New Insight regarding Legionella Non- Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene

The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes

The relevance of molecular genotyping to allocate cases in a suspected outbreak of Legionella pneumonia in patients with prolonged immunosuppressive therapy

Three cases of pneumonia caused by Legionella pneumophila serogroup 1 (Lp1) in immunosuppressed patients with repeated hospitalization were suspected as a healthcare-associated cluster. The environmental investigation did not reveal the presence of legionellae in the hospital patient rooms. Water samples collected from the homes of two patients were also negative for Legionella spp. In the absence of environmental strains potentially involved in the infections, we proceeded to genotype environmental Lp1 strains isolated in the hospital during routine water sampling during the decade 2009–2019 and recovered after long-term storage at −20 °C. These 'historical' strains exhibited a high grade of similarity and stability over time, regardless of the disinfection systems. The different molecular profiles shown among the clinical and environmental strains excluded a nosocomial outbreak. The study suggests that the application of molecular typing may be a useful tool to discriminate hospital vs community-acquired cases, mostly for severely immunosuppressed patients in whom the symptomatology could be insidious and the incubation period could be prolonged. Moreover, the genotyping allowed us to exclude any link between the cases. Keywords: Legionnaires' disease, Immunosuppressed patients, Sequence-based typing, Cluster, Environmental strains, Clinical strain

Serratia marcescens in a neonatal intensive care unit: two long-term multiclone outbreaks in a 10-year observational study

We investigated two consecutive Serratia marcescens (S. marcescens) outbreaks which occurred in a neonatal intensive care unit (NICU) of a tertiary level hospital in North Italy in a period of 10 years (January 2003-December 2012). Risk factors associated with S. marcescens acquisition were evaluated by a retrospective case-control study. A total of 21,011 clinical samples was examined: S. marcescens occurred in 127 neonates: 43 developed infection and 3 died. Seven clusters were recorded due to 12 unrelated clones which persisted for years in the ward, although no environmental source was found. The main epidemic clone A sustaining the first cluster in 2003 reappeared in 2010 as an extended spectrum ?-lactamase (ESBL)-producing strain and supporting the second epidemic. Birth weight, gestational age, use of invasive devices and length of stay in the ward were significantly related to S. marcescens acquisition. The opening of a new ward for non-intensive care-requiring neonates, strict adherence to alcoholic hand disinfection, the timely identification and isolation of infected and colonized neonates assisted in containing the epidemics. Genotyping was effective in tracing the evolution and dynamics of the clones demonstrating their long-term persistence in the ward

Pulmonary disease caused by Mycobacterium marseillense, Italy

Mycobacteriummarseillense was recently described as a new species belonging to the Mycobacterium avium complex (MAC).We describe a case of pulmonary disease caused by M. marseillense in an immunocompetent patient. All strains isolated from the patient were preliminarily identified as M. intracellulare; however, a retrospective molecular analysis corrected the identification to M. marseillense

Improvement of Legionnaires' disease diagnosis using real-time PCR assay: a retrospective analysis, Italy, 2010 to 2015

AimTo evaluate real-time PCR as a diagnostic method for Legionnaires' disease (LD). Detection of Legionella DNA is among the laboratory criteria of a probable LD case, according to the European Centre for Disease Prevention and Control, although the utility and advantages, as compared to culture, are widely recognised.MethodsTwo independent laboratories, one using an in-house and the other a commercial real-time PCR assay, analysed 354 respiratory samples from 311 patients hospitalised with pneumonia between 2010-15. The real-time PCR reliability was compared with that of culture and urinary antigen tests (UAT). Concordance, specificity, sensitivity and positive and negative predictive values (PPV and NPV, respectively) were calculated.ResultsOverall PCR detected eight additional LD cases, six of which were due to Legionella pneumophila (Lp) non-serogroup 1. The two real-time PCR assays were concordant in 99.4% of the samples. Considering in-house real-time PCR as the reference method, specificity of culture and UAT was 100% and 97.9% (95% CI: 96.2-99.6), while the sensitivity was 63.6% (95%CI: 58.6-68.6) and 77.8% (95% CI: 72.9-82.7). PPV and NPV for culture were 100% and 93.7% (95% CI: 91.2-96.3). PPV and NPV for UAT were 87.5% (95% CI: 83.6-91.4) and 95.8% (95% CI: 93.5-98.2).ConclusionRegardless of the real-time PCR assay used, it was possible to diagnose LD cases with higher sensitivity than using culture or UAT. These data encourage the adoption of PCR as routine laboratory testing to diagnose LD and such methods should be eligible to define a confirmed LD case

