Browsing by Author "Reis, Lucia"
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- Biofilm-forming bacteria on the pharyngeal tonsil in a pediatric populationPublication . Rodrigues, João; Reis, Lucia; Subtil, Joao; Jordão, LuísaObjective: The main goal of this study is to evaluate the existence of a link between biofilm assembly on pharyngeal tonsil and the incidence of recurrent infections within a paediatric population. Methods: Nasal and pharyngeal tonsils scrubs as well as a biopsies of pharyngeal tonsils tissue were collected (n=70) and processed for bacterial identification using standard microbiologic methods. Bacteria were identified at the species level using a VITEC apparatus. Another pharyngeal tonsil biopsies were processed for electron microscopy. Initially, a screening for the presence of biofilms was performed using scanning electron microscopy (SEM) being the positive samples further processed for transmission electron microscopy (TEM). Biofilm assembly assay ability in vitro was evaluated by the microtiter-plate test. The assay was performed at 37ºC in Muller-Hinton broth for different periods of time. Results: As expected a vast number of bacterial species were identified in the samples. The majority were aerobic and a balance between Gram negative and positive bacteria was observed. A good correlation between the floras of the three samples from the same patient was found. The presence of biofilms pharyngeal tonsils was assessed by SEM. The data collected so far suggest that in at least 30% of the samples biofilms were present. In vitro all the microorganisms evaluated were able to assemble biofilms. Conclusion: Biofilm formation can be part of the mechanism involved both in onset and propagation of infections. Further studies must be performed to validate the thesis that pharyngeal tonsils function as a reservoir for microorganisms.
- 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.
- Pathogens in ornamental waters: a follow up studyPublication . Duarte, Maria; Morgado, Patrícia I.; Rodrigues, Joao; Reis, Lucia; Matias, Rui; Carvalho, Patricia A.; Duarte, Aida; Jordão, LuisaOrnamental waters of easy access and populated with animals are quite attractive and can hide threats to human health. Here we evaluated the microbiota of ornamental waters in a Lisbon park. Water and biofilm samples where collected, in 2 lakes (L1-L2) and ornamental fountains (L3-L4) in February/2015. In May/2015 and monthly during a year (starting March/2016) samples from L4 where collected. Microbiota identification was performed as described previouslya. Biofilm assembly was monitored by crystal violet assay and SEMb and antibiotic susceptibility was performed by conventional methods. The results of the first water sampling (Feb/2015) revealed the presence of Enterobactereaceae and non-fermentative oxidase-positive bacteria. Fountains and lakes presented different microbota being the highest diversity found in L1 hosting a duck population. This result suggested the existence of an interplay between animal inhabitants and microbiota which was confirmed by the second sampling of L4 (May/2015). Between the 2 sampling events a fish population was introduced and the microbiota was completely altered with the appearance of a typical fish pathogen (Aeromonas spp). This tendency was also confirmed over 2016. K. pneumoniae and Aeromonas spp., present as planktonic and biofilm organized bacteria in 2015 showed an enhanced ability to assemble biofilms in vitro at 25 °C than at 37 °C. Bacteria recovered from biofilm showed an increased antibiotic resistance compared to planktonic counterparts. The pilot study conducted during 2015 and the follow up study (still in progress) support a periodic control of ornamental water microbiota as simple preventive measure to avoid potential health issues.
- Prospective observational study of adenoidal biofilms in a paediatric population and their clinical implicationsPublication . Subtil, Joao; Bajanca-Lavado, Maria Paula; Rodrigues, Joao; Duarte, Aida; Reis, Lucia; Nogueira, Isabel; Jordão, LuisaINTRODUCTION: Adenoids are nasopharyngeal lymphoid tissue with a relevant role in host defence against infection of upper respiratory tract. Nevertheless, adenoids are also a reservoir of microorganisms that can cause infections of upper respiratory tract and otitis particularly in children. OBJECTIVE: Evaluate and compare the association between biofilm assembly on adenoids and the incidence of recurrent infections in a paediatric population submitted to adenoidectomy by either infectious or non-infectious indication. METHODS: Scanning electron microscopy was used to assess biofilms on adenoid surface; biofilm assembly in vitro was monitored by crystal violet assay; antibiotic susceptibility was assessed following EUCAST guidelines; Hinfluenzae capsular typing was performed by PCR. RESULTS: Biofilms were present in 27.4% of adenoid samples and no statistical difference was found between infectious and non-infectious groups. In vitro, the most clinically relevant bacteria, H.influenzae, S.aureus, S.pyogenes, S.pneumoniae and M.catarrhalis, were mostly moderate biofilm assemblers (71.7%). 55.3% of these bacteria were intermediate/resistant to at least one of the tested antibiotics. No association was found between the ability to assemble biofilms in vitro and the presence of biofilms on adenoids nor antibiotic resistance. All H.influenzae were characterized as non-typeable. CONCLUSION: The presence of biofilms on adenoid surface was independent from clinical sample background. Bacterial ability to assemble biofilms in vitro cannot be used to predict biofilm assembly in vivo. The lack of correlation between biofilm formation and infectious respiratory diseases found contributes to question the relevance of biofilms on the pathogenesis of infectious diseases.
- Relação entre biofilmes na amígdala faríngea e incidência de infeções recorrentes em população pediátrica - estudo observacional transversalPublication . Subtil, João; Jordao, Luisa; Freitas, Luis; Santos, A.; Araujo, J.; Duarte, Aida; Rodrigues, João Carlos; Reis, Lucia; Nogueira, Isabel; Carvalho, Patricia A; Macor, C.Objectivo: Avaliar a relação entre a formação de biofilmes na amígdala faríngea e a incidência de infecções respiratórias altas recorrentes na idade pediátrica. Métodos: Colheita de amostras da superfície do cavum faríngeo e meato médio por zaragatoas e biópsia da profundidade da amígdala faríngea de 70 indivíduos, analisados para identificar população bacteriana por métodos padronizados. A identificação foi obtida ao nível da espécie usado dispositivo VITEK2. Foram ainda colhidas biópsias superficiais da amígdala faríngea para processamento por microscopia electrónica. A avaliação inicial para identificar a presença de biofilmes foi feita por microscopia electrónica de varrimento, sendo as positivas avaliadas por microscopia electrónica de transmissão. A capacidade para formação de biofilme foi avaliada in vitro por teste em placa de micro-titulação. O ensaio foi efectuado a 37ºC em meio Muller-Hinton por durações diferentes. Resultados: Como esperado, um vasto número de bactérias foi identificado nas amostras. A maioria são espécies aeróbicas, entre Gram negativos e positivos. Foi encontrada correlação forte entre as floras das três localizações no mesmo indivíduo. A presença de biofilmes na amígdala faríngea foi identificada em 30% das amostras. Todos os micro-organismos avaliados mostraram in vitro capacidade para formação de biofilmes. Conclusão: A formação de biofilmes constitui aparentemente uma parte importante em ambos a instalação e propagação de infecções respiratórias altas. São necessários estudos posteriores para validar a tese de que a amígdala faríngea actua como reservatório de agentes infecciosos nestas infecções.
- Risk assessment for public health from human interaction with ornamental watersPublication . Duarte, Aida; Rodrigues, João Carlos; Reis, Lucia; Nogueira, Isabel; Carvalho, Patricia Almeida; Paulino, Sérgio; Sousa, Sara; Jordão, LuísaWater is essential to life; nevertheless ingestion of contaminated water could result in death caused by waterborne diseases such as cholera. Pathogens present in the water can cause diseases, other than those resulting from water ingestion, being registered an increase in the number of case reports in recent years. It is not clear if this increase is due either to a better case reporting system or to an increase in microorganism’s virulence. The generalized use of antibiotics in agriculture and animal farming contributed to their dissemination in the environment which promotes microorganism’s selection and emergence of resistant strains. This phenomenon can be enhanced by the ability of microorganism to persist within complex communities known as biofilms. In the present work we aim to characterize the microbial population present in ornamental waters and perform a risk assessment for public health resulting from human interaction with it. Samples were collected in the Lisboa area between December 2014 and February 2015. Bacteria were identified either by growth on non-selective/ selective media incubated at different temperatures or VITEC after filtration through a 0.45m membrane. Antibiotic susceptibility tests were performed and analyzed for planktonic bacteria according to EUCAST. Biofilm assembly, sample preparation for scanning electron microscopy (SEM) and minimal inhibitory concentration for amoxicillin (AMX) were performed as described previously. In all water samples more than 300 colony forming units (CFU) per 10 mL were found on plate count agar. The majority of the identified microorganisms were members of Enterobacteriacea family namely, Klebsiella peneumoniae (Kp), K. pneumoniae ozaenae, Enterobacter spp., Serratia marcescens, S. rubidea e S. odorífera. Non fermentative, oxidase positive bacteria such as Elizabethkingia meningoseptica, Stenotrophomonas maltophilia and Vibrio metschnikovii were also identified. The presence of a main etiological agent of healthcare associated infections (HAI) with high rates of antibiotic resistance among the isolates is an interesting and concerning result. Since Kp resistance to antibiotics is linked to biofilm assembly we decided to study biofilms present in the lakes inhabit by this bacterium. Kp biofilms were present only in one of the lakes. Further analysis of the Kp isolates rendered the results showed in table 1. All the bacteria were able to assemble biofilms in conditions mimicking those present in the environment (25ºC). This ability is decreased at 37ºC or even absent (Kp2). Bacteria fitness to environmental conditions might suggest a diminish ability to colonize human beings. The environmental fitness was further documented by analysis of the biofilms using SEM. The best organized biofilms were assembled on cement at 25ºC. Both temperature and surface changes lead to a decrease in biofilm assembly. In contrast to clinical isolates environmental Kp were susceptible to the 3 main groups of antibiotics: -lactamic, aminoglycosides and quinolones. In other words environmental Kp do not meet the criteria to be classified as multi-resistant bacteria. For this reason only biofilm susceptibility to amoxicillin was assayed. Biofilm assembly increased antibiotic resistance of Kp. Two isolates (Kp3 and 4) exhibited the same MIC value (500 µg/mL) which is significantly higher than the MICs founds for Kp2 (0.98 g/mL) and Kp3 (7.81g/mL). This result together with the fact that Kp3 and 4 were isolated from the same lake lead us to hypothesize that the two isolates might be part of the same biofilm being the presence of Kp3 in the water explained by biofilm dispersion. The low risk of environmental Kp for human health and the universal role played by biofilm assembly on antibiotic resistance are the main conclusions arising from this study.