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A multiplex PCR for the simultaneous detection of five virulence genes ( asa1 , gelE , cylA , esp , and hyl ) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher ( P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher ( P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent ( P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific ( esp -positive) VREF clones in Italy and one center-specific ( hyl -positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1 , gelE , cylA , esp , and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp -positive VREF clones (in Italy) and hyl -positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

NAI-603 is a ramoplanin derivative designed to overcome the tolerability issues of the parent drug as a systemic agent. NAI-603 is highly active against aerobic and anaerobic Gram-positive bacteria, with MICs ranging from 0.008 to 8 μg/ml. MICs were not significantly affected by pH (range, 6 to 8), by inoculum up to 10 8 CFU/ml, or by addition of 50% human serum. Against staphylococci and enterococci, NAI-603 was rapidly bactericidal, with minimum bactericidal concentration (MBC)/MIC ratios never exceeding 4. The frequency of spontaneous resistance was low at 2× to 4× MIC (≤1 × 10 −6 to ≤1 × 10 −8 ) and below the detection limit (about ≤1 × 10 −9 ) at 8× MIC. Serial subcultures at 0.5× MIC yielded at most an 8-fold increase in MICs. In a systemic infection induced by methicillin-resistant Staphylococcus aureus (MRSA), the 50% effective dose (ED 50 ) of intravenous (i.v.) NAI-603 was 0.4 mg/kg, lower than that of oral (p.o.) linezolid (1.4 mg/kg) and subcutaneous (s.c.) teicoplanin (1.4 mg/kg) or vancomycin (0.6 mg/kg). In neutropenic mice infected with vancomycin-resistant enterococci (VRE), the ED 50 s for NAI-603 were 1.1 to 1.6 mg/kg i.v., compared to 0.5 mg/kg i.v. of ramoplanin. The bactericidal activity was confirmed in vivo in the rat granuloma pouch model induced by MRSA, where NAI-603, at 40 mg/kg i.v., induced about a 2- to 3-log 10 -reduction in viable bacteria in the exudates, which persisted for more than 72 h. The pharmacokinetic (PK) profiles of NAI-603 and ramoplanin at 20 mg/kg show similar half-lives (3.27 and 3.80 h, respectively) with the maximum concentration ( C max ) markedly higher for NAI-603 (207 μg/ml versus 79 μg/ml). The favorable pharmacological profile of NAI-603, coupled with the absence of local tolerability issues, supports further investigation.

The activity of penems against methicillin-resistant Staphylococcus aureus (MRSA) was determined on strains representative of the four expression classes of resistance. Two families of compounds, characterized by a C2-(hetero)aromatic moiety connected by an ether (CH 2 O) or ester (CH 2 OCO) link, displayed superior activity in vitro.

In the present report we describe a rapid ans sensitive assay for mycoplasma detection in cell cultures. The assay is based on the ability of contaminated culture supernatants to modulate [ 3 H]TdR incorporation by unstimulated mouse splenocytes. Several mycoplasma species ( Mycoplasma orale , culturable and non-culturable strains of Mycoplasma hyorhinis ) inhibited [ 3 H]TdR incorporation and permitted the detection of some contaminated cell cultures that would otherwise have escaped detection in assays measuring [ 3 H]TdR incorporation by mitogen-stimulated splenocytes. On the other hand, several other mycoplasma species ( Mycoplasma arginini, Mycoplasma hominis ) strongly enhanced [ 3 H]TdR incorporation by unstimulated splenocytes. This enhancement was optimally detectable on day 2 after initiation of the cultures. The sensitivity of the assay was determined for a mycoplasma species (culturable M. hyorhinis ) that inhibited as well as for one ( M. arginini ) that enhanced [ 3 H]TdR incorporation. In both cases, the sensitivity was such that 1−3× 10 2 mycoplasma colony-forming units (CFU) could be detected.

