RESUMO
Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.
Assuntos
Micoses , Humanos , Inclusão em Parafina , Micoses/diagnóstico , Formaldeído , Reação em Cadeia da Polimerase , Fixação de Tecidos , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management.
Assuntos
Antibióticos Antituberculose/uso terapêutico , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/genética , Rifampina/uso terapêutico , Tuberculose/microbiologia , Humanos , Polimorfismo de Nucleotídeo Único , Tuberculose/tratamento farmacológicoRESUMO
OBJECTIVES: Implementation of an antibiotic resistance detection tool in Legionella daily surveillance at the French National Reference Centre for Legionella. METHODS: Systematic WGS of Legionella pneumophila isolates and bioinformatics detection of specific mutations linked to antibiotic resistance. Phenotypic validation of antibiotic resistance detected by WGS was performed by the broth microdilution method. RESULTS: More than 3000 L. pneumophila strains were screened for antibiotic resistance. A macrolide resistance-associated A2052G mutation in the 23S rRNA gene was identified in the genome of eight isolates from a hotel water network. High-level macrolide resistance (i.e. MICs of 1024-2048â mg/L for azithromycin and erythromycin) with no cross-resistance to other antimicrobials was phenotypically confirmed by antimicrobial susceptibility testing for the eight isolates. CONCLUSIONS: Systematic WGS of L. pneumophila is a powerful tool for first-line high-throughput screening of antibiotic resistance before phenotypic validation.
Assuntos
Legionella pneumophila , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , ÁguaRESUMO
Natural transformation (i.e., the uptake of DNA and its stable integration in the chromosome) is a major mechanism of horizontal gene transfer in bacteria. Although the vast majority of bacterial genomes carry the genes involved in natural transformation, close relatives of naturally transformable species often appear not competent for natural transformation. In addition, unexplained extensive variations in the natural transformation phenotype have been reported in several species. Here, we addressed this phenomenon by conducting a genome-wide association study (GWAS) on a panel of isolates of the opportunistic pathogen Legionella pneumophila GWAS revealed that the absence of the transformation phenotype is associated with the conjugative plasmid pLPL. The plasmid inhibits transformation by simultaneously silencing the genes required for DNA uptake and recombination. We identified a small RNA (sRNA), RocRp, as the sole plasmid-encoded factor responsible for the silencing of natural transformation. RocRp is homologous to the highly conserved and chromosome-encoded sRNA RocR which controls the transient expression of the DNA uptake system. Assisted by the ProQ/FinO-domain RNA chaperone RocC, RocRp acts as a substitute of RocR, ensuring that the bacterial host of the conjugative plasmid does not become naturally transformable. Distinct homologs of this plasmid-encoded sRNA are found in diverse conjugative elements in other Legionella species. Their low to high prevalence may result in the lack of transformability of some isolates up to the apparent absence of natural transformation in the species. Generally, our work suggests that conjugative elements obscure the widespread occurrence of natural transformability in bacteria.
Assuntos
Transferência Genética Horizontal , Legionella pneumophila/genética , Plasmídeos/genética , Pequeno RNA não Traduzido/genética , Transformação Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inativação Gênica , Estudo de Associação Genômica Ampla , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , RNA , Pequeno RNA não Traduzido/metabolismoRESUMO
We describe a March 2020 co-occurrence of Legionnaires' disease (LD) and coronavirus disease in France. Severe acute respiratory syndrome coronavirus 2 co-infections were identified in 7 of 49 patients from LD case notifications. Most were elderly men with underlying conditions who had contracted severe pneumonia, illustrating the relevance of co-infection screening.
