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PubMed2000

Analysis of bacterial DNA in skin and muscle of the Tyrolean iceman offers new insight into the mummification process.

About 80 sequences (16s ribosomal RNA gene) of bacterial DNA in samples of skin and muscle taken directly from the Tyrolean iceman (3350-3100 years B.C.) or recovered during the 1992 archaeological expedition at the Alpine site were analyzed to obtain clues to the natural mummification process that allowed the corpse of the Neolithic shepherd/hunter to be preserved for more than 5,000 years. The investigation was made more complex by the fact that the surface of the mummy had been swabbed with phenol soon after the discovery (September 19, 1991). Our results show that no trace of microbial DNA is left on the actual surface of the body, while the untreated skin still bears the remains of large numbers of bacteria belonging to the genera Sphingomonas, Afipia, Curtobacterium, Microbacterium, Agromyces, and others. Compared to the untreated skin, the iceman's muscle is also very rich in bacterial DNA. However, this DNA comes, with few exceptions, from the species Clostridium algidicarnis. The sharp difference in the bacterial DNA composition of skin and muscle suggests that the remains of the original cadaveric microflora of the latter have not disappeared during the iceman's taphonomic history. On the other hand, the massive presence of C. algidicarnis, a cold-adapted sporigenous, the DNA of which was previously (Ubaldi et al. [1998] Am. J. Phys. Anthropol. 107:285-295) found in the soft tissue of a naturally desiccated Andean mummy, indicates that the hypothesis that the iceman's corpse underwent rapid dehydration by the effect of a warm wind (föhn) is no longer plausible. The results best fit with the hypothesis (Bereuter et al. [1997] Chem. Eur. J. 7:1032-1038) that the body was first covered by snow and ice, and then underwent thawing and, finally, desiccation.

Adult↗
PubMed2003

Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-kappaB-independent pathway.

Recognition of bacterial products by the innate immune system is dependent on pattern-recognition receptors: toll-like receptor 9 (TLR-9) in the case of bacterial DNA. We hypothesized that bacterial DNA can directly affect enteric epithelial cells. RT-PCR revealed constitutive TLR-9 mRNA expression in three human colonic epithelial cell lines (T84, HT-29, Caco-2) and THP-1 monocytes. Epithelial cells, in six-well culture plates or on filter supports, were exposed to E. coli DNA (1-50 microg/ml), synthetic CpG-rich oligonucleotides, or calf thymus DNA for 6-48 h. Exposure to E. coli DNA resulted in an increase in IL-8 mRNA, and a time- and dose-dependent increase in IL-8 secretion. Also, CpG oligonucleotides induced epithelial IL-8 production, whereas calf thymus DNA did not. Exposure to E. coli DNA resulted in phosphorylation of ERK 1/2 MAPK and inhibitors of ERK activity (PD98059, UO126) significantly reduced the evoked IL-8 production. In contrast, inhibitors of NFkappaB activity (PDTC, SN50) did not block E. coli DNA-induced IL-8 production. Electrophoretic mobility shift assays revealed that E. coli DNA stimulated epithelial AP-1 but not NFkappaB activation. The barrier (i.e., transepithelial resistance) and ion transport parameters of epithelial monolayers (assessed in Ussing chambers) were unaltered following E. coli DNA exposure. Thus model gut epithelia express TLR-9 mRNA and, while maintaining their barrier function, can respond to E. coli DNA by increased IL-8 production.

Caco-2 Cells↗
PubMed2004

Detection of bacterial DNA in cardiac vegetations by PCR after the completion of antimicrobial treatment for endocarditis.

PCR with broad-range primers for prokaryotic 16S rRNA genes was used to identify bacterial DNA in tissue from patients undergoing valve replacements following a previous episode of infective endocarditis (IE). Of eight valves investigated, bacterial DNA was detected in three from patients for whom IE had been treated by antibiotic therapy 5, 12 and 18 months previously. The demonstration of bacterial DNA within resected heart valves suggests either recurrence of infection, treatment failure or the persistence of bacterial debris within the cardiac vegetation. There may also be implications for routine use of PCR in the diagnosis of infection.

Adult↗
PubMed1987

Use of on-section immunolabeling and cryosubstitution for studies of bacterial DNA distribution.

