JOURNAL OF BACrERIOLOGY, Sept. 1968, p. 596-605 Vol. 96, No. 3Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Neisseria gonorrhoeaeII. Colonial Variation and Pathogenicity During 35 Months In Vitro

DOUGLAS S. KELLOGG, JR., IRUN R. COHEN, LESLIE C. NORINS,ARNOLD L. SCHROETER, AND GILBERT REISING

Venereal Disease Research Laboratory, National Communicable Disease Center, Atlanta, Georgia 30333

Received for publication 15 June 1968

During 35 months of selective in vitro cultivation, Neisseria gonorrhoeae cells re-tained their virulence for humans and were shown to be closely related to a particu-lar colonial morphology. Saline-autoagglutinability was the only other characteristicdistinguishing virulent from avirulent cells. Human responses to challenge with cellsof the different colonial types were studied for their relationships to virulence oravirulence.

Since its initial cultivation in 1882, Neisseriagonorrhoeae has been extensively studied as anacademic challenge and as a practical medicaldiagnostic problem. In spite of such study, littlehad been ascertained about the immunologicalresponse of humans to a gonococcal infection.Immunity appeared to be transient or limited innature as evidenced by repeated reinfections andthe development of chronic conditions and car-rier states. Antibody production could be detectedby complement-fixation procedures using ex-tracted antigens; however, there was not a closecorrelation of serological reactivity with infection(13, 19). Studies of the antigenic mosaic of N.gonorrhoeae demonstrated antigenic variationamong strains and considerable sharing of anti-gens with other species of Neisseria as well asother genera of bacteria (24). Most studies of N.gonorrhoeae antigens were done with strains whichhad been carried in vitro long enough to obtain asufficient number of cells for antigenic analysis.In vitro cultivation of N. gonorrhoeae resulted in aconversion of normal saline autoagglutinabilityfrom rough to smooth, possibly due to an anti-genic alteration, and a loss of virulence. Sinceman was the only known host for N. gonorrhoeae,and usually was infected only by venereal contact,little was known about virulence of N. gonor-rhoeae except for the possible involvement of apotent endotoxin which avirulent strains wereknown to possess. Progress in ascertaining basicfacts about N. gonorrhoeae was hampered by adouble deficiency: lack of an experimental animaland lack of a marker associated with virulence. In1963, four clonal types were described for N.gonorrhoeae and a correlation was demonstratedbetween colonial morphology and virulence withtwo of the four colonial types (12). At that time

(after 69 selective transfers in vitro), cells of colo-nial type Tl were found to be virulent and cells ofcolonial type T4 were found to be avirulent forhuman volunteers. Virulence testing could not beattempted with cells of either colonial type T2or T3 after 69 selective transfers in vitro becauseof time and space limitations. At 38 transfers, in-fections resulted from inoculations of cells ofcolonial types Ti, T2, T3, and T4; however, theappearance of signs and symptoms was paralleledby an alteration in recovered colony types fromT2, T3, or T4 to T1. At that point, Ti coloniescould still be isolated from cultures of colonialtypes T2, T3, and T4. This paper presents theresults of further studies of virulence of N. gonor-rhoeae for humans, and certain characteristics ofthe infectious process and the organisms them-selves which may be related to their virulence andoverall interaction with the host.

MATERIALS AND METHODSN. gonorrhoeae strain F62 was originally isolated by

this laboratory in 1962 (12). Primary isolates were ob-tained at the Fulton County Health Department,Atlanta, Ga., through the assistance and cooperationof John H. Tiedemann. All colonial lines of strainswere passaged by loop transfer of individual coloniesselected by morphological characteristics observed bymeans of an AO Cycloptic dissecting microscope withdiffused, angled light transmitted from below upthrough the medium. Measurements of colonial di-ameter during iron studies were accomplished with20-fold magnification and an oculhr micrometer. Onthree separate occasions, approximately 100 well-isolated colonies were measured at each compoundlevel under study. All transfers were made after 16 to20 hr of incubation at 35 C under increased carbondioxide tension (candle extinction). G C Medium Base(GCB; Difco), enriched with a defined supplement(DSF), was used for the isolation and cultivation of

