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PubMed2000

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community.

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.

Journal Article↗

PubMed1999

Involvement of the Cpx signal transduction pathway of E. coli in biofilm formation.

In a genetic screening directed to identify genes involved in biofilm formation, mutations in the cpxA gene were found to reduce biofilm formation by affecting microbial adherence to solid surfaces. This effect was detected in Escherichia coli K12 as well as in E. coli strains isolated from patients with catheter-related bacteremia. We show that the negative effect of the cpxA mutation on biofilm formation results from a decreased transcription of the curlin encoding csgA gene. The effect of the cpxA mutation could not be observed in cpxR- mutants, suggesting that they affect the same regulatory pathway. The cpxA101 mutation abolishes cpxA phosphatase activity and results in the accumulation of phosphorylated CpxR. Features of the strain carrying the cpxA101 mutation are a reduced ability to form biofilm and low levels of csgA transcription. Our results indicate that the cpxA gene increases the levels of csgA transcription by dephosphorylation of CpxR, which acts as a negative regulator at csgA. Thus, we propose the existence of a new signal transduction pathway involved in the adherence process in addition to the EnvZ-OmpR two-component system.

Bacterial Adhesion↗

PubMed2006

Chronic Helicobacter pylori infection does not significantly alter the microbiota of the murine stomach.

We examined the impact of Helicobacter pylori infection on the murine gastric microbiota by culture and terminal-restriction fragment length polymorphism and found that neither acute nor chronic H. pylori infection substantially affected the gastric microbial composition. Interestingly, the total H. pylori burden detected by real-time PCR was significantly higher than that revealed by viable counts, suggesting that the antigenic load sustaining H. pylori-induced gastritis could be considerably higher than previously believed.

Animals↗

PubMed1993

Quantitative reverse sample genome probing of microbial communities and its application to oil field production waters.

This paper presents a protocol for quantitative analysis of microbial communities by reverse sample genome probing is presented in which (i) whole community DNA is isolated and labeled in the presence of a known amount of an added internal standard and (ii) the resulting spiked reverse genome probe is hybridized with a master filter on which denatured genomic DNAs from bacterial standards isolated from the target environment were spotted in large amounts (up to 1,500 ng) in order to improve detection sensitivity. This protocol allowed reproducible fingerprinting of the microbial community in oil field production waters at 19 sites from which water and biofilm samples were collected. It appeared that selected sulfate-reducing bacteria were significantly enhanced in biofilms covering the metal surfaces in contact with the production waters.

Journal Article↗

PubMed1998

A novel beta-N-acetylglucosaminidase from Streptomyces thermoviolaceus OPC-520: gene cloning, expression, and assignment to family 3 of the glycosyl hydrolases.

A beta-N-acetylglucosaminidase gene (nagA) of Streptomyces thermoviolaceus OPC-520 was cloned in Streptomyces lividans 66. The nucleotide sequence of the gene, which encodes NagA, revealed an open reading frame of 1,896 bp, encoding a protein with an Mr of 66, 329. The deduced primary structure of NagA was confirmed by comparison with the N-terminal amino acid sequence of the cloned beta-N-acetylglucosaminidase expressed by S. lividans. The enzyme shares no sequence similarity with the classical beta-N-acetylglucosaminidases belonging to family 20. However, NagA, which showed no detectable beta-glucosidase activity, revealed homology with microbial beta-glucosidases belonging to family 3; in particular, striking homology with the active-site regions of beta-glucosidases was observed. Thus, the above-mentioned results indicate that NagA from S. thermoviolaceus OPC-520 is classified as a family 3 glycosyl hydrolase. The enzyme activity was optimal at 60 degreesC and pH 5.0, and the apparent Km and Vmax values for p-nitrophenyl-beta-N-acetylglucosamine were 425.7 microM and 24.8 micromol min-1 mg of protein-1, respectively.

Acetylglucosaminidase↗

PubMed2000

Comparison of acid mine drainage microbial communities in physically and geochemically distinct ecosystems.

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of approximately 0.5 to approximately 1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. "Ferroplasma" spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength. Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated. Sulfobacillus spp. were detected as a prominent inhabitant in warmer ( approximately 43 degrees C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.

