Evaluation par des études in vitro et in vivo de l'implication des biofilms dans la virulence des staphylocoques à coagulase négative

Les staphylocoques à coagulase négative (SCN) sont parmi les germes le plus isolés des infections associées aux implants médicaux. L'aptitude des SCN à former des biofilms (B) semble jouer un rôle essentiel dans leur virulence. Le PIA (P) est l'un des polymères le mieux caractérisé des constituants des biofilms. Sa biosynthèse est encodée par l'opéron icaADBC (I). Après avoir constitué un souchier représentatif d'isolats cliniques et commensaux de SCN, nous avons étudié in vitro le lien existant entre les trois paramètres B, I et P. Seulement, un tiers des SCN sont B+. Les S. epidermidis commensaux, et contrairement à ceux d'origine clinique, sont P- et l'opéron ica est 2,5 fois moins fréquemment présent dans leurs génomes. Nous avons remarqué que certaines souches B+ mais P-, alors que d'autres P+ mais B-. L'étude de la structure chimique des constituants de la fraction déprotéinisée du biofilm a confirmé ces résultats. Nous avons ensuite évalué in vivo l'implication de B, I et P dans la virulence de certains SCN dans un modèle animal d'infection. La souche de référence RP62A du type (B+, I+, P+), et contrairement à la TM300 du type négatif (B-, I-, P-), maintient une infection dans ce modèle. Les isolats cliniques de S. epidermidis du type (B+, I+, P+) maintiennent également une infection. Curieusement, certains S. epidermidis cliniques et commensaux du type négatif sont capables de développer une infection. Dans ce modèle, l'aptitude à former du biofilm pourrait être indispensable pour la virulence de certaines souches. Cependant pour d'autres, cette aptitude n'est pas suffisante et impliquerait d'autre(s) facteur(s) de virulence qui restent à déterminer.Coagulase-negative staphylococci (CoNS) are among the most pathogens isolated in prosthetic device-related infections. Their ability to form biofilms (B) is believed to make them more resistant to antibiotic treatments and immune host defence system. Amongst the most clearly characterized components of staphylococcal biofilm is the PIA (P) which is synthesized by the icaADBC operon (I). PIA seemed to be essential for biofilm formation. We assessed in vitro the link between these three parameters (B, I, P) in clinical and commensal CoNS isolates. Only the third of clinical and commensal strains forms biofilm in vitro. Commensal strains of S. epidermidis were P- and they are 2.5 times less I+. Some strains were B+ but P- while others were P+ But B-. Structural analyses show that the biofilm of certain strains contains PIA and teichoïc acid (TA), while that of others contains only TA. Using an animal model we carried out the involvement of B, I and P parameters in the ability of CoNS to maintain an infection in vivo. We have shown that the model strain, S. epidermidis RP62A known as (B+, I+, P+) was able maintain an infection in vivo whereas, the other model strain, S. carnosus TM300 known as (B-, I-, P-) was not. Examined with other clinical S. epidermidis, the type (B+, I+, P+) confers the ability to maintain an infection in vivo. Surprisingly some clinical and commensal isolates having the negative profile were also able to maintain an infection in vivo. Depending on the strains, and more than the presence of ica locus and in vitro-biofilm formation ability, S. epidermidis probably needs an other potential virulent factor(s) to be able to maintain an infection in vivo.BOULOGNE-BU Droit Lettres (621602101) / SudocSudocFranceF

Etude comparative du rôle du Quorum Sensing dans la régulation de l'expression des facteurs de virulence chez un isolat clinique de Staphylococcus Aureus en culture planctonique et en biofilm

Staphylococcus Aureus est un pathogène opportuniste produisant un grand nombre de facteurs de virulence. Parmi ceux-ci, la toxine a excrétée, codée par hla, et la protéine A de surface, codée par spa, sont souvent utilisées comme modèle d'étude de la régulation de l'expression des facteurs de virulence. Le système de quorum sensing (QS), codé par le locus agr, intervient dans leur régulation in vitro. La capacité de S. Aureus à former des biofilm est aussi considéré comme facteur de virulence permettant d'aggraver et de rendre chronique les infections. L'un des composants du biofilm (le PIA) est synthétisé par les produits du locus ica. Le rôle du QS dans la régulation de l'expression de hla, de spa et ica a été étudié chez un isolat clinique issu d'une infection sur prothèse orthopédique et chez son mutant isogénique délété dans le gène agrC, au cours de la croissance en conditions planctonique et sessile dans le biofilm. La technique de PCR quantitative en temps réel associée à la méthode de quantification relative des transcrits desgènes d'interêt, en utilisant le transcrit 16S comme standard interne se sont révélées les mieux adaptées à l'étude de la transcription au cours de la croissance. Dans les deux conditions de culture, nous avons montré que le transcrit RNAIII, molécule effectrice du QS, était exprimée à un niveau basal en absence d'agrC, supposé être absolument requis pour son expression. Notre étude a également mis en évidence, pour la première fois une activation d'ica par le QS, alors que la production de biofilm est supérieure chez le mutant. Enfin, la régulation de l'expression d'hla et spa par le QS est très différente selon les conditions de culture.Staphylococcus aureus is an opportunistic pathogen producing a large number of virulence factors. Among these, the secreted alpha-toxin, encoded by hla, and the surface-associated protein A, encoded by spa, are often considered as reporters of virulence factors regulation studies. The quorum sensing (QS) system, encoded by agr, is involved in their regulation in vitro. The biofilm formation ability of S. aureus is also considered as a virulence factor, as it is the cause of heavier and chronic infections. One of the biofilm components (PIA) is synthesized by the products of the ica locus. QS involvement in the expression regulation of hla, spa and ica is studied in a clinical strain, isolated from an infected prosthesis, and in an isogenic mutant strain deleted in agrC gene, during planktonic and biofilm sessile growth. The real-time quantitative PCR technique associated with relative quantification method of target genes transcripts compared to 16S transcripts appeared to be the best method for transcription study during growth. In both growth conditions, we have demonstrated that the RNAIII transcript, the QS molecular effector, was expressed in a basal level in agrC mutant. In the same time, our study has for the first time outlined an ica activation by the QS, whereas the biofilm production by the mutant was more important . Finally, hla and spa expression regulation by QS is very different depending on growing conditions.BOULOGNE-BU Droit Lettres (621602101) / SudocSudocFranceF

