Evaluation of five different methods to prepare bacterial extracts for the identification of beta-lactamases by isoelectric focusing.
The X-PRESS, osmotic shock, chloroform treatment, lysozyme treatment and ultrasonic disruption methods to release five different plasmid-mediated beta-lactamases from Escherichia coli and one chromosomal beta-lactamase from Enterobacter cloacae were compared. The main activities of TEM-1, SHV-1, OXA-1, OXA-2, PSE-4 and chromosomal P99 beta-lactamases were found at the same isoelectric point irrespective of the method used. However, additional satellite bands were found with TEM-1, OXA-1, OXA-2 and PSE-4 beta-lactamases released by the lysozyme method. In addition, beta-lactamase released by osmotic shock treatment was found to be unstable during storage at -20 degrees C or during the 18 h period of iso-electric focusing at +4 degrees C. Chloroform treatment produced similar band patterns and at least as good an enzyme yield as ultrasonic disintegration and was equally simple and fast to perform.
Arstila
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T
Antimicrobial Research Unit, National Public Health Institute, Turku, Finland.
Jacoby
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GA
Huovinen
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P
D001419:Bacteria / Q000201:enzymology*; D001431:Bacteriological Techniques; D002725:Chloroform / Q000737:chemistry; D005069:Evaluation Studies as Topic; D007525:Isoelectric Focusing; D009113:Muramidase / Q000737:chemistry; D009997:Osmotic Pressure; D014465:Ultrasonics; D001618:beta-Lactamases / Q000302:isolation & purification*
D002725:Chloroform; D009113:Muramidase; C039404:beta-lactamase OXA-2; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1