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PubMed1998

Sulfate-reducing bacteria and their activities in cyanobacterial mats of solar lake (Sinai, Egypt).

The sulfate-reducing bacteria within the surface layer of the hypersaline cyanobacterial mat of Solar Lake (Sinai, Egypt) were investigated with combined microbiological, molecular, and biogeochemical approaches. The diurnally oxic surface layer contained between 10(6) and 10(7) cultivable sulfate-reducing bacteria ml-1 and showed sulfate reduction rates between 1,000 and 2, 200 nmol ml-1 day-1, both in the same range as and sometimes higher than those in anaerobic deeper mat layers. In the oxic surface layer and in the mat layers below, filamentous sulfate-reducing Desulfonema bacteria were found in variable densities of 10(4) to 10(6) cells ml-1. A Desulfonema-related, diurnally migrating bacterium was detected with PCR and denaturing gradient gel electrophoresis within and below the oxic surface layer. Facultative aerobic respiration, filamentous morphology, motility, diurnal migration, and aggregate formation were the most conspicuous adaptations of Solar Lake sulfate-reducing bacteria to the mat matrix and to diurnal oxygen stress. A comparison of sulfate reduction rates within the mat and previously published photosynthesis rates showed that CO2 from sulfate reduction in the upper 5 mm accounted for 7 to 8% of the total photosynthetic CO2 demand of the mat.

Bacteria↗

PubMed2001

Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish.

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10(2) to 10(8) CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.

Animals↗

PubMed2002

Occurrence and phenotypic characterization of Yersinia ruckeri strains with biofilm-forming capacity in a rainbow trout farm.

The presence of Yersinia ruckeri in a French fish farm was investigated. Y. ruckeri was isolated mainly from algae and sediment samples rather than from water. Twenty-two Y. ruckeri isolates were obtained, and three strains were distinguished by enterobacterial repetitive intergenic consensus PCR amplification. These strains were able to adhere to solid supports. This characteristic was correlated with flagellum-mediated motility. Killing experiments showed that sessile cells were more resistant to oxolinic acid than their planktonic counterparts. Our results demonstrate that surface colonization of fish farm tanks by Y. ruckeri biofilms is a potential source of recurrent infection for extended periods of time.

Amino Acid Sequence↗

PubMed2002

Cyclic AMP and acyl homoserine lactones increase the cultivation efficiency of heterotrophic bacteria from the central Baltic Sea.

The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated. Numbers of cultivated cells were determined by the most-probable-number (MPN) technique. Artificial brackish water supplemented with different carbon substrates at low concentrations (200 microM each) was employed as the growth medium. Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media). A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP), N-butyryl homoserine lactone, or N-oxohexanoyl-DL-homoserine lactone at a low concentration of 10 microM. The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts. From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP. Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community. This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities. Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP.

4-Butyrolactone↗

PubMed2003

Differential growth response of colony-forming alpha- and gamma-proteobacteria in dilution culture and nutrient addition experiments from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat.

Even though it is widely accepted that bacterioplankton growth in lakes and marine ecosystems is determined by the trophic status of the systems, knowledge of the relationship between nutrient concentrations and growth of particular bacterial species is almost nonexistent. To address this question, we performed a series of culture experiments with water from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat (northern Red Sea). In the initial water samples, the proportion of CFU was typically <0.002% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. During incubation until the early stationary phase, the proportion of CFU increased to 20% of the DAPI counts and to 2 to 15% of the DAPI counts in unenriched lake water and seawater dilution cultures, respectively. Sequencing of the 16S ribosomal DNA of colony-forming bacteria in these cultures consistently revealed an abundance of alpha-proteobacteria, but notable phylogenetic differences were found at the genus level. Marine dilution cultures were dominated by bacteria in the Roseobacter clade, while lake dilution cultures were dominated by bacteria affiliated with the genera Sphingomonas and CAULOBACTER: In nutrient (glucose, ammonium, phosphate) addition experiments the CFU comprised 20 to 83% of the newly grown cells. In these incubation experiments fast-growing gamma-proteobacteria dominated; in the marine experiments primarily different Vibrio and Alteromonas species appeared, while in the lake water experiments species of the genera Shewanella, Aeromonas, and Rheinheimera grew. These results suggest that major, but different, gamma-proteobacterial genera in both freshwater and marine environments have a preference for elevated concentrations of nutrients and easily assimilated organic carbon sources but are selectively outcompeted by alpha-proteobacteria in the presence of low nutrient concentrations.

