Arthur E. Girard

Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against gram-negative organisms

The macrolide antibiotic azithromycin (CP-62,993; 9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A; also designated XZ-450 [Pliva Pharmaceuticals, 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 Chlamydia spp. Azithromycin was significantly more potent versus many genera of the family Enterobacteriaceae; its MIC for 90% of strains of Escherichia, Salmonella, Shigella, and Yersinia was less than or equal to 4 micrograms/ml, compared with 16 to 128 micrograms/ml for erythromycin. Azithromycin inhibited the majority of gram-positive organisms at less than or equal to 1 micrograms/ml. 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.

CP-45,899, a Beta-Lactamase Inhibitor That Extends the Antibacterial Spectrum of Beta-Lactams: Initial Bacteriological Characterization

CP-45,899 {3,3-dimethyl-7-oxo-4-thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid, 4,4-dioxide, [2S-(2α,5α)]} is an irreversible inhibitor of several bacterial penicillinases and cephalosporinases. In the presence of low concentrations of CP-45,899, ampicillin and other β-lactams readily inhibit the growth of a variety of resistant bacteria that contain β-lactamases. CP-45,899 used alone displays only weak antibacterial activity, with the notable exception of its potent effects on susceptible and resistant strains of Neisseria gonorrhoeae. CP-45,899 appears to be somewhat less potent but markedly more stable (in aqueous solution) than the recently described β-lactamase inhibitor clavulanic acid. The spectrum extensions provided by the two compounds are similar. A 1:1 mixture of CP-45,899 and ampicillin displays marked antimicrobial activity in mice experimentally infected with ampicillin-resistant Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumoniae, and Proteus vulgaris.

Pharmacokinetic and in vivo studies with azithromycin (CP-62,993), a new macrolide with an extended half-life and excellent tissue distribution.

Azithromycin (CP-62,993), a new acid-stable 15-membered-ring macrolide, was well absorbed following oral administration in mice, rats, dogs, and cynomolgus monkeys. This compound exhibited a uniformly long elimination half-life and was distributed exceptionally well into all tissues. This extravascular penetration of azithromycin was demonstrated by tissue/plasma area-under-the-curve ratios ranging from 13.6 to 137 compared with ratios for erythromycin of 3.1 to 11.6. The significance of these pharmacokinetic advantages of azithromycin over erythromycin was shown through efficacy in a series of animal infection models. Azithromycin was orally effective in treating middle ear infections induced in gerbils by transbulla challenges with amoxicillin-resistant Haemophilus influenzae or susceptible Streptococcus pneumoniae; erythromycin failed and cefaclor was only marginally active against the H. influenzae challenge. Azithromycin was equivalent to cefaclor and erythromycin against Streptococcus pneumoniae. In mouse models, the new macrolide was 10-fold more potent than erythromycin and four other antibiotics against an anaerobic infection produced by Fusobacterium necrophorum. Similarly, azithromycin was effective against established tissue infections induced by Salmonella enteritidis (liver and spleen) and Staphylococcus aureus (thigh muscle); erythromycin failed against both infections. The oral and subcutaneous activities of azithromycin, erythromycin, and cefaclor were similar against acute systemic infections produced by Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans, or S. aureus, whereas azithromycin was more potent than erythromycin and cefaclor against the intracellular pathogen Listeria monocytogenes. The pharmacokinetic advantage of azithromycin over erythromycin in half-life was clearly demonstrated in prophylactic treatment of an acute mouse model of S. aureus infection. These properties of azithromycin strongly support the further evaluation of this new macrolide for use in community-acquired infections of skin or soft tissue and respiratory diseases.

In vivo efficacy of trovafloxacin (CP-99,219), a new quinolone with extended activities against gram-positive pathogens, Streptococcus pneumoniae, and Bacteroides fragilis.

The interesting in vitro antimicrobial activity and pharmacokinetics of the new quinolone trovafloxacin (CP-99,219) warranted further studies to determine its in vivo efficacy in models of infectious disease. The significance of the pharmacokinetic and in vitro antimicrobial profiles of trovafloxacin was shown through efficacy in a series of animal infection models by employing primarily oral therapy. Against acute infections, trovafloxacin was consistently more effective than temafloxacin, ciprofloxacin, and ofloxacin against Streptococcus pneumoniae and other gram-positive pathogens while maintaining activity comparable to that of ciprofloxacin against gram-negative organisms. In a model of murine pneumonia, trovafloxacin was more efficacious than temafloxacin, while ciprofloxacin failed against S. pneumoniae (50% protective doses, 2.1, 29.5, and >100 mg/kg, respectively). In addition to its inherent in vitro potency advantage against S. pneumoniae, these data were supported by a pharmacokinetic study that showed levels of trovafloxacin in pulmonary tissue of S. pneumoniae-infected CF1 mice to be considerably greater than those of temafloxacin and ciprofloxacin (twice the maximum drug concentration in serum; two to three times the half-life, and three to six times the area under the concentration-time curve). Against localized mixed anaerobic infections, trovafloxacin was the only agent to effectively reduce the numbers of recoverable CFU of Bacteroides fragilis ( >1,000-fold), Staphylococcus aureus (1,000-fold), and Escherichia coli ( >100-fold) compared with ciprofloxacin, vancomycin, metronidazole, clindamycin, cefoxitin, and ceftriaxone. The in vitro and in vivo antimicrobial activities of trovafloxacin and its pharmacokinetics in laboratory animals provide support for the ongoing and planned human phase II and III clinical trials.

