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- Unconventional Cohesin-Dockerin binding mechanisms reveal the complexity of cellulosome assemblyPublication . Santos, Marlene Rafaela Cardoso Duarte; Gomes, Pedro Miguel Bule Dias; Prates, José António MestreABSTRACT - The plant cell wall is a vast reservoir of carbon and energy, yet its recalcitrant nature presents a significant challenge to efficient biomass conversion. In order to overcome this hurdle, certain anaerobic bacteria have evolved sophisticated multi-enzyme complexes for the efficient degradation of plant cell wall polysaccharides, termed cellulosomes. Cellulosomes are assembled via calcium-dependent, high-affinity interactions between the cohesin (Coh) modules present in non-catalytic scaffoldins and the enzyme-borne dockerin (Doc) modules. The majority of these interactions display a dual-binding mode (DBM), which enhances the structural flexibility of the cellulosome by allowing Docs to bind Cohs in two opposing orientations, thereby minimizing steric hindrance. This study focuses on the Coh-Doc systems of Bacteroides cellulosolvens and Ruminococcus flavefaciens, two bacteria with highly intricate cellulosomal architectures. While B. cellulosolvens makes extensive use of the DBM, facilitating the assembly of up to 110 enzymes into a single complex, R. flavefaciens was traditionally thought to rely solely on single-binding mode interactions, forming smaller, albeit complex, cellulosomes. Additionally, Ruminococcus albus, a ruminal bacterium that possesses several Doc-encoding genes with high homology to R. flavefaciens, lacks the necessary cohesins to form canonical cellulosomes, raising questions about its cellulose degrading mechanism. In this work, the molecular mechanisms underlying the Coh-Doc interactions in these three microorganisms were explored, revealing several key insights. First, it was discovered that type II Coh-Doc interactions in B. cellulosolvens, previously thought to function only in cell anchoring, also exhibit DBM, underscoring the organism's reliance on this versatile mechanism to assemble its highly complex cellulosome. Contrary to earlier reports, R. flavefaciens was also found to incorporate DBM through the action of the adaptor scaffoldin ScaH, introducing structural flexibility to avoid steric clashes between cellulosomal units. Furthermore, a novel binding mechanism was uncovered in which a single Coh domain could bind two Doc modules simultaneously. This novel 2:1 Coh-Doc interaction mode involves smaller, naturally truncated Doc modules. The study also demonstrated interspecies hybridization potential, with cross species Coh-Doc interactions occurring between R. flavefaciens and R. albus, suggesting a cooperative role in the rumen ecosystem. Additionally, through structure guided protein engineering, enhanced Coh-Doc binding affinities were achieved by modulating the electrostatic profiles of the binding surfaces. This research advances our understanding of cellulosome assembly, with implications for the efficient conversion of lignocellulosic biomass into biofuels, as well as potential biotechnological applications in protein engineering and molecular biology RESUMO - A biomassa vegetal representa a maior fonte de carbono orgânico do planeta. Contudo, a parede celular vegetal é altamente recalcitrante, o que dificulta o acesso e ação das enzimas envolvidas na sua degradação. Estas enzimas, genericamente designadas de Cazymes (carbohydrate active enzymes), são produzidas por diversos microrganismos e desempenham um papel crucial na conversão da biomassa vegetal. Elas encontram se organizadas em dois sistemas distintos. Os microrganismos aeróbios secretam enzimas livres no espaço extracelular, onde estas encontram e degradam o seu respetivo substrato. Em contraste, os microrganismos anaeróbios, que habitam ambientes altamente competitivos, desenvolveram uma estratégia distinta ao secretarem complexos multi-enzimáticos, denominados celulossomas, que mantêm as enzimas funcionalmente integradas, trabalhando em sinergia e acopladas à parede celular. Este mecanismo promove uma degradação mais eficiente e coordenada da parede celular vegetal, destacando-se como uma estratégia evolutiva para maximizar a conversão da biomassa. A montagem do celulossoma, que se baseia em interações proteína-proteína, tem vindo a ser alvo de estudos de natureza estrutural e funcional que conduziram à elucidação da forma como os celulossomas se organizam. A montagem destes complexos é mediada por interações de alta afinidade dependentes de cálcio entre duas proteínas modulares: as coesinas (Cohs) e as doquerinas (Docs). As coesinas são componentes modulares repetidas localizadas em proteínas estruturais não catalíticas, denominadas escafoldinas, enquanto as doquerinas estão predominantemente presentes na extremidade C-terminal das CAZymes. A interação Coh-Doc serve para fixar firmemente as CAZymes às escafoldinas, que por sua vez também podem conter um módulo doquerina que facilita a ancoragem à parede celular ou a articulação com outras escafoldinas portadoras de coesinas, formando estruturas ainda mais complexas. [...].
