In vitro activity of LY146032 (Daptomycin) against selected aerobic bacteria

The in vitro activity of the new lipopeptide antibiotic LY146032 was generally four-fold greater (MIC 90 0. 5g/ml) than that of vancomycin against methicillin-susceptible or methicillin-resistantStaphybcoccus aureus and coagulase-negative species ofStaphylococcus. Enterococci,Streptococcus bovis, group B and viridans streptococci, andCorynebacterium group J-K isolates were inhibited by 4g/ml of LY146032, which represented activity equivalent to or greater than that of vancomycin. Unlike vancomycin, LY146032 was bactericidal forEnterococcus faecalis, Enterococcus faecium andListeria monocytogenes. Due to its bactericidal properties LY146032 appeared to represent an improvement over vancomycin and teicoplanin.     

In vitro efficacy and fungicidal activity of voriconazole againstAspergillus andFusarium species

Twenty-nineAspergillus isolates and 25Fusarium isolates underwent in vitro antifungal susceptibility testing by a broth macrodilution procedure adapted from the National Committee for Clinical Laboratory Standards guidelines. The MIC50s of both voriconazole and amphotericin B were 0.5 g/ml and 1 g/ml against species ofAspergillus andFusarium, respectively, while the MIC90s of both agents were 1 and 2 g/ml. Voriconazole was more active in vitro than amphotericin B: the geometric mean MICs of voriconazole and amphotericin B againstAspergillus spp. were 0.36 g/ml and 0.64 g/ml, respectively. Voriconazole also demonstrated fungicidal activity againstAspergillus spp., with 86% (24/29) of isolates exhibiting minimum lethal concentrations of 4 g/ml.     

In vitro activity of the aryl-fluoroquinolones A-56619 and A-56620 and evaluation of disk susceptibility tests

The activity of two new quinolones, A-56619 and A-56620, was compared in vitro to that of norfloxacin and ciprofloxacin against 6,699 bacterial isolates in four separate clinical laboratories. The overall percentage of strains susceptible to designated concentrations were as follows: 99.1% for norfloxacin (MIC4.0 g/ml), 96.1% for ciprofloxacin (MIC1.0 g/ml), 96.8% for A-56620 (MIC 2.0 g/ml) and 96.1% for A-56619 (MIC 4.0 g/ml). For disk diffusion susceptibility tests 10 g A-56619 disks are tentatively recommended with interpretive standards of 18mm for susceptibility and 13mm for resistance; 5 g A-56620 disks may be used with tentative standards of 19mm for susceptibility and 14mm for resistance.     

Netilmicin disk susceptibility tests: Effect of cations on the MIC correlates

When testingPseudomonas aeruginosa against netilmicin, MICs were markedly affected by the concentration of cations added to the test medium. A susceptible disk test result (zone15 mm) corresponded to MIC4.0g/ml in unsupplemented broth, 12g/ml in broth with half the usual amount of cations and 32 g/ml in broth with the recommended concentration of cations. Tests with 30g netilmicin disks best predicted susceptibility as determined by MICs in broth without added cations. When the MICs were determined in cation supplemented broth, the number of interpretive discrepancies increased to an unacceptably high level.     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Modulation of human neutrophil and monocyte oxidative burst byLegionella pneumophila sonic extract

The effect of Legionella pneumophila sonic extract on human neutrophil and monocyte oxidative burst was studied by Superoxide anion release and luminol-enhanced chemiluminescence assays.Legionella pneumophila sonic extract by itself did not stimulate neutrophils and monocytes. The sonic extract at 8–2000g/ml primed neutrophils for enhanced Superoxide release and, at 8–62.5 g/ml, for enhanced chemiluminescence. Monocytes were only primed for enhanced chemiluminescence at very low extract concentrations (below 16g/ml). Monocyte Superoxide release was suppressed by extract concentrations higher than 2000g/ml and the chemiluminescence response of neutrophils and monocytes by concentrations higher than 250 and 125 g/ml, respectively. The priming activity was heat stable and present in fractions below 5 kDa. On the basis of these findings it is suggested that enhanced production of oxygen metabolites by neutrophils in contact with legionella components at low concentrations could contribute to the lung tissue damage seen in Legionnaires' disease, whereas the suppression of phagocyte oxidative burst by higher extract concentrations may be one of the mechanisms by which Legionella pneumophila survives intracellularly.     

