JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 740-749Copyright ©3 1975 American Society for Microbiology

Vol. 124, No. 2Printed in U.S.A.

Inheritance of Low-Level Resistance to Penicillin,Tetracycline, and Chloramphenicol in Neisseria gonorrhoeae

P. FREDERICK SPARLING,* FELIX A. SARUBBI, JR., AND ELEANOR BLACKMANDepartments of Medicine and Bacteriology and Immunology, University ofNorth Carolina School of

Medicine, Chapel Hill, North Carolina 27514

Received for publication 5 May 1975

The genetics of low-level resistance to penicillin and other antibiotics in aclinical isolate and a multistep laboratory mutant of Neisseria gonorrhoeae wasstudied by transformation. Mutations at three loci affected sensitivity to penicil-lin. Mutation atpenA resulted in an eightfold increase in resistance to penicillinwithout affecting response to other antimicrobial agents. Mutation at ery re-sulted in a two- to fourfold increase in resistance to penicillin and similarincreases in resistance to many other antibiotics, dyes, and detergents. Muta-tion at penB resulted in a fourfold increase in resistance to penicillin andtetracycline, the phenotypic expression of which was dependent on the presenceof mutation at ery. The cumulative effect of mutations at penA, ery, and penBwas an approximate 128-fold increase in penicillin resistance, to a minimuminhibitory concentration of 1.0 ,ug/ml. Low-level resistance to tetracycline orchloramphenicol was due to similar additive effects between mutations at thenonspecific ery and penB loci and a locus specific for resistance to each drug (tetand chl, respectively). No evidence was found for penicillinases or other drug-inactivating enzymes.

We previously presented evidence that low-level resistance ofNeisseria gonorrhoeae to pen-icillin (Pen), tetracycline (Tet), chlorampheni-col (Chl), and erythromycin (Ery) was deter-mined by separate loci, designated penA, tet,chl, and ery, respectively (29). Introduction ofthese loci by transforming deoxyribonucleicacid (DNA) individually into a wild-type (sensi-tive) recipient resulted in phenotypic levels ofresistance considerably less than exhibited bythe multiply antibiotic-resistant donor strains,however (29). In contrast, introduction of locifor high-level resistance to rifampin (Rif), strep-tomycin (Str), or spectinomycin resulted inphenotypic resistance in the transformantsequal to the donor's (29). These observationssuggested that the genetic control of low-levelresistance to Pen and other drugs was complex,either due to additive effects between theknown loci or existence of other undescribedloci.

In this communication, we have utilized ge-netic transformation to show that low-level re-sistance to each of Pen, Tet, and Chl is acquiredin small increments as the result of additiveeffects between mutations at several previouslydescribed loci and a new locus, designatedpenB. Both ery andpenB are shown to result inlow-level cross-resistance between unrelateddrugs. Evidence is also presented that pheno-

74C

typic expression of mutation at penB dependson the presence of mutation at ery.

MATERIALS AND METHODSBacterial strains. Bacterial strains are listed in

Table 1. Several have been described previously (16,17, 29). Strain FA19 is strain Ceylon 3 from thecollection of A. Reyn. Strain FA48 is a two-stepmutant from FA19, initially selected for high-levelStr resistance, and subsequently for Pen resistance,as described (17). Strain FA5 was a clinical isolatefrom a patient in Los Angeles, obtained as strainP955 from the Center for Disease Control collection,Atlanta. Phenotypic levels of resistance (minimuminhibitory concentration [MIC]) of various strainsare indicated in the text by parentheses enclosing thesymbol for the appropriate drug and the MIC inmicrograms per milliliter for that drug. Thus, FA48(Pen 2.0) indicates that strain FA48 has an MIC forPen of 2.0 Ag/ml.

Transformation procedure. Transformation pro-cedure, preparation of transforming DNA, and othermethods were essentially as described previously(29, 31). Recipient cells of colony type 1 were grownfor 20 to 22 h at 36 C in 5% CO2 on GC base agar(Difco) medium containing 1% defined supplements(29) (GCBA-DS). They were suspended and vigor-ously agitated in GC base broth (29) to give a finaldensity of about 5 x 107 colony-forming units/ml andwere then exposed in a final volume of 1.0 ml tolimiting concentrations (0.01 ,ug/ml) of transformingDNA or to control DNA pretreated with 50 Mg ofpancreatic deoxyribonuclease (Worthington) per ml.

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VOL. 124, 1975

After incubation for 30 min at 37 C, the reaction wasterminated by addition of 50 jAg of deoxyribonu-clease per ml. Five minutes later, volumes of 0.1 mlof the undiluted and 10-1 and 10-2 dilutions of thereaction mixture (undiluted only for controls) weresuspended in 4 ml of soft GCBA-DS at 48 C and wereimmediately layered onto 20 ml of GCBA-DS plates.After incubation for 6 to 10 h at 36 C (5% C02), anadditional 4-ml overlay of GCBA-DS soft agar con-taining sufficient drug to give the desired final con-centration was applied. Ordinarily, several concen-trations of drug were employed to achieve the lowestconcentration that effectively inhibited the recipi-ent. Actual concentrations used are indicated asappropriate in the text. Colonies appearing after 3 to5 days of incubation at 36 C in 5% C02 on selectiveplates were considered transformants if they weredistant from occasional crescentic areas of uninhi-bited growth of the recipient, or if they were ob-viously larger than occasional diffuse backgroundgrowth ofthe partially inhibited recipient. Transfor-mation frequencies (percentage of exposed cellstransformed) were corrected for numbers of colonieson deoxyribonuclease-pretreated DNA controlplates (usually less than five per plate; spontaneousmutation frequency, <10-6). Oxidase reaction and oc-casionally carbohydrate utilization were used tocheck identity of transformants. All experimentswere performed at least twice, and duplicate plateswere used throughout. Transformants to be used asrecipient in another transformation were purified tocolony type 1 and were retested for antibiotic pheno-type prior to use.

