Browsing by Author "Sousa, Sara"
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- Bacterial biofilms: a story of persistence and invasionPublication . Sousa, Sara; Bandeira, Maria; Carvalho, Patricia ALmeida; Duarte, Aida; Jordão, LuísaBiofilms are described as colonies of microorganisms that are attached to each other and to a surface, in an irreversible mode. These structures are virtualy everywhere: natural and humanized environments, as well as within living beings (humans and animals). For a long time biofilms where regarded as a bacterial survival strategie. Nowadays, in the industrialized world, the impact of acute bacterial infections caused by rapidly proliferating planktonic cells have gradually decreased in comparison to chronic infections owing to environmental organisms growing as biofilms. The major concern in this field is the healthcare-associated infections (HAIs). In 2012, HAIs estimated cases reached 6.7 million either in long-term care facilities or acute-care hospitals from which result 37,000 deaths configuring a serious public health problem [1]. The etiological agents are diverse being often resistant to antimicrobials and able to assemble biofilms both in abiotic and biotic surfaces. Here we evaluated the ability of different bacteria to assemble biofilms on a model surface and materials mimicking surfaces present either on healthcare units or medical devices. All bacteria were able to assemble biofilmls although following different kinetics and exhibiting different structural features assessed by electron microscopy. Additionally a link was established between bacteria ability to assemble biofilms and increased antibiotic resistance [2]. 1. ECDC Europe; (2012), Annual epidemiological report 2011 2. Bandeira M; (2014), Pathogens (doi:10.3390/pathogens3030720)
- Estudo do papel da imunidade inata nas infeções por micobactériasPublication . Sousa, Sara; Jordão, Luísa; Telhada, Maria Margarida Blasques[PT] Desde os primórdios da humanidade que existem relatos de infecções respiratórias causadas por micobactérias. A incidência de infecções por Micobactérias Não-Tuberculosas (MNT), consideradas oportunistas, tem aumentado gradualmente atingindo sobretudo a população imunodeprimida. As MNTs são maioritariamente ambientais e ubíquas. Sendo o difícil diagnóstico, a resistência aos antibióticos e o escasso conhecimento da patogenicidade destas micobactérias, os principais impulsores do seu estudo. Os macrófagos alveolares, em infecções do sistema respiratório, são as primeiras células do sistema imunitário que contactam com as MNTs. Estes irão desencadear de imediato uma resposta imunitária inata. Este trabalho teve como principal objectivo avaliar a resposta imunitária durante uma infecção com MNTs utilizando células THP-1 como modelo de macrófagos alveolares humanos. Para tal, foi avaliada a sobrevivência de algumas MNTs no interior dos macrófagos, seguida do estudo de vários componentes da resposta imunitária: maturação do fagossoma; produção de molécula pró- inflamatórias; e indução da morte celular. Foi possível observar a existência de 3 perfis de persistência intracelular: M. smegmatis é eliminado; M. fortuitum ATCC6841 apresenta um perfil de latência; o M. fortuitum 747/08, M. abscessus 549/08, M. avium ATCC25291 e M.avium 60/08 apresentam um perfil de crescimento. A maturação dos fagossomas de MNT é bloqueada duma forma menos eficiente do que a dos fagossomas de M. tuberculosis. Apenas o M. avium 60/08 foi capaz de induzir a produção de NO e IL-10. E ainda que existe a indução da apoptose diferenciada entre as estirpes estudadas, sendo o M. avium 60/08 o melhor indutor.
- Innate immune response during NTM infectionsPublication . Sousa, Sara; Martins, Fatima; Jordão, LuísaBackground As tuberculosis incidence declines in industrialized countries, nontuberculous mycobacteria (NTM) infections gained relevance. Human infection with NTM became relevant with AIDS pandemic, being currently recognized as a cause of pulmonary infection in humans. Despite this fact little is known about NTM pathogenesis. In the present work the role of innate immune response during NTM infection using THP-1 cells as a model of alveolar macrophages was evaluated. Methods M.smegmatis mc2 155, 2 reference strains (M.avium ATCC25291; M.fortuitum ATCC6841) and 2 clinical isolates (M.avium 60/08;M. fortuitum 747/08) were used. Bacteria were grown until mid-exponential phase and stored at -80°C. Before each experiment analiquot was thawed and diluted in RPMI with 10% HI- FCS in order to reach an OD600nm of 0.1. The inoculums were titrated by CFU enumeration on 7H10 medium supplemented with 10% OADC. Briefly, 4x104 THP-1 cells were platted/well and incubated for 72h with 100nM PMA (37°C/5% CO2) then fresh medium without PMA was added being the cells incubated for further 24h. The cells were infected for 1 or 3h for fast or slow growers, respectively. The intracellular persistence was evaluated by CFU enumeration at different time points from 1 to 24h or 3 to 168h for fast and slow growers, respectively. Phagosome acidification was followed using confocal microscopy. The secretion of pro-inflammatory cytokines was assayed by ELISA, NO production using the Griess reagent and apoptosis was followed by flow cytometry and confocal microscopy. The ability of mycobacteria to persist at different pHs was evaluated using BACTEC-MGIT960. Results The mycobacteria experienced different fates within THP-1 macrophages. M.smegmatis and M.fortuitum ATCC6841 were cleared within 24h, whereas 747/08 and the two M.avium strains were able to replicate. Despite this fact for the latest mycobacteria more than 50% of acidified phagosomes were present during the experience. Mycobacteria survival at acidic pHs (6.6; 5.4 and 4.6) was then evaluated. With the exception of M.smegmatis all strains grew at acidic pH showing that other factors than phagosome acidification were involved in mycobacteria killing. Next, other components of the inflammatory response were evaluated. Measurable values of NO were present in supernatants of THP-1 infected for 3 days with 60/08 being this bacterium susceptible, to high concentrations of NO in vitro. Il-10 secretion was also assayed. For both fast growing NTM and M.avium ATCC25291 the production of IL-10 was not detectable. For 60/08 IL-10 production peaked at 3 days, decreasing afterwards until undetectable levels at 7 days. Another factor being explored is apoptosis induction by NTM. Our preliminary results point to differential induction of apoptosis by different NTM.
