[Comparative antimicrobial activity of RP 59,500 (quinupristin-dalfopristin), the first semisynthetic injectable streptgramin, against gram-positive cocci and other recent clinical pathogens].

RP 59,500 (Quinupristin-Dalfopristin) is the first semisynthetic injectable streptogramin antimicrobial agent, which is a combination of quinupristin and dalfopristin in a 30:70 ratio. The components of RP 59,500 act synergically to provide bactericidal activity through action at different sites on bacterial ribosomes. In the present study, the antimicrobial activity of RP 59,500 was compared with those of four macrolides (erythromycin, clarithromycin, azithromycin, roxithromycin). Susceptibility testing was carried out by microdilution method on 303 strains of 10 species, especially antibiotic-resistant Gram-positive cocci. RP 59,500 was active against a wide range of Gram-positive cocci including methicillin-resistant Staphylococci and penicillin-resistant Streptococcus pneumoniae. The MICs90 of RP 59,500 against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis were both 0.25 microgram/ml, although those of four macrolides were higher than 32 micrograms/ml. The MICs90 of RP 59,500 against penicillin-sensitive, -intermediate and -resistant S. pneumoniae were all 0.5 microgram/ml, although those of four macrolides against penicillin-resistant S. pneumoniae were higher than 32 micrograms/ml. RP 59,500 also exhibited equivalent activities to the four macrolides against strains of Streptococcus pyogenes. Streptococcus agalactiae and Moraxella catarrhalis. RP 59,500 exhibited the highest activities against Enterococcus faecalis, Enterococcus faecium and Enterococcus avium strains which are intrinsically resistant to most antimicrobial agents. No cross-resistance was observed between RP 59,500 and the four macrolides, which will merit attention in future clinical trials of the agent. The effect of human serum on the MIC of RP 59,500 was studied with strains of S. aureus, S. epidermidis and E. faecalis. The presence of 20% (V/V) serum had little or no effect on the MIC, although 50% (V/V) serum increased MICs by 4-8 folds. Laboratory-induced resistance to RP 59,500 occurred in a stepwise fashion in broth cultures of S. aureus, S. epidermidis and E. facalis strains and the induction rate was slow and no more than four fold increases were observed. Population analysis was performed on RP 59,500 and the reference macrolides against S. aureus ATCC 25,923 strain. Although low frequencies (less than 0.01%) of resistant sub-population were detected with EM, CAM, AZM and RXM, no RP 59,500-resistant sub-population was detected in this study.

Everninomicin, a new oligosaccharide antibiotic: its antimicrobial activity, post-antibiotic effect and synergistic bactericidal activity.

Antimicrobial activity of everninomicin (SCH) 27899) in comparison with two glycopeptides (vancomycin, teicoplanin) and six beta-lactam agents was evaluated against recent clinical isolates of Gram-positive bacteria. Everninomicin showed the highest activity against the species tested and MICs90% of everninomicin against Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium and Clostridium difficile were 0.1, 0.1, 0.2, 0.39, 0.1 microgram/ml, respectively. MICs90% of everninomicin against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) were 0.78 microgram/ml. Laboratory induced resistance to everninomicin in strains of S. aureus and E. faecalis occurred in a stepwise manner and at a very slow rate. Post-antibiotic effect against strains of S. aureus and E. faecalis were 1.8 and 2.6 h, respectively, and a little longer than that of vancomycin. Adherence to glass surface of an MRSA strain was strongly repressed by the addition of sub MIC of everninomicin in combination with fosfomycin.

[Antimicrobial activity of clarithromycin and its effect on bacterial adherence to medical material].