Sensitivity testing. Transformants werestreaked onto GCBA-DS plates containing just suffi-cient antibiotic to inhibit growth of the recipientstrain. After incubation for 20 to 24 h at 36 C in 5%C02, the once-purified transformants were emulsi-fied in 0.2 ml of minimal medium Davis (Difco) andinoculated by means ofa multi-pronged replica-inoc-ulating device onto chocolate agar (Difco) plates con-taining 1% supplement C (Difco) and twofold dilu-tions of antibiotic. The plates were not more than 48h old and were stored at 4 C until use. Final inocu-lum size on the plates was 102 to 103 colony-formingunits, which resulted in confluent growth on controlplates without drug. MIC was defined as the leastconcentration almost (less than five colonies) or to-tally inhibiting growth after 44 h of incubation at 36C in 5% C02. With attention to detail (recentlyprepared media, cells less than 24 h old, inoculumdensity), results were quite reproducible, nevervarying more than twofold on separate days. Forinstance, the resistant parent strains used for mostof these studies, FA48 and FA5, usually, but notinvariably, grew on chocolate agar containing 1.0 jAgof penicillin G per ml. The MIC for Pen for thesestrains is therefore near to, but slightly greaterthan, 1.0 jAg/ml. Although the MIC (Pen) of FA48was formerly indicated as 1.0 jig/ml (29), the MIC(Pen) of both FA48 and FA5 is shown as 2.0 ,ig/ml inTable 1 and Fig. 1, in keeping with the usual resultsof testing with twofold increments of drugs at thetime these experiments were performed. Control

PENICILLIN LOCI IN GONOCOCCUS 741

TABLE 1. Strains ofN. gonorrhoeae used

Strain Genotype Origin

FA19 Wild type 17, 29FA48 penA2 ery-2 str-7 17, 29

tet-2 chl-2 penB2FA50 spc-3 29FA102 penA2 Recombinant, FA48

x FA19FA136 penA2 ery-2 Recombinant, FA48

x FA102FA140 penA2 ery-2 penB2 Recombinant, FA48

x FA136FA147 penA2 ery-2 str-7 Recombinant, FA48

x FA136FA162 tet-2 Recombinant, FA48

x FA19FA163 chl-2 Recombinant, FA48

x FA19FA171 ery-2 Recombinant, FA48

x FA19FA192 penA2 ery-2 spc- Recombinant, FA50

x FA136FA198 penA2 ery-2 penB2 Recombinant, FA50

spc-3 x FA140FA212 ery-2 penB2 Recombinant, FA140

x FA171FA5 penAl ery-1 str-1 17, 29

tet-l chl-1 penBIFA143 penAl Recombinant, FA5 x

FA19FA164 penAl ery-1 Recombinant, FA5 x

FA143FA180 penAl ery-1 penBI Recombinant, FA5 x

FA143

strains of known sensitivity were included in eachset of plates.

In consideration of the potential twofold variabil-ity inherent in results determined by replica-inoc-ulation, as routinely performed, we have presentedthe MIC for large numbers of presumably genotypi-cally identical transformants both as the range ob-served (Table 2) and the mean (Table 2, Fig. 1). Themean is assumed to be the closest approximation ofthe true MIC of the class of transformant beingtested.

Single-cell sensitivities were determined for se-lected transformants by spreading 100 to 200 colony-forming units onto chocolate agar containing two-fold dilutions of antibiotic. The single-cell MIC wasdefined as the lowest concentration of drug whichreduced viable count by at least 30% after incuba-tion for 44 h at 36 C in 5% C02.

Assays for antibiotic-inactivating enzymes. In-activation of Pen, Ery, Chl, and Tet was tested withwhole cells and crude extracts of resistant and sensi-tive strains by bioassay, using Sarcina lutea as thetest organism (3). Crude extracts were preparedeither by sonication or passage through a Frenchpressure cell (29), or by lysis in 30 mMtris(hydroxymethyl)aminomethane - hydrochloride,

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742 SPARLING, SARUBBI, AND BLACKMAN

pH 7.1, containing 1 mM ethylenediaminetetraace-tate, with stirring at 22 C for 10 min. All crudeextracts were kept at 0 C and were tested on the dayof preparation. Protein concentration was 5 mg/mlin each reaction mixture. Incubations were for 60min at 37 C. Other conditions of the bioassay were as

described (29). The presence of ,3-lactamases was

also assayed by the microiodometric method of Nov-ick (21), using penicillin G sodium (0.1 mM) or ampi-cillin (0.1 mM) as substrate. Under the conditions em-ployed, the assay detected 0.1 U of penicillinase(Calbiochem) per ml. Final protein concentrationswere determined by the Lowry method (12).

Antibiotics. Antibiotics were from sources previ-ously described (17, 29), with the following addi-tions: fusidic acid was from Leo Pharmaceuticals;deoxycholate was from BDH Chemicals; Triton X-100 was from Sigma; and crystal violet was fromEastman. Other chemicals were of the highest avail-able purity.

