ANTUCROBIAL AGENTS AND CHEzOTHrAPY, Sept. 1975, p. 271-276 Vol. 8, No. 3 Copyright 0 1975 American Society for Microbiology Printed in U.S.A. Mecillinam (FL 1060), a 6,3-Amidinopenicillanic Acid Derivative: Bactericidal Action and Synergy In Vitro L. TYBRING* AND N. H. MELCHIOR Bacteriological Research Department, Leo Pharmaceutical Products, DK-2750 Ballerup, Denmark Received for publication 26 December 1974 A newly described 6d-amidinopenicillanic acid derivative, mecillinam (for- merly called FL 1060), showed a high in vitro activity against Enterobacteriac- eae. 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. The morphology of the cells changed towards large spheric forms (2 to 5 ,m) under the influence of mecillinam. Consequently a great discrepancy between the optical density and the viable count was seen. The morphologically abnormal cells could be protected against lysis in vitro by addition of ionized compounds such as sodium chloride. Abnormal cells were more sensitive to ampicillin than normal cells. As expected synergy could be demonstrated between mecillinam and ampicillin. This was marked under experimental conditions where the abnormal cells were protected against lysis. In a previous paper from this laboratory a new the liquid media was measured by the cryostatic group of penicillanic acid derivatives, 6,- method using a Knauer type M osmometer, and the amidinopenicillanic acids, with unusual in specific conductivity was measured as millisiemens at vitro antibacterial properties was described (4). 36 C using a conductivity meter, Radiometer type One member of CDM 2c. the group, mecillinam Bactericidal activity. The bactericidal effect of (proposed intemational nonproprietary name), mecillinam on a growing culture of Escherichia coli 6,B- [(hexahydro-lH-azepin-1-yl)-methylene- (Leo HA2) was determined by measuring the change amino ]-penicillanic acid (formerly called FL in viable count and optical density (OD) during the 1060), has been studied extensively, and it has first 6 h of incubation. Viable counts were performed been shown that this compound differs funda- in quadruplicate on NIH agar by plating 0.1 ml of mentally from the hitherto known penicillanic serial 10-fold dilutions in NIH liquid medium. The acid derivatives both in regard to antibacterial colonies were counted after 18 h at 36 C. OD was spectrum (4, 8) and its mode of action (3, 5-8). measured by means of a Vitatron MPS photometer with a tungsten light without a filter. In some The results presented in this paper deal with instances the total number of microbial cells was the bactericidal effect of mecillinam and its quantitatively determined by microscopy. synergistic action with ampicillin in vitro. In Fig. 1 to 4 the OD scale was adjusted to the viable count scale by measuring the OD of dilutions of MATERIALS AND METHODS an overnight culture of the test organism. Such a Antibiotics. Mecillinam was used as the hydro- culture consists of separate minor cells, and the chloride dihydrate and ampicillin as the trihydrate. &adjustment is therefore not entirely valid for a culture The concentrations of both compounds were calcu- in the log phase, where the cells are more voluminous lated on the basis of the anhydrous zwitterion. Solu- and many pairs or short chains occur, each represent- tions of the compounds were freshly prepared for each ing only one colony-forming unit. experiment. Antibacterial activity. Antibacterial activity was Culture media. NIH agar medium and the corre- determined by the serial dilution technique either in sponding liquid medium were used throughout the agar or fluid medium. Agar plates were simultane- study. The medium contains 0.5% yeast extract ously seeded with 12 to 14 different Enterobacteriac- (Difco), 1.5% casein hydrolysate (pancreatic), 0.1% eae from our culture collection, using a heavy inocu- dextrose, 0.25% sodium chloride, 0.005% L-cystine, lum of 3.5-,1l drops of undiluted overnight cultures. and 1% agar (Oxoid no. 1) in distilled water, pH 7.1, This inoculum produced 10-mm zones of growth on after autoclaving. In some experiments NIH agar or control plates. The inoculum in fluid medium was liquid media without sodium chloride was used as a about 104 or 106 cells per ml. The results were read basic medium to which membrane-filtered solutions after 18 h at 36 C as the minimum inhibitory concen- of different compounds were added. The osmolality of tration (MIC). 271 272 TYBRING AND MELCHIOR ANTIMICROB. AGENTS CHEMOTHER. 