Detection of Pseudomonas aeruginosa in sputum samples by modified fluorescent in situ hybridization

Pseudomonas aeruginosa is the most common and dominant infectious agent that causes chronic pneumonia in patients with cystic fibrosis (CF). Fluorescent in situ hybridization (FISH) is a powerfulmolecular method for the specific and rapid diagnosis of bacteria, including the detection of P. aeruginosa in sputum samples from CF patients. High background fluorescence of viscous sputumsamples obtained from CF patients may impede detection of microorganisms by FISH. The aim of this study was to test the application of biotin during FISH technique to reduce unspecific background fluorescence in sputum samples to facilitate and improve detection of P. aeruginosa. Sixty-three sputum samples from CF patients were tested by FISH to detect P. aeruginosa. All the 63 samples were also examined by a modified FISH procedure including biotin treatment. The FISH results were compared with those of conventional culture method. The specificity of FISH was 100%. The sensitivity of FISH for detection of P. aeruginosa from samples without biotin treatment was 83.3%, whereas in biotin-treated samples was 88.1%. Biotin reduced background fluorescence of 12 sputum samples of CF patients and it did not show any adverse effect on FISH results of the remaining sputum samples. Therefore, using of biotin in FISH procedure seems to facilitate and improve the detection of respiratorytract infections by P. aeruginosa in this population

Distribution, expression and long range mapping of legiolysin gene (lly) specific DNA sequences in Legionellae

The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal Uy-specific DNA probe was used in Southern hybridizations for the detection of Uy-specific DNA in the genomes of legioneUae and other gram-negative pathogenic bacteria. Under conditi9ns of high stringency, tlie Uy DNA probe specifically reacted with DNA fragments fr9m L. pneumophiüz isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bact~ria of other genera. The Uy genewas mapped by pulsed-field gel electrophoresis to the respective genomic Notl fragments of Legionelltz isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates

Seasonal variation of Pneumocystis jirovecii infection: analysis of underlying climatic factors

AbstractPneumocystis jirovecii causes severe pneumonia (PCP) in immunocompromised patients. Seasonal changes of PCP incidence may be associated with climate changes. In this first study using multiple linear regression statistics to assess monthly climatic data and Pneumocystis, PCP incidence was positively correlated with mean temperature, but not with rainfall or wind strength

Distributed natural gas venting offshore along the Cascadia margin

Widespread gas venting along the Cascadia margin is investigated from acoustic water column data and reveals a nonuniform regional distribution of over 1100 mapped acoustic flares. The highest number of flares occurs on the shelf, and the highest flare density is seen around the nutrition-rich outflow of the Juan de Fuca Strait. We determine similar to 430 flow-rates at similar to 340 individual flare locations along the margin with instantaneous in situ values ranging from similar to 6 mL min(-1) to similar to 18 L min(-1). Applying a tidal-modulation model, a depth-dependent methane density, and extrapolating these results across the margin using two normalization techniques yields a combined average in situ flow-rate of similar to 88 x 10(6) kg y(-1). The average methane flux-rate for the Cascadia margin is thus estimated to similar to 0.9 g y(-1) m(-2). Combined uncertainties result in a range of these values between 4.5 and 1800% of the estimated mean values

Intrinsic and Extrinsic Regulation of Type III Secretion Gene Expression in Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that is particularly problematic in the healthcare setting where it is a frequent cause of pneumonia, bloodstream, and urinary tract infections. An important determinant of P. aeruginosa virulence is a type III secretion system (T3SS). T3SS-dependent intoxication is a complex process that minimally requires binding of P. aeruginosa to host cells, injection of the cytotoxic effector proteins through the host cell plasma membrane, and induction of T3SS gene expression. The latter process, referred to as contact-dependent expression, involves a well-characterized regulatory cascade that activates T3SS gene expression in response to host cell contact. Although host cell contact is a primary activating signal for T3SS gene expression, the involvement of multiple membrane-bound regulatory systems indicates that additional environmental signals also play a role in controlling expression of the T3SS. These regulatory systems coordinate T3SS gene expression with many other cellular activities including motility, mucoidy, polysaccharide production, and biofilm formation. The signals to which the organism responds are poorly understood but many seem to be coupled to the metabolic state of the cell and integrated within a master circuit that assimilates informational signals from endogenous and exogenous sources. Herein we review progress toward unraveling this complex circuitry, provide analysis of the current knowledge gaps, and highlight potential areas for future studies. Complete understanding of the regulatory networks that control T3SS gene expression will maximize opportunities for the development of strategies to treat P. aeruginosa infections

Fish and habitat community assessments on North Carolina shipwrecks: potential sites for detecting climate change in the graveyard of the Atlantic