RESULTSFirst-step PenR: penA transformations. As

indicated previously (29), transformationcrosses between the relatively highly Pen-resist-ant donor strain FA48 (Pen 2.0) and Pen-sensi-tive recipient strain FA19 (Pen 0.007) resultedin first-step PenR transformants with an MICfor Pen of 0.06 ,ug/ml. The phenotypic level ofresistance of the transformants was thereforeapproximately eightfold greater than the recipi-ent, but less than 5% of the donor. The fre-quency of PenR transformants declined as theconcentration of Pen used in selection was in-creased, but the phenotypic level of PenR of thetransformants was unaffected (Table 2). Nofirst-step PenR transformants were obtainedwhen initial selection was with more than 0.06,ug of Pen per ml, even with saturating concen-

trations of DNA (10.0 ,ug/ml). Since FA48 andFA19 are isogenic, host differences in expres-

J. BACTERIOL.

sion of mutant genes seemed unlikely. Wetherefore considered it probable that more thanone gene was responsible for the (Pen 2.0) phe-notype of FA48. The locus for first-step PenR(Pen 0.06) was designated penA; it did not co-

transform (<1%) with any of several other stud-ied markers in FA48, including str-7, tet-2, chl-2, and ery-2 (29).

Second-step PenR: ery transformations. Ifincomplete transfer of parental PenR to a sensi-tive recipient is due to presence of other locifor low-level PenR in the donor, unlinked topenA, it should be possible to introduce them inturn into the first-step (penA) transformant un-

til the phenotype of the donor is reproduced.Accordingly, a typical first-step penA2 trans-

formant (FA102) (Pen 0.06) was used as recipi-ent for transforming DNA from FA48 (Pen 2.0),employing 0.06 to 0.12 ,ug of Pen per ml forselection. Results were inconsistent, with sev-

eral failures (transformation frequency<0.00001) even with saturating (10.0 jug/ml)concentrations of DNA. However, transform-ants were obtained by this method in one experi-ment (Table 2). These second-step transform-ants were two- to fourfold more PenR (Pen 0.12to 0.25), and all (19 of 19) were also 8- to 16-foldmore EryR (Ery 2.0 to 4.0) and twofold more

ChlR (Chl 1.0). When this experiment was re-

peated (FA48 x FA102), but with selection forEryR rather than PenR, reproducible transfor-mation frequencies of -0.01 were obtained. All(45 of 45) tested EryR transformants were alsoincreased by two- to fourfold in PenR and two-fold in ChlR and were thus identical to thesecond-step transformants selected with Pen.Since introduction ofery-2 from FA48 into FA19was noted previously to increase resistance toPen and Chl by twofold (29), it seemed likely

TABLE 2. Multistep transfer ofPen resistance from strain FA48

Trans-Selected formation No. Pen resistance Genotype of

phenotype frequency scored of transformants transformants(%)b

FA48 (Pen 2.0) x FA19 (Pen 0.007) PenR 0.015c 0.02 20 0.06d penA20.03 0.01 110 0.06 penA20.06 0.003 20 0.06 penA20.12 <0.00001

FA48 (Pen 2.0) x FA102 (Pen 0.06) PenR 0.12 0.0006 19 0.18 (0.12-0.25) penA2 ery-2EryR 0.5 0.01 45 0.18 (0.12-0.25) penA2 ery-2

FA48 (Pen 2.0) x FA136 (Pen 0.25) PenR 0.3 0.006 110 0.83 (0.5-1.0) penA2 ery-2penB2

aThe donor is listed first and the recipient is listed second. The Pen phenotype is indicated in parenthesesafter each parental strain.

° Percentage of exposed cells transformed.c Concentration of drug used (in micrograms per milliliter) to select transformants.d Mean MIC in micrograms per milliliter; range in parentheses.

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PENICILLIN LOCI IN GONOCOCCUS

that the locus for second-step (additive) in-crease in PenR was ery-2.Third-step PenR: penB transformations.

The second-step PenR transformants were stillconsiderably less PenR than the donor strainFA48. Therefore, transforming DNA fromFA48 (Pen 2.0) was introduced into second-stepPenR transformant FA136 (Pen 0.25), employ-ing 0.25 to 0.50 ,ug of Pen per ml in selection.Third-step transformants, obtained with a fre-quency of 0.001 to 0.006 with 0.01 ,ug ofDNA perml, were increased approximately fourfold intheir level of PenR, bringing them close to thephenotypic level of PenR of FA48 (Table 2).

All (110 of 110) third-step PenR transformantsfrom this experiment were also two- to fourfoldmore TetR than the recipient. Extensive pre-vious study had not disclosed a locus that con-ferred resistance to both Pen and Tet (29). More-over, experiments described below showed thatthe locus for third-step PenR had a unique mapposition. The locus for third-step increase inPenR was designated penB.When a third-step PenR transformant

(FA140) was used as a source of transformingDNA rather than the original mutant FA48,the same sequence of stepwise and independenttransfer of Pen resistance to FA19 was repeated

in all details (experiments not shown). This wastaken as confirmation that FA48 and FA140contained three unlinked loci (penA2, ery-2,and penB2), which additively produced an 128-fold increase in Pen resistance (Pen 0.007 to Pen1.0). Moreover, very similar results in everyrespect were obtained when the clinical isolateFA5 was used as the donor (Fig. 1). The onlydifferences in results obtained with the clinicalisolate were the slightly lower resistance of thefirst-step transformants (Pen 0.03) and higherresistance of the second-step transformants(Pen 0.25).Parent strains and various transformants

were tested for Pen sensitivity by single-celland replica-inoculating MIC methods, whichgave similar results, excepting an approxi-mately twofold greater sensitivity by the sin-gle-cell method (Table 3). The similarity of re-sults by the two methods confirmed the value ofthe replica-inoculum method, which because ofits greater convenience was used as the stand-ard method throughout.Inasmuch as FA48 contained three loci for

low-level PenR (penA2, ery-2, and penB2), itmight have been expected that selection forvery low-level PenR in transformation crossesbetween FA48 and FA19 (penA+ ery+ penB+)

1 2 32.0

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TRANSFORMATION STEPFIG. 1. Multistep development of Pen resistance in sensitive strain FA19 by transformation from its

isogenic derivative FA48 or from clinical isolate FA5. Data for transformation between FA48 and FA19 are

from Table 2. The phenotype of 10 transformants was analzyed at each step in similar experiments betweenFA5 and FA19. In the experiments between FA5 and FA19, a first-step PenR (penAl) transformant, FA143,was used as recipient for selection of the second-step PenR transformants, and second-step (penAl ery-1)transformant FA164 was used as recipient for selection of third-step PenR transformants. Figures above barsindicate mean MIC of transformants. (All tested transformants from experiments using FA5 as donor scoredat a single level ofPenR.) Genotype of transformants is indicated below the bars. Symbols: solid bar, donorFA48 or FA5; open bar, recipient FA19; dotted bar, PenR transformants from FA48 x FA19; striped bar, PenRtransformants from FA5 x FA19.