Assuntos
COVID-19 , Coinfecção , Legionella , Idoso , Coinfecção/epidemiologia , França/epidemiologia , Humanos , Legionella/genética , Masculino , SARS-CoV-2RESUMO
Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire's disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Doença dos Legionários/diagnóstico , Doença dos Legionários/tratamento farmacológico , Padrões de ReferênciaRESUMO
BACKGROUND: Rarely, Legionnaires' disease (LD) can progress into a slowly or nonresolving form. METHODS: A nationwide retrospective study was conducted by the French National Reference Center for Legionella (2013-2017) including cases of slowly or nonresolving LD defined as persistent clinical symptoms, computed tomography (CT) scan abnormalities, and Legionella detection in lower respiratory tract specimens by culture and/or real-time (RT) polymerase chain reaction (PCR) >30 days after symptom onset. RESULTS: Twelve cases of community-acquired slowly or nonresolving LD were identified among 1686 cases of culture-positive LD. Median (interquartile range [IQR]) age was 63 (29-82) years. Ten (83.3%) patients had ≥1 immunosuppressive factor. Clinically, 9 patients transiently recovered before further deterioration (median [IQR] symptom-free interval, 30 [18-55] days), 3 patients had uniformly persistent symptoms (median [IQR] time, 48 [41.5-54] days). Two patients had >2 recurrences. CT scan imagery found lung abscess in 5 (41.6%) cases. Slowly or nonresolving LD was diagnosed on positive Legionella cultures (n = 10, 83.3%) at 49.5 (IQR, 33.7-79) days. Two cases were documented through positive Legionella RT PCR at 52 and 53 days (cycle threshold detection of 21.5 and 33.7, respectively). No genomic microevolution and no Legionella resistance to antibiotics were detected. The median (IQR) duration of treatment was 46.5 (21-92.5) days. Two empyema cases required thoracic surgery. At a median (IQR) follow-up of 26 (14-41.5) months, LD-attributable mortality was 16.6% (n = 2). CONCLUSIONS: Slowly or nonresolving LD may occur in immunocompromised patients, possibly leading to lung abscess and empyema.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Antibacterianos/uso terapêutico , Humanos , Legionella/genética , Legionella pneumophila/genética , Doença dos Legionários/diagnóstico , Doença dos Legionários/tratamento farmacológico , Reação em Cadeia da Polimerase em Tempo Real , Estudos RetrospectivosRESUMO
We describe 2 cases of healthcare-associated Legionnaires' disease in patients in France hospitalized 5 months apart in the same room. Whole-genome sequencing analyses showed that clinical isolates from the patients and isolates from the room's toilet clustered together. Toilet contamination by Legionella pneumophila could lead to a risk for exposure through flushing.
Assuntos
Aparelho Sanitário , Infecção Hospitalar , Legionella pneumophila , Doença dos Legionários , França , Humanos , Legionella pneumophila/genética , Doença dos Legionários/diagnóstico , Doença dos Legionários/epidemiologiaRESUMO
BACKGROUND: Enterobacter cloacae species is responsible for nosocomial outbreaks in vulnerable patients in neonatal intensive care units (NICU). The environment can constitute the reservoir and source of infection in NICUs. Herein we report the impact of preventive measures implemented after an Enterobacter cloacae outbreak inside a NICU. METHODS: This retrospective study was conducted in one level 3 NICU in Lyon, France, over a 6 year-period (2012-2018). After an outbreak of Enterobacter cloacae infections in hospitalized neonates in 2013, several measures were implemented including intensive biocleaning and education of medical staff. Clinical and microbiological characteristics of infected patients and evolution of colonization/infection with Enterobacter spp. in this NICU were retrieved. Moreover, whole genome sequencing was performed on 6 outbreak strains. RESULTS: Enterobacter spp. was isolated in 469 patients and 30 patients developed an infection including 2 meningitis and 12 fatal cases. Preventive measures and education of medical staff were not associated with a significant decrease in patient colonisation but led to a persistent decreased use of cephalosporin in the NICU. Infection strains were genetically diverse, supporting the hypothesis of multiple hygiene defects rather than the diffusion of a single clone. CONCLUSIONS: Grouped cases of infections inside one setting are not necessarily related to a single-clone outbreak and could reveal other environmental and organisational problematics. The fight against implementation and transmission of Enterobacter spp. in NICUs remains a major challenge.