- Laparoscopic techniques for reproductive surgery in dogsPublication . Tavares, Inês Maria Tenreiro; Tenreiro Tavares, Inês; Rivera Barreno, Ramón; Luís, José Paulo Pacheco de SalesABSTRACT - Laparoscopic surgeries are already routine in human medicine. In companion animals, they are gradually being applied more and tutors are already requesting them. Sterilization is the most common surgery performed on companion animals. In this way, the interest in evaluating the sterilization of male and female dogs and the associated pain arose. In the first study, we compared the viability and the effects on pain and inflammation caused by a new technique for laparoscopic castration of male dogs with the classic technique. Surgical times, pain scale, blood and salivary cortisol and C-reactive protein were compared between the two groups. The laparoscopic group had significantly lower pain, cortisol, and C-reactive protein values than the classical orchiectomy group. The results suggest that this laparoscopic castration is a safe and beneficial alternative to traditional orchiectomy in dogs. Studies in human medicine have concluded that acetazolamide reduces pain associated with carbon dioxide insufflation during laparoscopic surgery. So far, no study has evaluated the action of acetazolamide for this purpose in companion animals. In the second study we compared the values of serum cortisol, salivary cortisol and the University of Melbourne Pain Scale in three groups of bitches subjected to: open ovariectomy, laparoscopic ovariectomy and laparoscopic ovariectomy with preoperative administration of acetazolamide. We found evidence that preoperative administration of acetazolamide may be beneficial in controlling postoperative pain in bitches after laparoscopic surgery. The third paper reports the successful resolution of an intra-surgical finding of teratoma in the left ovary during laparoscopic ovariectomy. This is the first reported case of a teratoma removed by laparoscopic ovariectomy
- Sharing more than friendship : dynamics of direct transmission of antimicrobial resistance between human families and their companion animalsPublication . Menezes, Juliana Cruz de Oliveira de; Pomba, Maria Constança Matias Ferreira; Fonseca, Andreia de Jesus Amaral Gomes BarbosaEnterobacterales that produce extended-spectrum β-lactamases (ESBLs), AmpC cephalosporinases and carbapenemases (CPE), as well as colistin-resistant strains, are important pathogens raising public health concerns due to their increasing prevalence. The prospective longitudinal study focusing on households in Portugal (PT) and the United Kingdom (UK), showed that ESBL/AmpC-producing Enterobacterales strains carriage in healthy companion animals occur at a significantly lower frequency (p-value <0.0001) compared to animals with active infections under antibiotic therapy. Whole genome sequencing (WGS) analysis identified the sharing of ESBL/AmpC-producing Enterobacterales strains between healthy companion animals and humans in two households from Portugal (n=41), involving Escherichia coli human pandemics lineages ST93, ST410 and ST457. Among companion animals undergoing antibiotic treatment due to urinary tract infection (UTI) or skin and soft tissue infection (SSTI), and their cohabiting humans, sharing of faecal ESBL/AmpC producing E. coli strains was observed in four households (PT= 2/43; UK= 2/22), with strains belonging to ST2015, ST617 and ST963. Additionally, one Portuguese household shared a multidrug-resistant (MDR) ACT-24-producing Enterobacter hormaechei subsp. hoffmannii strain. Notably, three other animals shared the clinical strains with their cohabiting humans: a ST556 Klebsiella pneumoniae, the high-risk clonal lineage ST131 E. coli strain and a ST2179 E. coli classified as Avian Pathogenic (APEC). CPE strains were only detected in animals under antibiotic treatment. These strains were positive for blaOXA-181 and blaNDM-5 genes, present in plasmids virtually identical to those found in isolates from humans, food, and the environment in other countries. MDR E. coli strains harbouring the plasmid-mediated colistin resistant gene, mcr-1, were recovered from faecal samples of companion animals and humans in Portugal, with no significant difference between healthy and infected groups (p-value=0.257). In two households with dogs diagnosed with SSTIs, sharing of mcr-1-positive ST744 E. coli strains with the owner were observed. The detection of MDR bacteria sharing between companion animals and their cohabiting humans, especially when it matched the animal's UTI/SSTI clinical strain, highlights the importance of considering household-level interventions in response to the spread of antimicrobial resistance in the community, reinforcing the need for active monitoring and stringent hygiene practices under a One Health approach