Nippostrongylus brasiliensis: occurrence of multiple protein kinases

The presence of cyclic AMP-dependent protein kinase and phosvitin kinases, with activity independent of cyclic nucleotides, was shown in the intestinal nematodeNippostrongylus brasiliensis. The activity of the cyclic AMP-dependent protein kinase was found to be enhanced about 8-fold in the presence of 10^–7 M cyclic AMP; the apparent K_m values were determined to be 20 M and 80 M for ATP and kemptide, respectively. The molecular weight of the holoenzyme was about 170000. Two phosvitin kinases could be isolated and distinguished by their molecular weights of 600000 and 40000. The activity of the high-molecular-weight phosvitin kinase was effectively inhibited by suramin and heparin. The apparent K_m values were found to be 30 M and 0.1 mg/ml for ATP and phosvitin, respectively. In the case of the low-molecular-weight phosvitin kinase the apparent K_m values for ATP and phosvitin were found to be 30 M and 0.6 mg/ml, respectively. The investigation of different developmental stages ofN. brasiliensis revealed a marked higher level of protein kinase activity in the L₄ larvae compared to L₃ larvae and adults.     

Provisional quality control parameters and interpretive criteria for testing susceptibility ofStreptococcus pneumoniae andHaemophilus influenzae to quinupristin/dalfopristin (RP59500)

Studies were undertaken to select tentative criteria for susceptibility testing of quinupristin/dalfopristin againstStreptococcus pneumoniae andHaemophilus influenzae. Against 612 isolates ofStreptococcus pneumoniae, MICs of quinupristin/dalfopristin were 1.0 g/ml for all but one strain. With a tentative MIC breakpoint of either 1.0 g/ml or 2.0 g/ml for susceptible, a disk diffusion zone diameter breakpoint of 19 mm embraced all but two of the susceptible pneumococci; 16 mm included all strains. ForHaemophilus influenzae, MICs of quinupristin/dalfopristin clustered near the tentative breakpoints; 91.5% of the MICs were 2.0 to 8.0 g/ml. This precluded satisfactory performance of the disk diffusion test in discriminating between resistant and susceptible isolates unless MIC breakpoints are modified for this species: clinical experience will be needed before that can be justified. Based on data from a multilaboratory study, the following quality control limits are proposed forStreptococcus pneumoniae ATCC 49619 when testing quinupristin/dalfopristin: 0.25 to 1.0 g/ml for broth microdilution tests and 19 to 24 mm for disk diffusion tests. For tests ofHaemophilus influenzae ATCC 29247, MIC limits are 2.0 to 16 g/ml; disk tests were very reproducible but are not yet recommended.     

Naegleria fowleri andN. lovaniensis: Differences in sensitivity to trimethoprim and other antifolates

The growth ofNaegleria fowleri cultures in a BCS medium was not affected either by trimethoprim at 400 g/ml or by aminopterine, 3,5-diaminopterine and methotrexate at 500 g/ml.N. lovaniensis propagation in the same medium was inhibited with 10 g/ml of trimethoprim, 50 g/ml methotrexate and 100 g/ml 3,5-diaminopteridine. Aminopterine was ineffective at a concentration of 500 g/ml. The inhibitory effect of trimethoprim onN. lovaniensis cultures depended on the medium composition and could be neutralized by an addition of folic or tetrahydrofolic acids and a suspension of heat-killedEnterobacter aerogenes. Thymine, thymidine, hypoxantine and 2-amino-4-hydroxy-6-(tatrahydroxybutyl)-pteridine did not have an adverse effect. Trimethoprim activity inN. fowleri cultures could not be enhanced by the addition of Triton X-100 and Polymyxine B. Cryolyzate ofN. fowleri amoebae did not influence the trimethoprim inhibition ofN. lovaniensis cultures. Deviation in dihydrofolatereductase chemical structure or thymine dependency seems to be the probable explanation forN. fowleri antifolate resistance.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Susceptibility to beta-lactam antibiotics in septicemia isolates from twenty-nine European laboratories

