Wolfgang Cullmann

Imipenem Resistance in Acinetobacter baumanii Is Due to Altered Penicillin-Binding Proteins

The comparison of a clinical Acinetobacter baumanii isolate (strain No. 4852/88) and its selected imipenem-resistant (IMR) clone exhibited a complex reorganization of the penicillin-binding proteins (PBPs) with diminished labelling of all PBPs except the 24-kD PBP which showed an increased binding of 14C-penicillin. This protein could not be saturated by preincubation of membranes with imipenem at 8-fold the MIC of imipenem, thus indicating PBP alterations responsible for imipenem resistance. In A. baumanii 4852/88 seven PBPs with the apparent molecular weights of 94, 84, 65, 61, 48, 40 and 24 kD could be detected. beta-Lactamase production was barely detectable in any case and could not be enhanced in the presence of various beta-lactams as the inducer. The outer membrane proteins were found identical in both the wild-type strain and the Im clone. So far, imipenem-resistant A. baumanii isolates have been isolated twice in our diagnostic laboratory; however, no implications on the future relevance of the above findings can be made.

Comparative evaluation of recently developed quinolone compounds: with a note on the frequency of resistant mutants

The antibacterial activity of the new quinolone compounds enoxacin, norfloxacin, ofloxacin and ciprofloxacin was evaluated in 300 Enterobacteriaceae, 50 Pseudomonas aeruginosa, 30 Acinetobacter spp., 15 Haemophilus influenzae, 50 Streptococcus faecalis, and 70 Staphylococcus aureus isolates and compared to that of nalidixic acid, gentamicin and various beta-lactam compounds. Moreover, the rate of spontaneous mutants resistant to quinolone compounds was evaluated. In concentrations only insignificantly exceeding the minimal inhibitory concentrations (MIC), mutants could be isolated rather frequently (approx. 10(-6) fold); in concentrations of at least 10 times the MIC resistant mutants were barely detectable. In general, the mutants exhibited a 4- to 8-fold increase of the MIC as compared to the wild strain. In S. faecalis mutants were not detectable, whereas they occurred in low frequency (less than 10(-8) fold) in S. aureus strains. In all mutants there was almost, but not entirely, complete cross-resistance between the quinolone derivatives.

Antibiotic Susceptibility and Outer Membrane Proteins of Clinical Xanthomonas maltophilia Isolates

Twenty clinical Xanthomonas maltophilia isolates were studied for their susceptibility to various antibiotics and for their outer membrane protein profiles and compared to Pseudomonas aeruginosa isolates. Among the antibiotics studied, fleroxacin and ciprofloxacin exhibited the greatest activity (MIC90 4 micrograms/ml). All the X. maltophilia isolates exhibited similar outer membrane protein profiles with 40, 23 and 21 major proteins, whereas the outer membrane of P. aeruginosa revealed at least 7 major outer membrane proteins, as is known from the literature. Thus, it is assumed that the outer membrane profile of X. maltophilia contributes to the resistance of this nosocomial pathogen to most antibiotics.

Heterogeneity of Beta-Lactamase Production in Pseudomonas maltophilia, a Nosocomial Pathogen

Twenty Pseudomonas maltophilia isolates were examined for susceptibility to beta-lactam antibiotics, including carbapenems, and for beta-lactamase production. All the isolates were resistant to imipenem (MICs 64-512 mg/l) and to a lesser extent, to meropenem (MICs 16-256 mg/l). None of the isolates produced significant amounts of beta-lactamase without induction. Among the beta-lactams studied imipenem proved to be the most potent inducer; meropenem was a weaker inducer. Interestingly, 6-amino-penicillanic acid, even in concentrations up to 100 mg/l, entirely lacked induction activity. In any case enzyme production was drug concentration dependent and transient. Isoelectric focusing revealed 6 different enzymes distinguished by their different isoelectric points (pH 6.2, 8.3, 8.5, 9.0, 9.2 and 9.4). This suggested the lack of a unique beta-lactamase profile in P. maltophilia. Addition of 5 mM cyclic AMP (cAMP) or 0.5 mM cAMP-N6, O2-dioctanoyl (a lipophilic derivative) resulted in a marked drop of beta-lactamase induction by imipenem as compared to the control assay. Monitoring of carbapenem hydrolysis by cell-free supernatants revealed inactivation of both carbapenems. Meropenem was inactivated about 5 times more rapidly than imipenem. Our studies revealed that beta-lactamase production in P. maltophilia as well as growth kinetics were influenced to a considerable extent by the nutrient medium employed.

CARBAPENEM RESISTANCE IN ENTEROBACTER AEROGENES IS DUE TO LIPOPOLYSACCHARIDE ALTERATIONS

The extensive characterization of 2 clinical Enterobacter aerogenes isolates resistant to all beta-lactam antibiotics including imipenem revealed that imipenem resistance could not be attributed to overproduction of the chromosomal beta-lactamase; moreover, it was lost after subcultivation and can be thus considered as unstable. The comparison of sensitive and resistant clones revealed that the beta-lactamase in the resistant clones was less inducible in the resistant clones and moreover, there was an altered 2-keto-3-deoxyoctonate/carbohydrate ratio in the resistant clones as compared to the imipenem-sensitive clones, thus suggesting alterations in the lipopolysaccharide (LPS). Neither enzymatic degradation of both imipenem and meropenem nor alterations of the outer membrane proteins could be observed. These findings make it apparent that this type of resistance is likely due to an impaired uptake of the agents due to LPS alterations.

