Several streptococcal strains had an uncharacterized mechanism of macrolide resistance that differed from those that had been reported previously in the literature. This novel mechanism conveyed resistance to 14- and 15-membered macrolides, but not to 16-membered macrolides, lincosamides or analogues of streptogramin B. The gene encoding this phenotype was cloned by standard methods from total genomic digests of Streptococcus pyogenes 02C1064 as a 4.7 kb heterologous insert into the low-copy vector, pACYC177, and expressed in several Escherichia coli K-12 strains. The location of the macrolide-resistance determinant was established by functional analysis of deletion derivatives and sequencing. A search for homologues in the genetic databases confirmed that the gene is a novel one with homology to membrane-associated pump proteins. The macrolide-resistance coding sequence was subcloned into a pET23a vector and expressed from the inducible T7 promoter on the plasmid in E. coli BL21(DE3). Physiological studies of the cloned determinant, which has been named mefA for macrolide efflux, provide evidence for its mechanism of action in host bacteria. E.coli strains containing the cloned determinant maintain lower levels of intracellular erythromycin when this compound is added to the external medium than isogenic clones without mefA. Furthermore, intracellular accumulation of [14C]-erythromycin in the original S. pyogenes strain was always lower than that observed in erythromycin-sensitive strains. This is consistent with a hypothesis that the gene encodes a novel antiporter function which pumps erythromycin out of the cell. The gene appears to be widely distributed in S. pyogenes strains, as demonstrated by primer-specific synthesis using the polymerase chain reaction.

The macrolide antibiotic azithromycin (CP-62,993; 9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A; also designated Zagreb, Yugoslavia]) showed a significant improvement in potency against gram-negative organisms compared with erythromycin while retaining the classic erythromycin spectrum. It was up to four times more potent than erythromycin against Haemophilus influenzae and Neisseria gonorrhoeae and twofold more potent against Branhamella catarrhalis, Campylobacter species, and Legionella species. It had activity similar to that of erythromycin against Chiamydia spp. Azithromycin was significantly more potent versus many genera of the family Enterobaeteriaceae; its MIC for 90% of strains of Escherichia, Salmonella, Shigella, and Yersinia was s4 ,ug/ml, compared with 16 to 128 ,ug/ml for erythromycin. Azithromycin inhibited the majority of gram-positive organisms at sl gg/mI. It displayed cross-resistance to erythromycin-resistant Staphylococcus and Streptococcus isolates. It had moderate activity against Bacteroides fragilis and was comparable to erythromycin against other anaerobic species. Azithromycin also demonstrated improved bactericidal activity in comparison with erythromycin. The mechanism of action of azithromycin was similar to that of erythromycin since azithromycin competed effectively for [14C]erythromycin ribosomebinding sites.Erythromycin has been regarded for many years as possessing a good spectrum of activity and safety record for the treatment of respiratory, skin, and soft tissue infections in both adults and children. Recent developments have tended to reinforce the importance of this antibiotic, as erythromycin is now the primary or secondary therapeutic agent for four-prominent infections in humans: Legionnaires disease, Mycoplasma pneumonia, Campylobacter diarrhea, and chlamydial urethritis. However, the potential of erythromycin as a general-use oral antibiotic is limited by its modest potency against Haemophilus influenzae and Neisseria gonorrhoeae and by a low and erratic level in blood following oral administration. More recently, novel formulations or esters of erythromycin have been introduced to improve its pharmacokinetic properties. Each of these has incremental advantages, but none provides the kinetic improvements sufficient to completely incorporate H. influenzae and N. gonorrhoeae into the erythromycin spectrum.Our research in this area has been aimed at identifying novel macrolide antibiotics with in vitro potency and pharmacokinetic properties that would incorporate activity against H. influenzae into the macrolide spectrum and allow for total lower doses. This paper reports the microbiological and biochemical properties of azithromycin (CP-62,993; also designated [Pliva Pharmaceuticals, Zagreb, Yugoslavia]), which differs from erythromycin chemically by a methyl-substituted nitrogen in the macrolide ring (Fig. 1). This difference produces improvements in spectrum and potency compared with erythromycin.( MATERIALS AND METHODS Antibiotics, microorganisms, and chemicals. ...