The Monitor National Marine Sanctuary (MNMS) was the nation’s first sanctuary, originally established in 1975 to protect the famous civil war ironclad shipwreck, the USS Monitor. Since 2008, sanctuary sponsored archeological research has branched out to include historically significant U-boats and World War II shipwrecks within the larger Graveyard of the Atlantic off the coast of North Carolina. These shipwrecks are not only important for their cultural value, but also as habitat for a wide diversity of fishes, invertebrates and algal species. Additionally, due to their unique location within an important area for biological productivity, the sanctuary and other culturally valuable shipwrecks within the Graveyard of the Atlantic are potential sites for examining community change. For this reason, from June 8-30, 2010, biological and ecological investigations were conducted at four World War II shipwrecks (Keshena, City of Atlanta, Dixie Arrow, EM Clark), as part of the MNMS 2010 Battle of the Atlantic (BOTA) research project. At each shipwreck site, fish community surveys were conducted and benthic photo-quadrats were collected to characterize the mobile conspicuous fish, smaller prey fish, and sessile invertebrate and algal communities. In addition, temperature sensors were placed at all four shipwrecks previously mentioned, as well as an additional shipwreck, the Manuela. The data, which establishes a baseline condition to use in future assessments, suggest strong differences in both the fish and benthic communities among the surveyed shipwrecks based on the oceanographic zone (depth). In order to establish these shipwrecks as sites for detecting community change it is suggested that a subset of locations across the shelf be selected and repeatedly sampled over time. In order to reduce variability within sites for both the benthic and fish communities, a significant number of surveys should be conducted at each location. This sampling strategy will account for the natural differences in community structure that exist across the shelf due to the oceanographic regime, and allow robust statistical analyses of community differences over time

Ocean Observatories as a Tool to Advance Gas Hydrate Research

Since 2009, unprecedented comprehensive long-term gas hydrate observations have become available from Ocean Networks Canada's NEPTUNE cabled ocean observatory at the northern Cascadia margin. Several experiments demonstrate the scientific importance of permanent power and Internet connectivity to the ocean floor as they have advanced the field of gas hydrate related research. One example is the cabled crawler Wally at Barkley Canyon, enabling live in situ exploration of the hydrate mounds and its associated benthic communities through the crawler's mobility and permanent accessibility throughout the year. Another example is a bubble-imaging sonar at Clayoquot Slope, revealing the strong relationship between ebullition of natural gas and tidal pressure, without apparent correlation to earthquakes, storms, or temperature fluctuations, in year-long continuous recordings. Finally, regular observatory maintenance cruises allow additional science sampling including echo-sounder surveys to extend the observatory footprint. Long-term trends in the data are not yet apparent but can also become evident from continuous measurements, as ocean observatories such as NEPTUNE are built for a 25-year lifetime, and expansion of the observatory networks makes these findings comparable and testable. \ud Plain Language Summary Natural gas near the ocean floor creates a rapidly changing environment where it is important to collect data continuously in order to determine the magnitude, speed, and potential mechanism of change. This long-standing challenge of year-round access to the deep ocean has been tackled by Ocean Networks Canada through cabling the northern Cascadia seafloor, providing power and Internet communication-ideal for power-hungry instruments, large data volumes, and real-time access. The presence of gas influences the shape of the seafloor, animal activity, and potential escape of methane, a potent greenhouse gas. A seafloor crawler Wally was operated around deep canyon mounds of gas hydrate (a solid gas-water composite) since 2009 and helped discover environmental changes influencing sea life. Further along the continental slope, an acoustic sonar monitored rising methane bubbles where the bubbling appears to be controlled neither by earthquakes, winter storms, nor subtle temperature changes but actually strongly by tidal pressure. Regular maintenance of the observatory by ship allows more data to be collected near the cabled seafloor sites, extending the observations to a larger area. Ocean observatories are built to last decades and therefore more data for more research can be collected, potentially detecting relatively slow processes as well

Haem-iron plays a key role in the regulation of the Ess/Type VII secretion system of <i>Staphylococcus aureus</i> RN6390

This study was supported by the Wellcome Trust (through Investigator Award 10183/Z/15/Z to T. P. and through Clinical PhD studentship support to C. P. H. through grant 104241/z/14/z), the Biotechnology and Biological Sciences Research Council and the Medical Research Council (through grants BB/H007571/1 and MR/M011224/1, respectively).The Staphylococcus aureus type VII protein secretion system (T7SS) plays important roles in virulence and intra-species competition. Here we show that the T7SS in strain RN6390 is activated by supplementing the growth medium with haemoglobin, and its cofactor haemin (haem B). Transcript analysis and secretion assays suggest that activation by haemin occurs at a transcriptional and a post-translational level. Loss of T7 secretion activity by deletion of essC results in upregulation of genes required for iron acquisition. Taken together these findings suggest that the T7SS plays a role in iron homeostasis in at least some S. aureus strains.Publisher PDFPeer reviewe

The Importance of Marine Research Infrastructures in Capturing Processes and Impacts of Extreme Events

Extreme events have long been underestimated in the extent to which they shape the surface of our planet, our environment, its ecological integrity, and the sustainability of human society. Extreme events are by definition rarely observed, of significant impact and, as a result of their spatiotemporal range, not always easily predicted. Extremes may be short-term catastrophic events such as tsunamis, or long-term evolving events such as those linked to climate change; both modify the environment, producing irreversible changes or regime shifts. Whatever the driver that triggers the extreme event, the damages are often due to a combination of several processes and their impacts can affect large areas with secondary events (domino effect), whose effects in turn may persist well beyond the duration of the trigger event itself. Early studies of extreme events were limited to opportunistic approaches: observations were made within the context of naturally occurring events with high societal impact. Given that climate change is now moving us out of a relatively static climate regime during the development of human civilization, extreme events are now a function of underlying climate shifts overlain by catastrophic processes. Their impacts are often due to synergistic factors, all relevant in understanding process dynamics; therefore, an integrated methodology has become essential to enhance the reliability of new assessments and to develop strategies to mitigate societal impacts. Here we summarize the current state of extreme event monitoring in the marine system, highlighting the advantages of a multidisciplinary approach using Research Infrastructures for providing the temporal and spatial resolution required to monitor Earth processes and enhance assessment of associated impacts.publishedVersio