Typage moléculaire de streptococcus agalactiae par multi locus variable number of tandem repeat analysis

TOURS-BU Médecine (372612103) / SudocSudocFranceF

A multi locus variable number of tandem repeat analysis (MLVA) scheme for <it>Streptococcus agalactiae </it>genotyping

<p>Abstract</p> <p>Background</p> <p>Multilocus sequence typing (MLST) is currently the reference method for genotyping <it>Streptococcus agalactiae </it>strains, the leading cause of infectious disease in newborns and a major cause of disease in immunocompromised children and adults. We describe here a genotyping method based on multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) applied to a population of <it>S. agalactiae </it>strains of various origins characterized by MLST and serotyping.</p> <p>Results</p> <p>We studied a collection of 186 strains isolated from humans and cattle and three reference strains (A909, NEM316 and 2603 V/R). Among 34 VNTRs, 6 polymorphic VNTRs loci were selected for use in genotyping of the bacterial population. The MLVA profile consists of a series of allele numbers, corresponding to the number of repeats at each VNTR locus. 98 MLVA genotypes were obtained compared to 51 sequences types generated by MLST. The MLVA scheme generated clusters which corresponded well to the main clonal complexes obtained by MLST. However it provided a higher discriminatory power. The diversity index obtained with MLVA was 0.960 compared to 0.881 with MLST for this population of strains.</p> <p>Conclusions</p> <p>The MLVA scheme proposed here is a rapid, cheap and easy genotyping method generating results suitable for exchange and comparison between different laboratories and for the epidemiologic surveillance of <it>S. agalactiae </it>and analyses of outbreaks.</p

Profile d'expression d'adhésines chez<em> Mycobacterium intracellulare</em>

National audienc

Analysis of the type II-A CRISPR-Cas system of [i]Streptococcus agalactiae[/i] reveals distinctive features according to genetic lineages

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I-C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I-C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage

Proteomic Biomarkers Associated with <em>Streptococcus agalactiae</em> Invasive Genogroups

<div><p>Group B streptococcus (GBS, <em>Streptococcus agalactiae</em>) is a leading cause of meningitis and sepsis in newborns and an etiological agent of meningitis, endocarditis, osteoarticular and soft tissue infections in adults. GBS isolates are routinely clustered in serotypes and in genotypes. At present one GBS sequence type (<em>i.e.</em> ST17) is considered to be closely associated with bacterial invasiveness and novel proteomic biomarkers could make a valuable contribution to currently available GBS typing data. For that purpose we analyzed the protein profiles of 170 genotyped GBS isolates by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI). Univariate statistical analysis of the SELDI profiles identified four protein biomarkers significantly discriminating ST17 isolates from those of the other sequence types. Two of these biomarkers (MW of 7878 Da and 12200 Da) were overexpressed and the other two (MW of 6258 Da and 10463 Da) were underexpressed in ST17. The four proteins were isolated by mass spectrometry-assisted purification and their tryptic peptides analyzed by LC-MS/MS. They were thereby identified as the small subunit of exodeoxyribonuclease VII, the 50S ribosomal protein L7/L12, a CsbD-like protein and thioredoxin, respectively. In conclusion, we identified four candidate biomarkers of ST17 by SELDI for high-throughput screening. These markers may serve as a basis for further studies on the pathophysiology of GBS infection, and for the development of novel vaccines.</p> </div

BLAST comparison of the four identifiedprotein biomarkers of <i>S. agalactiae</i> to other taxa.

*<p>Accession number on <a href="http://www.ncbi.nlm.nih.gov/protein/" target="_blank">http://www.ncbi.nlm.nih.gov/protein/</a>.</p>**<p>Sequence identified by LC/MS-MS and used in BLAST to generate the list of the first 100 sequences with closest homology or identity.</p><p><b>NF:</b> Absent from the list of the first 100 sequences compared by BLAST.</p><p>Software used: BLASTP 2.2.26+ (server - NCBI). Reference: Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, et al. (1997) Nucleic Acids Res. 25: 3389–3402.</p

Expression levels of p6258, p7878, p10464 and p12200 in 149 <i>S. agalactiae</i> isolates genotyped by multilocus sequence typing (MLST) and represented as a dendrogram showing genetic relationships among the different sequence types (STs).

<p>The dendrogram, based on MLST data, was constructed using BioNumerics 6.5 software (Applied Maths, Sint-Martens-Latem, Belgium). Cluster analysis was based on an unweighted pair group method using arithmetic averages (UPGMA). The nine main MLST groups (A to I) are indicated on the right of the dendrogram as vertical dotted lines. The allelic profiles corresponding to each sequence type (ST), the number of isolates belonging to each ST, the origin of the isolate, the serotype and the presence (+) or absence (−) of the biomarkers of interest are reported in detail. Within each ST, the presence of the biomarker is indicated as a ratio between the number of isolates with a detectable corresponding peak in SELDI and the number of all isolates in the ST. A grey scale also illustrates the prevalence of the biomarker (dark grey when the peak corresponding to the biomarker is detected in all isolates; light grey when the peak corresponding to the biomarker is partially detected).</p

Biomarkers selected by SELDI profiling of two groups of <i>S. agalactiae</i> strains, positive or negative for ST17.

*<p>Acquisiton protocol:</p><p>Protocol 1: Focus Mass 10 000, Laser Energy 2 500, Matrix Attenuation 2 500 ; 20 shots ;</p><p>Partition 1/4.</p><p>Protocol 2 : Focus Mass 15 000, Laser Energy 3 000, Matrix Attenuation 5 000; 20 shots.</p><p>Partition 2/4.</p>**<p>Signal/Noise ratio of detected peaks >2.</p

Aerosol Route to Administer Teicoplanin in Mechanical Ventilation: In Vitro Study, Lung Deposition and Pharmacokinetic Analyses in Pigs

International audienceBackround: Glycopeptides given intravenously achieve low airway concentrations. Nebulization of teicoplanin may be an efficient way of delivering a high concentration of this antibiotic to the lung. This multistep study assessed the feasibility of teicoplanin nebulization during mechanical ventilation by evaluating: the stability of its antibiotic effect; epithelial tolerance; lung deposition and systemic absorption in ventilated pigs. Nebulized and non-nebulized teicoplanin activity was tested on Staphylococcus aureus cultures. The cytotoxic effect of teicoplanin on human respiratory epithelial cells was assessed by measuring lactate dehydrogenase activity released, cell viability, and transepithelial electrical resistance. Volume median diameter of particles of nebulized teicoplanin was measured by laser diffraction during mechanical ventilation. The deposited mass of teicoplanin nebulized with a vibrating mesh nebulizer in ventilated piglets was assessed by scintigraphy. Blood pharmacokinetics of teicoplanin administered either intravenously or by nebulization was compared. No decrease of antibiotic activity was observed after nebulization. In vitro cytotoxicity of teicoplanin was only observed with 1000 times the dose recommended for intravenous administration. Volume median diameter of particles was 2.5±0.1 μm. Of the initial nebulizer charge of teicoplanin, 24±7% was present in the lungs of ventilated pigs after the nebulization. Amount absorbed in blood was low (3.4%±0.9%) after nebulization, and blood stream elimination half-life value was 25.4 h. Teicoplanin was administered efficiently by nebulization during mechanical ventilation, without any effect on its pharmacological properties or any cytotoxicity. The pharmacokinetic parameters are promising in view of its time-dependent killing process. All the results of our multi-step study highlighted the potential of teicoplanin to be nebulized during mechanical ventilation