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Results for “microbial detection”

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PubMed1997

Antibodies against stress proteins in sera of patients with dilated cardiomyopathy.

The pathogenesis of dilated cardiomyopathy (DCM) is as yet unknown. However, it is widely believed that autoimmune mechanisms contribute to the manifestations of the disease. In sera of patients with dilated cardiomyopathy, antibodies against different antigens, including heat shock protein (hsp) 60, were found. Antibodies against other stress proteins have not yet been reported. The aim of this study, therefore, was to screen sera of patients with DCM for the presence of antibodies against the major stress proteins. Lysate of stressed human endothelial cells was used as antigenic substrate in 1- and 2-dimensional immunoblot, since this cell type has recently been shown to express the major stress proteins. Antibodies against hsp60, hsp70, and heat shock cognate protein (hsc) 70 were detected in sera of patients with dilated cardiomyopathy as compared to healthy controls. Interestingly, antibodies against hsp70 and hsc70 were found in sera of patients younger than 30 years significantly more often than in older individuals. A correlation between the presence of antibodies against stress proteins and disease activity, clinical status, or histological findings was not detected. These findings support the view that DCM might be a consequence of an infectious disease, because stress proteins are immunodominant antigens in microbial agents and antibodies against stress proteins were detected in sera of patients with infectious diseases. Whether these antibodies are of pathogenetic significance or may be used as a disease marker will have to be elucidated in future experiments.

Adolescent↗

PubMed2026

Application of PathoChip to urine-derived nucleic acids for broad microbial profiling in men with suspected prostate cancer: setup of a methodological workflow and pilot feasibility study.

BACKGROUND: Urine-based liquid biopsy is an attractive non-invasive source of prostate cancer (PCa) biomarkers, but urinary microbiome studies have mainly relied on 16S rRNA sequencing or shotgun metagenomics. This pilot study optimized and evaluated a practical workflow using PathoChip - a broad-spectrum microarray designed to detect bacterial, viral, fungal, and parasitic signatures - for microbial profiling of urine sediments from men with suspected PCa, an application not previously established. METHODS: First-morning urine was collected without prostatic massage from 35 men scheduled for biopsy; 19 were diagnosed with PCa and 16 were biopsy-negative. Different urine volumes and extraction strategies were evaluated to optimize DNA/RNA recovery. A setup phase compared 25 ng versus 50 ng of urine DNA and RNA input. DNA/RNA isolated from human B cells was used as reference control. An analysis pipeline was developed to detect outlier probes and create a presence/absence matrix. Reproducibility was assessed via library yield, Pearson correlation, blank-control subtraction, outlier probe detection. Prevalence comparisons were performed between clinical groups. RESULTS: An 8 mL starting volume was chosen as consistently available from self-collected urine. Sequential DNA/RNA extraction using the AllPrep DNA/RNA Micro Kit from sediment provided the best balance between nucleic-acid recovery, purity, and clinical compatibility. Reducing the input from 50 ng to 25 ng preserved highly concordant hybridization profiles, with matched samples clustering together with strong correlations. Exploratory analysis revealed PCa- and grade-associated patterns involving Actinomycetaceae, Aerococcaceae, and Streptococcaceae, with Streptococcaceae enriched in PCa of higher grades (ISUP GG ≥ 2). Other signatures, including Mobiluncus, Prevotella, Rhodotorula, Hymenolepis, and JC polyomavirus, were broadly detected but not PCa-discriminating. CONCLUSIONS: PathoChip can be adapted to urine sediments, generating reproducible microbial profiles from limited DNA/RNA input without prostatic massage. This platform provides a quick and accessible approach to broad screening, extending beyond 16S rRNA sequencing by enabling simultaneous multi-kingdom detection. The observed PCa- and grade-associated patterns are hypothesis-generating and require validation in larger independent cohorts.

Pathochip↗

PubMed2024

Metagenomic Study of the MESA: Detection of Gemella Morbillorum and Association With Coronary Heart Disease.

