Identification Of Bacterial DNA Research Articles

Which Biomarkers Can Be Used as Diagnostic Tools for Infection in Suspected Sepsis?

The diagnosis of infection in patients with suspected sepsis is frequently difficult to achieve with a reasonable degree of certainty. Currently, the diagnosis of infection still relies on a combination of systemic manifestations, manifestations of organ dysfunction, and microbiological documentation. In addition, the microbiologic confirmation of infection is obtained only after 2 to 3 days of empiric antibiotic therapy. These criteria are far from perfect being at least in part responsible for the overuse and misuse of antibiotics, in the community and in hospital, and probably the main drive for antibiotic resistance. Biomarkers have been studied and used in several clinical settings as surrogate markers of infection to improve their diagnostic accuracy as well as in the assessment of response to antibiotics and in antibiotic stewardship programs. The aim of this review is to provide a clear overview of the current evidence of usefulness of biomarkers in several clinical scenarios, namely, to diagnose infection to prescribe antibiotics, to exclude infection to withhold antibiotics, and to identify the causative pathogen to target antimicrobial treatment. In recent years, new evidence with "old" biomarkers, like C-reactive protein and procalcitonin, as well as new biomarkers and molecular tests, as breathomics or bacterial DNA identification by polymerase chain reaction, increased markedly in different areas adding useful information for clinical decision making at the bedside when adequately used. The recent evidence shows that the information given by biomarkers can support the suspicion of infection and pathogen identification but also, and not less important, can exclude its diagnosis. Although the ideal biomarker has not yet been found, there are various promising biomarkers that represent true evolutions in the diagnosis of infection in patients with suspected sepsis.

Identification of bacterial DNA in the peripheral blood of patients with active hidradenitis suppurativa.

Bacterial translocation may have a role in the pathogenesis of several inflammatory conditions. A prospective analytical case-control study was designed to assess the presence of bacterial DNA in the peripheral blood of patients with hidradenitis suppurativa (HS). An age- and gender-matched control population was recruited from healthy blood donors. Demographic and HS-related data were also collected. We took fasting blood samples from each participant and determined the presence of bacterial DNA (including bacterial species identification) and levels TNF-α, IL-1β, and IL-17A. We included 50 patients with HS and 50 healthy controls. Bacterial DNA was present in 17 (34.0%) cases vs. 2 (4.0%) controls (P < 0.001); 14/17 (82.4%) bacterial species identified in HS patients were Gram-negative bacilli, especially Escherichia coli. The presence of bacterial DNA in patients with HS was associated with elevated levels of TNF-α (P < 0.001), IL-1β (P = 0.01) and IL-17 (P < 0.001); however, it was not associated with disease severity or disease location. BactDNA in the peripheral blood of patients with active HS is more common that in healthy controls, and it is associated with higher levels of proinflammatory cytokines. We hypothesized that BT from the skin/intestinal lumen may play a relevant role in the pathogenesis of HS.

The Diagnostic Role of Bacterial DNA in Ascitic Fluid Infection in Patients with Cirrhotic Ascites

Background: Patients with cirrhosis and ascites carry a high risk of ascitic fluid (AF) infection. Identification of bacterial DNA in patients with cirrhotic ascites may provide a rapid and accurate tool helping in diagnosis of AF infection when it is compared with culture-based methodology to initiate treatment in such cases. Objectives: This study was aimed to evaluate the efficacy of broad range 16 S ribosomal RNA gene polymerase chain reaction (PCR) in diagnosis of AF infection. Methdology: A total of 130 liver cirrhotic ascetic patients were subjected to thorough history, clinical examination, laboratory investigations including, AF samples analysis for polymorh nuclear leucocytic (PMN) count, bacterial culture and PCR- bacterial DNA detection. Results: The sensitivities of PMN count, culture and PCR in diagnosing AF infection were 73.8%, 31.5% and 80.1% respectively, while the accuracies were 77.6%, 41.5% and 83% respectively. Conclusion: Bacterial DNA in AF samples might be an alternative diagnostic method to AF bacterial culture and PMN count in early diagnosis and promp treatment of AF infection.

Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method - the observation of DNAemia

Blood is considered to be a sterile microenvironment, in which bacteria appear only periodically. Previously used methods allowed only for the detection of either viable bacteria with low sensitivity or selected species of bacteria. The Next-Generation Sequencing method (NGS) enables the identification of all bacteria in the sample with their taxonomic classification. We used NGS for the analysis of blood samples from healthy volunteers (n = 23) and patients with sepsis (n = 62) to check whether any bacterial DNA exists in the blood of healthy people and to identify bacterial taxonomic profile in the blood of septic patients. The presence of bacterial DNA was found both in septic and healthy subjects; however, bacterial diversity was significantly different (P = 0.002) between the studied groups. Among healthy volunteers, a significant predominance of anaerobic bacteria (76.2 %), of which most were bacteria of the order Bifidobacteriales (73.0 %), was observed. In sepsis, the majority of detected taxa belonged to aerobic or microaerophilic microorganisms (75.1 %). The most striking difference was seen in the case of Actinobacteria phyla, the abundance of which was decreased in sepsis (P < 0.001) and Proteobacteria phyla which was decreased in the healthy volunteers (P < 0.001). Our research shows that bacterial DNA can be detected in the blood of healthy people and that its taxonomic composition is different from the one seen in septic patients. Detection of bacterial DNA in the blood of healthy people may suggest that bacteria continuously translocate into the blood, but not always cause sepsis; this observation can be called DNAemia.

Identification of Bacterial DNA in the Peripheral Blood of Patients With Active Psoriasis.

Article Tables References Comments Psoriasis is a systemic autoimmune inflammatory disease that shares some immunological aspects with other inflammatory-based diseases, such as Crohn disease.1 Bacterial DNA (bactDNA) fragments have been shown to induce a systemic immunological response in Crohn disease and other settings.2,3 Although the results of most blood bacterial cultures are negative in patients with psoriasis, we hypothesized that the presence of bactDNA in the blood might act as a molecular trigger in disease outbreaks and induce a systemic inflammatory response in these patients.

Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA

Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.

Culture independent bacterial identification in post-mortem samples

Investigation of post-mortem samples for a bacterial aetiology is challenging. There may be pathological signs of inflammation and infection, but not always. Further complications arise via detection of organisms derived from agonal or post-mortem translocation from the colonised mucous membranes and contamination artificially introduced by collection processes. Attributing a terminal outcome to the finding of specific bacteria ranges from high certainty for pathogens not associated with the human microbiome, to low certainty when observing mixed bacterial species including normal flora. Clinical features of infection, known individual risk factors, particular bacterial virulence genes, and case histories are important pieces of the puzzle. Treatment with antimicrobials, peculiar nutritional and atmospheric requirements of bacteria, and delays in sample collection can all reduce the ability of traditional culture methods to recover causative organisms. In those circumstances recovery and specific identification of bacterial DNA has been an effective diagnostic augmentation for post-mortem bacteriology. Use of DNA encoding the bacterial ribosomes has been the primary culture independent approach. This technique will be discussed and local cases will be presented. The diagnostic value of 2 nd and 3 rd generation DNA sequencing instrument output and resultant bioinformatically reconstructed microbial genome content from clinical samples will be discussed.

AB0149 Identification and Characterization of A Synovial Fluid Microbial DNA in Rheumatoid Arthritis Patients

BackgroundThe preclinical phase of Rheumatoid Arthritis (RA) is characterized by an immunological disorder that predate the arthritis onset for several years, and is supposed to take birth in oral, respiratory...

Application of Qualitative and Quantitative Real-Time PCR, Direct Sequencing, and Terminal Restriction Fragment Length Polymorphism Analysis for Detection and Identification of Polymicrobial 16S rRNA Genes in Ascites

Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct sequencing were applied for rapid detection and identification of bacterial DNA (bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a mean of 3.24 log copies ml(-1). Direct sequencing of PCR products revealed 62% mixed chromatograms predominantly belonging to Gram-positive bacteria. Terminal restriction fragment length polymorphism (T-RFLP) results of a sample subset confirmed sequence data showing polymicrobial DNA contents in 67% of bactDNA-positive ascites samples.

