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At least 73 records · Page 4Linked to original sources
PubMed1991

Bacterial DNA supercoiling and [ATP]/[ADP] ratio: changes associated with salt shock.

When Escherichia coli K-12 was shifted from a medium lacking salt to one containing 0.5 M NaCl, both the [ATP]/[ADP] ratio and negative supercoiling of plasmid DNA increased within a few minutes. After about 10 min both declined, eventually reaching a level slightly above that observed with cells growing exponentially in the absence of salt. Since in vitro the [ATP]/[ADP] ratio influences the level of supercoiling generated by gyrase (H. Westerhoff, M. O'Dea, A. Maxwell, and M. Gellert, Cell Biophys. 12:157-181, 1988), the physiological response of supercoiling to salt shock is most easily explained by the sensitivity of gyrase to changes in the intracellular [ATP]/[ADP] ratio. This raises the possibility that the [ATP]/[ADP] ratio is an important factor in the control of supercoiling.

Adenosine Diphosphate↗
PubMed1988

Electron microscopic approaches to the study of bacterial DNA organization.

Electron microscopic techniques have been applied to the study of all aspects of structure, function and organization of prokaryotic and eukaryotic genomes. Today these powerful methods are mainly used in determining sequence relationships of large DNA molecules and in structural studies of nucleic acid-protein complexes. Some recent applications in the analysis of prokaryotic genomes are discussed.

Chromatin↗
PubMed2001

Bacterial DNA in synovial fluid cells of patients with juvenile onset spondyloarthropathies.

OBJECTIVE: To identify bacterial DNA in synovial fluid cells of patients with active juvenile onset spondyloarthropathy (SpA). METHODS: The main group of study constituted 22 patients with juvenile onset SpA. In addition, five patients with adult onset SpA and nine with rheumatoid arthritis (RA) were studied. Polymerase chain reaction (PCR) with either genus- or species-specific primers was performed on synovial fluid cells to detect DNA sequences of Chlamydia trachomatis, Yersinia enterocolitica, Salmonella sp., Shigella sp., Campylobacter sp. and Mycobacterium tuberculosis. The presence of antibacterial antibodies in sera and synovial fluid was also determined by enzyme-linked immunoassay. RESULTS: The synovial fluid of nine patients with juvenile onset SpA, three with adult onset SpA and one with RA contained bacterial DNA. Five juvenile onset SpA samples had DNA of one single bacterium; two juvenile onset SpA and three adult onset SpA had DNA of two bacteria and two juvenile onset SpA had DNA of three bacteria. Overall, Salmonella sp. DNA was detected in seven synovial fluid samples, Shigella sp., Campylobacter sp. and M. tuberculosis were found in four samples each, and C. trachomatis was found in two. The bacterial DNA findings correlated with neither diagnosis nor disease duration. One RA synovial fluid had DNA of Campylobacter sp. Neither serum nor synovial fluid antibacterial antibodies correlated with DNA findings or clinical diagnosis. CONCLUSION: In this study, single and several combinations of bacterial DNA were identified in the synovial fluid of patients with long-term undifferentiated and definite juvenile onset SpA and adult onset SpA. Of relevance is that bacterial DNA corresponds to bacteria producing endemic disease in our population.

Adolescent↗
PubMedDate not supplied

Comparative study of diagnosis of PD peritonitis by quantitative polymerase chain reaction for bacterial DNA vs culture methods.

