Browsing by Author "Matias, Rui"
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- Aeromonas spp. in Freshwater Bodies: Antimicrobial Resistance and Biofilm AssemblyPublication . Nascimento, Maria; Rodrigues, João; Matias, Rui; Jordão, LuisaAeromonas spp. are environmental bacteria able to infect animals and humans. Here, we aim to evaluate the role of biofilms in Aeromonas persistence in freshwater. Aeromonas were isolated from water and biofilm samples and identified by Vitek-MS and 16S rRNA sequencing. Antibiotic susceptibility profiles were determined according to EUCAST, and a crystal violet assay was used to assess biofilm assembly. MTT and the enumeration of colony-forming units were used to evaluate biofilm and planktonic Aeromonas susceptibility to chlorination, respectively. Identification at the species level was challenging, suggesting the need to improve the used methodologies. Five different Aeromonas species (A. salmonicida, A. hydrophila, A. media, A. popoffii and A. veronii) were identified from water, and one species was identified from biofilms (A. veronii). A. veronnii and A. salmonicida presented resistance to different antibiotics, whith the highest resistance rate observed for A. salmonicida (multiple antibiotic resistance index of 0.25). Of the 21 isolates, 11 were biofilm producers, and 10 of them were strong biofilm producers (SBPs). The SBPs presented increased tolerance to chlorine disinfection when compared with their planktonic counterparts. In order to elucidate the mechanisms underlying biofilm tolerance to chlorine and support the importance of preventing biofilm assembly in water reservoirs, further research is required.
- 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.
- Biofilmes de aeromonas sp na água e risco de infeção para o HomemPublication . Meneses, Madalena; Matias, Rui; Rodrigues, Joao; Jordão, Luisaintrodução: As Aeromonas spp. são bactérias oportunistas, responsáveis por infeções no Homem e nos animais. Estas bactérias, frequentemente isoladas do ambiente aquático, possuem diversos fatores de virulência sendo responsáveis pelas infeções intestinais e extraintestinais no ser humano. Os biofilmes são estruturas compostas por microrganismos envolvidos por uma matriz extracelular por eles secretada. No caso dos biofilmes ambientais, existe uma variedade de microrganismos, incluindo, muitas vezes, fungos, microalgas e protozoários. Os biofilmes podem, ou não, estar ligados a superfícies bióticas ou abióticas e diferem, em muitos aspetos, dos seres planctónicos. Em comparação com bactérias isoladas (planctónicas), as que residem em comunidade (biofilme) encontram-se mais protegidas contra vários stresses ambientais. Segundo a literatura, apesar de microrganismos isoladas serem relativamente suscetíveis à desinfeção, populações associadas em biofilmes podem sobreviver a altas doses de cloro.