Recovery and identification of bacterial DNA from illicit drugs

Bacterial infections, including Bacillus anthracis (anthrax), are a common risk associated with illicit drug use, particularly among injecting drug users. There is, therefore, an urgent need to survey illicit drugs used for injection for the presence of bacteria and provide valuable information to health and forensic authorities. The objectives of this study were to develop a method for the extraction of bacterial DNA from illicit drugs and conduct a metagenomic survey of heroin and methamphetamine seized in the Australian Capital Territory during 2002–2011 for the presence of pathogens. Trends or patterns in drug contamination and their health implications for injecting drug users were also investigated.Methods based on the ChargeSwitch®gDNA mini kit (Invitrogen), QIAamp DNA extraction mini kit (QIAGEN) with and without bead-beating, and an organic phenol/chloroform extraction with ethanol precipitation were assessed for the recovery efficiency of both free and cellular bacterial DNA. Bacteria were identified using polymerase chain reaction and electrospray ionization-mass spectrometry (PCR/ESI-MS).An isopropanol pre-wash to remove traces of the drug and diluents, followed by a modified ChargeSwitch® method, was found to efficiently lyse cells and extract free and cellular DNA from Gram-positive and Gram-negative bacteria in heroin and methamphetamine which could then be identified by PCR/ESI-MS. Analysis of 12 heroin samples revealed the presence of DNA from species of Comamonas, Weissella, Bacillus, Streptococcus and Arthrobacter. No organisms were detected in the nine methamphetamine samples analysed.This study develops a method to extract and identify Gram-positive and Gram-negative bacteria from illicit drugs and demonstrates the presence of a range of bacterial pathogens in seized drug samples. These results will prove valuable for future work investigating trends or patterns in drug contamination and their health implications for injecting drug users as well as enabling forensic links between seizures to be examined.

Identification of Bacterial DNA in Noninfectious Pleural Fluid with a Highly Sensitive PCR Method

Background: Bacterial DNA due to bacterial translocation has been identified in noninfectious ascitic fluid samples. Objective: This study investigated the possible presence of bacterial DNA in the pleural fluid of patients with pleural effusions of noninfectious origin, using a highly sensitive PCR-based method. Methods: Pleural fluid samples from 175 patients (average age ± SD: 69 ± 14 years) with noninfectious pleural effusion (62 transudates, 113 exudates) were analyzed. Bacterial DNA was detected using nested PCR with amplification of a fragment of the gene r16S, with 2 amplification protocols, i.e. low sensitivity (10 and 40 cycles) and high sensitivity (40 and 40 cycles). Results: With the less sensitive amplification process, only 1 sample was positive (Haemophilus parainfluenzae in a patient with hepatic hydrothorax). With the highly sensitive nested PCR method, bacterial DNA was identified in the pleural fluid, of both transudative and exudative origin, of 75 of the 175 patients (43%). In cases of isolation of a single bacterium, the more frequent were Escherichia coli, Salmonella enterica and Streptococcus pneumoniae.Conclusions: Regardless of its origin, bacterial DNA can be identified in almost half of noninfectious pleural effusions by using a highly sensitive PCR-based method. The possible clinical significance or prognostic value of these findings deserves to be evaluated.

Real-time PCR assays compared to culture-based approaches for identification of aerobic bacteria in chronic wounds

Chronic wounds cause substantial morbidity and disability. Infection in chronic wounds is clinically defined by routine culture methods that can take several days to obtain a final result, and may not fully describe the community of organisms or biome within these wounds. Molecular diagnostic approaches offer promise for a more rapid and complete assessment. We report the development of a suite of real-time PCR assays for rapid identification of bacteria directly from tissue samples. The panel of assays targets 14 common, clinically relevant, aerobic pathogens and demonstrates a high degree of sensitivity and specificity using a panel of organisms commonly associated with chronic wound infection. Thirty-nine tissue samples from 29 chronic wounds were evaluated and the results compared with those obtained by culture. As revealed by culture and PCR, the most common organisms were methicillin-resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae (Group B streptococcus) and Pseudomonas aeruginosa. The sensitivities of the PCR assays were 100% and 90% when quantitative and qualitative culture results were used as the reference standard, respectively. The assays allowed the identification of bacterial DNA from ten additional organisms that were not revealed by quantitative or qualitative cultures. Under optimal conditions, the turnaround time for PCR results is as short as 4–6 h. Real-time PCR is a rapid and inexpensive approach that can be easily introduced into clinical practice for detection of organisms directly from tissue samples. Characterization of the anaerobic microflora by real-time PCR of chronic wounds is warranted.