Bacteria↗

PubMedDate not supplied

Retinal pigment epithelial phagocytosis and metabolism differ from those of macrophages.

The purpose of this study is to compare primary human retinal pigment epithelium (RPE) cells with respect to particle uptake and further processing steps with immunological phagocytes for a better understanding of the possible role of RPE cells in triggering autoimmune diseases in the eye. We investigated the similarities of human RPE and monocytes/macrophages studying the uptake of fluorescein- and europium-labeled synthetic microparticles and microbial pathogens by human and bovine RPE cultures and a permanent RPE cell line (CRL). The uptake was monitored by laser scanning microscopy, flow cytometry and time-resolved fluorescence analysis; for comparison, macrophages and a macrophage-like cell line (MonoMac6) were used. A size-dependent uptake was seen in primary RPE cultures as well as in CRL, showing a preferential uptake of smaller beads followed by Staphylococcus aureus and Escherichia coli. Opsonization with serum caused a modest increase in bacteria uptake, but in contrast to macrophages, the classical complement receptors were not found on RPE cells. Living bacteria were also ingested in a time-dependent manner, but, as no intracellular overgrowth was observed, we further investigated the oxidative ability of RPE as a possible mechanism for microbial suppression. Unlike macrophages/granulocytes, no respiratory burst was detected in RPE cells, but, comparable to MonoMac6, IFN-gamma induced neopterin in the human RPE. Interestingly a diurnal rhythm of phagocytosis was observed which was influenced by light exposure suggesting that RPE cells maintain their circadian rhythm also in cell culture to a certain extent. This study further demonstrates that in addition to similar phagocytic properties the RPE still shows substantial metabolic differences in comparison to blood-derived phagocytes.

Animals↗

PubMed2003

Differential modulation of the induction of inflammatory mediators by antibiotics in mouse macrophages in response to viable Gram-positive and Gram-negative bacteria.

We have investigated effects of beta-lactam antibiotics on TNF-alpha, and iNOS production from mouse peritoneal macrophages following co-culture with Escherichia coli or Staphylococcus aureus bacteria. Ceftazidime and aztreonam enhanced TNF-alpha secretion from macrophages stimulated with E. coli; however, imipenem does not alter either the kinetics or magnitude of TNF-alpha in E. coli-treated macrophages. Similar treatments with S. aureus co-cultured with macrophages markedly altered profiles of TNF-alpha response characterized by apparent early TNF-alpha peak relative to untreated S. aureus. All antibiotics increased E. coli-induced iNOS expression as assessed by both mRNA and protein. These same antibiotics significantly reduced S. aureus-induced iNOS levels of RNA. Both ceftazidime and aztreonam enhanced LPS release from E. coli in comparison to low-level LPS release from imipenem-treated bacteria, consistent with observed differences in TNF-alpha release. Incubation of all three antibiotics with S. aureus similarly increased levels of the cell wall constituent protein A detected in supernatants at early time points indicating microbial lysis. In parallel, S. aureus culture supernatants from 2-h incubation with antibiotics enhanced TNF-alpha release. These results indicate that different cellular mechanisms contribute to antibiotic-mediated regulation of TNF-alpha and iNOS secretion in mouse macrophages in response to E. coli versus S. aureus.

Animals↗

PubMed2005

Usefulness of procalcitonin levels in community-acquired pneumonia according to the patients outcome research team pneumonia severity index.

