The Use of Analytical Isoelectric Focusing for Detection and Identification of  -Lactamases

Abstract

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 ...

Detection of plasmid-mediated beta-lactamases with DNA probes

“…Extracts were prepared by sonication from cultures grown exponentially in brain heart infusion broth (Difco Laboratories, Detroit, Mich.) and frozen at -20°C until use. Analytical isoelectric focusing on polyacrylamide gels was carried out as described by Matthew et al (17). P-Lactamases isolated from control strains were used as pI standards.…”

ATR‐FTIR spectroscopic investigation of E. coli transconjugants β‐lactams‐resistance phenotype

“…Bacterial extracts were obtained by sonication (Sonicator: Sonifier 200, OSI, Paris, France) as described by Matthew et al [2]. The pulsation time was 1 min with a sonication frequency of 30%/s.…”

“…These strains are responsible for serious nosocomial infections in French hospitals since 1985 and thus knowledge of the main resistance patterns of E. coli isolates to (5-lactam agents should help in the choice of therapeutic strategies. The characterisation of TEM and SHV phenotypes can be performed by studying the biochemical properties of (3-lactamases (extended-spectrum or extended broad-spectrum P-lactamases) by isoelectric focusing, by determination of substrate and inhibition profile [2,3] and by hybridisation or nucleotide sequencing [4]. However, access to a new tool that enables rapid and straightforward characterisation of various TEM and SHV phenotypes seems of importance.…”

Molecular characterization of a new plasmid-encoded SHV-type -lactamase (SHV-2 variant) conferring high-level cefotaxime resistance upon Klebsiella pneumoniae

“…P-Lactamase SHV-1, first detected by Pitton (1972) and designated as such by Matthew et al (1979), Abbreviations: CTX, cefotaxime; CTZ, ceftazidime; IEF, isoelectric focusing; ORF, open reading frame; pHMB, p-hydroxymercuri benzoate.…”