L. L. CAVALLI SFORZA and J. LEDERBERG
Genetics of resistance to Bacterial inhibitors
SYMPOSIUM
INIBITORI DI CRESCITA E CHEMIOIERAPIA
GROWTH INHIBITION AND CHEMOTHERAPY
7-11 SETTEMBRE 19.53
ISTITUTO SUPERIORE DI SANITA'
ROMA -  WALE REGINA MARGHERITA, 299 - ANN0 1953

L. I,. CAVALLI-SFORZA
ISTITUTO SIEROTERAPICO MILANESE cx- lSTlTUT0 DI MICROBIOLOGl.4
                             J
           DELL'UNIVERSITA, MILAN0
                   and
                J .  LEDERRER( ;
DEPT. OF GENETICS OF THE UNIVERSITY OF WISCONSIN, MADISON, WIS. (")
GENETICS OF RESISTANCE TO BACTERIAL INHIBITORS.
I. - IN'l'ROI~U~:T10X,
  The development of neiv antibacterial agents has brought to the fore-
ground a new aspect of evolution in microorganisms, viz. drug adapta-
tion. The practical import,ance of this phenomenon is so great, that much
study has been dedicated to the problem of its origin, at a time when
genetic analysis of microorganisms is in its infancy and the prejudice
that their genetic systems are thoroughly different from those of the
more fully investigated higher organisms is still widespread. Thus it is
perhaps not too surprising, that the issue has from the beginning been
a highly controversial one, and that th(> discussion has been centered
around motives \vhich are essentially- similar to those prevailing in the
last, century on theories of evolution in general.
  It may be added that present, kno\\-ledge allows one to discriminate
much of t,he current, prejudice on microbial genetics and thus to settle
a fairly large portion of the controversy on drug adaptation.
  Most microorganisms can, in fact, show at least some degree of
adaptation to most of thl> drugs, to whose action t,hcy are subjected.
Rarely adaptation reaches a very high degree (i.e., to high concentration
of the drug) after one or few exposures to the drug of a bacterial popula-
tion.  More commonly, adaptat.ion dew,lops gradually, but, even so it
may often result. at the ond of a long process, in a high level of resist,ance
being attainod in the adapted strain.  Whether adaptat,ion occurs gra-
  (*) The work at Madison was supported in part by a research grant (E72-54) from
the National Microbiological Institute, National Institute of Health, United States Public
Health Service, by the Rockefeller Foundation, and by the Research Committee, Graduate
School. University of Wisconsin with funds provided by the Wisconsin Alumni Research
Foundation. This constitutes Paper No 5 r7 in the series of the Genetics Departn-:?t.

- 109 -
dua'lly or suddenly depends mostly on the drug, a,nd presumably there-
fore on the mechanisms of its action; too lit,tle of which is usually known,
ho\vever, to permit, further speculation along these lines. Complete single
step adaptation is observed wit,h streptomycin and, perhaps less through-
ly, with isoniazide: gradua,l adaptation Was first. dexribed in genetical
terms for penicillin by Demrrec (4945, 1949) and henc,e called by this
aut,hor the ((penicillin pattern 1) of drug-adaptation as contrasted I\-ith the
(cst,reptomvcin pattern)).
        Y
  Another angle from which the facts concerning drug-resistance can
be viewed is the variability of the resistance level gained by a given type
of microorganisms in a single exposure to the drug. Survivors of a bac-
t,erial population plated in the presence of a suitable concentration of
the drug may show-. on successive subculture, an increasccl resistance,
its compared with t,hat of the original strain; the measure of the resistamx
level thus acquit 11 by one csI~osu~c (often called first-step-resistance)
may show differenc,rs between independent isolates.  The variability of
first-step-resistance is of course likely to be highest for drugs of the
streptomycin pattern, these being defined as permitting full resistance
to be, develope,d with t,hc first st,ep.  However, a variation of the degree
of first-step-resistance is observed, even if less impressively, for drugs
to which only gradual adaptat,ion is possible, and, apart from \-ariability
of t,he level of resistance acquired, morphological, biochemical, genetical
variat,ion can be detected  amon g first-st#cp-resistants to a given drug.
Only \vith one agent, and this perhaps because of insufficient analysis.
is no important, variabilit,y of first-step-resist,ance encountered, namely
a.zide-resistance in E. coli.
  A technical det,ail of some import,ance is whether drug adaptation
is carried out in solid or in liquid media. containing th(x drug a.t suitable
concentrat,ions. hdapt,ation in liquid medium for drugs of the ((penicillin
pat,tern)), by means of successive subcultures in increasingly higher con-
centrations of t,he drug may result in the bacterial population showing
an almost continuous rise in the resistance level, although stepwise in-
creases are also observed. Increase in steps is forcefully observed when
adapt,ation to such drugs is carried out in solid media, since every suc-
cessive transfer on a drug-agar plate with a suitably high inoculum and
drug concentration is likely to give rise to a definite increase in resistance
of some of the survivors.
  Fina.lly, observat,ions may bear on the stability of resistance thus
acquired, and here a full range of stability degrees may be observed,
though carefully isolated resist,a,nt survivors which show some degree of

-
- 110 -
resistance on a successive subculture tend to keep this resist,ance more
or less unaltered over a number of transfers.
  This oversimplified summary of the main facts may serve as an in-
troduction to the explanation which have been offered for the behaviour
exhibited by microorganisms in the presence of antimicrobial drugs.
First let' us consider the explanation which is provided by standard
genetical theory for adaptive changes in the rea,ction of an individual
or a population t,o a given environment.  T\vo mechanisms \v-hich are
not mutually exclusive may come into pla?-:
  A) Ph?ysiologicul adaptation : rverv individual of a given genotype
                                 ,
can adapt to a certain range of environmental variation.  The ne\\- en
vironment, often directly induces the changes, as iu enzyme adaptation ;
on return to the old env-ironmcnt the change usually disappears both
rapidly and entirely.  AdaptnCou and deadaptation leave the hereditary
background unaltered ever] if carried out repeatedly.
  11) Genc!ic adnpfntio~n.  Individuals of ;I gi\-en genotype are unable
to cope effectively wit,h the situation created by a new environment.
Mutation or other genetic chnnpw pw)vidt~ the hasia of ne\\ heritable
\-ariation.  If out or mnrc mutant!: car propacC qte themselves in the
ue\~- environment. they \\ ill initiate c!one. q >,A-hich will e\-untually be sub-
stit,uted for t,he old population.  Thus the en\-ironment acts hv selec,ting
fitter variants, endowed with n hereditary capacity to withstand the nev\
conditions. (Genetic adaptation is eswntialt?- I~~~e~~d;~l~tntion. in cout~rast
\I\-ith the physiological kiud f A) \\-hich i.; txostadaptive. The change is not
readily rcversiblr as in the preccdin, rr iI1Ftillll?t?. hwnusc~ re\-ersion can Or-
cur only by bac,k mutation or mutation at auothcr locus v<it'h modifying
effects on the primary mutation, and these kinlls of genetic changes prove
to be generally v-cry rare.
  These t:;-o mechanisms of adapti\-e c tianpe ha.\-r been fairly well
ascertained in a number of instances and some models have been esamin-
ed in detail (Monad, 1952; Ryan, /SR2. to quote an example of each type).
Formal genetic, analysis can asccrtnin vvliich of these two mechanisms
opera& by an analysis of individual behnviour from homogeneous pop-
ulations, i.e. essentially clones of cells. Clones of sufficient size for muta-
tion to have occurred in one or more of its mrmbrrs \vill form an inci-
piently heterogeneous population on \vhich selection may act. Deliberate
mixture of genetically different line. s vvill provide means of analysis b>
genetic transfer in order to anal>se the genet,ic nature of the heritable
changes. The effort of the geneticist may therefore be direc,ted to 011~
dctec,tion of the relative importance of the two nnxhanisms in the deter

- 111 -
mination of drug-resistance, i.e. to the control of pre- and p&-adaptive
changes, by the use of such techniques.
A variety of a,lternative theories of drug adaptation ha\-cx been pro-
posed; just to summarise the most important ones:
C) Direct induction of genetic change by the drug (Linz 1950, others).
The change to resistance is a truly genetic one, i.e. heritable etc. but is
induced by the drug itself, and is hence of a postadaptive nature, which
has no parallel in the above scheme.
D) SelE-adjustment, of enzyme-balances to permit growth in tfhe ne\\
conditions without nuc1ea.r change or selection (Hinshelwood 1946). Al-
though this theory is not, substantially different from what has been call-
ed physiological adaptation, t>his author has pushed its application SO far
as to cance,l or minimise the role of selection or mutation.  It may be
noted that this expectation may represent an oversimplification of the
genetic theory, as it tends to draw no distinct,ion between the individual
and a population. This theory could therefore be formulated in terms
which would include the preadapt,at.ion theory.  If Ihe population is
studied as a whole, and no attempt made to resolve its elements, the
enzyme balance of the mass will doubtless be found in the long run
to show adapt,ive, adjustmentIs of the kind subsumed under this hypo-
thesis. For an understanding of the genetic basis of such changes, how-
ever, we must examine the adapt,ivc responses of individual cells, and
we shall find that the best studied cases of apparently directed, post
adaptive changes of whole populations can be interpreted in terms of
pre-adaptive changes of small numbers of its constituent cells.
Both these theories demand a post-adaptive change and ignore pre-
adaptation. lZn attempt to compromise was recently advanced by Yud-
kin, under the name of ((clonal variation with sele,ction)): where it is
(implicitly) assumed that phenotype and genotype of a cell arc identical,
and variation originat,es from the distribution in unequal parts at cell
division of the systems responsible for resistance. Actual cell resistance
determines the average resistance of the progeny (phenotype = genotype);
selection act,s in the same way as in genetic adaptation.  Here changes
would be preadnptive.  It may be added already at. this stage that the
knowledge on bacterial genetics is strongly at variance \\-ith such a.s-
sumptions. Again, however, Yudkin's approach may be considered as
an aversimplification of the genetic theory, assuming an inordinately
extreme polygenic picture.

Fig. I. - Models of drug resistance. Explanation in text

- 11:-1 -
II. - THE ANALYSIS OF INDI\`IDl~AL CHANGES.
  A visual aid to the forma.1 analysis of t,he various interpretations is
given in figure 1.  137r can be satisfied, at, t.his stage, \\-ith the analysis
of tw.o, or better t\vo pairs of models taken to explain the origin of drug
resistance. The first (1 -4) she\\-s strict gcnet,ic preadaptation. The change
occurs prior to addiCon of the drug, and is also phenotypically expressed
belore it. The second rrlodel (1 B) differs from the first in t,hnt mutation
dcter~ruines only the potential capaci1.y to I\-ithstand the drug; the neces-
sary enzymatic systems lvill ho\\-ever. he manufactured or adjusted only
in thy. prrsence of thr antibacterial agent.  Ilere, genetic adaptation is
coul~lcd \:-ith physiological adaptation.
  The third and fourth models (2 3 and 2 B) show t\vo varieties of
(1 physiological ,adaptation 1) lviih 101~ and high survil-al of the mass of
the population respecti\-cly.  In the third scheme, each cell has the sami.
probahilitv of giving rise to a. resistant clone folio\\-ing the addition of
the drug, or if the probabilities differ from cell to cell. there is uo
simple relationship bet\\-ccn the probabilil,y of survival of different cells
and their common ancestry. In both models hn\ve&r. it is nwumc~l that
heritable resistance is postadaptive.
  The eswmial difference bet\wen inodels 1 .\, 1 B nn one sitlc itIlt
2 A, 2 B on the other is \\hether or not prwulaptnt~ion plays a role.  If
it does, t\vo verifiable consequences follow from this: 1) resistants \vill
appear in clones; 2) it \vill be possible to accomplish selection of resistant
mutants in the absence of the drug. Both these methods ha\-e been used
e~xperinicntally, \\.ith results to be sunrmarised now.  It may be nddecl
that, the diffrreuce hetlveen schemes 1 A and 1 1~ Ii-ill demand physiolo<-
ical in\-cstigntions subsidiary to t,he major issue; and that even if the
difference betlvcen models 2 h and 2 B, as well as between 2 B and
the remainder may seem obvious on the basis of the ratio of output
of rcsistants to input of sensitives, the analysis of this ratio may de-
mand a more careful scrutiny than one may on first sight be led to
believe. Thus situations in nhich e\en 199% of t,he cells seem to adapt
may be entirely deceptive, as we shall SW later, \vith incompletely bac-
t,eriost,atic drugs, or in other conditions.  If it is ascertained that, this
ratio is lo\\-. 3s is usually the case, discussion may be confined to distin-
guishing bet\\-ecu models ,t A ior 1 H) and 2 A.
I'edigrrr analysis by isolation of cells after each division has bern
attempted hy Zellc (2942) in the cast of the smooth to rough variatiou

