Daptomycin (LY146032) caused a calcium-dependent dissipation of the membrane potential (At) in Staphylococcus aureus without noticeably affecting the chemical gradient (ApH) across the membrane. The effect of daptomycin on membrane energization may account for many of the inhibitory effects on macromolecular biosyntheses and membrane function reported for this antibiotic. Our evidence indicates that the bactericidal activity of daptomycin is dependent on an available At.Previous studies have demonstrated effects of daptomycin (LY146032), a calcium-requiring cyclic lipopeptide antibiotic, on both peptidoglycan synthesis and membrane integrity in gram-positive bacteria (4,7,11,12). No specific step in cell wall formation sensitive to inhibition by daptomycin has been identified, but membrane disruption as evidenced by leakage of intracellular potassium (6, 7) activity against an L form of Staphylococcus aureus (4), and the calciumdependent interaction between daptomycin analogs and phospholipid vesicles (14, 15) suggests a membrane-associated target for this antibiotic.In the present study, we examined the effects of daptomycin on membrane energetics (i.e., the electrochemical proton gradient) in S. aureus under conditions where daptomycin is bactericidal. The results of this study and a preliminary report (3) indicate that daptomycin can dissipate membrane potential (At) without affecting the chemical gradient (ApH). These findings provide, for the first time, a coherent explanation for the multiplicity of antibacterial effects attributed to this antibiotic, including inhibition of peptidoglycan biosynthesis (6 added to the upper chamber after an additional 15 min of incubation. The total volume of the reaction mix in the upper chamber was 0.5 ml; the volume of the lower chamber was 1.0 ml. Two-milliliter fractions were collected during the course of the experiment from the lower chamber at a flow rate of 2 ml/min. Radioactivity was determined from 1-ml aliquots mixed with 9 ml of Aquassure (DuPont) counted in a Beckman model LS3811 liquid scintillation counter.In some experiments, steady-state distribution data from the

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A total of 2,811 clinical isolates of Haemopbilus influenzae were obtained during 1986 from 30 medical centers and one nationwide private independent laboratory in the United States. Among these, 757 (26.9%) were type b strains. The overall rate of 3-lactamase-mediated ampicillin resistance was 20.0%. Type b strains were approximately twice as likely as non-type b strains to produce (-lactamase Antimicrobial resistance among clinical isolates of Haemophilus influenzae has become an increasingly prevalent problem (G. V. Doern, Antimicrob. Newsl. 5: [28][29][30][31][32][33][34] 1986). In a national collaborative study conducted in 1984, 15.2% of a large number of strains of H. influenzae produced Ilactamase (6). The problem of ampicillin resistance is complicated by recent descriptions of clinical isolates of H. influenzae that are resistant to ampicillin by mechanisms other than the production of a TEM-type P-lactamase (10,11,14). In addition, chloramphenicol resistance has now been reported (2,15), as has resistance to a variety of alternative agents commonly used to treat Haemophilus infections (Doern, Antimicrob. Newsl. 5:28-34). The intent of this investigation was to define systematically the prevalence of antimicrobial resistance among clinical isolates of H. influenzae in the United States. Rates of P-lactamase production and the activities of 12 antimicrobial agents were assessed. These agents included ampicillin, chloramphenicol, cefamandole, cefaclor, cephalothin, cephalexin, tetracycline, rifampin, erythromycin, sulfisoxazole, and the combinations erythromycin-sulfisoxazole and trimethoprim-sulfamethoxazole (TMP-SMX). MATERIALS AND METHODSStudy centers. A total of 30 hospital-based microbiology laboratories and 1 national, private, independent laboratory participated in the study (Table 1) by the laboratories listed in Table 1. All isolates were recovered from different patients and were randomly selected for inclusion in the study. After being characterized in study center laboratories, isolates were subcultured to chocolate agar slants (GIBCO Diagnostics, Madison, Wis.), which were incubated overnight in a CO2 atmosphere and then mailed, with selected patient demographic information, to one of two coordinating study centers for further characterization. The coordinating study centers were the Department of Clinical Microbiology, University of Massachusetts Medical Center, Worcester, and the Department of Pathology, University of Texas Health Science Center, San Antonio. Upon receipt in the coordinating study centers, growth from slants was transferred into 10% sterile skim milk and frozen at -70°C in 1-dram (ca. 3.7-ml) plastic freezer vials.Isolate characterization. Frozen stock suspensions were thawed, and aliquots were subcultured to chocolate agar plates (GIBCO) which were incubated overnight at 35°C in 5 to 7% CO2. Individual isolated colonies were then subcultured to a second chocolate agar plate which was incubated under identical conditions. Growth from the second plate was used for the fol...