Assuntos
Enterobacter cloacae/patogenicidade , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/prevenção & controle , Controle de Infecções/métodos , Surtos de Doenças/prevenção & controle , Enterobacter cloacae/genética , Enterobacter cloacae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Fezes/microbiologia , Feminino , França , Humanos , Higiene , Recém-Nascido , Unidades de Terapia Intensiva Neonatal/estatística & dados numéricos , Masculino , Sepse Neonatal/epidemiologia , Sepse Neonatal/microbiologia , Estudos Retrospectivos , Sequenciamento Completo do GenomaRESUMO
Legionella pneumophila is an environmental bacterium and the leading cause of Legionnaires' disease. Just five sequence types (ST), from more than 2000 currently described, cause nearly half of disease cases in northwest Europe. Here, we report the sequence and analyses of 364 L. pneumophila genomes, including 337 from the five disease-associated STs and 27 representative of the species diversity. Phylogenetic analyses revealed that the five STs have independent origins within a highly diverse species. The number of de novo mutations is extremely low with maximum pairwise single-nucleotide polymorphisms (SNPs) ranging from 19 (ST47) to 127 (ST1), which suggests emergences within the last century. Isolates sampled geographically far apart differ by only a few SNPs, demonstrating rapid dissemination. These five STs have been recombining recently, leading to a shared pool of allelic variants potentially contributing to their increased disease propensity. The oldest clone, ST1, has spread globally; between 1940 and 2000, four new clones have emerged in Europe, which show long-distance, rapid dispersal. That a large proportion of clinical cases is caused by recently emerged and internationally dispersed clones, linked by convergent evolution, is surprising for an environmental bacterium traditionally considered to be an opportunistic pathogen. To simultaneously explain recent emergence, rapid spread and increased disease association, we hypothesize that these STs have adapted to new man-made environmental niches, which may be linked by human infection and transmission.
Assuntos
Evolução Molecular , Legionella pneumophila/genética , Doença dos Legionários/microbiologia , Humanos , Legionella pneumophila/classificação , Legionella pneumophila/isolamento & purificação , Legionella pneumophila/patogenicidade , Mutação , Filogenia , Polimorfismo de Nucleotídeo Único , Seleção Genética , Virulência/genéticaRESUMO
BACKGROUND: While Legionella is a common cause of pneumonia, extrapulmonary infections like arthritis are scarce. Here, we describe a case of monoarthritis due to Legionella bozemanii, with no history of pneumonia. We provide a literature review of the 9 previously published Legionella arthritis and highlight a dichotomous epidemiology suggesting different physiopathological pathways leading to joint infection. CASE PRESENTATION: A 56-year old woman under immunosuppressive treatment by oral and intra-articular corticosteroids, methotrexate, and tocilizumab for an anti-synthetase syndrome was hospitalized for worsening pain and swelling of the left wrist for 3 days. Clinical examination showed left wrist synovitis and no fever. The arthritis occurred a few days after an accidental fall on wet asphalt responsible for a cutaneous wound followed by a corticosteroid intra-articular injection. Due to both the negativity of conventional culture of articular fluid and suspicion of infection, 16S rRNA and specific PCRs were performed leading to the identification of L. bozemanii. Legionella-specific culture of the articular fluid was performed retrospectively and isolated L. bozemanii. The empiric antibiotic therapy was switched for oral levofloxacin and rifampin and the patient recovered after a 12-week treatment. CONCLUSION: We report a case of L. bozemanii monoarthritis in an immunosuppressed woman, following a fall on wet asphalt and intra-articular corticosteroid injection. The review of the literature found that the clinical presentation reveals the mode of infection and the bacterial species. Monoarthritis more likely occurred after inoculation in patients under immunosuppressive therapy and were associated with non-Legionella pneumophila serogroup 1 (Lp1) strains that predominate in the environment. Polyarthritis were more likely secondary legionellosis localizations after blood spread of Lp1, the most frequently found in pneumonia. In both settings, 16S rRNA and Legionella-specific PCR were key factors for the diagnosis.
