A variant of the microtitre GM1-ELISA for Escherichia coli heat-labile enterotoxin was studied. The test was improved by both reducing the assay time from 2 1/2 d to 8 h and by determining the most appropriate GM1 coating concentration. Coating the plates with greater than or equal to 3 micrograms of GM1/ml yielded a maximal sensitivity and ensured a linear relationship between the enterotoxin concentration and the extinction observed when using the final assay-procedure. Thus an optimal accuracy was obtained. This ELISA was 4- to 8-times more sensitive than the Vero cell monolayer assay. The sensitivity of this ELISA and of the chinese hamster ovary cell monolayer assay were identical.

Out of 15 selected enterobacterial strains resistant to ampicillin, 12 were able to transfer resistance to mecillinam to Escherichia coli K-12. This resistance to mecillinam was found to be coupled to the presence of beta-lactamase. One strain contained a beta-lactamase characterized as a class IV beta-lactamase, whereas the other 14 strains possessed a class HI (TEM-like) beta-lactamase. The specific activity of the class IV beta-lactamase against mecillinam was 55%, and those of the class III beta-lactamases were 180% of the specific activities against benzylpenicillin. In view of this high beta-lactamase sensitivity of mecillinam, the minimal inhibitory concentrations were lower than might be expected. However, after enzymatic hydrolysis of mecillinam, no antibacterial activity was found. At increasing salt or buffer concentrations the minimal inhibitory concentrations of mecillinam increase to a varying extent for all strains, independently of betalactamase production. This study indicates that the increase in miniimal inhibitory concentration is dependent on the salt concentration. The study also shows that this increase is not due to salt-mediated hydrolysis or to stimulation either of beta-lactamase activity or of beta-lactamase production. To explain the difference between ampicillin and mecillinam resistance in the beta-lactamase-positive strains, a hypothetical model is presented and discussed.Mecillinam (6-beta-amidinopenicillanic acid) shows a remarkably high activity against gramnegative bacteria, whereas its activity against staphylococci and streptococci is relatively low (6). At low concentrations of this antibiotic Escherichia coli becomes spherical; at higher concentrations (>10 ,tg/ml) cells tend to lyse (7). Previous studies of this antibiotic include antibacterial activity (2, 6), mechanism of action (4,7,8,12), and emergence of resistance (1,3,7).Three factors may contribute to bacterial resistance to beta-lactam antibiotics: (i) the specific antibiotic susceptibility of the target, (ii) the specific penetration barrier function of the cell wall, and (iii) the presence of a beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6). A combination of these factors may occur.In vitro Enterobacteriaceae may readily become resistant to mecillinam (2). During successive passages in media containing mecillinam, an Aerobacter aerogenes strain developed resistance to mecillinam more rapidly than against ampicillin, cloxacillin, or cephazolin (11). Crossresistance between mecillinam and ampicillin is minimal (1). These data and the fact that the spontaneous mutation frequency from mecillinam-susceptible to -resistant strains is quite high (10-5 to 10-4) for beta-lactamase-negative E. coli (3) strongly suggest that these kinds of mecillinam resistance are all due to chromosomal mutations of the mecillinam target or of other components of the cell wall.Richmond showed that a number ofpenicillins were impeded in penetrating the periplasmic space of E. coli UB 1005, whereas a penetration barrier...

Comparison of the activity of cefoperazone, cefamandole, cefotaxime, cephalothin and moxalactam against Enterobacteriaceae showed cefoperazone to be twofold (Escherichia coli, Klebsiella) to eigthfold (Enterobacter) more active than cefamandole. The lowes MIC values were found for cefotaxime (0.03–0.25 µg/ml) followed by moxalactam (0.06–0.25 µg/ml). Cefoperazone stood out in activity against Pseudomonas aeruginosa (MIC₅₀ 4 µg/ml). Cephalothin resistance affected the MIC values of cefoperazone and moxalactam only to a small degree. From beta-lactamase susceptibility studies it was concluded that cefoperazone may be hydrolyzed by TEM type beta-lactamases, but that the cephalosporinases (class I) and the chromosomal broad-spectrum beta-lactamases (class IV) only have little effect on this antibiotic. Moxalactam was not degraded by any of the beta-lactamase preparations tested.

A Proteus-typing method based on proticin production and proticin susceptibility (c.f. Senior, 1977) has been modified to increase its sensitivity Proticins were prepared in fluid medium and applied to agar-plates shortly before seeding the plates with indicator bacteria. A given 10 proticin producer strains, which are responsible for the susceptibility patterns of the indicator-bacteria (S-types), form the foundation for this typing method. Using this producer-set and indicator-set (28 strains) was selected which was suitable for the typing of strains with different proticin activities (P-types). Standardization of the temperature for proticin production proved to be necessary. The degree of similarity between proticins was further elaborated by testing all indicator strains for susceptibility to proticin titrations. In the group of 148 clinical Proteus-isolates (four species) used for the development of the typing system 28 S-types and 34 P-types were observed. By combining the S-and P-type parameters 86 S-P-types were obtained for the 4 species combined. Seven strains were not typable. A separate group of 100 clinical Proteus-isolates was tested in order to prove the usefulness of the method. 39 new S-P types were found. Repeated isolations from the same patients yield the same patterns. Proteus S-P-typing is a useful method for the typing of Proteus vulgaris and Proteus mirabilis, but proves inadequate for the typing of Proteus rettgeri and Proteus morganii.

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