Macrolides: structures and microbial targets.

The macrolide class of antibiotics is well established and often recommended for use in the treatment of community-acquired respiratory tract infections. A number of agents with varying antimicrobial activity have been developed via chemical modification of the core macrolide structure, a macrocyclic lactam ring. Although structurally diverse, the macrolides share a common ability to bind to the bacterial 50S ribosome subunit and inhibit protein synthesis, thereby preventing bacterial multiplication. Resistance in the clinic is due to modification of the 50S subunit in the area of the peptidyl transferase center or to an efflux pump. The newer macrolides, and in particular azithromycin, with their broad-spectrum microbiological profile have extended the therapeutic uses of this class of antibiotics and ensured that they remain an integral part of the clinician's armamentarium.

Susceptibility and resistance emergence studies with macrolides.

An extended elimination half-life and good tissue penetration enable oral azithromycin to attain high and prolonged concentrations in infected tissues, yielding high antibacterial activity in vivo. It has been suggested, however, that prolonged subinhibitory concentrations of azithromycin from 2 to 4 weeks after therapy may lead to the emergence of azithromycin resistance in vivo, compared with other macrolide antibiotics. Data from two types of in vitro susceptibility studies, an animal tissue infection model, and a clinical pediatric study demonstrate that prolonged tissue concentrations of azithromycin are unlikely to lead to the emergence of resistance in the clinical setting. Further, data from in vitro susceptibility studies indicate that resistance to macrolides, and azithromycin in particular, is significantly over-estimated for bacterial strains incubated in the presence of CO2.

In vitro microbiological characterization of novel cyclic homopentapeptides, CP-101,680 and CP-163,234, for animal health use.

Two cyclic homopentapeptides, CP-101,680 and CP-163,234 [6a-(3',4'-dichlorophenylamino) analogs of viomycin and capreomycin, respectively], were identified as novel antibacterial agents for the treatment of animal disease, especially for livestock respiratory disease. The in vitro microbiological characterization of both CP-101,680 and CP-163,234 was carried out using their parent compounds, viomycin and capreomycin, as controls. This characterization included antibacterial spectrum, influence of media, inoculum size, pH, EDTA, polymixin B nonapeptide (PMBN), serum, cell-free protein synthesis inhibition, and time-kill kinetics. Our results indicated that the capreomycin analog, CP-163,234, showed slightly improved in vitro potency over the viomycin analog, CP-101,680. Both analogs showed very potent cell-free protein synthesis inhibition activity and were bactericidal against Pasteurella haemolytica, P. multocida and Actinobacillus pleuropneumoniae at the level of 4 times and 8 times MICs. CP-163,234 was bactericidal at the level of 4x and 8x MIC against E. coli, but re-growth was observed after 24 hours incubation at both concentrations of CP-101,680.

In vitro microbiological characterization of novel macrolide CP-163,505 for animal health specific use.

A novel 16-membered-ring macrolide agent (CP-163,505, a reductive amination derivative of repromicin) was identified as an antibacterial against Pasteurella haemolytica, P. multocida and Actinobacillus pleuropneumoniae, important etiological agents of livestock respiratory disease. In vitro MIC50/90 analysis revealed that CP-163,505 was more potent (4x) than tilmicosin against P. multocida, and equivalent to tilmicosin against P. haemolytica and A. pleuropneumoniae. In time kill kinetic studies, CP-163,505 showed bactericidal activity against P. haemolytica, P. multocida and A. pleuropneumoniae and bacteriostatic activity against E. coli at 8 times its MIC. In vitro, CP-163,505 was more potent in alkaline pH (16 approximately 32 x ) and less potent in the presence of excess cations (Mg+2 and Ca+2, 4x). EDTA and PMBN increased CP-163,505 potency against E. coli (4x) but not against the other species. Similar results were obtained with erythromycin A and tilmicosin, which were used as controls. From our data, we hypothesize that Pasteurella and Actinobacillus have an outer membrane significantly different from that of the typical enteric Gram-negative bacterium E. coli.

Resistencia con azitromicina y cómo se relaciona con su farmacocinética / Azithromycin resistance related to its pharmacokinetics