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743VOL. 124, 1975

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744 SPARLING, SARUBBI, AND BLACKMAN

would have resulted in three phenotypicclasses: one eightfold more resistant to penicil-lin, but not resistant to other drugs (penA2); a

second slightly (twofold) more resistant to Penbut also resistant to Ery (ery-2); and a thirdwhich was fourfold more resistant to Pen andtwo- to fourfold more resistant to Tet as well(penB2). In fact, selection for PenR resulted inonly penA2 transformants in many experi-ments (29).One reason for this apparent discrepancy

may have been the use of concentrations of atleast 0.015 ,g of Pen per ml when PenR trans-formants were selected in FA19, which was

twice the MIC or four times the highest concen-

tration of Pen on which FA19 would grow.

Therefore, there probably was selective biasagainst the very low-level increases in PenRdue to ery-2; only the eightfold more PenRpenA2 transformants survived. Note that therewas no difficulty in selecting ery-2 transform-ants in FA19 if initial selection was with Eryrather than Pen (29). The probable reason forfailure to selectpenB2 transformants in FA19 isdiscussed below.

Nonspecific resistance: ery-2 and penB2.Since both ery-2 and penB2 resulted in nonspe-

TABLE 3. Sensitivity of Pen-resistant transform-antsto Pen by single-cell and replica-inoculum methods

Pen sensitivity(,ug/ml)

Strain GenotypeSingle- Replica-

cell inoculum

FA19 0.004 0.007FA102 penA2 0.03 0.06FA136 penA2 ery-2 0.06 0.25FA140 penA2 ery-2 penB2 0.50 1.0FA48 Polygenic (Table 1) 1.0 2.0

cific low-level resistance to certain antibiotics,their effect on sensitivity to other compoundswas tested. Results showed that ery-2 causedlow-level resistance to many agents, includingvarious antibiotics, detergents, and dyes (Table4). The ery-1 locus from FA5 had the same effect(not shown). In contrast, penB2 (and also penBlfrom FA5) increased resistance to Pen and Tet,and to a slight degree to Chl, but had no effecton sensitivity to a variety of the other drugs(Table 4).The complicated phenotype of EryR (ery-2)

transformants was apparently due to mutationat a single locus, since it proved impossible(within the limits of accuracy of testing forsmall differences in sensitivity) to geneticallyseparate ery-2 transformants into variousclasses. Thus, 19 of 19 PenR (0.12) transform-ants from FA48 x FA102 were also EryR, and 45of 45 EryR (0.5) transformants from the same

cross were PenR (Table 2). In addition, 97 of 100EryR transformants from FA48 x FA19 were

resistant to acridine orange and 116 of 116 wereresistant to Triton X-100 and Rif (RifR). Selec-tion for Triton X-100-resistant (15 mg/ml) trans-formants (FA48 x FA19) resulted in 17 of 17which were also EryR, ChlR, and acridine or-

ange resistant.Mapping penB2. Transformation between

donor strain FA48 (str-7 tet-2 chl-2 penB2) andrecipient FA136 (str+ tet+ chl+ penB+) showedapproximately 1% co-transformation betweenstr-7 andpenB2 and 7 to 13% between chl-2 andpenB2, which suggested that penB2 might belocated in or near the linked cluster of genesaround str-7 and spc-3 (29). This was confirmedin other experiments with derivative strainscarrying various combinations of the spc andpenB loci, which showed 18 to 20% co-transfor-mation between spc-3 and penB2 (Fig. 2). In

TABLE 4. Nonspecific resistance due to ery-2 and penB2 mutations

AntibioticaStrain Genotype

Pen Ery Tet Chl Rif Fus Doc Trxb AO CV

FA19 0.007 0.25 0.25 0.50 0.12 0.12 0.12 0.5 100 2FA102 penA2 0.06 0.25 0.25 0.50 0.12 0.12 0.12 0.5 100 2FA136 penA2 ery-2 0.25 4.0 0.25-0.50 1.0 0.5 1.0 0.25 >16.0 400 8FA140 penA2 ery-2 penB2 1.0 4.0 1.0 2.0 0.5 1.0 0.25 >16.0 400 8FA171 ery-2 0.015 4.0 0.25-0.50 1.0 0.5 1.0 0.25 >16.0 400 8FA212 ery-2 penB2 0.06 4.0 1.0 1.0 NT NT NT NT NT 8Fold increaseDue to ery-2 2-4 16 1-2 2 4 8 2 >32 4 4Due to penB2 4 c 2-4 1-2

° Abbreviations: Fus, fusidic acid; Doc, sodium deoxycholate; Trx, Triton X-100; AO, acridine orange;CV, crystal violet; NT, not tested.

b MIC to Triton X-100 in milligrams per milliliter.e, No effect.

