1. A strain of Escherichia coli highly resistant to chlortetracycline and partially cross-resistant to tetracycline has been isolated. 2. The nitro-reductase system of the resistant cells was inhibited to a smaller extent by chlortetracycline than was the corresponding enzyme of sensitive cells. 3. The incorporation of leucine in vitro into the ribosomal protein of cell-free preparations from sensitive and resistant cells was equally inhibited by chlortetracycline. 4. Resistant cells accumulated much less chlortetracycline and tetracycline than did sensitive cells when both were cultured in the presence of these drugs. 5. The uptake of tetracycline by both sensitive and resistant E. coli was dependent on the presence of glucose in the medium. 6. Fractionation of cells cultured in medium containing [(14)C]chlortetracycline indicated that the largest proportion of radioactivity in sensitive cells was in the fraction consisting mainly of cell-wall material. There was no concentration of radioactivity in any one fraction of the resistant cells. 7. No evidence could be obtained for a specific tetracycline-excretion system in the resistant cells. 8. The significance of these results in relation to current theories of the antibiotic action of and resistance to the tetracycline drugs is discussed.

The emergence of ,B-lactam-resistant strains of Pseudomonas aeruginosa in a cystic fibrosis patient treated with high-dose tobramycin and piperacillin was studied. Two serotypes, M and K, were present before treatment and persisted, with changes in their /8-lactam resistance spectra, during treatment. The resistance was correlated with changes in the penicillin-binding proteins (PBPs) in both serotypes. In the low-level-resistant serotype K organism, PBP-3 either was absent or had lost the ability to bind ['4C]penicillin G. Two serotype M strains, one with low-and one with high-level resistance to several antipseudomonal ,8-lactam antibiotics, were isolated at progressively later stages of therapy. Several differences were noted between the PBP patterns of the resistant M and the susceptible M strains. The affinity for ['4C]penicilhin G was reduced in both resistant strains. PBP bands, with the exception of PBP-6 in the most resistant M type, were barely or not detectable at a ['4C]penicillin G concentration of 39 ,ug/ml. The graduated decrease in affinity for [14C]penicillin G was correlated with increasing 16-lactam resistance and with an increase in the quantity of the protein corresponding to PBP-6. The emergence of the low-level-resistant strains midway through, and of the highly resistant strain in the final stages of, the reported treatment strongly suggested that the resistance resulted from mutation in those strains present before treatment selected for by the high-dose piperacillin treatment.

Pseudomonas pseudomallei, the causative agent of melioidosis, is generally susceptible to some of the newer extended-spectrum cephalosporins or to combinations of a ,-lactam and clavulanic acid, a ,-lactamase inhibitor. Resistance to these agents may, however, emerge during treatment. We report on alterations in the chromosomal D-lactamase associated with the development of resistance. Three resistance patterns resulted from three different mechanisms in the strains investigated. Derepression of the chromosomal enzyme resulted in a general increase in the MICs of all of the f8-lactams tested. The second mechanism observed was an insensitivity to inhibition of the D-lactamase by clavulanic acid. In this case, the level of susceptibility to ,I-lactams as independent entities remained unchanged. The final "resistance" pattern occurred in a patient treated with ceftazidime and resulted in a I8-lactamase that was capable of hydrolyzing this antibiotic at detectable levels, but with reduced efficacy against other I-lactams. The net result was a strain that was generally susceptible to all of the I8-lactams tested except ceftazidime. In all cases, the level of susceptibility to antibiotics other than P-lactams remained unchanged. Such variability found within one genus over a relatively short time course suggests that treatment of infections caused by this organism should be carefully monitored to detect susceptibility alterations to the chosen therapy.

Four ,-lactam-resistant permeability mutants of Pseudomonas aeruginosa PA0503 were studied. The resistance phenotypes were correlated to changes within the lipopolysaccharide. Two of the mutants, PCC1 and PCC19, were shown to differentiate between I-lactams on the basis of relative hydrophobicity. The more hydrophilic antibiotics were less effective at inhibiting these strains. This phenotype was correlated to the presence of mannose, in measurable quantities, in lipopolysaccharide isolated from these strains. The other two strains, PCC23 and PCC100, differentiated between cephem antibiotics on the basis of electrical charge. The presence of a positive charge markedly increased the relitive efficiency of an antibiotic. This correlation did not hold for penam derivatives, with the lower-molecular-weight, dianionic molecules being the most effective. Mutants of this type were changed in the amount of "side chain" sugars or, to a minor extent, in their outer membrane protein profiles.The outer membrane of Pseudomonas aeruginosa has long been considered a barrier against ,B-lactam antibiotics (5,23,24). These compounds are believed to permeate through hydrophilic pores in the membrane (10). Hancock et al. (8) have estimated the exclusion limit of the pseudomonal pores to be between 6,000 and 9,000 daltons, in contrast to the pores in Escherichia coli and other gram-negative organisms (19) that exclude molecules larger than 600 daltons. A pore size of 9,000 daltons should allow entry of all known Plactam antibiotics, which range in size from 350 to ca. 800 daltons. P. aeruginosa is, however, resistant to a large number of classical P-lactams (3,4), and the search continues for antibiotics that have usable activity on this organism (4).Part of the innate resistance in P. aeruginosa lies in the possession of an inducible ,-lactamase carried chromosomally by most strains (25). The contribution to innate resistance of the outer membrane has been demonstrated by Zimmermann (31), who used hypersensitive mutants to show an increased ,-lactam sensitivity spectrum.The role played by the outer membrane in acquired resistance to ,-lactam antibiotics is at the moment based largely upon supposition (1, 9, 10). In an attempt to increase understanding in this area, we investigated a series of nonsibling P-lactam-resistant mutants of P. aeruginosa which could not be explained in terms of changes or lack of affinity (5, 6) in the P-lactam targets. MATERIALS AND METHODSBacterial strains. P. aeruginosa PA0503 (met-9011) (B. W. Holloway, Monash University, Australia) was used as the parent.Other strains were derived by mutagenesis (see below) with selection for resistance to P-lactams. The methionine requirement of each mutant was confirmed by growth around a methionine disk on minimal agar.Mutagenesis. A logarithmic-phase culture of PA0503 was treated with 0.05 ml of ethane methanesulfonate per ml for 1 * Corresponding author. h at 37°C. The washed, treated culture was plated onto brain heart infusion (BHI) agar (Difco Laboratories) su...

The potential pathogenicity of a microorganism is a major concern for Health Canada evaluators, who will be processing new biotechnology products under the Canadian Environmental Protection Act. Potential pathogenicity is generally predicted by the results of animal pathogenicity studies. In an attempt to define surrogate data for an animal model, this study was initiated. Pseudomonas aeruginosa isolates from clinical and environmental sources were screened for their pilus type, serotype, lipopolysaccharide type, ability to evade host responses, and production of toxin A, exoenzyme S, elastase, phospholipase C, and total protease. The 50% lethal dose (LD50) of the same isolates was determined in the neutropenic mouse model of infection. An attempted correlation was drawn between each (or combinations) of the virulence determinants and the LD50. Stepwise linear regression showed that the presence of high levels of exoenzyme S in association with elastase or phospholipase C, or to a minor extent toxin A, was correlated with low numbers of bacteria required to elicit an LD50. No correlation between any of the other factors examined and virulence was detected. The data suggest that an in vitro high level of exoenzyme S production could be used as surrogate information for neutropenic mouse modelling; however, the levels of all of the extracellular enzymes should be considered when making such an assessment.