- 114 -
in Salmonella iyphimurkm and she\\-ed tha.t the mutants appeared in
clones. C'nfortunately there is a complete bar to this direct approach in
drug-resistance of bacteria, owing to the extremely low frequency of
mutation for resistance, so that millions of isolation of cells imme-
diately after division would be necessary.  It is not surprising, there-
fore, if that various statistical devices have been elaborated to meet thi*
deadlock.
  1) A detailed analysis \\as first given by- Luria and Delbrtick
11943). They considered that, if there is preadaptation and t,herefore re-
sistant, mutants are to be found in clones, the number of resistant mu
tants in parallel cultures each started from a single sensitive cell and
grown under identical conditions should 1)~ highly variable ant1 their
distribution should indeed be predicta.ble , given certain easy assumption5
on t,he probabilit,y of mutation per cell, and the go\\-th rate and lag of
mutants.
The method was cstensivcly used b?- a number of authors in (1 crude
\vay. by comparin, m the variance of obsewed numbers of resistaut mu-
tants in pa.rallel cultures ~it,h that cxpccted under model 2 R, assuming
this to give rise to a Poisson dist,ribution, as vould indeed be the cas11
if, in model 2 B, c\-cry cell had the same probahilil,y of adaptin?, intle-
pendently of t,he ancestral history of the cell. In a Poisson distribution
the \-ariance should be equal to the mean; in all but, three series of
experimental data observed \-arianccs of numbers of resistant mutants
haI-e instead been much higher than the respective means. This was
taken to imply that model 2 H \vas untenable, and therefore to be dis-
carded in favor of models 1 A or 1 B.  This crude analysis is often re-
ferred to under the name of c(fluctuation test)). The three exceptions
quoted are: streptomycin dependence in E. roli (Newcombe and Ha\\irkn
1949) nhrre clones of dependents obviously cannot form in absence of
the drug, so t,hat the test is inapplicable; streptomycin-resistance in
Staph. aureus observed by Welsch (1952). who also showed the es-
ist,ence of a strong select,ion against the resistant mutants, so that this
case a.pproaches that of streptomycin-dependence; finally, penicillin-
resistance in Staphylococcus aureus (Erikscn 3949) where among other
diffhxlties the use of small samples from t,he cultures made the statistical
tests inefhcicnt. These exceptions cannot therefore he taken as proofs
of model 2 A.
It must be stressed that the analysis offered by the eocalled ((fluctua-
iion test)) is open to tn-o sources of error. One. \\.hich \\-a~ pointed cfut
by Eriksen (1949) and by Dean a.nd Hinshelxnod (1952), is based on the
fact. that no check is available of uncontrollable differences of en-

- 115 -
vironmental origin hetn-ecn parallP1 cult,urrs.  \l;e shall VP later how
this objection can be met.
For a more satisfact,ory support of n~c)del 1 I h or R) a comparison
of the observed distribut,ion of resistant colonies \\Ath that expected un-
der one of these models \vould therefore stem essential. The mathema-
tic,al theory was provided by Lea. and (Joulson (1949). and ext,endcti b>
Armitage (1952) to cover certain necessary- side assumpt,ions (phenotypic
delay. back mutat,ion. unequal growth rate of mutant and parent). .4na-
lysis of data published by Luria and Delbriick. and by Newcombe (l94!3)
on phagc resistance showed a tolerably pood fit with the theory; a good
fit \\-as obtained by Ryan (1952) for mutat.ion to lactose-adaptabilitv- (mo-
del 2 U) in E. coli. As to drug-resistance proper. only one example (New-
combe and Hawirko, 1949: streptomycin-resistance in E. coli) was given,
and she\\-ed a good fit with model 1.
  21 A method, which may be called the method of statistical clones
II-as proposed by Newcomhe il949). It consists of the comparison be-
tween the number of resistant colonies arising from plates on which
bacteria have been allowed to grow into microcolonies prior to the appli-
cation of the antibact,erial agent (`and this is administered in such way
as to keep all the cells in their original location) with the number of
resistant colonies arising in parallel plates in \\-hich microcolonies had
been spread just before addition of the agent. Clones of resistant cells
\vould t,hus be spread over t,hr pla.te, and I\-hile each clone \\-ould give
rise no more than a. resistant colony in the ((unspreadl) plates, its mem-
bers would all ,aivcJ rise to independent resistant colonies in the ((spread,)
rise to no more than a rcsist,ant colony in thr rrunspread)) plates, its
members \\-ould all give rise t,o independent resistant colnnies in the
respreads, plates.  Assuming equal size of t,he sensitive microcolonies,
and calling: r' = Yo. of microcolonies per plat,e cont,aining at least one
resistant mutant, i.e. equal to No. of resistant colonies per (cunspreadl)
plate; n = Total No. of microcolonies per plate: no = Mean number of
mutations per microcolony : the value of nt could be calculatetl from
r - ~l--e-~ giving m=-In 1-L
      ! )  This is approriniatc~l!-    L
n                                 n                        l?X=m
wben rin is small. In such conditions. therefore, the mean number of mu-
tations per plate would be n m =T.  Thus if the total number of resistant
colonies per ~~spread~~ plat,r is called H. the ratio R/r will gi\-e the aver-
aQ*` number of individuals per clonll.  It must hc higlltr than unity if
models 1 -1 or R are correct.
In Sc\\.comhct'< I+Ypt:rimcant the ant,ihactrrial auflnt uiecl \\a5 phage,

which was nkbulized 011 t,he preincubated plates, both spread and un-
spreacl.  Rornschein et al. tested chloramphenicol resistance in E. coli
by this method, using membrane filters as a tool for transfer of the bac-
t.erial inoculum from the preincubation plate bo the drug-agar-plate (1951).
Roth experiments yielded high ratios R/r, qualitatively at least in agrer'-
nlent wit,h the theory of genetic adaptation. This result does not suffer
from the objections raised to the fluctuation test, though it may be claim-
ed that model 2 A is not excluded by this criterion if it is assumed that
in ((spread)) plates, where cells are not packed in microcolonies as in
((unspreadj) plates, resistance may arise more easily.
   3) .4nother approach makes use of the seconda.ry differences br-
twen independent mutations for rpsista.nce to the same drug which
can hr* brought under observation by further analysis of the resistant
colonies.  Such differences, no mat,ter whether of a morphological, bio-
chemical, genetical c,haracter, or simply of degree of resistance should
be found, if resistants appear in clones. only between members of diffe-
rent clones and not, between members of the same clone.
  Since resistants from parallel cult.ures will have arisen from inde-
pendent mutations, among which some difference ma.y be detected, while
resistants from the same culture are likely to be members of the same
clone, the comparison of variability for some suitable character between
resistant,s from independent, cultures with that between resistants from
the same culture will permit a test of the clonal origin of mutants, under
the form of a test of the correlatio?t between relatives. If the character
tested is quantitative in nature, (e.g., degree of resistance) the test. will
take the slatistical form of a study of intraclass correlation and thus
may be performed as a straight analysis of variance by comparing the
variance between cultures with t,hat within cult,ures.  This method has
been applied to chloramphenicol-resistance in E. cnli (Cavalli 4952) where
a high correlation between relatives was found using degree of resistance
as the differential character. The same approach &s used by Mitchison,
and pursued further by him in the course of study of streptnmycin-
resista,nce in E. coli (1953): here, using Larious differential characters it
was possible to recognize clones within cultures and show that thf> num-
ber of clones was distribuled at, random among the various wlturw.
  This test is exposed to the objectinn of confusion bet\veon environ-
mental and genetic variability in a \vay somewhat different from tha.t
to which ihe fluctuation test is subjected, if it is assumed that uncon-
trollable cnvironmc~ntal variation bet\\ern cultures may favor anti or
other t,ypr. form or degree of r&stance, varying with the cultuw.

   4) A\lthough we may now be a.sking too much of the potentialities
nf environmental variation if we want t,o explain all the above results
hy model 2 A plus envirnnment,a.l variation between cultures, and to
consider the agreement with the genetical model as purely coincidental,
iI fest 01 tke rnvironmental cw%t~on may be useful at this point. Such
tests can be devised along lines such as those tried for chloramphenicol-
resistance in E. coEi, in which both t,he number of resistant colonies and
the ave.rage resistance of a sample of t,hem from each of a series of
parallc 1 cultures were tested and the parallel cultures were found to
differ significantly both for number of resi&ants (fluctuation test) and
for ayerage resistance (correlat,ion betwren relatives). If such differ-
ences were due to en\-irnnmcntal variation between cultures, it, would
be expected that factors favoring the appea.rance of a high number
of mutants in mc culture should also increase their average degree of
resistance a.nd hence these two variables should be correlated. Instead
a correla,tion of zero \vas found, (Cavalli i982), showing no evidence of
environmental variation.  This example encourages one t,o believe that
\arintion of cnvironmrntal origin between parallel cultures may be ne-
glipibl+. although tests for this snurce of error should be provided whcn-
el-4.r lpossible.
   S .a technical de\-ice knn\\n as replica-plat,ing permits one to
she\:. ihca clone1 origin of resistant mutant,s in a more direct way than
an!- ol lh(-, nbn\-e met,hnds. This rntxthnd also a.llows one to obtain the
swonrl kind of proof postulat,ed above, namely the establishme,nt, of re-
sistant strains in the absence of the drug (by sib-selection). It \\-ill there-
Iorc htb dcwribed under the follow-ing sectinn.
  Let us consider, h>l~~thetically, the types of rxpwimcnt that, are
capable of se.parating the rn\Gonmcnta,l and gcnetical components of
variance. To do this, (only a simple extension of the design of the fluc-
t,uation experiments is required. Let us, as before, consider groups of
c,ult,ures, prepared from small inocula, in a uniform medium. The initial
series \vill be considered as a parental group, whose descendants will
he compared in order to demonstrate the hereditary c,omponent of vari-
ance. For simplicity7 let us consider only the t.\\-o extreme cultures of
the parental series, those from which sa,mples give the highest and lowest
assays, respect,i\-ely. It has been urged t,hat suc,h extremes, instead of
being the expected result of pre-adapti\re mutation and clonal growth of
resistant wlls, in the absence of any drug, may instead reflect uncon-
trolled physiological differences between the cultures, which influence

