Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potentialperinatal pathogens. Their relationships between genotypes and pathogenesis ofGBS and ureaplasma infection were still not well understood, one of the reason isthat both of them are still short of a very practical genotyping system. In the study,to solve the above problem we developed genotyping systems for the organisms (thesecond section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasmaspecies (U. parvum and U. urealyticum). Further, based on the heterogeneity ofureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotypingsystems showed that the genotyping systems were practical alternative assays forthe conventional serotyping and they will be useful to further explore therelationships between genotypes and pathogenesis of GBS and ureaplasmainfection. In the study, we introduced novel data and tools into GBS and ureaplasmastudies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based onthe U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied thetwo published full genomes and exposed some new problems or possible future newresearch fields. In particular we found the two finished and one ongoing GBSgenomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integratedstudies of the two potential or conditional perinatal pathogens, from the viewpointof evolution, would provide a new understanding angle of the pathogenesis of thetwo organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host(by losing most of its virulence genes); however, GBS tried to increase its invasiveabilities (by getting more virulence genes) to fight against the human host attack.

Adhesion of bacteria is a key event in biofilm formation and is mediated by bacterial adhesins recognising host or bacterial partner receptors. In oral biofilm formation, primary Actinomyces and Streptococcus colonizers adhere to salivary pellicle proteins such as proline-rich proteins (PRPs) as well as to mucosal surfaces. Subsequently, Actinomyces and Streptococcus strains and other bacteria, such as Veillonella, Fusobacterium and Porphyromonas, adhere to each other. The nature of this community is highly important for the health or disease status, although specific pathogenic species may also have been implicated. The aim of this thesis was to study key players in early oral colonisation, Actinomyces and Streptococcus species, and more specifically the nature of their adhesins and ligands. A further aim was to study the function of the salivary PRP proteins and an innate peptide derived thereof on bacterial adhesion, proliferation and regulation of pH, i.e. key factors in biofilm formation. In paper I and II, adhesion, proliferation and pH affecting features of the RGRPQ (arginine-glycine-arginine-proline-glutamine) peptide, derived from PRP-1, were demonstrated. By use of an alanine-scan (I), motifs for adhesion inhibition and desorption of Actinomyces naeslundii, and proliferation stimulation, ammonia production and inhibition of sucrose induced pH drop by Streptococcus gordonii were indicated. The RGRPQ peptide also stimulated S. gordonii colonisation in vivo. In paper II, a more sophisticated quantitative structure-activity relationship (QSAR) study, using statistical molecular design (SMD) and multivariate modelling (partial least squares projections to latent structures, PLS), further narrowed down the RGRPQ peptide motifs. The R and Q amino acids were crucial for activity. For proliferation a hydrophobic and large size third position amino acid was crucial, while adhesion inhibition and desorption needed a small hydrophilic second position amino acid. All functions depended on a low polarity hydrophobic fourth position. Accordingly, activities could be optimized separately, with decreased function in the others. In paper III and IV, focus was on the bacterial adhesins and their binding epitopes. The genes for FimA major subunit proteins of type-2 fimbriae were sequenced from A. naeslundii genospecies 1 and 2 and Actinomyces odontolyticus, each with unique carbohydrate binding specificities (III). Three major subtypes of FimA proteins were found that correlated with binding specificity, including a novel fimA gene in A. odontolyticus. All subtypes contained a pilin, LPXTG and E box motif. In paper IV, multiple PRP binding patterns for Actinomyces and Streptococcus strains were mapped using a hybrid peptide construct. The two most deviating binding groups deviated in type-1 fimbriae mediated binding to milk and saliva protein ligands. In conclusion, differences in bacterial adhesins and their ability to utilise salivary proteins may render bacteria tropism for different niches. Peptides derived from protein receptors, such as RGRPQ, may be important modulators of biofilm formation, giving commensal bacteria a competitive edge in the bacterial community.

