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.

Contamination of a Water Stream and Water Drainage Reaching Matosinhos Beach by Antibiotic-Resistant Bacteria.

Antibiotic-resistant bacteria represent a major public health concern, especially impacting medical care centers and hospitals, thereby challenging the effectiveness of current infection treatment protocols. The emergence and persistence of antimicrobial resistance in the environment have been thoroughly researched, with a focus on the aquatic environment as a potential reservoir of these bacteria in areas with anthropogenic contamination. Having this in mind, this work aims to investigate the water streams of Riguinha and Brito Capelo Street, both of which ultimately flow into Matosinhos Beach in Portugal, to determine the potential presence of fecal contamination. Six water samples were collected and analyzed within twenty-four hours from these two water streams. A phenotypic characterization was performed in various volumes on MacConkey agar with antibiotics. Randomly selected lactose-fermenting gram-negative bacteria underwent antimicrobial susceptibility tests using the agar diffusion method following EUCAST guidelines, covering ß-lactam and non-ß-lactam antibiotics. The isolates were analyzed through Polymerase Chain Reaction. The findings of this study confirm that both water streams were contaminated by multidrug-resistant bacteria such as Enterobacteriaceae, including Escherichia coli, the KESC group, and Pseudomonas, exhibiting extended-spectrum ß-lactamases (ESBL), AmpC ß-lactamases, and carbapenemases. These indicate the presence of fecal contamination with relevant antimicrobial-resistant threats.

Occurrence and Phylogenetic Analysis of Zoonotic Enteropathogenic Protist Parasites in Asymptomatic Domestic Ruminants from Portugal.

Enteropathogenic parasites are of significant concern for public health due to their zoonotic potential and their impact on human and animal health. In this study, we investigated their occurrence and characterized these enteropathogens in asymptomatic domestic ruminants from Portugal. A total of 302 stool samples were collected from cattle (n = 166), sheep (n = 73), and goats (n = 63) in various regions of Portugal and tested for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Blastocystis sp., and Balantioides coli by PCR. The occurrence of Cryptosporidium spp. was found to be 12.7% (8/63, 95% confidence interval [CI]: 5.65-23.5) in goats; however, no sample was found to be positive for Cryptosporidium spp. in cattle and sheep. For E. bieneusi, 6.35% (4/63; 95%CI: 1.76-15.47) of goats were found to be positive; however, no cattle or sheep were found to be positive. Blastocystis sp. was found in sheep (9.59%; 7/73; 95% [CI]: 0.394-18.76) and goats (12.70%; 8/63; 95% [CI]: 5.65-23.50) but none was found in cattle. No positive results for G. duodenalis or B. coli were detected in this study. This study provides essential baseline information for understanding the silent shedding and epidemiology of these enteropathogens in Portugal, contributing to overall livestock health and related occupational safety. Raising awareness among consumers, veterinarians, and farm owners is crucial to minimize the risk of transmission and promote effective disease control strategies.

Effect of Different Time/Temperature Binomials on the Chemical Features, Antioxidant Activity, and Natural Microbial Load of Olive Pomace Paste.

Olive pomace is a by-product from olive oil production that can be further processed to obtain olive pomace paste. In this work, the influence of different time/temperature binomials (65 °C/30 min; 77 °C/1 min; 88 °C/15 s; and 120 °C/20 min) on the nutritional quality, chemical composition, and efficiency on control/elimination of natural microbial load of olive pomace paste was ascertained. The treatments significantly impacted the contents of ash, fat, vitamin E, phenolics (including hydroxytyrosol), flavonoids, and antioxidant activity, but not the fatty acids profile. The binomial 88 °C/15 s showed the greatest potential since it better preserved the phytochemical and antioxidant properties as well as the protein and fiber contents. This binomial is also faster and easy to be implemented at an industrial level, allowing the obtention of a safe functional ingredient to satisfy consumers' demands for novel sustainable products, simultaneously, responding to food safety and food security concerns.

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.

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.

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.

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.

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.

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.

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.

Epidemiology of Porcine Circovirus Type 2 Circulating in Wild Boars of Portugal during the 2018-2020 Hunting Seasons Suggests the Emergence of Genotype 2d.

