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- Using whole genome sequencing to understand host-nontuberculous mycobacteria interactionPublication . Sousa, Sara; Borges, Victor; Faria, Sónia; Carneiro, Catarina; Vieira, Luís; Gomes, João Paulo; Jordão, LuísaBackground: Nontuberculous mycobacteria (NTM) are a large group of Mycobacterium species that don’t belong to the Mycobacterium tuberculosis (Mtb) complex. These bacteria are mostly environmental being regarded as etiological agents of opportunistic infection in humans mainly immunocompromised. Distinguishing NTM from Mtb is still a challenge and identification at the species level is essential for an accurate diagnostic and effective treatment. The growth rate of NTM is very important for the onset of treatment being these bacteria divided into rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM). Mycobacterium fortuitum, M. abscessus and the model organism M. smegmatis are RGM whereas M. avium is SGM that take more than 7 days to form CFU on growth media. The knowledge of NTM infections is still reduced being needed more studies to understand the host-pathogen interaction during the infectious process in order to establish more effective therapeutic schemes. Recently our group conducted a study using human alveolar macrophages as a model. The obtained data showed that both RGM (747/08) and SGM (60/08) were able to persist and even replicate within this macrophages whereas others do not (M.smegmatis and M.abscessus)1. Here we used NGS as a tool to identify bacterial factor responsible for the observed outcome. Materials and Methods: Three reference strains (M.fortuitum ATCC6841, M.avium ATCC25291, M.smegmatis (ATCC700084) and 3 strains from Ricardo Jorge mycobacterial collection isolated from patients (M.fortuitum 747/08, M. avium 60/08 and Mtb70/09) were used. For DNA extraction bacteria were grown in Middlebrook 7H9 supplemented with 10% OADC and 0.05% Tween80. Full genome sequence was performed using NGS platform MiSeq (Illumina Inc., San Diego,CA, USA) according to the manufacturer’s instructions. Data analysis: RAST (www.rast.nmpdr.org) and MAUVE platforms were used for annotation and multiple alignments, respectively.3 Results: Data analysis suggest a link between mycobacteria growth rate and genome size with RGM (6.7 million bp) having longer genomes than SGM (4.8 million bp) what might reflect bacteria adaptation to the host(s). Mtb with an exclusive host has a shorter genome (4.3 million bp) than NTM which exhibit a wider host tropisms and the ability to persist within the environment Therefore, we can find genes associated to the Lactate fermentation like MAV_2543 and Methanogenesis xfp that cannot be found in Mtb. The two NTM (747/08 and 60/08) strains sequenced and that show intramacrophage persistence displayed also a longer size, which is associated to the persistence related genes. In specific gene subsystems were observed intra-species differences between clinical and reference NTM. Fatty acid metabolism cluster and cell envelope related genes subsystems illustrate this result. Conclusion: The preliminary results suggest a link between genome size and intracellular persistence and support the fact that clinic NTM share virulence factors with Mtb. However, only a transcriptional analysis could ensure the evolvement of these genes in intracellular persistence.
- Iron gene expression profile in atherogenic Mox macrophagesPublication . Marques, L.; Negre-Salvayre, A.; Costa, L.; Canonne-Hergaux, F.The role of macrophage iron in the physiopathology of atherosclerosis is an open question that needs to be clarified. In atherosclerotic lesions, recruited macrophages are submitted to cytokines and oxidized lipids which influence their phenotype. An important phenotypic population driven by oxidized phospholipids is the Mox macrophages which present unique biological properties but their iron phenotype is not well described.
