N. F. Azevedo

Using wavelets to decompose the time–frequency effects of monetary policy ☆

Central banks have different objectives in the short and long run. Governments operate simultaneously at different timescales. Many economic processes are the result of the actions of several agents, who have different term objectives. Therefore, a macroeconomic time series is a combination of components operating on different frequencies. Several questions about economic time series are connected to the understanding of the behavior of key variables at different frequencies over time, but this type of information is difficult to uncover using pure time-domain or pure frequency-domain methods.

Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH).

Background Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data. Methodology/Principal Findings We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm2) for 48 h biofilm: E. coli 2,1×108 (±2,4×107); L. monocytogenes 6,8×107 (±9,4×106); and S. enterica 1,4×106 (±4,1×105)]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. Significance While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.

Detection of Escherichia coli in Biofilms from Pipe Samples and Coupons in Drinking Water Distribution Networks

ABSTRACT Fluorescence in situ hybridization (FISH) was used for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal, and Latvia, and E. coli was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, polyvinyl chloride, or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after 1 to 6 months exposure to the drinking water. In order to increase the signal intensity, a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of E. coli on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of β-d-glucuronidase) methods. An additional verification was made by using PCR. Culture method indicated presence of E. coli in one of five pipes, whereas all pipes were positive with the FISH methods. E. coli was detected in 56% of the coupons using PNA FISH, but no E. coli was detected using culture or enzymatic methods. PCR analyses confirmed the presence of E. coli in samples that were negative according to culture-based and enzymatic methods. The viability of E. coli cells in the samples was demonstrated by the cell elongation after resuscitation in low-nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but nonculturable state, unable to grow on agar media. E. coli contributed to ca. 0.001 to 0.1% of the total bacterial number in the samples. The presence and number of E. coli did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g., temperature, chlorine, or biodegradable organic matter concentration). We show here that E. coli is present in the biofilms of drinking water networks in Europe. Some of the cells are metabolically active but are often not detected due to limitations of traditionally used culture-based methods, indicating that biofilm should be considered as a reservoir that must be investigated further in order to evaluate the risk for human health.

Coccoid form of Helicobacter pylori as a morphological manifestation of cell adaptation to the environment

ABSTRACT After characterization of preferred conditions for Helicobacter pylori survival in the sessile state, it was observed that the bacterium transforms from spiral to coccoid under mild circumstances, whereas under extreme ones it is unable to undergo shape modification. This strongly supports the view that transformation into the coccoid form is an active, biologically led process, switched on by the bacterium as a protection mechanism.

Persistence of Helicobacter pylori in heterotrophic drinking-water biofilms.

ABSTRACT Although the route of transmission of Helicobacter pylori remains unknown, drinking water has been considered a possible transmission vector. It has been shown previously that, in water, biofilms are a protective niche for several pathogens, protecting them from stressful conditions, such as low carbon concentration, shear stress, and less-than-optimal temperatures. In this work, the influence of these three parameters on the persistence and cultivability of H. pylori in drinking-water biofilms was studied. Autochthonous biofilm consortia were formed in a two-stage chemostat system and then inoculated with the pathogen. Total numbers of H. pylori cells were determined by microscopy using a specific H. pylori 16S rRNA peptide nucleic acid probe, whereas cultivable cells were assessed by standard plating onto selective H. pylori medium. Cultivable H. pylori could not be detected at any time point, but the ability of H. pylori cells to incorporate, undergo morphological transformations, persist, and even agglomerate in biofilms for at least 31 days without a noticeable decrease in the total cell number (on average, the concentration was between 1.54 × 106 and 2.25 × 106 cells cm−2) or in the intracellular rRNA content may indicate that the loss of cultivability was due to entry into a viable but noncultivable state. Unlike previous results obtained for pure-culture H. pylori biofilms, shear stress did not negatively influence the numbers of H. pylori cells attached, suggesting that the autochthonous aquatic bacteria have an important role in retaining this pathogen in the sessile state, possibly by providing suitable microaerophilic environments or linking biomolecules to which the pathogen adheres. Therefore, biofilms appear to provide not only a safe haven for H. pylori but also a concentration mechanism so that subsequent sloughing releases a concentrated bolus of cells that might be infectious and that could escape routine grab sample microbiological analyses and be a cause of concern for public health.

The epidemiology of Helicobacter pylori and public health implications.

This article presents a review of the literature on the epidemiology and public health implications of Helicobacter pylori infection published from April 2008 through to March 2009. The authors used MeSH terms “Helicobacter infections epidemiology,”“Helicobacter infections prevention and control” to search multiple databases (PubMed, Embase, Cochrane, Cochrane Library, EBMR, BIOSIS), and independently searched PubMed using the term “Helicobacter” with “Epidemiology,”“Transmission,”“Prevalence” or “Environment.” Articles without topical relevance were excluded. Two additional papers known to the authors were added. The identified literature is summarized by subtopic: reviews; prevalence; incidence; transmission; risk factors; and public health policy.

DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA). In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD) and recognition of individual genes (RING) FISH.

Critical review on biofilm methods

Abstract Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

Shear Stress, Temperature, and Inoculation Concentration Influence the Adhesion of Water-Stressed Helicobacter pylori to Stainless Steel 304 and Polypropylene

ABSTRACT Although molecular techniques have identified Helicobacter pylori in drinking water-associated biofilms, there is a lack of studies reporting what factors affect the attachment of the bacterium to plumbing materials. Therefore, the adhesion of H. pylori suspended in distilled water to stainless steel 304 (SS304) coupons placed on tissue culture plates subjected to different environmental conditions was monitored. The extent of adhesion was evaluated for different water exposure times, using epifluorescence microscopy to count total cell numbers. High shear stresses—estimated through computational fluid dynamics—negatively influenced the adhesion of H. pylori to the substrata (P < 0.001), a result that was confirmed in similar experiments with polypropylene (P < 0.05). However, the temperature and inoculation concentration appeared to have no effect on adhesion (P > 0.05). After 2 hours, H. pylori cells appeared to be isolated on the surface of SS304 and were able to form small aggregates with longer exposure times. However, the formation of a three-dimensional structure was only very rarely observed. This study suggests that the detection of the pathogen in well water described by other authors can be related to the increased ability of H. pylori to integrate into biofilms under conditions of low shear stress. It will also allow a more rational selection of locations to perform molecular or plate culture analysis for the detection of H. pylori in drinking water-associated biofilms.

Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples

ABSTRACT A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 109 ± 5 × 108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 107 ± 5 × 106 CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4′,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.