- Biofilms and catheter related bloodstream infection: a tale of two kigdomsPublication . Borges, Vítor; Wenner, Sigurd; Nogueira, Isabel; Faria, Isabel; Pessanha, Maria Ana; Verissimo, Cristina; Sabino, Raquel; Rodrigues, Joao; Matias, Rui; Martins, Filomena; Carvalho, Patricia; Gomes, Joao Paulo; Jordão, LuísaBackground: Biofilm-associated infections are a public health concern in the context of healthcare-associated infections (HAI) such as catheter-related bloodstream infections (CRBSI). Here, we studied two top ten CRBS etiological agents, Enterobacter cloacae and Candida parapsilosis, isolated from a patient with CRBSI in order to understand the role played by biofilms on this HAI. Materials/methods: E.cloacae and C.parapsilosis were isolated from CVC and peripheral blood by standard procedures. EUCAST guidelines were followed for antimicrobial susceptibility evaluation. Single and/or mixed biofilms were assembled on different materials in Mueller-Hinton broth with 2% glucose. Biofilm assembly was assessed by crystal violet assay and scanning electron microscopy (SEM). Fluorescence in situ hybridization (FISH) was used for identification and to assess microorganisms distribution within the biofilm (3D reconstruction). In addition, Focus Ion Beam (FIB)-SEM was used to assess biofilms assembled on inner and outer surfaces of CVCs and construct tomograms. CVC and hemoculture (HC) isolates were subjected to whole-genome sequencing (WGS). Results: All Enterobacter and Candida isolates were antimicrobial resistant. Of note, E. cloacae-CVC revealed an additional resistance (ceftolozame-tazobactam) in comparison to the HC- isolate. Both microorganisms assembled biofilms on glass, polystyrene and polyurethane. Mixed biofilms were denser when both microorganisms were present from the beginning. Biofilm phenotype was not dependent of biofilm initiation by E.cloacae or C.parapsilosis. FISH and SEM analysis showed that biofilm bottom layer was in all cases richer in E.cloacae. Environmental isolates of the same species were also tested, showing that this biofilm phenotype is not a general feature. Using polyurethane catheters (shape/material factor), we observed denser mixed biofilms richer in EPS. FIB-SEM preliminary results suggest that biofilms assembled on inner and outer catheter surface might differ on microorganisms’ distribution. WGS confirmed the genetic identity of the CVC/HC pairs while corroborating the virulence potential and antimicrobial resistant character of the CRBSI-driving pathogens. Conclusions: The results suggest that biofilms allow interaction and adaptation of microorganisms belonging to different kingdoms (Bacteria and Fungi). Adaptation might affect virulence in a transitory or permanent fashion, with potential impact on microorganisms’ potential to cause CRBSI.
- Caracterização da população bacteriana em barragens na bacia hidrográfica do SadoPublication . Sousa, Maria; Morgado, Patrícia; Rodrigues, João; Matias, Rui; Nogueira, Isabel; Jordão, LuísaAs barragens são fundamentais para a gestão dos recursos hídricos. Estas infraestruturas permitem regular os caudais e aproveitar a água para diversas atividades como a agricultura, pecuária, captação para consumo humano e turismo (p.e. praias fluviais e despor tos aquáticos). Neste estudo, foi avaliada a flora bacteriana (planctónica e organizada em biofilmes) presente em 4 barragens da bacia hidrográfica do rio Sado. A caracterização da flora bacteriana presente para além de permitir a caracterização do ecossistema permite inferir sobre potenciais riscos para a saúde humana decorrentes dos diversos usos da água.
- Catheter related bloodstream infection caused by E. cloacae and Candida parapsilosis: Are biofilms guilty?Publication . Štefánek, Matúš; Borges, Vítor; Wenner, Sigurd; Nogueira, Isabel D.; Pinto, Miguel; Faria, Isabel; Pessanha, Maria Ana; Veríssimo, Cristina; Sabino, Raquel; Rodrigues, João; Matias, Rui; Carvalho, Patrícia Almeida; Gomes, João Paulo; Bujdáková, Helena; Jordao, LuisaBiofilm-associated infections is a public health concern in the context of healthcare associated infections (HAI) such as catheter related bloodstream infections (CRBSI). Here the dynamics of two top ten etiological agents of CRBSI, Enterobacter cloacae and Candida parapsilosis isolated from a CRBSI’s patient, were studied to get insights on the role played by biofilms on this HAI. Antimicrobial susceptibility of CVC and HC’s isolates was evaluated according to EUCAST guidelines. Single and/or mixed biofilms assembled on different materials in Mueller-Hinton broth with 2% glucose were assessed by crystal violet assay