ABSTRACT: Antibiotic resistance in Streptococcus pneumoniae is often the result of horizontal gene transfer events involving closely related streptococcal species. Laboratory experiments confirmed that S. mitis DNA functions as donor in transformation experiments, using the laboratory strain S. pneumoniae R6 as recipient and chromosomal DNA of a high level penicillin resistant S. mitis B6 strain. After four transformation steps, alterations in five penicillin-binding proteins (PBP) were observed, and sequence analysis confirmed recombination events in the corresponding PBP genes. In order to detect regions where recombination with S. mitis DNA has occurred we analyzed the S. pneumoniae transformants by microarray analyses, using oligonucleotide microarrays designed for the S. pneumoniae genome and the S. mitis B6 genome as well.
Project description:In order to appreciate the presence of surface protein gene homologues in commensal species S. mitis and S. oralis, comparative genomic hybridization studies using DNA microarrays were performed with 8 S. mitis and 11 S. oralis from different geographic locations. The oligonucleotide microarray was designed based on the genomes of S. pneumoniae R6 and TIGR4 as well as S. mitis B6 to include genes of 63 cell surface proteins. The denatured genomic DNA of the S. mitis and S. oralis strains was labeled with Cy3-dCTP and control S. mitis B6 DNA was labeled with Cy5-dCTP. Hybridization was performed following the manufacturers recommendations using an hybridization temperature of 40C for 16 h. For data processing, microarrays were scanned on the laser scanner Pro Scan Array GX (PerkinElmer) with the low resolution of 50 µm using ScanArrayExpress Software version 4.0. Photomultiplier tube was adjusted to balance the two fluorescence channels and biochips were scanned with a resolution of 10 µm. After elimination of background values fluorescence intensity was determined. Signals that showed an intensity ratio of 0.3 and above were considered to be positive.
Project description:Alterations in genes for penicillin-binding proteins (pbp) are well-known determinants for the resistance of Streptococcus pneumoniae to B-lactam antibiotics. Surprisingly, some mutations in non-pbp genes were also found to contribute to B-lactam resistance. Two of them discovered in the piperacillin resistant mutants P106 and P104, affect the expression of cpoA (encoding a glycosyltransferase) and of the rgtABCDHR cluster (encoding two small membrane proteins, an ABC transporter and a regulatory two-component system), respectively. cpoA and rgtABCDHR are involved in maintaining the synthesis and the proper ratio of the two major membrane glycolipids, and deletions in these genes led to complex phenotypes. In attempts to identify genetic determinants for these phenotypes, the global trancription patterns of the deletion mutants R6 delta cpoA, R6 delta rgtA and R6 delta rgtD were compared to that of the parent strain R6.
Project description:Investigation of whole genome gene expression level changes in S. pneumoniae KCTC 5080T, S. mitis KCTC 3556T, S. oralis KCTC 13048T, and S. pseudopneumoniae CCUG 49455T. This proves that transcriptional profiling can facilitate in elucidating the genetic distance between closely related strains. A one chip study using total RNA recovered from S. pseudopneumoniae CCUG 49455T with three strain. For the the transcriptome of S. pseudopneumoniae CCUG 49455T was analyzed using the S. pneumoniae R6 microarray platform and compared with those of S. pneumoniae KCTC 5080T, S. mitis KCTC 3556T, and S. oralis KCTC 13048T strains.
Project description:Diagnostic primer extension assay to serotype Streptococcus pneumoniae. Assay validation. Background: Monitoring of Streptococcus pneumoniae serotype epidemiology is essential since serotype replacement is a concern when introducing new polysaccharide-conjugate vaccines. To simplify S. pneumoniae serotyping, a novel PCR-based automated microarray assay was developed to assist in the tracking of the serotypes. Results: Autolysin (lytA), pneumolysin (ply) and eight genes located in the capsular operon (cps) were amplified using multiplex PCR. This step was followed by a tagged fluorescent primer extension step targeting serotype-specific polymorphisms. The tagged primers were then hybridized to a microarray. Results were exported to an expert system that transforms genetic typing data into capsular serotype identification. The assay was validated on 166 cultured S. pneumoniae samples from 63 different serotypes as determined by the Quellung method. In addition, the assay was tested on clinical specimens including 43 cerebrospinal fluid samples from patients with meningitidis and 59 nasopharyngeal aspirates from bacterial pneumonia patients. The assay presented with no cross-reactivity for 24 relevant bacterial species found in these types of samples. The limit of detection for serotyping and S. pneumoniae detection was 100 genome equivalent per reaction. Conclusion: This automated assay is amenable to clinical testing and does not require any culturing of the samples. The assay will be useful for the evaluation of serotype prevalence changes after new conjugate vaccines introduction. 166 quellung serotyped strains and two negative controls
Project description:Investigation of transcription profile of spontaneous Cefotaxime resistant R6 mutants containing mutations in pbp2x, ciaH, pbp2a and mutants with different mosaic pbp2x, pbp1a genes.
Project description:Seven streptococcal isolates from the Mitis group were analysed for the presence of pneumococcal gene homologues using comparative genomic hybridization studies with microarrays based on open reading frames from the genomes of Streptococcus pneumoniae TIGR4 and R6. The diversity of pneumolysin (ply) and neuramindase A (nanA) gene sequences was explored in more detail in a collection of 14 S. pseudopneumoniae and 29 Mitis group isolates respectively. The Mitis group isolates used in the microarray experiments included a type strain (NCTC 12261), 2 S. mitis isolates from the nasopharynx of children, one S.mitis from infective endocarditis, one S. mitis isolate from a dental abscess, plus one S. oralis isolate and one S. pseudopneumoniae isolate from the nasopharynx of children. The results of the microarray study showed that the 5 S. mitis isolates had homologues to between 67-82% of pneumococcal virulence genes, S. oralis hybridised to 83% and S. pseudopneumoniae to 92% of identified pneumococcal virulence genes. Comparison of the pneumolysin, mitilysin (mly) and newly identified pseudopneumolysin (pply) gene sequences revealed that mly and pply genes are more closely related to each other than either is to ply. In contrast, the nanA gene sequences in the pneumococcus and streptococci from the Mitis group are closely clustered together sharing 99.4-99.7% sequence identity with pneumococcal nanA alleles. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-82
Project description:Chromatin immunoprecipitation and hybridization to a chromosome-wide DNA tiling array (ChIP-chip)was performed to compare the distribution pattern of H3K9me2 between nrpd1a-4_nrpd1b-11 and wild type. Experiments were done using two independent biological replicates.<br>
Project description:Chromatin immunoprecipitation and hybridization to a chromosome-wide DNA tiling array (ChIP-chip)was performed to compare the distribution of H3K4me2 in nrpd1a-4_nrpd1b-11 double mutant and in the wild type. Experiments were done using two independent biological replicates.<br><br>