may have differential host specificity.

Effect of Environmental and Anthropic Conditions on the Development of Solanum peruvianum: A Case of the Coastal Lomas, Lima-Peru.

Land degradation and the effects of climate change are increasing arid lands, accelerating desertification, and leading to the loss of ecosystem services worldwide. This research focused on evaluating how human impact and environmental factors affect the development of Solanum peruvianum in its natural habitat of coastal lomas. The study was carried out in the coastal lomas of Mangomarca-Peru, where phenotypic and ecological data on the plants were collected. Information was also gathered on human impacts on the nutritional characteristics of the soils. Then, five types of organic amendments were used to improve the physical and chemical characteristics of the degraded soil, and the development and photosynthetic activity of S. peruvianum were evaluated. As a result, under the study conditions, it was found that S. peruvianum was established approximately 33.74 cm from the rocks, in a range of 300 to 650 m asl. The maximum height of the plants was 90 cm, with a stem diameter at ground level of 2 cm. S. peruvianum produced fruits between January and July, with a seed germination rate of 36% in 25 days. On the other hand, the anthropogenic impact on the soil reduced 58% of organic material (OM), 71% of nitrogen, 40% of P2O5, and 13% of K2O and increased the concentration of magnesium oxide, calcium oxide, pH, and electric conductivity (EC). The organic amendments bokashi, compost, and biochar, when mixed with the degraded soil, increased the pH, OM, N, P, and EC; however, the plants died after 25 days. On the other hand, the application of the Premix5 substrate for 100 days favored the growth of 52.84 cm and 38.29 cm in the preserved soil and 23.21 cm in the black soil mixed with blond peat, and it should be noted that the substrates presented an acid pH and EC > 0.1. Regarding photosynthetic phenotyping, S. peruvianum plants grown in their natural habitat and in Premix5 showed a higher proton flux (vH+), linear electron flow (LEF), and maximum quantum yield (Fv'/Fm'). On the contrary, they showed a lower NPQt value than plants grown in preserved and black soil mixed with blond peat.

Experimental approach and analysis of the effectiveness of a tubular helical flow flocculator for water supply in developing communities.

The main objective of this study was to evaluate the impact of the length and retention time of a tubular helical flow flocculator (THFF) on the elimination of turbidity and color from raw water, to obtain quality treated water for consumption in areas rural. For this, a large-scale field experimental system was used, the THFF was built with 4-inch diameter polyethylene hose and coupled to a sedimentation and filtration process. For the different experimental tests, aluminum sulfate was chosen as the coagulant. To find the optimal dose of coagulant, jar tests were previously carried out. For the tests the length of the THFF was varied (50 m and 75 m), flow rates of 0.25, 0.5, 0.75, 1 and 2 L/s and turbidity ranges of <10, 10-20, 21-50, 51-100 and > 100 NTU of raw water were tested. An evaluation of the hydraulic behavior of the THFF was carried out through an analysis of the temporal distribution curve of the concentration of a tracer, applying the Wolf-Resnick model. The average results revealed a haze and color removal efficiency of 98.07 % and 98.50 %, respectively. The residence time and velocity gradient exhibited variations in a range of 2.25-35.0 min and 3.64 to 56.94 s-1, respectively. It was evident that the operation and effectiveness of THFF are directly influenced by the turbidity of the raw water, the residence time and the velocity gradient. These findings indicate that THFF could play a valuable role as a flocculation unit in a purification system, mainly the existence of a plug-type flow was observed. The findings indicate that THFF, complemented by settling and filtration processes, could be a valuable tool for implementation in rural areas.

An exploratory analysis of PM 2.5 /PM 10 ratio during 2016-2018 in Metropolitan Lima.