and scanning electron microscopy (SEM). Fluorescence in situ hybridization (FISH) was used for identification purposes and to assess microorganisms distribution within the biofilm (3D reconstruction) complemented with Focus Ion Beam (FIB)-SEM to assess biofilms assembled on the inner/outer CVC’s surfaces (tomograms). Whole-genome sequencing (WGS) was performed for all isolates. All isolates were antimicrobial resistant. Of note E.cloacae resistance to collistin and an additional resistance of the CVC compared to HC-isolate (ceftolozame-tazobactam) probably linked to a mutation in rpoB gene. Candida resistance to fluconazol might be explained by ERG11 gene mutation. Enterobacter and Candida assembled biofilms on glass, polystyrene and polyurethane being mixed biofilms denser when both microorganism were present from the beginning. FISH and SEM analysis showed that biofilm bottom layer was in all cases richer in E.cloacae. Using environmental isolates of the same species we showed that this biofilm phenotype is not a general feature. Using polyurethane catheters (shape/material factor), denser mixed biofilms richer in EPS were observed. A distinct phenotype was present on the patient’s CVC by SEM and FIB/SEM. WGS confirmed the genetic identity of the pair CVC/HC isolates, while corroborating the virulence potential and observed antimicrobial resistant character of the studied CRBSI-driving pathogens. The results suggest that biofilms allow interaction and adaptation of microorganisms belonging to different kingdoms (Bacteria and Fungi). Adaptation might affect virulence in a transitory or permanent fashion, with potential impact on microorganisms’ potential to cause CRBSI.
- Cephalosporin-resistant Neisseria gonorrhoeae isolated in Portugal, 2019Publication . Pinto, Miguel; Matias, Rui; Rodrigues, João Carlos; Duarte, Sílvia; Vieira, Luís; Gonçalves, Inês; Gonçalves, Maria João; Ramos, Maria Helena; Gomes, João Paulo; Borrego, Maria JoséWe report a multidrug resistant Neisseria gonorrhoeae exhibiting resistance to ceftriaxone and cefixime, isolated in Portugal in 2019. Whole-genome sequencing was performed for typing and identification of genetic determinants of antimicrobial resistance. Due to its antimicrobial susceptibility profile, awareness should be raised for the circulation of this strain.
- Diversity of bacterial communities and genes encoding AMR in different environmental compartments along the food/feed chainPublication . Manageiro, Vera; Cano, Manuela; Dias, Elsa; Rosado, Tânia; Vieira, Patrícia; Reis, Lígia; Matias, Rui; Rodrigues, João; Coelho, Rodrigo; Menezes, Carina; Ferreira, Eugénia; Sequeira, António; Moreira, Olga; Caniça, ManuelaAim: The aim of this study was to determine the microbial biodiversity and naturally occurring antibiotic resistance (AR)-encoding genes background load along the food/feed chain in an open-air agricultural testing catchment (HOAL). Methods: Samples were collected in two time points during the year 2020 from various – but interconnected – environmental compartments of the food/feed chain within an HOAL catchment, such as air, pig feces, manure, soil, water, crops, and feed. All samples were homogenized, diluted and plated in selective media. Colonies were selected andidentification made by MALDI-TOF. Antibiotic susceptibility (AST) was assessed by disc diffusion and/or MIC methods. DNA was extracted for further molecular and genomic characterization. Results: A total of 753 Gram-negative strains were isolated. The highest number of bacteria were from families of Enterobacteriaceae(44.1%), Morganellaceae(15.5%), Moraxellaceae(15.5%), and Pseudomonadaceae(9.3%). Enterobacteriaceae and Pseudomonadaceae were present in all but one of the tested compartments (ground water and feed, respectively). Of notice, 43.1% of Enterobacteriaceae identified were isolated from a selective medium supplemented with colistin 0.5mg/L and 8.7% were selected with cefotaxime 2mg/L. Extended-spectrum -lactamase (ESBL)-producing strains were identified. Regarding Gram-positive bacteria, we identified mainly Enterococcaceae (46.7%), Bacillaceae (30.6%), and Streptococcaceae (13.9%). Pig faces and manure were the compartments with higher bacterial biodiversity. Phenotypic AST revealed the presence of resistances against several AR classes. Based on this data, strains were classified as multidrug-resistant. Conclusions: The investigation of clinically important AR-encoding genes by PCR-amplification and whole-genome-sequencing of selected strains will contribute to clarify the resistome and microbial biodiversity in the tested environmental compartments.
