Unveiling a Listeria monocytogenes Outbreak in a Rabbit Farm: Clinical Manifestation, Antimicrobial Resistance, Genomic Insights and Environmental Investigation.

Listeria monocytogenes poses a threat to both human and animal health. This work describes an L. monocytogenes outbreak in a Portuguese rabbit farm, detailing the isolates' clinical manifestations, necropsy findings, and phenotypic and genomic profiles. Clinical signs, exclusively observed in does, included lethargy and reproductive signs. Post-mortem examination of does revealed splenomegaly, hepatomegaly with a reticular pattern, pulmonary congestion, and haemorrhagic lesions in the uterus, with thickening of the uterine wall and purulent greyish exudates. Positive L. monocytogenes samples were identified in fattening and maternity units across different samples, encompassing does and environmental samples. Core-genome Multi Locus Sequence Typing (cgMLST) analysis confirmed the outbreak, with the 16 sequenced isolates (lineage II, CC31, and ST325) clustering within a ≤2 allelic difference (AD) threshold. Antimicrobial susceptibility testing for five antibiotics revealed that 15 out of 19 outbreak isolates were resistant to sulfamethoxazole-trimethoprim (SXT). Concordantly, all SXT-resistant sequenced isolates were found to exclusively harbour a plasmid containing a trimethoprim-resistance gene (dfrD), along with loci linked to resistance to lincosamides (lnuG), macrolides (mphB), and polyether ionophores (NarAB operon). All sequenced outbreak isolates carried the antibiotic resistance-related genes tetM, fosX, lin, norB, lmrB, sul, and mprF. The outbreak cluster comprises isolates from does and the environment, which underscores the ubiquitous presence of L. monocytogenes and emphasizes the importance of biosecurity measures. Despite limited data on listeriosis in rabbit farming, this outbreak reveals its significant impact on animal welfare and production.

An Exploratory Study on Spoilage Bacteria and Listeria monocytogenes in Fresh Salmon: Extending Shelf-Life Using Vacuum and Seasonings as Natural Preservatives.

A growing population increases the demand for food, but short shelf-lives and microbial hazards reduce supply and increase food waste. Fresh fish is highly perishable and may be consumed raw, such as salmon in sushi. This work aims to identify strategies to improve the shelf-life and safety of fresh salmon, using available methods (i.e., vacuum) and exploring the use of natural preservatives (i.e., seasonings). Vacuum packaging and good hygiene practices (which reduce initial flora) extended shelf-life up to 20 days. Carnobacterium maltaromaticum was dominant in vacuum packaging conditions and showed potential for inhibiting Listeria monocytogenes. For natural preservatives, L. monocytogenes required higher inhibitory concentrations in vitro when compared to the 10 spoilage bacteria isolated from fresh salmon fillets, presenting a minimum inhibitory concentration (MIC) of 0.13% for oregano essential oil (OEO), 10% for lemon juice, 50 mg mL-1 for garlic powder, and >10% for NaCl. A good bacteriostatic and bactericidal effect was observed for a mixture containing 5% NaCl, 0.002% OEO, 2.5% lemon juice, and 0.08 mg mL-1 garlic powder. Finally, using the salmon medium showed an adequate correlation with the commercial culture medium.

Occurrence of Multidrug-Resistant Bacteria Resulting from the Selective Pressure of Antibiotics: A Comprehensive Analysis of ESBL K. pneumoniae and MRSP Isolated in a Dog with Rhinorrhea.

