Comparison of 99mTc- and 18F-ubiquicidin autoradiography to anti-Staphylococcus aureus immunofluorescence in rat muscle abscesses.

UNLABELLED: 99mTc-ubiquicidin (UBI) 29-41 is under clinical evaluation for discrimination between bacterial infection and unspecific inflammation. We compared the distribution of 99mTc-UBI 29-41, the potential PET tracers 18F-UBI 29-41 and 18F-UBI 28-41, and 3H-deoxyglucose (DG) in rat muscle abscesses to that of anti-Staphylococcus aureus immunofluorescent imaging. METHODS: Calf abscesses were induced in 15 CDF-Fischer rats after inoculation of Staphylococcus aureus. One to 6 d later, either 18F-UBI 29-41 and 3H-DG (n = 5) or 18F-UBI 28-41 and 3H-DG (n = 6) or 99mTc-UBI 29-41 and 3H-DG (n = 4) were injected simultaneously. Dual-tracer autoradiography of the abscess area was compared with the distribution of bacteria and macrophages. RESULTS: The UBI derivates exhibited increased uptake in the abscess area that partly matched 3H-DG uptake and macrophage infiltration but showed no congruity with areas that were highly positive for bacteria. CONCLUSION: A specific binding of UBI derivatives to Staphylococcus aureus in vivo could not be confirmed in this study.

Differential uptake of O-(2-18F-fluoroethyl)-L-tyrosine, L-3H-methionine, and 3H-deoxyglucose in brain abscesses.

UNLABELLED: The amino acid O-(2-(18)F-fluoroethyl)-L-tyrosine ((18)F-FET) has been shown to be a useful tracer for brain tumor imaging. Experimental studies demonstrated no uptake of (18)F-FET in inflammatory cells but increased uptake has been reported in single cases of human brain abscesses. To explore this inconsistency, we investigated the uptake of (18)F-FET in comparison with that of L-[methyl-(3)H]methionine ((3)H-MET) and D-(3)H-deoxyglucose ((3)H-DG) in brain and calf abscesses in rats. METHODS: Abscesses were induced in the brain (n = 9) and calf (n = 5) of Fisher CDF rats after inoculation of Staphylococcus aureus. Five days later, (18)F-FET and (3)H-MET (n = 10) or (18)F-FET and (3)H-DG (n = 4) were injected intravenously. One hour after injection the rats were sacrificed, and the brain or calf muscle was investigated using dual-tracer autoradiography. Lesion-to-background ratios (L/B) and standardized uptake values (SUVs) were calculated. The autoradiograms were compared with histology and immunostaining for glial fibrillary acidic protein (GFAP), CD68 for macrophages, and CD11b for microglia. RESULTS: (18)F-FET uptake in the area of macrophage infiltration and activated microglia at the rim of the brain abscesses was low (L/B, 1.5 +/- 0.4). In contrast, high uptake was observed for (3)H-MET as well as for (3)H-DG (L/B, 4.1 +/- 1.1 for (3)H-MET vs. 3.1 +/- 1.5 for (3)H-DG; P < 0.01 vs. (18)F-FET). Results for calf abscesses were similar. In the vicinity of the brain abscesses, slightly increased uptake was noted for (18)F-FET (L/B, 1.8 +/- 0.3) and (3)H-MET (L/B, 1.8 +/- 0.4), whereas (3)H-DG distribution was normal (L/B, 1.2 +/- 0.2). Anti-GFAP immunofluorescence showed a diffuse astrocytosis in those areas. CONCLUSION: Our results demonstrate that there is no accumulation of (18)F-FET in macrophages and activated microglia in experimental brain abscesses, whereas (3)H-MET and (3)H-DG exhibit high uptake in these cells. Thus, the specificity of (18)F-FET for gliomas may be superior to that (3)H-MET and (3)H-DG. Increased (18)F-FET uptake in human brain abscesses appears to be related to reactive astrocytosis.

Formation of volutin granules in Corynebacterium glutamicum.

Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.

A filtration, incubation and staining reactor including a new protocol for FISH.

A newly developed device for performing fluorescence in situ hybridization (FISH) is described. An adapted procedure was compared with two typical FISH protocols. Tests were performed with Pseudomonas cells and the gene probe EUB338. With the novel procedure, we obtained a better recovery of cells and less variability in results.

Enumeration of soil bacteria with the green fluorescent nucleic acid dye Sytox green in the presence of soil particles.

Total counts in soils are usually determined using fluorescent dyes, such as DAPI or Sybr green, due to fluorescence enhancement if they are bound to nucleic acids. Unfortunately, these commonly used dyes stain soil particles as well. Therefore, besides fluorescence enhancement, sufficient spectral differentiation is also required. We present a new procedure that overcomes the problems of visualising bacteria on surfaces in soil and avoids the separation of soil particles to a large extent. Spectral differentiation between bacteria and soil matrix is achieved by using Sytox green and a suboptimal excitation wavelength. Bacteria exhibit a bright green fluorescence, while soil particles fluoresce blue or red. Slight homogenisation and sedimentation of the sand and coarse silt that were too big for microscopic investigations were the only separation steps required. We compared the proposed Sytox green staining with Sybr green staining. The recovery of Sybr green-stained cells amounted to 38%, whereas in samples stained by Sytox green 81% of the spiked cells were counted. Sytox green can also be combined with fluorescence in situ hybridisation (FISH) using deep red dyes such as Cy5.

Determination of soluble and granular inorganic polyphosphate in Corynebacterium glutamicum.

Corynebacterium glutamicum forms inorganic polyphosphate (poly P) that may occur as soluble (cytosolic) poly P and/or as volutin granules. A suitable method for monitoring soluble and granular poly P in C. glutamicum was developed and applied to C. glutamicum cells cultivated under different growth conditions. Under phosphate-limiting conditions, C. glutamicum did not accumulate poly P, but it rebuilt its poly P storages when phosphate became available. The poly P content of C. glutamicum growing on glucose minimal medium with sufficient phosphate varied considerably during growth. While the poly P content was minimal in the midexponential growth phase, two maxima were observed in the early exponential growth phase and at entry into the stationary growth phase. Cells in the early exponential growth phase primarily contained granular poly P, while cells entering the stationary growth phase contained soluble, cytosolic poly P. These results and those obtained for C. glutamicum cells cultivated under hypo- or hyperosmotic conditions or during glutamate production revealed that the poly P content of C. glutamicum and the partitioning between cytosolic and granular forms of poly P are dynamics and depend on the growth conditions.