The mechanism of Pseudomonas aeruginosa resistance to imipenem in five imipenem-susceptible clinical isolates and in their resistant counterparts was investigated. The frequency for selecting imipenem-resistant variants ranged from 2.7 x 10-5 to 2.1 x 10-8 and was comparable to those for other ,-lactams.Cross-resistance between imipenem and other 13-lactam compounds was not observed. In all imipenemresistant variants, induction of chromosomal j3-lactamase by imipenem was markedly diminished compared with that in the susceptible parent strain. This was not the case for other inducers such as ampicillin or cefoxitin, suggesting an impaired uptake of imipenem as an explanation for resistance. Analysis of the outer membrane proteins revealed a marked decrease of either a 46-or a 45-kilodalton protein. The lipopolysaccharide of the outer membrane in the imipenem-resistant variants was not altered.Within the last 2 years, several reports have emphasized the development of resistance to imipenem in clinical Pseudomonas aeruginosa isolates (1,8,10,19,22,24). Emergence of resistance was observed especially in patients suffering from either cystic fibrosis or nosocomial lower respiratory tract infections (1,19,22). Consequently, development of cross-resistance between imipenem and other ,B-lactam compounds must be evaluated.Enzymatic inactivation of imipenem has been reported thus far only for Pseudomonas maltophilia (21). Since resistance to newer cephalosporins and broad-spectrum penicillins is often chromosomally mediated, we studied five imipenem-susceptible clinical isolates of P. aeruginosa and their derived imipenem-resistant counterparts for the production of imipenem-inactivating enzymes and for outer membrane alterations. As it was not clear whether mutation or selection of a resistant subpopulation occurred, the term "variants" will be used for the resistant counterparts.MATERIALS AND METHODS Strains. The P. aeruginosa strains used were recent clinical isolates from our laboratory and were identified by standard procedures (12). Strain FRA was kindly supplied by G. Seibert, Frankfurt, Federal Republic of Germany. None of the strains exhibited resistance to antipseudomonal P-lactam compounds.MICs. MICs were determined in Mueller-Hinton broth (E. Merck AG, Darmstadt, Federal Republic of Germany) by twofold serial dilutions representing a final inoculum of 5 x 105 CFU/ml (microdilution procedure).Selection of resistant variants. Resistant variants were selected by plating 0.05 ml of an overnight culture in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) onto Mueller-Hinton agar plates (Merck) containing the antibiotic in twofold serial dilution steps above the MIC. The frequency of resistant variants was expressed as the ratio of CFU grown in the presence of the antibiotic to the CFU of the control (grown without an antibiotic). All assays were carried out in triplicate. * Corresponding author.Kinetics of j3-lactamase production. ,B-Lactamase production was studied during exponential growth. Ove...

No abstract

The fimbrial (pili) profile of a single strain of Escherichia coli 07:K1:H6 (WF96) was evaluated. Fimbriae were isolated by sucrose density gradient ultracentrifugation, purified from flagellae by the use of 0.4% sodium dodecyl sulfate (SDS), and separated into distinct fimbrial types. Analysis of the purified WF96 fimbriae by SDS-polyacrylamide gel electrophoresis revealed two polypeptide bands with molecular weights of 16,000 and 21,000. Treatment of the fimbrial mixture with saturated guanidine hydrochloride resulted in the appearance of a third band with a molecular weight of 19,500. The relative susceptibilities of the WF96 fimbrial types to disrupting chemicals (octyl-glucoside, urea, SDS, and guanidine hydrochloride) were assessed by exposure of the fimbrial mixture to each agent, separation of the depolymerized fimbriae from intact fimbriae by gel filtration on Sepharose CL-4B, and identification of the disaggregated fimbrial types by SDS-polyacrylamide gel electrophoresis of column fractions. The physicochemical heterogeneity of the three fimbrial types coexpressed on WF96 was exploited to develop a method for separation of individual fimbriae.

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.

No abstract