INTRODUCTION: Peritonitis remains one of the main complications that afflict peritoneal dialysis patients. We conducted a pilot study to determine the feasibility and potential advantages of quantitative PCR (qPCR) assays for the presence of bacterial DNA in this clinical scenario. METHODS: 14 patients attending with 'cloudy bags' had PD fluid analyzed in accordance with Renal Association Standards. In addition, quantitative bacterial DNA analysis was performed on 50 mL samples of PD fluid. DNA was extracted using a Qiagen kit. Quantitative PCR assays using primers and probes targeted at 16S rDNA were used to measure the levels of bacterial DNA. Samples from 13 patients attending the department for other reasons served as negative controls. Laboratory staff were blinded to clinical details at the time of analysis. RESULTS: We determined a threshold of bacterial DNA whereby 11 of 13 negative controls were 'negative'. Significant bacterial DNA was found in 6 of 9 culture positive' peritonitis cases (p < 0.05 by chi(2). The 3 cases of 'no growth' peritonitis had 'insignificant' bacterial DNA. Serial DNA analysis was performed in 8 patients. Of the 6 patients who were 'cured' with standard antibiotic therapy, only 1 showed a rise in bacterial DNA from Day 1 to 5. But the 2 patients who relapsed after antibiotics had marked rises in bacterial DNA (p < 0.05 by chi(2). DISCUSSION: We showed that results from quantitative bacterial DNA PCR assays correlate with current microbiological tests despite the small size of this study. We also suggest that this technology might be clinically useful as an adjunct for cases of 'no growth' peritonitis and to identify those patients likely to relapse despite apparent clinical improvement with standard antibiotic therapy.

Bacteria↗
PubMed1998

Effects of bacterial DNA on cytokine production by (NZB/NZW)F1 mice.

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-gamma compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-gamma secreting cells, there was a significant rise in serum IFN-gamma levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-gamma while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.

Animals↗
PubMed1975

Uptake of bacterial DNA by Chlamydomonas reinhardi.

Escherichia coli [3H]DNA supplied to vegetative cultures of wild-type (mt+) and CW15 (mt+;mutant lacking the cell wall) Chlamydomonas reinhardi could bind to the cell wall of the wild-type and to the cell membrane of CW15 mutant cells. The extent of this binding decreased with time and was to a large degree (over 90%) DNA-ase-sensitive. Nevertheless, about 0.01% of the bacterial DNA remained irreversibly associated with the cells when they reached stationary phase. The irreversible binding of the donor bacterial DNA to Chlamydomonas cells could be increased by treatment of the cultures with polycations such as DEAE-dextran, poly-L-lysine and poly-L-ornithine. Although the CW15 cells rapidly degraded bacterial DNA in the culture medium wild-type cells showed only a small effect on the molecular weight of the donor DNA. The acid-insoluble radioactivity irreversibly bound to WT (+) cells consisted mainly of oligonucleotides with a small proportion present as less depolymerized donor DNA. No radioactivity, however, was found to be associated with the recipient high molecular weight Chlamydomonas DNA. No labeled donor DNA could be recognized in the cells given bacterial [3H]DNA in early stationary phase. Instead, radioactivity found in Chlamydomonas DNA corresponded to reutilization of [3H]thymine derivatives released as a result of [3H]DNA degradation. No evidence for the integration of detectable amounts of donor DNA sequences into the host cell DNA was obtained.

Bacillus subtilis↗
PubMed1998

Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction.

AIM:To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture.METHODS:DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP-PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification.RESULTS:The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11(11/14) patients, and more than 90% in 10(10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones.E.coli-related DNA fragments appeared in the stones of 8 (26.67%) patients; propionibacteria type DNA in 7 (23.33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes. A more heterogenous sequence collection was found in 7 (23.33%) patients, which could belong to multiple bacterial infections.Two (6.67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria.CONCLUSION:Most cholesterol gallstones harbor bacterial DNA.It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation.

Journal Article↗
PubMed2006

Usefulness of a molecular strategy for the detection of bacterial DNA in patients with severe sepsis undergoing continuous renal replacement therapy.

