M. H. Nicolas

Before 1985 at the Pitié-Salpêtrière Hospital in Paris (2,400 beds), resistance to cefotaxime in clinical isolates of Enterobacteriaceae involved only species producing inducible class 1 beta-lactamase. Between November 1985 and April 1987, however, 62 isolates (57 of Klebsiella pneumoniae and five of Escherichia coli) showed decreased susceptibility to cefotaxime (mean MIC, 8-16 micrograms/mL). The transferability of cefotaxime resistance in E. coli K12 was demonstrated for 15 of 16 selected isolates. By isoelectric focusing using iodometric detection with 20 mg of ceftriaxone/100 mL and determination of substrate and inhibition profiles, three beta-lactamases mediating cefotaxime resistance were identified as SHV-2 (isoelectric point [pI] 7.6), CTX-1 (pI 6.3), and "SHV-2-type" or SHV-3 (pI 6.98). The three beta-lactamases hydrolyzed penicillins and cephalosporins (including cefotaxime and ceftriaxone) and were therefore designated "extended broad-spectrum beta-lactamases" (EBS-Bla). The enzymes conferred to derivatives a high level of resistance to amoxicillin, ticarcillin, piperacillin, and cephalothin and a decreased degree of susceptibility (i.e., MICs increased by 10- to 800-fold) to cefotaxime, ceftriaxone, ceftazidime, and aztreonam. These beta-lactamases did not affect the activity of cephamycins (cefoxitin, cefotetan, moxalactam) or imipenem. Synergy between clavulanate or sulbactam (2 micrograms/mL) and amoxicillin was greater against derivatives producing EBS-Bla than against those producing TEM-1, TEM-2, or SHV-1; this synergy was greater with clavulanate than with sulbactam against derivatives producing SHV-2 and the SHV-2-type enzyme but was similar with clavulanate and sulbactam against those producing CTX-1. A double-disk synergy test performed with cefotaxime and Augmentin disks (placed 30 mm apart, center to center) seemed a useful and specific test for the detection of strains producing EBS-Bla.

A clinical isolate of Enterobacter cloacae, strain NOR-1, exhibited resistance to imipenem and remained susceptible to extended-spectrum cephalosporins. Clavulanic acid partially restored the susceptibility of the strain to imipenem. Two beta-lactamases with isoelectric points (pI) of 6.9 and > 9.2 were detected in strain E. cloacae NOR-1; the higher pI corresponded to AmpC cephalosporinase. Plasmid DNA was not detected in E. cloacae NOR-1 and imipenem resistance could not be transferred into Escherichia coli JM109. The carbapenem-hydrolyzing beta-lactamase gene was cloned into plasmid pACYC184. One recombinant plasmid, pPTN1, harbored a 5.3-kb Sau3A fragment from E. cloacae NOR-1 expressing the carbapenem-hydrolyzing beta-lactamase. This enzyme (pI 6.9) hydrolyzed ampicillin, cephalothin, and imipenem more rapidly than it did meropenem and aztreonam, but it hydrolyzed extended-spectrum cephalosporins only weakly and did not hydrolyze cefoxitin. Hydrolytic activity was partially inhibited by clavulanic acid, sulbactam, and tazobactam, was nonsusceptible to chelating agents such as EDTA and 1,10-o-phenanthroline, and was independent of the presence of ZnCl2. Its relative molecular mass was 30,000 Da. Induction experiments concluded that the carbapenem-hydrolyzing beta-lactamase biosynthesis was inducible by cefoxitin and imipenem. Subcloning experiments with HindIII partial digests of pPTN1 resulted in a recombinant plasmid, designated pPTN2, which contained a 1.3-kb insert from pPTN1 and which conferred resistance to beta-lactam antibiotics. Hybridization studies performed with a 1.2-kb HindIII fragment from pPtN2 failed to determine any homology with ampC of E. cloacae, with other known beta-lactamase genes commonly found in members of the family Enterobacteriaceae (bla(TEM-1)) and bla(SHV-3) derivatives), and with previously described carbapenemase genes such as those from Xanthomonas maltophilia, Bacillus cereus, Bacteroides fragilis (cfiA), and Aeromonas hydrophila (cphA). This work describing the biochemical properties of a novel chromosome-encoded beta-lactamase from E. cloacae indicates that this enzyme differs from all the previously described carbapenemases. This is the first reported cloning of a carbapenem-hydrolyzing gene from a member of the family Enterobacteriaceae.

Carbapenem resistance was studied in a clinical isolate of Enterobacter cloacae, strain 201 (MIC of imipenem and meropenem, 16 micrograms/ml). This strain was analyzed comparatively with the carbapenem-susceptible parent strain 200, an equally susceptible revertant, 201-Rev, and in vitro-selected mutants with different levels of carbapenem resistance. All strains produced similarly high amounts of the same cephalosporinase (pIapp = 8.8). Strain 201 apparently lacked two major outer membrane proteins of ca. 37 and 38 kDa, while 201-Rev produced only the 37-kDa protein. The permeability coefficient, determined with cephaloridine, was reduced up to ninefold in the resistant strains which also showed a substantial reduction in the uptake of [14C]meropenem. The introduction of the plasmid-borne ampD gene (whose product decreases the expression of ampC) resulted in almost complete cessation of cephalosporinase production in all strains and a substantial decrease in the MICs of the carbapenems which remained, however, 8- to 16-fold higher than those determined for the susceptible strains containing the ampD gene. This "residual" resistance was attributed to reduced outer membrane permeability. The contribution of cephalosporinase production was verified in a reverse experiment, in which the introduction of ampC into a low-level cephalosporinase producer resulted in a fourfold increase in the carbapenem MICs. From these results, we infer that reduced outer membrane permeability and high-level cephalosporinase production can operate in conjunction in clinical isolates of E. cloacae to confer imipenem resistance.