CP-45,899 in combination with penicillin or ampicillin against penicillin-resistant Staphylococcus, Haemophilus influenzae, and Bacteroides.

CP-45,899 is a new, semisynthetic beta-lactamase inhibitor. When tested alone, CP-45,899 displayed only weak antibacterial activity, with the notable exception of its potent action against penicillin-susceptible and -resistant Neisseria gonorrhoeae. A combination of 3.12 microgram of CP-45,899 per ml with 3.12 microgram of ampicillin per ml, tested in broth cultures, inhibited ca. 90% of resistant Staphylococcus and Haemophilus influenzae strains; similar data were obtained in a variety of media. The same combination of CP-45,899 with ampicillin or penicillin G inhibited 90% of Bacteroides fragilis as interpreted from agar dilution minimal inhibitory concentrations. Inhibitory concentrations of CP-45,899-ampicillin were bactericidal against H. influenzae strains and were as bactericidal as nafcillin or cephalothin against S. aureus. Ampicillin-resistant S. aureus, H. influenzae, and B. fragilis strains did not develop resistance to CP-45,899-ampicillin when transferred as many as six passages in the presence of a sublethal concentration of the combination.

Influence of Subtherapeutic Levels of Oxytetracycline on Salmonella typhimurium in Swine, Calves, and Chickens

Subtherapeutic levels of oxytetracycline in animal feeds have been evaluated to determine their influence on the relative quantity, prevalence, shedding, and antibiotic susceptibility of Salmonella typhimurium in swine, calves, and chickens, when compared with nonmedicated controls. The medicated groups were fed rations containing oxytetracycline commencing 5 days prior to oral inoculation with S. typhimurium and continuing through a 28-day post-inoculation period. Colonization of S. typhimurium occurred in all three animal species as evidenced by clinical signs of infection and/or colony counts in feces measured on seven separate occasions over the 28-day observation period. The accumulated data demonstrate that the subtherapeutic use of oxytetracycline did not bring about any increases in the quantity, prevalence, or shedding of S. typhimurium in swine, calves, and chickens. In fact, the medication generally brought about a decrease in the percentage of animals carrying S. typhimurium during the study period. In contrast to results in swine and calves, there was a significant occurrence of S. typhimurium resistance to oxytetracycline in chickens. Resistant colonies were isolated from chickens sporadically but never on more than two consecutive test periods. These isolates were also resistant to streptomycin, but not to the other six antibiotics tested. The population of resistant S. typhimurium isolated from medicated chickens was no larger than that of susceptible S. typhimurium isolated from the nonmedicated animals. It is concluded that no evidence has been obtained which would relate the continuous low-level feeding of oxytetracycline for a 4-week period to an increased incidence of disease in animals or as a hazard to humans.

Effects of environmental factors on the in vitro potency of azithromycin

The effects of media, pH, cations, serum, CO2 or anaerobic atmosphere, inoculum size and time of incubation on the in vitro potency of azithromycin were determined. The potency of azithromycin against all genera was particularly sensitive to changes in pH. The MIC forStaphylococcus aureus strains ranged from 50 µg/ml at pH 6 to ≤ 0.025 µg/ml at pH 8; for erythromycin the MIC change was less (1.6 to 0.05 µg/ml). Incubation for 18 h in 5 % CO2 or an anaerobic atmosphere (10 % CO2, 10 % H2, 80 % N2) lowered the pH by approximately 0.8 units with gram-negative organisms and 0.4 units with gram-positive organisms. This resulted in an MIC eight times greater than the aerobic MIC. In addition, the MIC100 for azithromycin and erythromycin againstBacteroides strains growing in Wilkins-Chalgren broth fell from 3.1 µg/ml in the anaerobic atmosphere to 0.2 and 0.4 µg/ml, respectively, when using the Oxyrase enzyme system to remove oxygen. With the Oxyrase system, the pH of the medium at the MIC remained at 7.2, while it fell to 6.7 in the anaerobic gas mixture. An increase in potency for both agents was also observed with other anaerobic species when using the Oxyrase system. The addition of serum produced an increase in potency of azithromycin and erythromycin that correlated with an increase in pH during incubation, despite the use of buffered media. Adding cations to Mueller-Hinton broth resulted in increased MICs for gram-negative organisms; the highest increases observed were four-fold forEscherichia coli. The activity of control antibiotics was not affected to the same degree as that of azithromycin. Increasing the incubation period from 24 to 48 h did not change the MIC values of azithromycin forStaphylococcus aureus orEscherichia coli; however, the MBC values were lower at 48 h and equalled the MIC values. Inoculum size or manner of preparation had no significant effect on the potency of azithromycin.

