GENETIC STUDIES ON MICROBIAL CROSS RESISTANCE TO TOXICAGENTS

I. CROSS RESISTANCE OF ESCHERICHIA COLI TO FIFTEEN ANTIBIOTICS' 2

WACLAW SZYBALSKI AND VERNON BRYSON

The Biological Laboratory, Cold Spring Harbor, New York

Received for publication April 2, 1952

It has been observed often that bacteria made resistant to a specific antibioticmay develop simultaneously new properties, including changes in sensitivityto other chemotherapeutic agents. Resistant strains may show lower, equal, orhigher sensitivity than the parent culture to any antibiotic not used in theirisolation. The comparative sensitivity of resistant bacterial strains to fifteenantibiotics forms the subject of this investigation. One primary goal has been toestablish a basis for biological classification of antibiotics on the assumption thatthose of similar chemical composition or mode of action should form naturalgroups related by cross resistance. A second objective has been to distinguish thesignificance of bacterial type as affecting the cross resistance pattern. Representa-tive types chosen include Escherichia coli, Micrococcus pyogenes var. aureus, andMycobacterium ranae. The favorable cultural characteristics and rapid growthrate of E. coli have permitted earlier completion of the analysis with this or-ganism.

MATERIALS AND METHODS

Materials. Two strains of E. coli were used in the study of cross resistance:the radiation-sensitive strain B, and its radiation-resistant variant B/r (Witkin,1947). Bacterial stocks were prepared in aerated nutrient broth (Difco). Thefollowing antibiotics have been employed: streptothricin, catenulin, neomycin,viomycin, vinactin, dihydrostreptomycin, hydroxystreptomycin, aureomycin,chloromycetin, terramycin, netropsin, penicillin, bacitracin, polymyxin B, andcirculin. Stock solutions of all antibiotics with the exception of chloromycetin andbacitracin were prepared at concentrations of 10 mg per ml. The chloromycetinsolution had a concentration of 2 mg per ml due to the low solubility of thissubstance; and bacitracin was made to a concentration of 100 mg per ml becauseof its low inhibitory power against E. coli. Demineralized water was used as asolvent, with adjustment of pH for penicillin (6.5) and terramycin (1.5). TheSeitz-filtered sterile solutions were adequately stable with few exceptions at atemperature of 1 C. Stock solutions of less stable antibiotics were kept in thefreezer compartment (-15 C) until the day of actual use.

1 This study was made possible by a grant from the Committee on Medical Research andTherapy of the American Trudeau Society, Medical Section of the National TuberculosisAssociation.

2 Presented at the Joint Session with Investigators, Committee on Medical Research,American Trudeau Society, Medical Section of the National Tuberculosis Association, NewYork, New York, October 10-11, 1951.

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All plating was done using nutrient agar supplemented with 0.2 per cent yeastextract (Difco) and 2 per cent glycerol. Ten ml portions of the supplementedmelted agar (pH -7) were distributed to 1 oz screw cap prescription bottles(Armstrong-Cork Company, GX-1) using an automatic pipetting machine, auto-claved, and kept in the water bath (50 C). Appropriate amounts of antibioticsolution were added to these bottles, and the contents were mixed and pouredinto petri dishes as required.

Development of resistant strains. Resistant strains were prepared by means ofthe gradient plate technique (Szybalski, 1952). This method allows a considerablereduction in the quantity of antibiotic required for experiment, a significantfactor when the supply is limited. Only one plate may be required to produce a

1:16

Figure 1. Preparation of a gradient plate. Details of method described in text.

