antibodies only one segregant was found. This was susceptible to chloramphenicol and streptomycin but was resistant to tetracycline and kanamycin.Discussion. It is impossible to give a precise rate for the transfer of the R factor but clearly it was able to spread epidemically and, eventually, infect a high proportion of K12. This occurred without the selective pressure of antibiotic treatment. However, the rate of spread was much slower than in vitro. Eighteen hours after feeding AK to the mice, only O.OOS./, of K12 had acquired antibiotic resistance. In zlitro 1 . 6 v acquired drug resistance in 18 hours. Only after more than 2 weeks did more than 1C/o of the K12 in the gut acquire the R factor. Nonetheless, the rate of acquisition of the R factor was much higher than the rate of segregation.I t is uncertain to what extent we may extrapolate these findings to conventional animals. There are many differences between germ-free and conventional animals. We cannot be certain that observation in mice contaminated only with Enterobacteriacae would be similar in conventional mice who are contaminated with many species of bacteria. For example, the bacterial density of Enterobactcriacae a t the sites in the gut where the milieu is suitable for R factor transfer may be much different in mono-contaminated germfree mice and in conventional animals. Also, subtle differences in the physical and chemical makeup of intestinal contents could profoundly affect the efficiency of R factor transfer. In vitro, R factor transfer is greatly inhibited by anaerobic growth and by inhibitors of oxidative phosphorylation (6). If the intestinal contents of mono-contaminated germ-free mice were less anaerobic than the gut contents of conventional mice, the rate of transfer of the R factor would, in all probability, be greater. Further work is required to explore these factors.Summary. An antibiotic resistant Klcbsiclla pneumoniae containing an R factor was fed to germ-free mice previously colonized with an antibiotic susceptible E . coli. Within a month, 21% of the E. coli acquired multiple drug resistance without the selective pressure of antibiotic therapy. However, the rate of spread of the R factor was significantly slower than in vitro.LVe thank Miss Dorinne Kan for her able assistance.
West Indian spiny lobsters, Panulirus argus, synthesized a hemolymph bactericidin after being injected with killed suspensions of gram-negative bacillus EMB-1 isolated from the normal gut of this lobster. To study differences between the primary response and secondary response, animals were given a primary antigen injection of EMB-1 followed by a second injection of the same antigen 22 to 51 days later. As a rule, secondary bactericidal responses were enhanced over the primary in a manner reminiscent of specific anamnesis in mammalian immunoglobulin synthesis. Immunological memory was also suggested when tertiary responses were compared to secondary and by the persistence of residual titers for many days or weeks without additional antigenic stimulation.
This report continues comparative studies of inducible bactericidins of invertebrates. The bactericidin of the sipunculid worm Dendrostomum zostericolum Chamberlain was induced by intracoelomic injections of killed bacteria. With the assay system used, optimal results were obtained with 90 min of incubation at 20 to 35 C. Bactericidal titers in coelomic fluids from noninjected worms were either low or nonexistent. After injections of gram-negative bacteria, bactericidal titers of up to 1:1280 were reached within 7 days. Considerable individual variations were noted. Optimal stimulation was achieved with 4 x 108 bacteria per injection. The response was relatively nonspecific as measured by cross-immunization and absorption experiments. Injections of sterile sea water produced no response. Coelomic fluids could be inactivated by heating at 50 or 56 C for 20 min. Activity was not restored by a pool of unheated coelomic fluid from nonimmunized worms. The significance of these observations was discussed with reference to immunity of sipunculids and to comparative immunology.
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