Browsing by Author "Castro, Rita"
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- Assessment of the Transmission Dynamics of Clostridioides difficile in a Farm Environment Reveals the Presence of a New Toxigenic Strain Connected to Swine ProductionPublication . Alves, Frederico; Nunes, Alexandra; Castro, Rita; Sequeira, António; Moreira, Olga; Matias, Rui; Rodrigues, João Carlos; Silveira, Leonor; Gomes, João Paulo; Oleastro, MónicaThe recent increase in community-acquired Clostridioides difficile infections discloses the shift in this bacterium epidemiology. This study aimed at establishing a transmission network involving One Health components, as well as assessing the zoonotic potential and genomic features of dominant clones. Samples were collected from different compartments of animal, human and environmental origin, from an animal production unit. C. difficile isolates were characterized for toxigenic profile by multiplex-PCR, while genetic diversity was evaluated by PCR-ribotyping and whole genome-based analysis. The overall C. difficile prevalence was 37.2% (70/188), and included samples from environmental (58.3%, 35/60) and animal (31.5%, 35/111) compartments; human samples (n = 17) taken from healthy workers were negative. A predominant clone from RT033 was found in almost 90% of the positive samples, including samples from all compartments connected to the pig production unit, with core-genome single nucleotide variant (SNV)-based Analysis supporting a clonal transmission between them (mean distance of 0.1 ± 0.1 core-SNVs). The isolates from this clone (herein designated PT RT033) were positive for all C. difficile toxin genes (tcdA, tcdB, cdtA/cdtB). The phyloGenetic positioning of this clone was clearly distinct from the classical RT033 cluster, suggesting a different evolutionary route. This new clone shares genomic features with several RTs from the clade 5 Sequence Type (ST) 11, including a complete pathogenicity locus (PaLoc) that is more similar to the one found in toxigenic strains and contrasting to the less virulent classical RT033 (tcdA-, tcdB-, cdtA + /cdtB +). The presence of a tcdA gene truncated into two ORFs, not previously described, requires further evaluation concerning toxin functionality. We hypothesize that the unique combination of genetic elements found in the PT RT033 clone may contribute to host tropism and environmental dissemination and maintenance. This study constitutes the first report of a toxigenic RT033 clone and adds to the overall knowledge on Clade 5 sequence type 11, considered the C. difficile evolutionary lineage with the highest zoonotic potential. The presence of this clone in all compartments associated with the pig production unit suggests a transmission chain involving these animals and contributes to unveil the role played by animal and environmental reservoirs in this pathogen epidemiology.
- Caracterização genotípica e fenotípica de estirpes de Escherichia coli patogénicas, Salmonella spp. e Campylobacter spp. isoladas de aves em liberdade em Portugal continentalPublication . Batista, Rita; Saraiva, Margarida; Lopes, Teresa; Silveira, Leonor; Coelho, Anabela; Furtado, Rosália; Castro, Rita; Correia, Cristina Belo; Rodrigues, David; Henriques, Pedro; Lóio, Sara; Soeiro, Vanessa; Martins da Costa, Paulo; Oleastro, Mónica; Pista, ÂngelaAs aves são potenciais portadoras de microrganismos patogénicos que afetam os seres humanos, e podem ser disseminadoras de perigos no ambiente de produção primária de géneros alimentícios de origem vege- tal e animal. Este trabalho teve como objetivo avaliar a ocorrência, em fezes de aves em liberdade, em Portugal, de três bactérias zoonóticas causadoras de infeções no Homem. Para tal, foi avaliada a presença de Escherichia coli (E. coli), Salmonella spp. e Campylobacter spp. em 108 amostras individuais de fezes de aves e em uma amostra em pool de 50 amostras de fezes de gaivotas. Foi efetuada a caracterização fenotípica dos isolados (serotipagem e perfis de resistência a antibióticos) e dete- tados genes específicos associados à patogenicidade e à resistência a antimicrobianos, por PCR e/ou sequenciação total do genoma (WGS). Isolados de E. coli patogénicos, Salmonella spp. e Campylobacter spp. foram detetados em 8,9%, 2,8% e 9,9% das amostras, respetivamente. A resistência a antimicrobianos foi testada em 54 isolados de E. coli, tendo sido detetada em 14 (25,9%). Onze destes isolados revelaram a presença de fatores de virulência, E.coli patogénicos. Dez dos isolados de E. coli revelaram ser resistentes a múltiplos antimicrobianos (MDR) e sete eram produtores de β-lactamases de espectro alargado (ESBL). Re- lativamente aos isolados de Salmonella spp. (n=3) e Campylobacter spp. (n=9), apenas uma estirpe de Campylobacter jejuni foi identificada como MDR. A maioria dos serotipos e/ou Sequence Types (ST) identificados já tinham sido referenciados como associados a doença humana. Estes resultados mostram que as aves que fazem parte da fauna portuguesa podem ser portadoras de bactérias patogénicas capazes de causar do- ença humana, algumas delas resistentes a antimicrobianos críticos.