2 to 5 fold increase in the resistance of a strain. Thus, in favorable cases only 3plates have been needed for increasing bacterial resistance over 100 times.The method employs a pressed-bottom petri plate (100 mm) with two layers

of agar (figure 1). The lower layer consists of 10 ml of plain nutrient agar and isallowed to harden with the plate slanted just sufficiently to cover the entirebottom. After placing the dish in the normal horizontal position, another 10ml of agar is added containing an appropriate concentration of the antibiotic(about 3 to 10 times higher than the inhibitory concentration for a given micro-organism). During subsequent incubation, the downward diffusion of drugresults in its dilution proportional to the thickness ratio of agar layers, establish-ing a uniform concentration gradient.When a heavy suspension of E. coli is streaked or spread over the surface of

the agar, only the resistant cells are able to develop colonies beyond the boundaryof confluent growth (figure 2). As seen in the figure, these colonies are streakedout until additional growth is obtained in the regions of highest antibiotic con-centration. Then the resistant colonies are resuspended in broth and streaked onanother gradient plate with a 2 to 5 times higher antibiotic concentration in theupper layer. The entire procedure is repeated until strains are obtained of the

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desired degree of resistance, limited only by the supply of antibiotic, its solu-bility, and the adaptive or genetic capacities of the organism.

Determination of comparative resistance. The gradient plate technique was alsoused in the determination of comparative resistance. If a bacterial suspeinsion isstreaked over the agar surface, parallel to the axis of the concentration gradient,the length of the growing streak is a direct measure of the inhibitory concentra-tion. This is because the concentration of antibiotic at any particular place onthe surface of a vertically equilibrated plate is roughly proportional to the ratio

Figure 2. Growth of Escherichia coli, strain B/r, on a gradient plate. Resistant coloniesare seen beyond the boundary delimiting the inhibition concentration for the majority ofcells. Two resistant colonies have been streaked out, resulting in the production of second-step resistant colonies at the termination of the streaks. The plate was prepared by adding20 ,ug/ml penicillin in the upper agar layer, as in figure 1, and spreading the bacterial sus-pension over the surface.

of thicknesses of the upper and lower agar layers at this position. In this respect,the assay does not depend on the diffusion coefficient of an antibiotic. All otherdiffusion assays (cylinder or paper disc methods, "guitter plate" assay, etc.)depend on the diffusioni coefficient of the drug tested and require a special cali-bration curve for every new drug.To make the determination more accurate, two plates were poured with anti-

biotic incorporated in both layers. The first plate was prepared with the antibioticteii times more concentrated in the upper layer than in the lower oile. In thesecond plate the layers were reversed. By interpolating betweein the length of thestreaks growiing on both dishes it was possible to eliminate any error due to slowdoNvttward diffusion of somr antibiotics and consequent poor vertical equilibra-

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492WACLAW SZYBALSKI AND VERNON BRYSON

tion. If the length of correspondinig streaks on both plates is identical, this servesto confirm the theoretical distribution of the concentratioin gradient, and indi-cates that the use of one plate only is adequate in this case.For a comparisoin of senisitivities, up to ten parallel streaks of standardized

E. coli suspensions (about 106 and 109 cells per ml) have been placed across thecentral part of a preincubated gradient plate, usinig small painting brushes pre-viously autoclaved. Square petri dishes3 are more suitable buit are not at presentavailable.The length of the growinig streaks has been measured as indicated in figure 3.

The isolated resistant colonies, bevond the boundary of confluent growth, have

9cm

AXIS OF CONCENTRATION GRADIENT

0 2 3 4 5 6 7 8 9 10cm

Figure 3. Measurement of bacterial sensitivity. Antibiotic is layered in two concentra-tions, x and lOx ,g/ml. In the example, the inhibitory level is 6 times the lower coincentration(6 ,ug/ml) .

not been included wheni measuring the length of the streaks. It was often observedthat a strain resistant to one antibiotic shows the same sensitivity to anotherdrug as does the parent strain, but differs from the parent strain in respect tothe proportioni of resistanit cells able to form coloniies beyond the boundary ofcomplete inhibitioni. This phenomenon may easily lead to an incorrect determina-tion of comparative seiisitivity by the method of stepwise dilution assay, espe-cially in liquid media.

This inivestigation deals onily with cross resistances as measured by growvth ofthe majority of cells in a bacterial population. Incidenital changes in the smallproportioii of more highly resistanit cells within those large populations will notbe considered.

3Hellige, Inc., Long Island City, New York.