In 1984 the European Study Group on Antibiotic Resistance (ESGAR) consecutively collected gram-negative bacilli and staphylococci blood isolates and performed susceptibility testing with 11 antibiotics using the microdilution method. In all 2,578 isolates were collected: 68% gram-negative bacilli and 32% staphylococci. The MICs of ampicillin and cefazoline for the susceptible gram-negative bacilli were 1–8g/ml; of piperacillin0.5–4; of Sch 34343, cefotaxime, moxalactam, ceftazidime and aztreonam0.5–2g/ml; of cefoxitin, cefuroxime and cefamandole0.5–8g/ml. For susceptible staphylococci the MICs of cefazoline and cefuroxime were0.5–1g/ml, and of cefoxitin, moxalactam, ceftazidime and cefotaxime,0.5–32 g/ml. The resistance levels varied between laboratories and countries, being lower in Northern Europe. In clinical protocols on patients with gram-negative septicemia from whom cefazoline-resistant strains were isolated, cefotaxime was the beta-lactam most commonly used (12%). In protocols on patients with staphylococcal septicemia from whom gentamicin-resistant or cefazoline-resistant strains were isolated, the most commonly used beta-lactam was cloxacillin (6%).     

Susceptibility testing quality control studies with fosfomycin tromethamine

Multiple studies were performed to define the quality control ranges for susceptibility tests of fosfomycin tromethamine. For disk diffusion tests, the limits proposed are 22 to 30 mm forEscherichia coli ATCC 25922 and 25 to 33 mm forStaphylococcus aureus ATCC 25923. Broth microdilution tests were not reproducible. For agar dilution tests, ranges proposed are 0.5 to 2.0 g/ml forEscherichia coli ATCC 25922, 0.5 to 4.0 g/ml forStaphylococcus aureus ATCC 29213; 2.0 to 8.0 g/ml forPseudomonas aeruginosa ATCC 27853, and 32 to 128 g/ml forEnterococcus faecalis ATCC 29212.     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Comparison of disk diffusion,the E test,and detection ofmecA for determination of methicillin resistance in coagulase-negative staphylococci

The aim of this study was to find a reliable, fast, and simple alternative to the methicillin disk method for determination of methicillin resistance in coagulase-negative staphylococci, since results of this method are often difficult to read due to growth within the zone of inhibition. The sensitivity of 319 strains of coagulase-negative Staphylococci to a 5 g methicillin disk on Mueller-Hinton agar using an incubation period of 48 h was compared with that of 1 (1 g and 5 g oxacillin disks on Mueller-Hinton agar with or without 2% NaCl, using an incubation period of 24 h. The detection ofmecA (MecAgen) by the polymerase chain reaction was used as a standard. Minimum inhibitory concentrations were determined by means of the E test. Of the 225mecA-positive strains, 190, 215, and 193 were resistant to 5 g methicillin, 1 g oxacillin and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 216, 218, and 223 were resistant on Mueller-Hinton agar with 2% NaCl. Of the 94mecA-negative strains, 89, 93, and 94 were susceptible to 5 g methicillin, 1 g oxacillin, and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 92, 93, and 94 were susceptible on Mueller-Hinton agar with 2% NaCl. Using breakpoints of 2 g/ml for oxacillin resistance and 8 g/ml for methicillin resistance, the E test yielded sensitivities of 99.6 and 99.1% and specificities of 97.9 and 98.9% after 48 h of incubation. The 5 g oxacillin disk was faster and easier to read than the methicillin disk and correlated better with detection ofmecA than the methicillin disk or the 1 g oxacillin disk.     