BACKGROUND: Inflammation is a feature of coronary heart disease (CHD), but the role of proinflammatory microbial infection in CHD remains understudied. METHODS AND RESULTS: CHD was defined in the MESA (Multi-Ethnic Study of Atherosclerosis) as myocardial infarction (251 participants), resuscitated arrest (2 participants), and CHD death (80 participants). We analyzed sequencing reads from 4421 MESA participants in the National Heart, Lung, and Blood Institute Trans-Omics for Precision Medicine program using the PathSeq workflow of the Genome Analysis Tool Kit and a 65-gigabase microbial reference. Paired reads aligning to 840 microbes were detected in >1% of participants. The association of the presence of microbe reads with incident CHD (follow-up, ~18 years) was examined. First, important variables were ascertained using a single regularized Cox proportional hazard model, examining change of risk as a function of presence of microbe with age, sex, education level, Life's Simple 7, and inflammation. For variables of importance, the hazard ratio (HR) was estimated in separate (unregularized) Cox proportional hazard models including the same covariates (significance threshold Bonferroni corrected P<6×10-5, 0.05/840). Reads from 2 microbes were significantly associated with CHD: Gemella morbillorum (HR, 3.14 [95% CI, 1.92-5.12]; P=4.86×10-6) and Pseudomonas species NFACC19-2 (HR, 3.22 [95% CI, 2.03-5.41]; P=1.58×10-6). CONCLUSIONS: Metagenomics of whole-genome sequence reads opens a possible frontier for detection of pathogens for chronic diseases. The association of G morbillorum and Pseudomonas species reads with CHD raises the possibilities that microbes may drive atherosclerotic inflammation and that treatments for specific pathogens may provide clinical utility for CHD reduction.

Humans↗

PubMed2005

Design, development, and use of molecular primers and probes for the detection of Gluconacetobacter species in the pink sugarcane mealybug.

Molecular tools for the species-specific detection of Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens from the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae) were developed and used in polymerase chain reactions (PCR) and in fluorescence in situ hybridizations (FISH) to better understand the microbial diversity and the numerical significance of the acetic acid bacteria in the PSMB microenvironment. The presence of these species in the PSMB occurred over a wide range of sites, but not in all sites in sugarcane-growing areas of Queensland, Australia, and was variable over time. Molecular probes for use in FISH were also designed for the three acetic acid bacterial species, and shown to be specific only for the target species. Use of these probes in FISH of "squashed" whole mealybugs indicated that these acetic acid bacteria species represent only a small proportion of the microbial population of the PSMB. Despite the detection of Glac. sacchari, Glac. diazotrophicus, and Glac. liquefaciens by PCR from different mealybugs isolated at various times and from various sugarcane-growing areas in Queensland, Australia, these bacteria do not appear to be significant commensals in the PSMB environment.

Animals↗

PubMed2001

Enumeration and factors influencing the relative abundance of a denitrifier, Pseudomonas sp. JR12, entrapped in alginate beads.

The relative abundance of the denitrifier, Pseudomonas sp. JR12, was examined in an alginate-based entrapment complex under non-sterile, denitrifying conditions. Immuno-labeling of the Pseudomonas inoculant followed by flow cytometry (FCM) was used for determination of the relative abundance of this bacterium under the various incubation conditions. Additional information on the relative abundance of the inoculant was obtained by a quantitative enzyme-linked immunosorbent assay (ELISA) and results obtained by FCM and ELISA were compared. Ambient nitrate levels controlled the successful, long-term proliferation of the inoculant. At low ambient nitrate levels, Pseudomonas sp. remained the dominant microorganism during incubation. Higher ambient nitrate concentrations, attained by either decreasing the inoculum size of Pseudomonas sp. or raising inlet nitrate concentrations of the medium supplied to the incubation vessels, resulted in a gradual shift toward other, nitrite-accumulating denitrifiers. Thus far, most studies on the use of entrapped microorganisms for bioremediation purposes have been conducted under controlled laboratory conditions. Based on this study, conducted under non-sterile laboratory conditions, it is concluded that in-situ bioremediation using entrapped target microorganisms is bound to fail without a proper understanding of the factors that cause the target microorganism to out-compete undesired microbial invaders. Furthermore, based on the close agreement between the two detection methods used, it is concluded that flow cytometry provides a rapid and accurate tool for the detection of the relative abundance of immuno-labeled target organisms in heterogeneous microbial populations.