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EXPERIMENTAL RESULTS

Using the gradient plate technique, cultures of E. coli, strains B and B/r, havebeen made resistant to the following antibiotics: streptothricin, catenulin, neo-mycin, viomycin, dihydrostreptomycin, hydroxystreptomycin, aureomycin, chlo-romycetin, terramycin, netropsin, penicillin, bacitracin, polymyxin B, and cir-culin. In addition, a strain of B/r resistant to vinactin was prepared, but due to avery small supply of this antibiotic and its low inhibitory power against E. coli,it was not possible to develop a high degree of resistance.

Subsequently, the sensitivity of each resistant strain was tested against theentire series of antibiotics. The numbers in tables 1 and 2 represent the "factorof resistance"; i.e., the sensitivity ratio of the resistant and parent strains. Thenumber 1.1 represents a slightly increased resistance. Increased sensitivity isshown by a fraction. The bottom row indicates the concentration of an antibioticlisted at the top of the table, required to inhibit the parental strain B (table 1)or B/r (table 2). For example, it is seen from table 1 that strain B of E. coli re-sistant to chloromycetin (130 times) is 80 times more resistant to penicillin and50 times more sensitive to circulin than is the parent strain. Resistance to radia-tion (r) or to a specific antibiotic (abbreviation in the first column of tables 1 and2) is conventionally represented by a bar. For example, B/r/AU is a strainresistant to radiation and aureomycin.

Certain facts may be observed by exanminig both tables of cross resistances.The cross resistance patterns are similar for strains B and B/r. Minor differenceswill be discussed. Furthermore, the antibiotics tested can be divided into fourmain groups showing marked similarities.The first group consists of viomycin, vinactin, neomycin, catenulin, and

streptothricin. Within this group a high degree of reciprocal cross resistance isnoted. Streptomycin is related to this group because the strains resistant to thepreceding antibiotics also show a marked increase in resistance to streptomycin.In the reciprocal test it is seen that the strains resistant to streptomycin, i.e.,B/SM and B/r/SM, are only 2 to 5 times more resistant to streptothricin, caten-ulin, and neomycin and show almost no cross resistance to viomycin and vinac-tin. We have observed complete cross resistance, cross dependence, and similarpatterns of resistance development for hydroxystreptomycin, dihydrostrepto-mycin, and streptomycin, and, therefore, we refer in the tables and in the dis-cussion to streptomycin only. Neomycin and catenulin are almost indistinguish-able in this test and are apparently more closely related to streptothricin thanto other antibiotics of this group. Similarly viomycin and vinactin appear to beidentical substances from the microbiological point of view (Mayer and Eisman,1952).Polymyxin B and circulin, two cyclic bacterial polypeptides, show almost

complete reciprocal cross resistance. Strains that have developed considerableresistance to these antibiotics (beyond the first step of about 2-fold resistance)grow very slowly and are readily outgrown by less resistant mutants, giving aselective reversion to sensitivity. Strains resistant to polymyxin B and circullin

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WACLAW 5ZYBALSKI AND VERNON BRYSON

TABLE 1Cross resistance relatiomhip of certain antibiotics as tested with strain B of Escherichia coli

BACTERIA

POLYPEPTIDES

STREPTOMYCES

z~~~~~~~~z z z z

X z o > 0 Zo~~z 0 Z F oIr.I t-

2~~~~~~~~~ICDIL. to z z~~~~~~~~~'

1/20 1/20 /1.5 1/1.5 1.1 I

,.~~ ~~ 4 4 3 to

1l 1/10'S2 1/2 1/2 1 2

01/. 1/2 V2 I 1.1 1.1 1.1. 1.12 1/2 1/2 I 1.1 1.1 1 I.5

2 1/40 I/SO 1.1 1.1 1.1 I 21.1 1.1

2

2l

1.5 1.5 1.5 1i51/2 1 Is

I I I 1/.5I 1/1.1 I IA)

