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. We assessed the groundwater microbiome's taxonomic and functional features from samples collected at two locations in the spring and summer. All datasets comprised 436-557 genera with Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Cyanobacteria accounting for > 95% of microbial DNA sequences. The aforementioned species constituted less than 18.3% of relative abundance. Escherichia and Salmonella were mainly detected in Hot Springs, relative to Jefferson, while Clostridium and Pseudomonas were mainly found in Jefferson relative to Hot Springs. Multidrug resistance efflux pumps and BlaR1 family regulatory sensor-transducer disambiguation dominated in Hot Springs and in Jefferson. These initial results provide insight into the detection of specified microorganisms and could constitute a framework for the establishment of comprehensive metagenomic analysis for the microbiological evaluation of pharmaceutical-grade water and other non-sterile pharmaceutical products, ensuring public safety.

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

Salmonella 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 Salmonella spp. can persist and go undetected when present in low numbers. In this study, we assessed the viability of 12 serotypes of Salmonella enterica subsp. enterica for 160 days in nuclease-free water at 4 and 25°C using flow cytometry and Tryptic Soy Agar (TSA) plate counts. The results show that all 12 serotypes remain viable after 160 days in distilled water using flow cytometry, whereas traditional plate counts failed to detect ten serotypes incubated at 25°C. Moreover, the findings demonstrate that 4°C constitutes a more favorable environment where Salmonella can remain viable for prolonged periods without nutrients. Under such conditions, however, Salmonella exhibits a higher susceptibility to all tested antibiotics and benzalkonium chloride (BZK). The pre-enrichment with Universal Pre-enrichment Broth (UP) and 1/10 × Tryptic Soy broth (1/10 × TSB) resuscitated all tested serotypes on TSA plates, nevertheless cell size decreased after 160 days. Furthermore, phenotype microarray (PM) analysis of S. Inverness and S. Enteritidis combined with principal component analysis (PCA) revealed an inter-individual variability in serotypes with their phenotype characteristics, and the impact of long-term storage at 4 and 25°C for 160 days in nuclease-free water. This study provides an insight to Salmonella spp. long-term survivability at different temperatures and highlights the need for powerful tools to detect this microorganism to reduce the risk of disease transmission of foodborne pathogens via nuclease-free water.

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

Burkholderia cepacia 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 Kef probe that specifically binds a KefB/KefC membrane protein sequence within BCC. Optimal conditions of a 1 nM Kef probe concentration at a 60 °C hybridization temperature for 30 min were determined and applied for the flow cytometry assay. The true-positive rate (sensitivity) and true-negative rate (specificity) of the Kef probe assay were 90% (18 positive out of 20 BCC species) and 88.9% (16 negative out of 18 non-BCC), respectively. The detection limit for B. cenocepacia AU1054 with the Kef probe flow cytometry assay in nuclease-free water was 1 CFU/mL. The average cell counts using the Kef probe assay from a concentration of 10 µg/mL chlorhexidine gluconate and 50 µg/mL benzalkonium chloride were similar to those of the RAPID-B total plate count (TPC). We demonstrate the potential of Kef probe flow cytometry as a more sensitive alternative to culture-based methods for detecting BCC in non-sterilized pharmaceutical raw materials and products with regards to water-based environments.

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. Twenty BCC strains were each suspended (1, 10, 100, and 1000 CFU/ml) in autoclaved nuclease-free water, 10 µg/ml CHX, and 50 µg/ml BZK. Five replicates of each strain were tested at each concentration (20 strains × 4 concentrations × 5 replicates = 400 tests) to detect BCC using the aforementioned four methods. We demonstrated the potential of ddPCR and flow cytometry as more sensitive alternatives to culture-based methods to detect BCC in autoclaved nuclease-free water and antiseptics samples.

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. Both the estimation and imaging parameters were optimized to reduce the estimation error ( E, %) to close to ±5%. The method was validated against conventional microbiological techniques such as the use of optical density, flow cytometry, and quantitative real-time PCR (qPCR). It could easily be tailored to estimate different species of pathogens, such as Escherichia coli O157, Listeria innocua, Staphylococcus aureus, Enterococcus faecalis, and Bacillus anthracis, on samples similar to those in real-time contamination scenarios. The present method is sensitive enough to detect ∼100 bacterial CFU/mL but has the potential to estimate even lower concentrations with increased imaging and computation times. Overall, this imaging-based method may greatly complement any signal-based pathogen-detection technique, especially in negating false signals, and therefore may significantly contribute to the field of analytical microbiology and biochemistry.

