PROJECT REPORT COMMITTEE ON FOOD RESEARCH KX8BABCR AND DEVELOPMENT BRANCH MILITARY PLANNING DIVISION OFFICE OF THE QUARTERMA8TER GENERAL >,QUARTERMASTER FOOD AND CONTAINER INSTITUTE FOR THE ARMED FORCES CHICAGO ILLINOIS c66p ERAT.I NG LkSTI TUTJ ON 'University of Texas LOCAL ITT Austin, Texas olv,sAHs & Sciences OEP ARTM EM 5Jacteri'>logy offi cwjvillfesy^ssoR COLLABORATORS- - - REP0RNoV*10 FILE NMr-900 pra*PP^gtt-70178 TAng £ R Oo D- N 1, 1947 I N I T I A J-T»E 1946 Tl TL E: PROGRESS REPORT PHASE REPORT ANNUAL REPORT TERMINATION REPORT Mode of Aotion of Certain Antibacterial Agents. SUMMARY The attack cn the problem of the inhibition of bacteria by chemical agents has generally involved a search for more active compounds suitable for this purpose. We have shown that it is possible under some conditions to modify the resistance of the organism to the chemict agent. 1 CQHD FORM 5 Ap r I I 4 6 12-121 (Seviaed) Many antibacterial agents are unsuitable inhibitors because of the resis- tant individuals which occur as spontaneous mutations in the microbial popula- tion. These individuals grow in the presence of the inhibitor and establish a resistant culture against which the inhibitor is ineffectual. We have devoted considerable study to the occurrence of such individuals. The recent work on di- rected mutations suggested that it may be possible to modify the resistance of a bacterial population by use of such techniques. The work at the Rockefeller In- stitute on the transformation of the pneumococcus and the studios of Bcwin and collaborators (conpte, rend. soc. biol. 139. 1047, 1943) have shown that certain characteristics of a bacterial population can bo modified by the use nf a thymo- nucleic acid extract of another strain. If this method of inducing mutations is a general one it might be put tc practical use in the field of bacterial resis- tance to inhibitory agents. These studies were made with our laboratory strain of E. coli (designated as the sensitive strain) and a sulfonamide resistant strain developed from, it, A quantity of cells of both strains was harvested and extracts from each were- prepared. The final procedure adopted is reported in table 4, but the oari.v studies were made with the crude extracts and with the nucleoprotcin r.thcr than with the purified nucleic acids. These materials were sterilized by per- mitting them to stand under 70% ethanol overnight. They wore then removed from the alcohol, dissolved in sterile buffer, and added to a series of test tubes which contained a synthetic L. coli medium and several concentrations of sulfa- nilamide. The experiment was set up in triplicate with rno series containing no nucleoprotcin extract, a second containing extract from the resistant strain and a third with extract from the sensitive strain. Sterility controls for the- ex- tract were included and al_ the other tubes were inoculated with a small inocu- lum from a young culture of the sulfonamide resist,ant strain. The results in table 1 shew that the resistant culture grew slightly in the M-900 #10 presence of 3C *-g sulflnilsmide ana that the addition to the medivan of ;.n ex- tract of the resistant culture had no effect on this resistance, the addition of an extract from a sensitive culture, ■ owever, prevented the growth by 30 nigh and even by 20 mgh of sulfanilamide. The experiment reported in table 2 was set up in an identical manner but was inoculated with the sensitive strain of jjj. coli. It is observed that this or- ganism is completely inhabited by 5 a - of fulfanilamide both in the control series and where an extract of the sensitive strain was added to the medium. The addition of extract from the resistant strain permitted slight growth even in 10 mg;, of sulfanilamide. A study of the quantitative changes in the population is reported in table 3. Here the organisms were grown in the presence or absence of the bacterial extracts as indicated but without any sulfanilamide added. After 24 hours the mature cultures plated in agar containing varying amounts of sulfanilamide to determine the distribution of resistance in the resulting population. As can be seen from the plate counts the presence of an extract from a resistant culture ii.creases the average resistance of a sensitive population. .ui<» more surprising the presence of extract from sensitive colls decreased the average resistance in the resistant population. xn experiment utilizing the -.urixied nicleic acid extracts prepared as in- dicated in table 4 s owed that these contained the “transforming principle". The most obvious explanation of this conception assumes that mutations re- sult fr -i inexact replications of the genetic mechanism of the microbe end that these “errors" are found in the nucleic acid,- Under some conditions or- ganisms may assimilate these preformed fragments of their genetic control mechanism if the; are added to the medium. If the fragments are slightly dif- ferent from those ordinarily buixt by the organisms the result is a mutation. M-900 #10 From the data presented here it -must be assumed that the highly re.vistrnt or- ganisms of the resistant population prefer to absorb the nucleic acid from the normal culture rather than duplicate their own genetic, mechanism which in ab- normal as compared with the main population. Table 1. ilT kl 1\> ■. w* oil. "0 jii Iuu H-lb j.oi ai 1 OulI K'ucleoprotein iXtract w.iu fa nil amide none resistant sensitive (mg y ) 0 + • 4 + 444 4 4444 2 4>-44 4444 4444 5 4 4 4444 4444 10 - 4* 4 + 4444 44 20 4-44 444 - 30 4 4 mm M-900 #10 Tcble 2. I*. u'lbiiUl j. iv. v. ji.jui'O.iiAi;SlTXV.j £• (JUJ j'lucleoprotein extract Sulfanilcmde None . . Resisant Sensitive 0 ++++ ♦+++ 1 +-M-+ ♦+++ ++++ 2 +++ ++ 3 «► +++ + 5 - ++ - 7 - ♦ 10 & a. Table 3« KLAT- COUNTS OF S. .,uxJ *•, AGAR CUiTTAI :IMG ViUlYIRG SULFAiJIiAi lDJi COdCEnTIUTiUHS l'.n Sulfanilamide Strain .-ocfcract 0 2 5 10 2Q oensitive none 130 21 2 11 900 0 0 sensitive resistant 120 K 7 M 20 T 100 0 Resistant none 140 M 112 a 8 M 800 T 900 Resistant sensitive 140 K 100 ii 420 T 2 T 0 M-900 #10 5 Table 4. PRbfAR/JIuII uF iIUCLEIC ACID 1 gram wet cells - M Na citrate in ,2h ha desoxycholate. Heat to 50° C. for xu minutes. Centrifuge, cell debris discard crude extract + 4 volumes of KtoH other precipitate and dissolved substance fibrous precipitate (crude nucleoprotein) dissolve in ii NaCl shake with 1/10 vol amyl alcohol and 1/3 vol chloroform protein nucleic acid M-900 #10 6