Clustering of Staphylococcus aureus bovine mastitis strains from regions of Central-Eastern Poland based on their biochemical and genetic characteristics.

Staphylococcus aureus strains were isolated from mastitic milk of cows with infected mammary glands. The animals were living in 12 different farms near Lublin, in Central-Eastern Poland. A biochemical identification method based on enzymatic assay was performed, followed by haemolytic and proteolytic tests. PCR-RFLP targeted on the gap gene allowed the genetic identification of strains at the species level and verified phenotypic identification results. A molecular typing method using triplex PCR was performed to recognize the genetic similarity of the analyzed strains. DNA microarray hybridization (StaphyType, Alere Technologies) was used for detection of antibiotic resistance and virulence associated markers. The results obtained indicate high genetic similarity in strains isolated from the same sites. High genetic similarities were also detected between strains isolated from cows from different farms of the same region. A slightly lower similarity was noted however, in strains from various regions indicating that the strains are herd specific and that the cow's infections caused by S. aureus were of a clonal character. In 21 representative isolates selected for DNA-microarray testing, only fosfomycin (fosB) and penicillin resistance markers (blaZ, blaI, blaR) were detected. The presence of genes coding for haemolysins (lukF, lukS, hlgA, hla, hld, hlb), proteases (aur, sspA, sspB, sspP), enterotoxins (entA, entD, entG, entI, entJ, entM, entN, entO, entR, entU, egc-cluster), adhesins (icaA, icaC, icaD, bbp, clfA, clfB, fib, fnbA, map, vwb) or immune evasion proteins (scn, chp, sak) was common and, with exceptions, matched triplex PCR-defined clusters.

Evaluation of DEPMPO as a spin trapping agent in biological systems.

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO3*- spin adduct of the spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (DEPMPO), was investigated, and the results were compared with those of the widely used spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Similar to DMPO, DEPMPO was quickly taken up (<15 min) after intraperitoneal injection, and distributed evenly in the liver, heart, and blood of the mice. In the presence of ascorbate the in vitro stability of the adduct DEPMPO/SO3*- was 7 times better than DMPO/SO3*-. Under in vivo conditions, the spin adduct DEPMPO/SO3*- was 2-4 times more stable than DMPO/ SO3*-, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO3*- radical both with DMPO and DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct signal at a concentration as low as 1 mM, as compared to 5 mM for DMPO. We conclude that DEPMPO is potentially a good candidate for trapping radicals in functioning biological systems, and represents an improvement over the commonly used trap DMPO.

Rate of oxygen consumption of hamster melanoma cells as a factor influencing their radioresistance.

It has been reported in recent years that the level of radiosensitivity of neoplastic cells in vivo and of sphaeroids in vitro can be modified by controlling their rate of oxygen consumption. Thus, an attempt was made to compare this rate in the case of the melanotic and amelanotic lines of Bomirski hamster melanoma in vitro, as it is known that these two lines distinctly differ in their reactivity to ionizing radiations. The measurements carried out by the use of a new ESR method revealed that pigmented and pigmentless cells consume oxygen at significantly different rates. This means that oxygen utilization may contribute to the overall level of radioresistance of melanoma cells.

Evidence that divalent cations bind to diphtheria toxin: an ESR approach.

We have used electron spin resonance (ESR) techniques to study the binding of divalent cations to diphtheria toxin (DT). Addition of DT to Mn(II) solution at a stoichiometry of 2:1 (DT:Mn) induces a 79% loss in the intensity of the ESR spectrum of Mn(II) suggesting a strong binding of Mn(II) to DT. Inclusion of Ca(II) at a ratio of 1:2:1 (Ca:DT:Mn) in the reaction mixture restores the intensity of the Mn(II) signal to 64%. This indicates that Ca(II) and Mn(II) share same binding site(s) in DT. The results presented in this communication suggest that DT is a Ca(II) binding protein.

Diphtheria toxin induces leakage of acidic liposomes.

Diphtheria toxin (DT) induces the leakage of dipalmitoylphosphatidic acid (DPPA) membranes but not neutral dipalmitoylphosphatidylcholine (DPPC) membranes. Cholesterol incorporated into liposomes enhances the membrane leakage induced by DT in acidic DPPA membranes but not in neutral DPPC membranes. Membrane leakage was determined by assaying the release of TEMPOcholine, a cationic spin probe from the multilamellar vesicles by using electron spin resonance methods. The effect of DT on membrane leakage is noticeable at 3 micrograms/ml concentrations, and reaches a plateau of about 20% leakage at 20 micrograms/ml. This saturation phenomenon led to the postulation that DT binds to the first shell of DPPA membranes and induces the leakage of TEMPOcholine limited to this layer of DPPA multimellar vesicles.

Gloxy: an oxygen-sensitive coal for accurate measurement of low oxygen tensions in biological systems.

This paper describes the characteristics of a new oxygen sensitive, paramagnetic material that has some significant advantages for measurements of tissue pO2 by in vivo EPR. This paramagnetic component of Welsh coal, termed "gloxy" was found to have valuable EPR features that allow accurate measurement of low oxygen tensions in vivo; these include large oxygen-dependent changes in linewidth, a high number of paramagnetic spin centers (resulting in high signal amplitude), and stability in tissue allowing repeated pO2 measurements to be made in vivo with high precision. Renal pO2 was measured deep in the medulla region of isolated perfused kidneys and found to be lower than that in the cortex (1.7 +/- 0.05 and 7.1 +/- 0.3 mm Hg, respectively). The quality of the EPR signal obtained from the renal outer medulla and also from tumors in mice was such that the pO2 measurements were obtained with a precision of +/-3% of the measured pO2 (Kidney: 1.7 +/- 0.05 mmHg; Tumor: 1.37 +/- 0.04 mmHg). In vitro tests on the viability of cells and in vivo studies using Gloxy demonstrate the stability and inertness of this oxygen-sensitive material.