In vitro activity of CP-65,207, a new penem antimicrobial agent, in comparison with those of other agents.

CP-65,207 is a new parenteral penem antibiotic with a broad spectrum that includes gram-positive, gram-negative, and anaerobic microorganisms, with MICs for 90% (MIC90s) of the majority of 1,101 clinical pathogens tested being less than or equal to 1 microgram/ml. The compound was from 10- to 100-fold more active than cefoxitin and broad-spectrum cephalosporins against gram-positive bacteria and anaerobes. CP-65,207 was less active than imipenem for staphylococci, group A streptococci, and Enterococcus faecalis. Against members of the family Enterobacteriaceae, CP-65,207 was in general 100-fold more active than cefoxitin, 5- to 10-fold more active than broad-spectrum cephalosporins, and 2-fold more active than imipenem. Fresh clinical isolates that were resistant to broad-spectrum cephalosporins were highly susceptible to CP-65,207 and imipenem (MIC90, 1 microgram/ml). Isolates of Enterococcus faecalis, Serratia marcescens, and anaerobic Peptococcus spp. had MIC90s of 8, 2, and 3.12 micrograms/ml, respectively. CP-65,207 was not very active against methicillin-resistant staphylococci or Pseudomonas aeruginosa. Killing kinetics showed that against some strains CP-65,207 is rapidly bactericidal at concentrations well below those required to achieve a similar degree of killing with cefotaxime, ceftazidime, and ceftriaxone. CP-65,207 was only slightly susceptible to hydrolysis by type I cephalosporinases and TEM-1, SHV-1, and PSE-2 plasmid-encoded enzymes. It had the highest affinity for penicillin-binding proteins 2, 1A, 1B, and 3 in cell-free preparations of Escherichia coli W-7. Images

Influence of Subtherapeutic Levels of a Combination of Neomycin and Oxytetracycline on Salmonella typhimurium in Swine, Calves, and Chickens

Subtherapeutic levels of oxytetracycline plus neomycin in animal feeds did not bring about increases in the quantity, prevalence, or shedding of Salmonella typhimurium in swine, calves, or chickens. In fact, the medication generally reduced the proportion of animals carrying S. typhimurium. The medicated groups were fed rations containing oxytetracycline plus neomycin commencing 5 days prior to oral inoculation with S. typhimurium and continuing through a 28-day postinoculation period. Colonization of S. typhimurium occurred in all three animal species, as evidenced by clinical signs of infection and/or colony counts in feces. Only from swine and on only one occasion was a single resistant colony isolated. It is concluded that no evidence has been obtained which would implicate the continuous low-level feeding of oxytetracycline and neomycin for a 4-week period to a potential increased incidence of disease in animals or as a hazard to humans.

Lack of emergence of significant resistance in vitro and in vivo to the new azalide antibiotic azithromycin

In vitro experiments were performed in which 6 to 12 strains ofStaphylococcus aureus, Streptococcus pyogenes, Haemophilus influenzae andEnterobacteriaceae were passaged nine times in sub-lethal concentrations of azithromycin or control antibiotics.Streptococcus pyogenes andStaphylococcus aureus quickly became resistant to rifampin as the MIC90 increased from 0.1 to > 50 µg/ml for both species. The MIC90 of azithromycin, erythromycin, amoxicillin and cefaclor increased by three dilutions forStaphylococcus aureus. The MIC values of azithromycin forStreptococcus pyogenes, Haemophilus influenzae andEnterobacteriaceae strains did not change significantly. However, forHaemophilus influenzae and theEnterobacteriaceae strains, the MIC values of erythromycin and oral cephalosporins increased four-fold. In the in vivo experiments, mice infected withStaphylococcus aureus orEscherichia coli contaminated sutures were administered azithromycin for three days, and on day 6 viable bacterial cells were recovered from the infection site. The sustained tissue concentrations of azithromycin indicated that the organisms would have been continuously exposed to azithromycin at the site of infection. Colonies isolated from azithromycin-treated and non-treated mice were cultured and their susceptibility to azithromycin compared. The azithromycin MIC values forStaphylococcus aureus cultures from treated and non-treated animals were identical. The azithromycin MICs forEscherichia coli recovered from treated animals were on average, less than one dilution higher than for control cultures. Emergence of significant resistance to azithromycin in the laboratory was not observed with the pathogens tested.