the likelihood of an adapti\-e response t,o exposure to the drug (Dean and
Hinshcl\\-ood, 19%). On this ph,siolo$cal hypothesis, a second Cycle
of gro\\ tlI must br cspect,ed to smoothe out any transient differences in
inocula taken from the estrume cult,ures, so that whatever their own
fluctuation, cclIigh)j and U~O\\-II filial series should she\\- no Correlation
with their parents in the awrage assay of resistant cells. On the genetic
hypothesis, however, the result- will depend on the size of the inoculum
\\-hich is taken from the parental 0high)8 and ctlo\v)) rcspectivcly, and
distributetl to the t\\o series of filial cultures.  In particxlar, the filial
high series should again display the sa~nc high average assay, and with
relatSi\-ely litllc further fluctuation, provided each sample inoculated was
l:lrg,a enough to contain at least 0nt wsistant (as cnlculatetl from the
arsa) of the parcntnl high). On the othc~r hillld. if fhc inocnlum sample
is In;Idc so small as to exclude the likclitiood of intrclducing a resistant
CC11 into tllcl Gliill Culturc~. thcl genetic component of variance could not
bc propagntcd. ant1 the> parental and t\vo filial series should shox\- thr
S;llllP iI\-el'il~l? ilSS;LYS. I\-ith a high \-nri:rncc: from culture to culture.  I11
princi~~l~~ I tltc~ugtl this \\-onld bc c!IIitcs IlvlioIlr in fact) 0Ilv Could iIdjUSt
1he inoculnm Siw so that. 03 the penl4ic hypnthr3is, :I rcvjistant cell is
inoculated info only an occasional filial tuhc. \\ hich is thrn detected bj
its high nssa~-. Repetition of thi? J~I~~~uI~~~ \\-ould nccc)mylish a gradual
COllCf?IltIYltiOIi of the Iwistilnt cells. rtkl;Lti\-r to the sensiti\-c, and ultim-
ntcly a pnrely indireci selection and purification of the resistant mutant.
To our knowlcdpe. ttrt5c c~sp~riIiicIlts !I;%\ c IIot ~-vi he11 csccutctl in just
this fashion. ;~lthough the tlcsign shoultl pro\-itle a tlcfinite test. \I-ithout
serious tlcpnrturcs in thcx pIqriinI of c~sy)cr'ime~~tntioll. of the criticisms
of tt1c r oeIiCti(* inlcI~l)I"tntioIl of flucluiltic~n analysis.  These hypothetical
c~spc~rimcnts nw discussed here) in or(11v to introduce a radicall>- different
ilr,J1I'OclC'JI ?\ iliCh is. ho\\ wcr, based on ttrc snmc theoretical foundation
for the wI);IrntioIi of c~IIviroIimeIit;tl and genetic \-nriability. `I'hc hypo-
thetical c~sp(~rinIc~nts in\.ol\-ing the tctlious manipulations of many indi-
\-iclI~;Il tulw culturc5 can 1)~ csecntctl much more espeditiouslv on ag.33i`
plntrs I))- tI1rans of a twhnicnl clrtilirc. wplirn plating.
Jh'l.i(: \ 1'1, \TISV(;.
  l'ho IIIost s!LI*iou-: linIi!;rtic~n io t tI(b treatmc~nt of thew problems ob-
I-iouslv ~~~~nccrns thv laurels- technical difiiculty of handling wry large
nuIi11~~~1~ of ~~11s and faniiliw nndcr drquate control. Uy previous Inc-
Phods. t hv clones of resistant bnctc\ri;r have been identifiablr only as
statisiicnl eniitics, or c~lsc b!- the destrlIction of their preponderant sensi-
ti\-ca n1~iptIhour5.  `I'hc ttvhniquv 01' rpplic;r ljlating gwatly mitigates this

difficulty.  Samples from innumerable clones, immobilized on an aga,r
surface. can be trasferred or replicated to a series of copy plates without
seriously disturbing their spaCal a.rrangement,. This is accomplished by
using a sheet of velveteen, wit,h its dense pile of vertical fibers as the
vect,or for the inoculation. In practiw, the initial agar plate, with its
pattern of surface bacterial growth, is inverted with slight, pressure onto
A sheet of velveteen, mounted on a c,glindric,al support. The initial plate
is then removed, and replaced by a series of fresh plates. Each of these
will pick up an impression of bact.eria from the velvet \\-hich subsequ-
ently develop into a remarkably faithful rendit,ion of the original pattern
of bacterial growth.
  The accurate replication of this pattern is most obvious when it con-
sists of discrete colonies, but t,he principle applies equally \\-ell when
the gro\vt,h on the initial plate is c,onfluent owing to the character of its
inoculum. The original plate is left suffKently undisturbed by taking
a velvet replica that, it can br used repeatedly for t,he sa.me or other
purpwes.
  Two adaptive responses haye been studied in detail by means of
rcldica plating t,echnique, resistance to streptomycin and resistance to
t,h!% phage T, in Escherichia coli strain K-12 (Lederberg & Lederberg,
1952>, with concordant, results.  Plain nutrient, agar plates were spread
with several thousands of sensitive bacteria, so that a smoot,h: confluent,
grxwth developed after 24 hours incubation, while no resistants were in-
cludcd in the inowlum. A series of replicas was then made to plates of
siicptomycin agnr. In order to ensuw objective registration of the plates,
pins were included in the support, blocks t,o mark the agar surfaces.  Aft,er
24 hours, rwistant colonies \~ould often develop on the replica plates.
More than half of these were ohscwwl to occ,upy supcrimposahle posi-
tions on the successi\-e replicas!  This could be explained only by the
deriva.tion of these resistant colonies from localized clusters of cells on
the original plain aga.r plate.  `l'hwc c,lusters are, of course, the clones
subsumed by genetic' theory; the physiological theory can offer no plaus-
ible explanation for srwh clustc>ra in a uniform film of go\\-th. Rather
than argue this point more fully, ho\\cver. we procectl to the next and
more conclusive stage of the csprriments.
It will be recalled, and must be emphasized here! t.hat thr original
plate has not been dnruagerl. except for t,he rrmo\-al of a sample of its
growth, nor has it had n.ny knowledge of the streptomycin to which only
the snmple on the \-rlwt has been expose,d. Nevertheless, t'he location:
of many l*esista.nt clones has been re\-ealed by the congruent patterns
of colons rlevclopmcnt on the replicas on streptnrn~cin-a,ar.  Let uc

-- 120 -
JIOW assume that t.he conditions of growth on the original plate have
been adjusted so as to permit the development of just one clone per
platl:. on the a,verage.  Let us assume also that the registration of the
plates and the t,echnique of transferring inocula permits us to pick an
area one-hundredth of the total film of grnivth {i. e. about 60 mm2)
and still inc'lude the entire resistant clone.  It is evident that by thus
retaining all of the resistant 1~11s on a plate, and discarding !XIy$, of
the sensitives we could accomplish a IOO-fold enrichment for the resi-
stant relative to the sensitive bacteria (provided of course that, as on
the genetic hypothesis, these bact,eria are actually present prior to
treatment with any drug). This is, in fact, precisely what was observed,
as told from the assays of cultures grown from such indirectly selected
inocula.  The practical enrichment factor is ac,tually closer to 4 : 1000
(6 mm2) than I : 100, but this is counterbalanced by the difficulty of
controlling conditions to permit just one detectable clone per plate.
However, the advantage of this procedure and one which obviates the
ne,cessity for quoting any numerical details is that it can be reiterated,
by plating appropriate dilutions of the first enrichment culture, locat-
ing t'he resistant clones by replica plating, and picking the correspond-
ing sites for a furt,her cycle of enrichment. It is evident that even with
the very low incidence of a.pproximately one streptomycin-resistant,
cell per billion bacteria, it requires (and actually did require) only three
cycles of indirect selection t,o increase the proportion to one per thous-
and. At this point, the inoculum for thy nest stage may be diluted tc)
t,he point where discret,e colonies will appear on the plain agar, and yet,
one or more of them will be resistant. This colony is quickly identified
by a final round of replica plat,ing and having been located, yields to
conventional methods of purification.  In this \\ay-, streptomycin and
virus-resistant cultures, respectively, were isolated by purely- indirect se-
lection. The replica platin g technique \vas also put to work to facilitate
tests of the unifermity and stability of these resistant cultures after they
were once purified.
The one detail of t,he forcFoing account that. must be kept, in min(l
is that the indirectly selected ba,cterin !or rather their ancestors) ha\-e
never been reposed t,o tbr drug. Tbc drug \\`as used exclusively to locate
the resistant c,lones by tests of their sibs, transferred via velvet. In fact,
t,be manipulations to which the selection line has been exposed consist
only of several cycles of platin g on plain agar, the removal of a sample
by a sheet of vclvctren. and after an appropriate site has been revealed
by comparison of the replica lblates, picking \\-ith a platinum loop to plain

- 124 -
broth. The same manipulations lcad, in one case to a streptomycin-re-
&ant pure culture, in t.he ot,her to a virus-resistant, depending 011 the
auxiliary procedures which do not. directly affect the main selechion line.
There is no opportunity whatsoe\;er for any chemical reac,tion of th(b
drug to play a role in the establishment of the resistant mutant, We do
not address ourselves to the question whether individual resistant bac-
tcria may not in fac,t he only uniformly but potentially resistant until
they actually encounter the drug.  \Ye note, hokyever. that each cell of
the indirect,l!; selected culture promptly develops into a colony on thca
streptomycin agar, \\.hich destroys all but a minute fraction of t,he pa-
rental cultuw. In addition, this potent,ial is inherited I'm an inrlefinitr
number of generations to all of the progeny. prior to any exposure oE
these cells to the drug. When taken together \I-ith the evidence of clo-
nal occurrcncc from the first stage of the experiment, thv success of
indirec,l selection pro\-itles incontrovertible e\ idrncr for the gvnel ic theor?
for those sj-stems to xhich it has been applied.
  14s in previous sections. \\I? must pi\-e some CO11Sidc~ilti011 to tIllI
possibilit)ics of a spurious negative result. The resistant mutant must not
be drastically wlected n.gainst on plain agar -- we ha\--e nn hope. for
example, nf isolating a streptomycin dependent mutnnt by this n~ctlrocl.
It is also csscntinl that single resistant cells frnm the initial plntv (as-
suming the gcnelic hypolhesis~ he unequivocally itlentiliable on tli(> SPlc(:-
t,i\Te replica plate. and these not hc confused lx-it11 mutants that nliglrt
ultima telp develop from populations pro\\-+ on the latter. whvrc wlcc~-
tion is insuCliciciill~- tirilstiC.  If indirect srlection sll0uld Eilil \\ llcrl np
plied to an nntreatetl population. but succeed in extracting added rcsi-
stnnt, bacteria, cwiginnll~ isola.ted bv direct selection, ii I\-ould 1~ nwxh-
sary to concctlr that direct selection is relevant to the dc~velopnicnt of
resistancr.  hmonp the most obvious possihilitiw that ~0~1~1 11av-r~ to be
exluded by further analysis is sequential selection. in two stages, for
casts v.hcn only the first step of resistance is likely to be present as SIIC~I
in unselected populntic~n. However, the positive results nlrvady obtainc>c!
are quite unnmhiguous, and further discussion of spurious negative rc-
suits may he deferrr~rl until specific details arc submitted for adjudication.
  Drug-resistant \-arinnts have also been secured in the absence of the
drug hy selection for correlated characters (English k Yc Coy. 1981.
Gale, 19/L9; Goldstein, ,1%;2).  This approach also conlirms thr> notion
of preadaptation, but is not a rigorous proof of it, as the unusual phy-
sinlogical contlit,ions cnuld be regarded as the specific inciters.