Selection and properties ofPseudomonas aeruginosa variants resistant to beta-lactam antibiotics

The relation between basal and inducibleβ-lactamase production and resistance toβ-lactam compounds was studied in five clinicalPseudomonas aemginosa 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 mostβ-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 ofβ-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 ofβ-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 theβ-lactamase-independent resistance results in increased resistance to allβ-lactams with the single exception of imipenem.

Treatment of abdominothoracic actinomycosis with imipenem

The probably successful treatment of a 42 year old diabetic patient suffering from relapsing abdominothoracic actinomycosis with imipenem is reported. Several earlier attempts to cure the patient by combination of surgical procedures (including cysto-jejunostomy and pancreatic tail resection) in combination with four or six week courses of intravenously administered penicillin G had failed.

Comparable Evaluation of Orally Active Beta-Lactam Compounds in Ampicillin-Resistant Gram-Positive and Gram-Negative Rods: Role of Beta-Lactamases on Resistance

The antibacterial activity of the recently developed cephems cefixime and cefetamet-pivoxyl was evaluated in 408 gram-positive and gram-negative rods, all isolated recently from clinical specimens, and compared to that of other orally active agents such as ampicillin, amoxycillin + clavulanic acid, cefaclor, cefuroxime-axetil and to ceftriaxone. With regard to ampicillin-resistant Enterobacteriaceae ceftriaxone proved to be the most active agent, followed by cefixime and cefetamet, whereas cefuroxime was less active. Cefaclor and amoxycillin + claculanic acid were active against ampicillin-resistant Escherichia coli, Klebsiella pneumoniae, and Proteus ssp. isolates. All beta-lactam compounds exhibited poor activity against Acinetobacter anitratus isolates, but were highly active against Haemophilus influenzae with the exception of cefaclor. Both cefixime and cefetamet were poorly active against Staphylococcus aureus, but highly active against beta-hemolytic streptococci. Moreover, both compounds remained unaffected by the production of plasmid-mediated beta-lactamases such as the TEM or OXA enzymes. Resistance to both agents was observed in Enterobacteriaceae that produced large amounts of chromosomally mediated enzymes; their affinity to the class I enzyme from Enterobacter cloacae was somewhat lower than that of other third-generation cephalosporins. However, in contrast with these agents breakdown of cefixime and cefetamet by a class IIIa enzyme form Proteus vulgaris was marginal. In methicillin-resistant S. aureus isolates there was a complete cross-resistance between all beta-lactam compounds included in this study.

Inducible β-Lactamases are Principally Responsible for the Naturally Occurring Resistance towards β-Lactam Antibiotics in Proteus vulgaris

The role of inducible chromosomally mediated β-lactamases was studied in 22 Proteus vulgaris isolates by monitoring enzyme induction in the presence of various inducers such as ampicillin, cefalothin, cefuroxime, cefsulodin, 6-aminopenicillanic acid (6-APS), and imipenem. 20 of the isolates exhibited resistance to ampicillin, cefalothin, and cefuroxime, whereas 2 isolates were susceptible to these compounds. In all resistant isolates marked inoculum effects could be observed. Enzyme production proved to be transient, i.e. maximum of enzyme production was achieved after 2 or 3 h. In both sensitive isolates enzyme production did not exceed 0.021 U β-lactamase/mg protein of the cell-free supernatant even after induction with 6-APS or imipenem, whereas it ranged from 0.46 to 6.3 U in the resistant ones. Moreover, enzyme induction was found to be concentration-dependent, as revealed by the extensive study of one of the isolates (No. 4917). Three different enzymes could be distinguished by means of isoelectric focusing with isoelectric points at 7.4, 8.8, and approximately 9.5. In the presence of 2.5 mg/l clavulanic acid even strains known to be strong enzyme producers became as susceptible as the sensitive ones: moreover, the inocolum effect was markedly reduced. These findings make it apparent that it is above all the production of inducible enzymes that is responsible for resistance to β-lactam antibiotics in P. vulgaris.

Evidence for nonspecific induction of beta-lactamase in overproducing variants ofEnterobacter cloacae andCitrobacter freundii

Induction of chromosomally mediatedβ-lactamases was studied in clinical isolates ofEnterobacter cloacae andCitrobacter freundii. Whereas isolates resistant to ampicillin and cefoxitin exhibited an inducible enzyme, those sensitive to both agents did not. Cefoxitin and above all imipenem proved the most efficacious enzyme inducers. Variants among these inducible isolates which produced large amounts ofβ-lactamase could be selected in the presence of cefamandole. In each of these selected variants constitutive enzyme production was markedly enhanced by induction, even with compounds lacking induction potency for the corresponding wild strains. Moreover, these variants could also be induced non-specifically, i.e. by non-β-lactam compounds. Production of large amounts of enzyme was associated with resistance to all availableβ-lactam compounds.