Because of public health concerns, much greater scrutiny is now placed on antibiotic use in pets, especially for antimicrobial agents that have human analogs. Therefore, this study aimed to characterize the phenotypic and genotypic profiles of multidrug-resistant bacteria isolated from nasal swabs samples taken from a one-year-old male Serra da Estrela dog with rhinorrhea that was treated with amikacin. An extended-spectrum ß-lactamases (ESBL) Klebsiella pneumoniae was isolated in the first sample taken from the left nasal cavity of the dog. Seven days later, methicillin-resistant (MRSP) Staphylococcus pseudintermedius was also isolated. Nevertheless, no alterations to the therapeutic protocol were performed. Once the inhibitory action of the antibiotic disappeared, the competitive advantage of the amikacin-resistant MRSP was lost, and only commensal flora was observed on both nasal cavities. The genotypic profile of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae revealed the same characteristics and close relation to other strains, mainly from Estonia, Slovakia and Romania. Regarding MRSP isolates, although resistance to aminoglycosides was present in the first MRSP, the second isolate carried aac(6')-aph(2″), which enhanced its resistance to amikacin. However, the veterinary action was focused on the treatment of the primary agent (ESBL K. pneumoniae), and the antibiotic applied was according to its phenotypic profile, which may have led to the resolution of the infectious process. Therefore, this study highlights the importance of targeted therapy, proper clinical practice and laboratory-hospital communication to safeguard animal, human and environmental health.

Microplastics altered cellular responses, physiology, behaviour, and regeneration of planarians feeding on contaminated prey.

Freshwater benthic environments are among the major sinks of microplastics (MPs, < 5 mm) sourced on inland anthropogenic activities. The ecotoxicological effects of MPs on benthic macroinvertebrates have been assessed preferably in collectors, shredders, and filter-feeders, but resulting in insufficient knowledge on the potential trophic transfer and its effects on macroinvertebrates with predator behaviour such as planarians. This work evaluated the behavioural (feeding, locomotion), physiological (regeneration) and biochemical responses (aerobic metabolism, energy reserves, oxidative damage) of the planarian Girardia tigrina after consuming contaminated live prey Chironomus riparius larvae previously exposed to microplastics of polyurethane (PU-MPs; 7-9 µm in size; 375 mg PU-MPs/kg). After the feeding period (3 h), planarians consumed 20 % more contaminated prey than uncontaminated prey, probably related to increased curling/uncurling movements of larvae (that might be more appellative to planarians). Histological analysis revealed planarians' limited intake of PU-MPs, mainly detected near the pharynx. The consumption of contaminated prey (and intake of PU-MPs) did not result in oxidative damage but slightly increased the aerobic metabolism and energy reserves which show that the consumption of more prey was sufficient to cope with the potential adverse effects of internalized MPs. Moreover, no effects were observed in the locomotion of planarians in good agreement with the hypothesis of sufficient energy acquired by the exposed planarians. Despite the previous, it seems that the energy acquired was not allocated for planarians' regeneration since a significant delay in the regeneration of the auricles was observed for planarians feeding on contaminated prey. Therefore, further studies should be performed considering the potential long-term effects (i.e., reproduction/fitness) and the effects of MPs that might result from continuous feeding on contaminated prey, representing a more realistic exposure scenario.

Microplastics in Terrestrial Domestic Animals and Human Health: Implications for Food Security and Food Safety and Their Role as Sentinels.

Terrestrial domestic animals are exposed to microplastics, therefore, contaminating the food chain, in the case of livestock, or acting as sentinels for human exposure, in the case of companion animals. The aim of this review was to address the importance of terrestrial domestic animals on human exposure to microplastics. Animal products may already show some microplastics contamination, which may occur during their lifetime, possibly also compromising productivity, and during processing, originating from equipment and packaging. Moreover, release of microplastics in animal feces (or manure) leads to the contamination of agricultural fields, with possible impacts and internalization in plants. Therefore, microplastics pose a threat to food security, compromising food productivity, and food safety, by being a foreign material found in animal products. Conversely, in urban environments, companion animals (cats and dogs) may be relevant sentinels for human exposure. While oral exposure may vary in pets compared to humans, due to indiscriminate ingestion and chewing or licking behaviors, airborne exposure is likely to be a good indicator for human exposure. Therefore, future studies should address the importance of terrestrial domestic animals for human exposure of microplastics, both in the food chain and as sentinels for environmental exposure.

Mechanisms influencing the impact of microplastics on freshwater benthic invertebrates: Uptake dynamics and adverse effects on Chironomus riparius.