Antimicrobial activity of clarithromycin (CAM) in comparison with other 6 macrolides was determined against 419 recent clinical isolates including Streptococci, Enterococci, Staphylococci, Moraxella, Haemophilus and Bacteroides strains. MICs80's of CAM against Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus agalactiae were 0.78, 0.10 and 0.10 microgram/ml, respectively. MICs80's against Moraxella catarrhalis and Haemophilus influenzae were 0.05 and 1.56 micrograms/ml, respectively. Laboratory-induced resistance to CAM in strains of S. pneumoniae, Enterococcus faecalis or methicillin-sensitive Staphylococcus epidermidis occurred in stepwise fashion and at a very low rate. Adherence to silicon filter of strains of Pseudomonas aeruginosa or S. epidermidis was strongly repressed by the addition of sub-MIC of CAM or other macrolides. Although tosufloxacin (TFLX) alone had no bactericidal activity against the sessile cells of P. aeruginosa strains tested, TFLX showed synergistic bactericidal activity when combined with sub-MICs of CAM or erythromycin.

A comparative study on the convulsant activity of carbapenems and beta-lactams.

Beta-lactams are well known to have potent convulsant activity. Imipenem, the first carbapenem antibiotic introduced in the clinical field, has been reported to induce convulsions. The neurochemical mechanism of the convulsions induced by carbapenems and cephalosporins were studied. Intraventricular injection of cefazolin, cephaloridine and imipenem, and of panipenem (a new carbapenem), induced convulsions in a dose-dependent manner in mice. They inhibited the receptor binding of gamma-aminobutyric acid (GABA), an inhibitory transmitter in the mammalian central nervous system. These in-vitro and in-vivo results suggest that carbapenems and cephalosporins might induce convulsions through the inhibition of GABA receptor binding when they are accumulated in the central nervous system.

In vitro activity of cefotaxime against clinically significant pathogens.

The present in vitro antibacterial activities of cefotaxime and 8 other cephalosporins (cefoperazone, cefmenoxime, cefpiramide, latamoxef, cefamandole, cefmetazole, cefotiam and cephazolin) were evaluated simultaneously in 384 strains of Gram-positive cocci, 595 strains of Enterobacteriaceae, 240 strains of non-fermenters and 143 strains of anaerobes and miscellaneous organisms. The results were expressed as minimum inhibitory concentration (MIC) range, MIC50 and MIC90. Of the beta-lactams, cefotaxime and latamoxef exhibited the highest activity against a wide variety of Gram-positive and Gram-negative bacteria. MIC90 of cefotaxime, however, for species of Pseudomonas aeruginosa, Xanthomonas maltophilia, enterococci, Bacteroides sp. and Clostridium difficile were more than 100 mg/L. Cefpiramide and cefoperazone were generally less active than these 2 agents. All strains were tested for beta-lactamase production by the cefinase disc method and the relationship of susceptibility to beta-lactams was evaluated in each species. The need was demonstrated for periodic susceptibility testing to be performed to better guide empirical antimicrobial therapy.

Correlation between susceptibility to chloramphenicol, tetracycline and clindamycin, and serogroups of Clostridium difficile.

A total of 114 Clostridium difficile strains were analysed for a possible correlation between serological susceptibility to chloramphenicol, tetracycline and clindamycin, and serogrouping. All 17 chloramphenicol-resistant strains belonged to serovar I, while none of 32 tetracycline-resistant strains belonged to serovar I. All strains included in serovar III were tetracycline- and clindamycin-resistant.

Protoplast dehydration correlated with heat resistance of bacterial spores.

Water content of the protoplast in situ within the fully hydrated dormant bacterial spore was quantified by use of a spore in which the complex of coat and outer (pericortex) membrane was genetically defective or chemically removed, as evidenced by susceptibility of the cortex to lysozyme and by permeability of the periprotoplast integument to glucose. Water content was determined by equilibrium permeability measurement with 3H-labeled water (confirmed by gravimetric measurement) for the entire spore, with 14C-labeled glucose for the integument outside the inner (pericytoplasm) membrane, and by the difference for the protoplast. The method was applied to lysozyme-sensitive spores of Bacillus stearothermophilus, B. subtilis, B. cereus, B. thuringiensis, and B. megaterium (four types). Comparable lysozyme-resistant spores, in which the outer membrane functioned as the primary permeability barrier to glucose, were employed as controls. Heat resistances were expressed as D100 values. Protoplast water content of the lysozyme-sensitive spore types correlated with heat resistance exponentially in two distinct clusters, with the four B. megaterium types in one alignment, and with the four other species types in another. Protoplast water contents of the B. megaterium spore types were sufficiently low (26 to 29%, based on wet protoplast weight) to account almost entirely for their lesser heat resistance. Corresponding values of the other species types were similar or higher (30 to 55%), indicating that these spores depended on factors additional to protoplast dehydration for their much greater heat resistance.