- 122 -
III. - DIRE(;T GENETIC EFFECTS OF DRl~-1;s
   The proven role of mutation and selection in the development of
drug-resistnncc in bacteria, and t,he vague contrary evidence that has
heen offered serve only to accentuate interest in a fen- clearcut cakes of
specific direct genetic effects of antibacterial agents on microorganisms.
We do not, refer here to the possibility that some antibiotics ma?- have
some incidental mutagenic activity of a nonspecific character (Ne\\ cornhe
s- MC (hTgor, ISM).  Streptomycin cscrts a most, remarkable influence
on t,hc unicellular alga, Euglrna g~acilis (Pro\-asoli, Hutnrr and Pintner,
1981; cf. Pringsheim, 1952) as ~-cl1 as on some higher plants. Euglenn
cult,ures tsposed to streptomycin suffer a rapid and irrel-ersiblc bleach-
ing: which results from the destruction of the chlnroplasts. Fortunately,
Euglena can bc cultured heterotrophically, but the apochlorosis is lxrma-
nent, and the chloroplasts are never restored. The organisms which have
hecn found to respnnd to streptomycin in this way are very limited in
number, perhaps for incidental reasons, and none are quite suitahle for
genetic analysis, so that it cannot be asserted I\-here the streplomycin
acts. It seems most likely ho\\-ever that this is a direct destruction of the
chloroplasts, I\-hich have been believed to possess considerable genetic
autonomy from other (not entirely conclusire) experiments. In a sense,
the apochlorosis is a result of chemotherapy, the clcuring)) of the plant of
its plnstitls (WC discussion in Lcderbcrg, Z952). In somewhat analogorls
fashion, the csposurc of yeast IO acriflal-ine induces the development of
small colony yarinnts, \I-llich ha\-ce c\-identlg lost part of their imitci-
chondrinl ?) respiratory apparatus.  In this case, morphological analysis
is incapable of demonstrating the direct locus of effect of the acrifln\.ine.
\\ hich ma> be ruorc complex than the simple destruction of the targrt
organelle, (Ephrussi, 1951). The end effect, like that of streptoniycin, is
helie\-cd to he loss of a genetically aut,onomous particle, in this case POPI!-
lation analyses haw confirmed that, the chemical effects are in fact indui,-
tivc, and not selcctil-e. This has been confirmed for the yeast by hea~lliful
single cell cspcriments, in \\-hich indi\-idual buds engendered in the
prrsencr nf acriflnxinc I\-erf slio\~ii to de\-elop almost consistjentl>. into
the small colon>- variant. A third example concerns t,he parabasal botl?
of hcmoEla~ellntcs: long kno\\n to disappear during the development of
~hemotlIc~r:lpcutic rcsistnncc to trypaflnvine. Circumstantial evidence fa-
yors the cl)nclusinn that the aparnbasal \-ariants are induced by the drug.
perhaps by interference with the division of the parabasal granule dur-
ing cell fission c,Pickarski, 1949), but, the problem has not been closeI>
studied from a genetic \-ielypoint.  There is no e\.idence in any of thcv

- 123 -
tixamples that t,he induced variant enjoys any selective advantage over
the normal wild t'ype, a circumstance that contributes to the clarity of
the proof that they are induced. It is therefore doubtful bvhether t,hesc
variants can be regarded as adaptive or drug-resistant, and their discus-
sion is therefore not directly pertinent to our paper.
  :I final example lies on the semant,ically confused boundary of para-
sitology and genetics (Lederberg, l952). Kappa, a determinant of t,he
killer trait in Pnrarmcium awclia shares many attributes of a cyto-
plasmic hereditary determinant on the one hand, and of an intracellular
symbiotic microorganism on the other (Sonncborn, 1950). It has been
found that kappa is destrolied by culti\-sting paramecia carrying them
in the presence of chloromycetin or of ljurine analogues (Bra\\-n. 1950;
Jacobson, l!E2). This is no more decisi\-e for the <C genetic quality H of
kappa, than is the effect of strept,om\Cn for the chloroplasts.  The four
esamples share the principle that an apparent,ly aut,onomous. but some-
ho\\- dispensable, hereditary partic,le is lost under the influence of t,he
drug, and illustrate a surprisin g applicat,ion of chemotherapeutic princi-
ples to the problems of genetics. The imporba.nce of such cases provides
all t,hc more reason why possible example, 9 in the bacteria should he
supported by ample proof.  It is difficult to quote any at the present
time which arc not beclouded by the undisqualified possibilities of mu-
tative, selected c,hanye. Three \A-hich perhaps dcser\-e closer attention
are t,hc attenuation of \-irulence of t,he crown-gall bacterium in the prc-
sence of amino acids (van Lanen et al., 1952); the development of small
colony variants of E. coli in the presence of naphthaquinone and other
chemicals idol\\-ell. 1946) and antigenic changes in streptococci pronn in
the absence of aceiatc iMcTlrog, Aselrod & Slellon. 1948).
  For want of more informat,ion, Voureka's findings may also bc sum-
marized at this point.  Her first finding \\-as that, a penicillin resistant,
strain of Siflphyl0coccus~ became sensitive when exposed to DNA ex-
tra& of sensitive bacteria. .it the time, this \vas t,hought to be possi-
bly analogous to the pneumococcus transformation. It was later found,
however., that extracts of the resistant bact,eria had the same effect. Most
recently, Voureka j1952) has reported a single cell analysis of staphylo-
cocci exposed to various injurious agents such as chloramphenicnl, ptni-
cillin, nikngen mustard, and hydrogen peroxide, each of which seems to
induce the same syndrome, \I-bich inclu!lcs increased sensitivity t,o peni-
cillin and streptomycin. and a loss of coagulase formation, mannitol
fermentation, and virulrnce.  The mechanism of these changes is ob-
scure, but in vie\\- of the non-specificity of the inciters is clearly not
adapti\-e. Vourelia nntcs that the cultures have also lost their phage-

- 124 -
specificity and this, together \vith some of the circumstances noted for
the development of colonies from single treated cells suggests t'hat tht~
phenomenon may be related to t.he induction of lysis by latent bacte-
riophage (Lw-off, 408-2). The report of morphological changes induced
by t.reatmcnt with chloramphenicol +antiserum was confirmed by Man-
ten and Ro\\-le; working on E. coEi K-12 !1953). Again these changes had
no adaptivc value.
IV. - THE HOLE OF PIlYSIOLO~~ICAI, AL).APTATIODj
The experiments \vhich \vere referred to in section 11 giw strong
experimental support to the belief tha.t genetic adaptation plays a major
role in the origin of drug resistance. They \vere not, however, designed
to test effects predicted by schemes of the kind postulated under 2B.
because t,hey deal essentially wi6h cases where only a small minority of
cells survive the action of the drug or at least develop, in its presence,
into a Gsible colony. \\-hich has acquired some st,able degree of resistanw
as shown by subcult,urin, m in the absence of t,he drug. Such experimc>nts
can discriminate bet\\-een models 1 (4 or B) and 2 A, in the sense that
6he first, t\\-n give rise t,o a prec.ise hypothesis which can be accurately
tested, and \vhich has not, been contradicted by any of the availahlr,
critically collected data. Complete exclusion of model 21\ would demantl
a perfect fit of model 1 and for t,his more \\-ork may be necessary, at least
for some systems. It is of import,ance, howeverY t,hat none of the analy-
ses sofar reported are capable of throwing doubt on the existence of
mechanisms of type 1.
Model 2 4 may arise either as a consequence of direct induction of
the genetic change by the drug, or as an ext,ension of model 2B under
consideration of simultaneous partial killing. Model 2B has the ad\-an-
tage that it can bc more easily put to a straight experimental test than
model 221. It is a classical basis for t,he description of enzyme: ildapta-
tion to carbon sources, it should be noted, however, that, in nll c,ases
analyscd the rate of deadirptation w~ultl sccn~ to be of the same order
of magnitude as the adaptation rntc. A brilliant experimental confirma-
tion that cells adapt has been rec.entl!; given by Benzer (1953) for the
adaptive synthesis of beta galactosidase in E. coli ML.  Experiments
showing that this is possible for drllp resistance are scanty. Baskett (1952)
\\-as able to increase resistance to proflavine in B. Zartis a.erogenes by
adding repeatedly small sublethal amounts of the drug to n growing
culture. Cells could thus bc induced lo grow at a drug concentration
which, \\-hen applicvl to unI)rimctl cultures would cause a killing of

- 125 -
1 - 10.") Cells, i. e. a lcvcl at vvlrich one ma)- hope to select some re-
sistant mutants, if anv \I-cre avnilahlr. .\daptntinn vvas rapidly lost in
~ubculturc on drug free medium. This could he a good example of stan-
(lard physiological adaptation.
Eagle, Fleischman and Levy (1952) found somewhat 111oro stable
changes in rcsistancc by selecting srirvivnrs in plntes containing sublc-
thal concentrations, i.e. just aho\-e the threshold for some observable
inhibitory action.  The change in resisiancc of the survivors seemed to
be correlated with the lc\-el of drug used for selection. Heritability of
such changes should bc tested brforc conclusions on their nature can be
reached.  This result is, incidentally, not entirely in agreement with
observations on similar lines by TiuFhes (t!%2), Bemerec (f94.5), New-
combc and Tla\\.irko (1949).
  It may bc useful to note, at thi:: point, that when incompletely bac-
terinstatic drugs, or dru g concentrations. are cmploycd, the applicability
of model 2B may be crroncousl~- claimed.  Sub\-isible go\\-th of the cells
seeded may result in an actual input of sensitive cells far higher t,han
that assumed on the basis of the inoculum employed.
One clear cut case of nda1~tiv.c response to drugs is penicillinase for-
iiiat,ioii in R. ccrcrfs (Pollock). In this case, it is obvious that addition
of subminimal amounts nf penicillin vv-ould protect a culture from the
later addition of grrater amounts of the drup. Knox and Collard were
able to she\\ that growth at higher temperatures 142'9 which blocks
penicillinasc adaptation is firr more easily inhibited by penicillin than
(rro\v-th at a tcmperaiurc permitting adaptation iat So); prcadaptntion
r
of the cullirre grc>atly incrcasctl resistance at 42'.
  On the basis nf the early \v-ark on drug-resistance in Paramecia it
has also been claimed that stable chnngcs in resistance vi-ithout the in-
tervention of nuclear changes arc possible ~:nnzc~?-monifikntioncn). The
recent \\,nrl; on induced antigenic, changes in Pn~nrccinnr (rrrrclin (Son-
neborn, Bea.le), and ot,her Protozoa (Ior a review, Harrisonj is in formal
agreement \\~ith model 2B, although here the change induced environ-
mentally, and cvtoplasmic in nature, is not, or not clearly, adaptive. On
the ot.her hand, &rapolation to bac,tcria of the conclusions reached in
Paramecium may be dangerous.  The limited knovvlcdgc a\-ailable for
both types of organisms indicates differences in the immediacy of nu-
clear control ov-er cytoplasmic activity.  It I\ ould not. be too surprising
if the stabilit,y of cytnplasmic systems and the latitude of variation under
c~nvironmentnl influence and outside nuclear control were, on ax.-erage.
tlifferent in the two categories of unicellular organisms, though the pre-
sent evidencS> is insufficient tn warrant such a generalization.

- 226 -
  Summing up this scnnt.y e\.idcncc. bolls7 /ic!e physiolopical 3,diilltFl-
tion to drugs can hardly be expected to be missing, althoug convincing
evidence t,hat physiological adaptation of a high degree can bc reached
is lacking. Conditions usually employed for selecting resistant mutants,
as for insta.nce high drug concentrations with large inocula are of course
of t,hc worst type both for detrctin,rr physiological variation, and for al-
lowing it, to pIa) freely.  However one finds, especially at, lower drug
concentrations. a spread of individual \-ariaCon in the ability to grow
visible colonies, the range of this variation depending on the particular
drugs and oqanisms employed.  Part of this \-ariation may be stoc,ha-
st,ic, i. e. the variation in the total amount of residual growth of a cell
in the presence of a drug may be due to random events of a physiological
nature; part may be due t,o the variation in the initial physiological con-
ditinns of genetically homogeneous cells; part to small penetic differen-
c,es, which are not e,asily measured.  TUO facts should be stressed: I)
that no genetical theory could reject t,he effects of environmental varia-
t,ion, or pre\-ious history of the cells (as in penicillinnse adaptation); 2)
that the existence of physiological variation, both immediate and C( pn-
terltlal )) (adnpt,ive) does not conflict \\it,h the fact that higlr and stable
levels of resistance can bc reached, and haw been pro\-ed to be reached,
hy mutation and sclect,ion.
  (;cnct,ic transfers from one individual to nnothcr, i.e. the genetical
consequences of sesunlitv in higher organisms. has been clearly- dc-
monstratcd in a variety of microorganisms.  \Vhilc in Fungi and Prtr-
tozoa, in \I-hich s;trains \\it,h a sexual cycle arc available, the process
parallels closclv wxaality of hiphcr organisms. in se\-era1 bacterial spe-
cies a ire\\- kind of genetical interaction has been clisclosed, in \\-hich one
&rain can cffcct genetic transfers to another (\vith a rclnti\-ely low fre-
q""ncy ( hut at any ra.te higher than spontaneous mutation in the recci-
ver) xvithout direct, inleraction bctwecn the cells of the donor and receiver.
:\n!; ch:iI'acter diffcrencc tested is transmissible, althoqh as far as lBl'e-
sent experience goes, the transfer seems to affect, only OIIC genetic charac-
ter at time. This kind of transfer has bcrn named fra~sdzrclion (Lcdcr-
berg, l!WZ). In SnlmonPEln typhi~nwictn~ the agent of transfer! which
acts purely as a passive c.arrCer. has been found tn be a phagc. The term
transduction is also meant to include an earlier described type of gene-
t.ic interaction, carrying traditionally the name of tmnsformation: which

