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In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.

Plasmid-mediated 3-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observed for HMS-1 and PSE-1 ,B-lactamases. The HMS-1 sample produced two highly resolvable enzyme bands in agarose gels rather than the single faint enzyme band observed on polyacrylamide gels. The PSE-1 sample showed an isoelectric point shift of 0.2 pH unit between polyacrylamide and agarose gel (pl 5.7 and 5.5, respectively). The short focusing time, lack of toxic hazard, and ease of formulation make agarose a practical medium for the characterization of P-lactamases.Analytical isoelectric focusing (IEF) for the detection of P-lactamase enzymes from gramnegative bacteria has been done since the mid1970s (7). This method allows for the characterization and differentiation of many types of Plactamases by visual comparison of the banding patterns and by the isoelectric points of the constituent proteins. Currently, ,-lactamases are characterized by IEF in polyacrylamide gel, a procedure which is a modification of the original work of Matthew et al. in 1975 (7). This experimental procedure requires 18 h of IEF to achieve adequate identification of most ,B-lactamase types. The resulting banding patterns and isoelectric points are consistent, although the origins and chemical composition of the patterns have not been clearly delineated.One alternative to polyacrylamide is agarose, a purified galactan hydrocolloid which was recently introduced as a medium for analytical IEF. Agarose media offer a macroporous matrix of considerable strength at concentrations 51% (4). The macroporous matrix and the low ionic character of agarose minimize the problems of electroendosmosis and molecular sieving associated with polyacrylamide. Agarose is also simpler in gel formulation and is nontoxic, in contrast to the reported toxicity of unpolymerized t Reprint requests should be directed to Antone A. Medeiros, Miriam Hospital, 176 Summit Avenue, Providence, RI 02906.polyacrylamide (10). These factors led us to explore the use of agarose gel IEF for the identification of ,-lactamase enzymes and to compare these results with those established by polyacrylamide gel IEF. MATERIALS AND METHODSBacterial strains. The Escherichia coli and Pseudomonas aeruginosa strains containing plasmid-mediated P-lactamases used in this study were kindly supplied by Margaret Matthew, R. W. Hedges, and George A. Jacoby. E. coli K-12 strains included R6K(TEM-1) (2, 5); RP1(TEM-2) (5); RGN238(OXA-1) (1, 5); R46(OXA-2) (1, 5); R57b(OXA-3) (1, 5); R997(HMS-1) (8); and R1O1O(SHV-1) (8). P. aeruginosa PU21 strains contained plasmids RPL11(PSE-1) (9); R151(PSE-2) (6); Rmsl49(PSE-3) (1...

Epidemiologic studies of plasmid-mediated resistance at the Cleveland Veterans Administration Medical Center revealed that related plasmids had disseminated among members of the family Enterobacteriaceae. We studied the P-lactamases encoded by these plasmids in Escherichia coli C600 transformants or transconjugants.Substrate and inhibition profiles of the enzymes determined by two of these plasmids suggested an activity resembling TEM-1; however, isoelectric focusing revealed a pl of 7.0. These two plasmids were originally found in a Serratia marcescens (pDS076) and an Enterobacter cloacae (pDS075) strain isolated from the same sink in the medical intensive care unit and later, in an Enterobacter cloacae (pDS142 identical to pDS076) isolate colonizing a patient in the same unit. The plasmids also carried the aminoglycoside resistance determinant, 2 -aminoglycoside nucleotidyl transferase. A 2-kilobase AvaI restriction endonuclease digestion fragment of pDS075 known to carry the P-lactamase determinant was used as a molecular probe. This probe did not recognize sequences of any plasmid-mediated P-lactamase tested including the recently described determinants ROB-1, TLE-1, and OXA-4-7. A TEM-1 probe derived from the 0.7-kilobase PstI-EcoRI fragment of pBR322 failed to recognize the new l-lactamase gene. Four additional Enterobacter cloacae and two Enterobacter aerogenes strains isolated in Columbus, Ohio, have been shown to produce a pI 7.0 l-lactamase and to carry plasmids recognized by the 2-kilobase probe. These data suggest dissemination of a novel plasmid-mediated I(-lactamase among members of the family Enterobacteriaceae in Ohio and demonstrate the development and utility of a molecular probe for the new determinant. We suggest that the novel I-lactamase be named OHIO-1.