Aerosols (PM 2.5 and PM 10 ) represent one of the most critical pollutants due to their negative effects on human health. This research analyzed the relationship of PM and its PM 2.5 /PM 10 ratios with climatic variables in the austral spring (2016-2018) in Metropolitan Lima. Overall, there was an average PM 2.5 /PM 10 ratio of 0.33 with fluctuations from 0.30 to 0.35. However, there have also been high point values that reached ratios greater than one. This situation indicates a moderate condition of contamination by particulate matter with a predominance of coarse aerosols in spring, with an increasing trend over the years. The locations Ate and Villa Maria del Triunfo, especially Ate, presented poor quality conditions. Thursdays showed outstanding pollution peaks by PM 10 , and a decrease is visible on Sundays. On the other hand, the PM 2.5 showed a similar pattern every day, including Sundays. The maximum peaks occurred in the morning and night hours. The increase in anthropogenic emissions associated with the formation of secondary aerosols has been evident, being the case of the location Campo de Marte, the one that had a significant increase in ratios PM 2.5 /PM 10 , which confirms a greater intensity of secondary formations of carbonaceous particles from industrial oil sources, vehicle exhaust, as well as aerosols from metal smelting and biomass burning. There were negative correlations of the ratios with PM 10 , temperature, wind speed, and direction, and positive correlations with PM 2.5 and relative humidity. Contour lines were successfully developed that demonstrated the interaction of climate with PM 2.5 /PM 10 ratios. This will deepen the exploration of emission sources and modeling, which allows for optimizing air quality indices to control emissions and adequately manage air quality in Metropolitan Lima.

Survey of Zoonotic Diarrheagenic Protist and Hepatitis E Virus in Wild Boar (Sus scrofa) of Portugal.

Enteropathogenic parasites and viruses have been frequently reported in swine and can infect a wide range of mammals, including humans. Among the wide variety of parasites infecting swine, diarrhoeagenic protists are among those that cause significant morbidity. Hepatitis E virus (HEV) has also been reported both in domestic pigs and wild boar and is known to have an important public health significance. These agents share the fecal−oral transmission route, but data on their fecal shedding and circulation pathways are still lacking or incomplete. Hence, the aim of the present study was to characterize the presence of microeukaryotes and HEV in the wild boar of Portugal. Wild boar stool samples (n = 144) were obtained during the official hunting seasons (October to February) in 2018/2019, 2019/2020, and 2021/2022 and tested for Cryptosporidium spp., Balantioides coli, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi and HEV by molecular assays, followed by sequencing and phylogenetic analysis. We have detected Cryptosporidium scrofarum (1.4%, 95% CI: 0.2−4.9), B. coli (14.6%, 95% CI: 9.2−21.4), Blastocystis ST5 (29.2%, 95% CI: 21.9−37.2) and HEV genotype 3 (2.8%, 95% CI: 0.7−6.9; subgenotypes 3e and 3m). Co-infections were observed in thirteen animals where two were positive for both HEV and B. coli, one was positive for both C. scrofarum and Blastocystis ST5, and ten were positive for both B. coli and Blastocystis ST5. Giardia duodenalis and E. bieneusi were not detected in the surveyed wild boar population. As far as we know, this is the first report describing protist infections by Cryptosporidium spp., B. coli, and Blastocystis sp., as well as the first identification of the emerging HEV genotype 3m in wild boar of Portugal. The present work shows that potentially zoonotic protozoa and HEV are circulating in wild boar populations in Portugal. Awareness and epidemic-surveillance network implementation measures targeting wild boar are needed to prevent the spread of these pathogenic agents to humans.

Children with extended oligoarticular and polyarticular juvenile idiopathic arthritis have alterations in B and T follicular cell subsets in peripheral blood and a cytokine profile sustaining B cell activation.

OBJECTIVES: The main goal of this study was to characterise the frequency and phenotype of B, T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in peripheral blood and the cytokine environment present in circulation in children with extended oligoarticular juvenile idiopathic arthritis (extended oligo JIA) and polyarticular JIA (poly JIA) when compared with healthy controls, children with persistent oligoarticular JIA (persistent oligo JIA) and adult JIA patients. METHODS: Blood samples were collected from 105 JIA patients (children and adults) and 50 age-matched healthy individuals. The frequency and phenotype of B, Tfh and Tfr cells were evaluated by flow cytometry. Serum levels of APRIL, BAFF, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-17A, IL-21, IL-22, IFN-γ, PD-1, PD-L1, sCD40L, CXCL13 and TNF were measured by multiplex bead-based immunoassay and/or ELISA in all groups included. RESULTS: The frequency of B, Tfh and Tfr cells was similar between JIA patients and controls. Children with extended oligo JIA and poly JIA, but not persistent oligo JIA, had significantly lower frequencies of plasmablasts, regulatory T cells and higher levels of Th17-like Tfh cells in circulation when compared with controls. Furthermore, APRIL, BAFF, IL-6 and IL-17A serum levels were significantly higher in paediatric extended oligo JIA and poly JIA patients when compared with controls. These immunological alterations were not found in adult JIA patients in comparison to controls. CONCLUSIONS: Our results suggest a potential role and/or activation profile of B and Th17-like Tfh cells in the pathogenesis of extended oligo JIA and poly JIA, but not persistent oligo JIA.