Escherichia coli cells were very rapidly frozen and substituted at a low temperature with 3% glutaraldehyde in acetone. Infiltration and embedding with Lowicryl K4M were carried out at -35 degrees C. This procedure resulted in good structural preservation of both the nucleoid morphology and its DNA plasm, such that immunolabeling with the protein-A gold technique could be carried out. With antibodies specific for either double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA), it was shown that dsDNA was present throughout the nucleoid but that ssDNA was located on the nucleoid periphery. Chloramphenicol-treated cells, in which protein synthesis but not DNA replication is stopped, produced a characteristic ringlike nucleoid shape and had both dsDNA and ssDNA present throughout the annular section of the DNA plasm. The relationship between metabolically active DNA and overall bacterial genome organization is discussed.

Antibodies, Monoclonal↗
PubMed1990

Typing of Campylobacter pylori by bacterial DNA restriction endonuclease analysis and determination of plasmid profile.

Campylobacter pylori isolates from 37 symptomatic patients and 3 asymptomatic volunteers were examined by chromosomal DNA restriction endonuclease analysis and determination of plasmid profile. Restriction digests with HindIII, HaeIII, PvuII, and BglII produced clear and reproducible results that permitted discrimination between different strains. Only 35% of C. pylori isolates were found to have plasmid DNA. Isolates from different patients, including those from two pairs of siblings, had unique restriction patterns and plasmid profiles. Consecutive isolates obtained 1 year apart from each of two asymptomatic volunteers had identical restriction patterns and plasmid profiles, suggesting persistence of the same strain. A pair of isolates obtained one year apart from the third volunteer differed in plasmid DNA content but had similar chromosomal DNA restriction patterns. Plasmid profile determination and bacterial DNA restriction endonuclease analysis provide a reliable means of discriminating between different strains of C. pylori and may be useful for typing these organisms in epidemiologic studies.

Campylobacter↗
PubMedDate not supplied

Bacterial DNA and CpG-containing oligodeoxynucleotides activate cutaneous dendritic cells and induce IL-12 production: implications for the augmentation of Th1 responses.

BACKGROUND: Unmethylated CpG sequences in bacterial DNA act as adjuvants selectively inducing Th1 predominant immune responses during genetic vaccination or when used in conjunction with protein Ag. The precise mechanism of this adjuvant effect is unknown. Because dendritic cells (DC) are thought to be crucially involved in T cell priming and Th1/Th2 education during vaccination via skin, we characterized the effects of bacterial DNA and CpG-containing oligodeoxynucleotides (CpG ODN) on cutaneous DC. METHODS AND RESULTS: Stimulation with CpG ODN 1826 (6 micrograms/ml) induced activation of immature Langerhans cell (LC)-like DC as determined by an increased expression of MHC class II and costimulatory molecules, loss of E-cadherin-mediated adhesion and increased ability to stimulate allogeneic T cells. Composition-matched control ODN 1911 lacking CpG sequences at equal concentrations was without effect. In comparison to LPS and ODN 1911, CpG ODN 1826 selectively stimulated DC to release large amounts of IL-12 (p40) and little IL-6 or TNF-alpha within 18 h and detectable levels of IL-12 p70 within 72 h. Stimulation with Escherichia coli DNA, but not calf thymus DNA, similarly induced DC maturation and IL-12 p40 production. Injection of CpG ODN into murine dermis induced enhanced expression of MHC class II and CD86 by LC in the overlying epidermis and intracytoplasmic IL-12 p40 accumulation in a subpopulation of activated LC. CONCLUSION: Bacterial DNA and CpG ODN stimulate DC in vitro and in vivo and may preferentially elicit Th1-predominant immune responses because they can activate and mobilize DC, inducing them to produce IL-12.

Animals↗
PubMed2004

Polymerase chain reaction can detect bacterial DNA in aseptically loose total hip arthroplasties.