The present investigation was undertaken to characterize a number of strains of Lactobacillus bulgaricus and Streptococcus thermophilus intended for use by a commercial starter supply company. Thorough characterization of each culture was required in order to combine compatible strains so that their usefulness in Mozzarella cheese manufacture would be maximized. In this regard, cocci were assayed for formate and carbon dioxide production, rods for proteolysis, and both types for salt and phosphate tolerance as well as rate of acid production. In addition, certain combinations of cocci and rods were assayed as mixtures for these characteristics. Analyses of the various strains of lactobacilli and S. thermophilus were performed. Proteolysis, as determined by the Church method, for the rods (L. bulgaricus , L. helveticus and L. lactis ) varied from as low as 11.3 to as high as 34.7 mM when incubated for six hours. Proteolysis analyses for S. thermophilus also revealed a wide range of values from a low of 18.5 to a high of 46.4 mM. However, when strains were incubated for 16 hours, rods were shown to be nearly twice as proteolytic as cocci. When mixed cultures were tested for proteolysis, results were dependent on strain synergism. Values ranged from a low of 5.1 mM to 70.5 mM in mixed cultures. Various strains of S. thermophilus and mixed cultures were assayed for formate production. The S. thermophilus strain values were from a low of 4.2 to as high as 20.3 mg/L. Formate production in mixed cultures varied from traces of formate in one culture to quantities two and a half times that produced by the single S. thermophilus strains tested. Carbon dioxide production for the rods (L. bulgaricus , L. helveticus , and L. lactis ) varied from as low as 0 μl to as high as 376 μl when incubated for six hours at 44 °C. Carbon dioxide production for S. thermophilus ranged from 5 μl to 1259 μl. Also, S. thermophilus strains produced significantly more carbon dioxide than rod cultures, with only three exceptions. All mixtures were weak producers of carbon dioxide. Nine of 19 L. bulgaricus strains were stimulated by 0.1% phosphate ion and one strain showed stimulation at 0.3% phosphate ion. Thirteen of 19 strains were severely inhibited by 0.5% phosphate. Three of 10 L. helveticus strains were stimulated by 0.1% phosphate and another three strains were unaffected. All strains were inhibited by 0.5% phosphate. Two L. lactis strains showed stimulation at 0.1% phosphate, but inhibition at 0.3% and 0.5%. Acid production by strains of S. thermophilus was inhibited in 11 of 13 cases at 0.1% phosphate. The two strains not inhibited were slightly stimulated by 0.1% and 0.3% phosphate and unaffected by 0.5% phosphate. The mixed cultures of L. bulgaricus CR 14/ S. thermophilus 2 and L. bulgaricus Ql S. thermophilus 2 were not inhibited by 0.1% phosphate, but inhibition occurred at higher concentrations. Mixed cultures of L. bulgaricus C, E/ S. thermophilus 7, 12 and L. bulgaricus C, G/ S. thermophilus 4, 12 were stimulated by all three concentrations of phosphate salts tested. Sodium chloride produced toxic effects on the rods at concentrations ranging from 2.5% to 3.0%, and acid production was stimulated 7 of 32 strains by low salt concentrations(0.5%). In general, cocci were more sensitive to NaCl, with 6 of 13 strains showing sensitivity at 0.5%. Sensitivity to salt was a more gradual effect in the cocci as revealed by a gradual reduction in rate of acid production as NaCl concentrations increased. Mixed cultures were more tolerant to NaCl with no inhibition occurring at concentrations of 1.0%. Culture L. bulgaricus C, GIS. thermophilus 4, 7 were stimulated at concentrations through 1.5%. The synergistic properties of the mixed strains increased NaCl tolerance. / Graduation date: 1993

Streptococcus suis es un importante patógeno que causa grandes pérdidas económicas en la industria porcina a nivel mundial, siendo también un importante agente zoonótico. Aunque son varias las aproximaciones que se han desarrollado mediante vacunas vivas o recombinantes para prevenir las enfermedades provocadas por S. suis, los esfuerzos para controlar su infección se ven dificultados por la falta de herramientas efectivas contra este patógeno.Diferentes tipos de transportadores implicados en la captación de cationes divalentes y asociados a la pared celular, entre ellos los transportadores ABC, se relacionan con la virulencia bacteriana y presentan propiedades inmunogénicas contra las especies bacterianas de las que derivan. Atendiendo a todas estas características, se han desarrollado estrategias para producir vacunas