A Five-Species Transcriptome Array for Oral Mixed-Biofilm Studies

BACKGROUND: Oral polymicrobial interactions and biofilm formation are associated with initiation and progression of caries, gingivitis, and periodontitis. Transcriptome studies of such interactions, allowing a first mechanistic insight, are hampered by current single-species array designs. METHODOLOGY/PRINCIPAL FINDINGS: In this study we used 385 K NimbleGene™ technology for design and evaluation of an array covering the full genomes of 5 important physiological-, cariogenic-, and periodontitis-associated microorganisms (Streptococcus sanguinis, Streptococcus mutans, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis). Array hybridization was done with cDNA from cultures grown for 24 h anaerobically. Single species experiments identified cross-species hybridizing array probes. These probes could be neglected in a mixed-species experimental setting without the need to exclude the whole genes from the analysis. Between 69% and almost 99% of the genomes were actively transcribed under the mono-species planktonic, monolayer, and biofilm conditions. The influence of Streptococcus mitis (not represented on the array) on S. mutans gene transcription was determined as a test for a dual-species mixed biofilm setup. Phenotypically, under the influence of S. mitis an increase in S. mutans biofilm mass and a decrease in media pH-value were noticed, thereby confirming previously published data. Employing a stringent cut-off (2-fold, p<0.05), 19 S. mutans transcripts were identified with increased abundance, and 11 with decreased abundance compared to a S. mutans mono-species biofilm. Several of these genes have previously been found differentially regulated under general and acid stress, thereby confirming the value of this array. CONCLUSIONS/SIGNIFICANCE: This new array allows transcriptome studies on multi-species oral biofilm interactions. It may become an important asset in future oral biofilm and inhibitor/therapy studies

Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA

<p>Abstract</p> <p>Background</p> <p><it>Francisella (F.) tularensis </it>is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, <it>F. tularensis </it>was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories.</p> <p>Results</p> <p>The complete 23S rRNA genes of representative strains of <it>F. philomiragia </it>and all subspecies of <it>F. tularensis </it>were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes.</p> <p>Different FISH protocols allowed the positive identification of all 4 <it>F. philomiragia </it>strains, and more than 40 <it>F. tularensis </it>strains tested. By combination of different probes, it was possible to differentiate the <it>F. tularensis </it>subspecies <it>holarctica, tularensis, mediasiatica </it>and <it>novicida</it>. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different <it>F. tularensis </it>strains in infected cells or tissue samples. In blood culture systems spiked with <it>F. tularensis</it>, bacterial cells of different subspecies could be separated within single samples.</p> <p>Conclusion</p> <p>We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of <it>F. tularensis </it>isolates from both laboratory cultures and clinical samples.</p

Serotype- and strain- dependent contribution of the sensor kinase CovS of the CovRS two-component system to Streptococcus pyogenes pathogenesis

<p>Abstract</p> <p>Background</p> <p>The <it>Streptococcus pyogenes </it>(group A streptococci, GAS) two-component signal transduction system CovRS has been described to be important for pathogenesis of this exclusively human bacterial species. If this system acts uniquely in all serotypes is currently unclear. Presence of serotype- or strain-dependent regulatory circuits and polarity is an emerging scheme in <it>Streptococcus pyogenes </it>pathogenesis. Thus, the contribution of the sensor kinase (CovS) of the global regulatory two-component signal transduction system CovRS on pathogenesis of several M serotypes was investigated.</p> <p>Results</p> <p>CovS mutation uniformly repressed capsule expression and hampered keratinocyte adherence in all tested serotypes. However, a serotype- and even strain-dependent contribution on survival in whole human blood and biofilm formation was noted, respectively.</p> <p>Conclusions</p> <p>These data provide new information on the action of the CovS sensor kinase and revealed that its activity on capsule expression and keratinocyte adherence is uniform across serotypes, whereas the influence on biofilm formation and blood survival is serotype or even strain dependent. This adds the CovRS system to a growing list of serotype-specific acting regulatory loci in <it>S. pyogenes</it>.</p

Setup of an In Vitro Test System for Basic Studies on Biofilm Behavior of Mixed-Species Cultures with Dental and Periodontal Pathogens

BACKGROUND: Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic in vitro single and mixed-species culture model for oral bacteria combining three complimentary methods. The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible. METHODS: Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups. FINDINGS: S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability. CONCLUSIONS: This setup shows safranin staining, microscopic analysis and viable counts together are crucial for basic examination and evaluation of biofilms. Our experiment generated meaningful results, exemplified by the noted S. mitis influence, and allows a fast decision about the most important bacterial interactions which should be investigated in depth