Environmental Monitoring↗

PubMed1999

Detection of the human 70-kD and 60-kD heat shock proteins in the vagina: relation to microbial flora, vaginal pH, and method of contraception.

The expression of the 60-kD and 70-kD heat shock proteins (hsp60 and hsp70) in the vaginas of 43 asymptomatic women of reproductive age with or without a history of recurrent vulvovaginitis (RVV) were compared. Vaginal wash samples were obtained and assayed by enzyme-linked immunosorbent assay (ELISA) for human hsp60 and hsp70. Heat shock protein 70 was not detected in any of the 19 women with no history of RVV, and hsp60 was present in only one woman in this group. In contrast, in the RVV group, 11 (45.8%) were hsp60-positive and eight (33.3%) were hsp70-positive. The presence of either heat shock protein in the vagina was associated with an elevated vaginal pH (>4.5). Bacterial vaginosis or Candida was identified in some of the asymptomatic subjects; their occurrence was significantly higher in women with vaginal hsp70 than in women with no heat shock proteins. Oral contraceptives were used by 35.7% of subjects who were negative for vaginal heat shock proteins, as opposed to only 12.5% of women who were positive for hsp70 and 8.3% who were positive for hsp60. Expression of heat shock proteins in the vagina may indicate an altered vaginal environment and a susceptibility to vulvovaginal symptoms.

Adult↗

PubMed2006

Preparation of anti-pefloxacin antibody and development of an indirect competitive enzyme-linked immunosorbent assay for detection of pefloxacin residue in chicken liver.

Pefloxacin has been increasingly used in veterinary medicine to treat microbial infections. To avoid using a labor-intensive instrumental method to detect the residue of pefloxacin in food, a simple and convenient indirect competitive enzyme-linked immunosorbent assay method has been developed in this study. The antibody generated from immunogen cationized bovine serum albumin-pefloxacin showed high sensitivity toward pefloxacin with an IC50 value of 6.7 ppb in buffer and was suitable for a screening assay to detect the residue of pefloxacin in food products. The antibody has been assessed using rapid enzyme immunoassays to exploit its specificity. The antibody prepared shows cross-reactivity with a few other (fluoro)quinolones including fleroxacin (116%), enrofloxacin (88%), and ofloxacin (10%). The assay measured drug residue in chicken liver spiked with pefloxacin with an interassay coefficient of variation of 13.6% or less and an intra-assay coefficient of variation of 10.9% or less. The average recovery rates at 0.5, 5, 10, 50, and 100 ppb were in the range of 86-106% for interassay and in the range of 87-103% for intra-assay, respectively.

Animals↗

PubMed1997

Faecal microbial flora and disease activity in rheumatoid arthritis during a vegan diet.

To clarify the role of the faecal flora in the diet-induced decrease of rheumatoid arthritis (RA) activity, 43 RA patients were randomized into two groups: the test group to receive living food, a form of uncooked vegan diet rich in lactobacilli, and the control group to continue their ordinary omnivorous diets. Based on clinical assessments before, during and after the intervention period, a disease improvement index was constructed for each patient. According to the index, patients were assigned either to a group with a high improvement index (HI) or to a group with a low improvement index (LO). Stool samples collected from each patient before the intervention and at 1 month were analysed by direct stool sample gas-liquid chromatography of bacterial cellular fatty acids. This method has proved to be a simple and sensitive way to detect changes and differences in the faecal microbial flora between individual stool samples or groups of them. A significant, diet-induced change in the faecal flora (P = 0.001) was observed in the test group, but not in the control group. Further, in the test group, a significant (P = 0.001) difference was detected between the HI and LO categories at 1 month, but not in the pre-test samples. We conclude that a vegan diet changes the faecal microbial flora in RA patients, and changes in the faecal flora are associated with improvement in RA activity.

