Caracterización molecular de los genes capsulares de "Streptococcus pneumoniae" serotipo 3

Tesis de la Universidad Complutense de Madrid, Faculta de Ciencias Biológicas, Departamento de Microbiología III, leída el 18-06-1996Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu

SdrF, a Staphylococcus epidermidis Surface Protein, Contributes to the Initiation of Ventricular Assist Device Driveline–Related Infections

Staphylococcus epidermidis remains the predominant pathogen in prosthetic-device infections. Ventricular assist devices, a recently developed form of therapy for end-stage congestive heart failure, have had considerable success. However, infections, most often caused by Staphylococcus epidermidis, have limited their long-term use. The transcutaneous driveline entry site acts as a potential portal of entry for bacteria, allowing development of either localized or systemic infections. A novel in vitro binding assay using explanted drivelines obtained from patients undergoing transplantation and a heterologous lactococcal system of surface protein expression were used to identify S. epidermidis surface components involved in the pathogenesis of driveline infections. Of the four components tested, SdrF, SdrG, PIA, and GehD, SdrF was identified as the primary ligand. SdrF adherence was mediated via its B domain attaching to host collagen deposited on the surface of the driveline. Antibodies directed against SdrF reduced adherence of S. epidermidis to the drivelines. SdrF was also found to adhere with high affinity to Dacron, the hydrophobic polymeric outer surface of drivelines. Solid phase binding assays showed that SdrF was also able to adhere to other hydrophobic artificial materials such as polystyrene. A murine model of infection was developed and used to test the role of SdrF during in vivo driveline infection. SdrF alone was able to mediate bacterial adherence to implanted drivelines. Anti-SdrF antibodies reduced S. epidermidis colonization of implanted drivelines. SdrF appears to play a key role in the initiation of ventricular assist device driveline infections caused by S. epidermidis. This pluripotential adherence capacity provides a potential pathway to infection with SdrF-positive commensal staphylococci first adhering to the external Dacron-coated driveline at the transcutaneous entry site, then spreading along the collagen-coated internal portion of the driveline to establish a localized infection. This capacity may also have relevance for other prosthetic device–related infections

Recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections

Sequence Diversities of Serine-Aspartate Repeat Genes among Staphylococcus aureus Isolates from Different Hosts Presumably by Horizontal Gene Transfer

BACKGROUND: Horizontal gene transfer (HGT) is recognized as one of the major forces for bacterial genome evolution. Many clinically important bacteria may acquire virulence factors and antibiotic resistance through HGT. The comparative genomic analysis has become an important tool for identifying HGT in emerging pathogens. In this study, the Serine-Aspartate Repeat (Sdr) family has been compared among different sources of Staphylococcus aureus (S. aureus) to discover sequence diversities within their genomes. METHODOLOGY/PRINCIPAL FINDINGS: Four sdr genes were analyzed for 21 different S. aureus strains and 218 mastitis-associated S. aureus isolates from Canada. Comparative genomic analyses revealed that S. aureus strains from bovine mastitis (RF122 and mastitis isolates in this study), ovine mastitis (ED133), pig (ST398), chicken (ED98), and human methicillin-resistant S. aureus (MRSA) (TCH130, MRSA252, Mu3, Mu50, N315, 04-02981, JH1 and JH9) were highly associated with one another, presumably due to HGT. In addition, several types of insertion and deletion were found in sdr genes of many isolates. A new insertion sequence was found in mastitis isolates, which was presumably responsible for the HGT of sdrC gene among different strains. Moreover, the sdr genes could be used to type S. aureus. Regional difference of sdr genes distribution was also indicated among the tested S. aureus isolates. Finally, certain associations were found between sdr genes and subclinical or clinical mastitis isolates. CONCLUSIONS: Certain sdr gene sequences were shared in S. aureus strains and isolates from different species presumably due to HGT. Our results also suggest that the distributional assay of virulence factors should detect the full sequences or full functional regions of these factors. The traditional assay using short conserved regions may not be accurate or credible. These findings have important implications with regard to animal husbandry practices that may inadvertently enhance the contact of human and animal bacterial pathogens

Recommendations for design and conduct of preclinical in vivo studies of orthopedic device-related infection

Orthopedic device-related infection (ODRI), including both fracture-related infection (FRI) and periprosthetic joint infection (PJI), remain amongst the most challenging complications in orthopedic and musculoskeletal trauma surgery. ODRI has been convincingly shown to delay healing, worsen functional outcome and incur significant socio-economic costs. To address this clinical problem, ever more sophisticated technologies targeting the prevention and/or treatment of ODRI are being developed and tested in vitro and in vivo. Amongst the most commonly described innovations are antimicrobial-coated orthopedic devices, antimicrobial-loaded bone cements and void fillers, and dual osteo-inductive/antimicrobial biomaterials. Unfortunately, translation of these technologies to the clinic has been limited, at least partially due to the challenging and still evolving regulatory environment for antimicrobial drug-device combination products, and a lack of clarity in the burden of proof required in preclinical studies. Preclinical in vivo testing (i.e. animal studies) represents a critical phase of the multidisciplinary effort to design, produce and reliably test both safety and efficacy of any new antimicrobial device. Nonetheless, current in vivo testing protocols, procedures, models and assessments are highly disparate, irregularly conducted and reported, and without standardization and validation. The purpose of the present opinion piece is to discuss best practices in preclinical in vivo testing of antimicrobial interventions targeting ODRI. By sharing these experience-driven views, we aim to aid others in conducting such studies both for fundamental biomedical research, but also for regulatory and clinical evaluation

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence.

BACKGROUND: The polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur. RESULTS: Here, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater. CONCLUSIONS: We identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential

Pathogenic Mechanisms and Host Interactions in Staphylococcus epidermidis Device-Related Infection

Staphylococcus epidermidis is a permanent member of the normal human microbiota, commonly found on skin and mucous membranes. By adhering to tissue surface moieties of the host via specific adhesins, S. epidermidis is capable of establishing a lifelong commensal relationship with humans that begins early in life. In its role as a commensal organism, S. epidermidis is thought to provide benefits to human host, including out-competing more virulent pathogens. However, largely due to its capacity to form biofilm on implanted foreign bodies, S. epidermidis has emerged as an important opportunistic pathogen in patients receiving medical devices. S. epidermidis causes approximately 20% of all orthopedic device-related infections (ODRIs), increasing up to 50%in late-developing infections. Despite this prevalence, it remains underrepresented in the scientific literature, in particular lagging behind the study of the S. aureus. This review aims to provide an overview of the interactions of S. epidermidis with the human host, both as a commensal and as a pathogen. The mechanisms retained by S. epidermidis that enable colonization of human skin as well as invasive infection, will be described, with a particular focus upon biofilm formation. The host immune responses to these infections are also described, including how S. epidermidis seems to trigger low levels of pro-inflammatory cytokines and high levels of interleukin-10, which may contribute to the sub-acute and persistent nature often associated with these infections. The adaptive immune response to S. epidermidis remains poorly described, and represents an area which may provide significant new discoveries in the coming years