Evaluation of a new presumptive medium for group D streptococci.

A new medium designated as D streptococcus-enterococcus broth was formulated and evaluated for the enrichment and isolation of strains of serological group D streptococci. This medium was made by modifying Todd-Hewitt broth. Most-probable-number multiple-tube and membrane filter techniques were employed to estimate the numbers of enterococci in known cultures, wastewater, and other samples. Preliminary most-probable-number counts with this medium were as much as 3 logs higher than those counts obtained from four other media with which it was compared. The methodology for using this medium to estimate the numbers of group D streptococci in water is discussed.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing

Background

Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo.

Methodology and Principal Findings

To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression.

Conclusions

In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.     

Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684

Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1?→?4)-β-d-GlcpNAc-(1?→?6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1?→?4)-[3-O-acetyl]-β-d-GlcpNAc-(1?→?6)-α-d-GlcpNH(2)-(1→.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Evaluation of a Most-Probable-Number Technique for the Enumeration of Pseudomonas aeruginosa

A most-probable-number (MPN) technique was evaluated for detecting and enumerating Pseudomonas aeruginosa in water and wastewater. Both the presumptive and confirmatory media, as described in the 13th edition of Standard Methods for the Examination of Water and Wastewater, as well as modifications of these media were included in evaluations. Various samples of water were tested, namely chlorinated tap water, creek water, and influent to a wastewater treatment plant. Modified media repeatedly gave higher estimated MPNs of P. aeruginosa than media listed in Standard Methods. P. aeruginosa was detected and recovered from all creek water and wastewater samples, but not from tap water samples tested. This organism was determined to be present in as large numbers as the fecal coliforms and in even greater quantities than the fecal streptococci in all samples, whenever MPN estimations were determined from those positive tubes containing the modified confirmatory medium.     

Mutations in SACPD-C Result in a Range of Elevated Stearic Acid Concentration in Soybean Seed

Soybean oil has a wide variety of uses, and stearic acid, which is a relatively minor component of soybean oil is increasingly desired for both industrial and food applications. New soybean mutants containing high levels of the saturated fatty acid stearate in seeds were recently identified from a chemically mutagenized population. Six mutants ranged in stearate content from 6–14% stearic acid, which is 1.5 to 3 times the levels contained in wild-type seed of the Williams 82 cultivar. Candidate gene sequencing revealed that all of these lines carried amino acid substitutions in the gene encoding the delta-9-stearoyl-acyl-carrier protein desaturase enzyme (SACPD-C) required for the conversion of stearic acid to oleic acid. Five of these missense mutations were in highly conserved residues clustered around the predicted di-iron center of the SACPD-C enzyme. Co-segregation analysis demonstrated a positive association of the elevated stearate trait with the SACPD-C mutation for three populations. These missense mutations may provide additional alleles that may be used in the development of new soybean cultivars with increased levels of stearic acid.     

Antimicrobial efficacy of a silver-zeolite matrix coating on stainless steel

A silver- and zinc-containing zeolite matrix (AgION) used as a coating for stainless steel was tested for antimicrobial efficacy against Escherichia coli 25922, Staphylococcus aureus 25923, Pseudomonas aeruginosa 27853, and Listeria monocytogenes 7644. Assays were performed on flat coupon surfaces and in formed steel cups. AgION reduced microbial colony-forming units when compared to uncoated steel surfaces under all conditions tested. Percent reductions ranged from 84.536 to 99.999 after 4 h exposure, and from 99.992 to 100 after 24 h in all cases. The durability of the coatings declined most markedly when the coating had been applied with a wet process and scrubbed between uses with a test tube brush. Powder-coated surfaces cleaned with a towel retained a high degree of activity after five cycles of use. Electronic Publication