J. BACTERIOL.

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PENICILLIN LOCI IN GONOCOCCUS 745

these and related experiments, donor and recipi-ent strains were penA2 ery-2, so that selectionor scoring for Pen' was specific forpenB2. Recip-rocal three-factor transformation crosses estab-lished the order str-7 ... spc3 ... penB2 (Table5). On the basis of this and previously publishedstudies (29), there are thus six linked genes inthe gonococcus for antibiotic resistance, ar-ranged in the linear order rif-1 ... str-7 . . . tet-2 ... chl-2 ... spc-3 . .. penB2. ThepenBl locusin clinical isolate FA5 showed 10 to 16% co-

str-7 tet-2 chl-2 spc-3 DenB2I

0.99 'I (2 /190)I 0.99 ! (3/356)

0.91

0.87(7/56)______ 1 0.93 (16/231)(12/133)L

0.82(17/92) i-0.80(26/125) !

FIG. 2. Recombination frequencies (1 - co-trans-formation frequency) between adjacent loci in two-fac-tor transformation crosses involving penB2. Arrowspoint from the selected to the scored unselectedmarker. Numbers in parentheses are (number oftransformants receiving unselected marker/numberof transformants scored). Data are from Table 5 andother experiments are not shown. All donors and recip-ients were penA2 ery-2. Selection was with 037 pg ofPen per ml for penB2, 1.5 pg of Chl per ml for chl-2,300 pg of Str per ml for str-7, and 200 pg ofSpc perml for spc-3 transformants. Ninety-nine percent oftransformants selected for penB2 were also at leasttwofold TetR.

transformation with spc-3, but none with str-7,in experiments similar to those in Fig. 2 andTable 5 (data not shown).FA48 was previously shown to contain a locus

(tet-2) specific for TetR, which was linked toboth spc-3 and str-7 (29). The tet-2 locus there-fore maps close topenB2 and has similar pheno-typic effects, excepting the fourfold increase inPenR due to penB2. There is little doubt thatpenB2 and tet-2 are separate loci, however.Three- and four-factor transformation crossesshowed that tet-2 mapped to the left ofspc-3 andwas closely linked to str-7 (29), whereas thepresent experiments showed thatpenB2 was on

the right side of spc-3 in the convention em-ployed and was only equivocally linked to str-7.Moreover, experiments described below showedadditive TetR due to sequential introduction ofpenB2 and tet-2.

Permissive effect of ery-2 for expressionof penB2. It was not clear why PenR TetR(penB2) transformants could be selected whenpenB2 DNA from FA48 was introduced intoFA136 (Tables 2 and 5) but could not be whenthe recipient was FA19 (29). Preliminary experi-ments suggested that ery-2 was required in therecipient before penB2 transformants could beselected, since introduction of tet-2 penB2 DNAinto either F19 or FA102 (both ery+) resulted inonly TetR Pens (tet-2) transformants, whereasintroduction of the same DNA into ery-2 recipi-ents FA171 or FA136, again selecting for TetR,resulted in both TetR Pens (tet-2) and TetR PenR(penB2) transformants (data not shown).To more adequately test whether ery-2 was

TABLE 5. Mapping penB2 by reciprocal three-factor transformation crosses

No. of re-Recombinant classes° No. of Possible combinants

Crossa recom- marker ordersc in quadruplePen Spc Str binants neorm crossover

classes

1. Donor FA198 str+ spc-3 penB2; recipi- 1 1 1 3 str-7 spc-3 penB2 0ent FA147 str-7 spc+ penB+; selected 1 1 0 23 str-7 penB2 spc-3 e

phenotype: PenR d 1 0 0 99 spc-3 str-7 penB2 231 0 0 99

2. Donor FA48 str-7 spc+ penB2; recipi- 1 1 1 0 str-7 spc-3 penB2 1ent FA192 str+ spc-3 penB+; selected 0 1 1 12 str-7 penB2 spc-3 12phenotype: StrR d 1 0 1 1 spc-3 str-7 penB2

0 0 1 120

a All strains were derived from FA19 and were penA2 ery-2 in addition to markers shown (see Table 1).b Donor and recipient phenotype referred to as 1 and 0, respectively. Spc, Spectinomycin.c Cross 1: suggested order, str-7{PenB2. Cross 2: suggested order, 8str7 penB2; probable order, str-7 spc-3

penB2.d PenR transformants were selected with 0.3 pg of Pen per ml. StrR transformants were selected with 300

Ag of Str per ml.-, Undetectable quadruple crossover classes.

0.89 1'. (10/92Ir_ -1

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746 SPARLING, SARUBBI, AND BLACKMAN

permissive for penB2, the same experimentswere repeated using FA140 (tet+ penA2 ery-2penB2) as donor. FA140 contains only one locus(penB2) that confers significant TetR, and there-fore selection for TetR transformants should bespecific for penB2. Results are shown in Table6. It was possible to select TetR transformants,all of which were also fourfold more PenR andtherefore were presumably penB2, in FA136and FA171 (both ery-2), but not in FA19 or

FA102 (both ery+). These results strengthenedthe idea that ery-2 was required in the recipientfor selection ofpenB2 transformants.

Preliminary experiments suggest that penB2can be taken up and integrated by ery+ recipi-ents but is only phenotypically expressed in thepresence of ery-2. This is based on the observa-tion that transforming DNA prepared from one

TetR Pens transformant arising from the cross

FA48 (tet-2 penB2) x ery+ recipient FA19 (tet+penB+) was capable of transferring resistanceto either Tet alone (tet-2) or Tet and Pen(penB2) to ery-2 recipient FA136. Further exper-iments are necessary to substantiate this hy-pothesis, however.

