Cephamycins, a New Family of β-Lactam Antibiotics II. Isolation and Chemical Characterization

Cephamycins A and B were isolated from the same fermentation broth of various actinomycetes by adsorption and ion-exchange methods. The antibiotics were separated from each other by column chromatography on a dextran-based ion-exchange resin. Cephamycin C was isolated from a different fermentation broth by ion-exchange and gel-filtration methods. The chemical characteristics of these new antibiotics were determined.     

Cephamycins, a New Family of β-Lactam Antibiotics. III. In Vitro Studies

Cephamycins A, B, and C are naturally produced cephalosporin-type antibiotics. Although A and B were found to be more active than C against gram-positive organisms, they were not so active against such strains as are cephalosporin C or the semisynthetic antibiotics cephaloridine and cephalothin. Against gram-negative organisms, cephamycin C was more active than A or B and, in general, was as active as the cephalosporins. In addition, cephamycin C was active in vitro against clinically isolated strains resistant to the cephalosporins, such as Proteus, Providencia, and Escherichia coli. The in vitro antibacterial activity of cephamycin C, cephalothin, and cephaloridine is primarily bactericidal. A 10,000-fold increase in inoculum of a strain of Proteus mirabilis resulted in 200-fold or greater increases in minimal inhibitory and minimal bactericidal end points of cephalothin and cephaloridine, but only 10- and 16-fold increases, respectively, for cephamycin C. After 15 passages through antibiotic-containing broths, during which time a culture of E. coli showed an increase in minimal inhibitory concentrations of streptomycin of >1,000-fold, end points for cephamycin C increased 4-fold, for cephalothin, 1.5-to 6-fold, and for cephaloridine, 128-fold.     

Cephamycins, a New Family of β-Lactam Antibiotics. IV. In Vivo Studies

Cephamycin A was found to be more active in vivo than cephamycin B. In comparison with cephamycin C, cephamycin A was more active against gram-positive organisms but less active against gram-negative organisms. Given subcutaneously, cephamycin C had good in vivo gram-negative activity, comparing favorably with cephalothin and cephaloridine against cephalosporin-susceptible organisms. In general, against the gram-negative organisms, it was more active than cephalothin or cephalosporin C and about as active as cephaloridine. In addition, cephamycin C protected mice against beta-lactamase-producing Proteus cultures, including clinically isolated strains. The compound is remarkably nontoxic. Cephamycin C was detected in the serum and recovered from the urine of treated mice to about the same extent as cephaloridine. Like cephaloridine and cephalosporin C, cephamycin C must be excreted mainly by glomerular filtration, because the use of probenecid did not enhance the therapeutic effectiveness nor concentrations of these agents in the sera of treated mice.     

Cephamycins, a New Family of β-Lactam Antibiotics I. Production by Actinomycetes, Including Streptomyces lactamdurans sp. n

A number of actinomycetes isolated from soil were found to produce one or more members of a new family of antibiotics, the cephamycins, which are structurally related to cephalosporin C. The cephamycins were produced in submerged fermentation in a wide variety of media by one or more of eight different species of Streptomyces, including a newly described species, S. lactamdurans. These antibiotics exhibit antibacterial activity against a broad spectrum of bacteria which includes many that are resistant to the cephalosporins and penicillins.     

Inhibition of β-Lactamases by β-Lactam Antibiotics

The inhibitory properties of a selected number of beta-lactam antibiotics were studied, with the use of three distinct types of beta-lactamases. The three enzymes were found to be distinguishable on the basis of their susceptibility to inhibition. Not one of the potential inhibitors tested was found to be a potent inhibitor of all three enzymes, but nafcillin possessed the broadest inhibitory activity. The enzyme isolated from Enterobacter cloacae was found to be the most susceptible. In some cases, the degree of inhibition varied with the time of incubation, and, depending upon the time chosen, widely different observations could be made. It is suggested that, in studies such as these, every consideration should be given to the period of incubation and to the concentration of inhibitor employed. Mixtures of inhibitor and cephaloridine did not always act synergistically against growing bacteria, and a number of reasons for failure are suggested.     

Microiodometric Determination of β-Lactamase Activity

The product of β-lactamase activity (a penicilloic acid in the case of a penicillin) is stoichiometrically oxidized by iodine. Hence, the β-lactamase activity can be measured as decolorization of the blue starch-iodine complex. Since the decolorization is a slow process, the rate of decolorization gave an underestimate of the rate of penicillin hydrolysis until a steady state was obtained after 15 to 20 min. If the reaction mixture (3 ml) contained no more than 0.001 unit of enzyme, a correct determination of β-lactamase activity was obtained from the microiodometric method described. The method is sensitive and some applications are mentioned.     

Yersinia enterocolitica: Comparative In Vitro Activities of Seven New β-Lactam Antibiotics

Minimum inhibitory concentrations of seven new β-lactam derivatives were determined against 35 isolates of Yersinia enterocolitica. Ceftizoxime and ceftriaxone were the most active of the antimicrobial agents tested.     

Comparative Stability of Newly Introduced β-Lactam Antibiotics to Renal Dipeptidase

Renal dipeptidase purified from swine kidney hydrolyzed N-formimidoyl thienamycin, carpetimycins A and B, and Sch29482, but not azthreonam, penicillin G, or cephaloridine.     