STUDY OBJECTIVES: To evaluate the usefulness of procalcitonin serum levels as a predictor of etiology and prognosis in adult patients with community-acquired pneumonia (CAP) when they are stratified according to severity. DESIGN: One-year, population-based, prospective study. SETTING: University teaching hospital. PATIENTS: All adult patients who received a diagnosis of CAP throughout the study period. INTERVENTIONS AND MEASUREMENTS: An extensive noninvasive microbiological workup was performed. In patients who gave informed consent, a blood sample was collected at the time the diagnosis of CAP was established to measure biological markers. Procalcitonin levels were measured by a commercially available monoclonal immunoluminometric assay (limit of detection, 0.1 microg/L). Patients were classified according to microbial diagnosis, Patients Outcome Research Team pneumonia severity index (PSI), and outcome measures, and procalcitonin levels were compared among groups. RESULTS: Of 240 patients who received a diagnosis of CAP during the study period, procalcitonin concentrations were measured in 185 patients (77.1%). Levels were higher in patients with high-severity risk classes (PSI classes III-V) [p = 0.01] and in those with complications (p = 0.03) or death (p < 0.0001). Among patients classified into PSI low-severity risk classes (classes I-II), levels tended to be higher in those with bacterial etiology (p = 0.08); in this group, a serum procalcitonin level > or = 0.15 microg/L was more frequently found in patients with bacterial pneumonia than in those with nonbacterial pneumonia (p = 0.03). In patients with higher-severity risk classes, no significant differences were observed in procalcitonin levels among etiologic groups, but higher concentrations were associated with development of complications (p = 0.01) and death (p < 0.0001). CONCLUSIONS: Procalcitonin contribution to the evaluation of CAP varies according to severity. While procalcitonin may have a role to predict the microbial etiology in patients with a low PSI score, in patients classified within high PSI risk classes, it is a prognostic marker rather than a predictor of etiology.

Adult↗

PubMed2006

Weathering and toxicity of marine sediments contaminated with oils and polycyclic aromatic hydrocarbons.

Many sediments are contaminated with mixtures of oil residues and polycyclic aromatic hydrocarbons (PAHs), but little is known about the toxicity of such mixtures to sediment-dwelling organisms and the change in toxicity on weathering. In the present study, we investigated the effects of a seminatural, two-year weathering period on PAH/oil chemistry and toxicity in a marine sediment that had been spiked with three different oils (a gas oil, a lubricating oil, and a crude oil; all tested at five concentrations). Toxicity of bioavailable, pore water-accommodated oil/PAH fractions was quantified using a bacterial (Vibrio fischeri) assay and the in vitro chemical-activated luciferase expression assay (DR-CALUX; using conditions to detect PAHs). Results of chemical analyses pointed to (microbial) degradation of all three oils: Sediment oxygen demand during weathering increased with increasing oil concentration, total oil concentrations decreased to between 17 and 29% of initial levels, and resolved n-alkanes were depleted in weathered oil fractions. Furthermore, a shift in the relative importance of different boiling-point fraction ranges of the oils was observed on weathering. Generally, the lowest fraction range (C10-C16) disappeared, whereas the relative proportion of the highest (C28-C40) fraction range increased considerably. Remarkably, for the gas oil, this fraction shift was dependent on the oil concentration in sediment. Similarly, degradation of PAHs was strongly affected by the sedimentary oil content, indicating that the presence of oil stimulated PAH degradation. This phenomenon applied to both low- and high-molecular-weight PAHs, although the first group (3- and 4-ring PAHs) was degraded most. Results from the V. fischeri and DR-CALUX assay showed that in most cases, pore-water toxicity decreased on weathering. Combining the assay responses with chemical data indicated that the observed toxicity probably was not caused by the analyzed PAHs but, rather, by specific oil constituents instead.

Aliivibrio fischeri↗

PubMed2000

Results of a longitudinal study of the prevalence of Escherichia coli O157:H7 on cow-calf farms.

OBJECTIVE: To describe the frequency and distribution of Escherichia coli O157:H7 in the feces and environment of cow-calf herds housed on pasture. SAMPLE POPULATION: Fecal and water samples for 10 cow-calf farms in Kansas. PROCEDURE: Fecal and water samples were obtained monthly throughout a 1-year period (3,152 fecal samples from 2,058 cattle; 199 water samples). Escherichia coli O157:H7 in fecal and water samples was determined, using microbial culture. RESULTS: Escherichia coli O157:H7 was detected in 40 of 3,152 (1.3%) fecal samples, and 40 of 2,058 (1.9%) cattle had > or = 1 sample with E coli. Fecal shedding by specific cattle was transient; none of the cattle had E coli in more than 1 sample. Significant differences were not detected in overall prevalence among farms. However, significant differences were detected in prevalence among sample collection dates. Escherichia coli O157:H7 was detected in 3 of 199 (1.5%) water samples. CONCLUSIONS AND CLINICAL RELEVANCE: Implementing control strategies for E coli O157:H7 at all levels of the cattle industry will decrease the risk of this organism entering the human food chain. Devising effective on-farm strategies to control E coli O157:H7 in cow-calf herds will require an understanding of the epidemiologic characteristics of this pathogen.