Chironomids inhabit freshwater benthic ecosystems which are prone to microplastic contamination. This work aimed at understanding the factors and mechanisms influencing microplastic uptake and related adverse effects on Chironomus riparius, by exploring an extensive project database, conducting a literature review, and performing an agent-based model to explore trends in data. Results reveal that high concentrations of small microplastics fill the gut of fourth instar C. riparius (99.7 %). Ingested microplastics had an average size of 38-61 µm, presenting slower elimination rates than undigested organic or mineral particles. Ingestion rates of microplastics depend mainly on encounter rates, and therefore on available concentrations, until reaching a plateau corresponding to the maximum gut volume. Short-term toxicity of microplastics seems to result from damage to gut epithelium, with inflammatory reactions, production of reactive oxygen species, and a negative energy balance exacerbated by the lack of food (organic matter). Long-term toxicity is characterized by a reduction in larval body length and increase in mean time to emergence, seemly from increased energy costs rather than a decrease in nutrient absorption. Wild chironomids already present microplastics in their guts and environmental concentrations in hotspots may already exceed no effect concentrations. Therefore, environmental exposure to microplastics may induce adverse effects to wild C. riparius in freshwater benthic ecosystems, which could compromise their ecologic role as deposit-feeders (e.g., reducing their nutrient cycling ability) and key-stone species in aquatic food webs.

Gulls in Porto Coastline as Reservoirs for Salmonella spp.: Findings from 2008 and 2023.

Gulls act as intermediaries in the exchange of microorganisms between the environment and human settlements, including Salmonella spp. This study assessed the antimicrobial resistance and molecular profiles of Salmonella spp. isolates obtained from fecal samples of gulls in the city of Porto, Portugal, in 2008 and 2023 and from water samples in 2023. Antimicrobial susceptibility profiling revealed an improvement in the prevalence (71% to 17%) and antimicrobial resistance between the two collection dates. Two isolate collections from both 2008 and 2023 underwent serotyping and whole-genome sequencing, revealing genotypic changes, including an increased frequency in the monophasic variant of S. Typhimurium. qacE was identified in 2008 and 2023 in both water and fecal samples, with most isolates exhibiting an MDR profile. The most frequently observed plasmid types were IncF in 2008 (23%), while IncQ1 predominated in 2023 (43%). Findings suggest that Salmonella spp. circulate between humans, animals, and the environment. However, the genetic heterogeneity among the isolates from the gulls' feces and the surface water may indicate a complex ecological and evolutionary dynamic shaped by changing conditions. The observed improvements are likely due to measures to reduce biological contamination and antimicrobial resistance. Nevertheless, additional strategies must be implemented to reduce the public health risk modeled by the dissemination of pathogens by gulls.

Motivation of Portuguese-Speaking Pet Owners Seeking Online Pet Health and Husbandry Information.

Internet use for pet health information is increasing in popularity among pet owners. So far, evidence on motivations and topics of interest is limited to surveys on English speaking countries. The objective was to evaluate the motivations of Portuguese-speaking pet owners when visiting a pet information website, written by a certified veterinarian. In situ needs and motivations of visitors were evaluated by classifying online questions (n = 202) and conducting a short questionnaire in the website sidebar (n = 453). Most pet owners were looking for general advice (73.9%), especially on systems with visible disease signs, being motivated by the lack of access to veterinary services due to costs (17.7%), support from other pet owners (11.3%), and complementing the insufficient information provided by the veterinarian (7.7%). Brazilians reported more lack of veterinary access due to costs compared to Portuguese respondents. Thus, Portuguese-speaking pet owners engaged in online searches to seek affordable veterinary care, better information, and emotional support.

Survey of Pet Owner Attitudes on Diet Choices and Feeding Practices for Their Pets in Portugal.