I I I I

I I I 1/1.II I 1/1.5 V/II I .1 I1

1/2 1/2 1/1.5 VII0 101

30 2I

d22i50,,

(MCVg/mI) 700 20 60 50 4 2 6 2 8 I I I S

Figures represent the factor of resistance (fold increase) compared with the parentstrain set as unity (first row). Figure 1.1 designates slightly increased resistance; otherfigures are rough estimates. Increased sensitivity (collateral sensitivity) is shown by a

fraction. The bottom row indicates the concentration of antibiotic, heading each verticalcolumn, required to inhibit the parental strain. Hatched or cross hatched areas emphasizerespectively a lower or higher degree of cross resistance within groups of similar antibiotics.Nonhatched frames indicate nonreciprocal cross resistance. Heavily outlined boxes on

the diagonal show the resistance factor of each experimental strain to the antibiotic usedin its selection. Resistant derivatives of strain B are listed in the first vertical column, inthe same sequence as the antibiotics heading the table. Resistance to radiation (r) or to a

specific antibiotic (abbreviation in the first column) is conventionally represented by a bar.

exhibit a 2- to 6-fold increase in resistance to streptothricin, neomycin, and caten-ulin, but the reverse relationship does not hold true.

Bacitracin, the third bacterial polypeptide, does not show significant cross

resistance with other antibiotics tested. It is not difficult to isolate stable strainsresistant to this drug, but the high natural resistance of E. coli to bacitracinmakes the isolation of strains with more than a 20-fold increase in resistanceimpractical.

FUNGI

ANTIBIOTIC

STRAIN

B

B/v'BC'PB/CR/vua/ST

I/NN/CT

1st

/NT/aU

/PNSENSITIVITY

Of

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1952] MICROBIAL CROSS RESISTANCE TO TOXIC AGENTS 495

Strains resistant to aureomycin, chloromycetin, and terramycin show a highdegree of reciprocal cross resistance. They are also more resistant to netropsinand penicillin. Strains B and B/r resistant to penicillin and netropsin behavemore irregularly; only B/NT and B/r/PN show highly increased resistance toaureomycin, chloromycetin, and terramycin. B/PN and B/r/NT are onlyslightly more resistant. The radiation-resistant strain B/r shows a small but

TABLE 2Cross resistance relationship of certain antibiotics as tested with strain B/r of Escherichia coli

BACTERIALY

POLYPE PTIDES

SIREPTOMYCES FUNGI

1 ~~~~~~~~~~~~~~~~zz

I z~~~~~~~~~~~~~~~x>ANTIBIOTIC Z z = z Z ° Xx z 2 Z -j 0 r 2a

STRAIN- I ~~~~~~~~~~~~ ~ ~~ a.~$ z a. 0 0 4 r

-_ _ _

W W<

w4 0 - - w

zn w

I} I/2 I/201/20 1/10

1.5 )

2 2I/5 I/S

I I/S 1/10

1 1/10 1/10I I II 1.5 I.5

1/1.5 I/1oo I/501/1.5 /lOO 1/40

1.5 l/lOO 1/401.1 1/1001/300

1/21.1

1.5

1/21.1

1.1

Lv'15

Li II I

I 1.1

I 1/1.5

' 1 15IS~~~~~~'k

-5

K106400

1.1

I:SJ .1

15

5/,1003030

I I

I 1.1I 1.1

1.1 1.5 I.$ 1.50.S 3 4 3

1.53

I I

II I

1.5 21.1 1/1.1I

2 1/1.1

1.1 1/1.11.1 1/1.1

I 1/1.1UQfl 2

1/1.1

3I/1.l

1/2

1/1.51/2

1/1.1

I.S

1/1.1I/.1/1.1

1.1

1/1.1

I/1/21/2

1/1.1

I/LI

I.$

1.5

so IT.i6F 01.soii.Io200 \2\ l

?-O I5 'IS~ '08

700 20 60 50 .200 4 2 6 2 a 1.5 1.5 1.5

For explanation see table 1.

definite increase in resistance (1.5 times) to aureomycin, chloromycetin, terra-mycin, and penicillin. Witkin (1947) has cited an increased resistance of the B/rstrain to penicillin.