- Use of minimally invasive techniques in the diagnosis and characterization of lymphoid hematopoietic tumors in dogsPublication . Valente, Pâmela Cristina Lopes Gurgel; Duarte, António José de Freitas; Correia, Jorge Manuel de Jesus; Pomba, Maria Constança Matias FerreiraABSTRACT - Lymphoma is the most common hematopoietic tumor in dogs, presenting a variety of clinical and morphological forms originating from lymphoid cells. In contrast, multiple myeloma is considered rare. The introduction of minimally invasive techniques in human medicine has had a significant impact, allowing for the accurate diagnosis, screening, and monitoring of neoplasms in humans. The potential for applying these techniques to domestic animals, such as dogs with hematopoietic neoplasias, still lacks the necessary additional studies to assess their efficacy and applicability in veterinary medicine. The main objective of this work was the implementation and use of minimally invasive diagnostic techniques, such as cytology, cell block, immunocytochemistry and PCR for antigen receptor rearrangements (PARR), in the diagnosis of dogs with suspected lymphoid hematopoietic tumors. Initially, the research focused on evaluating and comparing various minimally invasive techniques used in cytological aspirates to diagnose multicentric lymphomas in 38 dogs. It was shown that these approaches make it possible to partially diagnose and characterise multicentric lymphoma in dogs, suggesting the feasibility of obtaining the necessary diagnostic material in a single consultation, which leads to significant gains in terms of cost and time savings without compromising diagnostic accuracy. This work then reinforces the importance of using cell blocks in the diagnosis of lymphoid cell neoplasms by demonstrating their potential in aiding the diagnosis of canine multiple myeloma (MM). Faced with the hypothesis of lymphoid neoplasms with a plasma cell phenotype in dogs, cell blocks from aspiration punctures of the spleen and bone marrow were taken for immunohistochemical analysis. The results of the immunophenotyping allowed the diagnosis of MM and excluded other lymphoid neoplasms. Finally, the applicability of the cytoblock and clonality assays in the diagnosis of a complex case of gastrointestinal lymphoma was evaluated. In this clinical case, the initial diagnosis observed by cytology and cytoblock techniques of liver aspirates was suggestive of lymphoma with hepatic dissemination and indicated rare co-expression of CD3 and CD20 positive lymphocytes, which was confirmed in histopathology with immunohistochemistry of endoscopic biopsies. The final diagnosis of T-cell lymphoma associated with type 2 enteropathy with hepatic infiltration was established by PARR following the detection of the monoclonal T-cell rearrangement. These results, therefore, demonstrate the utility and accuracy of minimally invasive methods in diagnosing and characterizing diferents types of lymphoid hematopoietic tumors in dogs
- Characterization of insect bite hypersensitivity in a population of Lusitano horses : contribution for future implementation of skin prick tests (SPT) in IBH diagnosisPublication . Pessoa, Vera Purificação Carvalho; Tilley, Paula Alexandra Botelho Garcia de Andrade Pimenta; Ferreira, Manuel BrancoInsect Bite Hypersensitivity (IBH) is a recurrent seasonal pruritic dermatitis affecting many horses worldwide. It is mainly a type I hypersensitivity, but type IV may also occur, to allergens present in the saliva of Culicoides spp. It is a multifactorial disease affecting different breeds, with a variable prevalence. Treatment options are very limited, but being an allergic disease IgE-mediated, there is the possibility of developing specific immunotherapy. This study aimed to characterize IBH in Lusitano horses living in Portugal mainland, through a case control study involving 30 IBH-affected animals and 30 healthy controls. Questionnaires were performed for all the horses involved in the study in order to characterize the studied population. In vivo allergen testing was evaluated by performing skin tests, both skin prick tests (SPT) and intradermal tests (IDT), using 14 specific allergens, including 13 different recombinant (r-) proteins from Culicoides nubeculosus (Cul n) or Culicoides obsoletus (Cul o) salivary glands (termed Cul n 1 to Cul n 11, Cul o 1P and Cul o 2P) as well as Cul n whole body extract (WBE). Moreover, a cluster of 12 of these horses, 6 IBH-affected and 6 from the control group, were also tested with allergens produced in insect cells (Cul n 3Bac, Cul n 4Bac) and barley (Cul n 3 Bar, Cul n 4Bar) as well as Cul o 3 and Cul o whole body extract (WBE). Furthermore, in