The new phylogenesis of the genus Mycobacterium

Abstract Phylogenetic knowledge of the genus Mycobacterium is based on comparative analysis of their genetic sequences. The 16S rRNA has remained for many years the only target of such analyses, but in the last few years, other housekeeping genes have been investigated and the phylogeny based on their concatenated sequences become a standard. It is now clear that the robustness of the phylogenetic analysis is strictly related to the size of the genomic target used. Whole genome sequencing (WGS) is nowadays becoming widely accessible and comparatively cheap. It was decided, therefore, to use this approach to reconstruct the ultimate phylogeny of the genus Mycobacterium . Over 50 types of strains of the same number of species of Mycobacterium were sequenced using the Illumina HiSeq platform. The majority of the strains of which the whole sequence was already available in GenBank were excluded from this panel with the aim of maximizing the number of the species with genome available. Following assembling and annotation with proper software, the phylogenetic analysis was conducted with PhyloPhlAn and the pan-genome analysis pipeline. The phylogenetic three which emerged was characterized by a clear-cut distinction of slowly and rapidly growing species with the latter being more ancestral. The species of the Mycobacterium terrae complex occupied an intermediate position between rapid and slow growers. Most of the species revealed clearly related and occupied specific phylogenetic branches. Thanks to the WGS technology, the genus Mycobacterium is finally approaching its definitive location

Sadržaj

Metastasis is a multi-step process in which direct crosstalk between cancer cells and their microenvironment plays a key role. Here, we assessed the effect of paired tumor-associated and normal lung tissue mesenchymal stem cells (MSCs) on the growth and dissemination of primary human lung carcinoma cells isolated from the same patients. We show that the tumor microenvironment modulates MSC gene expression and identify a four-gene MSC signature that is functionally implicated in promoting metastasis. We also demonstrate that tumor-associated MSCs induce the expression of genes associated with an aggressive phenotype in primary lung cancer cells and selectively promote their dissemination rather than local growth. Our observations provide insight into mechanisms by which the stroma promotes lung cancer metastasis

Dynamic changes in prefrontal cortex involvement during verbal episodic memory formation

During encoding, the neural activity immediately before or during an event can predict whether that event will be later remembered. The contribution of brain activity immediately after an event to memory formation is however less known. Here, we used repetitive Transcranial Magnetic Stimulation (rTMS) to investigate the temporal dynamics of episodic memory encoding with a focus on post-stimulus time intervals. At encoding, rTMS was applied during the online processing of the word, at its offset, or 100, 200, 300 or 400 ms thereafter. rTMS was delivered to the left ventrolateral (VLPFC) or dorsolateral prefrontal cortex (DLPFC). VLPFC rTMS during the first few hundreds of milliseconds after word offset disrupted subsequent recognition accuracy. We did not observe effects of DLPFC rTMS at any time point. These results suggest that encoding-related VLPFC engagement starts at a relatively late processing stage, and may reflect brain processes related to the offset of the stimulus

Le rôle des cellules Natural Killer humaines dans les réponses immunes anti-tumorales