Arthritis, Rheumatoid↗

PubMed1996

Estimating biodegradative gene numbers at a JP-5 contaminated site using PCR.

We have utilized a most-probable-number polymerase chain reaction (MPN-PCR) procedure to estimate gene numbers and biodegradative potential at a jet fuel (JP-5) contaminated site undergoing the first phase of bioremediation. Nucleic acid analysis was used to determine whether a lack of genetic potential for bioremediation was responsible for low levels of oxygen utilization at the site. Total community DNA was extracted and analyzed by PCR for genes (nahAc,alkB, and xylE) known to be involved in the degradation of certain JP-5 constituents. Results indicate that significant aromatic biodegradative potential exists at the site and outlying areas not subjected to engineered remediation, suggesting that physical and/or chemical factors are inhibiting oxygen delivery. xylE and nahAc were often present in significant portions of the microbial community, whereas alkB was rarely detected. This study illustrates the utility of molecular techniques in evaluating biodegradative potential in the field during active bioremediation.

Base Sequence↗

PubMed2005

Markers of periodontal infection and preterm birth.

OBJECTIVE: This study was undertaken to explore the relationship between clinical, microbiologic, and serologic markers of periodontitis and preterm birth (PTB). STUDY DESIGN: We compared women with a singleton gestation giving birth before the 37th week (cases, n = 83) with term delivery controls (n = 120). Periodontal examination and collection of dental plaque and blood samples were performed within 48 hours after delivery. Microbial levels and maternal immunoglobulin G titers to oral bacteria were analyzed. Multivariate regression models were fitted controlling for common covariates. RESULTS: Cases showed greater mean attachment loss (1.7 vs 1.5 mm, P = .003) and higher prevalence of periodontitis (30.1% vs 17.5%, P = .027). No differences in microbial or serum antibody levels were detected between the groups. Logistic regression revealed that PTB was associated with attachment loss (adjusted odds ratio: 2.75, 95% CI: 1.01-7.54). Linear regression indicated a significant ( P = .04) association between attachment loss and low birth weight (LBW). CONCLUSION: The data support the notion that periodontitis is independently associated with PTB and LBW.

Adult↗

PubMed2006

Confocal microscopy for Nocardia keratitis.

PURPOSE: To assess the role of confocal microscopy for in vivo detection of Nocardia in patients with microbial keratitis. DESIGN: Retrospective interventional case series. PARTICIPANTS: Three patients with microbiologically proven Nocardia keratitis. INTERVENTION: Confocal microscopy. METHODS: We performed confocal microscopy in microbiologically proven cases of Nocardia asteroides keratitis. A masked observer examined the images. For better understanding of the images, we also performed confocal microscopy on a blood agar culture plate with Nocardia growth. RESULTS: Confocal microscopy of infiltrated cornea revealed highly reflective, short, thin branching filaments with bright inflammatory cells in the background. The filaments were seen clearly at the edge of the infiltrate. In scans with faint images, these filaments became more visible on inversion of bright and dark components. Confocal microscopy of cultured organisms also revealed filamentous beaded structures with a morphology identical to that of those seen in vivo. CONCLUSION: Nocardia, a filamentous bacterium, produces a distinct image on confocal microscopy. This in vivo examination technique may be useful in cases of deep-seated infiltrates where routine microbiology workup does not yield positive results.

Adult↗

PubMed1999

Species-specific detection of hydrocarbon-utilizing bacteria.