contra las bacterias patógenas basadas en la sobreexpresión en la superficie celular bacteriana de transportadores de cationes divalentes inducidos mediante agentes quelantes o a través de la construcción de cepas deficientes en los represores de la transcripción de estos transportadores. En este contexto, el objetivo del presente trabajo ha sido estudiar los mecanismos de captación de cationes divalentes de S. suis, así como su papel en la virulencia y su posible uso para el desarrollo de herramientas eficaces contra este patógeno. Para abordar el objetivo propuesto, se identificaron in silico diversos transportadores de S. suis implicados en la captación de zinc y hierro y también sus posibles reguladores (AdcR y Fur, respectivamente). Además, se clonó el gen adcR de S. suis, que codifica un posible regulador de los transportadores implicados en la captación de Zn2+ y/o Mn2+ en Streptococcus spp., se purificó la proteína AdcR y mediante ensayos con DNasaI (footprinting) y de movilidad electroforética se demostró, por primera vez, que dicha proteína reconoce y se une específicamente a la secuencia TTAACNRGTTAA. Asimismo, también se ha demostrado que in vitro se requiere Zn2+ o Mn2+ para establecer dicha unión y que la proteína AdcR controla la expresión de los genes que codifican las proteínas SsuiDRAFT 0103 y SsuiDRAFT 1237, componentes de transportadores ABC implicados en la captación de zinc y/o manganeso. Por otra parte, se clonó el gen fur de S. suis, que codifica el posible regulador de los transportadores implicados en la captación de Fe2+, y se sobreexpresó su producto en Escherichia coli. Ensayos de movilidad electroforética con extracto crudo de esta cepa de E. coli mostraron que la proteína Fur de S. suis controla la expresión de los genes feoAB, implicados en la captación de hierro.Seguidamente, se obtuvieron mutantes mediante la deleción de los genes adcR y fur en una cepa virulenta de S. suis con el objetivo de caracterizar ambos regulones. Varios transportadores implicados en la captación de cationes aparecieron desreprimidos en las cepas mutantes cuando la expresión génica fue comparada con la de la cepa salvaje a través de ensayos de RT-PCR a tiempo real. En concordancia con ello, los ensayos de movilidad electroforética mostraron que estos reguladores se unen específicamente al promotor de dichos genes. Asimismo, la ausencia de los genes adcR y/o fur en un estreptococo patógeno mostró, por primera vez, una importante atenuación de su virulencia en el modelo animal de ratón.Finalmente, se abordaron estudios de inmunogenicidad y protección. Para ello, se purificaron tres proteínas periplásmicas de S. suis implicadas en la captación de cationes divalentes (SsuiDRAFT 0103, SsuiDRAFT 0174 y SsuiDRAFT 1237), resultando ser todas ellas inmunogénicas, aunque sólo SsuiDRAFT 0103 confiere una protección significativa contra S. suis en el modelo animal de ratón. Además, las proteínas Ssu0309 y Ssu1103 asociadas a la pared celular, que están sobreexpresadas en el mutante adcR, fueron identificadas mediante espectrometría de masas como factores de virulencia pertenecientes a la familia de proteínas Pht (Pneumococcal histidine triad). Asimismo, se estudiaron las propiedades protectoras de las cepas mutantes demostrándose que aunque enteras no confieren protección contra S. suis en el modelo animal de ratón, las proteínas asociadas a la pared celular del doble mutante adcR fur sí que inducen una protección significativa ante una infección con la cepa virulenta de S. suis 89/1591 en dicho modelo. / Streptococcus suis is an important pathogen that causes significant economical losses in the swine industry worldwide and it is also an important zoonotic agent. Although several approaches to develop either live or recombinant vaccines to prevent S. suis-mediated disease have been tested, efforts to control the infection are hampered by the lack of effective weapons against this pathogen.Different cell-wall-associated transporters involved in divalent-cation uptake, including ABC transporters, have been shown to be involved in bacterial virulence and have immunogenic properties against the bacterial species from which they are derived. Accordingly, several strategies have been developed to produce vaccines against this pathogenic bacterium. One of them involves overexpression on the bacterial cell surface of divalent-cation-uptake transporters induced by chelator agents or by the construction of deficient strains in the cation-uptake repressors. In this context, the aim of this work has been to study the S. suis-cation-uptake mechanisms and their role in virulence as well as their