- Environmental biofilms, consequences for health and diseasePublication . Morgado, Patrícia I.; Reis, Lucia; Matias, Rui; Grossinho, Jose; Rebelo, Helena; Rodrigues, Joao; Jordao, LuisaOrnamental waters of easy access and populated with animals are quite attractive and can hide threats to human health. Here we evaluated, during one year, the microbiota of ornamental waters in a Lisbon park in order to assess the risk for human health. A total of 26 different bacterial species were identified during the study. Among these are potential human pathogens such as Aeromonas, E. coli, K. pneumoniae, Pseudomonas and Raoutella. Aeromonas sobria is a resident bacterium whereas other species such as A. hydrophila and A. veronii were sporadically identified. A possible explanation for this result is the presence of a fish population. The presence of E. coli indicates water contamination with fecal materials. We first isolate this bacterium in June when the water temperature increased and then after it was a resident despite the natural temperature drop in the autumn. This might be explained by the replace of the fish population by ducks in October which lead to an increase in water tubirdity and total carbon content. Pseudomonas fluorescens, P. putida and P. oryzihabitans have been considered low-virulence bacteria but recently have been identified as ethiological agents of healthcare assotiated infections (HAI) mostly in immunosupressed individuals. Another recognized HAI agent present is K. pneumoniae. The same bacteria species were isolated from biofilm and water suggesting the existence of an equilibrium between planktonic and biofilm organized bacteria. The role played by biofilm assembly on the emergence of antibiotic resistance is still ongoing for the isolated bacteria, as well as the possible interaction between water chemical composition and bacteria isolates. Potential pathogens (biofilm or planktonic) represent a significant cause of acute bacterial infections mainly in specific populations (paediatric and geriatric). These results support a periodic control of ornamental water microbiota as simple preventive measure to avoid potential health issues.
- Epidemiological Data and Antimicrobial Resistance of Campylobacter spp. in Portugal from 13 Years of SurveillancePublication . Duarte, Andreia; Pereira, Luísa; Lemos, Maria-Leonor; Pinto, Miguel; Rodrigues, João Carlos; Matias, Rui; Santos, Andrea; PTCampyNet; Oleastro, MónicaThis study extensively analyzed campylobacteriosis surveillance in Portugal from 2009 to 2021, aiming to investigate demographic shifts, seasonal variations, and antimicrobial resistance (AMR) within Campylobacter isolates. Surveillance network and sentinel laboratory-based system data revealed a substantial under-notification of campylobacteriosis cases, suggesting an underestimated disease burden. Notification rates exhibited a paradigm shift, with a notable prevalence among the pediatric population, particularly in children aged 1-4 years, diverging from European reports. Additionally, an emerging trend of Campylobacter infections in younger adults (15-44 years) was observed. The study unveiled a unique seasonal distribution of cases, defying typical summer peaks seen elsewhere. AMR analysis revealed high resistance to ciprofloxacin and tetracycline, in both C. jejuni (93.7% and 79.2%, respectively) and C. coli (96.5% and 93.2%, respectively), stable throughout the studied period (2013-2021). C. coli exhibited significantly higher resistance to erythromycin, gentamicin, ampicillin and ertapenem compared to C. jejuni (p < 0.001). Multilocus Sequence Typing (MLST) data demonstrated the distribution of resistance markers across diverse sequence types, challenging the notion of a clonal origin for multidrug-resistant isolates. In conclusion, the study highlights the need for enhanced surveillance and raises concerns about alarming AMR levels, recommending the implementation of whole-genome sequencing (WGS)-based surveillance for a deeper comprehension of disease patterns and an evolving AMR landscape.