Additive effects of ery-2 and penB2 withother loci. Previous study showed that selec-tion of low-level TetR transformants from FA48(Tet 4.0) x FA19 (Tet 0.25) resulted in onlypartial transfer of donor TetR: all first-step TetR(tet-2) transformants were Tet 1.0 (29). Sinceery-2 slightly increased TetR, and since penB2also increased TetR by two- to fourfold (Table 4),it seemed likely that the (Tet 4.0) phenotype ofFA48 was due to additive effects between thesegenes. A donor strain carrying only tet-2(FA162) was used to introduce tet-2 into recipi-

TABLE 6. Permissive effect of ery-2 on selection ofTetR (penB2) transformantsa

Recipien strain Selected pheno- Transformationtype (jg/ml) frequency (%)b

FA19 EryR (0.5) 0.1TetR (0.37)Y <0.00001

FA102 (penA2) TetR (0.37) <0.00001FA136 (penA2 ery-2) TetR (0.50) 0.02dFA171 (ery-2) 1IetR (0.50) O.OO1d

I Donor: FA140 penA2 ery-2 penB2; donor DNAconcentration saturating (10.0 Mg/ml).

I Percentage of exposed cells transformed. TheEry' (ery-2) transformants of FA19 were included as

a positive control of the competence of the strain.r Numbers in parentheses indicate concentration

of drug used in selection of transformants. Use of0.25 ,g of Tet per ml failed to select against recipi-ent strains FA19 or FA102.

d All TetR transformants (45 tested from each ofFA136 and FA171) were also fourfold more PenR.

ents carrying various combinations of ery-2 andpenB2. Results showed additive effects betweenthe three mutations, which cumulatively re-sulted in a 16-fold increase in TetR (Table 7).Similar experiments also showed additive ef-fects between chl-2, ery-2, andpenB2, resultingcumulatively in a 16-fold increase in ChlR (Ta-ble 8). Other experiments (not shown) estab-lished the same sort of additive relationshipsbetween the chl-1, tet-1, ery-1 andpenBi loci inclinical isolate FA5.We previously reported that introduction of

ery-1 or ery-2 into FA19 resulted in incomplete

TABLE 7. Additive effects of tet-2, ery-2, and penB2mutations in determination of low-level

resistance to Tet

MIC of Tet (gg/ml)aRecipient Genotype tet+ tet-2straintt trans-

recipient formant

FA19 0.25 1.0FA162 penA2 0.25 1.0FA136 penA2 ery-2 0.50 1.0FA140 penA2 ery-2 penB2 1.0 40

a All recipients were tet+. The tet-2 transformantswere prepared by transformation with DNA fromtet-2 donor strain FA162, which has an MIC (Tet) of1.0 ,zg/ml. TetR transformants were selected with 0.5jig of Tet per ml (FA19, FA102, FA136) or 1.5 Mg ofTet per ml (FA140).

bThe level of Tet resistance attained by the ery-2penB2 tet-2 transformants (MIC, 4.0 ,ug/ml) wasequal to that of the original parental donor strainFA48.

TABLE 8. Additive effects of chl-2, ery-2, and penB2in determination of low-level resistance to Chl

MIC of Chl (pLg/ml)aRecipient Genotype chl-2strains Genoe

trans-recipient formant

FA19 0.5 2.0FA102 penA2 0.5 2.0FA136 penA2 ery-2 1.0 4.0FA140 penA2 ery-2 penB2 2.0 8.0b

a All recipients were chl+. The chl-2 transform-ants were prepared by transformation with DNAfrom chl-2 donor strain FA163, which has an MIC(Chl) of 2.0 ,ug/ml. ChlR transformants were selectedwith 1.0 Ag of Chl per ml (FA19, FA102, FA136) or2.0 ,ug/ml (FA140).

b The level of Chl resistance attained by the ery-2penB2 chl-2 transformants (MIC, 8.0 MLg/ml) wasequal to that of the original parental donor strainFA48.

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transfer of the donor's phenotypic level of EryR(29). With more attention to details of inoculumsize and age of cells, most EryR transformantswere shown to acquire the donor's phenotypiclevel of EryR (4.0) in a single step.Lack of penicillinase. Strains FA5, FA48,

and FA19 were each assayed for the presence off3-lactamase by the microiodometric method(21) and for inactivation of Pen, Tet, Ery, andChl by a sensitive bioassay method (3, 29). Noevidence was found for ,¢lactamases or for de-struction of the biological activity of any drugby any strain by the methods employed.

DISCUSSIONThe results clearly showed that resistance to

each of Pen, Tet, and Chl was transferred instepwise fashion, due to additive effects be-tween several genes (Table 2, 7, and 8; Fig. 1).These results are therefore consistent with thehistorically gradual emergence of resistance tothese drugs which has been noted among clini-cal isolates (32) and with the multistep develop-ment of resistance noted in vitro (14, 17, 18).They are also consistent with prevalence stud-ies of Pen sensitivity among clinical gonococcalisolates, which have often shown discrete peaks(up to five) separated in Pen sensitivity by stepsof approximately fourfold (5, 22).Remarkably similar stepwise development of

resistance of pneumococcus to Pen in vitro wasstudied by Hotchkiss (9), and later by Shockleyand Hotchkiss (30) by transformation. Theyshowed that the combined effects of mutationsat four loci resulted in a 12-fold increase in Penresistance (30). Multistep development in vitroof resistance to Pen was also described in Sal-monella typhimurium (2, 20) and Staphylococ-cus (8). Additive effects between genes for &lactamase production and others which affectthe permeability of the cell envelope to Pen andother drugs have been extensively studied inEscherichia coli (4, 13, 19). Complex, polygeniccontrol of resistance of many laboratory mu-tants ofpneumococcus to Ery (24), sulfonamides(10), and Str (6) has also been described.Our studies failed to disclose evidence of 3-

lactamases or other drug-inactivating enzymesin the gonococcus. We have only studied oneclinical isolate of gonococcus carefully to date(FA5), but results with it have been nearlyidentical to the laboratory mutant (FA48). Toour knowledge, this is the first demonstrationof multistep, non-penicillinase-mediated resist-ance to Pen in any clinical bacterial isolate.Our earlier study of resistance in gonococcus

by transformation failed to disclose evidence formutation at penB in either FA48 or FA5 (29).

