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(1) Lasers have been used for the treatment of dentinal hypersensitivity and bacterial reductions in periodontology. The purpose of this in vitro study was to evaluate the effect of Carbon Dioxide (CO2) and Erbium-doped Yttrium Aluminum Garnet (Er:YAG) lasers with chlorhexidine (CHX), hydrogen peroxide (H2O2), sodium hypochlorite (NaOCl), or sodium fluoride (NaF) on the viability of oral bacteria associated with root caries. (2) Streptococcus mutans, Streptococcus sanguinis, and Enterococcus faecalis were grown in Brain Heart Infusion (BHI) broth, diluted to an OD660 of 0.5, and treated with antiseptics with or without simultaneous irradiation with the Er:YAG and CO2 lasers for 30 s repeated three times. The treatment groups consisted of 1: no treatment, 2: 0.5% H2O2 alone, 3: 0.5% NaOCl alone, 4: 0.12% CHX alone, 5: 2% NaF alone, 6: laser alone, 7: laser with 0.5% H2O2, 8: laser with 0.5% NaOCl, 9: laser with 0.12% CHX, and 10: laser with 2% NaF for both lasers. The microbial viability was determined through plating and viable colonies were counted, converted into CFU/mL, and transformed into log form. The statistical analysis was performed using a two-tailed paired t-test. (3) The use of CO2 and Er:YAG lasers alone failed to show statistically significant antibacterial activity against any of the bacteria. The only effective monotreatment was CHX for S. mutans. The combined treatment of 0.5% NaOCl with Er:YAG produced the greatest reduction in overall viability. (4) The combination of the Er:YAG laser with 0.5% NaOCl resulted in the largest reduction in bacterial survival when compared to monotherapies with antimicrobial solutions or lasers. [ABSTRACT FROM AUTHOR]

Completion of the genome sequence of Streptococcus sanguinis SK36 necessitates tools for further characterization of this species. It is often desirable to insert antibiotic resistance markers and other exogenous genes into the chromosome; therefore, we sought to identify a chromosomal site for ectopic expression of foreign genes, and to verify that insertion into this site did not affect important cellular phenotypes. We designed three plasmid constructs for insertion of erm, aad9 or tetM resistance determinants into a genomic region encoding only a small (65 aa) hypothetical protein. To determine whether this insertion affected important cellular properties, SK36 and its erythromycin-resistant derivative, JFP36, were compared for: (i) growth in vitro, (ii) genetic competence, (iii) biofilm formation and (iv) virulence for endocarditis in the rabbit model of infective endocarditis (IE). The spectinomycin-resistant strain, JFP56, and tetracycline-resistant strain, JFP76, were also tested for virulence in vivo. Insertion of erm did not affect growth, competence or biofilm development of JFP36. Recovery of bacteria from heart valves of co-inoculated rabbits was similar to wild-type for JFP36, JFP56 and JFP76, indicating that IE virulence was not significantly affected. The capacity for mutant complementation in vivo was explored in an avirulent ssaB mutant background. Expression of ssaB from its predicted promoter in the target region restored IE virulence. Thus, the chromosomal site utilized is a good candidate for further manipulations of S. sanguinis. In addition, the resistant strains developed may be further applied as controls to facilitate screening for virulence factors in vivo.

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-foid. [[sup.3]H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin [B.sub.12] biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.

Streptococcus mutans belongs to the viridans group of oral streptococci, which is the leading cause of endocarditis in humans. The LraI family of lipoproteins in viridans group streptococci and other bacteria have been shown to function as virulence factors, adhesins, or ABC-type metal transporters. We previously reported the identification of the S. mutans LraI operon, sloABCR, which encodes components of a putative metal uptake system composed of SloA, an ATP-binding protein, SloB, an integral membrane protein, and SIoC, a solutebinding lipoprotein, as well as a metal-dependent regulator, SIoR. We report here the functional analysis of this operon. By Western blotting, addition of Mn to the growth medium repressed SloC expression in a wild-type strain but not in a sloR mutant. Other metals tested had little effect. Cells were also tested for aerobic growth in media stripped of metals then reconstituted with Mg and either Mn or Fe. Fe at 10 [micro]M supported growth of the wild-type strain but not of a sloA or sloC mutant. Mn at 0.1 [micro]M supported growth of the wild-type strain and sloR mutant but not of sloA or sloC mutants. The combined results suggest that the SloABC proteins transport both metals, although the SloR protein represses this system only in response to Mn. These conclusions are supported by (55) Fe uptake studies with Mn as a competitor. Finally, a sloA mutant demonstrated loss of virulence in a rat model of endocarditis, suggesting that metal transport is required for endocarditis pathogenesis.

Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a ΔssaACB deletion mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP-family manganese transporter. Transcriptomic analysis of fermentor-grown cultures of SK36 WT and ΔssaACB strains identified pH-dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its ΔssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis. [ABSTRACT FROM AUTHOR]

Streptococcus sanguinis is an important cause of infective endocarditis. In strain SK36, the ABC-family manganese transporter, SsaACB, is essential for virulence. We have now identified a ZIP-family protein, TmpA, as a secondary manganese transporter. A tmpA mutant had no phenotype, but a ΔssaACB ΔtmpA mutant was more attenuated for serum growth and for virulence in a rabbit model than its ΔssaACB parent. The growth of both mutants was restored by supplemental manganese, but the ΔssaACB ΔtmpA mutant required twenty-fold more and accumulated less. Although ZIP-family proteins are known for zinc and iron transport, TmpA-mediated transport of either metal was minimal. While ssaACB appears ubiquitous in St. sanguinis, tmpA was present in a majority of strains and a mntH gene encoding an NRAMP-family transporter was identified in relatively few, including VMC66. As in SK36, deletion of ssaACB greatly diminished VMC66 endocarditis virulence and serum growth, and deletion of tmpA from this mutant diminished virulence further. Virulence was not significantly altered by deletion of mntH from either VMC66 or its ΔssaACB mutant. This and the accompanying paper together suggest that SsaACB is of primary importance for endocarditis virulence while secondary transporters TmpA and MntH contribute to growth under differing conditions. [ABSTRACT FROM AUTHOR]

Mutational analysis of Pseudomonas syringae aids the identification of the gacA gene, which encodes the response regulator for lemA sensor kinase. The suggested GacA protein is a type of FixJ response regulator and is identical to the Escherichia coli UvrY protein. Gene transcription causes polarity, leading to the UV sensitivity of the gacA mutant.

The recombination of the variable major protein (Vmp) gene (vmp) in Borrelia hermsii was characterized by investigating the number of copies of the spirochete's chormosome and two linear plasmids. Borrelia can undergo serotype switching to elude the host's immune system. This is done by organizing complete genomes out of the DNA present in the cell. Serotype switching is then accomplished by coexpression of Vmp, suppression of transcription or partition expression of vmp in the different plasmids.

Streptococcus sanguinis is a primary colonizer of teeth and is associated with oral health. When it enters the bloodstream, however, this bacterium may cause the serious illness infective endocarditis. The genes required for survival and proliferation in blood have not been identified. The products of these genes could provide a rich source of targets for endocarditis‐specific antibiotics possessing greater efficacy for endocarditis, and also little or no activity against those bacteria that remain in the mouth. We previously created a comprehensive library of S. sanguinis mutants lacking every nonessential gene. We have now screened each member of this library for growth in human serum and discovered 178 mutants with significant abundance changes. The main biological functions disrupted in these mutants, including purine metabolism, were highlighted via network analysis. The components of an ECF‐family transporter were required for growth in serum and were shown for the first time in any bacterium to be essential for endocarditis virulence. We also identified two mutants whose growth was reduced in serum but not in saliva. This strategy promises to enable selective targeting of bacteria based on their location in the body, in this instance, treating or preventing endocarditis while leaving the oral microbiome intact. [ABSTRACT FROM AUTHOR]

Blends of arabinoxylan ferulate (AXF) and gelatin (GEL) at 1:1, 2:1 and 4:1 mass ratios were electrospun into composite fibrous mats as a wound-healing drug delivery platform. The composite fibers were characterized in terms of morphology, tensile properties, pore size, porosity and molecular composition. The composite fibers showed excellent cytocompatibility. Silver was impregnated into GEL-AXF nanofibers, and it was slowly released, resulting in bacterial growth inhibition as confirmed by the Kirby–Bauer disk-diffusion assay. This work establishes an electrospun arabinoxylan fibrous material platform with the potential to treat chronic diabetic wounds. [ABSTRACT FROM AUTHOR]

InS. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in theGalleria mellonellainvertebrate model. To further characterize Cnm as a virulence factor, thecnmgene fromS. mutansstrain OMZ175 was expressed in the non-pathogenicLactococcus lactisNZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall ofL. lactis. Similar toS. mutans, expression of Cnm inL. lactisenabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence inG. mellonella. Using anex vivohuman heart tissue colonization model, we showed that Cnm-positive strains of eitherS. mutansorL. lactisoutcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitatedL. lactisadhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute ofS. mutansand confirm the usefulness of theL. lactisheterologous system for further characterization of bacterial virulence factors. [ABSTRACT FROM AUTHOR]