putative use as a tool to achieve broad protection against this pathogen. To achieve this purpose, several transporters involved in zinc and iron uptake and their putative regulators (AdcR and Fur, respectively) have been identified in silico. Furthermore, the S. suis adcR gene, which encodes a predicted regulator of Zn2+ and/or Mn2+ uptake in streptococci, was cloned and its protein product was purified. Footprinting and electrophoretic mobility shift assays with purified S. suis AdcR protein showed, for the first time, that the AdcR-DNA binding sequence corresponds to the TTAACNRGTTAA motif. In addition, the requirement for either Zn2+ or Mn2+ to establish in vitro binding of AdcR to its target sequence and the ability of AdcR to control the genes codifying the ABC-transporter components SsuiDRAFT 0103 and SsuiDRAFT 1237, involved in zinc and/or manganese uptake, were demonstrated.Besides, the S. suis fur gene, which encodes a predicted regulator of Fe2+ uptake, was cloned and its protein product was overexpressed in Escherichia coli. Electrophoretic mobility shift assays with crude extract of this E. coli strain showed that S. suis Fur protein controls the feoAB genes, involved in ferric uptake. In addition, both adcR and fur genes were deleted in a virulent S. suis strain in order to charecterize these regulons. Several cation-uptake transporters appeared desrepressed in the knockout strains when gene expression was compared with wild-type strain through real-time RT-PCR analyses. Accordingly, EMSA results showed that these regulators specifically bind to the promoter of these genes. Moreover, the absence of adcR and/or fur genes in pathogenic streptococci showed, for the first time, an important attenuation of its virulence in mice.Finally, immunogenic and protective analysis were carried out with the products of three genes encoding putative divalent-cation-binding lipoproteins of S. suis (SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237), being all of them immunogenic although only one (SsuiDRAFT 0103) induces a significant protective response against a virulent S. suis strain in mice. Moreover, the overexpressed cell wall-associated proteins Ssu0309 and Ssu1103 of the adcR mutant, were identified by mass spectrometry as putative virulence factors belonging to the Pht (Pneumococcal histidine triad) family. Likewise, protective abilities of mutant strains were analyzed showing that although mutant cells are not effective to confer protection in mice, the combination of adcR- and fur-regulated cell wall-associated proteins confers a significant protection against S. suis 89/1591 challenge to mice vaccinated with them.

Streptococcus pyogenes és un patogen humà responsable d'un ampli ventall d'infeccions que varien des d'infeccions superficials com faringitis i impetigen a formes sistèmiques greus com fascitis necrosant (FN) i síndrome del xoc tòxic estreptocòccic (SSTS). El ressorgiment i persistència de formes invasives greus descrit des de mitjans dels anys 1980 ha motivat una intensa recerca sobre els aspectes epidemiològics, microbiològics i clínics d'aquestes infeccions. S'ha realitzat un estudi retrospectiu de base hospitalària que inclou 126 soques de S. pyogenes (27 procedents d'infeccions invasives i 99 d'infeccions no invasives) aïllades entre gener de 1999 i juny de 2003. Les soques de S. pyogenes es van caracteritzar en base a la distribució de tipus i subtipus emm i els perfils genètics de superantígens (SAgs) (speA-C, speF-J, speL, speM, ssa i smeZ). Tanmateix, es va determinar la prevalença i els mecanismes de resistència a macròlids, tetraciclina i levofloxacino. Les formes clíniques més freqüents d'infecció invasiva van ser les infeccions de la pell i teixits tous (40,7%). La SSTS es va registrar en quatre (14,8%) dels casos invasius i es va associar a FN en la meitat dels casos. La majoria dels pacients afectats de quadres invasius eren adults, en particular d'edat avançada i de mitjana edat, i una elevada proporció presentaven factors predisposants, destacant l'alteració de la barrera cutània, la infecció per HIV, l'ús de drogues per via parenteral, i les neoplàsies. En la col·lecció de 126 soques analitzada es van identificar un total de 29 tipus emm amb una distribució encapçalada pel tipus emm1 (17,5%), seguit d'emm3 (8,7%), emm4 (8,7%), emm12 (7,1%), emm28 (7,1%), emm11 (6,3%) i emm77 (6,3%). Aquests set tipus van constituir el 61,9% del total de soques. No es van observar diferències significatives en la distribució de tipus emm entre soques aïllades d'infeccions invasives i no invasives amb l'única excepció del tipus poc freqüent emm25 que es va