Porcine circovirus type 2 (PCV-2) is associated with several syndromes affecting swine, also known as porcine-circovirus-associated diseases, of which post-weaning multi-systemic wasting syndrome stands out due to its high economic impact on swine production. Recent data suggest the increasing circulation of the PCV-2d genotype in several countries worldwide. To provide updated data on PCV-2 genotypes currently circulating in swine in Portugal, we screened wild boar stools collected from several districts across Portugal, during the 2018-2020 hunting seasons, for PCV-2 and genetically characterized detected strains. From a total of 76 stool samples of wild boar tested by PCR for the partial PCV-2 ORF2 gene, two sequences were obtained (2/76; 2.6%, 95% confidence interval: 0.032-9.18). Bidirectional sequencing showed that the sequences were 100% identical and both of the PCV-2d genotype, showing for the first time the presence of this genotype in Portugal. Monitoring wild PCV-2 reservoirs is important for both veterinary public health and economic reasons, since PCV-2 infection has a strong economic impact on the swine industry.

The role of spray-drying atmosphere on fridericia chica (bonpl.) L.G. Lohmann standardized extract production for wound healing activity.

Fridericia chica (Bonpl.) L.G. Lohmann (synonym Arrabidaea chica Verlot) is widely used in Brazilian folk medicine. Considering overcoming pitfalls of scaling up production of plant extracts, herein the effects of N2 atmosphere for extract spray-drying process is reported. Samples were monitored by in vitro antioxidant activity and microbiological evaluation. The drying atmosphere influenced 3-deoxyanthocyanines content when using air as atomizing gas, decreasing carajurin (37.5%) content with concomitant increase in luteolin yield (24.1%). Both drying processes preserved the pharmacological activity. In the cell migration test with HaCaT cells, the extract dried under air flow (5 µg/mL) promoted wound closure by 78% (12 hours) whereas the extract dried using N2 flow promoted 49% (12 hours), with 98% closure (12 hours) for the positive control. The antimicrobial evaluation for Staphylococcus aureus did not differ within drying atmospheres, with MIC (minimum inhibitory concentration) at 0.39 mg/mL. Therefore, the drying process reported herein did not interfere with the biological activity's outcome.

KPC-Producing Enterobacterales from Douro River, Portugal-Persistent Environmental Contamination by Putative Healthcare Settings.

Carbapenemase-producing Enterobacterales (CPE) are a growing concern, representing a major public health threat to humans, especially in healthcare settings. In the present study, we evaluated the persistent contamination by carbapenem-resistant Enterobacterales in water from Douro River, Portugal. KPC-producing Enterobacterales were detected in five water samples separated chronologically by 15 days each. Susceptibility testing was performed by disk-diffusion-method according to Clinical and Laboratory Standards Institute (CLSI), phenotypic carbapenemase activity was evaluated by carbapenem inactivation method, presumptive identification of the isolates was performed by CHROMagar orientation and confirmed by API-20E. Carbapenemase genes were screened by PCR and the clonality of all isolates was assessed by XbaI-Pulsed Field Gel Electrophoresis (PFGE). Fifteen KPC-producing Enterobacterales isolates were selected, identified as multidrug-resistant and showed a resistance profile to non-beta-lactam antibiotics: sulfamethoxazole + trimethoprim (7/15), ciprofloxacin (3/15), fosfomycin (3/15) and chloramphenicol (2/15). Isolates were identified as (6) Escherichia coli and (9) Klebsiella pneumoniae. Our results suggest a punctual contamination with KPC-producing Enterobacterales continued through the time. The absence of clonality between the isolates suggests a circulation of mobile genetic element harbouring KPC gene in the origin of contamination. This work provides a better understanding on the impacts of water pollution resulting from human activities on aquatic environments.

First Global Report of Plasmid-Mediated mcr-1 and Extended-Spectrum Beta-Lactamase-Producing Escherichia coli from Sheep in Portugal.