- 127 -
has reproducibly been obtained in two bacterial species, Pneumococcus
{Griffith 1928; Avery, McLeod, McCarthy 1943) and HernophiEus (Alexan-
der and Leidy t953). In this particular type of transduction no special
carrier appears t,o be involved ot,her than what is believed t,o be the
specific inducer of the genetic change, i. H. a highly polymrrised DNA.
  Another type of genet,ic transfer has been demonst,rat,ed in bacteria,
and specifically in e. coli (Lederberg and Tatum, 1946): namely recombi-
nation. Here direct interaction bet\veen the cells of the t\vo strains is
necessary.  Both t,ransduction and recombination occur as a rule with
low frequency, so that the progeny. i.e. t,tic result of the interaction
must usually be selected from a mass of unchanged parents. The main
formal difference between transduction and recombinatjion is t,hat. while
in transduction only t,he character specifically selcctecl for is recovered,
in combination the transfer of the selected character is usually accompa-
nied by the simultaneous f,ransfer of other unselected markers according
to a comples, but well reproducible pattern of reciprocal linkages. At
present the genetic system of the more completely analysed strain of E.
coli is best interpreted as formed by two chromosomes, reconrhination
being the result of mating bet,\+-een compatibles cells, followed by a pola-
rised segregation which tends to eliminate l)r'ferentially scgment,s from
one mating type. Ileterozypous indivitlual~ seg repating for various mark-
                                          _
ers have been recovered. as welt as apparently fully diploid individuals.
The diploid state is usually unstabh~, and the normal condition of t,hc
cell is belie\-ed to bt haploid (though often multinuclrntr). (Lrderberg
et al.? 1952: (Javalli, Lederberg and Ledcrberg, 1953).
  %7rilc the type of analysis permitt,ed by recombination in E. coli
K-12 closely al~prosimatcs mendetian anal)-sis in higher organisms, trans-
duction offers opl)ortunities for another. morr rcstric.ted type of genet,ic
analysis \\ hich nia~-, ho\\-c\-l;r. sho\i certain advnntngcs over' tht> rnorp
strictly mrrrdelian nne.
  It is important to distinguish betbveen what may be expected, from
a mendelian analysis, of drug-resistances which she\\ the streptomycin
pattern, and those showing t,he penicillin pattern. Or, more exactly,
the distinction must be dra\\-n bet,ween inherit,ance of first-step-resistancr
(if truly such) and that of multi-step-resistance (for drugs to which gra-
dua.1 adapt,at,ion is the rule). In thr first instance \ve expect, if the t.heory
of genetic adaptat,inn is correct, that the genetic change to resistance is
inherited as an all-or-none character, i.e. the progeny of first-step-resi-
stants crossed \\it,h sensitik-e should give rise only to sensitives and resist-

- 128 -
ants, the progeny rembling closely either one or the other parent in
respect of drug-resistance. Crosses of resist,ant x resistant will give rise
entirely t,o resist,ant progeny: unless of course the changes in resistance
represented in the two parent,s have originated through independent
mutation at different loci. i.e. the two changes are not allelic.  In the
case of multistep-resistance, successive adaptation must have been due
to the accumulat,ion of successive mutations, presumablg at a number
of different loci. This polygenic picture \\as early advanced by Demerec
to explain penicillin resistance in staphilococci; the later availability
of crossing techniques in E. coli allowed our it) test this hypothesis in
the latter organism.  At least five cases of drug-resistances hal-c beer1
nnalysed in some detail. t\vo of the-ru folio\\-ing the streptomycin pattern
and three the penicillin pattern.
  Slrepicm~ci~7. - Various types of chauges to strel,tclJllycin-resistance,
inr dependence) are possible in E. coli. all appearing as first-step muta-
tions. Very lo\v dqrees of resistance, v+-hich are considered adaptive by
(Gibson and Gibson, have not been fnllo\ved genetically, except in au
incidental obwrvatiou by iYe\\-combe and Ryholm, on a fully resistant-
strain which was presumably a second-step mutant. Differences are dctec-
table between strains cndowd \sith high resistance, and Mitchison (1953)
distinguish& four types on the basis of gro\\th rate, stability and beha-
viour at high concentrations.  Demerec (I950 a, b) found a variety of
changes mostly of a nutritional type accompanying resistance (Sr) and
dependence i8d).  It may be noted that changes correlated lyith the
character primarily selected 111ay be accidental. i. e. due to concomitant
mutation: in whic,h case primary and secondary character, might be sepn-
rated by recombination. Unless such an analysis is available, the estima-
tion of the number of different alleles or loci cannot be considered as
valid, but is likely to be excessi\-e.  Smith, Oginski and Cvmbreit (2949)
reported that Pr mutants sho\ved se\-era1 physiological de\-iations, includ-
ing a. failure to she\\- improvement of pro\\-th under aerobic conditions.
AS this is reminiscent of the small-colony variants of yeast, this effect
\vas looked for in our OWJI Sr cultures. All of the Sr mutants \\-hich w-erc
examined, in our o\\-n stocks as well as in the initial culture of the cited
authors have displayed a typical respolise to aeration (Lederber?, un-
published). It is possible t,hat the non-aerobic behaviour of the Smith-
Oginski-Umbreit culture resulted from a coincidental mutation.   The
pyrwic acid metabolism of our Sr strains, a defect of \\-hich is claimed
to be a more consistent concnmitant of strcptornvrin-resistance has not
been studied.
Mendeliau o.nalysis nf changes to full resistance, perform& h!- w-

Vera1 authors (Newcoinhe and Syholm 1950, Ucnierec, 1950, 1951. etc.);
showed: 2) clear cut segregation into scnsit,i\-es and resistants in the
cross sensitil-c x resistant; the frequency of segregation, \\-hich depends
on the nrnting-tvpc of the pawnts ipolarit,y of the cross) and on linkage
of the resistance markers with the markers used for selecting recombi-
nants need not concern us here; 2) 1)`rfect rrvrrsal of the ratin sensit,ive/
resistant in the progeny of crosses in \\-hich the resistance marker is swit-
ched n\-er from one p?rcnt to I.hr otlrcy, keeping polarity unaltered (Tue\\--
combe and Yyholm); this is an importan requirement nf the mendelinn
theory: 3) allelism of all the various <I' mutations tested iI+merec); 4) al-
lclisrn of Sr and Sd, nlthouph some room for pseudoalle~lism may still
remain !Nr\vcombc and Nyhnln!. IIenrwrc~: 5) linkage \I-ith the methio-
nine n~nrkcr. \vhich has rcccntly hcen c~onlirm~cvl iny,if IX of t!rc clifficuli icy
met in mapping tbis locus iFried and Lctlcrbcrg. 1X%); G) streptomycin
wsistanw is co~i;j)letely recessi\-c to srnsiti\-it\- iI,rrlerlwg, 1951); at-
tempts to sccuw i:eterozygotw i) f Sd \\-ith Ss or Sr ha\-e tailed. perhaps
because of l~hysiolopical incompatibility lwtwecn these alleles.
  The mntntion ratr to rtrrptom,vcin wsistance has been tletc~rminctl
at about 1O-1" per bacterial I generation in a nnmbcr of bactcrinl strains and
species. Il. P. Treffers (personal cnnrmunicntion) ohsewrd that a bio-
tin-~~hen~lnlnni~~~~ requiring mutant cof E. coli K-12 displayc(l a much
hi?hcr ratrx of nlutation to 518 Inbout 1W per pncration). Crossing cxpe-
rinlr>nls ha\-c ;;ho\vn that I) thi-: high Yi\tC i5 unwlatcll to the nutritional
IllIltilt ions in the stock: 2) tllcl nmtation i)ccurs at tlltl SalllP 5lc)CllS; 3) the
high rate is go\-erned 1)~ a mutability mndificr, separable from the S lo-
ws by recombination !P. I). Sknnr and Lederhcrg, unpublished). -`I morr~
tlctailcd study of thaw effects ir heing made in Proof. Treffrr's lahrwatory.
  .l:itlv. - E. iwli K-12 can mutalc in a ainglc slep to il small degree
of wsistnnco to azide. This mutaticon \\-a~ used l)~- TACrberg (1950) as a
wlwtivr Il~~~l~liP~ f~jr iwonibinntion. in ns9ocintion \\-ith streptomycin.
Ilutatiou-rntv to nxidc wsistancc~ iabout 10~`) is fairl!- high. and the effi-
cirnr: of 1 his crossing tcrhniquc caa be incrrawd 1)~ using T, phage
wsistnncc~ in coupling {\-ii II .\F; the Sr parent can thus hc fully elimi-
nntrcl iCa\,nlli, unpublisli~d).  This association \\-a~ supFcstt:d by thr
~10~ lirllinpc of .\77 aatl q. .b= having been nIappw1 bet\\-ccn T; and
fhr thrconinc- Icucinv markrre.  Only one .\F locus has been found;
also hew. the switching over of AZ= from one parent t,o the other, keep-
ing polarity unnltc~rc~c! L'j\-l'G I'rnS;ultc iv il!?l.l'~`lllC1ll vt it!) nlc~n!lcli:!:l rzpw~-
tations (Cayalli, 3952).
  Ch~ol.n~,,~,hr~-nic~ol. - Hcsistanw to thic: drug in E. coli K-12, obtained
throuprh !zrntlual ntlaptatinn \\a? nnal!;srd \\ ith ln~~ndrlinn techniques 1~;~

- 130 -
Cavalli and Maccacaro 1.1X1. 1932). Crossing first-step resistants to sen-
sitives gal-P a seprega.tion into sensiti\-es and resistants, without inter-
mediate dctgwz of rwistnnw in thr progeny.  Crossing tirst-step-resi-
atants brtwcctn thcmsel\-cs wsu1tet-l practically always in the reappea-
rance of a prol~~Jrtion of wnsitivcs in the progeny, shovkg that first-
step resistance is here due to mutation at a number of different loci four
or five at ll?ilSt). ;il>mc crosser between first-steps ga\-P rise tn progeny
\\hich shn\\ ~(1 wdisitancc: higher than that of eithtr lji~rent, i.e. rrccln\bi-
nation nlc~tli;rtc~cl thcx nccumula t ion of thv resistance acquirecl 1~). indr-
JWIldCTli ~Illlt~liOllS i>t rliffl-:`PlIt loci.  In a mnw fully anal!-wd crews of
this kind. cloubl!- wsistant mut:\nts arosc in approsimatel,v the espcctctl
proportion, calculated from the proportion of resistants in the crow3
of the parental first-steps to the original sensiti\-es.  Crossing a second
step to n ?ensiti\-c. intermediate resistance bd\wcn that of either parent
nppeared.  On crossing multi-step resistants. obt,ained I~>- prolonged
v tmining 1) in liquid medium, \\-ith sensitil-cq. the progeny \\-as founcl
to cshibit dcgrces of resistance x-nryin, w over almost the full range from
full sensitik-it\- to the> full resistance of tb,s resistant parent. Linkage of
rcsistanw \\-ill1 the ortlinnr>- cl~ron~osomc ruarkcr~ could be detected iu
this progcu~. as \\oulcl lw c\-petted if sweral loci \\-ere in\-ol\-cd. L\l-
though thcrc \\;Is e\-idence for at least some activii!- in a number of chrn-
mosomc rcpions , the nlaximum concrnl ration of genetic a(+\-it>- \\a<
IlCW Lx.  Crossing Illulti-stc~p-r~ristants bet\\-een tbemselv~es ditl n1)1
sho\x- positiw interncticjn, P iu the sense of increased resistance above that
of either parent, perhaps bccnusr the physiological limit. to resistan
had nlrcady bwn reach4 1)~ sclcction.  There \\-as instcall ;I sprexl
towards the lo\\ \-alucs ol` resistance. anti SOIIIP fully sensitil-c indil-i-
duals v-erc rccovcretl among this progeny.  This sho\\s 1) that full 0~' nt
least hi@ resistance can be acquired in gradual adaptation through se-
veral independent paths; 2) that interaction bet.\vecn independant mut:r-
tions is not nccessnril?- adtlil-ive.  Indeed. after the first resistance- step.
all successi\-c steps acquired by selection may well be. in theory at Icast.
due to modifiers, i. e. genes havin, cr no direct cffwt on resistance by
themselx-es. but intc>rncting \vith other loci to gi\-r augmented rcsistnncc.
The possiblit>- of direct accunlulation of independently obtainrd resistnn-
ces has been observed, however.
  `I'e?mtL~y~irl. - This drug is kno\\n to sllc~\\ a fairly high degrw
of cross-resistanw to chloramphenicol. In the anal)-& by recombination
of t\\-o strains of k'. coli K-12 \\-hich had adapted to high concentrations
of terrnm?-tin nftcr a long (( training )) period, resistance of the progeny
to both drugs \\-a~ tested. Correlation was still apparent in the progeny.

