Resistance to cefotaxime (CTA) and ceftriaxone (CTR) in Enterobacter cloacae and Pseudomonas aeruginosa was investigated in several strains which are susceptible or resistant to these agents. All strains produced a chromosomally mediated cephalosporinase of the Richmond type I. P-Lactamases in susceptible strains were inducible, whereas resistant strains produced the enzymes constitutively. CTA and CTR were very poor substrates but potent inhibitors of all enzymes. Binding to, rather than hydrolysis by, ,B-lactamases was assumed to be a major reason for resistance, and combination experiments supported this assumption. Dicloxacillin, which did not inhibit the growth and which was a poor inducer but a strong inhibitor of these P-lactamases, exerted strong synergistic activity when combined with CTA or CTR in strains which produced large amounts of Plactamase constitutively. Cefoxitin, on the other hand, poorly active alone, but a good inducer, strongly antagonized CTA or CTR in susceptible strains producing inducible enzymes. In marked contrast to CTA and CTR were the findings with cefsulodin. Cefsulodin was active against CTA-and CTR-resistant Pseudomonas, and its activity was hardly influenced by dicloxacillin or cefoxitin. Since cefsulodin was found to have a very low affinity for all cephalosporinases, these findings corroborate the assumption that binding of nonhydrolyzable cephalosporins, rather than hydrolysis by cephalosporinases, may play an important role in resistance to these agents and other newer cephalosporins in Enterobacteriaceae, as well as in other gram-negative bacteria.

Mutants of Enterobacter cloacae, selected in vitro with ceftriaxone, ceftazidime, carumonam, or aztreonam, fell into several distinct classes. Three mutants highly resistant to nearly all beta-lactam antibiotics were stably derepressed for beta-lactamase production. Although no other changes could be detected, virulence in a mouse septicemia model was decreased in two of these mutants. One mutant, 908-Ssi, showed selectively decreased susceptibility to ampicillin and cefotetan. A change in beta-lactamase expression was thought to be responsible for this. Alterations in the production of two outer membrane proteins with molecular sizes of 36.5 and 39 kilodaltons were responsible for multiple antibiotic resistance in two mutants, both of which acquired a low level of resistance to beta-lactam antibiotics. Whereas one of the mutants, AMA-R, simultaneously acquired resistance to chloramphenicol and trimethoprim, the other, AZT-R, became hypersusceptible to these and other hydrophobic agents. Both strains had drastically reduced virulence in mice.

6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases. Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated. 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1. The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin. Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15. Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme. All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.

Ro 09-1428, a new parenteral cephalosporin with a catechol moiety attached at position 7 of the cephalosporin ring, showed high in vitro activity against Escherichia coli, Kkbsielia pneumoniae, Proteus mirabilis, Proteus vulgaris, and Streptococcus pyogenes, with MICs for 90% of strains tested (MIC9s) of c0.39 ,ug/ml.MorganeUa morganii, Providencia reftgeri, Citrobacterfreundii, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae were inhibited with MIC90s of <3.13 ,ug/mi. Serratia marcescens was less susceptible to Ro 09-1428, with a MICg of 25 ,ug/ml. The most distinctive feature of Ro 09-1428 was its potent activity against Pseudomonas aeruginosa and Acinetobacter caloaceticus, with MIC90s of 0.39 and 6.25 ,Ig/ml, respectively. Most of the ceftazidime-resistant strains of P. aeruginosa, E. cloacae, and C. freundii were inhibited by Ro 09-1428, while those of S. marcescens were resistant at a concentration of 12.5 ,ug/ml. Ro 09-1428 was more active than ceftazidime against staphylococci. PBP 3 was the most sensitive target in E. coli and P. aeruginosa. The response to ferric iron in growth medium suggests that Ro 09-1428 may be taken up by transport mechanisms similar to those of other catechol cephalosporins. In accordance with its in vitro activity, Ro 09-1428 activity was equal to or greater than ceftazidime activity in efficacy against experimental septicemias in mice. The results indicate that Ro 09-1428 is a broad-spectrum cephalosporin with advantages over ceftazidime in its activity against P. aeruginosa, staphylococci, and ceftazidime-resistant strains of C. freundii and E. cloaceae.In spite of a considerable number of cephalosporins on the market, few compounds have useful activity against Pseudomonas aeruginosa. Although ceftazidime and cefpiramide are cephalosporins which inhibit the growth of P. aeruginosa at therapeutically attainable concentrations in blood, this activity leaves room for improvement. Recently, several cephalosporins containing catechol substituents have been reported as having superior antipseudomonal activities compared with those of other P-lactam antibiotics (1-9, 16).Ro 09-1428, (6R, (Fig. 1), is a parenteral cephalosporin that has a catechol moiety in the 7-acylamino side chain and showed high activity against gram-negative rods, particularly P. aeruginosa. Furthermore, the compound was found to be active against strains refractory to broad-spectrum cephalosporins. In this paper, we evaluated the in vitro and in vivo activities of Ro 09-1428 compared with those of ceftazidime. In addition, stability to ,-lactamases and affinity for penicillin-binding proteins (PBPs) are described.(This work was presented in part at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 17 to 20 September 1990.) MATERIALS AND METHODS Antibiotics. Ro 09-1428 and ceftriaxone were synthesized in the research laboratory of Nippon Pharmaceutical Devel-* Corresponding author. opment Institute, Hokkaido, Japan, and F. Hoffmann-La Roche A...

Resistant variants of three clinical Pseudomonas aeruginosa isolates were obtained in the presence of aztreonam. The variants exhibited a four- to eightfold increase in the minimal inhibitory concentrations to β-lactam antibiotics (except imipenem) to quinolones, such as norfloxacin and fleroxacin, chloramphenicol and tetracycline, but not to gentamicin and polymyxin B. β-Lactamase production was barely detectable in both wild-type strains and the resistant clones. Only ampicillin, cefoxitin and imipenem increased the production of β-lactamase, whereas various other β-lactams did not. Penicillin-binding proteins remained unchanged in the aztreonam-resistant clones. The analysis of the outer membrane proteins did not reveal differences in the outer membrane proteins between the wild-type strains and the aztreonam-resistant clones. Two of the three antibiotic-resistant isogenic clones contained less lipopolysaccharides (LPSs) than their corresponding wild-type strains. Moreover, it could be demonstrated that the ratio of 2-keto-3-deoxy octonate to carbohydrate of the LPS changed in any case between the wild-type strains and the aztreonam-resistant clones. These alterations were accompanied by a decrease in surface hydrophobicity of the resistant clones as compared to the wild-type strains. Therefore, quantitative as well as qualitative alterations in the LPS may provide an explanation for the resistant phenotype observed.