La eficacia de azitromicina se correlaciona con las altas y sostenidas concentraciones que alcanza en los tejidos y estos niveles se incrementan, además, por la liberación del fármaco desde los fagocitos, en el sitio de la infección. Sin embargo, existe cierta preocupación de que las bajas concentraciones séricas y las altas concentraciones tisulares de azitromicina puedan favorecer la aparición de resistencia. ¿Qué hay de cierto en esto? Estudios estándares, realizados in vitro en los cuales se hicieron cultivos sucesivos -en nueve ocasiones- en medios que contenían concentraciones séricas menores o equivalentes a las alcanzadas después de la administración de una dosis de 500 mg, no favorecieron la aparición de resistencia en ninguna de las cepas evaluadas (13 cepas de especies de Streptococcus; 10 cepas de Straphylococcus aureus y 12 cepas de Haemophilus influenzae). La resistencia apareció en algunas cepas expuestas a cefaclor. En un modelo de infección localizada con S. aureus, azitromicina produjo un efecto terapéutico significativo, y por lo tanto, ejerció presión selectiva para resistencia. Sin embargo, al comparar la susceptibilidad de las células de S. ureus tomadas del sitio de la infección en ratones control, y en ratones tratados con azitromicina, ésta fue equivalente; así pues, no apareció resistencia. En dos extensos estudios clínicos en niños con faringitis causada por Streptococcus pyogenes, la susceptibilidad de 58 cultivos persistentes, tomados el día 30 posterior al tratamiento en niños que había recibido azitromicina, fue similar a la susceptibilidad encontrada en los cultivos del día cero. Solamente 1.7 por ciento de los cultivos obtenidos a los 30 días, en niños que habían recibido previamente azitromicina, fueron resistentes a este antibiótico, comparado con 3.9 por ciento en el grupo que recibió penicilina V en el mismo estudio. Estos trabajos sugieren que el singular perfil farmacocinético de azitromicina no favorece la aparición de resistencia. Azitromicina tiene características que pueden reducir la presión de selección cuando se compara con otros macrólidos. Tiene la mejor actividad en contra de haemophilus, y no se observó resistencia a un estudio mundial de vigilancia. Su dosificación, menos frecuente y durante un periodo corto favorece su cumplimiento. También su baja dosis total puede dar como resultado menor presión de selección sobre los ecosistemas ambientales

Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes.

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.

Glycylcyclines bind to the high-affinity tetracycline ribosomal binding site and evade Tet(M)- and Tet(O)-mediated ribosomal protection.

N,N-dimethylglycylamido (DMG) derivatives of 6-demethyl-6-deoxytetracycline and doxycycline bind 5-fold more effectively than tetracycline to the tetracycline high-affinity binding site on the Escherichia coli 70S ribosome, which correlates with a 10-fold increase in potency for inhibition of E. coli cell-free translation. The potencies of DMG-doxycycline and DMG-6-demethyl-6-deoxytetracycline were unaffected by the ribosomal tetracycline resistance factors Tet(M) and Tet(O) in cell-free translation assays and whole-cell bioassays with a conditional Tet(M)-producing E. coli strain.

Influence of immunosuppression on the pharmacokinetics and pharmacodynamics of azithromycin in infected mouse tissues.

Azithromycin has been shown to preferentially distribute to infection loci. Due to the potential contribution of phagocytes as transporters of drug to these sites, there has been some concern that immunosuppression of the cellular arm of the host defence system would greatly reduce the delivery of azithromycin to sites of infection and hence impair efficacy. Therefore, we evaluated the pharmacokinetics and pharmacodynamics of azithromycin in a Staphylococcus aureus intramuscular infection model in normal and immunosuppressed mice, employing therapeutic and prophylactic regimens. Immunosuppression was induced by daily doses of cyclophosphamide that culminated in leucopenia with an underlying granulocytopenic condition, with circulating peripheral granulocytes numbering from < or = 0.1-0.3 x 10(9)/L. Azithromycin tissue levels were not reduced in infection loci in granulocytopenic mice but moderate increases in Cmax and AUC values were observed, relative to similar tissues from normal mice. The tissue half-life of azithromycin in infected tissues in a therapeutic mode (75 h) was three-fold longer than in a prophylactic mode (25 h); this correlated with the degree of inflammation (therapy was withheld until inflammation was evident; i.e., prophylaxis reduced inflammation). Histological examination of infected tissues from normal and leucopenic mice was indistinguishable despite a 70%-85% reduction in circulating granulocytes. Compared with untreated infected controls, bactericidal activity was noted following prophylaxis with azithromycin and bacteraemia was suppressed in mice receiving azithromycin therapeutically. In summary, these data indicate that azithromycin delivery and efficacy in a moderately immunosuppressed animal model are unimpaired.

Efficacy of azithromycin for treating Babesia microti infection in the hamster 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.

Comparison of azithromycin, roxithromycin, and cephalexin penetration kinetics in early and mature abscesses.

During the process of abscess formation, a myriad of changes are observed histologically that impede the penetration of antimicrobial agents into infection loci. A Staphylococcus aureus foreign body abscess, developed in rats, was employed to evaluate the penetration kinetics of azithromycin, roxithromycin and cephalexin at various stages of abscess development; the progressive patho-histological changes of abscess formation were also characterized in this model. In an early abscess (18 h post-challenge), azithromycin penetration into inflammatory fluid was enhanced (AUC of 351 vs 130 mg.h/kg) and residence prolonged relative to an inflammation control (half-life of 88 vs 27 h). In contrast, roxithromycin and cephalexin penetration into, and residence in, inflammatory fluid were unaltered in the early abscess. However, penetration into, and egress from, a mature abscess (ten days post-challenge) were impeded for all three antimicrobials (P < or = 0.03). The penetration kinetics of azithromycin into inflammatory fluid in an early abscess were independent of the dose regimen, but dependent on the total dose. The persistently high concentrations of azithromycin in inflammatory fluid within abscess were associated with the infiltration of phagocytic cells and encapsulation by fibrous tissue. These data are consistent with a phagocytic delivery mechanism for azithromycin, whereby the presence of high concentrations of azithromycin in inflammatory fluid are a consequence of augmented drug distribution via the release of accumulated intracellular drug from the infiltrating phagocytic cells and fibroblasts associated with abscess formation.