trobar associat a infeccions invasives en addictes a drogues per via parenteral. Es va trobar una forta correlació entre el patró de SAgs i el tipus emm independentment del tipus d'infecció. La resistència a eritromicina va mostrar un increment anual progressiu del 16,6% (1999) al 38,8% (2003) i va estar causada per soques pertanyents a 11 tipus emm. Les soques mef(A) positives dels tipus emm4, emm12 i emm75 i erm(B) positives dels tipus emm11 i emm25 constituïren el 80% de les soques resistents. La freqüència de resistència a tetraciclina va fluctuar durant el període estudiat (màxim 34,6% el 2002 i mínim 15,8% el 2001) i va ser superior en les soques resistents a eritromicina que en les soques sensibles (42,8% vs 18,7%). En les soques resistents a tetraciclina el gen tet(M) va ser el predominant i es va trobar en soques pertanyents a 14 tipus emm, mentre que el gen tet(O) només es va trobar en soques emm77. No es van observar diferències significatives en la prevalença de resistència a eritromicina ni a tetraciclina en el grup invasiu respecte del no invasiu. La prevalença de resistència a levofloxacino fou del 3,2%, incloent quatre soques amb sensibilitat reduïda o resistència intermèdia (CIM 2-4 µg/ml) i dues soques amb resistència d'alt nivell (CIM >32 µg/ml). La resistència de baix nivell es va associar a substitucions únicament en ParC (Ser80Pro, Ser79Ala, Ser79Phe i Ala121Val), mentre que la resistència d'alt nivell es va relacionar amb mutacions en ParC (Ser79Phe i Ala121Val) i GyrA (Ser81Tyr). / Streptococcus pyogenes (GAS) is a human pathogen responsible for a wide array of infections, ranging from pharyngitis and impetigo to severe invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The resurgence and persistence of severe forms of GAS diseases reported since the mid 1980s have motivated intensive research on epidemiological, microbiological and clinical aspects of these diseases. A retrospective hospital-based study was conducted including 126 GAS isolates (27 from invasive infections and 99 from non-invasive infections) collected from January 1999 to June 2003. GAS isolates were characterized by emm type and subtype and superantigen (SAg) gene profile (speA-C, speF-J, speL, speM, ssa and smeZ). The prevalence and mechanisms of macrolide, tetracycline and levofloxacin resistance were also determined. The most common clinical presentations of invasive cases were skin and soft-tissue infections (40.7%). SSTS occurred in four cases (14.8%) and was associated to NF in half of the cases. Most invasive cases were found in adults, in particular among the elderly and the middle-aged, and a large proportion had underlying conditions, the most frequent being skin lesions, HIV infection, injection drug use, and malignancy. A total of 29 emm types were identified among the 126 isolates; the most prevalent were emm1 (17.5 %), followed by emm3 (8.7 %), emm4 (8.7 %), emm12 (7.1 %), emm28 (7.1 %), emm11 (6.3 %) and emm77 (6.3 %). These seven emm types accounted for 61.9 % of isolates. There were no differences in the emm type distribution between invasive and non-invasive infections, except for emm25 isolates, which were associated with invasive infections in injecting drug users. The SAg gene profiles were closely associated with the emm type and were independent of the disease type. The prevalence of erythromycin resistance showed an annual progressive increase from 16.6% (1999) to 38.8% (2003) and was caused by isolates belonging to 11 emm types. mef(A)-positive emm types 4, 12 and 75, and erm(B)-positive emm types 11 and 25 were responsible for up to 80% of the erythromycin-resistant isolates. The prevalence of tetracycline resistance fluctuated over the period studied (maximum 34.6% in 2002 and minimum 15.8% in 2001) and was higher in erythromycin-resistant isolates than in susceptible isolates (42.8% vs 18.7%). Among the tetracycline-resistant isolates, the tet(M) determinant was the most prevalent and was distributed in isolates belonging to 14 emm types, whereas tet(O) was only found in emm77 isolates. No significant differences in resistance rates to erythromycin or tetracycline were found between invasive and non-invasive isolates. The rate of resistance to levofloxacin was 3.2%, encompassing four isolates with reduced susceptibility or intermediate resistance (MIC 2-4 µg/ml) and two isolates with a high level of resistance (MIC >32 µg/ml). Low-level resistance was associated with alterations in ParC (Ser80Pro, Ser79Ala, Ser79Phe and Ala121Val), while high-level resistance was associated with alterations involving both ParC (Ser79Phe and Ala121Val) and GyrA (Ser81Tyr).