In vitro activity of the two new 4-quinolones A56619 and A56620 againstNeisseria gonorrhoeae,Chlamydia trachomatis,Mycoplasma hominis,Ureaplasma urealyticum andGardnerella vaginalis

The in vitro activity of tetracycline, ciprofloxacin and two recently developed 1-aryl-fluoro-quinolones, A56610 and A56620, was tested against 65 beta-lactamase-negative and 35 betalactamase-positive Neisseria gonorrhoeae strains, 12 Chlamydia trachomatis,50 Mycoplasma hominis,28 Ureaplasma urealyticum and 50 Gardnerella vaginalis strains. In the case of Chlamydia trachomatis and Mycoplasma hominis both the MIC and the MBC were determined. The MIC90 of ciprofloxacin for Neisseria gonorrhoeae was 0.008 g/mland of A56619 and A56620 0.03 g/ml.No difference was observed between the activity against beta-lactamase-negative and beta-lactamase-positive strains. The MIC90 values of ciprofloxacin and A56620 for Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum were identical, the values being 2 g/ml, 1g/mland 4 g/mlrespectively. The MIC90 of A56619 for Chlamydia trachomatis and Ureaplasma urealyticum was 0.5 g/mland 1 g/mlrespectively. The MBC90 values of the three quinolones for Chlamydia trachomatis and Mycoplasma hominis were 2 g/ml.The activity of the quinolones against Gardnerella vaginalis was rather low, the MIC90 being 4 g/ml.It is concluded that A56619 and A56620 might be useful for single-dose therapy of gonococcal infections.     

Potentiation of antifungal activity of amphotericin B by azithromycin againstAspergillus species

The potential role of azithromycin in combination with amphotericin B against 25 clinical isolates ofAspergillus was assessed. The MIC of amphotericin B was 1 g/ml for 44% of the isolates, 0.5 g/ml for 48%, and 0.25 g/ml for 8%. All isolates were resistant to azithromycin. Synergism, defined as a twofold reduction in the MIC of both drugs upon combination, was demonstrated between amphotericin B and azithromycin for all 25 isolates. To prove that azithromycin exerts its antifungal effect by inhibiting protein synthesis, we studied [³⁵S]-methionine incorporation into protein inone Aspergillus isolate. Neither amphotericin B at 0.125 g/ml (fourfold below its MIC) nor azithromycin at 16 g/ml ( 16-fold below its MIC) had any effect on protein synthesis when tested alone. Upon combination, however, a 68% inhibition in protein synthesis was evident by the inhibition of [³⁵S]-methionine incorporation.     

Effect of praziquantel on the free-living stages ofSchistosoma mansoni

Summary The effect of the new schistosomicide praziquantel (2-cyclohexyl-carbonyl-1,2,3,6,7,11b-hexahydro-2H-pyrazino[2,1a]isoquinolin-4-one) on the miracidia and cercariae ofSchistosoma mansoni was investigated.In vivo praziquantel inhibits hatching of miracidia for 24 h after administration of 500 mg/kg to infected mice. In vitro a concentration of 10 g/ml inhibits subsequent hatching in drug-free water. Free swimming miracidia are rapidly killed by 1 g/ml. Even 0.01 g/ml is still partially effective.In a solution of 0.03 g/ml cercariae lose their ability to swim within 10 min. This effect is reversible in drug-free water. Morphological damage to cercariae incubated in 0.1 g/ml is clearly evident. However, cercariae are fully infective when given subcutaneously to mice after a 3-h incubation period. Incubation in 1 g/ml reduces the infection rate by 80%. A 2-h incubation in 0.1 g/ml almost completely inhibits the percutaneous infection through the abdominal skin. The number of cercariae that develop to schistosomules is reduced by more than 90%. After a 2-h incubation in a concentration of 0.01 g/ml the swimming ability of cercariae is impaired in such a way that the number of cercariae penetrating in the tail immersion test and developing to schistosomules is reduced by half.Praziquantel is a more potent protective agent than the molluscicides copper sulphate, sodium pentachlorophenate and Bayluscide^® or cadmium and zinc ions.