In addition to the observation of cross resistance, the reverse phenomenon of"collateral sensitivity" was noted. In this case, a strain resistant to one antibioticexhibits a considerably higher sensitivity than the parent strain, to another. Theeffect is very pronounced when certain resistant strains are tested against poly-myxin B and circulin. Many strains resistant to other unrelated antibiotics are

more sensitive to these two agents. Both the B and B/r strains resistant tochloromycetin show a high degree of collateral sensitivity to polymyxin B and

I I I

I I 1/1.5

4 6 6 8

2 2 2 3

S/ r

8/r/BC/As/CRAm/VI/ST

/SM

AU/TM

/CmAN

SENSITIVITYOF

B/r(mcg/ml)

.1

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WACLAW SZYBALSKI AND VERNON BRYSON

circulin. But there are rather considerable differences in the degree of collateralsensitivity between strains B and B/r resistant to streptothricin, catenulin,neomycin, aureomycin, terramycin, penicillin, and bacitracin. With few excep-tions, strains derived from B/r show a higher degree of collateral sensitivity.The preliminary experiments made with strain B indicate that small initial

increases in resistance to chloromycetin cause a sudden increase in sensitivityto polymyxin B. Further increase in resistance to chloromycetin induces onlyslightly higher sensitivity. The strains, within B/CM populations, selected forhigher polymyxin B resistance are found to be again more chloromycetin sensi-tive than the parent strain B/CM. Molho and Molho-Lacroix (1951) have re-cently observed that a "chloromycetin adapted" strain of E. coli becomes moresensitive to DL-serine. However, their experimental procedures exclude the possi-bility of quantitative comparison with our results.Another phenomenon, which may be described as the indirect development

of resistance, is evident from table 1. A strain with a 50-fold increase in resistanceto streptothricin shows a 20-fold increase in viomycin resistance although it haspreviously never been in contact with viomycin. By direct isolation from mediacontaining viomycin it was difficult to obtain this degree of resistance and stillhave a viable and vigorously growing strain. A similar relationship pertainsbetween chloromycetin and netropsin. Also evident in table 2 is another type ofindirectly developed resistance: a strain 15 times more resistant to viomycinshows an even higher degree of resistance to streptothricin (20 times) with whichit has previously never been in contact.

DISCUSSION

Systematic studies on cross resistance are both of theoretical and practicalsignificance. They provide information relevant to the problem of genetic andbiochemical mechanisms in the development of resistance. If a strain made re-sistant to one antibiotic shows highly increased resistance to another, it indicatesthat biological and possibly chemical similarity may exist between the two anti-biotics. This cross resistance phenomenon has often been reported for chemicallysimilar antibiotics and other chemotherapeutic agents. The extensive literaturewill be reviewed elsewhere.

Certain relationships previously undescribed in the available papers areevident in referring to tables 1 and 2. The resemblance between strains resistantto aureomycin, chloromycetin, and terramycin, and netropsin and penicillinresistant strains has apparently not been observed. Our preliminary experimentsindicate that similar relationships may not pertain to micrococci and mycobac-teria. Also, the high degree of reciprocal cross resistance developed in strainsexposed to streptomycin, neomycin, catenulin, and to the actinomyces anti-biotics has been described only for some members of this group.Another observation evident from the data is the close microbiological simi-

larity of circulin and polymyxin B as demonstrated by a considerable degree ofreciprocal cross resistance between these two drugs. Both act similarly on strainsresistant to other antibiotics, particularly in respect to the phenomenon of

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"collateral sensitivity". These biological likenesses are in accord with analogiesin chemical structure as described by Koffler (1950).A one-way cross resistance exists also between these two bacterial polypeptides

and some -actinomyces antibiotics (streptothricin, catenulin, and neomycin).Thus, strains resistant to circulin and polymyxin B show 2 to 8 times higherresistance to these three related actinomyces antibiotics and also to streptomycin.The reverse relationship, however, does not hold true. Bacitracin, a third bac-terial polypeptide, seems to be completely unrelated to other polypeptides andnonpolypeptide antibiotics, constituting at present the sole member of an inde-pendent group.

