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.

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.

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.

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.

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.

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.

Temporal and geographical research trends of antimicrobial resistance in wildlife - A bibliometric analysis.

Antimicrobial resistance (AMR) is a complex and global problem. Despite the growing literature on AMR in the medical and veterinary settings, there is still a lack of knowledge on the wildlife compartment. The main aim of this study was to report the global trends in AMR research in wildlife, through a bibliometric study of articles found in the Web of Science database. Search terms were "ANTIMICROBIAL" OR "ANTIBIOTIC" AND "RESISTANT" OR "RESISTANCE" and "WILDLIFE" "MAMMAL" "BIRD" "REPTILE" "FERAL" "FREE RANGE". A total of 219 articles were obtained, published between 1979 and 2019. A rising interest in the last decades towards this topic becomes evident. During this period, the scientific literature was distributed among several scientific areas, however it became more multidisciplinary in the last years, focusing on the "One Health" paradigm. There was a geographical bias in the research outputs: most published documents were from the United States, followed by Spain, Portugal and the United Kingdom. The most productive institutions in terms of publication number were located in Portugal and Spain. An important level of international collaboration was identified. An analysis of the main keywords showed an overall dominance of "AMR", "E. coli", "genes", "prevalence", "bacteria", "Salmonella spp." and "wild birds". This is the first study providing a global overview of the spatial and temporal trends of research related to AMR in wildlife. Given the growth tendency over the last years, it is envisaged that scientific production will expand in the future. In addition to offering a broad view of the existing research trends, this study identifies research gaps both in terms of geographical incidence and in relation to unexplored subtopics. Unearthing scientific areas that should be explored in the future is key to designing new strategic research agendas in AMR research in wildlife and to inform funding programs.

Epidemic spread of IncI1/pST113 plasmid carrying the Extended-Spectrum Beta-Lactamase (ESBL) blaCTX-M-8 gene in Escherichia coli of Brazilian cattle.

INTRODUCTION: The prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide and the Agri-Food sector acts as a reservoir of clinically relevant ESBL genes. Our study aimed at detecting and characterizing ESBL-producing Enterobacteriaceae responsible for intestinal colonization of Brazilian bovines. MATERIAL AND METHODS: ESBL-producing E. coli isolates were recovered from fecal samples of healthy cattle in Northwest Brazil. Antimicrobial susceptibility was determined by disk diffusion. Resistance and virulence genes were identified by PCR and amplicons were sequenced, clonality was assessed by PFGE and MLST, and plasmids were characterized by replicon typing, S1-PFGE and Southern blot hybridizations. Transferability of ESBL genes was assessed by conjugation assay. RESULTS: A total of 40 ESBL-producing E. coli were characterized, which originated from 34/191 animals (17.8 %) and 15/22 farms (68.2 %). The blaCTX-M-8 gene was the most frequent ESBL gene (62.5 %), followed by blaSHV-2a (20.0 %), blaCTX-M-2 (10.0 %), and blaCTX-M-15 (7.5 %). The blaCTX-M-8 gene was localized on the IncI1/pST113 plasmid in multiple E. coli sequence types across unrelated animals and farms. DISCUSSION: We report the first characterization and a high prevalence of ESBL-producing E. coli in the beef cattle sector in Brazil, which is mainly supported by the spread of an epidemic IncI1/pST113/blaCTX-M-8 plasmid. Since Brazil is one of the biggest beef meat exporters worldwide, the spread of this ESBL plasmid across other sectors, countries and continents should be considered with attention.

CLSI - EUCAST: Comparison of antibiotic-susceptibility profile of Enterobacteriaceae of animal origin according to the standards.

Antimicrobial resistance is a relevant "One Health" issue that shows the need of comparison of isolates of different origins. In this way, guidelines for antimicrobial-resistance evaluation in animals are relevant in relation to human sources. This work aims to compare antimicrobial-resistance results of animal isolates considering CLSI and EUCAST guidelines. The comparison shows considerable differences in the results, which include antibiotics used as primary options in hospital infections. EUCAST showed the higher number of samples with resistance profiles than CLSI that indicates a more efficient scenario to the EUCAST to screen antibiotic-resistant bacteria. EUCAST was more consonant to the expected phenotype for ESBL producers, with higher index of resistance to oxyimino-beta-lactam antibiotics. The study shows that there are differences in the interpretative results using different guidelines, where the susceptibility test results concerning Enterobacteriaceae of animal origin are not always coincident in CLSI and EUCAST. EUCAST has proved to be the most reliable alternative for profile screening of antibiotic resistance, when compared to CLSI. We might say the same with respect to the ESBL-producing Enterobacteriaceae, in which EUCAST has proved to be more efficient about the demonstration of expected resistance profiles for the ESBL producers. These differences show that guideline selection might influence the therapeutic option.

Draft genome of a ST443 mcr-1- and blaCTX-M-2-carrying Escherichia coli from cattle in Brazil.

OBJECTIVES: Colistin is used in Brazil for the treatment of food-producing animals. The colistin resistance gene mcr-1 has already been reported from chicken and swine in this country. Here we report the draft genome of an Escherichia coli isolate presenting both an extended-spectrum ß-lactamase (ESBL) gene and the mcr-1 gene in a healthy cow in Brazil. METHODS: Whole genomic DNA from E. coli E12 was extracted and 2× 150-bp paired-end reads were generated using Illumina sequencing technology. De novo genome assembly was performed using SPAdes v.3.11 and the draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Further analyses were performed using Center for Genomic Epidemiology databases. Southern blots were performed to characterise plasmid location. RESULTS: The 5024393-bp genome displayed several resistance genes, including the mcr-1 and blaCTX-M-2 genes. These two genes were located on different plasmids (mcr-1 on an IncX4 plasmid and blaCTX-M-2 on an IncF plasmid). CONCLUSION: The genome sequence reported here can be compared with previously published genomes for mcr-1-producing isolates. This will ultimately help to understand the routes of dissemination of the resistance genes.