Rapid detection and quantitative assessment of specific microbial species in environmental samples is desirable for monitoring changes in ecosystems and for tracking natural or introduced microbial species during bioremediation of contaminated sites. In the interests of developing rapid tests for hydrocarbon-degrading bacteria, species-specific PCR primer sets have been developed for Pseudomonas aeruginosa, Stentrophomonas (Xanthomonas) maltophilia, and Serratia marsescens. Highly variable regions of the 16S rRNA gene were used to design these primer sets. The amplification products of these primer sets have been verified and validated with hemi-nested PCR and with ligase chain reaction (LCR) techniques, and have been applied to the analyses of environmental water samples. These species-specific primer sets were also chosen to amplify in conjunction with a universal set of PCR primers chosen from highly conserved neighboring sequences in the same gene. These multiplex or competitive PCR procedures enable testing with an internal marker and/or the quantitative estimation of the relative proportion of the microbial community that any one of these species occupies. In addition, this universal PCR primer set amplified the same size amplicon from a wide spectrum of procaryotic and eucaryotic organisms and may have potential in earth biota analyses.

Base Sequence↗

PubMed2002

Monitoring of ethanol during fermentation using a microbial biosensor with enhanced selectivity.

The present study is concerning the construction of ferricyanide-mediated Gluconobacter oxydans cell ethanol biosensor. The size exclusion effect of a cellulose acetate membrane was used for elimination of glucose interferences during ethanol assays in real samples. A typical response time of the biosensor was 13 s with a high sensitivity of 3.5 microA mM(-1). The microbial biosensor exhibits a very low detection limit of 0.85 microM and a wide linear range from 2 to 270 microM. The operational stability was excellent. During 8.5 h of repetitive ethanol assays, no decrease in the sensor sensitivity was observed. The biosensor was successfully used in the off-line monitoring of ethanol fermentation with a good agreement with HPLC measurements (R(2)=0.998).

Biosensing Techniques↗

PubMedDate not supplied

Comparison of broth and filtration methods for culturing of intravenous fluids.

Ninety-six specimens of intravenous fluid solutions (D5/025 NS) were inoculated with S. aureus, E. coli, P. aeruginosa, K. pneumoniae, E. agglomerans, or C. albicans in concentrations of .1, 1, 10, or 10(2) organisms/ml. They were cultured in tubes containing 5 ml of double enriched broth and after passage through a .45 mu pore membrane filter. After 24 hours of incubation, broth cultures were 68% as sensitive as the filter cultures (p less than .001). At the lowest concentration (.1 organism/ml) broth cultures wer only 45% as sensitive as the membrane filter technique after 24 hours of growth (p less than .001). Membrane filters provide a rapid method to accurately detect and quantitate the presence of microbial contamination even at very low levels of concentration. The simplicity and accuracy of the filtration method offers the clinician a valuable adjunct in managing suspected cases of intravenous fluid related sepsis.

Bacteria↗

PubMedDate not supplied

Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization.

Botulinum neurotoxin (BoNY) type A, a causative agent of botulism, is a di-chain protein (molecular weight 140,000) from Clostridium botulinum, and the most neurotoxic substance known. Some cases of sudden infant cot deaths have been attributed to such a neuroparalytic condition. BoNT inhibits irreversibly the release of acetylcholine from peripheral nerves in a highly selective manner. Hence, it is potentially an invaluable probe for studying the mechanism of transmitter release. Here we demonstrate specific labelling of murine motor nerve terminals with neurotoxic, 125I-labelled BoNT (type A) by autoradiography. We observed saturable, temperature-sensitive binding of BoNT to sites which reside solely on the nerve terminal membrane; these were distributed on all unmyelinated areas, at an average density of 150-500 per micron2 of membrane. The binding was mediated by the larger subunit of the toxin and was inhibited partially by tetanus toxin, another microbial protein. No specific binding was detectable on any other cell types examined, including noradrenergic terminals. Following binding, internalization of radioactivity was observed; this process was energy-dependent as it could be prevented totally by azide or dinitrophenol (DNP). This direct demonstration of separable steps, including highly selective binding and acceptor-mediated internalization, is reconcilable with the unique potency and the multiphasic inhibitory action of BoNT on transmitter release, as shown electrophysiologically.

Animals↗

PubMed2006

Selection for Staphylococcus aureus small-colony variants due to growth in the presence of Pseudomonas aeruginosa.