- Escherichia coli patogénica, Salmonella spp. e Campylobacter spp. em dois Centros de Conservação da Vida Selvagem em Portugal: caracterização genotípica e fenotípicaPublication . Pista, Ângela; Silveira, Leonor; Ribeiro, Sofia; Fontes, Mariana; Castro, Rita; Coelho, Anabela; Furtado, Rosália; Lopes, Teresa; Maia, Carla; Mixão, Verónica; Borges, Vítor; Sá, Ana; Soeiro, Vanessa; Correia, Cristina Belo; Gomes, João Paulo; Saraiva, Margarida; Oleastro, Mónica; Batista, RitaA coexistência entre humanos e animais selvagens pode aumentar o risco de transmissão direta de agentes patogénicos zoonóticos emergentes ou reemergentes para humanos. Este trabalho teve como objetivo avaliar a ocorrência de três importantes agentes patogénicos de origem alimentar em animais selvagens de dois centros de conservação da vida selvagem, em Portugal. Para tal, foram testadas 132 amostras fecais para a presen- ça de Escherichia coli (E. coli produtora de toxina Shiga (STEC) e não pro- dutora de toxina Shiga (não-STEC)), Salmonella spp. e Campylobacter spp.. Foi realizada a caracterização genotípica (pesquisa de genes de vi- rulência, pesquisa de genes de resistência a antimicrobianos (AMR), se- quenciação total do genoma (WGS)) e fenotípica (serotipagem e perfis de AMR) de todos os isolados de interesse. No geral, 62 amostras testaram positivo para pelo menos uma das espé- cies analisadas: 27,3% para STEC, 11,4% para não-STEC, 3,0% para Sal- monella spp. e 6,8% para Campylobacter spp. Foi detetada resistência a antimicrobianos em quatro isolados de E. coli e no único isolado de Cam- pylobacter coli. A análise de WGS revelou que 57,7% (30/52) das E. coli patogénicas integram agrupamentos genéticos de isolados fortemente relacionados (muitas vezes envolvendo diferentes espécies de animais), indicando a existência de circulação e transmissão de diferentes estirpes patogénicas de E. coli nas áreas estudadas. Estes resultados apoiam a ideia de que a saúde dos seres humanos, dos animais e dos ecossistemas são interdependentes, reforçando a importân- cia de uma abordagem One Health (Uma Só Saúde) para melhor monitori- zar e controlar as ameaças em saúde pública
- Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland PortugalPublication . Batista, Rita; Saraiva, Margarida; Lopes, Teresa; Silveira, Leonor; Coelho, Anabela; Furtado, Rosália; Castro, Rita; Correia, Cristina Belo; Rodrigues, David; Henriques, Pedro; Lóio, Sara; Soeiro, Vanessa; da Costa, Paulo Martins; Oleastro, Mónica; Pista, AngelaBirds are potential carriers of pathogens affecting humans and agriculture. Aiming to evaluate the occurrence of the top three most important foodborne pathogens in free-living birds in Portugal, we investigated 108 individual fecal samples from free-living birds and one pooled sample of gull feces (n = 50) for the presence of Escherichia coli (pathogenic and non-pathogenic), Salmonella spp. and Campylobacter spp. Virulence- and antimicrobial resistance- (AMR) associated genes were detected by PCR and Whole-Genome Sequencing (WGS), and phenotypic (serotyping and AMR profiles) characterization was performed. Overall, 8.9% of samples tested positive for pathogenic E. coli, 2.8% for Salmonella spp., and 9.9% for Campylobacter spp. AMR was performed on all pathogenic isolates and in a fraction of non-pathogenic E. coli, being detected in 25.9% of them. Ten of the tested E. coli isolates were multidrug-resistant (MDR), and seven of them were Extended-spectrum β-lactamase (ESBL) producers. Among Salmonella (n = 3) and Campylobacter (n = 9), only one strain of C. jejuni was identified as MDR. Most of the identified serotypes/sequence types had already been found to be associated with human disease. These results show that free-living birds in Portugal may act as carriers of foodborne pathogens linked to human disease, some of them resistant to critically important antimicrobials.