vitro diagnostic tests have been performed, namely, serum IgEs that were measured by ELISA for the allergens Cul n 3, 4, 9, 10 and Cul o 2, 3, as well as Cul o1P and Cul o2P and in vitro sulfidoleukotriene (sLT) release assay which were carried out with Cul n WBE and Cul o WBE. In our study SPTs presented a higher discriminatory diagnostic potential than IDTs, and should be preferred, being a potential breakthrough in the establishment of more suitable allergen avoidance measures in IBH diagnosis. Regarding the in vitro tests, even though serology measurement of specific IgEs showed that IBH-affected horses had higher serum IgE levels for the significant allergens, sLT release assay performed best. It was also possible to study and identify Culicoides spp found in the stud farms, which may be related to IBH in the studied horses. The most frequently found were Obsoletus group species, C. nubeculosus and C. imicola. This study contributed to improve IBH diagnosis, representing a step forward for the future implementation of locally relevant diagnostic allergen panels and eventually for the establishment of patient tailored, component resolved specific immunotherapy
- New approaches to the study of neurorehabilitation protocols in dogs and cats with acute or chronic spinal cord injury with or without deep pain sensation and possible spinal shock signsPublication . Martins, Ângela Paula Neves Rocha; Ferreira, António José de AlmeidaABSTRACT - Intensive neurorehabilitation protocols (INRP) with rehabilitation modalities and weight supported treadmill training (BWSTT), are suggested as treatment to obtain ambulation in dogs and cats with complete (DPP-), discomplete and incomplete (DPP+) compressive or non-compressive spinal cord injury (SCI), similarly to what is performed in human medicine.The first study is a cohort, prospective, controlled and blinded study that was performed in 22 dogs with T11-L3 Hansen type I, revealing ambulation in 100% of the BWSTT group, within a mean of 4.6 weeks. One other study, a retrospective controlled clinical study, was developed in 367 acute post-surgical dogs, with T10-L3 Hansen type I. A new functional neurorehabilitation scale (FNRS-DPP-) was performed to evaluate the DPP- or discomplete dogs, that were able to achieve spinal reflex locomotion (SRL). A strong significance between groups was verified in the DPP+ (p<0.001), with 99.4% of ambulation. The same difference was seen in the DPP- (p=0.007) with 58,5% of ambulation and a tendency (p=0.058) was observed in regard to DPP recovery, with 37.2% achieving SRL, within a maximum of 3 months. INRP was demonstrated to be safe and ambulation recovery achieved earlier. The same population was included in another study, on 16 dogs with incomplete recovery 3 months after surgery. DPP- were under INRP associated with 4-aminopyridine administration, achieving 78% of SRL at day 45 and automatic micturition within a mean of 62 days. Also, 100% of ambulation in the DPP+ within a mean of 47 days and positive follow-up evolution. Ambulatory status was achieved in 88%, establishing this INRP as a therapeutic option to reduce euthanasia. Non-compressive myelopathies with contusive patterns were also referred in a prospective study of 9 cats that revealed 56% (n=5) of ambulation and 44% (n=4) of SRL, showing that INRP should be considered to improve quality of life and the well-being of our patients. Some dogs may develop spinal shock following SCI, including in acute noncompressive nucleus pulposus extrusion. Thus, a cohort prospective study applied a spinal shock scale as a monitoring tool, suggesting spinal shock as a negative factor for a quick recovery. INRP was shown to be safe, tolerable and feasible, allowing 32% of ambulation within 7 days and 94% within 60 days. Follow-ups until 4 years revealed a positive evolution. These studies should be continued, considering each limitation
- Retinal erythropoietin distribution and neuroprotective effect in a nanoparticulate drug delivery system after subconjunctival and topical administration in an animal glaucoma modelPublication . Silva, Beatriz Rosa Fernandes Duarte da; Delgado, Esmeralda Sofia da Costa; São Braz, Berta Maria Fernandes Ferreira; Gonçalves, Lídia Maria DiogoABSTRACT - Glaucoma is a neurodegenerative ocular disease with substantial impact in public health, as it causes retinal ganglion cells (RGC) degeneration and irreversible blindness. Neuroprotective strategies have been a focus of research in glaucoma, and the use of erythropoietin and its recombinant forms, like epoetin beta (EPOβ), have shown anti-apoptotic effects on RGC. We aimed to create a nanoformulation carrying EPOβ, adequate for topical ocular administration, that could provide neuroprotection to the