Several interpretations of the cause of cross resistance may be advanced, withthe reservation that an explanation valid for one example may not apply toanother. The simplest and most probable cause of cross resistance would dependon identity or close chemical similarity of toxic agents, resulting in parallelbiological effects. Reciprocal cross resistance would indicate similar configurationof the biologically active moiety in the drug molecule. A second interpretationwould be that chemically dissimilar agents may interfere with the same meta-bolic pathway. For example, one agent may block an enzyme system, whereasanother may form a complex with intermediary products in the same chain ofbiochemical events. Obviously, bacterial mutants or adapted cells resistantthrough use of an alternative pathway would be immune to the effects of toxicagents related by intracellular site of action, rather than by common structure.A similar interpretation is that bacteria may become cross resistant to certainsubstances by nonspecific biological change. For example, selection by the toxicagent for decreased permeability would afford some degree of simultaneous pro-tection (cross resistance) against antibacterial agents dependent upon freediffusion into the cell. Another explanation is that the development of resistancein a specific case would be governed by mutation at one or a few genetic loci andthat these mutations always result in several phenotypic changes related inorigin but diverse in effect (pleiotropy). One physiological change would make thecell resistant to a specific antibiotic; another correlated but different changewould provide protection from an unrelated agent.

Closely related is the problem of simple biological identification of antibiotics.The use of resistant strains for this purpose was advocated by Stansly (1946;

1948), Eisman et al. (1946), and others. Then it was believed generally that re-sistant strains are very specific and that one resistant strain would be adequatefor exclusive identification of the corresponding antibiotic. Although cross re-sistance makes the situation more complex, knowledge of the exact patterns ofcross resistance for a few bacterial species is a very useful and simple tool forthe preliminary identification of any new antibiotic developed in the course of ascreening program. Tables 1 and 2 give examples of two almost indistinguishablepairs of antibiotics: catenulin-neomycin and viomycin-vinactin, which may even-tually prove to be identical.Another problem of interest, from both a theoretical and practical point of

view, is "collateral sensitivity" (formerly "induced sensitivity", Szybalski and

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Bryson, 1952) in which a strain made resistant to one antibiotic becomes con-siderably more sensitive to another. This phenomenon may be regarded as theresult of a selective process. Reference to tables 1 and 2 shows that in the isolationof strains possessing resistance to one antibacterial substance (e.g., chloromyce-tin), we may at the same time select associated characters producing highersensitivity to another agent (e.g., polymyxin B). If these characters are asso-ciated in an obligatory manner, it will enable the facile selection of back muta-tions from resistance to sensitivity, at present a formidable task. Preliminaryexperiments show that in the case of polymyxin B and chloromycetin it is im-possible to have a strain of E. coli simultaneously more resistant to both than isthe parent strain. Whenever one antibiotic can be found that is particularlyeffective against bacteria resistant to another, it might be proved useful incombating disease and in permitting the application of antibiotics in a rationalsequence when more than one is to be employed. Thus, the exact study of bothcollateral sensitivity and cross resistance may help in designing a proper programof multiple chemotherapy.

ACKNOWLEDGMENTS

We are gratefully indebted to Dr. H. B. Woodruff of Merck & Company fordihydrostreptomycin sulfate (lot no. 1295) and streptothricin hydrochloride(4R3135); to Dr. H. A. Nelson of The Upjohn Company for circulin (res. lot177-HAN-14) and neomycin sulfate (res. no. 9291-3); to Dr. R. J. Hickey ofCommercial Solvents Corporation for bacitracin (lot 276-A); to Dr. N. E. Riglerand M. A. Darken of Heyden Chemical Corporation for penicillin (crystallinepotassium penicillin G-lot no. FF-377); to Dr. P. C. Eisman of Ciba Pharma-ceutical Products for vinactin sulfate; to Dr. J. C. Sylvester of Abbott Labora-tories for hydroxystreptomycin (NA 232-Mi); to Dr. R. G. Benedict and Dr. F.H. Stolola of Northern Regional Research Laboratory for hydroxystreptomycin(NRRL 156-A); to Dr. F. W. Tanner, Jr. and Dr. G. L. Hobby of Chas. Pfizer &Company for viomycin sulfate (lot no. N509142), catenulin (lot 588-206), netrop-sin hydrochloride (lot 208), polymyxin B sulfate (4KF514519), and terramy-cin hydrochloride (WBW 507019); to Dr. J. Ehrlich of Parke, Davis & Com-pany for chloromycetin (Rx 151770) and to Lederle Laboratory Division foraureomycin hydrochloride (lot No. 4443-20018).We also wish to acknowledge the continued interest of Dr. M. Demerec during

the progress of these investigations.

