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Because a multiresistant K. pneumoniae outbreak detected in an intensive care unit of a parisian hospital, combined to the production of the plasmid-encoded cephalosporinase ACC-1, a probable importation via a patient was suggested from another country (Tunisia). The investigation was conducted to examine 35 clinical strains of enterobacteria resistant to ceftazidime without synergy towards Augmentin. Other test of synergy with two inhibitors, BRL 42715, Ro 48-5545 was performed by diffusion method and deposit of 10 micrograms of inhibitor on disks containing ceftazidime, cefoxitin and cefotetan. Synergies were obtained suggesting a probable production of ACC-1 type among six isolates of K. pneumoniae (two), Proteus mirabilis (one) and Salmonella (three) issued from different units. The isoelectric focusing on gel revealed at least one band of beta-lactamase activity at 7.8 but also demonstrated the simultaneous production of several probable beta-lactamases including TEM-type, SHV-2 and ACC-1 among S. enterica ser. Livingstone. The PCR of the gene blaacc-1 was positive. The sequencing (1160 pb) of two products showed high identity (99-100%) with the gene blaacc-1 deposited in 1999. Finally the ACC-1 type reported in Tunisia was probably imported in France via a patient. Because a simultaneous synthesis of ESBL and ACC-1 type, its presence may be invisible and need more investigation.

We analyzed the beta-lactamase production of a Serratia fonticola isolated for its resistance to cefuroxime (Minimum Inhibitory Concentration > 256 mg/l) in December 1993 from a patient hospitalized in Meaux. The wild strain was resistant to amoxycillin but sensitive to augmentin, that suggested the production of a beta-lactamase susceptible to clavulanic acid. For the wild strain, beta-lactamase production was inducible and only one enzyme with an isoelectric point of 8.12 was detected. beta-lactamase production was 16 mU/mg for non-induced extracts and ranged from 100 to 230 mU/mg in the presence of inducing beta-lactams (enzyme activity was measured with penicillin G as substrate). On a Szybalski gradient a constitutive strain was obtained. Its enzyme production was 13,000 mU/mg. The kinetics and isoelectric points of the enzymes produced by the two strains were identical. This beta-lactamase hydrolyzes penicillins (amoxycillin: Vm = 60 relative to penicillin G = 100, ticarcillin: 15), first generation cephalosporins (cephalothin Vm = 930). However, this enzyme hydrolyzes efficiently oxyimino-cephalosporins: cefuroxime (Vm = 70) and cefotaxime (Vm = 120), but cephamycins are not substrates. Clavulanic acid has a very good affinity for this beta-lactamase (Ki = 0.09 microM) which is inactivated progressively (I50 = 0.045 microgram/ml). These properties shows some similarities with those of the class A beta-lactamases of P. vulgaris RO104 (pI = 8.3), P. penneri 14HBC (pI = 6.65) and the plasmid-mediated extended-spectrum MEN-1 (pI = 8.4).

P. penneri produces an inducible cephalosporinase, as many Enterobacteriaceae. Nevertheless this betalactamase is susceptible to clavulanic acid which is an exception also encountered for P. vulgaris. The authors studied the enzyme produced by P. penneri 14HBC resistant to cefotaxime (MIC 16 mg/l) isolated in Spain in 1992. This betalactamase of isoelectric point 6.65 hydrolyzes first generation cephalosporins, amoxycillin and poorly ticarcillin as it occurs for all cephalosporinases. However, this enzyme hydrolyzes strongly oxyimino-cephalosporins: cefuroxime, cefotaxime, cefepime, cefpirome as it occurs with extended-spectrum betalactamases. Cephamycins and imipenem are not substrates. Clavulanic acid has a very good affinity for this betalactamase which is inactivated progressively. These properties are similar to those of the enzyme of P. vulgaris Ro104 of isoelectric point 8.3 which, contrarily to other cephalosporinases, belongs to the structural Ambler's class A.

Affinity columns able to purifie beta-lactamases have been prepared either by linking covalently reversible inhibitors or substrates to agarose beds. The enzyme is eluted with a gradient of sodium chloride or released with the substrate. This method is a pertinent one for the purification of these enzymes and for the study of bacteria harbouring more than one beta-lactamase.

This paper is dealing with the enzymatic problem raised by two strains of Ps. aeruginosa resistant to classical beta lactam antibiotics including carbenicillin. These two strains hydrolyse all these antibiotics. In both cases, we have shown the simultaneous biosynthesis of two enzymes: an inducible and chromosome cephalosporinase frequently found in this germ, and a constitutive beta lactamase, with a penicillinase activity which has been identified with the extrachromosomic beta lactamase R-TEM. These two enzymes have been separated by affinity chromatography, characterized by their kinetic constants given by computerized microacidimetry, and their isoelectric points which are respectively 9.2 for the cephalosporinase and 5.40 for the penicillinase R-TEM. Isoelectric focussing also shows the separation of these two enzymes.