Feeding practices and perceptions of pet owners determine consumer decisions on pet foods and influence the health of companion animals. The objective of the survey was to study emerging trends on pet food diets in Portugal by conducting a preliminary survey. A survey of 74 pet owners revealed that most fed pets with commercial diets (67.6%) bought in supermarkets (40.3%), spending a monthly average of EUR 30, following healthy practices (e.g., ≥30 min of exercise), which translated to an average self-reported pet body condition score of 3. Information about pet foods mainly originates from the animal's veterinarian (64.9%), followed by the internet (16.2%). A trend for a growing interest in alternative diets (e.g., natural and organic) was identified, being already the second most consumed diet type (19.0%), perceived as being of a higher quality, and as a topic of interest (38.6%). This interest likely originates from a higher weighting of the ingredient list (31.5%) in consumer choices and beliefs that commercial diets should consist of a higher proportion of meat (29.3%) and less carbohydrates (38.7%). Therefore, more research is needed on the nutritional adequacy of alternative diets and uncommon ingredients, the pet food industry will have to adapt to changing consumer behaviors, and veterinarians should be available to discuss and oversee novel dietary practices in companion animals.

Microplastics in Internal Tissues of Companion Animals from Urban Environments.

Companion animals living in urban areas are exposed to environmental contaminants, which may include microplastics. A preliminary study was conducted by collecting postmortem samples from the internal tissue (lungs, ileum, liver, kidney, and blood clots) of 25 dogs (Canis familiaris) and 24 cats (Felis catus) living in an urban environment in Porto metropolitan area, Portugal. Suspected microplastics were found in 80 samples from 35 animals (18 cats and 17 dogs), often occurring in more than one tissue of the same animal (71.4%), primarily under small sizes (50.3% as 1-10 µm). Micro-Raman spectroscopy confirmed a fraction of particles as common polymer types (e.g., polyethylene terephthalate). However, the number of particles was very low. This study highlights the possibilities of the internalization and distribution of microplastics in the internal tissues of terrestrial vertebrates.

A straightforward method for microplastic extraction from organic-rich freshwater samples.

The extraction of microplastics from organic-rich freshwater samples is challenging and limited information is available in the literature. This study aims at developing efficient methods for water volume reduction and organic matter removal in freshwater samples, while focusing on the reduction of the economic and environmental costs, maintaining microplastics integrity and avoiding contamination. For the water volume reduction approach, centrifuging freshwater samples (water, sediment, algae, leaves, driftwood, fish tissue) at different speeds (3500, 6000 rpm) and times (5, 10 min) showed that 3500 rpm for 5 min was efficient to settle the mineral and organic material, while preserving the polymers and showing high microplastic recovering rates (93 ± 6%). These recovery rates were significantly higher than the traditional sieving approach (77 ± 22%). The posterior minimal consumption of reagents resulting from the reduction of water volume helped to reduce the economic and environmental costs of the devised methodology, becoming more aligned with green chemistry principles. For biogenic organic matter removal, four digestion solutions were tested on freshwater samples, namely 10% potassium hydroxide, Fenton reagent (30% H2O2 + Fe(II)), 7% and 10% sodium hypochlorite (NaClO), under 3 periods of time (1, 6 and 15 h), at 50 °C. Both 7% and 10% NaClO showed the highest rates of organic matter removal (86 ± 1% and 90 ± 1%, respectively), after 6 h at 50 °C. Exposure of virgin and aged polymers (polyethylene, polypropylene, polystyrene, polyvinyl chloride, nylon, polyethylene terephthalate) to NaClO showed no weight, visual, surface structure, Fourier transform infrared spectra and carbonyl index changes, except for nylon, although not to an extent that affected its identification. This method resulted in high recovery rates of polymers (92 ± 6%). Thus, 7% NaClO at 50 °C for 6 h (or overnight) may be efficiently used for microplastic analysis in organic-rich freshwater samples.

The road to sustainable use and waste management of plastics in Portugal.