retina in cases of glaucoma, with absent or residual secondary effects and the advantage of promoting patients’ compliance to the treatment. Therefore, chitosan and hyaluronic acid (CS/HA) nanoparticles for topical ocular deliver of epoetin beta (EPOβ) were developed, aiming to deliver EPOβ to the retina in a sustained profile. Firstly, in vitro and ex vivo studies of the physicochemical stability, cytotoxicity, release and permeation profiles and mucoadhesive strength of the CS/HA-EPOβ nanoparticles were performed. These nanoparticles allowed EPOβ permeation through ocular membranes in ex vivo assays, with no in vitro cytotoxicity. Afterwards, healthy Wistar Hannover rats were used for subconjunctival and topical administrations of CS/HA-EPOβ nanoparticles, to assess the formulation’s local and systemic influence, its biological tolerance and safety, its effect in retinal electrophysiology, and EPOβ’s distribution in ocular tissues. A sustained EPOβ delivery to the retina was observed using both routes of administration, with no side-effects. Finally, we explored the topical ocular delivery of CS/HA-EPOβ nanoparticles using a rat glaucoma model. We assessed retinal morphological and physiological changes in response to the nanoformulation applied through topical ocular route, using electroretinography and histological evaluation, which comprised immunofluorescence, hematoxylin and eosin staining and apoptosis assessment (cleaved caspase-3). Topical ocular administration of CS/HA EPOβ nanoparticles in glaucomatous rats allowed EPOβ permeation, including by transcorneal pathway, reaching the inner retina. Improvements in retinal electrical activity and thickness, and in apoptosis reduction occurred earlier and more significantly in the treatment group. In conclusion, EPOβ reached the retina, where its neuroprotective action was observed, thus demonstrating the feasibility of topical administration of neuroprotective agents in nanoformulations, targeting the posterior ocular segment. Results were promising and contribute to the development of new therapeutic strategies to preserve the visual acuity of patients with glaucoma or other neurodegenerative ocular diseases
- Cytological endometritis in dairy cattle : new insights into pathogenesis, diagnosis and treatmentPublication . Pereira, Gonçalo da Silva; Silva, Maria Elisabete Tomé Sousa; Costa, Luís Filipe Lopes daABSTRACT - In postpartum dairy cows, subclinical endometritis (SCE) is characterized by persistent endometrial inflammation, with profound detrimental effects on subsequent fertility. Despite the known role of adipokines regulating metabolism and inflammation, the association of adipokine signaling with SCE is still poorly understood. Moreover, the effect of SCE on endometrial transcription was mainly determined from biopsy-derived whole tissue, thus masking effects on cell type-specific gene transcription. In addition, despite the recognized improvement in immune function following n-3PUFA supplementation, the therapeutic potential of these fatty acids for SCE control is still to be determined. Therefore, the main objectives of this work were to assess the relationship between adipokines and SCE (Chapter III), to elucidate the effects of progesterone and SCE in the transcription profiles of endometrial glandular, luminal and stromal cells (Chapters IV, V), and the effects of feeding rumen-protected n-3PUFA on endometrial homeostasis and fertility in postpartum dairy cows (Chapter VI). The results showed that adiponectin in plasma and uterine fluid in addition to chemerin uterine fluid have high discriminatory power for the diagnosis of SCE. This work additionally evidenced that progesterone and SCE affect endometrial transcriptomic profiles in a cell type-specific manner. This work also evidenced that the previous presence of immune cells is still impacting the transcriptome of endometrial cells at the end of the voluntary waiting period and that recovery or persistence of inflammation is associated with transcription patterns involved not only in immune function but also in tissue remodeling, cell adhesion, and uterine receptivity. Furthermore, despite having no apparent effect on the endometrial inflammatory status at the end of the voluntary waiting period, dietary supplementation with n-3PUFA decreased calving to conception interval. Overall, this thesis establishes adiponectin as a suitable biomarker of SCE, able to predict the risk of persistence of inflammation in postpartum dairy cows, provides new insights into the persistence and recovery of SCE, thus presenting putative alternative therapeutic strategies. Moreover, this work substantiates dietary n-3PUFA as valid nutraceuticals to improve reproductive parameters in postpartum dairy cows.