0 3 I.X \ s~~~~ @'^*.,V.Q Mecillinam FIG. 2. Growth of E. coli with and without 1 ,g of mecillinam per ml, recorded as viable count (VC) and OD with an inoculum of 4.9 x 106 cells per ml. i1 2 3 i 6hrs. FIG. 1. Growth of E. coli with and without 1,gg of mecillinam per ml, recorded as viable count (VC) and OD with an inoculum of 1.2 x 106 cell per ml. RESULTS AND DISCUSSION Bactericidal activity. The growth of E. coli (Leo HA2) was compared in broth with and without mecillinam. The changes in viable count and in cell mass (OD) were measured. Figure 1 shows the effect of 1 Ag of mecillinam per ml on an E. coli culture with an initial cell FIG. 3. Growth of E. coli uwith and without 1 Ag of count of 1.2 x 106 per ml (0.2% of an overnight mecillinam per ml, recorded as viable count (VC) and culture). The viable count declined in 6 h to less OD with an inoculum of 2.6 x 107 cells per ml. than 100 per ml. The OD, however, was only slightly less than that of the control culture for ,gm and thereafter by lysis (4, 6). Furthermore the first 2 h. After 2.5 h the OD decreased due to the viable count declined faster than the total lysis of the cells. cell count (determined by microscopy) indicat- This dicrepancy between viable count and ing an increasing number of nonviable cells. cell mass agrees with previous observations by The same low final viable count was observed us that growing cells of E. coli respond to the with various concentrations of mecillinam down action of mecillinam by first forming swollen to 0.06 ,g/ml. At a contration of 0.02 fg/ml the and later spherical cells with diameters up to 5 final count was 1,500 per ml. VOL. 8, 1975 BACTERICIDAL ACTION OF MECILLINAM 273 oxygen was introduced continously and the 11: culture was stirred, the final count increased F 15-fold, i.e., a culture with the identical inocu- lum as above could now accomplish 8 doublings ,IC instead of 4.3 doublings. Mecillinam (1 Ag/ml) had a marked bactericidal effect on such a culture (Fig. 4), as the initial count was reduced more than 10,000-fold in 6 h. The OD was still high after 6 h due to the remains of lysed cells. Mecillinam only exhibits its bactericidal ef- fect when the cells are growing under the influence of the substance for relatively long periods of time. To ensure suitable growth o.Dn conditions for a dense population over a pro- Mecillinam 0 1 longed period a simple chemostat was used. A II steady-state culture containing 2 x 108 E. coli II I cells per ml responded to 1 qg of mecillinam per II ml with marked lysis. II II Table 1 summarizes the results of these II experiments and shows the correlation between I the number of cell doublings accomplished by II the control culture and the bactericidal effect of mecillinam, expressed as the reduction ratio, 0 i.e., the ratio of initial count to count after 6 h. The more cell doublings possible the higher the 0 reduction ratio at the same concentration. When the compound was added to an exponen- tially growing culture the same correlation be- tween the reduction ratio and the number of cell doublings of the control was seen. The inoculum and the experimental condi- V.G Mecillinam tions determine the number of possible cell doublings. As the lytic effect of mecillinam depends on the time growing cells are exposed FIG. 4. Growth of E. coli with and without 1 ,g of to the compound, an apparent strong inoculum mecillinam per ml, recorded as viable count (VC) and effect can be seen. However, the inoculum effect OD, with an inoculum of 3.3 x 107 cells per ml, is not determined by the initial number of cells oxygenated, and stirred culture. but by the initial cell mass relative to that of the maximum stationary phase. Figure 2 shows the bactericidal effect of 1 yg Alterations in the composition of the medium of mecillinam per ml against 4.9 x 106 orga- influenced the results. When the 0.25% sodium nisms per ml. This inoculum gave a measurable chloride was omitted from the normal medium, OD from the beginning of the experiment. After a steep decline in viable counts occurred after 60 3 h the entire cell mass had reached a level close min instead of after 90 min. Furthermore, the to that of the maximum stationary phase. reduction ratio was 2.5 x 104 even when the Consequently the lysis and decline in viable initial count corresponded to 5% of that of an count over the next few hours were much less overnight culture. pronounced. Compared to ampicillin the bactericidal ef- When an inoculum of 2.6 x 107 cells was used, fect of mecillinam on E. coli sets in rather late.