Identifying low-grade infection in failed total hip arthroplasties is an important but difficult task. This study investigated the ability of the polymerase chain reaction to identify low-grade infection during revision of total hip arthroplasties that failed from aseptic causes. One hundred thirteen specimens from 31 total hip arthroplasties revised for aseptic loosening were compared with 105 control specimens from 28 primary total hip arthroplasties. All surgeries were done in laminar flow operating rooms. No primary or revision specimen had positive microbiologic cultures. No revision specimen had histologic evidence suggestive of infection. Using the polymerase chain reaction with a detection threshold of 10 organisms per cubic centimeter of specimen, bacterial DNA was identified in 39 of 85 revision tissue specimens (46%) compared with 18 of 84 primary tissue specimens (21.4%). Bacterial DNA was identified in the synovial fluid of three specimens taken from 28 revision total hip arthroplasties (10.7%) and in two specimens taken from 21 primary total hip arthroplasties (9.5%). As multiple specimens were sent for each hip, a maximum of 16 of 31 revision total hip arthroplasties (52%) and eight of 28 primary total hip arthroplasties (29%) were considered to be infected. Bacterial DNA can be found in many specimens obtained from revised total hip arthroplasties considered to be aseptically loose. Because bacterial DNA identified at primary total hip arthroplasty was assumed to be attributable to contamination rather than present in healthy tissues, the overall specimen contamination rate of 19% and case contamination rate of 29% indicate that the polymerase chain reaction has poor specificity at this sensitivity level for diagnosing infection in revision total hip arthroplasty.

DNA, Bacterial↗
PubMed2005

[The synergistic effects of lipopolysaccharide, bacterial lipoprotein and bacterial DNA on mouse alveolar macrophage activation].

OBJECTIVE: To investigate the synergistic effects of lipopolysaccharide (LPS), bacterial lipoprotein (BLP), and bacterial DNA on the expression of pattern recognition receptors (PRRs) on the cell surface of mouse alveolar macrophages and cellular activation at the level of receptor and its possible mechanism. METHODS: Mouse alveolar macrophages were isolated, cultivated and randomly divided into 7 groups: control group, LPS group, CpG oligonucleoetide (CpG-ODN) group, BLP group, LPS + BLP group, LPS + CpG-ODN group, and LPS + BLP + CpG-ODN group. Six hours later the supernatants were collected to detect the level of tumor growth factor alpha (TNFalpha) by ELISA. RT-PCR was used to detect the expression of the main PRRs: CD14, SR, TLR2, TLR4, and TLR9. RESULTS: The TNFalpha levels in the supernatant were 234 pg/ml +/- 30 pg/ml in the LPS group, 274 pg/ml +/- 30 pg/ml in the BLP group, and 308 pg/ml +/- 28 pg/ml in the CpG-ODN group, all significantly higher than that in the control group (92 pg/ml +/- 27 pg/ml, P < 0.01 or P < 0.01). The TNFalpha levels in the supernatant were 483 pg/ml +/- 31 pg/ml in the LPS + BLP group, and 511 pg/ml +/- 46 pg/ml in the LPS + CpG-ODN group, both significantly higher than those of the groups of the 3 factor alone (all P < 0.05). And the TNFalpha levels in the supernatant was 665 pg/ml +/- 24 pg/ml in the LPS + BLP + CpG-ODN group, significantly higher than those of the LPS + ODN group and LPS + BLP group (both P < 0.05). LPS, BL, and CpG-ODN alone, combinations of any 2 of them, and the combination of the three all up-regulated the expression of CD14 mRNA more and more strongly in sequence. LPS, BLP, and CpG-ODN alone all up-regulated the expression of SR mRNA (all P < 0.01), however, the combinations of any 2 factors or of the 3 factors failed to further up-regulate the expression of SR. LPS and BLP up-regulated the expression of TLR2 mRNA (both P < 0.05), LPS combined with BLP showed a stronger up-regulation of TLR2 mRNA (P < 0.05) than those by LPS and BLP alone. CPG-ODN alone failed to up-regulate the expression of TLR2 mRNA (P > 0.05) but significantly increased the up-regulation by LPS (P < 0.05). In comparison with the combinations of any 2 factors, LPS and BLP with CPG-ODN together up-regulated the expression of TLR2 mRNA more strongly (all P < 0.05). LPS, BLP, and CpG-ODN alone did not significantly up-regulate the expression of TLR4 mRNA (P > 0.05), LPS + BLP significantly regulated the expression of TLR4 mRNA than the groups of any factor alone (all P < 0.05). LPS + BLP + CpG-ODN further up-regulated the expression of TLR4 mRNA. LPS and CpG-ODN, especially LPS + CpG-ODN significantly up-regulated the expression of TLR9 mRNA (all P < 0.05). BLP failed to up-regulate the expression of TLR9 mRNA (P > 0.05) and did not coordinate the upregulation by LPS, however, in comparison with any combinations of the 3 factors, the combination of LPS, BLP, and ODN up-regulated the expression of TLR9 mRNA the most strongly (all P < 0.05). CONCLUSION: Bacterial LPS, BLP and bacterial DNA not only up-regulate the expression of PRRs of each other, but also synergistically increase the each other's effects on the cell surface of mouse alveolar macrophages.

