Selected aminothiazolyl-oxime cephalosporin congeners substituted at C-3' with a catechol moiety were used to probe the basis of the enhanced antibacterial activity against Escherichia coli K-12 often associated with chemical modifications of this type. Evidence is presented for a tonB-dependent illicit transport of the compounds across the outer membrane of E. coli K-12, the process involving jointly and specifically the Fiu and Cir iron-regulated outer membrane proteins. Thus, both tonB and flu cir mutants showed a comparably reduced susceptibility to the probe compounds, whereas mutants singularly lacking any one of the six iron-regulated outer membrane proteins (Fiu, FepA, FecA, FhuA, FhuE, and Cir) or lacking any combination of any two of these proteins (except Fiu plus Cir) did not show this resistance. Mutants devoid of all six iron-regulated outer membrane proteins were no more resistant to the probe compounds than fiu cir or tonB strains. In addition to the latter genes, the products of the exbB and possibly the exbC loci were necessary for maximal antibacterial potency. A dependence of antibacterial activity on the level of expression of the uptake system components was noted. Comparison of penicillin-binding protein target affinity with antibacterial activity suggested a possible periplasmic accumulation of active compounds by E. coli K-12. Free vicinal hydroxyl groups of the catechol residue were a primary chemical requirement for recognition by the uptake pathway and thus for high antibacterial activity.Recent reports have described the enhanced antibacterial potency against members of the family Enterobacteriaceae and pseudomonads of ,B-lactam antibiotics variously substituted with catechol, derivatized catechol, and related moieties (2, 6, 29-31, 33, 34, 45). Using cephalosporin E-0702 (featuring a catechol derivative at C-7), Watanabe et al. (45) reported the isolation of spontaneous Escherichia coli K-12 mutants specifically resistant to this compound owing to a defect at the tonB locus. From the known functionality of the tonB gene product in the transport of iron-chelated siderophores by E. coli K-12, it was hypothesized that the potency of E-0702 resulted from the incorporation of the compound by a tonB-dependent siderophore transport route (45). Unpublished studies in this laboratory have shown that such (tonB) mutants of E. coli K-12 can be isolated irrespective of the chemical diversity of the catechol-substituted cephalosporin (catechol-cephalosporin) used. These mutants, like tonB mutants independently isolated as multiply colicintolerant variants of sensitive strains (3,24,25), are specifically cross resistant to catechol-cephalosporins as a chemical class (unpublished observations and this report).In view of the pivotal importance of the tonB gene product in determining the sensitivity of E. coli K-12 to these agents, we attempted to identify whether any of the known tonBdependent uptake processes of E. coli K-12 could be implicated in catechol-cephalosporin activity.The tonB gene pro...

A temperature-conditional, cell-division mutant of Escherichia coli K-12 possessing a thermolabile penicillin-binding protein (PBP) 3 was isolated. The mutant phenotype was due to a lesion in the pbpB gene. This mutant, and leu+ pbpB co-transductants of E. coli C600 grew as rods at 30 degrees C but were converted to filaments at 42 degrees C upon denaturation of PBP3 and concomitant cessation of cell division. These strains have been used to study the consequences of the specific inhibition of PBP3 of E. coli K-12 upon growth, viability and outer membrane integrity. Our results indicate that the singular inhibition of PBP3 is bactericidal in E. coli K-12, even though the turbidimetric response of the bacteria in broth culture suggests bacteriostasis. Furthermore, filament formation is accompanied by disruption of outer membrane barrier function, as witnessed by the rapid leakage of periplasmic beta-lactamase. This latter finding was confirmed by observing the lytic effect of a sub-inhibitory concentration of cefsulodin on filaments of E. coli K-12 induced by PBP3-specific beta-lactams. The impact of these results upon the testing of beta-lactam sensitivity of E. coli K-12 is discussed.

Diseases, General Hospital, Newcastle upon Tyne NE4 6BESUMMARY. Motile curved rods seen in vaginal secretions have been isolated on Columbia agar supplemented with 5% human blood and vitamin K. Growth occurred anaerobically and in 5% oxygen but not in more aerobic conditions. There were two distinct groups of these organisms, distinguishable by morphology, biochemical activity and susceptibility to metronidazole. All isolates were sensitive to a wide range of antimicrobial agents, with the exception of nalidixic acid and polymyxin, but one group was resistant to metronidazole. There was little difference between the results of tests of susceptibility to aminoglycosides or to metronidazole performed in anaerobic and microaerophilic conditions. Motile curved rods were isolated from 18 of 80 patients with a clinical diagnosis of non-specific vaginitis, but from only two of 39 without the disease.

Mutants, showing either constitutive (depressed) or non-inducible expression of chromosomally-mediated Type I beta-lactamase were obtained from clinical isolates of Enterobacter cloacae, Ent. aerogenes, Citrobacter freundii, Providencia stuartii, Morganella morganii, Serratia marcescens and Pseudomonas aeruginosa. The wild-type and mutant strains were compared for susceptibility to a range of beta-lactam antibiotics. Derepression of beta-lactamase synthesis generally, but not always, resulted in a marked reduction in susceptibility to the agents tested, including the '3rd generation' cephalosporins. In many cases, the observed resistance would preclude, or severely compromise, the therapeutic efficacy of the drugs. In this context, depressed mutants of Enterobacter spp., Citro. freundii and Ps. aeruginosa could be of primary concern although those of Ser. marcescens, Prov. stuartii and Morg. morganii often exhibited equally high resistance levels to older beta-lactams. Comparison of the susceptibilities of the non-inducible mutants with that of their inducible parents suggested variation in the beta-lactamase inductive potency of different compounds in different organisms. For example, cefoxitin was a powerful inducer in Ent. cloacae, Citro. freundii and one strain of Ps. aeruginosa; similarly cefazolin and cefuroxime were good beta-lactamase inducers in Ser. marcescens and Morg. morganii. Aminothiazolyl-oxime cephalosporins and ureido-penicillins were generally poor inducers. From such comparisons, the contribution of inducible Type I beta-lactamase to resistance phenotype could be ascertained.

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