As a European Union (EU) member, Portugal must comply with reductions in plastic waste. In Portugal, the 330 items/100 m of beach litter, comprising up to 3.9 million pieces and of which 88% is plastic, is higher than the EU median (149 items/100 m) and must be reduced to 20 items/100 m (94%). Integrative measures are needed to reduce littering and improve plastics' use and disposal under the circular economy. Of this 414 kt of plastic packaging waste, 163 kt were declared plastic packaging, 140 kt subjected to recycling, and 94 kt to energy recovery. The current recycling rate of plastic packaging (34%) should be improved to reach EU recycling averages (42%) and goals and to provide widespread benefits, considering revenues of 167 €/t. As a net importer of waste, Portugal could benefit from the valorization of imported waste. Besides increased recycling, pyrolysis and gasification could provide short-term alternatives for producing value-added substances from plastic waste, such as hydrogen, consistent with the National Plan of Hydrogen and improving ongoing regulations on single-use plastics. This manuscript provides an integrative view of plastics in Portugal, from use to disposal, providing specific recommendations under the circular economy. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available in the online version of this article at 10.1007/s11783-021-1439-x and is accessible for authorized users.

Effects of virgin and weathered polystyrene and polypropylene microplastics on Raphidocelis subcapitata and embryos of Danio rerio under environmental concentrations.

Microplastics are ubiquitous contaminants of freshwater ecosystems. However, few ecotoxicity assays have been conducted on freshwater organisms using environmentally relevant concentrations of virgin and weathered microplastics. This work assessed the adverse effects of virgin and artificially weathered fragments of polystyrene and polypropylene on the microalga Raphidocelis subcapitata (72 h growth inhibition assay) and on embryos of the fish Danio rerio (96 h fish embryo assay) under environmentally relevant concentrations (2000-200,000 MP L-1) and high concentrations (12.5-100 mg L-1). Sizes of microplastics were measured as tens (polystyrene) to hundreds (polypropylene) of micrometers, while aging was assessed by measuring the carbonyl index. In the microalga, the tested high concentrations promoted growth, while environmentally relevant concentration induced either growth inhibition or promotion. In zebrafish embryos, environmentally relevant concentrations decreased body length and heart rates. No relevant effects were observed in organisms exposed to high concentrations for mortality, malformations, hatching rates, and swimming bladder inflation. Virgin microplastics presented slightly higher toxicity but direct comparison was hindered by the lack of a linear dose-response curve. Despite the lack of a clear pattern, adverse effects were often observed in the lowest environmentally relevant concentrations, raising concerns over the impacts of microplastics on freshwater ecosystems.

Suspected microplastics in Atlantic horse mackerel fish (Trachurus trachurus) captured in Portugal.

Microplastics have been found in fish, but most studies have focused on the digestive system without considering additional organs. Herein, the objective was to assess the presence of microplastics in internal organs (gills, guts, kidney, heart) of the Atlantic horse mackerel (Trachurus trachurus) captured of the coast of Portugal (Northeast Atlantic Ocean). Suspected microplastics were present in all organs, with particles of larger size (i.e., equivalent diameter) found in the gut and those of lower size in the heart and its luminal blood. Suspected microplastics of 1-10 µm were the most abundant (65.4%), more likely to translocate, owing to their minute size, but more difficult to properly characterize. These results highlight the need to expand the analytical work on organs and tissues for assessing microplastics in organisms, but also emphasize the actual need for developing analytical methods that allow for an accurate isolation, identification, and characterization of microplastics in biota.

Risks of Covid-19 face masks to wildlife: Present and future research needs.

The use of disposable face masks became essential to fight against the COVID-19 pandemic, resulting in an unprecedented rise in their production and, unfortunately, to a new form of environmental contamination due to improper disposal. Recent publications reported the abundance of COVID-19-related litter in several environments, wildlife interaction with such items, and the contaminants that can be released from such protective equipment that has the potential to induce ecotoxicological effects. This paper provides a critical review of COVID-19 face mask occurrence in diverse environments and their adverse physiological and ecotoxicological effects on wildlife. It also outlines potential remediation strategies to mitigate the environmental challenge impose by COVID-19-related litter.

Preparation of biological samples for microplastic identification by Nile Red.