Animals↗
PubMed1999

Elimination of bacterial DNA from Taq DNA polymerases by restriction endonuclease digestion.

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications.

DNA, Bacterial↗
PubMed2004

Bacterial DNA within granulomas of patients with Crohn's disease--detection by laser capture microdissection and PCR.

OBJECTIVES: We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohn's disease. While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects. The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohn's disease. METHODS: Archival tissue from 15 surgical cases of Crohn's disease and 10 non-Crohn's granulomatous bowel disease controls were examined. Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E. coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene. RESULTS: E. coli DNA was detected in microdissected granulomas in 12/15 Crohn's disease patients and in 1/10 non-Crohn's control granulomas (p < 0.001). Also, E. coli DNA was detected in 8/15 Crohn's full-thickness sections and in 4/10 control full-thickness sections. CONCLUSIONS: E. coli DNA may be detected more frequently in Crohn's granulomas than in other non-Crohn's bowel granulomas. This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells. In light of previous detection of M. paratuberculosis DNA in Crohn's granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohn's disease.

Crohn Disease↗
PubMed2002

A comparison of five methods for extraction of bacterial DNA from human faecal samples.

The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type.

Bacteria↗
PubMed1995

Use of Multiscreen plates for the preparation of bacterial DNA suitable for PCR.

A rapid and inexpensive method for isolating bacterial DNA for use in PCR is described. The method is based on the guanidinium thiocyanate (GuSCN)-lysis method of Boom et al. (J. Clin. Microbiol. 28:495-503, 1990) and enables a multiple of 96 samples to be prepared in only one hour. We use Multiscreen plates and a vacuum manifold from Millipore. Clinical samples are lysed and washed in the wells of a Multiscreen plate, and DNA is eluted in a standard microplate. Purified DNA was recovered with high yields (over 25%). The method allows multichannel or robotic pipetting for both the sample preparation as well as for the PCR step. The method has been applied successfully to detect pathogenic Streptococcus suis type 2 in nasal and tonsil swab specimens of pigs.

Animals↗
PubMed1998

Cyclosporin A enhances IL-12 production by CpG motifs in bacterial DNA and synthetic oligodeoxynucleotides.

Certain sequences of nucleotides (CpG motifs) in bacterial DNA or synthetic oligonucleotides (CpG DNA) promote the production of proinflammatory cytokines, including TNF-alpha, IFN-gamma, IL-6, and IL-12. Here we demonstrate that the immunosuppressant cyclosporin A (CsA) unexpectedly enhanced CpG DNA-induced IL-12 production in murine splenocytes. CsA did not inhibit CpG DNA-induced TNF-alpha or IL-6 production, but decreased the production of IFN-gamma by CpG DNA. Upon examining mechanisms by which CsA increases IL-12 production, we found that CpG DNA can also induce IL-10 production in B cells and that this production was sensitive to CsA. IL-10 has anti-inflammatory effects and can reduce the production of IL-12. To determine the possible role of CsA-modulated IL-10 production in mediating the increased IL-12 levels, splenocytes from IL-10 gene-disrupted mice (IL-10 -/-) and splenocytes cultured in anti-IL-10 Ab were studied. CpG DNA-stimulated IL-10 (-/-) splenocytes demonstrated no increase in IL-12 levels in the presence of CsA. Anti-IL-10 Ab treatment of normal splenocytes increased the magnitude of CpG DNA-induced IL-12 production to that seen with CsA. These results suggest that CpG DNA induces CsA-sensitive IL-10 production in B cells and that IL-10 acts as a negative feedback regulator of CpG DNA-induced IL-12 production.

Animals↗