Opportunistic infections are often polymicrobial. Two of the most important bacterial opportunistic pathogens of humans, Pseudomonas aeruginosa and Staphylococcus aureus, frequently are coisolated from infections of catheters, endotracheal tubes, skin, eyes, and the respiratory tract, including the airways of people with cystic fibrosis (CF). Here, we show that suppression of S. aureus respiration by a P. aeruginosa exoproduct, 4-hydroxy-2-heptylquinoline-N-oxide (HQNO), protects S. aureus during coculture from killing by commonly used aminoglycoside antibiotics such as tobramycin. Furthermore, prolonged growth of S. aureus with either P. aeruginosa or with physiological concentrations of pure HQNO selects for typical S. aureus small-colony variants (SCVs), well known for stable aminoglycoside resistance and persistence in chronic infections, including those found in CF. We detected HQNO in the sputum of CF patients infected with P. aeruginosa, but not in uninfected patients, suggesting that this HQNO-mediated interspecies interaction occurs in CF airways. Thus, in all coinfections with P. aeruginosa, S. aureus may be underappreciated as a pathogen because of the formation of antibiotic-resistant and difficult to detect small-colony variants. Interspecies microbial interactions, analogous to those mediated by HQNO, commonly may alter not only the course of disease and the response to therapy, but also the population structure of bacterial communities that promote the health of host animals, plants, and ecosystems.

Cell Proliferation↗

PubMed2002

Cloning and characterization of a novel human alcohol dehydrogenase gene (ADHFe1).

There are three types of NAD(P)(+)-dependent alcohol dehydrogenase: "medium-chain" zinc-containing enzymes, "short-chain" zinc-lacking enzymes and iron-activated enzymes. Although the medium-chain family and the short-chain family have been characterized in human, the iron-activated alcohol dehydrogenase family seems to exist only in microbial organisms. We have now cloned and characterized an iron-activated alcohol dehydrogenase gene, Fe-containing alcohol dehydrogenase 1 (ADHFe1) in human. The cDNA was isolated from the human fetal brain cDNA library. It contains a long open reading frame, which is not homologous with the other alcohol dehydrogenases isoenzymes characterized in human. The hypothetical alcohol dehydrogenase does, however, show strong homology to the iron-activated alcohol dehydrogenases from microbial organisms. Northern blotting analysis only detected expression in adult liver tissue. At least two different splicing variants were screened by PCR using the multiple tissue cDNA panels as templates.

Alcohol Dehydrogenase↗

PubMed1995

Pyridoxine and pyridoxine-5'-beta-D-glucoside exert different effects on tissue B-6 vitamers but similar effects on beta-glucosidase activity in rats.

Pyridoxine glucoside is a partially available form of vitamin B-6 present in plant-derived foods. In this set of three studies, rats were fed diets containing different concentrations of either pyridoxine or pyridoxine glucoside in the presence or absence of a fixed concentration of pyridoxine for 2 wk. The distribution of B-6 vitamers and beta-glucosidase activity in tissues was examined to determine the metabolic effects of chronic consumption of pyridoxine glucoside. Rats fed pyridoxine glucoside either with or without pyridoxine exhibited a significant increase in the amount of hepatic pyridoxine 5'-phosphate (not detected in rats fed pyridoxine alone), whereas hepatic pyridoxal 5'-phosphate was decreased with increasing dietary pyridoxine glucoside. The activity of cytosolic beta-glucosidases in small intestine and kidney was affected by the dietary concentration of both pyridoxine and pyridoxine glucoside. Enzymatic activity capable of hydrolyzing pyridoxine glucoside was found in mucosal and intraluminal fractions of small intestine and in the kidney. Other tissues examined, including liver, spleen and stomach, did not hydrolyze pyridoxine glucoside in detectable quantities. These findings indicate that microbial and mucosal enzymes can participate in the intestinal hydrolysis of pyridoxine glucoside and that the kidney may contribute to postabsorptive hydrolysis. These findings further support the observations that dietary pyridoxine glucoside influences vitamin B-6 metabolism.

Animals↗