- Bovine genital campylobacteriosis : new insights into the molecular diagnosis and pathogenesisPublication . Silva, Marta Filipa Serra da; Mateus, Luísa Maria Freire Leal; Silva, Maria Elisabete Tomé Sousa; Duarte, Ana Isabel Simões PereiraC. fetus subsp. venerealis is the pathogen responsible for the Bovine Genital Campylobacteriosis (BGC), a cattle’s venereal disease whose diagnosis relies on the accurate identification of Cfv. Despite its relevance for BCG control and management, the applicability of the molecular methods on the disease diagnosis is controversial whereas the mechanisms underlying the pathogenesis of BGC remain unclear. Therefore, the main objectives of this work were to assess the suitability of different real-time PCR assays to be used in bovine preputial samples for Cfv detection and to further elucidate the virulence potential of Cfv through the genomic characterization of Cfv field isolates. The results showed that the currently used molecular targets for Cfv identification, the parA gene and the insertion sequence ISCfe1, originate a high rate of false-positive results (up to 50 %) in clinical samples. This lack of specificity was also evidenced by the identification of a novel bacterial species in the bovine prepuce, Campylobacter portucalensis, with sequences homologous to parA (98 % identity) and ISCfe1 (94 % identity) sequences of Cfv, hindering the molecular diagnosis of BGC. On the other hand, the absence of the parA gene in a high proportion of Cfv strains (77 %), also invalidate the use of this gene as a diagnostic target for Cfv identification. The identification of three novel Sequence Types (ST) in venerealis subspecies, previously characterized by a high genetic stability, questions the use of Multilocus Sequence Typing (MLST) for subspecies identification. This work additionally evidenced differential features between Cfv and its biovar intermedius, namely in whole-genome Single Nucleotide Polymorphisms (SNPs) and genes encoding genus-specific proteins families. Moreover, the results suggest that a type IV secretion system, previously indicated as involved in Cfv virulence, does not play a crucial role in the pathogenesis. In vitro antimicrobial resistance to streptomycin, penicillin, tetracycline and enrofloxacin was not detected, even in strains encoding two multidrug efflux pumps, CmeABC and YkkCD, revealing that its sole presence in the genome is not enough to provide in vitro antimicrobial resistance. Overall, these results have a significant impact on the molecular diagnosis of BGC and revealed new insights into the Cfv genetic diversity and virulence potential.
- Extended–spectrum–beta-lactamases, cephalosporinases and carbapenemase-producing Escherichia coli in the human-dog interfacePublication . Belas, Adriana; Pomba, Maria Constança Matias Ferreira; Carriço, João André Nogueira CustódioExtended–spectrum–beta-lactamases (ESBLs), cephalosporinases (encoded by the ESBLs and Ampc genes, respectively) and carbapenemase–producing Escherichia coli have become a major public health concern to both human and animal health. Urinary tract infections (UTI) are one of the most frequent bacterial infections in both human and companion animals. Uropathogenic E. coli (UPEC), belonging to extraintestinal pathogenic E. coli (ExPEC), is the most common bacterium isolated from companion animals. Moreover, the close contact of companion animals with humans creates opportunities for interspecies transmission of resistant bacteria and genes. E. coli from companion animals with UTI were found to harbour important antimicrobial resistance mechanisms and to belong to high-risk human clonal lineages, namely third-generation cephalosporin (3GC)-resistant E. coli O25b:H4-B2-ST131-H30/H30Rx, CC23 and ST648. In this work E. coli O25b:H4-ST131-H30/H30Rx was described for the first time in Europe in companion animals. Furthermore, the blaCMY-2 producing E. coli ST648 is the most common high-risk clonal lineage causing UTI in companion animals from the Lisbon area. Companion animals also seem to be reservoirs of bacteria and clinically important resistance genes, such β-lactams genes (classe A and C) which supports their role as reservoirs. The detection of faecal high-risk clone OXA-181-producing- E. coli ST410 strains that were closely related to uropathogenic clinical human strains was also an important finding and to our best knowledge was the first description in Portugal and Europe. These studies highligth the importance of companion animals as reservoirs of pathogenic E. coli harbouring important antimicrobial resistant genes. The emergence and spread of multidrug-resistant (MDR) E. coli in the natural environment by companion animal faecal contamination is also a concern towards animal and human health. These results point to need for control measures to prevent the dissemination of MDR ESBLs/AmpC and carbapenemases – producing bacteria from companion animals.