The explanation for this is almost certainly theuse of ery+ recipient FA19 for the earlier study,since penB2 transformants could only be se-lected in strains carrying mutations at ery-2(Table 6). In this sense, ery-2 is a modifier ofpenB2. An analogous problem was studied ear-lier by Shockley and Hotchkiss, who found thatexpression of mutations at two of the four Penresistance loci in pneumococcus appeared to de-pend on mutation at the other two (30). Bryanalso reported that certain Str resistance muta-tions in pneumococcus were greatly influencedby modifier mutations, which had little effectby themselves (6). The molecular basis for theseinteractions was unknown. In the present exam-ple, understanding will undoubtedly requirebiochemical definition of the ery-2 and penB2gene products.The highest levels of resistance to each of

Pen, Tet, and Chl required the nonspecific eryand penB mutations, as well as mutation at alocus specific for each drug (penA, tet, and chl,respectively) (Tables 2, 7, and 8). This helps toexplain the clinical observation that correla-tions of low-level resistance to various drugsare greatest among the strains with highestlevels of resistance to any one (1, 17, 23, 25-28).In addition, the ery-1 and ery-2 mutations ac-counted fully for the low-level resistance of FA5and FA48 to several drugs, including Rif, fu-sidic acid, acridine orange, deoxycholate, andTriton X-100. The observed correlations of re-sistance to most of these in clinical isolates (17)is therefore probably due to the frequent occur-rence ofery mutations in other clinical isolates.It should be noted that ery only results in low-level resistance, and that there are separateloci in the gonococcus for high-level resistanceto each of Rif (29) and fusidic acid (T. Sox and P.F. Sparling, unpublished data). Sequential se-lection of mutations of the type described herewould also explain the multiple-drug resistancephenotypes of mutants of gonococcus selected invitro for progressively greater resistance toeither Pen or Tet (14, 17).There were many pragmatic difficulties in

attempting to analyze the genetics of low-levelantibiotic resistance. Because we only testedfor twofold increments in sensitivity, we mayhave missed minor effects of certain mutations,and may have missed other mutations alto-gether. We felt this was necessary, since it wasdifficult to accurately determine less than two-fold differences. The fact that mutations at dif-ferent loci sometimes produced similar pheno-typic effects (penB2 and tet-2 both resulted inan approximate fourfold increase in TetR) wasan additional complication, although this was

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748 SPARLING, SARUBBI, AND BLACKMAN

usually surmountable by complete phenotypicanalysis and by scoring for proximity to nearbyloci for high-level spectinomycin resistance andStrR (Fig. 2). It would, of course, be advanta-geous if auxotrophic markers linked to some ofthe drug resistance loci could be obtained. Pre-liminary results indicate that none of the locistudied was linked to naturally occurring (7)Pro- or Arg- markers (G. Biswas and P. F.Sparling, unpublished observation).Another sort ofproblem was presented by the

eventual demonstration that many ultraviolet(UV)-induced mutants were actually complexmultisite mutants. For example, FA48 differsfrom its original parent strain FA19 by no lessthan six mutations, which arose after only twoexposures to mutagenic doses of UV light (17).The mechanism for this was not investigated,but it may be pertinent that UV-sensitive "mu-tator" strains have been described in N. menin-gitidis (11). Our earlier designation of two phen-otypic classes of StrR mutations ("StrRA" andthe more pleiotropic "StrRB") (17) was in error,because transformation has shown that all strmutations result in resistance to Str only (29).Since nearly all "StrRB" mutations arose afterUV mutagenesis (17), the complicated pheno-type of such mutants was probably due to induc-tion of multiple mutations with a single vigor-ous exposure to UV light. Similar considera-tions apply to the pleiotropic, UV-induced PenRStrR mutants formerly designated "PenRB" (17).It should be emphasized, however, that somemutations of the gonococcus, which by transfor-mation appear to be due to alteration of a singlesite, do have quite pleiotropic effects, as shownhere for both ery-2 and penB2.

After submission of this manuscript, a paperby Maier et al. (15), which described a locus mtrfor nonspecific resistance in the gonococcus, ap-peared. It is likely that mtr and ery are sepa-rate symbols for the same locus, but futurestudy is needed to establish this.

ACKNOWLEDGMENTS

G. Biswas gave helpful advice. D. Walstad performed theassays for 3-lactamase and antibiotic inactivation.

This work was supported by Public Health Service grantAI10646 from the National Institute of Allergy and Infec-tious Diseases, by a grant from the John A. Hartford Foun-dation, Inc., and by Public Health Service Research CareerDevelopment Award A133032 to P.F.S. from the NationalInstitute of Allergy and Infectious Diseases. Part of thiswork was done while P.F.S. was working in the Depart-ment of Bacteriology, Bristol, England. Thanks are ex-pressed to M. Richmond and E. Lewis of Bristol, who helpedin various ways.

LITERATURE CITED1. Amies, C. R. 1969. Sensitivity of Neisseria gonorrhoeae

to penicillin and other antibiotics. Br. J. Vener. Dis.45:216-222.

2. Banic, S. 1959. Transduction to penicillin and chloram-phenicol resistance in Salmonella typhimurium. Ge-netics 44:449-457.

3. Barza, M., M. G. Bergeron, J. Brusch, and L. Wein-stein. 1973. Selective filtration of antibiotics throughcolloidion membranes. Antimicrob. Agents Chemo-ther. 4:337-342.

4. Bowman, H. G., K. Nordstrom, and S. Normark. 1974.Penicillin resistance in Escherichia coli K12: syner-gism between penicillinase and a barrier in the outerpart of the envelope. Ann. N.Y. Acad. Sci. 235:569-585.