- Next generation sequencing: The key to understand Klebsiella pneumoniae biofilms?Publication . Jordão, Luísa; Borges, Victor; Vieira, Luís; Gomes, João Paulo; Duarte, AidaBackground: The incidence of healthcare-associated infections (HAI) is determined by underlying disease conditions and exposure to high risk medical interventions. In Portugal since 1980s K. pneumoniae is a recognized etiological agent of epidemic and endemic infections in healthcare units. An increasing rate of K. pneumoniae strains resistant either to extended cephalosporins or carbapenems has been observed and one of the mechanisms responsible for the emergence of drug resistance could be the biofilm assembly. The capacity of K. pneumoniae to form biofilm was first described in the 1980s for abiotic surfaces and ten years later on biotic surfaces. The antibiotic failure to penetrate through the biofilm layers, the emergence of mutations which might be easily transferred horizontally, and quorum sensing have been pointed as responsible for the increased antibiotic resistance of bacteria within biofilms. The main objective was to study the biofilm structure and the kinetic assembly associated to antibiotic resistance profile in K. pneumoniae strains capsulate or not, and identify the genes involved in biofilm assembly on full genome sequencing of studied strains. Material and Methods: Twoo K. pneumoniae isolates collected in 1980 (Kp45, Kp703) and one (Kp2948) in 2011 were studied. Kp703 was encapsulated and the remaining had capsular type K:2. The bacterial ability to assemble biofilms on cell culture plates was evaluated. For SEM analysis, biofilms were allowed to form on six wells cell culture plates (Nunc) for 12h at 37ºC. DNA was extracted using QiAamp DNA mini kit following the manufactures instructions. Full genome sequence was performed using next-generation sequencing platform MiSeq (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. The RAST platform was used for annotation and MAUVE platform for multiple alignments. Results: The three isolates were able to assemble biofilms although following different kinetics. K. pneumoniae strains Kp703 and Kp45 followed similar kinetics with identical biomass increase nevertheless these bacteria differ in capsule expression. Full-sequencing and annotation of genomes of isolates was performed in order to explain the differences found in biofilm assembly. Preliminary data already revealed that the K. pneumoniae strains displaying enhanced biofilm-forming ability is genetically different from the others, and, in particular, present some specific features enrolling genomic regions believed to be biofilm-related in other bacteria (an intact prophage, genes coding for a filamentous haemoagglutinin, a haemolysin expression modulating protein and an YdeA protein). Conclusion: K. pneumoniae lacking capsule, regarded as less virulent, have a better performance as biofilm assembler and exhibited the highest increase in antibiotic resistance when organized within biofilms. The analysis of the full genome sequence will allow reachingon K.pneumoniaebiofilms and provide novel opportunities to exploit the overall fitness ofK. pneumoniaeunder antibiotic stress.
- Multilocus sequencing typing as a tool to investigate childhood Haemophilus influenzae invasive disease in PortugalPublication . Bettencourt, Célia; Borges, Vitor; Gonçalo-Marques, José; Cunha, Florbela; Bajanca-Lavado, Maria Paula; on behalf of Portuguese Study Group of Invasive Haemophilus influenzae Disease of the Pediatric Infectious Disease SocietyBackground: Haemophilus influenzae is an important cause of serious childhood invasive disease despite the use of the vaccine against serotype b strains (Hib). Six capsular types, a-f, have been identified to date, although most of strains are non-capsulated (NC). Multilocus Sequencing Typing (MLST) is a powerful method that allows a precise and unambiguous characterization of H. influenzae genotypes. A partnership between the National Institute of Health and the Society for Paediatric Infectious Diseases aimed to characterize invasive childhood infection in Portugal. The objective of this study was to genotype isolates by MLST to follow the epidemiology of disease. Material / Methods: This study was conducted between 1 January 2010 and 31 December 2015. During this period 41 H. influenzae strains were analysed, mostly isolated from blood (88%). Antibiotic resistance was assessed by the microdilution assay and β-lactamase production was determined with nitrocefin. Capsular status was characterized by polymerase chain reaction using primers and conditions described in the literature. MLST was performed by