Tetracyclines were discovered in the 1940s and exhibited activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. They are inexpensive antibiotics, which have been used extensively in the prophlylaxis and therapy of human and animal infections and also at subtherapeutic levels in animal feed as growth promoters. The first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated in 1953. Tetracycline resistance now occurs in an increasing number of pathogenic, opportunistic, and commensal bacteria. The presence of tetracycline-resistant pathogens limits the use of these agents in treatment of disease. Tetracycline resistance is often due to the acquisition of new genes, which code for energy-dependent efflux of tetracyclines or for a protein that protects bacterial ribosomes from the action of tetracyclines. Many of these genes are associated with mobile plasmids or transposons and can be distinguished from each other using molecular methods including DNA-DNA hybridization with oligonucleotide probes and DNA sequencing. A limited number of bacteria acquire resistance by mutations, which alter the permeability of the outer membrane porins and/or lipopolysaccharides in the outer membrane, change the regulation of innate efflux systems, or alter the 16S rRNA. New tetracycline derivatives are being examined, although their role in treatment is not clear. Changing the use of tetracyclines in human and animal health as well as in food production is needed if we are to continue to use this class of broad-spectrum antimicrobials through the present century.

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

In 1998 we updated earlier descriptions of the largest family of secondary transport carriers found in living organisms, the major facilitator superfamily (MFS). Seventeen families of transport proteins were shown to comprise this superfamily. We here report expansion of the MFS to include 29 established families as well as five probable families. Structural, functional, and mechanistic features of the constituent permeases are described, and each newly identified family is shown to exhibit specificity for a single class of substrates. Phylogenetic analyses define the evolutionary relationships of the members of each family to each other, and multiple alignments allow definition of family-specific signature sequences as well as all wellconserved sequence motifs. The work described serves to update previous publications and allows extrapolation of structural, functional and mechanistic information obtained with any one member of the superfamily to other members with limitations determined by the degrees of sequence divergence.

The major facilitator superfamily.

Dangerous, antibiotic resistant bacteria have been observed with increasing frequency over the past several decades. In this review the factors that have been linked to this phenomenon are addressed. Profiles of bacterial species that are deemed to be particularly concerning at the present time are illustrated. Factors including economic impact, intrinsic and acquired drug resistance, morbidity and mortality rates, and means of infection are taken into account. Synchronously with the waxing of bacterial resistance there has been waning antibiotic development. The approaches that scientists are employing in the pursuit of new antibacterial agents are briefly described. The standings of established antibiotic classes as well as potentially emerging classes are assessed with an emphasis on molecules that have been clinically approved or are in advanced stages of development. Historical perspectives, mechanisms of action and resistance, spectrum of activity, and preeminent members of each class are discussed.

Antibiotics and Bacterial Resistance in the 21st Century

A primary goal in organ transplantation is the prevention or effective treatment of infection, the most common life-threatening complication of long-term immunosuppressive therapy. The challenges involved in achieving this goal are several: a broad range of potential sources of infection ranging from latent viruses to pathogens of both community and hospital origin; immunosuppression-induced impairment of the inflammatory response, which attenuates the signs and symptoms of invasive infection; and the adverse effects of the antimicrobial drugs used for prophylaxis and therapy, which result both from the duration of therapy required and from interactions with the immunosuppressive drugs cyclosporine and tacrolimus. Our . . .

Infection in Organ-Transplant Recipients

The influence of natural products upon drug discovery.

Infection models were used to clarify the roles of serum and extravascular concentrations in the in-vivo efficacy observed with azithromycin. In-vivo experiments were designed to give serum concentrations well below the MIC and tissue levels generally above the MIC at time of challenge and during the course of infection. The efficacy of azithromycin against a Salmonella enteritidis oral challenge (a tissue-associated infection model) in mice correlated directly with azithromycin liver levels, but not serum concentrations. The significance of extravascular pharmacokinetics was observed in a comparative study of azithromycin and ciprofloxacin against the salmonella challenge. Ciprofloxacin has a greater than 100-fold in-vitro potency advantage over azithromycin against this organism, but azithromycin (5 mg/kg) produced a greater reduction in cfu than ciprofloxacin (100 mg/kg) at the primary site of infection (liver). In another model, extravascular fluid levels, measured by bioassay of implanted paper discs, were compared with plasma levels in relation to control of a localized Staphylococcus aureus infection in rats. Extravascular fluid levels of azithromycin were greater than the MIC of the strain used for five days after a 100 mg/kg dose, while erythromycin levels were less than 20% of the MIC at 30 h after a 200 mg/kg dose. Serum concentrations of both compounds were less than 20% of the MIC at the time of challenge. The antibiotic levels at the site of infection correlated with the reduction of Staph. aureus cfu (99% with azithromycin compared with controls, P less than 0.01; 0% with erythromycin) recovered from inoculated discs. The significance of extravascular concentrations of azithromycin was further supported in other models of localized infections induced with Escherichia coli or a mixture of Staph. aureus and Bacteroides fragilis.

