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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 beta-lactam antibiotics (except imipenem) to quinolones, such as norfloxacin and fleroxacin, chloramphenicol and tetracycline, but not to gentamicin and polymyxin B. beta-Lactamase production was barely detectable in both wild-type strains and the resistant clones. Only ampicillin, cefoxitin and imipenem increased the production of beta-lactamase, whereas various other beta-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.

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Antibiotics are known to exert an influence on the host-parasite relationship either by impairment of immunocompetent cells or by alteration of the bacterium, such as changes of surface properties or the production of toxins. The main problem in investigating the effect of antibiotics on the surface properties of bacteria consists in morphological changes of bacteria (round cell or filament formation) after treatment e.g. with beta-lactam antibiotics. These changes of morphology lead to problems in the comparison of such bacterial forms with untreated organisms. Therefore, in this study outer membrane vesicles from bacteria were used as a model to investigate the effect of antibiotics on the surface properties of Escherichia coli with regard to the interaction with mouse peritoneal macrophages tested by chemiluminescence reaction. It could be shown that these membrane vesicles induce a luminol dependent chemiluminescence response. Treatment of E. coli with different beta-lactams lead to an increase of the stimulating properties. The relative effectiveness of certain antibiotics depended on the particular E. coli strain. Analysis of the different adhesions involved in the stimulation of macrophages revealed that only mannose-sensitive adhesins were increased after treatment with beta-lactam antibiotics. No stimulation of the membrane-bound NAD(P)H-oxidase could be found following the reaction with outer membrane vesicles. Even the treatment of bacteria with antibiotics did not evoke such a reaction.

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The relation between basal and inducible beta-lactamase production and resistance to beta-lactam compounds was studied in five clinical Pseudomonas aeruginosa isolates and their corresponding resistant variants selected in the presence of either piperacillin, ceftazidime or aztreonam. In all wild-type strains enzyme levels were barely detectable in the uninduced state and most beta-lactams, including sulbactam and clavulanic acid, exhibited poor induction potency. Imipenem proved to be the most potent inducer in both these strains and their resistant variants. In the variants selected by either piperacillin or ceftazidime enzyme production amounted to 1.28 units/mg protein of the cell-free supernatants following the addition of beta-lactams as inducers. Additionally, these variants exhibited the phenomenon of "non-specific" induction, i.e. the increase of enzyme production by either a complex nutrient medium or by addition of vitamins. Enzyme production in the aztreonam-resistant variants was identical to that in the wild-type strains with a single exception, where the entire derepression of beta-lactamase production in one of the variants took place. Derepression of the chromosomally mediated enzyme affects the susceptibility to ureidopenicillins more than that to carboxy-penicillins and cephalosporins, whereas the beta-lactamase-independent resistance results in increased resistance to all beta-lactams with the single exception of imipenem.

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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 beta-lactams. Cross-resistance between imipenem and other beta-lactam compounds was not observed. In all imipenem-resistant variants, induction of chromosomal beta-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.

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The frequency of selection of resistant variants by 10 different broad-spectrum beta-lactam derivatives was evaluated for 10 clinical Enterobacter cloacae isolates. With respect to most penams or cephems, resistant variants could be selected up to 8- or 32-fold the MIC, respectively. However, with cefpirome as the selecting agent resistant variants were obtained only at twice the MIC, whereas resistant variants were barely detectable with temocillin and not detectable in any case with imipenem. The variants exhibited cross-resistance between penams and cephems including aztreonam, but not to temocillin and imipenem regardless of the beta-lactamase amount produced. Enzyme production of the variants ranged from 0.2 U to 19.0 U beta-lactamase/mg protein of the cell-free supernatants. Moreover, analysis of the outer membrane protein composition did not reveal marked alterations between wild strains and the corresponding variants. It is evident that 'overproduction of the chromosomal beta-lactamase' cannot explain entirely phenotypic resistance to broad-spectrum beta-lactam compounds, but a lack of porin production, i.e., major outer membrane proteins, cannot provide an explanation for the above findings.

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Imipenem-resistant variants can be selected from clinical Pseudomonas aeruginosa isolates in a frequency of 10(-8) to 10(-7) and 10(-5) in a single strain. In any case, there was no cross resistance between imipenem and other beta-lactams. In all IMI variants the induction potency of imipenem for the chromosomally-mediated Id beta-lactamase was markedly diminished as compared to the corresponding parent strains. Moreover, in all imipenem-resistant variants as well as in imipenem-resistant clinical isolates phenotypic expression of either a 46,000 dalton or a 47,000 dalton outer membrane protein was marginal; these proteins could be identified as proteins D1 and D2. These findings suggest a penetration barrier responsible for imipenem-resistance in Pseudomonas aeruginosa.

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Macrophages are known to release reactive oxygen species (O2-, 1O2, H2O2, OH.) in response to various membrane stimuli. However, our studies show that phagocytic stimulation of macrophages is not necessarily accompanied by a stimulation of the oxidative burst. Whereas IgG-opsonized erythrocytes were capable to induce phagocytosis and a chemiluminescence response, both being dependent on the number of IgG bound per erythrocyte, C3b-bearing erythrocytes were well ingested but failed to induce any chemiluminescence reaction. Furthermore, stimulation of macrophages, via the Fc-receptors, seems to alter their functional state in regard to the activation of a receptor, which enables them to recognize membrane lesions on the target erythrocyte. The presence of IgG and membrane lesions, e.g. the C5b-9-complex of complement, induced a marked increase in chemiluminescence compared with stimulation by IgG-bearing particles alone. The augmented response of macrophages was at least in part due to an additional release of H2O2, which was not liberated in response to IgG-bearing erythrocytes. This "lesion recognizing receptor" in the macrophage membrane could not be activated by stimulation of C3b-receptors, indicating its functional linkage to the Fc-receptors.

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The fimbrial (pili) profile of a single strain of Escherichia coli O7: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.

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