Alphaproteobacteria↗

PubMed2003

Distribution of microorganisms in the subsurface of the manus basin hydrothermal vent field in Papua New Guinea.

The distribution of microorganisms in the subsurfaces of hydrothermal vents was investigated by using subvent rock core samples. Microbial cells and ATP were detected from cores taken at depths of less than 99.4 and 44.8 m below the seafloor (mbsf), respectively. Cores from various depths were incubated anaerobically with a heterotrophic medium. Growth at 60 and 90 degrees C was ascribed to a Geobacillus sp. in the 448.6- to 99.4-mbsf cores and a Deinococcus sp. in the 64.8- to 128.9-mbsf cores, respectively, based on the 16S ribosomal DNA analysis.

Anaerobiosis↗

PubMed2005

PldB, a putative phospholipase D homologue in Dictyostelium discoideum mediates quorum sensing during development.

Quorum sensing, also known as cell-density sensing in the unicellular eukaryote Dictyostelium discoideum, is required for efficient entry into the differentiation and development segment of its life cycle. Quorum sensing is accomplished by simultaneously secreting and sensing the glycoprotein Conditioned Medium Factor, or CMF. When the density of starving cells is high, CMF levels are high, which leads to aggregation followed by development. Here, we describe the role of pldB, a gene coding for a putative phospholipase D (PLD) homologue, in quorum sensing. We find that in submerged culture, adding butanol, an inhibitor of PLD-catalyzed phosphatidic acid production, allows cells to bypass the requirement for CMF mediated quorum sensing and aggregate at low cell density. Deletion of pldB mimics the presence of butanol, allowing cells to aggregate at low cell density. pldB- cells also initiate and finish aggregation rapidly. Analysis of early developmental gene expression in pldB- cells reveals that the cyclic AMP receptor cAR1 is expressed at higher levels earlier than in wild-type cells, which could explain the rapid aggregation phenotype. As would be predicted, cells overexpressing pldB are unable to aggregate even at high cell density. Adding CMF to these pldB- overexpressing cells does not rescue aggregation. Both of these phenotypes are cell autonomous, as mixing a small number of pldB- cells with wild-type cells does not cause the wild-type cells to behave like pldB- cells.

Amino Acid Sequence↗

PubMed2006

Protective effect of the Nramp1 BB genotype against Brucella abortus in the water buffalo (Bubalus bubalis).

We tested 413 water buffalo cows (142 cases and 271 controls) for the presence of anti-Brucella abortus antibodies (by the skin test, the agglutination test, and the complement fixation test) and the Nramp1 genotype (by capillary electrophoresis). Four alleles (Nramp1A, -B, -C, and -D) were detected in the 3' untranslated region of the Nramp1 gene. The BB genotype was represented among only controls, providing evidence that this genotype confers resistance to Brucella abortus. The monocytes from the BB (resistant) subjects displayed a higher basal level of Nramp1 mRNA and a lower number of viable intracellular bacteria than did the monocytes from AA (susceptible) subjects. The higher basal level of the antibacterial protein Nramp1 most probably provides the BB animals with the possibility of controlling bacteria immediately after their entry inside the cell.

3' Untranslated Regions↗

PubMed2000

In vivo-induced genes in Pseudomonas aeruginosa.

In vivo expression technology was used for testing Pseudomonas aeruginosa in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight ivi proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called pvdI) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2).

Animals↗

PubMed1994

Genetic diversity of human immunodeficiency virus type 2: evidence for distinct sequence subtypes with differences in virus biology.

