RWJ-54428 (MC-02,479), a new cephalosporin with high affinity for penicillin-binding proteins, including PBP 2a, and stability to staphylococcal beta-lactamases.

RWJ-54428 (MC-02,479) is a new cephalosporin active against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The potency of this new cephalosporin against MRSA is related to a high affinity for penicillin-binding protein 2a (PBP 2a), as assessed in a competition assay using biotinylated ampicillin as the reporter molecule. RWJ-54428 had high activity against MRSA strains COL and 67-0 (MIC of 1 micro g/ml) and also showed affinity for PBP 2a, with a 50% inhibitory concentration (IC(50)) of 0.7 micro g/ml. RWJ-54428 also displayed excellent affinity for PBP 5 from Enterococcus hirae R40, with an IC(50) of 0.8 micro g/ml and a MIC of 0.5 micro g/ml. The affinity of RWJ-54428 for PBPs of beta-lactam-susceptible S. aureus (MSSA), enterococci (E. hirae), and Streptococcus pneumoniae showed that the good affinity of RWJ-54428 for MRSA PBP 2a and E. hirae PBP 5 does not compromise its binding to susceptible PBPs. RWJ-54428 showed stability to hydrolysis by purified type A beta-lactamase isolated from S. aureus PC1. In addition, RWJ-54428 displayed low MICs against strains of S. aureus bearing the four classes of staphylococcal beta-lactamases, including beta-lactamase hyperproducers. The frequency of isolation of resistant mutants to RWJ-54428 from MRSA strains was very low. In summary, RWJ-54428 has high affinity to multiple PBPs and is stable to beta-lactamase, properties that may explain our inability to find resistance by standard methods. These data are consistent with its excellent activity against beta-lactam-resistant gram-positive bacteria.

Activation of temperature-dependent flagellar movement of demembranated fowl spermatozoa: involvement of an endogenous serine protease.

In the presence of ATP, the motility of demembranated fowl spermatozoa was vigorous at 30 degrees C, but negligible at 40 degrees C. Motility could be restored at 40 degrees C by the addition of 10-100 ng trypsin ml-1. Chymotrypsin also stimulated the motility, but neither papain nor carboxypeptidase B appreciably affected motility. Conversely, at 30 degrees C sperm motility was inhibited by aprotinin or phenylmethylsulfonyl fluoride. These results suggest that endogenous protease, presumably serine protease, activity is instrumental in the regulation of fowl sperm motility. It seems likely that the site of action of this protease is axonemal, but a direct effect of added protease on dynein ATPase activity could not be demonstrated.

Effects of beta-lactamase-mediated antimicrobial resistance: the role of beta-lactamase inhibitors.

Production of beta-lactamase is the most common mechanism of bacterial resistance to beta-lactam antibiotics. Virtually all bacteria have the capability of synthesizing the enzyme. Microorganisms may already possess the native genetic information necessary for beta-lactamase production (i.e., chromosomal), or may acquire the capacity by transfer of DNA from another organism (i.e., plasmid-mediated). The level of beta-lactamase production may be stable and noninducible (constitutive enzyme production), or may be stimulated on exposure to selected beta-lactam antibiotics (inducible enzyme production). Inhibitors such as clavulanic acid and sulbactam prevent antibiotic degradation by the beta-lactamases of many clinically significant pathogens. Therefore, currently available beta-lactam-beta-lactamase-inhibitor combinations exhibit broad spectra of in vitro activity. Ticarcillin-clavulanate possesses clinically significant activity against many bacteria, including streptococci, Staphylococcus aureus, Bacteroides fragilis, and numerous Enterobacteriaceae. Amoxicillin-clavulanate and ampicillin-sulbactam demonstrate clinically significant activity against streptococci (including enterococci), S. aureus, B. fragilis, and some Enterobacteriaceae. Ticarcillin-clavulanate is indicated for treatment of serious infections, including septicemia. Amoxicillin-clavulanate is useful in the treatment of upper respiratory, urinary tract, and skin and soft tissue infections. Ampicillin-sulbactam may be used for treatment of intraabdominal, gynecologic, urinary tract, and skin and soft tissue infections.

The mechanism of action of penicillin. Penicillin acylates the active site of Bacillus stearothermophilus D-alanine carboxypeptidase.

Penicillin kills susceptible bacteria by specifically inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan. It was hypothesized (Tipper, D., and Strominger, J. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141) that 1) penicillin is a structural analog of the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan, and that 2) penicillin, by virtue of its highly reactive beta-lactam structure, irreversibly acylates the active site of the cell wall transpeptidase. Although the cell wall transpeptidase has proven elusive, a closely related penicillin-sensitive cell wall enzyme, D-alanine carboxypeptidase, has been purified from membranes of Bacillus stearothermophilus by penicillin affinity chromatography. By amino acid sequence analysis of 14C-labeled cyanogen bromide peptides generated and purified from this carboxypeptidase covalently labeled with either [14C]penicillin G or the substrate, [14C]diacetyl-L-lysyl-D-alanyl-D-lactate, it was shown that the penicillin and substrate were both bound as esters to a serine at residue 36. Therefore, the second hypothesis stated above was proven to be correct for D-alanine carboxypeptidase. Several new methods were developed in the course of this work, including 1) a rapid penicillin-binding assay, 2) use of hydroxylamine to protect peptides against carbamylation during ion exchange chromatography in concentrated urea solutions, and 3) gel filtration chromatography in 70% formic acid, a universal solvent for peptides.