Many methods have been used to isolate and identify microplastics from biological matrices. In biological samples, Nile Red can stain undigested residues, such as fats, soaps, and gels formed during organic matter removal, hindering the identification of fluorescent microplastics (≥2 µm). Thus, adjustments on sample preparation (e.g., fat removal) are required for the accurate identification of Nile Red stained microplastics. Multiples tests allowed to identify that digestion with 10% KOH at 60 °C for 24 h, followed by treatments with boiling water, acetone, and staining, produced good results in fourteen biological samples, including vertebrates and invertebrates. Digestion efficiencies were 94-100%, except for feces, which were 87%. Recovery rates of spiked microplastics were 97-100%, and few effects were observed in the infrared spectra and carbonyl index of seven polymers, with only the occasional yellowing suggesting surface changes. Filtration rates were improved by reducing the amount of sample. Small fluorescent microplastics could be identified in all samples under the microscope. Overall, the proposed method was efficient in removing natural organic matter from biological samples for Nile Red staining, requiring minimal sample handling, improving sample throughput, and allowing quantification of fluorescent microplastics in biological samples.

Considerations when using microplates and Neubauer counting chamber in ecotoxicity tests on microplastics.

The ubiquity of microplastics raises issues regarding contamination control measures and laboratory practices. The objective was to adapt the use of counting chambers and plastic microplates on the ecotoxicity evaluation of microplastics. Counting chambers, originally used to quantify cells, can also be used to count high concentrations of microplastics (<100 µm) used in laboratory assays. By decontaminating the chamber and mixing the test solution with Nile Red (1:1), fluorescent particles can be easily counted under optical microscopy. Microplate wells, due to their composition, can be contaminated or release microplastics to the test medium, which can interfere with the results of ecotoxicity assays or spectroscopy readings. A cleaning method based on ethanol was developed, which effectively removed particles by 91% without interfering with microalgae yield. Besides providing practical applications that can improve ecotoxicity assays, this work intends to raise awareness on the need to adapt laboratory practices when working with microplastics.

Selection of microplastics by Nile Red staining increases environmental sample throughput by micro-Raman spectroscopy.

Nile Red staining enables visual identification and quantification of fluorescent particles as a proxy to microplastics at low cost and high throughput, including those of small sizes (≥2 µm), when preceded by proper natural organic matter removal, but providing no chemical characterization. On the other hand, micro-spectroscopy methods allow chemical characterization of particles based on their spectra, essential for polymer identification, but are costly and time-consuming. This work addresses the combination of both Nile Red staining with micro-Raman spectroscopy for the identification of microplastics. Besides being useful for quantification, Nile Red staining can be advantageously used as an objective criterion for pre-selection of particles for micro-Raman spectroscopy, producing little interference. The use of the 442 nm laser in micro-Raman spectroscopy induces Nile Red luminescence thus allowing to target the specific suspected microplastics when using an orange filter, reducing the number of particles subjected to identification and improving sample throughput. Staining dyes could also be used for mapping suspected microplastics before targeted analysis by micro-Raman spectroscopy. Thus, coupling Nile Red with micro-Raman spectroscopy can be useful to improve time efficiency while using this equipment.

Microplastics on Barra beach sediments in Aveiro, Portugal.

Microplastic (MPs) pollution has been recognized as a serious threat to marine environment with the growing use of plastics. There is no sufficient data on the extent and characteristics of MPs pollution in the beach sediments and sand in the Atlantic Ocean. The coastal area is one of the main tourist zones in Aveiro in Portugal, thus, impacted by both tourism and maritime traffic, which are major sources of MPs. Considering this issue, 33 sediment samples were collected from the Praia da Barra beach in Aveiro. This pilot study showed that large quantities of MPs are accumulated on this beach with a median concentration of 100 MP kg-1 (15-320 MP kg-1), that is dominated by polyethylene (30%), polypropylene (27%), polystyrene (18%), nylon (12%), and polyester (6%). Size <1 mm constituted 99.5% of particles, mostly of transparent or black colors, with highest concentrations closest to the water line.