The virulence properties of human immunodeficiency virus type 2 (HIV-2) are known to vary significantly and to range from relative attenuation in certain individuals to high-level pathogenicity in others. These differences in clinical manifestations may, at least in part, be determined by genetic differences among infecting virus strains. Evaluation of the full spectrum of HIV-2 genetic diversity is thus a necessary first step towards understanding its molecular epidemiology, natural history of infection, and biological diversity. In this study, we have used nested PCR techniques to amplify viral sequences from the DNA of uncultured peripheral blood mononuclear cells from 12 patients with HIV-2 seroreactivity. Sequence analysis of four nonoverlapping genomic regions allowed a comprehensive analysis of HIV-2 phylogeny. The results revealed (i) the existence of five distinct and roughly equidistant evolutionary lineages of HIV-2 which, by analogy with HIV-1, have been termed sequence subtypes A to E; (ii) evidence for a mosaic HIV-2 genome, indicating that coinfection with genetically divergent strains and recombination can occur in HIV-2-infected individuals; and (iii) evidence supporting the conclusion that some of the HIV-2 subtypes may have arisen from independent introductions of genetically diverse sooty mangabey viruses into the human population. Importantly, only a subset of HIV-2 strains replicated in culture: all subtype A viruses grew to high titers, but attempts to isolate representatives of subtypes C, D, and E, as well as the majority of subtype B viruses, remained unsuccessful. Infection with all five viral subtypes was detectable by commercially available serological (Western immunoblot) assays, despite intersubtype sequence differences of up to 25% in the gag, pol, and env regions. These results indicate that the genetic and biological diversity of HIV-2 is far greater than previously appreciated and suggest that there may be subtype-specific differences in virus biology. Systematic natural history studies are needed to determine whether this heterogeneity has clinical relevance and whether the various HIV-2 subtypes differ in their in vivo pathogenicity.

Acquired Immunodeficiency Syndrome↗

PubMedDate not supplied

Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells.

Cyclic GMP-dependent protein kinase (cGMP kinase) is the major receptor protein for cGMP in vascular smooth muscle. Vascular smooth muscle cells (VSMC) isolated from the rat aorta express type I cGMP kinase at high levels, but expression decreases markedly upon passage of the cells. In primary or early passage, the expression of cGMP kinase is lowest when cells are plated at low density as assessed by immunological and Northern analyses. Expression increases at confluence and is maintained in postconfluent cultures. With repeated passaging, however, the levels of cGMP kinase decrease even in confluent and postconfluent cultures so that after several passages enzyme levels are undetectable. The decrease in expression in passaged cells is not due to exposure to serum-derived growth factors, but rather on the repeated exposure of cells to conditions in which cell density is reduced (i.e., subculturing). These results indicate that aortic VSMC grown at low density or those repetitively passaged have reduced expression of cGMP kinase, and thus may not represent appropriate cultures with which to investigate the role of nitric oxide and cGMP in VSMC function.

Animals↗

PubMed2004

Purification, characterization, and sequencing of novel antimicrobial peptides, Tu-AMP 1 and Tu-AMP 2, from bulbs of tulip (Tulipa gesneriana L.).

Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic bacteria and fungi were 2 to 20 microg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.

Amino Acid Sequence↗

PubMed2003

Metschnikowia bicuspidata and Enterococcus faecium co-infection in the giant freshwater prawn Macrobrachium rosenbergii.