Azithromycin 993

CP-45,899 {3,3-dimethyl-7-oxo-4-thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid, 4,4-dioxide, [2S-(2a,5a)]} is an irreversible inhibitor of several bacterial penicillinases and cephalosporinases. In the presence of low concentrations of CP-45,899, ampicillin and other f,-lactams readily inhibit the growth of a variety of resistant bacteria that contain ,-lactamases. 899 In vitro susceptibility studies were performed in brain heart infusion broth as the basal medium. The broth was enriched with 5% (vol/vol) sheep blood for studies with Streptococcus pyogenes and 5% Fildes plus 2% IsoVitaleX for Haemophilus influenzae. Tests with Neisseria gonorrhoeae were performed on gonococcus agar base (BBL) supplemented with hemoglobin and IsoVitaleX. Studies with Bacteroides fragilis were carried out in prereduced brain heart infusion as described in the Anaerobe Laboratory Manual (3); incubation was in an 80% N2-10% C02-10% H2 gas mixture either in an anaerobic chamber or in GasPak jars equipped with gas-exchange capability.Methods. Minimal inhibitory concentrations (MICs) of antibiotics in combination with CP-45,899 were determined using a 7-by-7-concentration grid protocol in broth culture, as described by Sabath (8), with an inoculum of-i0 colony-forming units per ml.Testing for synergy against B. fragilis was performed by an agar dilution method similar to the broth dilution method.p8-Lactamase studies. The hydrolysis of ampicillin and penicillin G was determined by the microiodometric method of Novick (5). Cephaloridine hydrolysis was measured by following the decrease in ultraviolet absorbance at 255 nm (6). Conditions for both assays were identical: 0.5 M potassium phosphate, pH 6.5, and 37°C. Reactions were initiated by the addition of the cell-free ,B-lactamase, except in the case of preincubation experiments in which the inhibitor and enzyme were incubated together in the assay mixture for 10 min before initiation of the reaction by addition of substrate. With the cell-free extracts of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, the substrate was

A series of erythromycin A-derived semisynthetic antibiotics, featuring incorporation of a basic nitrogen atom into a ring expanded (15-membered) macrocyclic lactone, have been prepared and biologically evaluated. Semisynthetic modifications focused upon (1) varied substitution at the macrocyclic ring nitrogen and (2) epimerization or amine substitution at the C-4" hydroxyl site within the cladinose sugar. In general, the new azalides exhibit improved Gram-negative potency, expanding the spectrum of erythromycin A to fully include Haemophilus influenzae and Neisseria gonorrhoeae. Whencompared to erythromycin A, the azalides exhibit substantially increased half-life and area-under-the-curve values in all species studied. The overall in vitro/in vivo performance of TV-methyl, C-4" epimers 3a and 9; and C-4" amine ll identify these compounds as the most interesting erythromycin Asuperior agents. Compound3a has been advanced to clinical study. 1029Erythromycin A is a widely used antibiotic in oral outpatient therapy, including pediatrics. It is frequently the agent of choice for treatment of respiratory, cutaneous, Chlamydia, and Campylobacter infections. However, erythromycin A is not indicated for the treatment of Haemophilus influenzae except with co-administration of sulfonamides. Erythromycin A is also unstable at gastric pH, and is poorly absorbed with oral dosing.In our effort to expand the antimicrobial spectrum and to improve upon the pharmacokinetic properties of erythromycin A, the syntheses of erythromycin A-derived 15-membered aza-macrolides depicted in Schemes 1 and 2 were undertaken. Herein are presented the antibacterial profiles of the series, which features varied alkyl substitution at the 9a-aza site within the macrocyclic ring, and modifications at the C-4" site within the cladinose sugar. Additionally, for selected compounds, antiinfective activity against Staphylococcus aureus in mice, and pharmacokinetic profiles in several species are presented.