- 131 -
but it was evident that while some changes had affected only, or mostly,
resisbance to one drug, others had affect,ed resistance to both. This shows
at once the existence of receptors within the cell which are u hit )) pre-
ferentially, or exclusively, bq one or other of the two drugs, as well
as of receptors (( hit )) by both approximately in the same way (Ca-
va.lli, 1952).
  F~adrozyl (SF 67). -- T. C. Nelson has aualysed, in unpublished
experiments, resistance to furadroxyl (B-nit#ro-2-furaldehyde-2-(2 hydrox-
yethyl)-semicarbazone) developed by E. coli K-12. At, least four steps can
be recognized: the inhibitory level for the parent is 4 pg/ml, and for
successive steps 20-30, 40-50, 90-100, 160-200 pg/ml.   The survival
curves are remarkably sharp, t,he threshold covering only a few y0 of
the dose. The mutation rat.es for each step are co. 1O-5 - 10e6, uncorrected
for plate mutations.  ,$ sat,ellite effect was prominent around resistant
colonies.  No mutant,s have been found to develop a t\vo-step level of
resistance in a single plating.  Crosses of Fx" x l-step F; showed a
linkage of this particular factor to TL, wit,h a sharp segregation into the
parental categories.  Different first-step mutants when crossed, yielded
occasional sensitive as well as two-step\\- level recombinants, along
with the parental types. It is apparent that multistep resistance is here
also controlled by a polygenic system.  The incidence of recombination
is so low, however, as t,o sunggest very close linkage of some of the
fact,ors.
Other drugs have been analysed to a smaller extent.  Mrogen-
mustnrd resistance in E. Coli Ii-12 is not, a single-step-resistance as in
l?. co2i B, and is not corrrlated \\-ith IW or X-ray rnsist,ance; it shows
an appare,nt,ly polyge,nic brhnviour on crossing (Cavalli. unpublished),
but, the instability of the drug prevents a detailed analysis. Valiue has
n ma,rked antibacterial effect on E. coli h-12 when t,ested in minimal
medium but none in nutrient agar? due to a.ntagonism \vith isoleucine
iTa.tum, unpublished; Iiokvley, 1953). In minimal mrdium, strains end-
owed \\-ith a fairly high degree of resistance can be isolated in one step
and, on crossing sho\v linkage with M (Cavalli and Bolzoni, unpublish-
cd). This marker can be used t,o select recombinants in minimal.
  Colicine- rcsist.ance ha.s been nnnlysed geneticall)- by Fredcricq and
Betz. Resistance to eight t,ypes of colicines was shown to segregate in
R mendelian fashion. Some discrepancy is observed in the segregations
given by reversed crosses, probably due to viability differences.  One of
the colicines studied? Ii. showed cross-resistance wit,h phage T,, and thr:
resistant locus showed. on mapping, to fall in a position comparable to
that ascertained for phage T,: resistance b> Lederberp id94,7).

- 132 -
  In Salmonella typhimwium Lederberg and Zinder (1952) obtained
the transfer of a number of genetic markers, ranging from nutritional
deficiencies of various types and sugar fermentations, to drug-resistance;
the only one tested here has been streptomycin-resistance. As no more
than one character at a time can be transmitted in these conditions,
proof of the transfer is given by the fact that the change to, say, strepto-
mycin-resistance is signihcantly higher than in untreated controls.  For
streptomycin resistance, transduction shows an increase of 100-1000 times
in the number of resist,ant colonies as compared with the number due to
spontaneous mutation in the untreated, sensitive controls. The transfer
can occur through a filter (which would bar any genetic exchange in
E. coli K-12) and, when the transducing agent. is purified, the carrier,
i.e. phage, is recovered. Sr is here apparently a single-step-resistance, as
could be inferred from t,he method of selecting the mutant.
  In llemophilus influenzae, Alexander and Leidy (1953) could obtain
transformations of a kind similar to those classically described for Pneu-
1110c0cclls.  The spontaneous mutation rate to streptomycin-resistance is
stated to be ca. 1O-`l per cell per generation, and the tolerance level is
increased from 3 to 1000 pg/ml in one step. DNA extracts prepared from
resistant cells were usctl for treating sensitive c,ells for 10 minutes, then
DNA -ase \yas applied.  If plating on streptomycin was carried out im-
mediately at this stage, transformation of sensitives to resist,ants could
bc observed only with a 1011 concentration of streptomycin !I0 &ml);
hut the resista.nts thus oht,ained showed the same degree of resistance as
the D>A-donor.  If treated cells were incubated for t\\-o hours bcforc>
plating on streptomyc'in, the. rate of transformation was idrnt,ical in pla-
tc>s \vith high and low concunt,ration of streptomycin. A single step of
resistance is therefore transduced, and some phenot.ypic lag ii: apparent
in the development of resist,ance.
  In PI~~~~EOCOCC~~S. Itot,chkiss (1951) has recently added to the already
tlsisting dat,a on nntigenic transformations. IIe has shown that transfor-
mation was not limited to nntigenic characters, but is possible for all
the: drug-resistances I\-hich \\.ere tested, namely streptomycin, penicillin
and sulpha drugs.  Pncumoc,occus transformations require a battery of
supplementary conditions beside specific D?GiA from donor cells. Even in
optimal condit,ions, transformation is 1imite.d to a few percent of the
cells treated, the bar to complete transform&ion remaining unknown. .
Of the drugs tested, streptomyc,in resistance is acquired by mutation. and
transmitted by transformation, in a single-step. Penicillin resist,ance is

- 133 -
.of greaI.>r interest since the donor strain had acquired resistance by long
training ;:nd hence by a number of steps (the number of which could not
bc determined exactly on the evidence of the adaptation process alone).
11X.4 extracted from the donor strain could impart resistance to the sen.
sitivc receiver only in three succrssive steps: first exposure provided
resistance from the initial lcvcl of 0.01 p,Irnl to 0.05 IL/ml; second ex-
posure of the first-step-transforlrlcd  strain increased this level to
0.12 t~/ml, and third exposure could siill raise t,his to 0.30 p/ml. The
polygcnic picture comes out vi-r>- prettily from this experiment, where
the peculiarity of the CC hybridisation N process (one factor transduced at
time) seems to split the complcs polypenic system developed by selection
into its c~lciilc~ninr~ components.  It \\-ould hc interesting to know if the
orda* \\,ith A\-hich these components are transduced is that follo\\ed by
wlcction, as \\-ould haptwn for instance if the changes due to later steps
\I-erc onl)- modifiers of t trc resistance resulting from the first-step without
n direct, cffcct on resistance. The publised data do not seem to warrant
R conclusion on this pain?.  The USC of tlonor strains \\-ith a knowi
IIu~nBcr of stcfls, nntl of \.,arious indefwndent first- or multistops as don-
ors or receivers miglrl certainly add, in an interesting way. to our
hIlo\\-letlp of tlbis polygc~rlic l~~lli~Vi~~l1~ in dru; rcsistanc*e.
* 1 *
  The roles of lrrutation and selection ha\-e been suffkient ty tlocu-
mcntetl b)- the above summarised investigations to convince us that drug

-- 134 -
adaptation is, at least to a large extent, a matter of genetical adaptation,
and in this way does not differ from other kinds of adaptive changes
observed in the same and other organisms.  Thus bacteria do not seem
to differ greatly from higher orga.nisms in their evolutionary mechanisms,
and unusual or complex hypothetical mechanisms, devised to predict
the adaptive response to drugs in bacteria, may have to fall under
Ockam's razor.
  Although the nat,ure of the mutat.ion process remains fundamentally
obscure, we know that it can provide a population with a great variety
of heritable changes, and in this way the demands of new environments
can be met in such a way that the survival of the population is guar-
anteed even against extreme conditions, through t,he survival of some
individual which can both \I-ithstand and propagate themselves within
the adverse environment.  If the change to the new environment is per-
manent, or at least long-lasting as compared I\-ith the generation time
of the organism, there will be opportunities for further genetic adapta-
tion, so as to raise the level from bare survival of the population to,
optimal or nearly optimal conditions of growth.  Jf the change is
transient, return to the original form by back mutation is possible and
if the original type had a selective advantage over the mutant in the
old conditions, as is usually the case, the population \vill shift back to
the primitive condition. The multiplicity of genetic changes effecting
a similar result, however, and the rapid accumulation of secondary
changes of adaptive significance will in most instances determine dif-
ferences between a populat.ion before, and after a cycle of genetic adapta-
tion to a new transient environment, follo\wd by deadaptation (or re-
adaptation) to the old enviroments.  The change may or may not be
easily detectable.  If no difference is detected after a cyc,le of genetic
adaptation and re,adaptation, we say that the change was unstable (and,
of course, an almost unlimited range of instability may exist). When a:
differenc.e is detected, which may or may not reflect a partial return
to the original drug-sensitive state, we have been faced with the irre-
versibility of evolution.
That, the variety of genetic changes available in a population is a
very great one is shown by a ~\ealtb of dire& or indirect information.
The variabilit,y of independent first-step-resistants provides evidence of
the variation that is directly available for selection to play upon. 11
number of apparently unrelated changes are accompanied by variation
in drug resistance.  Thus, when selecting for aminoacid deficient mu-
tants, an increase in penicillin sensitivity may be observed @`lough and
Grimm, 1949; Rowley, 1952); when test.ing individuals selected for resi-

- 1% -
stance to a drug, they often show changes in resistance to other drugs,
both increased (cross-resistance) and decreased (induced sensitivity, Szi-
balski and Bryson, 4952). Resistance for chloramphenicol has occasional-
ly been accompanied by the loss of penicillinasc formation. h great
variety of hereditary changes of this kind may be given, so that we can
confidently expect that a population \\-hich originated from a. single cell
some twe,ntl or thirty generations before may already contain a wealth
of new heritable variation. It is not too surprising, therefore, if different
isolates from a bacterial population show differences of sensitivity (Hu-
ghes, 1952), although minut,e difference< $ in resistance ma>- be overesti-
mated, if careful control by suitably planned experiments is not exercised
to check day-to-day variation.
  If, inspitz of the large suppI>- of ne\\ variabilit!-, bacterial pol~uln-
tions grown on a constant medium rarely show apparently spontaneous
changes in behaviour, at least for most of i.he characters analysed, the
reason can only be that most of the new variation has au adaptive value,
in respect of the standard environment. inferior to that of the ordinary
type.  Most mutants therefore will be outgro\\-n. This is simply the
consequence of the fact that the strain has reached some degree of genetic
equilibrium, i.e. it has conccnt'rated at least the most frequent changes
which are more favorable to reproduction in the standard environment.
Experience shohvs that these equilibria are largely dictated by the equi-
librium of mutation and selection, rather than by equilibrium between
forward and backward mutation rates.  Therefore it is not surprising
that most mutants have lower growth rate than the parental form, as
shown by Demerec (1950) for streptomycin-resistance.
  An altered environment. such as may result from the addition of n
drug, is bound t,o destroy this equilibrium.  If the change is not too
drastic, the original type may multiply- to SOI~P extent. and its fre-
quency bvill decrease front almost one to snmr \-alue intermediate be-
tween one and zero: if the change is \-cry drastic, ii \\-ill fall down to
zero. A special situation inay arise if the resistant mutants ma!
u help 1) the sensitij-e p,arents to sur\3w or e\-en multiply in the drug-
medium; an extreme example beiu g that of partial or complete destrw-
tion of the drug by the mutant, or the production of metabolites an-
tagonistic to the drug (PAI<-producing mutants in sulfa drug resistance.
for inst,ance). Very c,omples equilibria may be weated in bacterial po-
pulations in this way, and the fact that apparently resistant colonies,
obtained in selection in liquid, and even in solid clrug-media may give
rise to partially or fully sensitive subcultures. simulating re\-ersion
(Banic, 1953) may be explained b>- the partial sur\-i\-al and transfer of