Extracellular Carbohydrate-Containing Polymers of a Model Biofilm-Producing Strain, Staphylococcus epidermidis RP62A

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. It has been established that clinical isolates often produce a biofilm, which is involved in adherence to biomaterials and provides enhanced resistance of bacteria against host defenses and antibiotic treatments. It has been thought that the staphylococcal biofilm contains two polysaccharides, one responsible for primary cell adherence to biomaterials (polysaccharide/adhesin [PS/A]) and an antigen that mediates bacterial aggregation (polysaccharide intercellular adhesin [PIA]). In the present paper we present an improved procedure for preparation of PIA that conserves its labile substituents and avoids contamination with by-products. Based on structural analysis of the polysaccharide antigens and a thorough overview of the previously published data, we concluded that PIA from S. epidermidis is structurally identical to the recently described poly-β-(1→6)-N-acetylglucosamine from PS/A-overproducing strain S. aureus MN8m. We also show that another carbohydrate-containing polymer, extracellular teichoic acid (EC TA), is an essential component of S. epidermidis RP62A biofilms. We demonstrate that the relative amounts of extracellular PIA and EC TA produced depend on the growth conditions. Moderate shaking or static culture in tryptic soy broth favors PIA production, while more EC TA is produced in brain heart infusion medium

Structural Characterization of a Flavonoid-Inducible Pseudomonas aeruginosa A-Band-Like O Antigen of Rhizobium sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules

Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRΩfixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the fixF mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-α-l-Rhap-(1,3)-α-l-Rhap-(1,2)-α-l-Rhap-(1-](n), and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of Pseudomonas aeruginosa, except that it is composed of l-rhamnose rather than the d-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the P. aeruginosa cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection

Potential Use of Poly-N-Acetyl-β-(1,6)-Glucosamine as an Antigen for Diagnosis of Staphylococcal Orthopedic-Prosthesis-Related Infections▿

Staphylococcus aureus and coagulase-negative staphylococci are microorganisms most frequently isolated from orthopedic-implant-associated infections. Their capacity to maintain these infections is thought to be related to their ability to form adherent biofilms. Poly-N-acetyl-β-(1,6)-glucosamine (PNAG) is an important constituent of the extracellular biofilm matrix of staphylococci. In the present study, we explored the possibility of using PNAG as an antigen for detecting antibodies in the blood sera of patients with staphylococcal orthopedic-prosthesis-associated infections. First, we tested the presence of anti-PNAG antibodies in an animal model, in the blood sera of guinea pigs that developed an implant-associated infection caused by biofilm-forming, PNAG-producing strains of Staphylococcus epidermidis. Animals infected with S. epidermidis RP62A showed levels of anti-PNAG immunoglobulin G (IgG) significantly higher than those of the control group. The comparative study of healthy individuals and patients with staphylococcal prosthesis-related infections showed that (i) relatively high levels of anti-PNAG IgG were present in the blood sera of the healthy control group, (ii) the corresponding levels in the infected patients were slightly but not significantly higher, and (iii) only 1 of 10 patients had a level of anti-PNAG IgM significantly higher than that of the control group. In conclusion, the encouraging results obtained in the animal study could not be readily applied for the diagnosis of staphylococcal orthopedic-prosthesis-related infections in humans, and PNAG does not seem to be an appropriate antigen for this purpose. Further studies are necessary to determine whether the developed enzyme-linked immunosorbent assay method could serve as a complementary test in the individual follow-up treatment of such infections caused by PNAG-producing staphylococci

Poly-N-acetylglucosamine and poly(glycerol phosphate) teichoic acid identification from staphylococcal biofilm extracts using excitation sculptured TOCSY NMR

We report the successful application of selective excitation sculptured TOCSY NMR (SXS-TOCSY) to identify individual solution components from a heterogeneous system using selectively acquired 1 H NMR spin system patterns. SXS-TOCSY application is illustrated by detection of the simultaneous presence of poly-beta-(1,6)-N-acetylglucosamine (PNAG) and poly(glycerol phosphate) teichoic acid (TA) carbohydrate polymer components in crude biofilm extracts from Staphylococcus epidermidis without the need for further sample purification and component separation. Biofilms are implicated in the barriers for resistance of microbes toward antibiotics and immune responses, therefore efficient rapid detection and quantification of key components are important to assist in the design of a clinical infection response