In May 2001, an epizootic yeast and bacterial co-infection in the giant freshwater prawn Macrobrachium rosenbergii occurred in Taiwan causing a cumulative mortality of 25%. The diseased prawns had a yellowish-brown body color, milky hemolymph, opaque, whitish muscles, and were approximately 7 mo old with total lengths ranging from 8 to 10 cm. Histopathological examination showed marked edema, yeast infiltration, and necrotic lesions with inflammation in the muscles, hepatopancreas and other internal organs. We isolated 2 pathogens from the diseased prawns, one was a yeast (AOD081MB) and the other a gram-positive coccus (AOD081EF). The gram-positive coccus was identified as Enterococcus faecium by the API 20 Strepsystem, conventional biochemical tests, and it had 99% 16S rDNA sequence identity (GenBank Accession Number AJ276355) to E. faecium (GenBank Accession Number AF529204). The sequence of a PCR product from the D1/D2 domain of 26S rDNA (GenBank Accession Number AF529297) from the yeast gave 99% sequence identity to Metschnikowia bicuspidata (GenBank Accession Number U44822). Experimental infections with these isolates produced gross signs and histopathological changes similar to those observed in the naturally infected prawns. The lethal doses (LD50) for isolate E. faecium AOD081EF, M. bicuspidata AOD081MB and the co-infection were 4.7 x 10(4), 2.6 x 10(2), and 2.4 x 10(2) colony-forming units prawn(-1), respectively. This is the first report of a confirmed co-infection of M. bicuspidata and E. faecium in prawn aquaculture.

Animals↗

PubMed2005

Prevention of experimental autoimmune encephalomyelitis by transfer of embryonic stem cell-derived dendritic cells expressing myelin oligodendrocyte glycoprotein peptide along with TRAIL or programmed death-1 ligand.

Experimental autoimmune encephalomyelitis (EAE) is caused by activation of myelin Ag-reactive CD4+ T cells. In the current study, we tested a strategy to prevent EAE by pretreatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TRAIL or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and an MHC class II-associated invariant chain-based MOG epitope-presenting vector. Subsequently, double-transfectant ES cell clones were induced to differentiate to ES-DC, which expressed the products of introduced genes. Treatment of mice with either of the double-transfectant ES-DC significantly reduced T cell response to MOG, cell infiltration into spinal cord, and the severity of MOG peptide-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC-expressing TRAIL or PD-L1 had no effect in reducing the disease severity. In contrast, immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. The double-transfectant ES-DC presenting Ag and simultaneously expressing immune-suppressive molecules may well prove to be an effective therapy for autoimmune diseases without inhibition of the immune response to irrelevant Ag.

Adoptive Transfer↗

PubMed2003

A PCR method based on 16S rRNA sequence for simultaneous detection of the genus Listeria and the species Listeria monocytogenes in food products.

The genus Listeria comprises six closely related species, of which only Listeria monocytogenes is a human pathogen. The rapid and sensitive detection of L. monocytogenes is important in the food industry as well as in medical diagnosis. In this study, a PCR-based method for the rapid, specific, and sensitive detection of L. monocytogenes in food products was developed. The PCR is based on DNA sequences and primer pairs that are found within the 16S subunit of the rRNA gene and are specific to the Listeria genus and to L. monocytogenes within the Listeria genus. The primers for the Listeria genus and for L. monocytogenes were used in the same reaction mix for their simultaneous detection. In addition, a pair of bacterial primers universal to any bacterial DNA at the 16S subunit of the rRNA gene were developed as a positive control. For the detection of Listeria and L. monocytogenes in food products, the method includes selective enrichment for Listeria followed by DNA extraction and a specific PCR reaction. The method detects 1 to 5 CFU in a 25-g sample in < or = 24 h. It can be easily incorporated into the routine screening of diverse food products and readily adapted for clinical use.

Base Sequence↗

PubMed2005

Identification of factors involved in recovery of heat-injured Salmonella Enteritidis.