Avaliação da atividade antimicrobiana in vitro e determinação da concentração inibitória mínima (CIM) de extratos hidroalcoólico de angico sobre cepas de Staphylococcus aureus / Evaluation of the antimicrobial activity in vitro and determination of minimum the inhibitory concentration (MIC) of hidroalcoholic extracts of angico in strains Staphylococcus aureus

Plantas medicinais com propriedades antimicrobianas juntamente com a problemática da resistência microbiana são de grande relevância, principalmente em países de desenvolvimento,são uma ótima alternativa para investimentos em pesquisas, pois apontam para o uso de novos antibióticos que sejam eficazes diante dos patógenos emergentes. O objetivo desse estudo foi avaliar a atividade antimicrobiana in vitro do extrato hidroalcoólico da Anadenanthera macrocarpa (angico) e determinação da concentração inibitória mínima (CIM) sobre amostras de cepas de Staphylococcus aureus de origem humana. A atividade antimicrobiana e a CIM do extrato do angico frente a cepas de S. aureus foi determinada por difusão em placa com Ágar Mueller-Hinton usando o método de pour plate. Após solidificação do inóculo (1mL) adicionado ao ágar (25mL) foram feitas cavidades de 6mm, preenchidas com 50ul do extrato. Foram testadas 10 amostras de S. aureus e a atividade foi determinada pela presença de halo de inibição igual ou superior a 8mm. Todas as amostras monstraram-se sensiveis à ação do extrato de angico com halos de inibição variando de 19 a 25 mm. O extrato hidroalcoólico do angico apresentou atividade antimicrobiana e CIM de 3,12% (1:32) para todas as cepas de S. aureus testadas.

Avaliação da atividade antimicrobiana in vitro do extrato hidroalcóolico de Stryphnodendron adstringens(Mart) Coville sobre isolados ambulatoriais de Staphylococcus aureus / Evaluation of the antimicrobial activity in vitro of the hydroalcoholic extract Stryphnodendron adstringens against of Staphylococcus aureus

Staphylococcus aureus é um agente etiológico importante associado a infecções adquiridas, onde se destaca por frequencia elevada e sua patogenicidade, e que foi capaz de desenvolver rapidamente resistência aos antibióticos , constituindo uma séria ameaça à saúde´pública. As plantas tem sido uma fonte valiosa de produtos para manutenção da saúde humana e consideradas uma das melhores fontes para se obter uma variedade de drogas, entre elas, encontra-se o Stryphnodendron adstringens (basrbatimão) que apresenta uma entre outras ações, atividade antimicrobiana. A atividade antimicrobiana foi determinada pelo método de difusão em meio sólido, processo cavidade-placa e a concentração inibitória mínima foi calculada considerando a menor concentração do extrato capaz de inibir o crescimento bacteriano, após incubação a 37 graus Celsius durante 24 horas. O extrato hidroalcóolico de barbatimão foi testado frente ao extrato bruto (100%) e nas diluições referentes a concentrações 50%, 25%, 12,5%, 6,25%, 3,12%, 1,56% e 0,78%, onde foi observado que todas as cepas testadas foram sensíveis ao extrato, onde maior halo de inibição de crescimento bacteriano foi de 22mm, no extrato bruto (100%) e o menor de 8mm na diluição de 1:64 (1,56%)

Perfil de sensibilidade de Salmonella sp. de ambiente aquático a antimicrobianos comerciais e a extratos hidroalcoólicos de plantas medicinais / Profile of sensitivity of Salmonella sp. of water environment for commercial antimicrobials and the hydroalcoholic extracts of medical plants

Doenças de veiculação hídrica é a causa de grandes problemas de saúde pública no mundo, sendo a Salmonella sp. umrepresentante dessa classe de microrganismos patogênicos. A contaminação dos recursos hídricos vem acompanhada de outro fator agravante, o crescente aparecimento de cepas de Salmonella sp. resistentes aos antimicrobianos comerciais, demonstrando a importância de encontrar novas substâncias com atividade antimicrobiana. Este estudo objetivou verificar a sensibilidade de 28 cepas de Salmonella sp., isoladas de um rio impactado por esgotos em Campina Grande (PB), a antimicrobianos comerciais e analisar a atividadeantimicrobiana dos seguintes extratos hidroalcoólicos: Rosmarinus officinalis, Ximenia americana, Anadenanphera colubrina, Astronium uruncleura, Sprychnobendron abscringens e Anarcadium occidentale. As análises foram realizadas no Laboratório deMicrobiologia da Universidade Estadual da Paraíba (Campina Grande/PB). Na metodologia, foram usados métodos de difusão em placas com meio sólido Mueller-Hinton. Observou-se, diante dos antimicrobianos comercias, 6 (21,5) cepas resistentes à Tetraciclina,seguida por Cotrimoxazol com 1 (3,6) cepa resistente. Nenhuma resistência foi verificada à Amicacina, Amoxacilina, Aztreonam, Cefalotina, Cefoxitina, Cloranfenicol, Gentamicina, Imipenema e Lomefloxacina. Dos seis extratos hidroalcoólicos testados, todosapresentaram atividade antimicrobiana às cepas de Salmonella sp. testadas, apenas o Rosmarinus officinalis mostrou pouca atividade, sendo eficaz a 4 (14,3) das cepas testadas.