Germinability and heat resistance of spores of Clostridium difficile strains.

Out of 111 Clostridium difficile strains, 108 produced spores in numbers of more than 10(5)/ml and the remaining three did not produce any spores in brain heart infusion medium. The germination frequency in the medium without lysozyme varied widely from strain to strain, ranging from less than 10(-8) to 10(0), and in 77 of the 108 strains the germination frequency was 10(-5) or less. The spores, when treated with sodium thioglycollate and then inoculated into the medium containing lysozyme, germinated in all of the 108 strains at a frequency of 10(-0.5) or more. The spores of two strains germinated at a frequency of more than 10(-0.5) in all methods. Spores of C. difficile strains were fairly highly heat-resistant; D100C values ranged from 2.5 to 33.5 min.

Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers.

A variant strain that produced spores lacking exosporium was isolated from a culture of Bacillus megaterium QM-B1551. Two additional spore morphotypes were obtained from the parent and variant strains by chemical removal of the complex of coat and outer membrane. Among the four morphotype spores, heat resistance did not correlate with total water content, wet density, refractive index, or dipicolinate or cation content, but did correlate with the volume ratio of protoplast to protoplast plus cortex. The divestment of integument layers exterior to the cortex had little influence on heat resistance. Moreover, the divestment did not change the response of either the parent or the variant spores to various germination-initiating agents, except for making the spores susceptible to germination by lysozyme. The primary permeability barrier to glucose for the intact parent and variant spores was found to be the outer membrane, whereas the barrier for the divested spores was the inner membrane.

Variable sugar fermentation by clostridia.

Forty-two sugar fermentation characteristics recorded as "d" in the 8th edition of Bergey's Manual of Determinative Bacteriology were reinvestigated by using eight different peptone media and 205 strains of nine species of pathogenic clostridia. In the absence of sugar, the final pH of 7-day cultures in the basal medium varied widely depending on the peptone employed, the species tested, and even the strain of the species. In the presence of sugar, the final pH of 7-day cultures was markedly influenced by these factors. Since the sugars with reactions recorded as "d" were weakly fermented and, consequently, the fermentation reactions were easily affected by cultural variations resulting in strain instability, we employed the pH difference method (delta pH method) in which the pH difference of 0.5 between cultures with and without sugar was used as the critical level. Thirty-six (86%) of the 42 sugar reactions recorded as "d" were sorted into "+" or "-" by the delta pH method. Six sugars, however, still remained as "d" because of their extremely weak fermentation characters. The use of the delta pH method in any peptone medium not only minimizes incorrect evaluation but also can provide some distinct criteria for identification of clostridia.

Antimicrobial susceptibility of Clostridium difficile from different sources.

A total of 79 Clostridium difficile strains from healthy young and elderly adults, elderly patients without gastrointestinal disease, elderly patients receiving antibiotics without gastrointestinal complications, and elderly patients with antibiotic-associated diarrhea or pseudomembranous colitis were tested for their susceptibilities to 24 antimicrobial agents. All of the 79 strains were inhibited by low concentrations of rifampicin, metronidazole, fusidic acid, vancomycin, ampicillin, and penicillin G. Strains were highly resistant to aminoglycosides, trimethoprim, sulfamethoxazole, nalidixic acid, and cycloserine and often resistant to neomycin, cefoxitin, and cefalexin. Wide variations in the susceptibility of C. difficile strains to erythromycin, clindamycin, lincomycin, chloramphenicol, and tetracycline were found. Strains resistant to erythromycin, clindamycin, and lincomycin were more frequently found among strains isolated from elderly adults than those isolated from young adults, with particularly high frequency among strains isolated from elderly patients receiving antibiotics. None of the 23 strains isolated from healthy young adults was resistant to chloramphenicol. All of the 14 strains resistant to erythromycin, clindamycin, lincomycin, and chloramphenicol were sensitive to tetracycline and all of the 15 strains resistant to erythromycin, clindamycin, lincomycin, and tetracycline were sensitive to chloramphenicol. Only one out of 19 tetracycline, resistant strains was highly toxigenic, whereas 42 (70%) of the 60 sensitive strains were highly toxigenic.