Resistances to extended-spectrum cephalosporins (ESC) and colistin are One Health issues since genes encoding these resistances can be transmitted between all sectors of the One Health concept, i.e., human, animal, and the environment. Among food-producing animals, sheep farming has long been overlooked. To fill in this knowledge gap, we looked for ESC- and colistin resistance in 21 faecal samples collected from sheep in one farm in the south of Portugal. ESC-resistant isolates were selected on MacConkey agar plates supplemented with cefotaxime. Susceptibility testing was performed by the disk-diffusion method according to CLSI, while colistin MIC was determined by broth microdilution. ESC- and colistin-resistance genes were identified by PCR, and the clonality of all isolates was assessed by XbaI-PFGE. The replicon content was determined by PCR according to the PCR-based replicon typing (PBRT) scheme. Sixty-two non-duplicate ESC-resistant E. coli isolates were identified, which all presented an extended-spectrum beta-lactamase (ESBL) phenotype, mostly due to the presence of CTX-M genes. One CTX-M-1-producing E. coli was concomitantly colistin-resistant and presented the plasmid-mediated mcr-1 gene. Nearly all isolates showed associated resistances to non-beta-lactam antibiotics, which could act as co-selectors, even in the absence of beta-lactam use. The results showed a high proportion of ESBL-producing E. coli in sheep faeces. Their dissemination was very dynamic, with the spread of successful clones between animals, but also a large diversity of clones and plasmids, sometimes residing in the same animal. This study highlights the need for global surveillance in all food-producing sectors, in order to avoid the dissemination of genes conferring resistance to last-resort antibiotics in human medicine.

Mapping the scientific knowledge of antimicrobial resistance in food-producing animals.

Antimicrobial resistance (AMR) can be highlighted as one of the most significant health concerns among the last decades, for which antimicrobial drug use in food-producing animals has contributed as one of the major drivers. Food-producing animals are one of the most important and rapidly expanding commercial agricultural sectors worldwide but there is currently limited knowledge on the temporal and geographical distribution of scientific research on antimicrobial resistance in food-producing animals. We provide a global overview of the spatial and temporal trends of scientific knowledge on AMR in food-producing animals. Peer-reviewed papers of AMR on food-producing animals were retrieved from the Web of Science, systemized and dissected. The final validated dataset contained 1341 occurrences observations covering the 1957-2018 period. There has been a shift of research efforts, both geographically and temporally, emphasizing regional differences in food animal production and changing practices in the food production industry. It becomes evident that many regions have been poorly surveyed, wherein intensified sampling and testing efforts should be most valuable. This systematization of knowledge will be crucial in helping to determine how to optimally allocate limited resources available for AMR monitor and control, aiding in the prediction where the threat of new resistant infections will be greatest. AMR research in food-producing animals in developing countries is markedly growing, reflecting changes in food animals production systems but also posing a particularly significant threat, not only due to intensive animal production, but also exacerbated by poor sanitation. We highlight that the use of antibiotics in food producing animals is pervasive, calling for urgent action. These findings raise the possibility to finetuning key priorities on AMR global issues.

Chemical Composition and Antimicrobial Activity of a New Olive Pomace Functional Ingredient.

Olive pomace, an olive oil processing byproduct, can be upcycled and meet the current demand for natural and sustainable food ingredients. In this work, a patented process was used to obtain a functional ingredient from different olive pomaces. The nutritional, chemical and antioxidant profiles, as well as the antimicrobial activity against S. aureus, E. coli and C. albicans, were investigated for the first time. The amount of phenolics ranged between 3.1 and 3.8 g gallic acid eq./100 g in all samples and flavonoids between 2.0 and 3.2 g catechin eq/100 g. No significant differences were found regarding the antioxidant activity. The total fat varied between 5 and 11%, α-tocopherol being the major vitamer and oleic acid the main fatty acid. The protein and ash contents were 1-4% and 10-17%, respectively. The functional ingredient with a higher hydroxytyrosol content (220 mg/100 g) also presented the best minimal inhibitory concentration against the tested bacteria. No activity against C. albicans was verified. This new functional ingredient presents the potential to be used as a natural preservative or as a nutritional profile enhancer. Moreover, it can be an advantageous ingredient in food products, since it comprises specific lipid and hydrophilic bioactive compounds usually not present in other plant extracts.

Emergence of colistin resistance genes (mcr-1) in Escherichia coli among widely distributed wild ungulates.