Natural Killer cells are cytotoxic lymphocytes involved in the immune response against tumours and infections. We investigated the NK-mediated functions in response to clear-cell renal cell carcinoma (RCC) and metastatic melanoma, two human immunogenic tumours. We showed that certain VHL mutations increased RCC cell susceptibility to NK lysis. VHL loss of function correlated with lower expression levels of membrane HLA-I molecules on VHL-mutated RCC and a decreased triggering of inhibitory NK receptors compared to RCC with a functional VHL. In stage IV melanoma patients, we showed that blood NK cells displayed a unique NKp46dim/NKG2Adim phenotype and high lytic potential towards melanoma cells. Following chemotherapy, NK cell function was reduced and the phenotype modulated. To study melanoma-infiltrating NK cells, we have set up experimental conditions to characterise NK cells in metastatic LNs from stage III melanoma patients. Our preliminary data show that, compared to normal LNs, NK cells from metastatic LNs are altered. Our findings suggest that oncogenic-dependent immunogenicity, tumour-associated NK alterations and chemotherapy are important factors that must be taken into account in the choice of immunotherapeutic protocols based on NK cells.Les cellules Natural Killer (NK) sont des effecteurs cytotoxiques impliqués dans la réponse immune contre les infections et les tumeurs. Pendant ma thèse j’ai étudié la fonctionnalité des cellules NK humaines en réponse à des lignées cellulaires de carcinome rénal à cellules claires (RCC) et de mélanome métastatique, deux tumeurs immunogènes. Nos résultats montrent que certaines mutations de VHL augmentent la susceptibilité des lignées RCC à la lyse NK. La perte de fonction de VHL corrèle avec une expression membranaire diminuée des molécules HLA-I par les lignées RCC mutées pour VHL. Chez les patients atteints de mélanome métastatique de stade IV, nous avons décrit un phénotype particulier des NK sanguines (NKp46dim/NKG2Adim) qui leur confère une forte activité antitumorale. Après traitement des patients par chimiothérapie, la fonctionnalité NK était réduite et le phénotype modifié. Pour étudier les cellules NK infiltrant les mélanomes, nous avons mis au point des conditions expérimentales pour caractériser les cellules NK de ganglions métastatiques de patients de stade III. Nos résultats préliminaires montrent que, par rapport aux ganglions sains, les NK des ganglions métastatiques présentent un phénotype altéré et un potentiel fonctionnel diminué. Nos résultats suggèrent que d’une part l’immunogénicité dépendante des oncogènes et d’autre part les altérations NK induites par la tumeur et/ou par la chimiothérapie sont des facteurs importants à considérer dans le choix des protocoles d’immunothérapie basés sur les cellules NK

Le rôle des cellules Natural Killer humaines dans les réponses immunes anti-tumorales

Les cellules Natural Killer (NK) sont des effecteurs cytotoxiques impliqués dans la réponse immune contre les infections et les tumeurs. Pendant ma thèse j ai étudié la fonctionnalité des cellules NK humaines en réponse à des lignées cellulaires de carcinome rénal à cellules claires (RCC) et de mélanome métastatique, deux tumeurs immunogènes. Nos résultats montrent que certaines mutations de VHL augmentent la susceptibilité des lignées RCC à la lyse NK. La perte de fonction de VHL corrèle avec une expression membranaire diminuée des molécules HLA-I par les lignées RCC mutées pour VHL. Chez les patients atteints de mélanome métastatique de stade IV, nous avons décrit un phénotype particulier des NK sanguines (NKp46dim/NKG2Adim) qui leur confère une forte activité antitumorale. Après traitement des patients par chimiothérapie, la fonctionnalité NK était réduite et le phénotype modifié. Pour étudier les cellules NK infiltrant les mélanomes, nous avons mis au point des conditions expérimentales pour caractériser les cellules NK de ganglions métastatiques de patients de stade III. Nos résultats préliminaires montrent que, par rapport aux ganglions sains, les NK des ganglions métastatiques présentent un phénotype altéré et un potentiel fonctionnel diminué. Nos résultats suggèrent que d une part l immunogénicité dépendante des oncogènes et d autre part les altérations NK induites par la tumeur et/ou par la chimiothérapie sont des facteurs importants à considérer dans le choix des protocoles d immunothérapie basés sur les cellules NK.Natural Killer cells are cytotoxic lymphocytes involved in the immune response against tumours and infections. We investigated the NK-mediated functions in response to clear-cell renal cell carcinoma (RCC) and metastatic melanoma, two human immunogenic tumours. We showed that certain VHL mutations increased RCC cell susceptibility to NK lysis. VHL loss of function correlated with lower expression levels of membrane HLA-I molecules on VHL-mutated RCC and a decreased triggering of inhibitory NK receptors compared to RCC with a functional VHL. In stage IV melanoma patients, we showed that blood NK cells displayed a unique NKp46dim/NKG2Adim phenotype and high lytic potential towards melanoma cells. Following chemotherapy, NK cell function was reduced and the phenotype modulated. To study melanoma-infiltrating NK cells, we have set up experimental conditions to characterise NK cells in metastatic LNs from stage III melanoma patients. Our preliminary data show that, compared to normal LNs, NK cells from metastatic LNs are altered. Our findings suggest that oncogenic-dependent immunogenicity, tumour-associated NK alterations and chemotherapy are important factors that must be taken into account in the choice of immunotherapeutic protocols based on NK cells.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF