Applications of fluorescence in situ hybridization and molecular strategies for the diagnosis of bacterial infections

Une partie de ce travail de thèse a consisté à appliquer les méthodes de FISH pour l’étude de trois bactéries pathogènes intracellulaires. La viabilité de Bartonella henselae a été évaluée à partir de ganglions de patients atteints de la maladie des griffes du chat (CSD). Le faible taux d’ARN détecté par biologie moléculaire, la stérilité des cultures, l'absence de détection par analyses histologiques et FISH confirment que B. henselae n'est pas ou rarement viable dans les ganglions de patients atteints de CSD. Tropheryma whipplei, l’agent de la maladie de Whipple, a été identifié et localisé par FISH, dans les macrophages d’un ganglion et d’une biopsie pulmonaire, confirmant le diagnostic infectieux. Deux méthodes de FISH ont été testées pour détecter Coxiella burnetii dans des cas d’endocardites et d’infections vasculaires en utilisant des sondes oligonucléotidiques et des sondes PNA. Les résultats ont confirmé une meilleure efficacité des sondes PNA et démontré que les techniques de FISH sont plus sensibles que l’immunohistochimie pour le diagnostic des endocardites et des infections vasculaires à C. burnetii. Nous avons également évalué les stratégies moléculaires mises en place pour le diagnostic syndromique. Bien que la PCR conventionnelle à large spectre permette l'identification de micro-organismes fastidieux et anaérobies, la PCR spécifique en temps réel révèle une supériorité significative dans le diagnostic syndromique. En conclusion, ce travail a permis de démontrer l’efficacité et l’applicabilité de la FISH pour la détection bactérienne. Cette méthode peut être utilisée comme un outil complémentaire afin d'améliorer le diagnostic de microbiologie clinique.We applied FISH methods to the study of three intracellular pathogenic bacteria. The viability of Bartonella henselae was evaluated in a large series of lymph nodes from patients with cat scratch disease (CSD). The results obtained, associated with sterile cultures and negative histological analyzes and FISH, as well as the low level of RNA detected by molecular biology, provide evidence that B. henselae are not or are rarely viable in the lymph nodes of patients with CSD. Tropheryma whipplei has been identified by FISH in macrophages from one lymph node and for the first time in a pulmonary biopsy, confirming the diagnosis of infection. Two methods of FISH have been tested to detect Coxiella burnetii in cases of endocarditis and vascular infections using oligonucleotide and PNA probes. The results attested to the greater efficiency of PNA probes, and demonstrated that FISH were applicable for the diagnosis of C. burnetii endocarditis. We also evaluated the molecular strategies used for syndrome-driven diagnosis of infectious diseases. Although conventional broad-spectrum PCR allows for the identification of fastidious and anaerobic microorganisms, real-time specific PCR reveals a significant superiority in syndrome-driven diagnosis. The addition of specific PCRs in real time PCR would improve our molecular strategies, for example, in the case of the detection of Staphylococcus aureus for the diagnosis of lymphadenopathy. In conclusion, this work demonstrates the effectiveness and applicability of FISH for the identification of intracellular bacteria. This method can be used as an important complementary tool to the improvement of clinical microbiological diagnosis

Fluorescence in situ hybridization, a complementary molecular tool for the clinical diagnosis of infectious diseases by intracellular and fastidious bacteria

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Original sequence divergence among Pseudomonas putida CadRs drive specificity

International audienceBacteria, especially those living in soils, are in constant contact with metals. Transition metals like Fe or Zn, are required for proper growth. Some other metals like Cd or Hg are only toxic. Several systems exist to detoxify cells when these metals are present in concentrations harmful to biological systems. The expression of these systems is under control of specialized regulatory proteins able to detect metals and to regulate cognate detoxifying systems. In this work we report on the characterisation of the metallo-regulator CadR from P. putida KT2440. By using gene reporter assays, we investigated the repertoire of metals detected by CadR. We show that CadR is much more responsive to Hg than to Cd, as compared to CadR from P. putida 06909. CadR from P. putida KT2440 differs in only 3 amino-acids in its metal-binding domain with respect to CadR from P. putida 06909. We show that these residues are important determinants of metal selectivity by engineering a modified CadR

Fluorescence In Situ Hybridization (FISH) and Peptide Nucleic Acid Probe-Based FISH for Diagnosis of Q Fever Endocarditis and Vascular Infections

International audienceEndocarditis and vascular infections are common manifestations of persistent localized infection due to Coxiella burnetii, and recently, fluorescence in situ hybridization (FISH) was proposed as an alternative tool for their diagnosis. In this study, we evaluated the efficiency of FISH in a series of valve and vascular samples infected by C. burnetii. We tested 23 C. burnetii-positive valves and thrombus samples obtained from patients with Q fever endocarditis. Seven aneurysms and thrombus specimens were retrieved from patients with Q fever vascular infections. Samples were analyzed by culture, immunochemistry, and FISH with oligonucleotide and PNA probes targeting C. burnetii-specific 16S rRNA sequences. The immunohistochemical analysis was positive for five (17%) samples with significantly more copies of C. burnetii DNA than the negative ones (P = 0.02). FISH was positive for 13 (43%) samples and presented 43% and 40% sensitivity compared to that for quantitative PCR (qPCR) and culture, respectively. PNA FISH detected C. burnetii in 18 (60%) samples and presented 60% and 55% sensitivity compared to that for qPCR and culture, respectively. Immunohistochemistry had 38% and 28% sensitivity compared to that for FISH and PNA FISH, respectively. Samples found positive by both immunohistochemistry and PNA FISH contained significantly more copies of C. burnetii DNA than the negative ones (P = 0.03). Finally, PNA FISH was more sensitive than FISH (60% versus 43%, respectively) for the detection of C. burnetii. We provide evidence that PNA FISH and FISH are important assays for the diagnosis of C. burnetii endocarditis and vascular infections

Evaluation of 11 DNA Automated Extraction Protocols for the Detection of the 5 Mains Candida Species from Artificially Spiked Blood

International audienceThe molecular detection of Candida plays an important role in the diagnosis of candidaemia, a major cause of morbidity and mortality. The sensitivity of this diagnosis is partly related to the efficiency of yeast DNA extraction. In this monocentric study, we investigated the suitability of 11 recent automated procedures for the extraction of low and high amounts of Candida DNA from spiked blood. The efficacy of the DNA extraction procedures to detect Candida spp. in blood samples ranged from 31.4% to 80.6%. The NucliSENSTM easyMAGTM procedure was the most efficient, for each species and each inoculum. It significantly outperformed the other procedures at the lower Candida inocula mimicking the clinical setting. This study highlighted a heterogeneity in DNA extraction efficacy between the five main Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei). Up to five automated procedures were appropriate for C. krusei DNA extraction, whereas only one method yielded an appropriate detection of low amount of C. tropicalis. In the era of the syndromic approach to bloodstream infection diagnosis, this evaluation of 11 automated DNA extraction methods for the PCR diagnosis of candidaemia, puts the choice of an appropriate method in routine diagnosis within the reach of laboratories

Molecular strategy for the diagnosis of infectious lymphadenitis

International audienceMolecular methods have been considered to be the gold standard for the diagnosis of infectious lymphadenitis. However, culture remains critical in the case of low bacterial concentrations. We used molecular assays and culture to examine fresh lymph node biopsies from patients with suspected infectious lymphadenopathy. We analyzed 1762 lymph node biopsies of which 522 (30%) samples were found positive by real-time PCR; the most commonly amplified bacteria were Bartonella henselae (n = 438, 84%), Francisella tularensis (n = 46, 9%), and Mycobacterium spp. (n = 29, 6%). PCR amplification and sequencing of the 16S rDNA were positive for 359 (20%) lymph node specimens including mainly B. henselae (n = 167, 47%), Staphylococcus spp. (n = 77, 21%), and Streptococcus spp. (n = 41, 11%). In total, 351 lymph nodes were cultured on agar plates and 77 (22%) were positive. Significantly more lymph nodes infected by Gram-positive easy-growing agents were diagnosed by culture (n = 45) than by 16S rDNA PCR (p = 0.02). Culture remains critical for the diagnosis of easy-growing bacteria and mycobacteria; clinicians should be aware that a negative molecular result does not imply absence of infection

Bartonella infections diagnosed in the French reference center, 2014–2019, and focus on infections in the immunocompromised

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Case Report: Scalp Eschar and Neck Lymphadenopathy Associated with Bacteremia due to Coxiella-Like Bacteria

International audienceCoxiella-like bacteria have been recently proposed as human pathogens. Using molecular techniques, we detected Coxiella-like bacteria in the blood and serum samples of a patient with a scalp eschar, neck lymphadenopathy, severe urticaria, edema, fever, and arthralgia indicating that this organism can provide systemic complications

Fluorescent in situ hybridization can be used as a complementary assay for the diagnosis of Tropheryma whipplei infection

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Pushing the limits of nickel detection to nanomolar range using a set of engineered bioluminescent Escherichia coli

International audienceThe detection of nickel in water is of great importance due to its harmfulness for living organism. A way to detect Ni is the use of whole-cell biosensors. The aim of the present work was to build a light-emitting bacterial biosensor for the detection of Ni with high specificity and low detection limit properties. For that purpose, the regulatory circuit implemented relied on the RcnR Ni/Co metallo-regulator and its rcnA natural target promoter fused to the lux reporter genes. To convert RcnR to specifically detect Ni, several mutations were tested and the C35A retained. Deleting the Ni efflux pump rcnA and introducing genes encoding several Ni-uptake systems lowered the detection thresholds. When these constructs were assayed in several Escherichia coli strains, it appeared that the detection thresholds were highly variable. The TD2158 wild-type E. coli gave rise to a biosensor ten times more active and sensitive than its W3110 E. coli K12 equivalent. This biosensor was able to confidently detect Ni concentrations as little as 80 nM (4.7 μg l −1), which makes its use compatible with the norms governing the drinking water quality