INTRODUCTION: Sepsis is a major cause of morbidity and mortality in critically ill patients. Sepsis is associated with cell necrosis and apoptosis. Circulating plasma levels of DNA have been found in conditions associated with cell death, including sepsis, pregnancy, stroke, myocardial infarction and trauma. Plasma DNA can also derive from bacteria. We have recently implemented a method to detect bacterial DNA and, in the present study, we validated this technique comparing it to standard blood culture in terms of diagnostic efficacy. METHODS: We examined a cohort of 9 critically ill patients with a diagnosis of severe sepsis and acute renal failure requiring continuous renal replacement therapy (CRRT). We analyzed bacterial DNA in blood, hemofilters, and ultrafiltrate (UF) by polymerase chain reaction amplification of 16S rRNA gene sequence analysis. Standard blood cultures were performed for all patients. RESULTS: The blood cultures from 2 of the 9 (22%) patients were positive. However, bacterial DNA was identified in the blood of 6 patients (67%), including the 2 septic patients with positive blood cultures. In 9 (100%) patients bacterial DNA was found on the filter blood side, whereas in 7 (78%) subjects it was found in the dialysate compartment of the hemofilters. Bacterial DNA was never detected in the UF. CONCLUSIONS: Using the 16S rRNA gene, the detection of bacterial DNA in blood and adsorbed within the filter could be a useful screening tool in clinically septic, blood culture-negative patients undergoing CRRT. However, the identification of the etiologic agent is not feasible with this technique because specific primers for the defined bacteria must be used to further identify the suspected pathogenic organisms.

Acute Kidney Injury↗
PubMed2005

Peptidoglycan and bacterial DNA induce inflammation and coagulation markers in synergy.

Bacterial compounds signal the presence of foreign pathogens in the innate immune system. These microbial components are key players in infectious diseases and implicate toll-like receptors in the activation of inflammation and coagulation. Nevertheless, the existence of a synergistic relationship between peptidoglycan and bacterial DNA on these two physiological responses has not been investigated. The present study reports new findings on the regulation of tumor necrosis factor alpha and tissue factor in peripheral blood mononuclear cells by peptidoglycan and bacterial DNA. These were found to induce tumor necrosis factor alpha and tissue factor simultaneously and in a synergistic manner. These findings provide a new proinflammatory and procoagulant mechanism likely to play a role in sepsis pathogenesis.

Biomarkers↗
PubMed2006

[Bacterial DNA alteration by plasma generated atomic nitrogen. Real-time PCR detection contribution].

AIMS: Bacterial sterilization by the technology of plasma in post-discharge shows a growing interest. The main appeal of this new process resides in its action at dry and low temperature (60 degrees C). This technology would be therefore useful for the complex medical equipment, sensitive to the oxidization, humidity and/or requiring a temperature lower than 60 degrees C. The objective of this survey is to demonstrate the activity of an atomic flux emanating a plasma of pure molecular nitrogen on the bacterial DNA: does the plasma of nitrogen damage the genetic material? MATERIALS AND METHODS: The bacteria tested (Bacillus stearothermophilus, Staphylococcus aureus MRSA and Helicobacter pylori) are cultivated on suitable agar, and the bacterial DNA is extracted from every CFU by the technique of the columns (High Pure PCR Template, Roche). Every quantity of DNA extracted is diluted in 1 ml pure water. Then, 50 microl of each of these solutions of DNA are laid down in sterile Nunclon's plates holes, which undergo an advanced emptiness cycle during 60 minutes. The DNA residues will be then introduced during 40 minutes in a plasma sterilization surrounding wall (Plasmalyse), Satelec) where the debit of nitrogen, the pressure and the temperature are adjusted respectively to 1 L x min(-1), 5 Torrs and 60 degrees C. The DNA so ''plasmalysé'', is recovered then by aspiration in 500 mul pure water and processed to undergo an amplification/detection by Real-Time PCR (LightCycler2.0, Roche). The DNA ''plasmalysé'' will be compared to the intact DNA control(1), to the DNA control control(2) having undergone the cycle of emptiness solely, as well as to the DNA control(3) solely heated to 60 degrees C during 40 min. RESULTS: The amplification curves demonstrated that the only advanced emptiness and the only heat don't have any activity on the bacterial DNA. On the other hand, the DNA ''plasmalysé'' shows a deterioration of the amplified sequences. CONCLUSION: The genomic bacterial DNA, once extracted, is damaged by the gaseous flux of nitrogen plasma. A new sterilization process of the medical material will presumably impose itself in medium-term.

