DEVELOPMENT OF RESISTANCE 



109 



4. l^y conilHiiing" two antibiotics, such as 

 streptomycin with penicilhn, or an antibiotic 

 and a chemical agent, such as streptomycin 

 with p-aminosahcyhc acid or isoniazid, 

 resistance to any one agent can be delayed 

 if not prevented. 



5. Little is known concerning the mecha- 

 nism of de\'elopment of resistance. The fact 

 that treatment of sensitive cells with deoxy- 

 ribonucleic acid isolated from resistant cells 

 renders the sensitive cells resistant, and 

 similar observations, may suggest proper 

 approaches in o\'ercoming the development 

 of resistance. 



The background of the problem of devel- 

 opment of resistance of microorganisms to 

 drugs, comprising both natural and acquired 

 resistance, has been further analyzed in 

 detail by Abi'aham (11)53), Schnitzer and 

 Grunberg (1957), DiAIarco (1958), and 

 others. Abraham (1959) considered the 

 al)ility of bacteria to acquire resistance to 

 antibiotics as one of the man}' examples of 

 the adaptability of microorganisms, involv- 

 ing the basic problems of protein and nucleic 

 acid synthesis. The procedure of Lederberg 

 and Lederl)erg (1952), whereby samples of a 

 mass of bacteria are first transferred from 

 the surface of a nutrient agar plate to a 

 velvet pad and then printed on other plates 

 to give replicas of the first, suggested that 

 bacteria highly resistant to streptom3^cin 

 can be formed without anj^ contact with the 

 drug. The fact that successive prints from a 

 normal plate of E. coli to plates containing 

 streptomycin show a lack of multiplication 

 of most of the organisms, but give growth of 

 a small numl^er of colonies of resistant cells 

 suggests that resistant cells, formed bj^ rare 

 mutations, already exist on the normal plate 

 and are later selected in the presence of the 

 drug. Abraham added, however, that this 

 does not prove that random mutation repre- 

 sents the only mechanism by which resist- 

 ance to antibiotics develops. 



Lederberg in 1959, said: 



In some favorable instances the spontaneous 

 origin of drug-resistant mutants can be verified 

 unambiguouslj^ by contriving to isolate them 

 without their ever being exposed to the drug. 

 One method entails indirect selection. To illus- 

 trate its application, consider a culture of Esch- 

 erichia coli containing 10^ bacteria per milliliter. 

 By plating samples on agar containing strepto- 

 mycin, we infer that one per million bacteria or 

 10' per milliliter produce resistant clones. But to 

 count these clones they were selected in the pres- 

 ence of streptomycin which hypothetically might 

 have induced the resistance. We may, however, 

 dilute the original bacteria in jjlain broth to give 

 samples containing 10° per milliliter. Since lO""* 

 of the bacteria are resistant, each sample has a 

 mathematical expectation of 0.1 of including a 

 resistant bacterium. The individual bacteria being 

 indivisible by dilution, nine samples in ten will 

 include no resistants; the tenth will have one, but 

 now augmented to 10^^ Which one this is can be 

 readily determined by retrospective assay on the 

 incubated samples. The procedure can be reiter- 

 ated to enrich for the resistant organisms until 

 they are obtained in pure culture. The same result 

 is reached more conveniently if we spread the 

 original culture out on a nutrient agar plate rather 

 than distribute samples into separate test tubes. 

 Replica plating, transposing a pattern of surface 

 growth from plate to plate with a sheet of velvet, 

 takes the place of assaying inocula distributed in 

 tubes. Dilution sampling and replica plating are 

 then alternative methods of indirect selection 

 whereby the test line is spared direct contact with 

 the drug. 



According to Leidy ef at. (1956), strepto- 

 mycin resistance of a high degree can be 

 induced in sensitive populations of Hemophi- 

 lus influenzae and H. parainfluenzae by 

 deoxyribonucleic acids (DNA) derived 

 from streptomycin-resistant cells of at least 

 one heterologous species of Hemophilus. 

 Comparison of the activity of heterologous 

 and homologous DNA showed differences 

 within species and degrees of differences 

 among species not brought out by other 

 available methods. According to these re- 

 sults, H. influenzae is more closely related to 

 H. parainfluenzae than to H. suis, the rela- 

 tionship between the last two being remote. 

 The low proportion of cells in H. influenzae 