5. Bro-Jorgensen, A., and T. Jensen. 1973. Gonococcalpharyngeal infections. Report of 110 cases. Br. J.Vener. Dis. 49:491-499.

6. Bryan, B. E. 1961. Genetic modifiers of streptomycinresistance in pneumococcus. J. Bacteriol. 82:461-470.

7. Carifo, K., and B. W. Catlin. 1973. Neisseria gonor-rhoeae auxotyping: differentiation of clinical isolatesbased on growth responses on chemically defined me-dia. Appl. Microbiol. 26:223-230.

8. Demerec, M. 1945. Production of staphylococcus strainsresistant to various concentrations of penicillin. Proc.Natl. Acad. Sci. U.S.A. 31:16-20.

9. Hotchkiss, R. D. 1951. Transfer of penicillin resistancein pneumococci by the deoxyribonucleate derivedfrom resistant cultures. Cold Spring Harbor Symp.Quant. Biol. 16:457-461.

10. Hotchkiss, R. D., and A. H. Evans. 1958. Analysis ofthe complex sulfonamide resistance locus of pneumo-coccus. Cold Spring Harbor Symp. Quant. Biol.23:85-97.

11. Jyssum, K. 1968. Mutator factor in Neisseria meningiti-dis associated with increased sensitivity to ultravi-olet light and defective transformation. J. Bacteriol.96:165-172.

12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

13. Lundack, A. K., and K. Nordstrom. 1974. Mutations inEscherichia coli K- 12 decreasing the rate of streptomy-cin uptake: synergism with R-factor-medicated capac-ity to inactivate streptomycin. Antimicrob. AgentsChemother. 5:500-507.

14. Maier, R. W., H. R. Beilstein, and L. Zubrzycki. 1974.Multiple antibiotic resistance in Neisseria gonor-rhoeae. Antimicrob. Agents Chemother. 6:22-28.

15. Maier, T. W., L. Zubrzycki, and M. B. Coyle. 1975.Genetic analysis ofdrug resistance in Neisseria gonor-rhoeae: identification and linkage relationships ofloci controlling drug resistance. Antimicrob. AgentsChemother. 7:676-681.

16. Maness, M. J., G. C. Foster, and P. F. Sparling. 1974.Ribosomal resistance to streptomycin and spectinomy-cin in Neisseria gonorrhoeae. J. Bacteriol. 120:1293-1299.

17. Maness, M. J., and P. F. Sparling. 1973. Multiple anti-biotic resistance due to a single mutation in Neisseriagonorrhoeae. J. Infect. Dis. 128:321-330.

18. Miller, C. P., and M. Bohnhoff. 1945. Studies on theaction of penicillin. IV. Development of penicillinresistance by the gonococcus. Proc. Soc. Exp. Biol.Med. 60:354-356.

19. Monner, D. A., S. Johsson, and H. G. Bowman. 1971.Ampicillin-resistant mutants ofEscherichia coli K-12with lipopolysaccharide alterations affecting matingability and susceptibility to sex-specific bacterio-phages. J. Bacteriol. 107:420-432.

20. Nelson, B. W., and R. J. Roantree. 1967. Analysis oflipopolysaccharides extracted from penicillin-resist-ant serum-sensitive salmonella mutants. J. Gen. Mi-crobiol. 48:179-188.

21. Novick, R. P. 1962. Micro-iodometric assay for penicilli-nase. Biochem. J. 83:236-240.

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22. Olsen, G. A. 1973. Consumption of antibiotics in Green-land. IV. Changes in the sensitivity of N. gonor-rhoeae to antibiotics. Br. J. Vener. Dis. 49:33-41.

23. Phillips, I., D. Rimmer, M. Ridley, R. Lynn, and C.Warren. 1970. In-vitro activity of twelve anti-bacte-rial agents against Neisseria gonorrhoeae. Lancet1:263-265.

24. Ravin, A. W., and V. N. Iyer. 1961. The genetic relation-ship and phenotypic expression of mutations endow-ing pneumococcus with resistance to erythromycin. J.Gen. Microbiol. 26:277-301.

25. Reyn, A. 1961. Sensitivity ofN. gonorrhoeae to antibiot-ics. Br. J. Vener. Dis. 37:145-157.

26. Reyn, A., and M. W. Bentzon. 1968. A study of therelationships between the sensitivities of Neisseriagonorrhoeae to sodium penicillin G, four semi-syn-thetic penicillins, spiramycin, and fusidic acid. Br. J.Vener. Dis. 44:140-150.

27. Reyn, A., and M. W. Bentzon. 1969. Relationships be-tween the sensitivities in vitro of Neisseria gonor-

rhoeae to spiramycin, penicillin, streptomycin, tetra-cycline, and erythromycin. Br. J. Vener. Dis. 45:223-227.

28. Ronald, A. R., J. Eby, and J. C. Sherris. 1969. Suscepti-bility ofNeisseria gonorrhoeae to penicillin and tetra-cycline, p. 431-434. Antimicrob. Agents Chemother.1968.

29. Sarubbi, F. A., Jr., E. Blackman, and P. F. Sparling.1974. Genetic mapping of linked antibiotic resistanceloci in Neisseria gonorrhoeae. J. Bacteriol. 120:1284-1292.

30. Shockley, T., and R. E. Hotchkiss. 1970. Stepwise intro-duction oftransformable penicillin resistance in pneu-mococcus. Genetics 64:397-408.

31. Sparling, P. F. 1966. Genetic transformation of Neis-seria gonorrhoeae to streptomycin resistance. J. Bac-teriol. 92:1364-1371.

32. Sparling, P. F. 1972. Antibiotic resistance in Neisseriagonorrhoeae. Med. Clin. North Am. 56:1133-1144.

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