[Renal oncocytoma in pregnancy--an unusual presentation of secondary hypertension]. / Oncocitoma renal na gravidez--uma forma invulgar de hipertensão secundária.

INTRODUCTION: Renal oncocytoma accounts for 5-7% of primary renal neoplasms. It is usually, diagnosed in asymptomatic patients and is characterized by a benign behavior without invasion of adjacent tissues or metastasis. Diagnosis during pregnancy is uncommon and to date there have been only a few cases reported in the literature. CASE REPORT: The authors present the case of a 32-year-old nulliparous woman with uncontrolled hypertension diagnosed at seven weeks gestation. She was referred to our institution at 24 weeks with superimposed pre-eclampsia complicated by acute pulmonary edema and hemodynamic instability requiring mechanical ventilatory support, fetal growth restriction and stillbirth. Etiological study of the hypertensive disorder performed in the postpartum period was consistent with renal oncocytoma. CONCLUSION: The clinical behavior of renal oncocytoma remains poorly characterized during pregnancy and may lead to an adverse maternal and fetal outcome despite its theoretically benign behavior. It is essential to exclude a possible secondary cause of hypertension in cases that are difficult to control.

Methotrexate: Implications of pharmacogenetics in the treatment of patients with Rheumatoid Arthritis.

BACKGROUND: Methotrexate (MTX) is an anti-folate drug with anti-proliferative and anti-inflammatory effects. MTX proved to be the most highly effective, fast-acting disease modifying anti-rheumatic drug (DMARD), being widely used for the treatment of rheumatoid arthritis (RA). This review aims to describe the main genetic variants identified concerning proteins that play a role in methotrexate's kinetics and efficiency profile. METHODS: A literature review was conducted since January of 2000 until December 2020, by searching the PubMed and Embase bibliographic databases, employing the following MeSH terms: methotrexate, pharmacogenetics, pharmacokinetics, and rheumatoid arthritis. The search was limited to articles in English language. Two independent reviewers screened the titles and abstracts followed by a full-text review to assess papers regarding their eligibility. A total of 48 articles matched the research criteria and were analyzed. RESULTS: Reduced folate carrier 1 (RFC1), a constitutively expressed folate transport protein that has high affinity for MTX is responsible, almost exclusively, for the transport of folate and MTX into the cell. The most studied variant of the gene is the 80G>A variant, mapped within exon 2, on chromosome 21. It seems to improve RA responses to MTX, clinical efficacy with long disease remission. ABC transporters are involved in the efflux of MTX from cells. An increased expression and function of these transporters should decrease MTX concentrations in target cells, resulting in lack of therapeutic response. ABCB1 3435 C/T is a high frequency polymorphism, significantly associated with RA good responses, symptom remission and reduced adverse events, due to MTX treatment. Thymidylate synthase (TYMS) is involved in thymidine synthesis. MTX decreases TYMS activity by inhibition and decreasing the access to tetrahydrofolate (THF) cofactors. The most common genetic variant of the TYMS gene consists of a 28 bp tandem repeat, with double and triple number of repeats (2R and 3R). The 3R allele genotype was associated with decreased efficacy and increased toxicity. The 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme is indirectly inhibited by MTX. The most common SNPs of the MTHFR gene are C677T and A1298C. Both are associated with a decreased efficacy and an increased toxicity of MTX. CONCLUSION: MTX response is affected by many gene variants; the effect of each variant separately is likely to be small. Additionally, gene-gene interaction seems to enhance the potential role of linkage disequilibrium. This shows the emerging need for a better gene characterization and to improve the knowledge about variants distribution according to ethnicity, to explain different responses to MTX at an individual level.

A walk on the wild side: Wild ungulates as potential reservoirs of multi-drug resistant bacteria and genes, including Escherichia coli harbouring CTX-M beta-lactamases.

Extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales have been classified as critical priority pathogens by the World Health Organization (WHO). ESBL are universally distributed and, in 2006, were firstly reported on a wild animal. Understanding the relative contributions of wild animals to ESBL circulation in the environment is urgently needed. In this work, we have conducted a nationwide study in Portugal to investigate the occurrence of bacteria carrying clinically significant antimicrobial resistance genes (ARG), using widely distributed wild ungulates as model species. A total of 151 antimicrobial resistant-Enterobacterales isolates were detected from 181 wild ungulates: 50% (44/88) of isolates from wild boar (Sus scrofa), 40.3% (25/62) from red deer (Cervus elaphus), 41.4% (12/29) from fallow deer (Dama dama) and 100% (2/2) from mouflon (Ovis aries subsp. musimon). Selected isolates showed a diversified resistance profile, with particularly high values corresponding to ampicillin (71.5%) and tetracycline (63.6%). Enterobacterales strains carried blaTEM, tetA, tetB, sul2, sul1 or dfrA1 ARG genes. They also carried blaCTX-M-type genes, which are prevalent in human infections, namely CTX-M-14, CTX-M-15 and CTX-M-98. Strikingly, this is the first report of CTX-M-98 in wildlife. Almost 40% (n = 59) of Enterobacterales were multi-drug resistant. The diversity of plasmids carried by ESBL isolates was remarkable, including IncF, K and P. This study highlights the potential role of wild ungulates as environmental reservoirs of CTX-M ESBL-producing E. coli and in the spill-over of AMR bacteria and their determinants. Our findings suggest that wild ungulates are useful as strategic sentinel species of AMR in terrestrial environments, especially in response to potential sources of anthropogenic pollution, providing early warning of potential risks to human, animal and environmental health.

Worldwide Disseminated IncX4 Plasmid Carrying mcr-1 Arrives to Wild Mammal in Portugal.

The mcr-1 gene spread is worldwide recognized as a public health threat at multidrug-resistant infections therapy level. Here, we report for the first time, to the best of our knowledge, the detection of the globally distributed IncX4 plasmid carrying mcr-1 (mcr-1/IncX4) in Escherichia coli isolated from a wild mammal in Portugal and Europe. This plasmid was found in a ST533 E. coli isolate with a multidrug-resistant profile, virulence potential, and possibly phylogenetically related to human isolates. Our work contributes to highlight the importance of antimicrobial resistance (AMR) surveillance in wildlife, an important compartment of the whole ecosystem often overlooked in the fight against AMR. IMPORTANCE Colistin resistance mediated by plasmids is recognized worldwide as an emergency problem connected with the whole ecosystem, since is well described in the interface of the human-animal-environment. The plasmid IncX4 is reported as one of the most prevalent plasmids harboring the gene mcr-1. On an European scale the plasmid IncX4 carrying mcr-1 has been described in humans, the environment, and animals, including wildlife, but only in wild birds. This study shows the first report of the plasmid IncX4 harboring mcr-1 in a wild mammal in Portugal and Europe, identified in a ST533 E. coli commensal that is, curiously, more related to isolates from humans than from livestock. Our findings show that the plasmid IncX4 harboring mcr-1 is well established in a colistin resistance drive embracing the whole ecosystem.

A high-risk carbapenem-resistant Pseudomonas aeruginosa clone detected in red deer (Cervus elaphus) from Portugal.