- Molecular epidemiology of Clostridioides difficile in companion animals: Genetic overlap with human strains and public health concernsPublication . Alves, Frederico; Castro, Rita; Pinto, Miguel; Nunes, Alexandra; Pomba, Constança; Oliveira, Manuela; Silveira, Leonor; Gomes, João Paulo; Oleastro, MónicaIntroduction: The changing epidemiology of Clostridioides difficile reflects a well-established and intricate community transmission network. With rising numbers of reported community-acquired infections, recent studies tried to identify the role played by non-human reservoirs in the pathogen's transmission chain. This study aimed at describing the C. difficile strains circulating in canine and feline populations, and to evaluate their genetic overlap with human strains to assess the possibility of interspecies transmission. Methods: Fecal samples from dogs (n = 335) and cats (n = 140) were collected from two populations (group A and group B) in Portugal. C. difficile isolates were characterized for toxigenic profile and PCR-ribotyping. The presence of genetic determinants of antimicrobial resistance was assessed in all phenotypically resistant isolates. To evaluate the genetic overlap between companion animals and human isolates from Portugal, RT106 (n = 42) and RT014/020 (n = 41) strains from both sources were subjected to whole genome sequencing and integrated with previously sequenced RT106 (n = 43) and RT014/020 (n = 142) genomes from different countries. The genetic overlap was assessed based on core-single nucleotide polymorphism (SNP) using a threshold of 2 SNP. Results: The overall positivity rate for C. difficile was 26% (76/292) in group A and 18.6% (34/183) in group B. Toxigenic strains accounted for 50% (38/76) and 52.9% (18/34) of animal carriage rates, respectively. The most prevalent ribotypes (RT) were the toxigenic RT106 and RT014/020, and the non-toxigenic RT010 and RT009. Antimicrobial resistance was found for clindamycin (27.9%), metronidazole (17.1%) and moxifloxacin (12.4%), associated with the presence of the ermB gene, the pCD-METRO plasmid and point mutations in the gyrA gene, respectively. Both RT106 and RT014/020 genetic analysis revealed several clusters integrating isolates from animal and human sources, supporting the possibility of clonal interspecies transmission or a shared environmental contamination source. Discussion: This study shows that companion animals may constitute a source of infection of toxigenic and antimicrobial resistant human associated C. difficile isolates. Additionally, it contributes with important data on the genetic proximity between C. difficile isolates from both sources, adding new information to guide future work on the role of animal reservoirs in the establishment of community associated transmission networks and alerting for potential public health risk.
- Pathogenic Escherichia coli, Salmonella spp. and Campylobacter spp. in Two Natural Conservation Centers of Wildlife in Portugal: Genotypic and Phenotypic CharacterizationPublication . Pista, Angela; Silveira, Leonor; Ribeiro, Sofia; Fontes, Mariana; Castro, Rita; Coelho, Anabela; Furtado, Rosália; Lopes, Teresa; Maia, Carla; Mixão, Verónica; Borges, Vítor; Sá, Ana; Soeiro, Vanessa; Correia, Cristina Belo; Gomes, João Paulo; Saraiva, Margarida; Oleastro, Mónica; Batista, RitaHuman–wildlife coexistence may increase the potential risk of direct transmission of emergent or re-emergent zoonotic pathogens to humans. Intending to assess the occurrence of three important foodborne pathogens in wild animals of two wildlife conservation centers in Portugal, we investigated 132 fecal samples for the presence of Escherichia coli (Shiga toxin-producing E. coli (STEC) and non-STEC), Salmonella spp. and Campylobacter spp. A genotypic search for genes having virulence and antimicrobial resistance (AMR) was performed by means of PCR and Whole-Genome Sequencing (WGS) and phenotypic (serotyping and AMR profiles) characterization. Overall, 62 samples tested positive for at least one of these species: 27.3% for STEC, 11.4% for non-STEC, 3.0% for Salmonella spp. and 6.8% for Campylobacter spp. AMR was detected in four E. coli isolates and the only Campylobacter coli isolated in this study. WGS analysis revealed that 57.7% (30/52) of pathogenic E. coli integrated genetic clusters of highly closely related isolates (often involving different animal species), supporting the circulation and transmission of different pathogenic E. coli strains in the studied areas. These results support the idea that the health of humans, animals and ecosystems are interconnected, reinforcing the importance of a One Health approach to better monitor and control public health threats.