A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

A survey in eight European countries, including 13 hospitals, of vancomycin-resistant enterococci (VRE) in at-risk hospital wards (such as the ICU and the haematology ward) was performed in 2001, and the in vitro susceptibility of the isolates ramoplanin and other drugs was tested. A total of 1314 non-duplicate clinical enterococcal isolates were collected, and 38 (2.9%) were vancomycin resistant: 27 Enterococcus faecium and 11 Enterococcus faecalis; 35 VanA and three VanB phenotypes. Rates of VRE among clinical enterococcal isolates varied between 0 and 1.7% for the participating countries, except the UK (10.4%) and Italy (19.6%). One hundred and twenty-three (3.5%) VRE were found among 3499 stool samples tested for the presence of these organisms: 111 (3.2%) E. faecium and 12 (0.3%) E. faecalis; 114 (3.3%) VanA and nine (0.3%) VanB phenotypes. Rates of intestinal colonization with VRE varied between 0 and 1.2% for the participating countries, except Italy (7.5%) and the UK (32.6%). In vitro susceptibility testing showed that the Italian and UK VRE are multi-resistant (including resistance to ampicillin and high-level resistance to gentamicin and streptomycin), and that ramoplanin was active against all strains of VRE, with an MIC90 of 0.5 mg/L for clinical isolates. Pulsed-field gel electrophoresis showed that the high prevalence of VRE in the Italian and UK centres was related to the monoclonal emergence and spread of three centre-specific clones. This survey suggests that in some centres in Europe, a similar situation may be encountered to that in the USA (monoclonal spread of multi-resistant VRE in at-risk wards).

The activity of rifabutin against Toxoplasma gondii was investigated in vitro and in vivo. One set of in-vitro studies used either a low virulence or a high virulence T. gondii strain subcultured in drug-free conditions after exposure to various drug concentrations. No parasite growth was observed after subculture at 1 month after exposure to 6 mg/L of rifabutin for the low virulence and 12 mg/L for the high virulence strain. The IC50 of rifabutin was 26.5 mg/L in a different series of experiments using an enzyme-linked immunoassay) and the T. gondii high virulence strain. In similar experiments with clarithromycin, low virulence and high virulence parasites grew in drug-free subcultures following exposure to drug concentrations as high as 100 mg/L; the IC50 of clarithromycin exceeded 100 mg/L for the high virulence strain. In the acute toxoplasmosis murine model, mice received a 12 day treatment starting 5 days before infection with high virulence parasites. Survival was significantly improved compared with untreated controls in response to 50, 100 and 200 mg/kg/day rifabutin and 100 and 200 (but not 50) mg/kg/day clarithromycin, and with the combination of the two drugs at 25, 50 and 100 mg/kg/day. Survival was significantly improved when combination therapy was administered. The calculated ED50 values (mg/kg/day) were 160.5 for rifabutin, 119.4 for clarithromycin, and 114.8 for the combination. In the present experimental conditions rifabutin proved effective in vitro and in vivo against T. gondii and showed potentiation with clarithromycin in vivo.

The isosteric CH----N substitution in the class of 2-arylpenems results in improved antibacterial activity, with retention of the favorable characteristic of stability towards renal dehydropeptidase. High therapeutic efficacy was demonstrated in experimental mice septicemias with the 2-(3-pyridyl) derivative 2b and its orally absorbed acetoxymethyl ester prodrug 4n.

The reduction of tetranitroblue tetrazolium with cysteamine, mediated by a number of dyes, elemental sulphur, elemental selenium and selenide, under aerobic conditions, was inhibited to various extent upon addition of superoxide dismutase. A strict parallelism between the ability to produce O2- ions and the property of those compounds to act as cofactors for cysteamine-oxygenase, to yield hypotaurine, has been observed. Based on the fact that the autoxidation of cysteamine also gives rise to O2- formation, though to a minor extent, we propose a mechanism for cysteamine-oxygenase action. This mechanism was derived from the data obtained in the model system studied.