Identification of bacterial DNA in neutrocytic and non‐neutrocytic cirrhotic ascites by means of a multiplex polymerase chain reaction

Even though bacterial cultures of ascitic fluid are negative in up to 65% of the cases of spontaneous bacterial peritonitis (SBP); bacterial DNA (bactDNA) has been frequently detected in episodes of SBP as well as in culture-negative non-neutrocytic ascites. To evaluate multiplex polymerase chain reaction (PCR) for pathogen identification in SBP and to determine the prevalence of ascitic bactDNA and its prognostic relevance in hospitalized patients with liver cirrhosis. Ascitic fluid from 68 consecutive patients who underwent diagnostic paracentesis was analysed for polymorphonuclear leucocyte (PMN) count, bacterial culture and bactDNA. BactDNA was identified by gel analysis after multiplex PCR of selectively enriched prokaryotic nucleic acids. Correlations of bactDNA status with PMN count, bacterial culture result and 3-month mortality were determined for neutrocytic and for non-neutrocytic ascites. 11/68 patients presented with an elevated ascitic PMN count. BactDNA was detected in 5/5 culture-positive neutrocytic samples, in 1/6 culture-negative neutrocytic samples and in 8/56 culture-negative non-neutrocytic samples. Three-month mortality did not differ with respect to ascitic bactDNA status (7/14 vs. 14/47, P=0.162). 3-month mortality was increased in the presence of ascitic bactDNA for patients older than 65 years (4/5 vs. 4/14, P=0.046) and for patients with a model for end-stage liver disease score >15 (7/10 vs. 9/30, P=0.025). Identification of ascitic bactDNA is an appropriate alternative to bacterial ascite culture for pathogen identification in patients at risk for SBP. Its prognostic relevance as a proposed marker of bacterial translocation for certain risk groups has to be further evaluated.

M1840 Detection and Identification of Bacterial DNA in Jejunal Liquid and Blood from Patients with Acute Pancreatitis As Evidence of Bacterial Traslocation: Preliminary Results

M1840 Detection and Identification of Bacterial DNA in Jejunal Liquid and Blood from Patients with Acute Pancreatitis As Evidence of Bacterial Traslocation: Preliminary Results

Detection and identification of bacterial DNA in semen

To detect and identify bacteria in semen by sequencing polymerase chain reaction (PCR)-amplified ribosomal RNA gene regions (rDNAs). Bacterial rDNAs were detected by PCR amplification of semen DNA. Conditions were adjusted to detect only abundant organisms, no fewer than 20,000 bacteria/mL of semen. Clinical andrology laboratory and academic research laboratories. Men undergoing fertility evaluation (n = 29) or vasectomy (n = 5). None. MAIN OUTCOME MEAURE(S): Frequency of bacterial rDNA-positive specimens, relationship of rDNAs to bacteria in GenBank, and correlation with semen cells. Twenty-five (56%) of the specimens from 22 (65%) of the men were positive. A total of 141 bacterial rDNA sequences were compared with GenBank data for identification. The largest group matched gram-positive anaerobic cocci (Peptoniphilis, Anaerococcus, Finegoldia, Peptostreptococcus spp.) in 13 specimens, followed by Corynebacterium spp. in 10 specimens, Staphylococcus, Lactobacillus, and Streptococcus spp. in 7 specimens each, Pseudomonas spp. in 4 specimens, and Haemophilus and Acinetobacter spp. in 2 specimens each. The rDNA-positive specimens averaged 59 +/- 13 million sperm/mL, 46 +/- 5% of which were motile, not statistically different from the rDNA-negative specimens (77 +/- 16 million/mL, 47 +/- 5% motile). Normal sperm forms were lower in the rDNA-positive (10 +/- 1.1%) than in the rDNA-negative specimens (22 +/- 2%), and lymphocytes/monocytes were fivefold lower in the rDNA-positive specimens (0.4 +/- 0.2 million/mL) than in the negative specimens (1.9 +/- 0.7 million/mL). Abundant bacteria in semen are not commensal, arise from infection in the male genitourinary tract, may influence fertility, and may reflect an inadequate cellular immune response.

Demonstration by PCR and DNA sequencing of Corynebacterium pseudodiphtheriticum as a cause of joint infection and isolation of the same organism from a surface swab specimen from the patient

A case of infectious arthritis following arthroscopy is described. Real-time PCR, using universal bacterial primers targeting the 16S rRNA gene, and subsequent DNA sequencing of the PCR product demonstrated the presence of DNA from Corynebacterium pseudodiphtheriticum in the synovial fluid from the affected knee. Culture from a surface swab from the site of purulent discharge from the knee was initially reported as growing normal skin microbiota. Knowing the result of the DNA analysis, the specimen was re-examined and a diphtheroid bacterium was isolated. The DNA sequence of the isolated bacterium was identical to that of the DNA in the joint. The isolated bacterium was tested for susceptibility to relevant antibiotics. Demonstration and identification of bacterial DNA by PCR and gene sequencing may not by itself give information on important characteristics such as susceptibility to antibiotics of the infecting bacterium. The present case illustrates that the results obtained by the method can be used to isolate the relevant bacterium in culture from other sites and thereby characterize it. It furthermore demonstrates that C. pseudodiphtheriticum can cause severe arthritis when inoculated into joints.

