Browsing by Author "Carvalho, Patricia A."
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- Morphologic characterization of Mycobacterium tuberculosis circulating strains in a Lisbon hospitalPublication . Silva, Carla; Alverca, Elsa; Matos, António Pedro Alves; Carvalho, Patricia A.; Portugal, Isabel; Jordão, LuísaTuberculosis (TB) is one of the major causes of mortality and morbidity worldwide accounting for 3.1 million deaths per year. This disease, caused by Mycobacterium tuberculosis (M. tuberculosis) made a deadly comeback, during the 1990’s, triggered mainly by the emergence of acquired immunodeficiency syndrome (AIDS). More recently, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) M. tuberculosis strains, uncovered the most freighting face of this disease an incurable infection with the currently available therapeutic tools. Although Portugal is considered a medium incidence setting, annually are reported MDR and even XDR TB cases. The majority of these cases occur in the Lisbon area and the strains involved are genetically related being known as Lisboa family. In the present work a group of 283 M. tuberculosis isolates collected in a Lisbon hospital during a two years period (2008-2009) were studied. The morphology of colonies grown on Lowenstein-Jensen slants was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using previously described procedures. The aim of the study was the establishment of a link between mycobacteria drug susceptibility and structure. In the first part of the study approximately 20 isolates, with different drug susceptibility profiles ranging from pan-susceptible to XDR, were grown on Lowenstein-Jensen slants and their morphology was compared. Although all mycobacteria originated rough colonies their size differ with the drug susceptibility profile. The pan-susceptible strains generated larger colonies than drug resistant strains as shown in figure 1.These colonies were then processed for SEM analysis. The results obtained show that mycobacteria surface are distinct in susceptible and drug resistant strains as shown in figure 2.A and B. While drug susceptible mycobacteria have a homogenous surface (Figure 1A), drug resistant bacteria present a heterogeneous surface (Figure 2B) with small protrusions (Fig. 2B inset). In order to evaluate the existence of differences in the ultrastructure of circulating M. tuberculosis strains the colonies were processed and analysed by TEM. For this approach were selected only two isolates: the pan-susceptible R188/09 and the XDR HPV108/09. The results obtained by the analysis of at least 300 bacteria present in non consecutive sections show that mycobacteria cell width (0 350 nm) is similar for both bacteria (Table 1). Nevertheless, their cell length and cell envelope width are significantly different. The XDR strain is shorter (p=0.009) and has a ticker cell envelope (p=0.004) than the pan-susceptible strain. These results are in agreement with those published in the literature. Altogether our data clearly shows the existence of a link between mycobacteria ultrastructure and drug susceptibility. In order to better evaluate these differences a larger number of isolates must be studied. The use of other electron microscopy techniques, such as CEMOVIS, will avoid the formation of undesirable artefacts (e.g. mesosome) produced by dehydration and room temperature sectioning allowing a better characterization of mycobacteria ultrastructure.
- 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.