Vancomycin resistance in Enterococcus faecium 180, a clinical isolate from England, was studied. Resistance to vancomycin was transferable by conjugation to other enterococci. Expression of resistance was inducible and coincided with the appearance of a new membrane protein.

Certain derivatives of the glycopeptide antibiotic LY264826 with N-alkyl-linked substitutions on the epivancosamine sugar are active against glycopeptide-resistant enterococci. Six compounds representing our most active series were evaluated for activity against antibiotic-resistant, gram-positive pathogens. For Enterococcus faecium and E. faecalis resistant to both vancomycin and teicoplanin, the MICs of the six semisynthetic compounds for 90% of the strains tested were 1 to 4 micrograms/ml, compared with 2,048 micrograms/ml for vancomycin and 256 micrograms/ml for LY264826. For E. faecium and E. faecalis resistant to vancomycin but not teicoplanin, the MICs were 0.016 to 1 micrograms/ml, compared with 64 to 1,024 micrograms/ml for vancomycin. The compounds were highly active against vancomycin-susceptible enterococci and against E. gallinarum and E. casseliflavus and showed some activity against isolates of highly vancomycin-resistant leuconostocs and pediococci. The MICs for 90% of the strains of methicillin-resistant Staphylococcus aureus tested were typically 0.25 to 1 micrograms/ml, compared with 1 microgram/ml for vancomycin. Against methicillin-resistant S. epidermidis MICs ranged from 0.25 to 2 micrograms/ml, compared with 1 to 4 micrograms/ml for vancomycin and 4 to 16 micrograms/ml for teicoplanin. The spectrum of these new compounds included activity against teicoplanin-resistant, coagulase-negative staphylococci. The compounds exhibited exceptional potency against pathogenic streptococci, with MICs of < or = 0.008 microgram/ml against Streptococcus pneumoniae, including penicillin-resistant isolates. In in vivo studies with a mouse infection model, the median effective doses against a challenge by S. aureus, S. pneumoniae, or S. pyogenes were typically 4 to 20 times lower than those of vancomycin. Overall, these new glycopeptides, such as LY307599 and LY333328, show promise for use as agents against resistant enterococci, methicillin-resistant S. aureus, and penicillin-resistant pneumococci.

A54145 complex is made up of eight factors; A, A1? B, B1? C, D, E, and F which were active in vitro (MIC 0.25~> 32/ig/ml) against Gram-positive aerobic organisms. The complex, factors B and Bj were found to be active against two strains of Clostridium perfringens. A calcium dependence study on someof the factors showed that their in vitro antibacterial activity was greatly enhanced by the presence of calcium (50mg/liter) in the media. Resistance build-up was seen when Staphylococcus sp. and Streptococcus sp. were passed seven times in the presence of sublethal concentrations of A54145antibiotics. This resistance disappeared immediately when the resistant organisms were passed in the absence of the antibiotics. Factor A was very effective against Staphylococcus aureus and Streptococcuspyogenes infections in mice (sc ED5Osof 3.3~2.4 mg/kg x 2, respectively). Factor B was more active against S. pyogenes in vivo (sc ED50, 0.9mg/kg x 2). Acute mousetoxicities were determined with these antibiotics. Semisynthetic derivatives were evaluated.

A polymerase chain reaction (PCR)-based test was developed for the detection of mecA in staphylococci. To facilitate this process, a rapid cell lysis procedure was established for the release of DNA from staphylococcal strains. Primers based on the DNA sequence of the mecA gene from Staphylococcus aureus were used in PCRs to screen for the presence of this gene in a total of 98 staphylococcal isolates. Fifty-one isolates were mecA positive (17 S. aureus strains and 34 coagulase-negative staphylococci including S. epidermidis, S. haemolyticus, and S. simulans). Results obtained with PCRs were generally consistent with those of standard microbiological assays. PCRs designed to detect thefemA gene (factor essential for methicillin resistance) revealed the presence of the gene in all S. aureus strains examined regardless of the susceptibility profiles of the strains to methicillin. In contrast,femA could not be detected in coagulase-negative staphylococci by PCR with the same primers. Low-stringency hybridization suggested the presence of a gene structurally related tofemA in S. epidermidis and other coagulase-negative staphylococci examined.

Three new cephalosporin antibiotics, prepared by substitution of heterocyclic groups on 7-aminocephalosporanic acid, possess