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

Very low cell count detection of Escherichia coli 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 E. coli O157:H7. The challenge is to find the single cell in a complex matrix like raw spinach. To find that cell requires growing it as quickly as possible to a number sufficiently in excess of matrix background that identification is certain. The experimental design for this work was that of a U.S. Food and Drug Administration (FDA) In-House Level 3 validation executed in the technology's originating laboratory. Using non-selective enrichment broth, 6.5 h incubation at 42°C, centrifugation for target cell concentration, and a highly selective E. coli O157 fluorescent antibody tag, the cytometry method proved more sensitive than a reference regulatory method (p = 0.01) for detecting a single target cell, one E. coli O157:H7 cell, in 25 g of spinach. It counted that cell's daughters with at least 38× signal-to-noise ratio, analyzing 25 samples in total-time-to-results of 9 h.

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. The main objectives of this study were to facilitate fast detection (<30 min to analyze 6 samples, including preparation) and sub-species-level bacterial characterization based on pattern recognition of mass spectral fingerprints acquired from whole cells volatilized and ionized at atmospheric pressure. An AccuTOF DART mass spectrometer was re-engineered to permit ionization of low-volatility bacteria by means of Plasma Jet Ionization (PJI), in which an electric discharge, and, by extension, a plasma beam, impinges on sample cells. RESULTS: Instrumental improvements and spectral acquisition methodology are described. Performance of the re-engineered system was assessed using a small challenge set comprised of assorted bacterial isolates differing in identity by varying amounts. In general, the spectral patterns obtained allowed differentiation of all samples tested, including those of the same genus and species but different serotypes. CONCLUSIONS: Fluctuations of ±15% in bacterial cell concentrations did not substantially compromise replicate spectra reproducibility.

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. coli require an infective dose of as few as 10 cells (Schmid-Hempel and Frank, 2007). Although, according to the FDA, the infectious dose is unknown (Food and Drug Administration, 1993). Therefore, the inoculation level into the spinach, a total of 2.0±2.6 viable E. coli O157 cells, was specified to yield between 25% and 75% detection by the new method, out of 20 samples (10 positives and 10 negatives). This criterion was met in that the new method detected 60% of the nominally positive samples; the corresponding sensitivity of the reference method was 50%. For both methods the most likely explanation for false negatives was that no viable cells were actually introduced into the sample. In this validation study, the flow cytometry method was equal to the BAM in sensitivity and far superior in speed.

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. The need for a more broadly applicable and useful sample handling procedure is warranted. METHODS: Thymol was added to the suspension solvent of bacteria prior to MALDI analysis. The suspension solvent consisted of ethanol, water and TFA. The bacterium was added to the thymol suspension solvent and heated. An aliquot of the bacterial suspension was mixed directly with the matrix solution at a 9:1 ratio, matrix/bacteria solution, respectively. The mixture was then placed on the MALDI plate and allowed to air dry before MALDI analysis. RESULTS: The thymol method improved the quality of spectra and number of peaks when compared to other sample preparation procedures studied. The bacterium-identifying biomarkers assigned to four strains of E. coli were statistically 95% reproducible analyzed on three separate days. The thymol method successfully differentiated between the four E. coli strains. In addition, the thymol procedure could identify nine out of ten S. enterica serovars over a 3-day period and nine S. Typhimurium strains from the other ten serovars 90% of the time over the same period. CONCLUSIONS: The thymol method can identify certain bacteria at the sub-species level and yield reproducible results over time. It improves the quality of spectra by increasing the number of peaks when compared to the other sample preparation methods assessed in this study. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

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. anthracis during phagocytosis using murine macrophages. The effects of GCs were studied under three conditions: A) prior to, B) during, and C) following exposure of macrophages to B. anthracis individual toxin (protective antigen [PA], edema factor [EF], lethal factor [LF] or toxin complexes (PA-EF-LF, PA-EF, and PA-LF). We employed ex vivo phagocytosis and post-phagocytosis analysis including direct microscopic observation of macrophage viability, and macrophage activation. Our results demonstrated that macrophages are more prone to adhere to GC-altered PA-EF-LF, PA-EF, and PA-LF toxin complexes. This adhesion results in a higher phagocytosis rate and toxin complex neutralization during phagocytosis. In addition, GCs enhance macrophage viability, activate macrophages, and stimulate nitric oxide (NO) production. The present study may be helpful in identifying GC ligands with toxin-neutralizing and/or immunomodulating properties. In addition, our study could suggest GCs as new targets for existing vaccines and the prospective development of vaccines and immunomodulators used to combat the effects of B. anthracis.