- Nontuberculous mycobacteria pathogenesis and biofilm assemblyPublication . Sousa, Sara; Bandeira, Maria; Carvalho, Patricia Almeida; Duarte, Aida; Jordão, LuísaNontuberculous mycobacteria (NTM) are emergent pathogens whose importance in human health has been gaining relevance after being recognized as etiological agents of opportunist infections in HIV patients. Currently, NTM are recognized as etiological agents of several respiratory and extrarespiratory infections of immune-competent individuals. The environmental nature of NTM together with the ability to assemble biofilms on different surfaces plays a key role on their pathogenesis. In the present work the ability of three fast-growing NTM (M. smegmatis, M. fortuitum and M. chelonae) to persist within a model of human alveolar macrophages was evaluated. Most often human infections with NTM occur by contact with the environment. Biofilms can work as environmental reservoirs. For this reason, it was decided to evaluate the ability of NTM to assemble biofilms on different surfaces. Scanning electron microscopy was used to elucidate the biofilm structure. The ability to assemble biofilms was connected with the ability to spread on solid media known as sliding. Biofilm assembly and intracellular persistence seems to be ruled by different mechanisms.
- Nontuberculous Mycobacteria Persistence in a Cell Model Mimicking Alveolar MacrophagesPublication . Sousa, Sara; Borges, Vítor; João, Inês; Gomes, João Paulo; Jordão, LuisaNontuberculous Mycobacteria (NTM) respiratory infections have been gradually increasing. Here, THP-1 cells were used as a model to evaluate intracellular persistence of three NTM species (reference and clinical strains) in human alveolar macrophages. The contribution of phagosome acidification, nitric oxide (NO) production and cell dead on NTM intracellular fate was assessed. In addition, strains were characterized regarding their repertoire of virulence factors by whole-genome sequencing. NTM experienced different intracellular fates: M. smegmatis and M. fortuitum ATCC 6841 were cleared within 24h. In contrast, M. avium strains (reference/clinical) and M. fortuitum clinical strain were able to replicate. Despite this fact, unexpectedly high percentages of acidified phagosomes were found harbouring rab7, but not CD63. All NTM were able to survive in vitro at acidic pHs, with the exception of M. smegmatis. Our data further suggested a minor role for NO in intracellular persistence and that apoptosis mediated by caspase 8 and 3/7, but not necrosis, is triggered during NTM infection. Insights regarding the bacteria genomic backbone corroborated the virulence potential of M. avium and M. fortuitum. In conclusion, the phenotypic traits detected contrast with those described for M. tuberculosis, pointing out that NTM adopt distinct strategies to manipulate the host immune defense and persist intracellularly.
- On nontuberculous mycobacteria in human alveolar macrophagesPublication . João, Inês; Borges, Vítor; Sousa, Sara; Gomes, João Paulo; Jordão, LuísaBackground: Despite growing relevance of nontuberculous mycobacteria (NTM) infections, namely pulmonary infections, accurate data on incidence and pathogenesis is lacking. Iintracellular persistence of three NTM species in a human alveolar macrophages model. The contribution of particular innate immune mechanisms and NTM genomes to mycobacteria intracellular fate were investigated. Materials/methods: M.smegmatis mc2155, reference strains (M.avium ATCC25291; M.fortuitum ATCC6841) and clinical isolates (M.avium 60/08; M. fortuitum 747/08) were used. Mycobacteria intracellular persistence was assessed via: 4x104 THP-1 cells platted/well and incubated for 72h with 100nM PMA. Then fresh medium without PMA was added and the cells incubated for further 24h. The cells were infected for 1h (fast) or 3h (slow growers). Intracellular persistence was evaluated by CFU enumeration at different time points. NO production was evaluated using the Griess reagent, phagosome acidification and apoptosis was followed by confocal microscopy. Persistence ability of mycobacteria at different pHs was evaluated using BACTEC-MGIT960. NTMs virulence factors were characterized by whole-genome sequencing.
- Risk assessment for public health from human interaction with ornamental watersPublication . Duarte, Aida; Rodrigues, João Carlos; Reis, Lúcia; Nogueira, Isabel; Carvalho, Patricia; Paulino, Sérgio; Sousa, Sara; Jordao, LuisaWater 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 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.
- 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.
- Risk assessment for public health from human interaction with ornamental watersPublication . Duarte, Aida; Rodrigues, João Carlos; Reis, Lúcia; Nogueira, Isabel; Carvalho, Patricia; Paulino, Sérgio; Sousa, Sara; Jordão, LuisaWater 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 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.
- 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.