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Zusammenfassung Es wurde der Einfluß des neuen Schistosomenmittels Praziquantel (2-Cyclohexylcarbonyl-1,2,3,6,7,11b-hexahydro-2H-pyrazino[2,1 a]isochinolin-4-on) auf die Miracidien und Cercarien vonSchistosoma mansoni untersucht.In vivo verhindert Praziquatel das Schlüpfen von Miracidien aus Eiern für 24 h nach einer Behandlung infizierter Mäuse mit 500 mg/kg. Nach Inkubation von Eiern in 10 g/ml schlüpfen anschließend keine Miracidien in präparatfreiem Wasser. Frei schwimmende Miracidien werden durch 1 g/ml schnell abgetötet und noch 0,01 g/ml haben eine deutliche Wirkung.Cercarien werden in vitro durch 0,03 g/ml innerhalb von 10 min schwimmunfähig. Dieser Effekt ist in präparatfreiem Wasser reversibel. Nach 3stündiger Vorbehandlung in 0,1 g/ml sind die Cercarien deutlich morphologisch geschädigt. Trotzdem sind sie noch normal entwicklungsfähig, wenn sie Mäusen subcutan injiziert werden. Nach Inkubation in 1 g/ml ist der Infektionserfolg allerdings um 80% reduziert. Nach nur 2stündiger Inkubation in 0,1 g/ml ist die Zahl der Cercarien, die sich nach percutaner Infektion durch die Bauchhaut der Maus zu Schistosomulae entwickelt, um mehr als 90% reduziert. Nach 2stündiger Inkubation in nur 0,01 g/ml ist das Schwimmvermögen der Cercarien so beeinträchtigt, daß sich nach percutaner Schwanzinfektion nur noch halb so viele zu Schistosomulae entwickeln wie in der Kontrolle.Praziquantel ist in seiner infektionsverhindernden Wirkung den Molluskiziden Kupfersulfat, Pentachlorphenolnatrium und Bayluscid^® sowie auch Cadmium- und Zinkionen überlegen.

     

The effects of indomethacin and verapamil on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Histamine (300 M), bradykinin (2 M), prostaglandin E₂ (PGE₂) (30 M), or the leukotrienes (LT) C₄ and E₄ (1 M) but not D₄ (1 M) appliedin vitro have been shown to change the shape of endothelial cells lining the guinea pig isolated thoracic inferior vena cava. All caused the formation of inter-endothelial cell gaps. Pre-treatment with either indomethacin (100 M) or verapamil (20 M) reduced the effects of these compounds. It is suggested that indomethacin and verapamil act by reducing the amount of intracellular calcium available for the shortening of contractile protein filaments within endothelial cells.     

Inhibitory effects of E-5110 on interleukin-1 generation from human monocytes

Effects of E-5110, a novel non-steroidal antiinflammatory drug, on interleukin-1 (IL-1) generation from human monocytes were studiedin vitro. E-5110 reduced the amounts of extra- and intracellular IL-1 activity induced by lipopolysaccharide (LPS, 1 g/ml) in a dose-dependent manner (1–10M). E-5110 also inhibited the IL-1 generation induced by antigen-antibody complexes, opsonized zymosan and silica particles. It was suggested that the inhibition of IL-1 generation by E-5110 was independent of the inhibitory effects on arachidonate cyclooxygenase and/or lipoxygenase because indomethacin, piroxicam, BW755C and AA861 had no effects on IL-1 generation. Hydrocortisone (IC₅₀:0.084 M), aurothioglucose (11.5 M) and lobenzarit (75.0 M), which are clinically effective antirheumatic drugs, also inhibited IL-1 generation, like E-5110 (1.21 M). It is expected that E-5110 will be superior to classical non-steroidal antiinflammatory drugs in medical treatment of rheumatoid arthritis.