Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae 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.

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

BACKROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Proteomic biomarkers associated with Streptococcus agalactiae invasive genogroups.

Group B streptococcus (GBS, Streptococcus agalactiae) 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 (i.e. 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.

A multi locus variable number of tandem repeat analysis (MLVA) scheme for Streptococcus agalactiae genotyping.

BACKGROUND: Multilocus sequence typing (MLST) is currently the reference method for genotyping Streptococcus agalactiae 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 S. agalactiae strains of various origins characterized by MLST and serotyping. RESULTS: 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. CONCLUSIONS: 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 S. agalactiae and analyses of outbreaks.

Rapid detection of "highly virulent" Group B Streptococcus ST-17 and emerging ST-1 clones by MALDI-TOF mass spectrometry.

MALDI-TOF MS identified a 6250-Da protein specific to Sequence Type-1 (ST-1) strains and a 7625-Da protein specific to ST-17 strains when used for identification of Group B streptococci. The strains of these STs are major causes of meningitis and late-onset-disease in neonates. This rapid method of identification could thus be valuable in the evaluation of risk of neonatal diseases.

Identification of Streptococcus agalactiae isolates from various phylogenetic lineages by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Variations in proteins related to bacterial diversity may affect species identification performed using matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry. Using this method, we identified 110 Streptococcus agalactiae isolates characterized by serotyping and multilocus sequence typing. Serotype III and sequence type 23 strains expressed the widest variation in molecular weight of putative "species-identifying" biomarker ions. Recognition of the diversity of MALDI patterns observed in strains that represent all major intraspecies lineages assists in the constitution of an optimal reference database.