Preferential concentration of azithromycin in an infected mouse thigh model.

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.

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 for Staphylococcus aureus strains ranged from 50 micrograms/ml at pH 6 to less than or equal to 0.025 micrograms/ml at pH 8; for erythromycin the MIC change was less (1.6 to 0.05 micrograms/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 against Bacteroides strains growing in Wilkins-Chalgren broth fell from 3.1 micrograms/ml in the anaerobic atmosphere to 0.2 and 0.4 micrograms/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 for Escherichia coli.(ABSTRACT TRUNCATED AT 250 WORDS)

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 of Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenzae and Enterobacteriaceae were passaged nine times in sub-lethal concentrations of azithromycin or control antibiotics. Streptococcus pyogenes and Staphylococcus aureus quickly became resistant to rifampin as the MIC90 increased from 0.1 to greater than 50 micrograms/ml for both species. The MIC90 of azithromycin, erythromycin, amoxicillin and cefaclor increased by three dilutions for Staphylococcus aureus. The MIC values of azithromycin for Streptococcus pyogenes, Haemophilus influenzae and Enterobacteriaceae strains did not change significantly. However, for Haemophilus influenzae and the Enterobacteriaceae strains, the MIC values of erythromycin and oral cephalosporins increased four-fold. In the in vivo experiments, mice infected with Staphylococcus aureus or Escherichia 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 for Staphylococcus aureus cultures from treated and non-treated animals were identical. The azithromycin MICs for Escherichia 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.

(6R,8S)-(2-benzimidazolyl)hydroxymethylpenicillanic acids as potent antibacterial agents and beta-lactamase inhibitors.

(6R,8S)-(2-Benzimidazolyl)hydroxymethylpenicillanic acids (1a-1x) are potent antibacterial agents and beta-lactamase inhibitors against Gram-positive bacteria and Haemophilus influenzae. The corresponding (6R,8R)-isomers (2a-2x), the 6,6-spiro benzimidazole-penam alcohol (3), (7R,9S)-(2-benzimidazolyl)hydroxymethylcephalosporanic acid (4), and 6 beta-(2-benzimidazolyl)aminopenicillanic acid (5) are much less active as antibacterials or beta-lactamase inhibitors. The syntheses and structure-activity relationships of these compounds are discussed. Antibacterial activity and beta-lactamase inhibition data are presented.

Comparison of the effects of the new azalide antibiotic, azithromycin, and erythromycin estolate on rat liver cytochrome P-450.

Erythromycin and some other macrolide antibiotics can first induce a cytochrome P-450 isozyme similar to the one induced in rats by pregnenolone-16 alpha-carbonitrile and then inhibit it by forming a stable cytochrome P-450-metabolite complex. The purpose of this study was to compare azithromycin, a novel 15-membered ring azalide, and erythromycin estolate for the potential to cause hepatic microsomal enzyme induction and inhibition in Sprague-Dawley rats. The daily oral administration of 800 mg of erythromycin estolate per kg for 7 days resulted in statistically significant elevations of NADPH-cytochrome c reductase, erythromycin N-demethylase (3.2-fold), and total cytochrome P-450 content. Approximately 40% of cytochrome P-450 was complexed with erythromycin metabolite. In contrast, the daily administration of 200 mg of azithromycin per kg for 7 days caused significant elevations of N-demethylase (2.5-fold) only and did not produce any increases in total cytochrome P-450 content or NADPH-cytochrome c reductase. No complexed cytochrome P-450 was detected in the azithromycin-dosed rats despite liver concentrations of azithromycin that were 118 times greater than the liver concentrations of erythromycin estolate in erythromycin estolate-dosed rats. Although the short-term oral administration of azithromycin produced hepatic accumulation of the drug and elevated azithromycin demethylase activity, there was no other evidence of hepatic cytochrome P-450 induction or inactivation via cytochrome-metabolite complex formation. In contrast to erythromycin estolate, azithromycin is not expected to inhibit its own metabolism or that of other drugs via this pathway.

Cephalosporins to carbapenems: 1-oxygenated carbapenems and carbapenams.

The photo "Wolff" rearrangement of readily available 2-diazoceph-3-em oxides (1) directly affords carbapen-2-ems, allowing a facile entry into a ring system previously accessible only by total synthesis, lengthly semisynthesis or fermentation. The chirality of the cephalosporin is accurately translated into the corresponding carbapenem. The resulting 1-oxocarbapenems (2) were selectively transformed through reduction into 1-oxygenated carbapenems and carbapenams (3 and 4, respectively). On microbiological screening, a carbapenem (3c) was found to possess a broad spectrum of activity. An interesting antibacterial profile was discovered for a carbapenam (26).

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

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.

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

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.

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.