amplifying and sequencing internal fragments of the 7 housekeeping genes (adk, atpG, frdB, fucK, mdh, pgi and recA). Sequences were analyzed and submitted to the MLST website http://pubmlst.org/hinfluenzae/ for assignment of the sequence type (ST). To display the allelic distances between the obtained STs, we applied the goeBURST algorithm implemented in the PHYLOViZ platform. Results: Antimicrobial susceptibility testing showed that most strains were susceptible to all beta-lactams studied, with only three strains being ampicillin resistant due to beta-lactamase production. Most of invasive disease was due to the presence of NC strains (27/41; 66%), while 14 isolates (34%) were capsulated and characterized as follow: two serotype a (5%), 10 b (24%) and two f (5%). As expected, MLST typing revealed high genetic variability among 27 NC isolates, which had 24 (89%) different sequence types (STs), with four new STs represented by previously unidentified allele combinations. In opposition, capsulated isolates were very clonal: all 10 Hib were assigned to CC6 (eight strains ST6, one ST 1149, one ST 190), the two Hia strains were assigned to CC 23 (ST 23) and the two Hif belonged to CC124 (ST 124 and ST 1188) (Figure 1). Conclusions: Our data indicate that invasive disease among Portuguese children is now due to highly genetically diverse, fully susceptible NC strains, suggesting that no particular virulent clone is responsible for epidemiological change of disease, after vaccine implementation in the year 2000. Nevertheless, we are concerned about Hib disease (24% of the isolates) despite the higher vaccine coverage. MLST typing continues to show a high genetic diversity among NC strains and clonal relationships among capsulated isolates. In conclusion, in order to monitor the evolving dynamics of this pathogen and the epidemiology of invasive disease, ongoing surveillance is needed to monitor the true magnitude of this problem.
- Diagnosis and prevention of chromosomal disordersPublication . Simão, LaurentinoClinically significant chromosomal defects occur in 0.65% of all births, with high morbidity and mortality. Chromosome disorders form a major category of genetic disease. They account for a large proportion of all reproductive wastage, congenital malformations, and mental retardation and play an important role in the pathogenesis of malignancy. Specific chromosome abnormalities are responsible for more than 100 identifiable syndromes. The strategies followed in cytogenetics have changed over the past 25 years, moving from the exclusive use of classical cytogenetic methods (karyotyping) to the use of molecular biology methods (molecular cytogenetics and cytogenomics). The cytogenetic studies deserves a great attention since allows identifying the chromosomal abnormalities behind the congenital anomaly. This fact allows establishing genotype-phenotype relationships and the cause and prognosis of pathological pregnancies and population genetic diseases. The utility of studies in prenatal diagnosis (PND) and in patients with chromosomal disorders extends to the possibility of intervening in the diagnosis, prognosis and prevention of the genetic disease, as illustrated with the presentation of some case studies in the laboratory, that also assesses the value of new techniques implementation in a cytogenetic laboratory in order to obtain a complete result. In the other hand, the analysis of retrospective case series with an epidemiological approach contributes to the knowledge of the etiological cause of certain groups of chromosomal abnormality. For instance, Down Syndrome (DS) is the most common chromosomal disorder and the most common cause of moderate mental retardation. It is the most frequent abnormality in PND and is even one of the reasons for its implementation. We carried out a large study on the last 20000 PND we received in the laboratory. 2% showed karyotype compatible with DS. An increased number of karyotypes with DS were detected since chorionic villus sampling (CVS) started to be received in the laboratory. The implementation of rapid aneuploidy detection methods in PND also allowed a faster reporting time of trisomy 21. However, one question remains in discussion: pregnancies affected by DS have a great risk of spontaneous fetal loss during the first trimester and earlier second-trimester. Thus, some CVSs in DS cases will result in spontaneous miscarriages. These thematic show the role of chromosome abnormalities in the diagnosis and prevention of the genetic disease and also the complexity of the ways that we choose.
- Non-canonical translation initiation in the human UPF1 mRNAPublication . Lacerda, Rafaela