115: Detection and Identification of Bacterial DNA and HIV Gag in the Semen of HIV Infected Men

115: Detection and Identification of Bacterial DNA and HIV Gag in the Semen of HIV Infected Men

Detection and identification of bacterial DNA in patients with cirrhosis and culture-negative, nonneutrocytic ascites

The current pathogenic theory of spontaneous bacterial peritonitis (SBP) in patients with cirrhosis and ascites suggests that repeated episodes of bacterial translocation (BT) from intestinal lumen to mesenteric lymph nodes followed by systemic seeding are the key steps for the final development of infectious events. However, most of the episodes of systemic bacterial circulation remain undetected. Therefore, we investigated the hypothetical presence of bacteria in blood and/or ascitic fluid (AF) from patients with cirrhosis and sterile (culture negative) AF by means of bacterial DNA (bactDNA) detection and identification. Twenty-eight consecutively admitted patients with cirrhosis and presence of AF were included in the study. BactDNA was detected using a polymerase chain reaction (PCR)-based method. The corresponding bacteria were identified by nucleotide sequencing of purified PCR products. BactDNA was detected simultaneously in blood and AF in 9 patients (32.1%). DNA sequencing allowed the identification of Escherichia coli (n = 7) and Staphylococcus aureus (n = 2). In all cases, the similarity between the sequence found in AF and blood indicated that the bactDNA present in both locations originated from a single clone (single translocation event). Child-Pugh score and basic hemodynamic, clinical, endoscopic, and biochemical characteristics were similar among patients with or without the presence of bactDNA. In conclusion, we have detected bactDNA in serum and AF in 32% of all patients studied, and this likely represents single clone episodes of translocation and systemic seeding. E. coli is the most frequently identified bacteria. (H EPATOLOGY 2002;36:135-141.)

Chlamydia pneumoniae and atherosclerosis. Identification of bacterial DNA in the arterial wall.

The intracellular Gram-negative bacterium Chlamydia pneumoniae has been associated with atherosclerosis. The presence of Chlamydia pneumoniae has been investigated in fragments of the arterial wall with a technique for DNA identification. Arterial fragments obtained from vascular surgical procedures in 58 patients were analyzed. From these patients, 39 were males and the mean age was 65+/-6 years. The polymerase chain reaction was used to identify the bacterial DNA with a pair of primers that codify the major outer membrane protein (MOMP) of Chlamydia pneumoniae. The amplified product was visualized by electrophoresis in the 2% agarose gel stained with ethidium bromide, and it was considered positive when migrating in the band of molecular weight of the positive controls. Seven (12%) out of the 58 patients showed positive results for Chlamydia pneumoniae. DNA from Chlamydia pneumoniae was identified in the arterial wall of a substantial number of patients with atherosclerosis. This association, which has already been described in other countries, corroborates the evidence favoring a role played by Chlamydia pneumoniae in atherogenesis.

Body lice as tools for diagnosis and surveillance of reemerging diseases.

Body lice are vectors of three bacteria which cause human disease: Rickettsia prowazekii, the agent of epidemic typhus; Bartonella quintana, the agent of trench fever; and Borrelia recurrentis, the agent of relapsing fever. A recrudescence of body lice is being observed as the numbers of individuals living under social conditions which predispose individuals to infestation have increased. Because this phenomenon may lead to the reemergence of infections transmitted by body lice, we aimed to assess the occurrence and prevalence of the three agents described above in more than 600 body lice collected from infested individuals in the African countries of Congo, Zimbabwe, and Burundi, in France, in Russia, and in Peru. The presence of the three bacteria in each louse was determined by specific PCR amplification, and the identities of the organisms detected were confirmed by determination of the nucleotide base sequences of the amplification products. Using this approach, we were able to confirm the presence of R. prowazekii in lice collected from refugees in Burundi, among whom typhus was epidemic, and the presence of B. quintana in lice collected from all locations except the Congo. B. recurrentis was never found. Molecular approaches are convenient tools for the detection and identification of bacterial DNA in body lice and for the epidemiological study of louse-borne bacteria from countries where no medical and biological laboratory facilities are available.