The environment is considered a major reservoir of antimicrobial resistant microorganisms (AMR) and antimicrobial resistance genes (ARG). Colistin, a "last resort" antibiotic, is used for the treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. The global dissemination of mobile colistin resistance genes (mcr) in natural and non-natural environments is a major setback in the fight against antimicrobial resistance. Hitherto, there is a limited number of studies screening this resistance determinant in bacteria from wildlife. In this study, we describe for the first time the detection of plasmid-mediated colistin resistance in Escherichia coli from wild ungulates in Portugal, which are also widely distributed across Europe. This information is critical to identify the importance of ungulates in the dissemination of resistant bacteria, and their corresponding genes, across the environment. Here, 151 resistant-Enterobacteriaceae isolated from 181 samples collected from different wild ungulate species throughout Portugal were screened for mcr genes. Four mcr-1-positive Escherichia coli were detected from four fallow deer individuals that were sampled in the same hunting ground. These four isolates harboured mcr-1-related IncP plasmids belonging to sequencing types ST155, ST533 and ST345 (n = 2), suggesting bacterial and/or plasmid circulation. All mcr-1-positive E. coli also showed other resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. All mcr-1-positive E. coli show additional resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. Our findings are upsetting, highlighting the global dissemination of colistin resistance genes in the whole ecosystem, which, under the One Health framework, emphasizes the urgent need for effective implementation of AMR surveillance and control in the human-animal-environment interfaces.

Emergence and Spread of Cephalosporinases in Wildlife: A Review.

In the last decade, detection of antibiotic resistant bacteria from wildlife has received increasing interest, due to the potential risk posed by those bacteria to wild animals, livestock or humans at the interface with wildlife, and due to the ensuing contamination of the environment. According to World Health Organization, cephalosporins are critically important antibiotics to human health. However, acquired resistance to ß-lactams is widely distributed and is mainly mediated by extended-spectrum beta-lactamase and AmpC beta-lactamases, such as cephalosporinases. This work thus aimed to compile and analyse the information available on the emergence and dissemination of cephalosporinases in wildlife worldwide. Results suggest a serious scenario, with reporting of cephalosporinases in 46 countries from all continents (52% in Europe), across 188 host species, mainly birds and mammals, especially gulls and ungulates. The most widely reported cephalosporinases, CTX-M-1, CTX-M-14, CTX-M-15 and CMY-2, were also the most common in wild animals, in agreement with their ubiquity in human settings, including their association to high-risk clones of Escherichia coli (E. coli), such as the worldwide distributed CTX-M-15/ST131 E. coli. Altogether, our findings show that anthropogenic activities affect the whole ecosystem and that public policies promoting animal and environmental surveillance, as well as mitigation measures to avoid antimicrobial misuse and AMR spread, are urgently needed to be out in practise.

Multidrug-resistant bacteria as intestinal colonizers and evolution of intestinal colonization in healthy university students in Portugal.

Multidrug-resistant bacteria have been increasingly described in healthcare institutions, however community resistance also seems to be emerging. Escherichia coli an intestinal commensal bacteria, is also a pathogen and represents an important intestinal reservoir of resistance. Our aim was the study of the intestinal colonization and of the persistence of antibiotic resistant intestinal bacteria in healthy university students of Porto, in the north of Portugal. Samples from 30 university students were collected and analysed. Two E. coli isolates were randomly obtained from each student and Gram-negative bacilli resistant to antibiotics were studied. In addition, we evaluated changes in the Gram-negative intestinal colonization of ten university students in a short period of time. Molecular characterization showed a high presence of bla TEM in commensal E. coli . Gram-negative bacteria with intrinsic and extrinsic resistance were isolated, namely Pseudomonas spp., Enterobacter spp. and Pantoea spp. We isolated three ESBL-producing E. coli from two students. These isolates showed bla CTX-M group 1 (n=1), bla CTX-M group 9 (n=2), bla TEM (n=2), bla SHV (n=1) and tetA (n=2) genes. Additionally, they showed specific virulence factors and conjugational transfer of antibiotic resistance and virulence genes. One Pseudomonas spp. isolate resistant to carbapenems was detected colonizing one student. Our results confirm that healthy young adults may be colonized with commensals showing clinically relevant antibiotic resistance mechanisms, creating a risk of silent spread of these bacteria in the community.