SUMMARY

Strains of Escherichia coli have been made resistant to aureomycin, bacitracin,catenulin, chloromycetin, circulin, dihydrostreptomycin, hydroxystreptomycin,neomycin, netropsin, penicillin, polymyxin B, streptothricin, terramycin, vinac-tin, and viomycin. The factor of resistance (fold increase) was determined.The degree of cross resistance was established then by testing the resistant

strains against each antibiotic. Resistant bacteria were found to show equal,lower, or higher sensitivity to other antibiotics than did the parent culture.

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Specific examples of the indirect development of bacterial resistance to anti-biotics are cited, wherein strains exposed to one agent become highly resistant toboth this agent and others with which the bacteria have never been in contact.

Exploration of the resulting cross resistance patterns shows that the antibioticstested fall into four, internally related, major groups: Group 1; streptothricin,viomycin, vinactin (actinomyces polypeptides), catenulin, and neomycin. Bac-terial strains resistant to the foregoing antibiotics are also resistant to the strepto-mycins, but the inverse relationship does not hold true. Complete cross resistanceand cross dependence exist between hydroxystreptomycin and other strepto-mycins. Group 2; aureomycin, chloromycetin, and terramycin; penicillin andnetropsin are less closely related. Group 3; polymyxin B and circulin (cyclicbacterial polypeptides). Bacteria resistant to these two antibiotics show also aone-way cross resistance to streptothricin, catenulin, neomycin, and strepto-mycin. Group 4; bacitracin (bacterial polypeptide).

Increasing the sensitivity to one antibiotic by increasing resistance to anotheris termed collateral sensitivity. For example, strain B/r of E. coli in becoming300 times more resistant to chloromycetin simultaneously develops a hundred-fold increase in sensitivity to polymyxin B.

Isolation of resistant strains and tests for cross resistance were performed bythe gradient plate technique, a method permitting rapid and accurate develop-ment and quantitative comparison of resistance.

Theoretical advantages of cross resistance analysis as compared with thedetermination of bacterial inhibition spectra are discussed with reference to theclassification of new antibiotics and the planning of chemotherapeutic programs.

REFERENCESEISMAN, P. C., MARCH, W. S., AND MAYER, R. L. 1946 Differentiation of antibiotics by

resistant strains. Science, 103, 673-674.KOFFLER, H. 1950 Antibiological polypeptides as illustrated by circulin. Proc. Indiana

Acad. Sci., 60, 58-63.MAYER, R. L., AND EISMAN, P. C. 1952 Unpublished communication.MOLHO, D., ANa MOLHO-LACROIX, L. 1951 Etude comparee de l'accoutumance de

Escherichia coli A la chloromyc4tine et A la dl-s6rine. Compt. rend., 233, 1395-1397.STANSLY, P. G. 1946 The presumptive identification of antibiotics. Science, 103, 402-

403.STANSLY, P. G. 1948 Identification of antibiotics by means of resistant strains of bacteria.

J. Bact., 55, 721-726.SZYBALsKI, W. 1952 Gradient plates for the study of microbial resistance to antibiotics.

Bact. Proc., 1952, 36.SZYBALSKI, W., AND BRYSON, V. 1952 Cross resistance of Escherichia coli, Micrococcus

pyogenes var. aureus and Mycobacterium ranae to seventeen antibiotics. Bact. Proc.,1952, 40.

WITKIN, E. M. 1947 Genetics of resistance to radiation in Escherichia coli. Genetics,32, 221-248.

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