Animal Husbandry↗

PubMed1992

Quantification of lipopolysaccharides in human bile with or without gram-negative bacteria.

Lipopolysaccharide (LPS) was assayed in 78 samples of human common-duct bile, obtained at endoscopic retrograde cholangiography. The LPS was assayed by a chromogenic limulus amebocyte lysate (LAL) test, after dilution of bile samples in heparinized plasma and inactivation of inhibitors. The assay was not influenced by other biliary constituents, as demonstrated by the recovery of standards. Bile pigments did not influence the results. The LAL test was positive in 60 of the samples, 59 of which had a positive culture for gram-negative bacteria or Candida sp. The levels of LPS were significantly correlated to the total number of bacteria (n = 16, R = 0.55, p less than 0.05). The median LPS level was 35,250 ng/l and showed a very large variation (140 ng/l to 27.8 mg/l). In four of the samples gram-negative bacteria were present, but no LPS could be detected. The study demonstrates the presence of LPS in great quantities in human bile and supports the feasibility of using the LAL test on bile samples. The presence of LPS (within the detection limit) appears to be associated with local microbial colonization.

Adult↗

PubMed1993

Screening method for colony-stimulating factor inducers using a human bone marrow stromal cell line, KM-102.

A new screening method for inducers of colony-stimulating factors (CSFs) was established using KM-102, a human bone marrow stromal cell line as the producer. In this method, the assay system which uses CSF dependent cell lines is combined with the CSF production system. Interleukin-1 (IL-1), which is known to upregulate CSF production in many cell populations, was used as a positive control for production of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF). Induction in the positive controls was clearly detected within 24 hours. Activators of protein kinase C (PKC), protein phosphatase inhibitors and lipopolysaccharide (LPS) were positive in this assay system, but muramyl dipeptide (MDP) and Bestatin which are known macrophage activators, were negative. Inducers of CSFs were successfully detected using this assay method. Among 1,600 microbial strains tested, 2 actinomycete strains were found to produce active substances. One strain produces teleocidin-A, a strong activator of PKC, and the other strain produces a mixture of active compounds including three novel compounds. These three compounds do not induce terminal differentiation of HL-60 cells, suggesting that they are not teleocidin-like substances and form a new class of CSF inducers.

Bone Marrow↗

PubMedDate not supplied

A technique to recover tracer as carboxyl-carbon and alpha-nitrogen from amino acids in soil hydrolysates.

Isotope analysis of biochemical compounds provides an unequivocal means for detecting assimilation of tracer C and N into microbial biomass. A diffusion method recently developed to determine amino acid-N by ninhydrin oxidation of soil hydrolysates was modified to permit simultaneous collection of the CO2 liberated during this oxidation. In the technique described herein, this is accomplished after removal of (NH4+ + amino sugar)-N, by performing ninhydrin oxidation at 90 degrees C for 7 h in a 1.9 L Mason jar sealed with a lid equipped to support a petri dish containing 5 mL 0.2N NaOH. Recoveries of carboxyl-C and alpha-N ranged from 98 to 101% in evaluations with standard solutions of amino acids, whereas these recoveries exceeded 90% for 14C and 94% for 15N when soil hydrolysates amended with labeled amino acids were analyzed.

Alanine↗

PubMed2006

[Harmful impact of biological agents released at metalworking].