Assuntos
Artrite Infecciosa/imunologia , Artrite Infecciosa/microbiologia , Legionellaceae/isolamento & purificação , Legionelose/microbiologia , Acidentes por Quedas , Administração Oral , Corticosteroides/administração & dosagem , Corticosteroides/efeitos adversos , Corticosteroides/uso terapêutico , Artrite Infecciosa/tratamento farmacológico , Feminino , Humanos , Hidrocarbonetos , Hospedeiro Imunocomprometido , Injeções Intra-Articulares , Legionellaceae/genética , Levofloxacino/uso terapêutico , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Rifampina/uso terapêutico , Resultado do TratamentoRESUMO
Background: Legionnaires' disease is an important cause of hospital-acquired pneumonia and is caused by infection with the bacterium Legionella. Because current typing methods often fail to resolve the infection source in possible nosocomial cases, we aimed to determine whether whole-genome sequencing (WGS) could be used to support or refute suspected links between cases and hospitals. We focused on cases involving a major nosocomial-associated strain, L. pneumophila sequence type (ST) 1. Methods: WGS data from 229 L. pneumophila ST1 isolates were analyzed, including 99 isolates from the water systems of 17 hospitals and 42 clinical isolates from patients with confirmed or suspected hospital-acquired infections, as well as isolates obtained from or associated with community-acquired sources of Legionnaires' disease. Results: Phylogenetic analysis demonstrated that all hospitals from which multiple isolates were obtained have been colonized by 1 or more distinct ST1 populations. However, deep sampling of 1 hospital also revealed the existence of substantial diversity and ward-specific microevolution within the population. Across all hospitals, suspected links with cases were supported with WGS, although the degree of support was dependent on the depth of environmental sampling and available contextual information. Finally, phylogeographic analysis revealed that hospitals have been seeded with L. pneumophila via both local and international spread of ST1. Conclusions: WGS can be used to support or refute suspected links between hospitals and Legionnaires' disease cases. However, deep hospital sampling is frequently required due to the potential coexistence of multiple populations, existence of substantial diversity, and similarity of hospital isolates to local populations.
Assuntos
Infecção Hospitalar/epidemiologia , Genômica/métodos , Legionella pneumophila/classificação , Legionella pneumophila/genética , Doença dos Legionários/epidemiologia , Epidemiologia Molecular/métodos , Tipagem Molecular/métodos , Biologia Computacional/métodos , Infecção Hospitalar/microbiologia , Genótipo , Hospitais , Humanos , Legionella pneumophila/isolamento & purificação , Doença dos Legionários/microbiologia , Filogenia , Análise de Sequência de DNA/métodos , Microbiologia da ÁguaRESUMO
Monitoring the emergence of antibiotic resistance is a recent issue in the treatment of Legionnaires' disease. Macrolides are recommended as first-line therapy, but resistance mechanisms have not been studied in Legionella species. Our aim was to determine the molecular basis of macrolide resistance in L. pneumophila Twelve independent lineages from a common susceptible L. pneumophila ancestral strain were propagated under conditions of erythromycin or azithromycin pressure to produce high-level macrolide resistance. Whole-genome sequencing was performed on 12 selected clones, and we investigated mutations common to all lineages. We reconstructed the dynamics of mutation for each lineage and demonstrated their involvement in decreased susceptibility to macrolides. The resistant mutants were produced in a limited number of passages to obtain a 4,096-fold increase in erythromycin MICs. Mutations affected highly conserved 5-amino-acid regions of L4 and L22 ribosomal proteins and of domain V of 23S rRNA (G2057, A2058, A2059, and C2611 nucleotides). The early mechanisms mainly affected L4 and L22 proteins and induced a 32-fold increase in the MICs of the selector drug. Additional mutations related to 23S rRNA mostly occurred later and were responsible for a major increase of macrolide MICs, depending on the mutated nucleotide, the substitution, and the number of mutated genes among the three rrl copies. The major mechanisms of the decreased susceptibility to macrolides in L. pneumophila and their dynamics were determined. The results showed that macrolide resistance could be easily selected in L. pneumophila and warrant further investigations in both clinical and environmental settings.