- 136 -
the sensitive parent.  The careful, possibly repeated isolation of single
colonie,s from resistant mutants on drug free media, before any test of
stability, is carried out on them, seems therefore an elementary (though
a somewhat neglected) prec,aution.
  When rapid trainin g to high concentrations of drugs like chloram-
phenicol is carried out, t~he general \+ability of the strain, as shobvn by
growth rate or densit,y at saturation, ct,c., is usually decreased. T\\-enty
strains of E. coli K-42 independently trained to high chloramphenicol-
resist,ance she\\-ed a densit,y at saturation bet\\-een one third and one
half that, of the original sensitil-e strains, in t,he absence of the drug,
-and still smaller in its presence.  It is not surprising therefore t,hnt
these strains showed a tendency to re\-ert in the absence of bhc drug;
this n-odd indeed be inevitable, if back mutation is possible. Return
lo sensit,ivity. if it occurs, is usually gradual; full reversion of a highly
resistant strain may take six months or more of w-eekly transfers t,hough
it, may occasionally be more rapid; sometimes complete or even partial
reversion has not been ohserved in three of four years.
  Experimental proofs have heen given that return to sensitivity in
these well established cases of genetic resistance is due to (back) muta-
tion and selection.  Ptudving artificial mixtures of sensit.ives and rp-
                       . ,
sistant mut,ants one finds, as a rule, that the latter arp outgrown (Da-
merec, 1950; Welsch, 1952). It, might be argued t,hat such experiments
cannot rule out transformation unless the two strains carry suitable
genetic markers.  Spont,aneously occurring morphological changes were
used by Harm (1951) t,o follow the fate of population of staphilococci
,exposed to the action of phenol and formaldehyde; mixed cultures of
sensitives and resistants, marked by their colonial morphology, showed
continuous changes in the proportion of t,he t\\-o morphological types in
the sense of an increase in the frequency of the resistant type when the
drug was added, and of t,he se,nsiti\-e type when the drug was withdrawn.
Experiments with art,ificially marked strains \\-ere performed using strepto-
myc,in resistance in E. coli K-22 (Cavalli, unpublished) : mixtures of sensi-
tives and resistants marked by Lac fermentation were followed\\\- hy counts
on EMB Lac with and without streptomycin after every third transfer.
Resistants were outgrown in six to ten tra.nsfers starting with equal fre-
quencies. The genetic markers used (other markers were available in the
strains additional to Lac) each had its o\\-n effect on the rate of the elimi-
nation.  The rate of elimination due to the Ss/Sr difference only was
equivalent. t,o a difference of 140/, bet\\-een the growth rate of parent and
mut,ant: analysis of t'he growth rate of the two strains grobvn in&pen-
dent,ly confirmed this difference.  It is of interest that in a contra! 1:ulture

- 137 -
of this experiment, with only Sr individuals originally, at, the 9th transfer-
a clear cut discrepancy between plate counts xvith and without strepto-
mycin appeared: it was ascertained that only 3% of th? cells were re-
sistant. the other being fully sensitive.  This cult,ure on further transfers
sho\vrd a progressive> decrease of the proportion of resistants, with the
same rate of elimination as a comparable art,ificial mixture Iof sensitives
and resistants. Mut,ation to sensitivity must have occurred in this cul-
ture, a very rare event for st,rept,omycin-resistance in E.coZi. R.eversion
rates have been studied with great detail in the more favorable case of
st,reptomycin-dependents (Bertani, 1981; IIemerec. 4951) Sd can revert to
both Sr and Ps wit,h approximately equal ease. and rates which are
usually low but vary from to strain.
  The geneticist t,ends of course to deal preferentially with stable he-
reditary c,hanges. Apparently resistant colonies which, for some reasons
or other, very ea' ily revert, to sensitivit,y on subculture in the absence of
t,he drug will oh\ ;onsly be dixarded in viex, for instance, of mendelian
analysis. The objection map be raised t,hat this \\\-ill inevitably create a
selection of the mat,erial analysed. This xlect,ion cannot be very impor-
tant, since is is easy to sercurc a wealth of stable resistants for genetic
analysis with most systems.  On t,he other hand, adaptationists who
dismiss (II' minimise the rolrh of mutation and selec,tion claim that stable
resistants of non-mutative origin can be obtained at least b;\; prolonged
training; but. whene,ver suc,h material has come under genetic analysis,
it proved to contain a great deal of genetic variation. Also: the evidence
from Dauwmodifikat,ionen in Paramecium she\\ R that enyironment-
induced changes of ext.ra,nuclrar origin may be &able; so that the use of
stable rrsistant,s for analysis should not discard all estrauuclear changes,
if these nc,tually exist in bacteria.  It may be added that relatil-c insta-
bilit,y should be checked, at any rate, with frequent single colony isola-
tion and control of resistance of subcultures of isolates wit'hout re-
exposure to the drug of t,he strain, t,o avoid the selec,tion of other changes.
  The problem of considering the origin of unstable variants still re-
ma.ins. Full rapid reversion, demanding, on a Fenetic interpretation, a
hypothetically high back mutation rate, would certainly be more easily
interpret4 as due to physiological adaptation, and is at any rate dif-
ficult to submit to rsperiment~al analysis.  Such rapid reversions ho\\=
ever seem more frequent onl) \\-it,11 low degrees of resistance, at the
fringes of physiological variat,ion, or in tests for physiological adaptation
designed to exclude the int,ervention of selection.
  The relative importance of r crenetic and physiological adaptation in
                                          Y
t.he development of drug-resistance of bacterja seemx therefore to be in

- 1x3 -
favor of t,he genetic mechanism.  The reverse may well be true with
Protozoa or at, least some Protozoa; it would indeed be surprising if
such a delicate balancr 11.ere adjusted at exactly the same level for all
organisms and drugs.  It is also possible of c,ourse that for some drugs,
physiological adaptation may play a more important role than for others,
thus lcndiug \\orkers who use t,hese particular drugs to make undur
generalisations on the o\-es\\-helming role of physiologic,al adaptation.
  Whatever the role of physiological adaptat,ion, t.he exact limits of
which are difficult to assess al present, some slwculation concerning
it may be of interest.  It is clear that whenever physioloprical mechan-
isms can cover a wide ranac of adaptabilil,y. all mufants whose pheno-
typic efl'ect is smaller than this range will de damprd by the morr pff~-
cient phenocopy which will rapidly arise through physiological adap-
tation, or be already present in the form of non-genetic variation. A
large range of physiological variability, immediate or potential
therefore tend to suffocate genetic adaptation. On th. other l,&d"-it:
                                                t' I c
this range extends only to somewhere midbvay between the parc';lal
(average and the mutant, a\-erngc, physiological adaptation will smoothfl
the way to the genet,ic one, by favouriug partial growth of the tail of the
distribution c.urve and thus increasing the c.hnnces of mutation.
  Drug resistance has been b (riven a prominent place in the StLldy of
cvolut,ion of microorganisms essentially in viP\\, of its importanw for
chemotherapy, and, to a lesser extent, antisepsis.  Some practical con-
clusions may therefore be a.ppropriate.  That this kind of evolution can
aCEect the praxis of chemoterapy has been she\\-11 hy the accumulation
of failures of certain therapies due to the increasing frequency of drug
resistant pathogens : sulfona.mide resistant ~onococci, penicillin resist-
ant staphilocci, strept'omycin and isoniazide r'wistant tuberclc bacilli
being but the outstanding examples.
  The answer to this danger has hcen mostly found in the develop-
ment of new drugs unrelated to the old ones; in the enlployment of
mawive doses since the beginning and without, interruption, in tIlc>
a,ssoc.iatfion of local and general antibiotic therapy, and lastly and more
casil) bv the association of drugs. Genetics' otfcrs an ea.sy explanation
for thl. &X~SS of drug associations, since with drugs to \vhich no cross-
rr4auce (due to similarity of mechanism of a&ion) is observed,  one
drag will destroy the few resistant mutants left by the other. In this
counwiion, the importance of using simultaneously associated drugs.
.and not in sequence, hardly needs being, stressed. Genefic synergism,
i.c. al~cncc of cross-resistance, is to be kept, distinct from physroZogical
synergism between drugs.  A first foundation for a map of genetic

- 139 -
synergism was laid by Szibalski and Bryson, wha analysed strains
made resistant to each of 45 different antibiotics for all possible cross-
rf~sisiances.  The number of tests necessary in such investigations is
lessened by the use of a rapid technique of scoring for resistance which
tht,y have developed, namely the gradient, plate technique (Bryson SC
Szibalski).
  The interaction bet,ween tv,.o drugs at the physiological level may
range from true synergism (joint effect greater than expected on simple
addition of effects) to simple addition, indifferent effect , or antagonism.
Physiological synergism is, to some extent, negatively correlated with
genetic synergism (Klein and Schon, 1952). In fact an additive interac-
tion may he expected between related drugs like chloramphenicol and
terramycin, which show cross-resistance and therefore genetic antago-
nism. On the other hand unrelat,ed drugs like terramycin and penicillin
show genetic synergism and physiological antagonism (.Jawetz et al.,
1953; Klein and Schon, 4952) due presumably to the fact that penicillin
is actively bactericidal only on growing cells, and a bacteriostatic drug
would therefore greatly reduce its action.  In other cases, such as the
association of chloramphcnicol and sulpha drugs, or for the therapy of
tuberculosis, the asociation of isoniazide and strept,omycin, probably
both genetic and physiological synergism occur. At any rate, the rules
of physiological synergism are still largely unknown, and show varia-
tions form strain to strain. Therefore, any remark we can make must,
at this stage, be qualified by a number of reservations so that every
case of therapy must, in the end, be judged on its own merits.
LITERATURE
Alexander H. E. f4 Leidy G. (1953). Induction of streptomycin resistance in sensitive He-
  mophilus influenzae by extracts containing desoxyribonucleic acid from resistant He-
  mophilus influenzae. J. exp. Med. 97, r7.
Armitage P. (x952). The statistical theory of bacterial populations subject to mutation.
  J. Roy. Statistic. Sot. Series B., 16, I.
Avery 0. T., Mac Leod C. M. 8 MC Carty M. (1944). Studies on the chemical nature
  of the substance inducing transformation of pneumococcal types. Induction of transfor-
  mation by a desoxyribonucleic acid fraction isolated from pneumococcus type III.
  J. exp. Med., 79. 137.
Banic S. (1953). Ueber die Hypothese van den reversen Mutationen der Bakterien. Schweiz.
  Ztschr. allg. Path. Bakt. 16, I 12.
Baskett A. C. (1952). The resistance of Bact. lactis aerogenes to proflavine (2 : Il-diami-
  noacridine). 11: The direct induction of adaptation. Proc. Roy. Sot. B., 3 g. 25 I.
Beale G. H. (1952). Antigen variation in Paramecium aurelia variety 1.. Genetics, 37, 62.
Benzer S. (unpublished).
Bertani G. (rg5 I). A method for detection of mutations, using streptomycin dependence in.
  Escherichia cob. Genetics, 3 6, 598.
Bornschein H., Dittrich W. I H66ne G. (rg5 I). Zur Entstehung der Chemoresistenz bei
  Bakterien. Naturwiss. I 6, 383.