Proteins and genes involved in the recovery of heat-injured Salmonella Enteritidis were investigated. Salmonella Enteritidis cells cultured overnight in tryptic soy broth (TSB; nonselective medium) were suspended in citric acid-disodium hydrogen phosphate buffer (pH 6). After heat treatment at 55 degrees C for 15 min, the culturable counts measured by tryptic soy agar (TSA; nonselective medium) decreased from 10(8) to 10(7) CFU/ml. On the other hand, culturable counts measured by desoxycholate-hydrogen sulfite-lactose (DHL) agar (selective medium) were decreased from 10(8) to 10(4) CFU/ml by the same treatment. The results suggest that 99.9% of Salmonella Enteritidis detected on TSA were injured but recoverable. When injured Salmonella Enteritidis was incubated in TSB, the culturable count measured by TSA did not increase for 2 h, whereas that by DHL agar increased after incubation for 30 min. After incubation for 2 h, the culturable count measured by DHL agar reached a similar level with that by TSA, indicating that Salmonella Enteritidis had recovered. The two-dimensional polyacrylamide gel electrophoresis analysis revealed that elongation factor G (FusA) and pyruvate kinase (PykF) specifically increased in the cells just after heat treatment and in the recovery cells. The levels of transcription of 86 stress-inducible genes were also investigated by reverse transcription PCR. Nineteen heat-inducible (clpB, clpX, degP, dnaJ, fkpA, ftsJ, gapA, hflB, hslJ, hslU, hslV, htpG, htrA, lon, mopA, mopB, mreB, rpoE, and ppiD), and 12 oxidative-stress and DNA damage-inducible (ahpC, ahpF, fldB, fur, grxA, dinF, katG, mutM, recA, soxR, trxC, and zwf) genes were transcribed extensively during recovery in TSB. The results obtained in this study will be used to develop the media or culture conditions that will promote recovery for the detection of food poisoning bacteria, including injured cells from food products.

Animals↗

PubMed2006

Infectious keratoconjunctivitis in bighorn sheep, Silver Bell Mountains, Arizona, USA.

An infectious keratoconjunctivitis (IKC) epizootic in bighorn sheep (Ovis canadensis) occurred in the Silver Bell Mountains, Arizona, USA, from 1 December 2003 to 31 March 2004. We used standard culture methods and polymerase chain reaction (PCR) amplification of the 16S rRNA gene to test for the causative agents of IKC and other diseases reported to be associated with bighorn sheep populations. All bighorn sheep and domestic goat test results were negative except for Mycoplasma spp. and Branhamella spp. The culture and PCR results differed. Conjunctival swabs from four of 19 IKC-affected bighorn sheep tested by culture were positive for Mycoplasma spp., whereas 22 of 22 bighorn sheep samples tested by PCR were positive for Mycoplasma spp. None of 13 domestic goats tested positive by culture for Mycoplasma spp., whereas five of 16 tested positive by PCR. Three of 16 domestic goats and seven of 24 IKC-affected bighorn sheep tested positive for Branhamella spp. by culture. Bighorn sheep began showing clinical signs of IKC between 21 and 28 days following initial detection of domestic goats in bighorn sheep habitat. The IKC epizootic lasted 122 days, and individual bighorn sheep were blind for an average of 38.4 days. Given the clear potential for disease transmission to bighorn sheep, we recommend that land managers not allow the pasturing of domestic goats near occupied bighorn sheep habitat.

Animals↗

PubMed1999

Detection of GPI-anchored protein-deficient cells in patients with aplastic anaemia and evidence for clonal expansion during the clinical course.

The incidence of patients with aplastic anaemia (AA) who show a deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins on their peripheral blood (PB) or bone marrow (BM) cells is higher than that previously reported. We analysed the expression of CD55 or CD59 on PB and BM cells and those of colonies and bursts formed in cultures with marrow cells from AA patients. 4/21 (19%) AA cases later developed paroxysmal nocturnal haemoglobinuria (PNH). 7/17 (41. 2%) AA cases showed a subpopulation without GPI-anchored proteins. The defect characteristic for PNH was observed only on the colonies/bursts but not on the PB or BM cells in three cases. We found the affected colonies/bursts in cultures using thawed marrow cells which had been cryopreserved at the diagnosis of aplasia in one patient who developed PNH 3 years later. Two different mutations of the PIG-A gene were found in the colonies/bursts at the time of AA. The nucleotide sequences were identical between the colonies/bursts at the time of AA and those after the transition to PNH. However, one of the mutations was detected only at the haemopoietic progenitor level but not in PB granulocytes. There may be latent subclones with PNH abnormalities which could expand to the level of clinical detection, or which do not progress and remain indolent for a relatively long time, implying the different extents of clonal expansion among the affected progenitors.

Adolescent↗