Definition of the term "creativity" in the works of foreign authors

The universal general theory of creativity does not exist. Creativity has been analyzed by scientists for hundreds years. The term "creativity" in translation from Latin (creatio) means "creation". "Creativity" is a process of creative activity of a person. This activity is resultants a new innovative product. Creativity is manifestation of the creator. The creator is the person who induces creative activity. The creator is responsible for the product he has created. The most well- known researchers of "creativity" are J. Guilford (1953) and E. Torrance (1988). Other authors claim that there are three aspects of creativity: person, process and product (N.Aderson (1990), T. Amabile (1998), E.Barron (1981), R.Woodman (1993), N.King (1990), etc.)."Creativity" is an ability of a person to use knowledge, skills and abilities for creation of a product for a short time. Progressive way of development demands product creativity, as it is essential for further success of extension

Intracellular Persisting Staphylococcus aureus Is the Major Pathogen in Recurrent Tonsillitis

BACKGROUND: The two major indications for tonsillectomy are recurrent tonsillitis (RT) and peritonsillar abscess (PTA). Unlike PTAs, which are primarily treated surgically, RT is often cured by tonsillectomy only after a series of failed drug therapy attempts. Although the bacteriological background of RT has been studied, the reason for the lack of success of conservative therapeutic approaches is not well understood. METHODS: In a prospective study, tonsil specimens from 130 RT patients and 124 PTA patients were examined for the presence of extra- and intracellular bacteria using antibiotic protection assays. Staphylococcus aureus isolates from RT patients were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing and MSCRAMM-gene-PCR. Their ability for biofilm formation was tested and their cell invasiveness was confirmed by a flow cytometric invasion assay (FACS), fluorescent in situ hybridization (FISH) and immunohistochemistry. FINDINGS: S. aureus was the predominant species (57.7%) in RT patients, whereas Streptococcus pyogenes was most prevalent (20.2%) in PTA patients. Three different assays (FACS, FISH, antibiotic protection assay) showed that nearly all RT-associated S. aureus strains were located inside tonsillar cells. Correspondingly, the results of the MSCRAMM-gene-PCRs confirmed that 87% of these S. aureus isolates were invasive strains and not mere colonizers. Based upon PFGE analyses of genomic DNA and on spa-gene typing the vast majority of the S. aureus isolates belonged to different clonal lineages. CONCLUSIONS: Our results demonstrate that intracellular residing S. aureus is the most common cause of RT and indicate that S. aureus uses this location to survive the effects of antibiotics and the host immune response. A German translation of the Abstract is provided as supplementary material (Abstract S1)

M-protein and other intrinsic virulence factors of Streptococcus pyogenes are encoded on an ancient pathogenicity island

BACKGROUND: The increasing number of completely sequenced bacterial genomes allows comparing their architecture and genetic makeup. Such new information highlights the crucial role of lateral genetic exchanges in bacterial evolution and speciation. RESULTS: Here we analyzed the twelve sequenced genomes of Streptococcus pyogenes by a naïve approach that examines the preferential nucleotide usage along the chromosome, namely the usage of G versus C (GC-skew) and T versus A (TA-skew). The cumulative GC-skew plot presented an inverted V-shape composed of two symmetrical linear segments, where the minimum and maximum corresponded to the origin and terminus of DNA replication. In contrast, the cumulative TA-skew presented a V-shape, which segments were interrupted by several steep slopes regions (SSRs), indicative of a different nucleotide composition bias. Each S. pyogenes genome contained up to nine individual SSRs, encompassing all described strain-specific prophages. In addition, each genome contained a similar unique non-phage SSR, the core of which consisted of 31 highly homologous genes. This core includes the M-protein, other mga-related factors and other virulence genes, totaling ten intrinsic virulence genes. In addition to a high content in virulence-related genes and to a peculiar nucleotide bias, this SSR, which is 47 kb-long in a M1GAS strain, harbors direct repeats and a tRNA gene, suggesting a mobile element. Moreover, its complete absence in a M-protein negative group A Streptococcus natural isolate demonstrates that it could be spontaneously lost, but in vitro deletion experiments indicates that its excision occurred at very low rate. The stability of this SSR, combined to its presence in all sequenced S. pyogenes sequenced genome, suggests that it results from an ancient acquisition. CONCLUSION: Thus, this non-phagic SSR is compatible with a pathogenicity island, acquired before S. pyogenes speciation. Its potential excision might bear relevance for vaccine development, because vaccines targeting M-protein might select for M-protein-negative variants that still carry other virulence determinants

Проектирование технологического процесса изготовления шкива

Цель работы-проектирование технологического процесса изготовления детали "Шкив". В ходе выполнения данной работы было проделано : спроектирован технологический процесс и станочное приспособление, анализ ресурсоэффективности проекта, выявлены вредные факторы и меры их предотвращения.The purpose of the work is the design of the manufacturing process of the “Pulley” part. In the course of this work, the following was done: a technological process and a machine tool were designed, an analysis of the resource efficiency of the project, harmful factors and measures to prevent them were identified