Percorrer por autor "Borges, Victor"
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- 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.
- 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.