Pseudomonas aeruginosa is a ubiquitous bacterium, successfully exploiting a variety of environmental niches due to its remarkable metabolic versatility. The World Health Organization classifies P. aeruginosa as a "priority pathogen" due to its a great ability to overcome the action of antimicrobials, including carbapenems. Hitherto, most studies have focused on clinical settings from humans, but much less on animal and environmental settings, particularly on wildlife. In this work, we report the isolation of a carbapenem-resistant Pseudomonas aeruginosa strain recovered from the faeces of a red deer adult female sampled in a humanized area. This isolate was obtained during a nationwide survey on antimicrobial resistance in wildlife aimed to determine the occurrence of carbapenem-resistant bacteria among 181 widely distributed wild ungulates. This P. aeruginosa isolate was found to be a high-risk clone, belonging to the sequence type (ST) 274. The genomic analysis of P. aeruginosa isolate UP4, classified this isolate as belonging to serogroup O3, which was also found to harbour the genes blaPAO, blaPDC-24, blaOXA-486 (encoding resistance to beta-lactams), aph(3')-IIb (aminoglycosides resistance), fosA (fosfomycin resistance) and catB7 (chloramphenicol resistance). Antimicrobial susceptibility screening, according to EUCAST, showed resistance to imipenem and intermediate resistance to meropenem and doripenem. To our knowledge, this is the first description of carbapenem-resistant P. aeruginosa in deer in Europe. Our results highlight the importance of wild ungulates either as victims of human activity or amplifiers of AMR, either way with potential impacts on animal, human and ecosystem health, since excretion of AMR bacteria might directly or indirectly contaminate other animals and the surrounding environment, perpetuating the spill-over and chain dissemination of AMR determinants.

Antimicrobial resistance in commensal Staphylococcus aureus from wild ungulates is driven by agricultural land cover and livestock farming.

Staphylococcus aureus is a human pathobiont (i.e., a commensal microorganism that is potentially pathogenic under certain conditions), a nosocomial pathogen and a leading cause of morbidity and mortality in humans. S. aureus is also a commensal and pathogen of companion animals and livestock. The dissemination of antimicrobial resistant (AMR) S. aureus, particularly methicillin-resistant (MRSA), has been associated to its ability for establishing new reservoirs, but limited attention has been devoted to the role of the environment. To fill this gap, we aimed to characterize animal carrier status, AMR phenotypes, predominant clonal lineages and their relationship with clinical and food-chain settings, as well as to find predictors of AMR occurrence. Nasal swabs (n = 254) from wild boar (n = 177), red deer (n = 54) and fallow deer (n = 23) hunted in Portugal, during the season 2019/2020, yielded an overall carrier proportion of 35.8%, ranging from 53.7% for red deer and 32.2% for wild boar to 21.7% for fallow deer. MRSA from wild boar and phenotypically linezolid-resistant S. aureus from wild boar and red deer were isolated, indicating that resistance to antimicrobials restricted to clinical practice also occurs in wildlife. The most prevalent genotypes were t11502/ST2678 (29.6%) and t12939/ST2678 (9.4%), previously reported in wild boar from Spain. Clonal lineages reported in humans and livestock, like CC1, CC5 or CC8 (19.1%) and ST425, CC133 or CC398 (23.5%), respectively, were also found. The sequence type ST544, previously restricted to humans, is described in wildlife for the first time. We also identified that land use (agricultural land cover), human driven disturbance (swine abundance) and host-related factors (sex) determine resistance occurrence. These findings suggest that antibiotics used in clinical settings, agriculture and livestock farming, spill over to wildlife, leading to AMR emergence, with potential biological, ecological, and human health effects. This work is one of the most comprehensive surveys in Europe of S. aureus occurrence and determinants among widely distributed wild ungulates.

Balantioides coli Fecal Excretion in Hunted Wild Cervids (Cervus elaphus and Dama dama) from Portugal.

Balantioides coli is a zoonotic enteric protozoan parasite of public veterinary health relevance and a concern in animal production and food safety. While wild cervids are recognized reservoirs for several zoonotic pathogens, little is known about the occurrence of B. coli in deer species, especially in Europe. To fill this gap, a total of 130 fecal samples from legally hunted red deer (Cervus elaphus, n = 95) and fallow deer (Dama dama, n = 35) were passively collected during two hunting seasons (October to February; 2018-2019 and 2019-2020) in Portugal. After assessment by PCR assay targeting the complete ITS1-5.8s-rRNA-ITS2 region and the 3' end of the ssu-rRNA gene of the parasite, a prevalence of 4.2% (4/95, 95% CI: 0.2-8.3) in red deer and of 5.7% (2/35, 95% CI: 0.0-13.4) in fallow deer was found. Sequence and phylogenetic analyses allowed the identification of B. coli genetic variants A (in two red deer) and B (in two red deer and two fallow deer). This is the first molecular-based description of B. coli in European deer species, whose population have increased in density and geographical range in recent years. Continued monitoring of wild ungulates as potential vectors of parasitic infection diseases of zoonotic nature is crucial to safeguard public health and food safety.