Base Sequence↗
PubMed1998

Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells.

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as "nature's adjuvant" since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS-fractionated MHC class IIlow (termed immature DC) or MHC class IIhigh populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-alpha. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.

Adjuvants, Immunologic↗
PubMed2001

Role of the heat shock protein 90 in immune response stimulation by bacterial DNA and synthetic oligonucleotides.

To elucidate the mechanisms of immunostimulation by bacterial DNA and synthetic oligonucleotides, the effects of heat shock protein 90 (Hsp90) inhibitors on the activation of murine spleen cells and macrophages by these molecules were investigated. Murine spleen cells and J774 and RAW264.7 macrophages responded to a CpG-containing oligodeoxynucleotide (CpG ODN) and Escherichia coli DNA by increased production of interleukin 6 (IL-6), IL-12, tumor necrosis factor alpha, and nitric oxide (NO). Pretreatment with any of the three Hsp90 inhibitors geldanamycin, radicicol, and herbimycin A resulted in a dose-dependent suppression of cytokine production from the spleen cells and macrophages and of NO from macrophages stimulated with CpG ODN or E. coli DNA. These Hsp90 inhibitors, however, had no effect on Staphylococcus aureus Cowan strain 1-induced IL-12 production from either the murine spleen cells or macrophages. CpG ODN and E. coli DNA induced increased intracellular levels of phosphorylated extracellular signal-regulated kinases (ERK1 and -2), which are members of the mitogen-activated protein (MAP) kinase family, while geldanamycin and radicicol blocked the phosphorylation of ERK1 and -2 in J774 and RAW264.7 cells. These data indicate that DNA-induced activation of murine spleen cells and macrophages is mediated by Hsp90 and that Hsp90 inhibitor suppression of DNA-induced macrophage activation is associated with disruption of the MAP kinase signaling pathway. Our findings suggest that Hsp90 inhibitors may provide a useful means of elucidating the mechanisms of immunostimulation by bacterial DNA and CpG ODN as well as a strategy for preventing adverse effects of bacterial DNA as well as lipopolysaccharide.

Adjuvants, Immunologic↗
PubMed2004

Bacterial DNA activates cell mediated immune response and nitric oxide overproduction in peritoneal macrophages from patients with cirrhosis and ascites.

BACKGROUND AND AIMS: Translocation of intestinal bacteria to ascitic fluid is probably the first step in the development of episodes of spontaneous bacterial peritonitis in patients with cirrhosis. We have recently reported the detection of bacterial DNA in blood and ascitic fluid from patients with advanced cirrhosis, what we consider as molecular evidence of bacterial translocation. Several studies have shown the immunogenic role of bacterial DNA in vitro, and we hypothesised that the presence of bacterial DNA could activate the type I immune response in peritoneal macrophages from these patients, leading to greater cytokine synthesis (interleukin (IL)-2 and IL-12, tumour necrosis factor alpha, and interferon gamma) and effector molecules such as nitric oxide. METHODS: Peritoneal macrophages obtained from patients with cirrhosis and culture negative non-neutrocytic ascitic fluid were collected and characterised by flow cytometry. Inducible nitric oxide synthase, nitric oxide levels, and cytokine production were measured by immunoenzymometric assays in basal and harvested conditions according to the presence/absence of bacterial DNA. RESULTS: The ability of peritoneal macrophages to synthesise nitric oxide and levels of all cytokines were significantly increased in patients with bacterial DNA. There was a positive correlation between inducible nitric oxide synthase and nitric oxide levels. CONCLUSIONS: The presence of bacterial DNA in patients with decompensated cirrhosis is associated with marked activation of peritoneal macrophages, as evidenced by nitric oxide synthesising ability, together with enhanced cytokine production.

Aged↗