Carbohydrate fermentation by Clostridium difficile.

Biochemical properties of Clostridium difficile were reinvestigated for the practical identification of the organism in clinical laboratories. Bacterial growth in 2% proteose peptone medium supplemented with 0.01% L-cysteine.HCl and 0.1% agar supported sufficient growth to read the fermentation results just as well as did pre-reduced anaerobically sterilized medium. Incubation for 2 days was long enough for determining the ability to ferment fructose, glucose, mannitol, mannose, melezitose, and sorbitol. All of the 82 strains liquefied 2% but not 10% gelatin. The significance of mannitol fermentation and gelatin liquefaction is stressed since C. difficile is the only species fermenting mannitol among the gelatin-liquefying species of clostridia having subterminal spores.

Identification of taurine occurring sporulating cells of Bacillus subtilis.

An unidentified ninhydrin-positive substance was detected in trichloroacetic acid extract from the sporulating cells of Bacillus subtilis NRRL B558. The substance was isolated as the corresponding 2,4-dinitrophenyl (DNP) derivative, the structure of which was identified with a pyridinium salt of DNP-taurine, based on spectral and synthetic evidence. a pyridine molecule should be introduced into a taurine molecule during the isolation procedure and hence, the original amino acid was assigned to taurine. Taurine was accumulated into the cells from the medium during the early stages os sporulation, and thereafter metabolized or excreted.

Agglutination, toxigenicity and sorbitol fermentation of Clostridium difficile.

A total of 79 Clostridium difficile strains from different sources (50 strains from the fecal specimens of healthy adults, 13 from patients receiving antibiotics without gastrointestinal complications, 13 from antibiotic-associated pseudomembranous colitis (PMC) or diarrhea patients, and three strains from ATCC) were investigated for agglutinability, using formol-treated cells as antigen, in relation to toxigenicity. C. difficile strains tested were divided into four serovars, I, II, III, and IV, by the cross-agglutination test. The agglutinin absorption test revealed that strains of serovar I, agglutinable with high titers (5,120-10,240) to antiserum prepared against a highly toxigenic C. difficile strain, ATCC 17859, possessed the serovar-specific antigen. All of the strains of serovar I were highly toxigenic and all 13 strains isolated from the fecal specimens of antibiotic-associated PMC or diarrhea patients belonged to this serovar, whereas 19 (38%) out of 50 strains from healthy adults and four (30.8%) out of 13 strains from patients receiving antibiotics without gastrointestinal complications possessed this antigen. None of the strains of other clostridial species than C. difficile were agglutinated by the three reference antisera used. Further study on the sugar fermentation test disclosed that sorbitol-fermenting property of C. difficile if very closely related to the toxigenicity and agglutinability.

Isolation of Clostridium difficile from the feces and the antibody in sera of young and elderly adults.

Attempts were made to isolate Clostridium difficile from a total of 431 fecal specimens from 149 young and 213 elderly healthy adults, and 69 elderly adults with cerebrovascular disease but no gastrointestinal disease. C difficile was isolated from 49 specimens, and the frequency of isolation was 15.4% in healthy young adults, 7.0% in healthy elderly adults, and 15.9% in elderly adults with cerebrovascular disease. Thirty-four (about 70%) of the 49 C. difficile strains isolated produced cytotoxin which was neutralized by Clostridium sordellii antitoxin in vitro; in both young and elderly adults approximately 30% of the C. difficile isolates were nontoxigenic. The mean concentration of C. difficile in feces was 10(4.1)/g in young adults and 10(4.6)/g in elderly adults, with a range of 10(2.0) to 10(6.9)/g. Antibody against C. difficile toxin was found in most of the sera obtained from young adults carrying toxigenic C. difficile, but not in sera of elderly adults, no matter how abundant was toxigenic C. difficile in the feces.