The plasma concentrations of FCE 22101 observed following slow intravenous infusions (1 and 4 h), intramuscular injection and after oral administration of the prodrug FCE 22891 were simulated in a glass chamber containing bacterial cultures. The bacteria used were Staphylococcus aureus ATCC 13709, Staph. aureus 2101 (methicillin-resistant), Streptococcus faecium ATCC 8043, Escherichia coli ATCC 12407, Enterobacter cloacae 1321E and Ent. cloacae P99 (cefazolin-resistant). Addition of FCE 22101 was found to result in a reduction in number of all bacterial strains except Str. faecium, with which only bacteriostasis was observed.

Publisher Summary Although immobilized enzymes and proteins have been studied mainly for technological applications, the behavior of proteins attached to solid matrices may help us to understand the general properties of these systems and to give us a better insight into their reactivity in solution. A comparison of the behavior of these systems in solution with those in immobilized forms may also be useful in interpreting structure-function relationships. Hemoproteins, especially myoglobin, hemoglobin, and cytochromes, are well suited for studying proteins in the semisolid state because their visible and near-ultraviolet absorption spectra can be easily observed and the reactions of these immobilized proteins can be followed spectrophotometrically. It is suggested that one of the major difficulties in interpreting the behavior of matrix bound vs soluble-state proteins is the distinction between effects such as immobilization and those such as the chemical modification involved in the linkage of the protein to the matrix. These difficulties can sometimes be overcome by appropriate control experiments on soluble proteins.

Publisher Summary This chapter describes the procedures for the isolation of globin in the native state and for its reconstitution with metalloporphyrins. Globins are isolated from the corresponding hemoproteins by the removal of the heme moiety at acid pH, under controlled experimental conditions. The quality of the globin samples obtained can be checked by titration experiments with hemin (ferric protoheme) and by studying the properties of the reconstituted hemoproteins. There are two known methods for the preparation of globin from the holoprotein; both are based on the decreased affinity of the heme for globin at acid pH values. The first method employs acid–acetone at low temperature to split the heme group from the globin, which precipitates and is subsequently redissolved in water. A second method is based on heme extraction in methyl ethyl ketone at acid pH; in this case, the apoprotein remains dissolved in the aqueous layer. Further purification of the globin is accomplished similarly in both methods. The chapter presents the physicochemical properties of globin.

The therapeutic activity of FCE 22891 was compared with that of two new oral cephalosporins, cefuroxime axetil and cefixime against Streptococcus pneumoniae respiratory infection and subcutaneous abscesses induced by mixed aerobes and anaerobes in mice. In experimental pneumonia FCE 22891 was the most active antibiotic. In aerobic abscesses FCE 22891 proved the most active agent in infections induced by methicillin susceptible and resistant Staphylococcus aureus while all three compounds were very active, against Str. pyogenes. In abscesses caused by Gram-negative bacteria, FCE 22891 showed good and constant efficacy. Cefixime was the most active drug against the two susceptible strains of Escherichia coli and Enterobacter cloacae and also against resistant Esch. coli but was inactive against a strain of Ent. cloacae that produced cephalosporinase. Cefuroxime axetil was less active than the other two drugs against Gram-negative bacteria with adequate efficacy only against a susceptible strain of Ent. cloacae. FCE 22891 was more effective than cefixime and cefuroxime axetil in preventing and reducing the size of abscesses induced by Bacteroides fragilis 101. We conclude that FCE 22891, despite its short half life of 6 min in mice, exerts comparable and sometimes better activity than the two oral cephalosporins characterized by longer half lives.

Ovotransferrin immobilized by covalent linkage to Sepharose 4B showed a bacteriostatic effect towards Escherichia coli similar to that of free ovotransferrin. The growth of the bacteria, after exposure to the gel-bound ovotransferrin and its removal, depended on the length of exposure. The results suggest that the antibacterial activity of transferrin is not due simply to the removal of iron from the medium.