A Comparative Metagenomic Analysis of Specified Microorganisms in Groundwater for Non-Sterilized Pharmaceutical Products.

In pharmaceutical manufacturing, ensuring product safety involves the detection and identification of microorganisms with human pathogenic potential, including Burkholderia cepacia complex (BCC), Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma spp., some of which may be missed or not identified by traditional culture-dependent methods. In this study, we employed a metagenomic approach to detect these taxa, avoiding the limitations of conventional cultivation methods.

A flow cytometric assay to detect viability and persistence of subsp. serotypes in nuclease-free water at 4 and 25°C.

spp. is one of the most isolated microorganisms reported to be responsible for human foodborne diseases and death. Water constitutes a major reservoir where the spp.

Specific Detection and Enumeration of Complex by Flow Cytometry Using a Fluorescence-Labeled Oligonucleotide Probe.

complex (BCC) contamination has resulted in recalls of non-sterile pharmaceutical products. The fast, sensitive, and specific detection of BCC is critical for ensuring the quality and safety of pharmaceutical products. In this study, a rapid flow cytometry-based detection method was developed using a fluorescence-labeled oligonucleotide probe that specifically binds a KefB/KefC membrane protein sequence within BCC.

A comparison of culture-based, real-time PCR, droplet digital PCR and flow cytometric methods for the detection of Burkholderia cepacia complex in nuclease-free water and antiseptics.

The presence of Burkholderia cepacia complex (BCC) strains has resulted in recalls of pharmaceutical products, since these opportunistic pathogens can cause serious infections. Rapid and sensitive diagnostic methods to detect BCC are crucial to determine contamination levels. We evaluated bacterial cultures, real-time PCR (qPCR), droplet digital PCR (ddPCR), and flow cytometry to detect BCC in nuclease-free water, in chlorhexidine gluconate (CHX) and benzalkonium chloride (BZK) solutions.

Estimating Bacterial Concentrations in Fibrous Substrates Through a Combination of Scanning Electron Microscopy and ImageJ.

Conventional signal-based microanalytical techniques for estimating bacterial concentrations are often susceptible to false signals. A visual quantification, therefore, may compliment such techniques by providing additional information and support better management decisions in the event of outbreaks. Herein, we explore a method that combines electron microscopy (EM) and image-analysis techniques and allows both visualization and quantification of pathogenic bacteria adherent even to complex nonuniform substrates.

Rapid Flow Cytometry Detection of a Single Viable O157:H7 Cell in Raw Spinach Using a Simplified Sample Preparation Technique.

Very low cell count detection of O157:H7 in foods is critical, since an infective dose for this pathogen may be only 10 cells, and fewer still for vulnerable populations. A flow cytometer is able to detect and count individual cells of a target bacterium, in this case O157:H7. The challenge is to find the single cell in a complex matrix like raw spinach.

Instrumental improvements and sample preparations that enable reproducible, reliable acquisition of mass spectra from whole bacterial cells.

Rationale: Rapid sub-species characterization of pathogens is required for timely responses in outbreak situations. Pyrolysis mass spectrometry (PyMS) has the potential to be used for this purpose.

Methods: However, in order to make PyMS practical for traceback applications, certain improvements related to spectrum reproducibility and data acquisition speed were required.

Level 2 validation of a flow cytometric method for detection of Escherichia coli O157:H7 in raw spinach.

The Bacteriological Analytical Manual (BAM) method currently used by the United States Food and Drug Administration (FDA) to detect Escherichia coli O157:H7 in spinach was systematically compared to a new flow cytometry based method. This Food and Drug Administration (FDA) level 2 external laboratory validation study was designed to determine the latter method's sensitivity and speed for analysis of this pathogen in raw spinach. Detection of target cell inoculations with a low cell count is critical, since enterohemorrhagic strains of E.

Thymol treatment of bacteria prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis aids in identifying certain bacteria at the subspecies level.

Rationale: The identification of bacteria based on mass spectra produced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has become routine since its introduction in 1996. The major drawback is that bacterial patterns produced by MALDI are dependent on sample preparation prior to analysis. This results in poor reproducibility in identifying bacterial types and between laboratories.

Neutralization of B. anthracis toxins during ex vivo phagocytosis.

Glycoconjugates (GCs) are recognized as stimulation and signaling agents, affecting cell adhesion, activation, and growth of living organisms. Among GC targets, macrophages are considered ideal since they play a central role in inflammation and immune responses against foreign agents. In this context, we studied the effects of highly selective GCs in neutralizing toxin factors produced by B.

Glycoconjugates prevent B. anthracis toxin-induced cell death through binding while activating macrophages.

Bacillus anthracis toxins may be attenuated if macrophages could neutralize toxins upon contact or exposure. Glycoconjugate-bearing polymers, which have been shown to bind to Bacillus spores, were tested for recognition and binding of protective antigen (PA), lethal factor (LF), and edema factor (EF) toxins. We have demonstrated modulation of macrophage activity following exposure to these toxins.

Improving proton MR spectroscopy of brain tissue for noninvasive diagnostics.

Purpose: To examine preprocessing methods affecting the potential use of Magnetic Resonance Spectroscopy (MRS) as a noninvasive modality for detection and characterization of brain lesions and for directing therapy progress.

Materials And Methods: Two reference point re-calibration with linear interpolation (to compensate for magnetic field nonhomogeneity), weighting of spectra (to emphasize consistent peaks and depress chemical noise), and modeling based on chemical shift locations of 97 biomarkers were investigated. Results for 139 categorized scans were assessed by comparing Leave-One-Out (LOO) cross-validation and external validation.

Killing of Bacillus spores is mediated by nitric oxide and nitric oxide synthase during glycoconjugate-enhanced phagocytosis.

Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity against various pathogens. Bacillus spores can produce serious diseases, which might be attenuated if macrophages were able to kill the spores on contact.

Defensive and simultaneous actions of glycoconjugates during spore decontamination.

An estimated $1 billion was lost in decontaminating areas exposed to anthrax in the 2001 attacks. To counter the threat of biological attacks, an effective 'green' decontaminant is vital to minimize the consequences of such attacks. The objective of our research was to study the ability of glycoconjugate ligands to decontaminate Bacillus cereus spores on hard surfaces.

Glycoconjugates enhanced the intracellular killing of Bacillus spores, increasing macrophage viability and activation.

Infections caused by Bacillus spores can be attenuated if the intracellular killing of the organism by macrophages can be enhanced. Glycoconjugate-bearing polymers, which selectively bind to Bacillus spores, were tested for modulation of intracellular killing when added prior to, during, and following macrophage exposure to B. cereus spores.

Polymeric glycoconjugates protect and activate macrophages to promote killing of Bacillus cereus spores during phagocytosis.

Diseases caused by Bacillus spores might be attenuated if macrophages were able to kill the spores on exposure. Glycoconjugate-bearing polymers, which have been shown to bind to Bacillus spores, were tested for modulation of phagocytosis of B. cereus spores.