778 Eur. J. Clin. Microbiol. Infect. Dis. tions of outbreaks. Reviews of Infectious Diseases 1986, 8: 682-692. 6. Van de Klundert, J. A. M., Van Gestel, M. H., Van Doom, E., Mouton, R. P.: Disc diffusion test for the determina- tion of semi-quantitative substrate profiles of beta- lactamases. Journal of Antimicrobial Chemotherapy 1986, 17: 471-479. 7. Lugtenberg,B., Meyers,J., Peters, R., Van tier Hoek, P., Van Alphen, L.: Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Letters 1975, 58: 254-258. 8. Harder,K.J., Nikaido, H., Matsuhashi, M.: Mutants of Escherichia coti that are resistant to certain beta-lactam compounds Iack the OmpF porin. Antimicrobial Agents and Chemotherapy 1981, 20: 549-552. 9. Piddoek, L. J.V., Wijnands,W. J.A., Wise, R.: Quino- lone]ureidopenicillin cross-resistance. Lancet 1987, ii: 907. Synergistic Effect of Dosage and Bacterial Inoculum in TEM-1 Mediated Antibiotic Resistance J. A. Reguera , F. Baquero 1 , J. C. Perez-Diaz 1 J. L. Martinez 1'2 The effect of inoculum size and gene dosage on the level of antibiotic resistance mediated by TEM-1 /3-1actamase was measured. From the results it seemed that gene dosage is a more efficient mechanism than inoculum size for increasing TEM-1 mediated resis- tance to fl-lactam antibiotics. It also seemed that the two mechanisms for enhancing antibiotic resistance are synergistic. The clinical implications of these results are discussed. Production of antibiotic-inactivating enzymes is one of the most common and clinically relevant antibiotic resistance mechanisms developed by bacteria (1). Epidemiological studies on the distribution of the different types of antibiotic-inactivating enzymes are useful to provide data for predicting the evolu- tion of the different patterns of antibiotic resistance. However, production of the same enzyme as a unique mechanism of resistance may result in different phenotypes, depending on the quantity of enzyme 1Servicio de Microbiologia, Hospital Ramon y Cajat, Ctra. Colmenar Km 9.100, 28034 Madrid, Spain. 2Instituto Investigaciones Biomedicas CStC, Arzobispo Morcillo 4, 28029 Madrid, Spain. produced by the organism (2, 3) and also on its inoculum size (3-5). In the case of/3-1actam anti- biotics, it has been clearly established that these two factors dramatically affect /3-1actamase mediated resistance. Furthermore, they can be responsible for unexpected mechanisms of antibiotic resistance such as the recently described TEM-1 mediated resistance to amoxycillin/clavulanate in Escherichia coli (6, 7). Using this enzyme as a model, we evaluated the in- fluence of the inoculum size and copy number (and hence, quantity of enzyme) on the resistance to different/3-1actams in Escherichia coil Materials and Methods. Experiments were performed using three isogenic Escherichia coli strains. As a fully antibiotic susceptible strain, Escherichia coli HB101 was used. Its derivative strains,Escherichia coli HB101 (pMM4::Tn3) and Escherichia coli HBIO1 (pUC8), were obtained by transformation with the plasmids pMM4::Tn3 and pUC8, respectively. Previous deter- ruination of the number of copies of both plasmids, carried out by the standard technique of cesium chloride-ethidium bromide equilibrium gradient ultra- centrifugation (8), revealed that plasmid pMM4::Tn3 was a monocopy whereas pUC8 yielded approximate- ly 100 copies per chromosome. Both plasmids encoded for the synthesis of TEM-1 /3-1actamase, but strains carrying the plasmid pUC8 produced 34 times more /34actamase than those carrying pMM4::Tn3 (23,000 mU/mg versus 675 mU/mg of bacterial protein, respectively) (3). MIC determinations were made in Mueller-Hinton broth in microtitre plates, using inocula of 103, l0 s and 107 CFU/ml. Results and Discussion. The MICs of different/34actam antibiotics were measured for three isogenic Eseheri- chia coli strains, which produced different quantities of TEM-1 /34actamase, using three different inocula. As Table 1 shows, only the MICs of penicillins (in- cluding piperaciUin) and, to a certain extent, first generation cephalosporins were affected by produc- tion of TEM-1 13-1actamase under conditions of a low inoculum and a single copy of the gene. The MICs of these agents and also of some second generation cephalosporins (such as cefamandole or LY163892) dramatically increased with both inoculum size and gene copy number. This increase was not seen in the case of poorly hydrolizabte second generation cepha- losporins (cefoxitin, cefuroxime, cefotetan), third generation cephalosporins, imipenem, carumonam and aztreonam. Various studies on the effect of inoculum size on the antibiotic resistance have been published (4, 5), but there are few data on the combination of the effects of inoculum size and gene dosage (3). From the results of Table 1, it is clear that gene dosage is a much more efficient mechanism for enhancement of antibiotic resistance than inoculum size. The ex-