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. Without glycoconjugate (GC) activation, murine macrophages were killed by Bacillus toxins. GCs were shown to have a protective influence, sparing macrophages from toxin-induced cell death, as shown by increased macrophage cell viability based on trypan blue assay. Increased levels of inducible nitric oxide (NO) production by macrophages in presence of GCs suggest that GCs provide an activation signal for macrophages and stimulate their function. Results hint to GCs that promote neutralization of Bacillus toxins, block toxin-induced macrophage death, while increasing macrophage activation. Polymeric GCs may suggest novel approaches to improve existing or develop new vaccines as well as immunotherapeutics.

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. RESULTS: For distinction of nine brain tissue categories, use of re-calibration, variance weighting, and biomarker modeling improved LOO classification of MRS spectra from 31% to 95%. External validation of the two best nine-category models on 47 unknown samples gave 96% or 100% accuracy, respectively, compared with pathological diagnosis. CONCLUSION: Preprocessing of MRS spectra can significantly improve their diagnostic utility for automated consultation of pattern recognition models. Use of several techniques in combination greatly increases available proton MRS information content. Accurate assignment of unknowns among nine tissue classes represents a significant improvement, for a much more demanding task, than has been previously reported.

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. Present research was carried out to study whether glycoconjugates stimulated NO and nitric oxide synthase (NOS2) production during phagocytosis killing of Bacillus spores. Murine macrophages exposed to glycoconjugate-treated spores induced NOS2 and NO production that was correlated with high viability of macrophages and killing rate of bacterial spores. Increased levels of inducible NOS2 and NO production by macrophages in presence of glycoconjugates suggested that the latter provide an activation signal directed to macrophages. Glycoconjugates were shown to exert a protective influence, sparing macrophages from spore-induced cell death. In presence of glycoconjugates, macrophages efficiently kill the organisms. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores. These results suggest that glycoconjugates promote killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level and NO and NOS2 production. Glycoconjugates suggest novel antimicrobial approaches to prevention and treatment of infection caused by bacterial spores.

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. Polyvalent glycoconjugates (also known as neoglycoconjugates) were tested during decontamination of B. cereus spores. Resulting colony forming units (CFU) of viable spores were a direct evidence of glycoconjugate decontamination efficacy. Our results indicate a substantial, decreasing CFU count due to defensive and simultaneous actions of glycoconjugates compared to spores only used as controls. Decontamination of B. cereus spores was most efficiently and consistently achieved using Galalpha1-->3GalNAcbeta-PAA-flu glycoconjugate under both defensive and simultaneous conditions. Atomic force microscopy (AFM) allowed us to visualize decontamination at the nanoscale level using glycoconjugates. AFM reveals the size of glycoconjugate agglomerates (clusters) and a noticeably different morphology of glycoconjugate-treated spores during decontamination. Morphological features of untreated spores disappear under a thin layer of glycoconjugate solution. This thin layer is formed due to the defensive action of glycoconjugates. Simultaneous action has shown agglomeration of glycoconjugates in solution with B. cereus spores in glycoconjugate suspensions. Glycoconjugates might be useful for the development of an environment-friendly decontaminant of bacterial spores.

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. In the absence of glycoconjugates, murine macrophages were ineffective at killing Bacillus spores. In presence of glycoconjugates, however, macrophages efficiently killed spores, whether the glycoconjugates were added to the cells prior to, during, and following spore addition. Glycoconjugates were shown to exert a protective influence on macrophages and increase their activation, as evidenced by viability and lactate dehydrogenase release assays. Increased levels of nitric oxide production by macrophages pretreated with glycoconjugates suggest that, under these conditions, glycoconjugates provide an activation signal to macrophages. These results indicate that glycoconjugates promote killing of Bacillus spores, while increasing macrophage activation level and viability. The selection of glycoconjugate ligands bearing immunomodulating properties could be exploited for vaccine and/or immunomodulator development and/or for the improvement of existing vaccines against B. cereus and B. anthracis.

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. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores during phagocytosis. In the presence of glycoconjugates, however, the macrophages efficiently killed the organisms. The glycoconjugates were shown to have a protective influence, sparing macrophages from spore-induced cell death. Very low concentrations of the glycoconjugates prevented macrophage cell death, as shown by lactate dehydrogenase (LDH) release and trypan blue assays. Increased levels of inducible nitric oxide (NO) production by the macrophages in the presence of glycoconjugates suggested that the glycoconjugates provide an activation signal to the macrophages. These results suggest that glycoconjugates promote the killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level. Polymeric glycoconjugates may suggest novel approaches to improve existing vaccines as well as prevent and treat infections incurred through either B. cereus or B. anthracis spores.