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Legionella pneumophila/genética , Mutação , RNA Ribossômico 23S/genética , Proteínas Ribossômicas/genética , Antibacterianos/farmacologia , Azitromicina/farmacologia , Proteínas de Bactérias/metabolismo , Células Clonais , Eritromicina/farmacologia , Sequenciamento de Nucleotídeos em Larga Escala , Legionella pneumophila/efeitos dos fármacos , Legionella pneumophila/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
Objectives: A previous study on 12 in vitro -selected azithromycin-resistant Legionella pneumophila lineages showed that ribosomal mutations were major macrolide resistance determinants. In addition to these mechanisms that have been well described in many species, mutations upstream of lpeAB operon, homologous to acrAB in Escherichia coli , were identified in two lineages. In this study, we investigated the role of LpeAB and of these mutations in macrolide resistance of L. pneumophila . Methods: The role of LpeAB was studied by testing the antibiotic susceptibility of WT, deleted and complemented L. pneumophila Paris strains. Translational fusion experiments using GFP as a reporter were conducted to investigate the consequences of the mutations observed in the upstream sequence of lpeAB operon. Results: We demonstrated the involvement of LpeAB in an efflux pump responsible for a macrolide-specific reduced susceptibility of L. pneumophila Paris strain. Mutations in the upstream sequence of lpeAB operon were associated with an increased protein expression. Increased expression was also observed under sub-inhibitory macrolide concentrations in strains with both mutated and WT promoting regions. Conclusions: LpeAB are components of an efflux pump, which is a macrolide resistance determinant in L. pneumophila Paris strain. Mutations observed in the upstream sequence of lpeAB operon in resistant lineages led to an overexpression of this efflux pump. Sub-inhibitory concentrations of macrolides themselves participated in upregulating this efflux and could constitute a first step in the acquisition of a high macrolide resistance level.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Legionella pneumophila/efeitos dos fármacos , Macrolídeos/farmacologia , Proteínas de Membrana Transportadoras/genética , Azitromicina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eritromicina/farmacologia , Genes Bacterianos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Óperon , RNA Ribossômico 23SRESUMO
A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.
Assuntos
Legionella pneumophila/classificação , Legionella pneumophila/genética , Microesferas , Tipagem Molecular/métodos , Automação Laboratorial , França , Ensaios de Triagem em Larga Escala , Humanos , Epidemiologia Molecular/métodosRESUMO
Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Legionella pneumophila/fisiologia , Fagócitos/microbiologia , Acanthamoeba castellanii/microbiologia , Proteínas de Bactérias/genética , Cátions Bivalentes/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Deleção de Genes , Legionella pneumophila/genética , Lobosea/microbiologiaRESUMO
Bacterial pneumonia is a considerable problem worldwide. Here, we follow the inter-kingdom respiratory tract microbiome (RTM) of a unique cohort of 38 hospitalized patients (n = 97 samples) with pneumonia caused by Legionella pneumophila. The RTM composition is characterized by diversity drops early in hospitalization and ecological species replacement. RTMs with the highest bacterial and fungal loads show low diversity and pathogen enrichment, suggesting high biomass as a biomarker for secondary and/or co-infections. The RTM structure is defined by a "commensal" cluster associated with a healthy RTM and a "pathogen" enriched one, suggesting that the cluster equilibrium drives the microbiome to recovery or dysbiosis. Legionella biomass correlates with disease severity and co-morbidities, while clinical interventions influence the RTM dynamics. Fungi, archaea, and protozoa seem to contribute to progress of pneumonia. Thus, the interplay of the RTM equilibrium, the pathogen load dynamics, and clinical interventions play a critical role in patient recovery.
Assuntos
Coinfecção , Microbiota , Pneumonia Bacteriana , Humanos , Pneumonia Bacteriana/diagnóstico , Sistema Respiratório , DisbioseRESUMO
Legionella pneumophila are host-adapted bacteria that infect and reproduce primarily in amoeboid protists. Using similar infection mechanisms, they infect human macrophages, and cause Legionnaires' disease, an atypical pneumonia, and the milder Pontiac fever. We hypothesized that, despite the similarities in infection mechanisms, the hosts are different enough that there exist high-selective value mutations that would dramatically increase the fitness of Legionella inside the human host. By comparing a large number of isolates from independent infections, we identified two genes, mutated in three unrelated patients, despite the short duration of the incubation period (2-14 days). One is a gene coding for an outer membrane protein (OMP) belonging to the OmpP1/FadL family. The other is a gene coding for an EAL-domain-containing protein involved in cyclic-di-GMP regulation, which in turn modulates flagellar activity. The clinical strain, carrying the mutated EAL-domain-containing homologue, grows faster in macrophages than the wild-type strain, and thus appears to be better adapted to the human host. As human-to-human transmission is very rare, fixation of these mutations into the population and spread into the environment is unlikely. Therefore, parallel evolution - here mutations in the same genes observed in independent human infections - could point to adaptations to the accidental human host. These results suggest that despite the ability of L. pneumophila to infect, replicate in and exit from macrophages, its human-specific adaptations are unlikely to be fixed in the population.
Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Legionella/genética , Doença dos Legionários/metabolismo , Macrófagos/microbiologiaRESUMO
Bacterial persisters are a transient subpopulation of non-growing, antibiotic-tolerant cells. There is increasing evidence that bacterial persisters play an important role in treatment failure leading to recurring infections and promoting the development of antibiotic resistance. Current research reveals that recurring legionellosis is often the result of relapse rather than reinfection and suggests that the mechanism of bacterial persistence may play a role. The development of single-cell techniques such as the Timerbac system allows us to identify potential persister cells and investigate their physiology. Here, we tested the persister forming capacity of 7 pairs of Legionella pneumophila (Lp) clinical isolates, with isolate pairs corresponding to two episodes of legionellosis in the same patient. We distinguished non-growing subpopulations from their replicating counterparts during infection in an amoeba model. Imaging flow cytometry allowed us to identify single non-growing bacteria within amoeba cells 17 h post-infection, thus corresponding to this subpopulation of potential persister cells. Interestingly the magnitude of this subpopulation varies between the 7 pairs of Lp clinical isolates. Biphasic killing kinetics using ofloxacin stress confirmed the persister development capacity of ST1 clinical isolates, highlighting enhanced persister formation during the host cell infection. Thus, persister formation appears to be strain or ST (sequence type) dependent. Genome sequence analysis was carried out between ST1 clinical isolates and ST1 Paris. No genetic microevolution (SNP) linked to possible increase of persistence capacity was revealed among all the clones tested, even in clones issued from two persistence cycle experiments, confirming the transient reversible phenotypic status of persistence. Treatment failure in legionellosis is a serious issue as infections have a 5-10% mortality rate, and investigations into persistence in a clinical context and the mechanisms involved may allow us to combat this issue.
Assuntos
Legionella pneumophila , Legionelose , Humanos , Legionella pneumophila/genética , Reinfecção , Antibacterianos/farmacologia , Células ClonaisRESUMO
Introduction: Severe Legionnaires' disease (LD) can lead to multi-organ failure or death in 10%-30% of patients. Although hyper-inflammation and immunoparalysis are well described in sepsis and are associated with high disease severity, little is known about the immune response in LD. This study aimed to evaluate the immune status of patients with LD and its association with disease severity. Methods: A total of 92 hospitalized LD patients were included; 19 plasmatic cytokines and pulmonary Legionella DNA load were measured in 84 patients on the day of inclusion (day 0, D0). Immune functional assays (IFAs) were performed from whole blood samples collected at D2 and stimulated with concanavalin A [conA, n = 19 patients and n = 21 healthy volunteers (HV)] or lipopolysaccharide (LPS, n = 14 patients and n = 9 HV). A total of 19 cytokines (conA stimulation) and TNF-α (LPS stimulation) were quantified from the supernatants. The Sequential Organ Failure Assessment (SOFA) severity score was recorded at D0 and the mechanical ventilation (MV) status was recorded at D0 and D8. Results: Among the 84 patients, a higher secretion of plasmatic MCP-1, MIP1-ß, IL-6, IL-8, IFN-γ, TNF-α, and IL-17 was observed in the patients with D0 and D8 MV. Multiparametric analysis showed that these seven cytokines were positively associated with the SOFA score. Upon conA stimulation, LD patients had a lower secretion capacity for 16 of the 19 quantified cytokines and a higher release of IL-18 and MCP-1 compared to HV. IL-18 secretion was higher in D0 and D8 MV patients. TNF-α secretion, measured after ex vivo LPS stimulation, was significantly reduced in LD patients and was associated with D8 MV status. Discussion: The present findings describe a hyper-inflammatory phase at the initial phase of Legionella pneumonia that is more pronounced in patients with severe LD. These patients also present an immunoparalysis for a large number of cytokines, except IL-18 whose secretion is increased. An assessment of the immune response may be relevant to identify patients eligible for future innovative host-directed therapies.