Autor(es):
Silva, Carla 
; Alverca, Elsa ; Matos, António Pedro ; Carvalho, Patricia A ; Portugal, Isabel ; Jordao, Luisa
Data: 2012
Identificador Persistente: http://hdl.handle.net/10400.18/1092
Origem: Repositório Científico do Instituto Nacional de Saúde
Assunto(s): Mycobacterium Tuberculosis; Microscopy; Infecções Respiratórias
Descrição
Abstrat publicado em: http://metallica.civil.ist.utl.pt/~patricia/SPMicros2012/book.pdf Tuberculosis (TB) is one of the major causes of mortality and morbidity worldwide accounting for 3.1 million deaths per year. This disease, caused by M. tuberculosis (Mtb) made a deadly, 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) Mtb strains, uncovered the most freighting face of this disease an incurable infection with the currently available therapeutic tools (1). 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 belong to a genetic related family of strains known as Lisboa family (2).
In the present work we studied a group of Mtb isolates collected in a Lisbon hospital during a two years period (2008-2009). The morphology of colonies grown on Lowenstein-Jensen (LJ) slants was studied by different electron microscopy (EM) techniques. The aim of the study was the establishment of a link between mycobacteria drug susceptibility and structure.
A total of 283 Mtb strains were collected during the period of the study. Among the isolates collected during 2008, 12% were resistant to at least one first line anti-bacillar drug, 5% were MDR and 5.2% XDR. During 2009, the percentage of XDR strains dropped to 2.8%, despite the increase of resistant and MDR strains to 19% and 5.6%, respectively.
In the first part of the study approximately 20 isolates, with different drug susceptibility profiles, were grown on LJ 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 scanning electron microscopy (SEM) analysis. The results obtained show that mycobacteria surface are distinct in susceptible and drug resistant strains as shown in figure 2.A-D. While drug susceptible mycobacteria have a homogenous surface (Fig. 1A), drug resistant bacteria present a heterogeneous surface (Fig. 2B) with small protrusions (Fig. 2C). In order to evaluate the existence of differences in the ultrastructure of circulating Mtb strains we started a study using transmission electron microscopy (TEM). For this approach were selected only two isolates (pan-susceptible: R188/09 and XDR: HPV108/09) which were chemically fixed, post-fixed with osmium tetroxide and further processed for epon embeeding.
The results obtained (Table 1) show that mycobacteria cell width ( 350 nm) is similar for both bacteria. 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 (3,4).
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 EM techniques, such as CEMOVIS, will avoid the formation of undesirable artefacts (e.g. mesossome) produced by dehydration and room temperature sectioning allowing a better characterization of mycobacteria ultrastructure (5).
The authors aknowledge the funding by Fundação para a Ciência e Tecnologia (SFRH/BD/73579/2010 and C2008-C2008_P2).
1. WHO 2011/ 2012 Tuberculosis Global Facts
2. Portugal I. Int J Tuberc Lung Dis 1999;3:207.
3. Takade A. Microb Immunol 2003; 47: 265.
4. Velayati AA. Chemotherapy 2009; 55:303.
5. Bleck CKE. J of Microscopy 2010; 237: 23.