- 140 -
Brown C. H. (rg5o). Elimination of kappa particles from cc killer )) strains of Parame-
  cium aurelia by treatment with chloromycetin. Nature, t 66, 527.
Bryson V. I Szybalski ( 1952). Microbial selection. Science I 16, 45.
Catlin B. W. (195 I). Response of Escherichia coli to ferrous ions. J. Bact. 65, 4 13.
Cavalli L. L. I4 Maccacaro G. ( rg5o). Chloromycetin-resistance in E. ~021. A case of
  quantitative inheritance in Bacteria. Nature, 166, 991.
Cavalli L. L. 8 Maccacaro G. A. ( 1952). Polygenic inheritance of drug-resistance in the
  bacterium Escherichio COIL Heredity, 6, part 3, 3 I I.
Cavalli L. L. ( I 952). Genetic analysis of drug-resistance. Bull World Hlth. Org., 6, r85.
Cavalli L. L., Lederberg J. ti Lederberg E. M. (1953). An infective factor controlling sex-
  compatibility in ~Qcterium cofi. J. gen. Microbial., 8, 89.
Colwell C. A. (1946). Small colony variants of Escherichia coli. J. Bact. 52, 4r 7.
Dean A. C. R. 8 Hinshelw-ood C. ( 1952). The resistance of Bact. Lactis aerogenes to
  proflavine (2 : 8-diaminoacridine). I: The applicability of the statistical fluctuation
  test., Proc. Roy. Sot. B., t3g. 236.
Demerec M. (1945). Production of Staphylococcus strains resistant to various concencra-
  tions of Penicillin. Proc. Nat. Acad. Sci. 3'. 16.
Demerec M. (1948). Origin of bacterial resistance to antibiotics. J. Bact., 56, 63.
Demerec M. (rg4g). Patterns of bacterial resistance to penicillin, aureomycin and strepto-
  mycin. J. Clin. Inv., 28. 8g I.
Demerec M. (rg5oa). Reaction of populations of unicellular organisms to extreme changes
  of environment. Amer. Naturalist, 84.
Demerec M. et al. ( I g5ob), The gene. Carnegie Inst. Yearbook, 49, I 44.
Demerec M. (1051). Studies of the streptomycin-resistance in system of mutation in E.
         ~ ;-
  coli. Genettcs, 36, 586.
Eagle H., Fleischman R. 8 Levy M. (1952). Development of increased bacterial resistance
  to antibiotics. I : Continuous spectrum of resistance to penicillin, chloramphenicol,
and streptomycin. J. Bact. 63, 623.
English A. R. 8 MC Coy E. (195 I). A study of streptomycin resistance in micrococcus
  var. Aureus. J. Bact. 6 I, 51.
English A. R. u Mc Cov E. (1951). Growth comparisons of streptomycin-sensitive and
streptomycin-resistant Micrococcus pyogenes var. aureus. J. Bact. 62, 19.
Ephrussi B. 8 Hottinguer H. (195 I). Cytoplasmic constituents of heredity. On an unsta-
  ble cell state in yeast. Cold Spring Harbor Symp. 16, 75.
Eriksen K. R. (1949). Studies on the mode of origin of penicillin resistant staphylococci.
  Acca Path. et Micr. Stand. 26, 269.
Friddricq P. ?4 Betz-Bareau M. (1952). Recombinants gXtiques de souches marquees
  par rhsistance aux colicines et aux bacthriophages. Ann. Inst. Pasteur 83, 283.
Fried P. S. 8 Lederberg J. Linkage in E. coli K-12. Genetics: 37, 582.
Gale E. F. (rg4g). The action of penicillin on the assimilation and utilization of amino-
  acids by gram positive bacteria. Selective Toxicity X4 Antibiotics, Symp. Sot. E. B.
  III, 235.
Gale E. F. I4 Rodwell A. W. (1949). The assimilation of amino acids by bacteria. 7. The
  nature of resistance to penicillin in Staphylococcus aureus. J. gen. Microbial. 3, 127.
Gibson M. I. 8 Gibson F. (195'). Development of resistance to dihydrostreptomycin by
  Bacterium coli. Nature 167, t r3.
Goldstein A. (1952). Effect of penicillin on streptomycin-dependent variants in Escheri-
  chia coli populations. Science t 16, I 13.
Graessle 0. E. F4 Pietrowski J. J. (1949). The in vitro effect of para-aminosalicylic
  acid (PAS) in preventing acquired resistance to streptomycin in Mycobacterium tuber-
  culosis. J. Bact. 57, 459.
Griffith F. ( 1928). The significance of pneumococcal types. J. Hyg. 27, I I 3.
Harm W. (1951). Untersuchungen zur Frage der Variabilitiit der Formaldehyd und Phenol-
  Emofindlichkeit von Micrococcus pyogenes var. aureus (Staphylococcus aureus). Arch.
  f. Mikrobiol. I 6, 3 19.        _. -
Harrison J. A. (1947). Antigenic variation in protozoa and bacteria. Ann. Rev. Micro-
biol. 19-42.
Hinshelwood C. M. (1946). The chemical kinetics of the bacterial cell. Oxford Univ.
  Press, 1946.
Hobby G. L. (1953). Symposium on the mode of action of antibiotics. Part. II: The
  mode of action of terramycin and eureomycin. Bact. Rev. r7, 29.
Hotchkiss R. D. (tg5 I). Transfer of pmiciiiin resistance in patumococci by the desoxy-
  ribonucleate derived from resistant cultures. Cold Spring Harbor Symp. 16, 457.

- 141 -
Hughes VJ. H. (rg5z). Variation in penicillin resistance in single-cell cultures of Staphylo-
  coccus aureus. J. gen. Microbial. 6, `75.
Jacobson W. E.. Williamson M. U Stock C. C. (1952). The destructive effect of 2.6-
  diaminopurine on kappa of stock 5 I killers, variety 4. of Paramecium aurelia. J. Exp.
  Zoology, tat, 505.
Jawetz E., Gunnison J. B., Bruff J. B. X4 Coleman W. R. (1953). Studies on antibiotic
  synergism and antagonism. J. Bact. 64, zag.
Klein M., f4 Schon. S. E. ( 1952). The role of bacterial resistance in antibiotic synergism
  and antagonism. J. Bact. 65: 454.
Knox R. 8 Collard P. ( 1952). The effect of temperature on the sensitivity of Bacillus
  cereus to penicillin. J. gen. Microbial. 6. 369.
Van Lanen J. M.. Baldw-in I. L. 8 Riker A. J. (1952). Attenuation of crown gall bac-
  teria by cultivation in medium containing glycine. J. Bact. 63, 7'5.
Lea D. E. l4 Co&on C. A. (1949). The distribution of the numbers of mutants in bacte-
  rial populations. J. Genetics, 49, 264.
Lederbcrg J. l4 Tatum E. L. (1946). Gene recombination in Escherichia coli. Nature.
  158. 558
Lederberg J. (1947). Gene recombination and linked segregations in Escherichia coli. Ge-
  netics 32. 5Oj.
Lederberg J. (r950). The selection of genetic recombination with bacterial growth inhi-
  bitors. J. Bact. 5g : a I I.
Ledcrberg J. i I 95 I). Streptomycin resistance: a genetically recessive mutation. J. Batter..
  61. 549.
Lederberg J., Lederberg E. M., Zinder N. D. %5 Lively E. R. ( I 95 I ). Recombination ana-
  lysis of bacterial heredity. Cold Spring Harbor Symp. I 6, 413.
Lederberg J. (1952). Cell genetics and hereditary symbiosis. Physiol. Rev. 32, 403.
Lederberg J. 8 Lederberg E. M. (1952). Replica plating and indirect selection of bacterial
  mutants. J Bact. 63, 399.
Leskowitz S.. Fox Ch. I.. jr. ?4 Raymond S. (1952). The isolation and identification of
  PABA produced by staphylococci resistant to sulfonamide. J. exp. Med., 95, 7.47,
Linz R. (`950). Sur le mecanisme de l'action de la Streptomycine. II: La rfsistance i la
  Streptomycine. Ann. Inst. Pasteur 78, tog.
Luria S. E. `d Delbriick M. ( r943). Mutations of bacteria from virus sensi:ivity to virus
  resistance. Genetics 28, 49 I.
Lwoff A. ( rg53). L'induction. Ann. Inst. Pasteur 84, 325.
Manten A. B Rowley D. ( 1953). Influence of chloramphenicol and specific antiserum on
  the variability of the K-rz strain of Bact. coli. Brit. J. Exper. Path., 34, 23.
Mc Ilroy A. I?.. Axelrod A. E. `?5 Mellon R. R. (r948). The influence of sodium acetate
  upon the dissociation of a strain of hemolytic streptococcus. J. Bact. 56, 547.
Mitchison D. A. ( ,953). The occurrence of independent mutations to different types of
  streptomycin resistance in Bacterium coli. J. gen. Microbial. 8, 168.
Monod J. ( I g I a). Le role des inducteurs spt%iques dans la biosynthese des enzymes. Symp.
  sur la bing&tese des prothines, p. 75. II Congris Int. de Bioch.. Paris.
Newcombe H. B. ( : 948) De!ayed phenotypic expression of spontaneous mutations in Escbe-
  richia coli. Genetics, 33. 447.
Newcombe H. B. (r949). Origin of bacterial variants. Nature, 164. I 50.
Newcombe H. B. 8 Hawirko R. (`949). Spontaneous mutations to streptomycin-resistance
  and dependence in Escherichia coli. J. Bact. 5 7, 565.
Newcombe H. B. ti MC Grcgor J. (195 I). On the nonadaptive nature of change to full
  streptomycin resistance in Fscherichia coli. J. Bact. 62. 539.
Newcombe H. B. 8 Nyholm M. H. (1950). The inheritance of streptomycin-resistance
  and dependence in crosses of Escherichia coli. Genetics 3 5, 603.
Piekarski G. ( 1949). Blepharoplast nnd Trypaflavin-wirkung bei Trypanosoma brucei.
  Zbl. Bact. Abt. I, I 53, rgo.
Plough H. H. l4 Grimm M. R. / t949). Reversal to penicillin sensitivity in a Tysteine
  requiring mutant of Salmonella. Science, 129, r73.
Pollock M. R. ( `953). Penicillinase adaptation and fixation of penicillin sulphur by Bacil-
  lus ceceus spores. J. gen. Microbial. 8. r86.
Potter V. R. (195:). Sequential blocking of metabolic pathyays in vitro. Proc. SOC. Exp.
  Biol. Med.. N. Y., 76, qt.
Pringsheim E. G. l5 Pringsheim 0 (1952). Experimental elimination of chromatophores
  and eyespot in Euglena gracilis. New Phytologist, 5 r, 65.

- 14,2 -
Provasoli L., Hutner S. H. ti Pintner I. J. (1951). Destruction of chloroplasts by strepto-
  mycin. Cold Spring Harbor Symp. 16, I 13.
Rowley D. (1953). Inhibition of E. coli strains by aminoacids. Nature. 17 I. 80.
Ryan F. J. (1952). Adaptation to use lactose in Escherichia coli. J. gen. Microbial. 7. 69.
Smith P. H., Oginsky E. L. 8 Umbreit W. ( I 949). The action of streptomycin. II: The
  metabolic properties of resistant and dependent strains. J. Bact. 58. 761.
Sonncborn T. M. (1950). The cytiplasm in Heredity. Heredity, 4, I I.
Sonneborn T. M. (1950). Cellular transformations. Harvey Lectures, 49. 145.
Szybalski W. 8 Brvson V. (1952). Genetic studies on microbial cross resistance :s toxl:
  agents. I: Cross resistance of Escherichia coli to fifteen antibiotics. J. Batter. c +. 489.
Voureka A. (1952). Induced variations in a penicillin-resistant staphylococcus. J. gen.
  Microbial. 6, 352.
Welsch M. ( I 952). Some aspectoi microbial resistance to streptomycin. Bull. Org Mond.
  Santi 6, `73.
Yang Y. N. 8 White P. B. (1934). Rough variation in V. cholerae and its relation IO
  resistance to cholera phage (type A). J. Path. Bact. 38, 187.
Yudkin J. (1953). Origin of acquired drug resistance in bacteria. Nature 171. 54 1.
Zelle M .R. (19423. Genetic constitution of host and pathogen in mouse typhoid. J. infect.
  Dis.. 71, 131.
Zinder N. D. u Lederberg J. ( 1952). Genetic exchange in Salmonella typhi mtirium. .I.
  Bact., 64, 679.