In metalworking, the temperature of the cutting edge can reach 700 degrees C, which can adversely affect either the surface of the metal processed or the cutting edge itself. To reduce the heat and friction, the metalworking fluids and coolants are used. The most common are the oil-in-water emulsions, which are a mixture of concentrated mineral oils, different additives, and water. The presence of water and organic substrates generates conditions for microbial growth. Bacterial endotoxins can also be frequently detected in metalworking fluids. Rapid rotation of the metalworking tools makes various biological agents to be released as droplet bioaerosols that can be inhaled by the workers, thus producing adverse health effects, mainly on the respiratory system. More than 130 species of bacteria (including pathogens), moulds and yeasts have so far been found in the metalworking fluids and coolants. This paper summarizes the outcomes of projects on the impact of biological agents present in metalworking fluids. Exposure to oil mist, bacteria and endotoxins as well as the variety of health effects resulting from that exposure have been discussed. The findings indicate that the most frequent symptoms reported by the workers include cough with phlegm, wheezing breath, chest tightness and throat irritation. The most common among the diagnosed diseases are chronic allergic bronchitis, asthma, hypersensitivity pneumonitis and acute respiratory dysfunction indicated by decreased FEV1 values. The main skin diseases include oil acne, bacterial infections, dermatomycosis and allergic reactions. The review made the authors conclude that the problem of occupational exposure to biological agents released with coolants during metalworking has not been well recognized in Poland.

Air Microbiology↗

PubMed1978

[Investigations on bloodcultures with three culture media containing Na-polyanetholsulfonate (aerobic iso- and hypertonic and anaerobic isotonic (author's transl)].

By using parallel culture techniques 330 blood specimens steming from patients of an intensive care unit were cultured simultanously in 3 media containing 0.05% Na-Poly-anetholsulfonate. Otherwise having an identical composition the media differed in the following respects : the isotonic medium A and the - by additional content of 10% sucrose - hypertonic medium B were used for aerobic culture. The anaerobic and isotonic medium C contained 2% Proteose-peptone, additionally. Without regards to the isolation of strictly anaerobic bacteria a total of 114 positive bloodcultures was obtained (tab. 1). The use of medium A resulted in 81 positive bloodcultures, that of medium B and C in 93 and 50, respectively (tab. 2). With the combined use of media A + B 103 positive bloodcultures would have been detected, with B + C 102, but with A + C 92 only (tab. 2). Of all 3 media the hypertonic medium B promotes bacterial growth most efficiently, but 13 strains grown in A did not grow in B (tab. 3), a fact that leads to the conclusion that isotonic as well as hypertonic aerobic media are employed in parallel advantageously. The effect of the hypertonic medium was expressed most distinctly with the isolation of grampositive bacteria (tab. 4). As during the period of this investigation the intensive care unit was afflicted by an outbreak of Serratia marcescens the predominance of this species among the other isolated microbes (tab. 5) is not surprising. While no difference of detection-time was observed between A and B, microbial growth in medium C seemed to be slower (fig). It was concluded that the use of 3 media, aerobic isotonic and hypertonic as well as anaerobic isotonic should be employed whenever possible.

Anaerobiosis↗

PubMedDate not supplied

[The development of a Proteus mirabilis macrocolony on a solid nutrient medium].

As revealed in this study, the macrocolony of Proteus mirabilis, formed on solid culture medium, may consist both of the main part and of sporadically appearing dissociating subcolonies, considerably differing one from another. The outer edge of the main part of the macrocolony of swarming cells is represented by bacteria located in three perpendicular directions. The next intermediate area consisting of two layers is represented by bacteria oriented, as a rule, in one direction. The center of the colony is made up of short microbial cells. Between the upper layer and the surface of agar an original subpopulation of microbial cells, forming a separate layer, has been detected; together they determine the planar sandwich-like architectonics of the macrocolony.

Culture Media↗

PubMed1977

Microbial flora of the vagina and cervix.

The microbial flora of the vagina and cervix was assessed qualitatively and semiquantitatively in 40 women attending an intrauterine contraceptive device clinic. Both sites harboured many types of macroorganism, the mean number of microbial types isolated being five from the vagina and four from the cervix. Typical lactobacilli were detected in 61% of vaginal and in 53% of cervical specimens; faecal bacteria, including anaerobes, were even more frequently found at both sites. No differences in the microbial populations at either the vagina or the cervix were detected after fitting of the devices, in the different weeks of the menstrual cycle, or with various previously used contraceptive methods.

Adult↗