S U M M A R YP-Lactamases (EC. 3 . 5 . 2 . 6 ) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of P-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate P-lactamase in mutants previously reported to lack the enzyme. R factor RTEM and RPI P-lactamases that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R I and RGNI1, with identical isoelectric focusing patterns, have the same biochemical properties. Chromosomal and R factor-mediated P-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing, R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of P-lactamases carried by R factors. I N T R O D U C T I O NIsoelectric focusing (Haglund, 1967) is a method of separation in which proteins align themselves as sharp bands at their isoelectric points (PI) in an electrophoretically-produced pH gradient. A high degree of resolution is obtained by the method, because focusing is caused by forces that act against diffusion and proteins are therefore concentrated during their separation. We have used the technique in the analytical mode for the separation of P-lactamases (EC. 3 . 5 . 2 . 6 ) on thin layers of polyacrylamide gel and have located the focused enzymes with a cephalosporin substrate which changes colour after degradation by P-lactamase. This technique of separation and specific staining of P-lactamases allows demonstration of low levels of activity and presents the different enzymes produced by various strains as patterns of bands that can easily be recognized and compared.The P-lactamases produced by Gram-negative bacteria are known to differ in their substrate specificities, inhibitor profiles, electrophoretic mobilities, biochemical properties and reactions with antisera ( Yamagishi et al. 1969). These enzymes have frequently been classified by the relative rates at which they hydrolyse penicillins and cephalosporins. Various methods have been used for estimating the activity of crude and pure enzyme preparations, and when a single strain produces two P-lactamases these have not always been separated before determining the substrate profile. Comparison of profiles determined in different laboratories may therefore be misleading. The use of isoelectric focusing of P-lactamases for direct comparison of different enzymes has been illustrated in this paper principally by reference to enzymes from Escherichia coli. This species was chosen because strains carrying R factors specifying well-characterized P-lactamases were available. METHODSStrains. These are listed in Table I. The clinical ...

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The efficacy of a preparation (Oterna—Glaxo Laboratories Ltd) for use in the treatment of otitis externa in small animals has been assessed bacteriologically. The aetiology of the disease was studied in 133 ears of cats and dogs; normal ears were examined by comparison. Pityrosporum canis and staphylococci were the organisms most commonly isolated; their incidence was much higher in clinically affected ears than in normal ears. Gram‐negative organisms were found only in diseased ears. Seventy‐three clinical cases of otitis in dogs and cats were treated with a preparation containing neomycin, monosulfiram and betamethasone. Bacteriological and clinical evaluations were made before and after treatment and the organisms isolated were tested for sensitivity to the antimicrobial constituents of the preparation. There was a 92 % correlation between the bacteriological and clinical results. A ‘good’ or ‘very good’ bacteriological response was recorded for 71% of the ears treated. Résumé. On a évalué bactériologiquement l'efficacité d'une préparation (Oterna—Glaxo Laboratories Ltd) pour l'usage dans le traitement de l'otite externe de petits animaux. L'étiologie de la maladie a été étudiée dans 133 oreilles de chats et chiens; des oreilles normales ont été examinées pour comparaison. Le Pityrosporum canis et des staphylocoques ont été les microorganismes le plus souvent isolés; leur incidence était beaucoup plus élevée dans les oreilles affectées cliniquement que dans les oreilles normales. Des micro‐organisms Gram‐négatifs ont été seulement trouvés dans les oreilles malades. Soixante‐treize cas cliniques d'otite chez les chats et les chiens ont été traités avec une préparation contenant de la néomycine, du monosulfiram et du bétaméthazone. Les évaluations bactériologique et clinique ont été faites avant et après le traitement et les micro‐organismes isolés ont été vérifiés pour la sensibilité aux constituants antimicrobiens de la préparation. Il y avait une corrélation de 92% entre les résultats bactériologiques et cliniques. On a enregistré une réaction bactériologique ‘bonne’ ou ‘très bonne’ dans 71% des oreilles traitées. Zusammenfassung. Die Wirksamkeit eines Präparats (Oterna—Glaxo Laboratories Ltd) für die Behandlung von äußerer Otitis bei kleinen Tieren ist bakteriologisch bestimmt worden. Die Ursachenforschung dieser Krankheit wurde an 133 Ohren von Katzen und Hunden durch‐geführt. Zum Vergleich wurden normale Ohren untersucht. Pityrosporum canis und Staphylokokken waren die am häufigsten abgesonderten Lebewesen. Ihr Vorkommen war bei klinisch betroffenen Ohren viel größer als bei normalen Ohren. Gram‐negative Lebewesen wurden nur in kranken Ohren vorgefunden. Dreiundsiebenzig klinische Fälle von Otitis bei Hunden und Katzen wurden mit einem Präparat behandelt, das Neomycin, Monosulfiram und Betamethasone enthielt. Bakteriologische und klinische Bewertungen wurden vor und nach Behandlung durchgeführt, und die abgesonderten Lebewesen wurden auf Empfindlichkeit gegen mikrobenbetreffende Bestandteile des...

A potent fl-lactamase (EC 3.5.2.6) produced by a strain of Klebsiella aerogenes (K. pneumoniae), 1082E, isolated from a hospital patient, has been examined. Its properties were different from those of most gram-negative f-lactamases previously reported. The enzyme has been partly purified, and its activity against a range of substrates has been compared with that of the enzyme from Enterobacter cloacae (Aerobacter cloacae) P99. The K. aerogenes enzyme, although predominantly a penicillinase, had a wide range of specificity. In addition to hydrolyzing the cephalosporins, it attacked the normally fl-lactamaseresistant compounds methicillin and cloxacillin as well as cephalosporin analogues with the same acyl substituents. The results obtained with the E. cloacae enzyme confirmed its cephalosporinase activity and showed that, unlike the enzyme from K. aerogenes, it was relatively inactive against the penicillins. The fl-lactamases produced by gram-negative bacteria differ considerably in their substrate specificities. Although most of the enzymes hydrolyze penicillin G, cephalothin, and cephaloridine, comparatively few hydrolyze methicillin or cloxacillin.

Su ary: Cephaloridine serum half-life was determined in 11 patients undergoing maintenance haemodialysis. Three of them were anephric. The mean cephaloridine half-life was 10.4 hours. There was an inverse correlation between cephaloridine half-life and the duration of maintenance haemodialysis treatment. Reasons for this are discussed. The effect of haemodialysis with the Kill dialyser on cephaloridine half-life was studied in three patients. Dosage recommendations for patients on maintenance haemodialysis are suggested.