Streptococcus pneumoniae is a major pathogen that causes Otitis Media infections and bacterial meningitis in children as well as community acquired pneumonia in adults. Clinical isolates of S. pneumoniae exhibiting resistance to Beta-lactam antibiotics are being isolated with increased frequency in many countries. Streptococcus pneumoniae strains resistant to Beta-lactam drugs have modified forms of penicillin-binding proteins that exhibit reduced affinity for binding to chemotherapeutic Beta-lactams. Penicillin binding proteins are membrane-bound enzymes that catalyze the terminal step in cell wall synthesis, and are targets for Beta-lactam drugs. Seventeen clinical isolates and six vaccine strains of Streptococcus pneumoniae were characterized using conventional phenotypic methods, susceptibility to antimicrobial agents, capsular serotyping, and by different biochemical and genotyping methods. One strain, Sp D2, was resistant to penicillin and other Beta-lactams used in the study, to erythromycin, and to Trimethoprim/Sulfamethoxazole. Sp D2 exhibited a unique protein profile in 1D SDS-PAGE gels of whole-cell proteins. Cells of Sp D2 were fractionated, and the cytoplasmic membrane fraction was obtained by ultracentrifugation and analyzed using a 1D SDS-PAGE gel. A protein band with a mass of ~50 kDa was excised and subjected to Trypsin In-Gel Digestion, followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. The resulting MALDI-TOF-MS data (peptide mass fingerprints) did not produce any significant matches with proteins in any of the published S. pneumoniae genome databases. The 50 kDa protein was further subjected to N-terminal and internal sequence analysis and database searching, and the protein could not be identified by significant matches. Sp D2 did not react with any anti-pneumococcal polysaccharide capsular antibodies, and is designated as a non-typeable strain. Sp D2 exhibited a positive reaction in the Bile Solubility Test, the Optochin Test, and also positive reactions in PCR assays for the presence of the pneumococcal surface protein gene (PsaA), the autolysin gene (LytA), and the pneumolysin gene (Ply); which confirms that Sp D2 is a strain of S. pneumoniae.

Biofilms are highly organized bacterial structures that are attached to a surface. They are ubiquitous in nature and may be detrimental, causing numerous types of illnesses in living organisms. Biofilms in the human oral cavity are the main cause of dental caries and periodontal diseases and can act as a source for pathogenic organisms to spread within the body and cause various types of systemic diseases. Streptococcus mutans is the primary etiological agent of dental caries, the single most chronic childhood disease. In many cases, quorum sensing (QS) is required for initial formation and subsequent development of biofilms and the signaling molecule autoinducer 2 (AI- 2) has been well studied as an inter-species QS signaling molecule. However, recent reports also suggest that AI-2-mediated signaling is important for intra-species biofilm formation in both Gram-negative and positive bacteria. Therefore, there is significant interest in understanding the role of different QS signals such as AI-2 in oral biofilm formation. Microfluidic devices provide biomimetic environments and offer a simple method for executing multiple stimuli experiments simultaneously, thus, can be an extremely powerful tool in the study of QS in biofilms. In this study, we report conditions that support the development of S. mutans biofilms in microchannel microfluidic devices, and the effects of extracellular addition of chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione (DPD; precursor of AI-2) on mono-species S. mutans luxS (AI-2 deficient strain) biofilm formation using a gradient generating microfluidic device. S. mutans wild type (WT) and luxS biofilms were developed in nutrient rich medium (25% brain heart infusion medium, BHI + 1% sucrose) for up to 48 h. Maximum biofilm formation with both strains was observed after 24 h, with distinct structure and organization. No changes in S. mutans luxS biofilm growth or structure were observed upon exposure to different concentrations of AI-2 in a gradient generating device (0 to 5 M). These results were also validated by using a standard 96-well plate assay and by verifying the uptake of AI-2 by S. mutans luxS. Our data suggest that extracellular addition of AI-2 does not complement the luxS deletion in S. mutans with respect to biofilm formation.

Thèse doctorat : Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2006. / Titre provenant de l'écran-titre. Bibliogr. 26 p.

Thèse doctorat : Génétique moléculaire : Nancy 1 : 2005. / Titre provenant de l'écran-titre.

Thèse doctorat : Génétique moléculaire : Nancy 1 : 2005. / Titre provenant de l'écran-titre.