Correlation of the extravascular pharmacokinetics of azithromycin with in-vivo efficacy in models of localized infection

Measurement of killing kinetics of azithromycin against strains of Streptococcus pneumoniae and Klebsiella pneumoniae in vitro showed that it had a limited bactericidal activity (greater than 90% kill) for the first eight hours of incubation, but developed complete bactericidal activity (greater than 99.9% kill) by 24 h incubation. Since high and sustained tissue levels of azithromycin occur in animals and humans, it was proposed that it might produce a bactericidal effect in vivo. This was demonstrated in a lung infection model in mice, designed to mimic the in-vitro killing studies. A 25 mg/kg dose of azithromycin given 24 h before intranasal challenge reduced the recoverable Str. pneumoniae population by greater than 99.9%, in comparison with untreated controls. Erythromycin did not produce a bactericidal effect at 100 mg/kg, and roxithromycin only reduced the viable count by 96%, at a dose of 50 mg/kg. Against a K. pneumoniae lung infection, a 50 mg/kg dose of azithromycin reduced the bacterial count by 99%. The bactericidal effect was correlated with lung tissue concentrations of azithromycin. In a proliferating Escherichia coli paper disc infection model, extravascular fluid concentrations of azithromycin were correlated with a 99.9% reduction in bacterial count, while corresponding serum concentrations were always less than the MIC. Dosing with azithromycin eradicated Haemophilus influenzae from the bulla (middle ear) of gerbils, as was not the case with erythromycin and roxithromycin. This effect was correlated with the antibiotic concentration in bulla lavage.

Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo

The possibility of augmentation of azithromycin delivery to infection loci was evaluated by the use of Staphylococcus aureus thigh infection models with CD-1 mice. The intramuscular infections that developed were characterized by rapid growth of bacteria and induction of a localized oedema that was assessed gravimetrically. Microscopic examination of infected thighs showed massive infiltration of polymorphonuclear leucocytes (viable and degranulated), when compared to saline-injected thighs, from 24 to > or = 72 h after infection. Azithromycin concentrations were enhanced significantly (P < or = 0.02) in infected thigh tissues compared with contralateral non-infected tissues, and correlated with oedema from 24-72 h after challenge and dosing. The azithromycin levels in infected tissue after a 5 mg/kg dose were sufficient to cause a significant reduction in the number of cfu. If azithromycin administration was delayed until inflammation was more severe, the result was an even greater preferential concentration of azithromycin into the infected thigh. Preferential concentration of azithromycin was not observed when extensive oedema was produced by injection of histamine. However, this oedema was not associated with a significant influx of polymorphonuclear leucocytes. In comparative studies, macrolide antibiotics known to be concentrated in phagocytes, such as erythromycin, roxithromycin, and clarithromycin, were not concentrated preferentially in infected tissues under the experimental conditions used; tissue levels were above or at the in-vitro MIC level for < or = 24 h. The data indicate that delivery of biologically available azithromycin to infected tissues is enhanced by cellular inflammatory processes.

Preferential concentration of azithromycin in an infected mouse thigh model

Because of its prevalence and severity, Babesia microti infection is an important public health problem. The current treatment of choice is clindamycin plus quinine. However, in some cases other treatments are needed because of drug intolerance or relapse. The activity of azithromycin was investigated for treatment of babesiosis in the hamster model. All animals received vancomycin to prevent antibiotic-associated colitis. Quinine (250 mg/kg/day), azithromycin (150 mg/kg/day), and the combination of azithromycin and quinine were compared. A significant suppression of parasitemia was found in all treatment groups (combination had the greatest effect, followed by azithromycin, then quinine; P < .05). The mean survival was significantly prolonged in the combination group (P < .05). Azithromycin as monotherapy in a higher dose (300 mg/kg/day) also resulted in a significant prolongation of survival (P < .05). Spirogermanium and ciprofloxacin, which have been reported to have antimalarial activity, had no effect on parasitemia or survival in this experimental babesiosis model.

Efficacy of Azithromycin for Treating Babesia microti Infection in the Hamster Model

Carbenicillin indanyl sodium, an orallyactive derivative ofcarbenicillin, isactive against a broadspectrum ofbacterial species. Although theesterhasinvitro antimicrobial activity persewhenevaluated inBrain HeartInfusion broth, theinvivo antibacterial activity seeninmiceandratsreflects primarily theefficient hydrolysis oftheester tocarbenicillin. Withan acutesystemic infection inmiceas a test system,orally administered carbenicillin indanyl sodium protected miceagainst lethal infections produced byEscherichia coli, Salmonella choleraesuis, Pasteurella multocida, Proteus vulgaris, Staphylococcus aureus,andStreptococcus pyogenes.Thedose thatprotected 50%oftheanimals against eachofthese infections was comparable tothatofparenteral carbenicillin. Against experimental urinary-tract disease in ratsproduced byE.coli, P.vulgaris, andPseudomonas aeruginosa, itwas again observed thatcarbenicillin indanyl sodiumprovided activity comparable tothat ofparenterallv administered carbenicillin. Because ofitsunique activity against Pseudomonasaeruginosa andindole-positive Proteus species usually resistant toampicillin, carbenicillinhasassumed animportant role asaparenteral chemotherapeutic agent fortheserious medical problems caused bythese microorganisms. Carbenicillin isadministered parenterally asdisodium carbenicillin. Thisformulation is,however, poorly absorbed oracid-labile, orboth, whengivenorally andisnottherapeutically effective. The5-indanyl esterofcarbenicillin (Fig. 1)isanacid-stable formofcarbenicillin. It iswellabsorbed fromthegastrointestinal tract andisrapidly hydrolyzed invivo tocarbenicillin. Laboratory studies presented inthis report demonstrate thattheesterprovides bacteriological activity comparable tothatofparenterally administered carbenicillin. MATERIALSAND METHODS

James A. Retsema

Papers

Correlation of the extravascular pharmacokinetics of azithromycin with in-vivo efficacy in models of localized infection

Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo

Preferential concentration of azithromycin in an infected mouse thigh model

Efficacy of Azithromycin for Treating Babesia microti Infection in the Hamster Model

Carbenicillin Indanyl Sodium, an Orally Active Derivative of Carbenicillin