Competition of Various β-Lactam Antibiotics for the Major Penicillin-Binding Proteins of Helicobacter pylori: Antibacterial Activity and Effects on Bacterial Morphology

The penicillin-binding proteins (PBPs) of helical (log-phase) Helicobacter pylori ATCC 43579 were identified by using biotinylated ampicillin. The major PBPs had apparent molecular masses of 47, 60, 63, and 66 kDa; an additional minor PBP of 95 to 100 kDa was also detected. The relative affinities of various beta-lactams for these PBPs were tested by competitive-binding assays. Only PBP63 appeared to be significantly bound to each of the competing antibiotics, whereas PBP66 strongly bound mezlocillin, oxacillin, amoxicillin, and ceftriaxone. Whereas most of the beta-lactams significantly bound two or more PBPs, aztreonam specifically targeted PBP63. The influence of sub-MICs of these beta-lactams on the morphologies of log-phase H. pylori was observed at both the phase-contrast and transmission electron microscopy levels. Each of the eight beta-lactams examined induced blebbing and sphere formation, whereas aztreonam was the only antibiotic studied which induced pronounced filamentation in H. pylori. Finally, studies comparing the PBPs of helical (log-phase) cultures with those of coccoid (7-, 14-, and 21-day-old) cultures of H. pylori revealed that the major PBPs at 60 and 63 kDa seen in the helical form were almost undetectable in the coccoid forms, whereas PBP66 remained the major PBP in the coccoid forms, although somewhat reduced in level compared to the helical form. PBP47 was present in both forms at approximately equal concentrations. These studies thus identified the major PBPs in both helical and coccoid forms of H. pylori and compared the relative affinities of seven different beta-lactams for the PBPs in the helical forms and their effects on bacterial morphology.     

In Vitro Activities of Thirteen β-Lactam Antibiotics Against Chlamydia trachomatis

The in vitro activity (minimal inhibitory concentration and minimal lethal concentration) of 13 β-lactam antibiotics against two laboratory strains of Chlamydia trachomatis was compared. No useful activity could be detected.     

Construction and Characterization of Mutants of the TEM-1 β-Lactamase Containing Amino Acid Substitutions Associated with both Extended-Spectrum Resistance and Resistance to β-Lactamase Inhibitors

Extended-spectrum TEM beta-lactamases (ESBLs) do not usually confer resistance to beta-lactamase inhibitors such as clavulanate or tazobactam. To investigate the compatibility of the two phenotypes we used site-directed mutagenesis of the bla(TEM-1) gene to introduce into the TEM-1 beta-lactamase amino acid substitutions that confer the ESBL phenotype: TEM-12 (Arg164-->Ser), TEM-26 (Arg164-->Ser plus Glu104-->Lys), TEM-19 (Gly238-->Ser), and TEM-15 (Gly238-->Ser plus Glu104-->Lys). These were combined with three sets of substitutions that confer inhibitor resistance: TEM-31 (Arg244-->Cys), TEM-33 (Met69-->Leu), and TEM-35 (Met69-->Leu and Asn276-->Asp). Introduction of the Arg244-->Cys substitution gave rise to inhibitor-resistant hybrid enzymes that either lost ESBL activity (TEM-12, TEM-15, and TEM-19) or had reduced activity (TEM-26) against ceftazidime. In contrast, the introduction of Met69-->Leu or Met69-->Leu plus Asn276-->Asp substitutions did not significantly affect the abilities of the enzymes to confer resistance to ceftazidime, although increased susceptibility to cefotaxime was observed with Escherichia coli strains that expressed the TEM-19 and TEM-26 beta-lactamases. With the exception of the TEM-12 beta-lactamase, introduction of the Met69-->Leu substitution did not give rise to enzymes with increased resistance to clavulanate compared to that of the TEM-1 beta-lactamase. However, introduction of the double substitution Met69-->Leu plus Asn276-->Asp in the ESBLs did give rise to low-level (TEM-19, TEM-15, and TEM-26) or moderate-level (TEM-12) clavulanate resistance. None of the hybrid enzymes were as resistant to clavulanate as the corresponding inhibitor-resistant TEM beta-lactamase mutant, suggesting that active-site configuration in the ESBLs limits the degree of clavulanate resistance conferred.     

Selection and Characterization of β-Lactam–β-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 β-Lactamase Gene

Mechanism-based inactivators of β-lactamases are used to overcome the resistance of clinical pathogens to β-lactam antibiotics. This strategy can itself be overcome by mutations of the β-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 β-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with β-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp β-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM β-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.     

Correlation Between the Binding of β-Lactam Antibiotics to Staphylococcus aureus and Their Physical-Chemical Properties

The rate of ¹⁴C-benzylpenicillin (penicillin G) binding to Staphylococcus aureus Oxford cells increased with increasing hydrogen ion concentration. The extent of inhibition of ¹⁴C-penicillin G binding caused by a competing ¹²C-β-lactam antibiotic is a function of hydrogen ion concentration and can be correlated both with net charge of a competing ¹²C-molecule and net charge of the S. aureus cell at a given pH. The ability of a β-lactam antibiotic to compete for ¹⁴C-penicillin G-binding sites can generally be correlated with its hydrophobic nature. It is proposed that, although semisynthetic cephalosporins are chemically less reactive than penicillins, they are superior to benzylpenicillin in their ability to permeate the outer surface of the Staphylococcus cell wall and irreversibly bind to reactive sites.