A newly described 6d-amidinopenicillanic acid derivative, mecillinam (formerly called FL 1060), showed a high in vitro activity against Enterobacteriaceae. The effect on Escherichia coli was bactericidal and was due to lysis of the cells. The longer the culture grew under the influence of mecillinam or the lower the inoculum, the greater the bactericidal effect. In a previous paper from this laboratory a new group of penicillanic acid derivatives, 6,-amidinopenicillanic acids, with unusual in vitro antibacterial properties was described (4). One member of the group, mecillinam (proposed intemational nonproprietary name), 6,B-[(hexahydro-lH-azepin-1-yl)-methyleneamino ]-penicillanic acid (formerly called FL 1060), has been studied extensively, and it has been shown that this compound differs fundamentally from the hitherto known penicillanic acid derivatives both in regard to antibacterial spectrum (4, 8) and its mode of action (3, 5-8).The results presented in this paper deal with the bactericidal effect of mecillinam and its synergistic action with ampicillin in vitro.MATERIALS AND METHODS Antibiotics. Mecillinam was used as the hydrochloride dihydrate and ampicillin as the trihydrate. The concentrations of both compounds were calculated on the basis of the anhydrous zwitterion. Solutions of the compounds were freshly prepared for each experiment.Culture media. NIH agar medium and the corresponding liquid medium were used throughout the study. The medium contains 0.5% yeast extract (Difco), 1.5% casein hydrolysate (pancreatic), 0.1% dextrose, 0.25% sodium chloride, 0.005% L-cystine, and 1% agar (Oxoid no. 1) in distilled water, pH 7.1, after autoclaving. In some experiments NIH agar or liquid media without sodium chloride was used as a basic medium to which membrane-filtered solutions of different compounds were added. The osmolality of the liquid media was measured by the cryostatic method using a Knauer type M osmometer, and the specific conductivity was measured as millisiemens at 36 C using a conductivity meter, Radiometer type CDM 2c.Bactericidal activity. The bactericidal effect of mecillinam on a growing culture of Escherichia coli (Leo HA2) was determined by measuring the change in viable count and optical density (OD) during the first 6 h of incubation. Viable counts were performed in quadruplicate on NIH agar by plating 0.1 ml of serial 10-fold dilutions in NIH liquid medium. The colonies were counted after 18 h at 36 C. OD was measured by means of a Vitatron MPS photometer with a tungsten light without a filter. In some instances the total number of microbial cells was quantitatively determined by microscopy.In Fig. 1 to 4 the OD scale was adjusted to the viable count scale by measuring the OD of dilutions of an overnight culture of the test organism. Such a culture consists of separate minor cells, and the &adjustment is therefore not entirely valid for a culture in the log phase, where the cells are more voluminous and many pairs or short chains occur, each representing only one colony-forming unit...