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VIRULENT N. GONORRHOEAE IN VITRO

all strains (22). The defined supplement was added tothe basal medium at 43 to 45 C just before pouring.When hemolyzed whole rabbit blood (5%) supple-ment was used, it was added with the defined supple-ment. In examinations of pharyngeal and rectal speci-mens, a selective medium was used (20). The fermen-tation medium utilized for confirmation of speciesidentity was described by White and Kellogg (23). Thesynthetic medium used was described by Gould, Kane,and Mueller (8). Demonstration of capsules was at-tempted by a technique in which suspensions of N.gonorrhoeae in 10% sterile skim milk are examinedwith fixed-phase optics (6). The fluorescent-antibody(FA) procedures applied to direct smears, and/orsmears of cultures, were described by White and Kel-logg (22). The precipitin procedure for serum anti-bodies and control sera production was that describedby Reising and Kellogg (16). The oxidase procedurewas performed as described in U.S. Public HealthService Publication 499 (21). Assays for the presenceof hemolysin, coagulase, and fibrinolysin were con-ducted according to standard procedures describedfor other microorganisms (9). Toxic potential of thecell types was assayed by intraperitoneal injection ofequivalent numbers of each cell type into CFW mice.Antibiotic susceptibilities of the strains used for volun-teer inoculation were determined by James D. Thayerof the Venereal Disease Research Laboratory.

After 17 months of passage, the four colony types ofN. gonorrhoeae (TI, T2, T3, and T4) were tested forvirulence with separate groups of four subjects. After35 months of passage, only the colony; type Ti wastested for virulence and a group of 10 subjects wasused.

All subjects were male volunteers at Atlanta, Ga.Before examination as a possible subject for the study,each volunteer provided assurance that he understoodthe purpose of the experimentation and the possiblerisks involved. Selection of the subjects for the studyrequired satisfaction of the following criteria. He musthave been between ages 21 and 45 years and availablefor a poststudy observation period of 6 months. Hemust have been acceptable from the standpoint ofmentality, psyche, and ability to cooperate. To be ac-ceptable, the subject must not have had a gonococcalinfection within the previous 3 months nor have beenan asymptomatic carrier of N. gonorrhoeae; neithercould he harbor an acute or chronic disease or psy-chogenic disorder. Freedom from gonococcal infectionor carrier state was determined by clinical and labora-tory examinations of urethra, prostate, rectum, andpharynx. A base-line laboratory examination includedwhite blood-cell count, differential count, and hemo-globin, as well as a Venereal Disease Research Labora-tory test and routine urinalysis.

Procedurally, each selected subject was placed in arestricted ward area the day before inoculation. Oninoculation day, a set of GCBDSF agar plates with a16- to 18-hr growth of the gonococcal strain to be usedfor inoculation was examined for the purity of colonialtype. Where individual colonies or areas of coloniesof other than the desired type were observed, thegrowth was marked with a dye. Immediately beforethe subject was inoculated, a sample of blood and a

loop sample of the urethral canal surfaces were ob-tained. Each subject received a full 2-mm bacteriologi-cal loop of the desired colonial type of cells pickedonly from areas free of contaminating colonial types.Each inoculum was inserted approximately 2 to 3inches (5 to 7.6 cm) into the urethra, and the subjectwas instructed not to urinate for several hours. Dailysamples for examinations were taken by inserting theloop into the urethra. Each daily specimen from eachsubject was examined by FA techniques and culturalprocedures (colony morphology, oxidase reactivity,sugar fermentations, and Gram reaction) to identifythe infecting organism as N. gonorrhoeae. After ter-mination of the infection study, each subject was ade-quately treated with either aqueous procaine penicillinG or oxytetracycline. The subject was examined 24,48, and 72 hr after treatment for signs of infection, andthe urethra, urine, and prostate were sampled for thepresence of N. gonorrhoeae by FA and cultural pro-cedures. Volunteers were not released from the studyward until three successive daily examinations werenegative for N. gonorrhoeae by all procedures. Bloodspecimens for serological examination were collectedat intervals over the next 3 months.

RESULTSColonial morphology in N. gonorrhoeae. The

four colonial types originally described have beenfound to represent the most stable morphologicalconfigurations for the conditions of their cultiva-tion. There are a variety of temporary morpho-logical variations which appear as a result ofenvironmental alterations, as well as some varia-tions which are capable of perpetuating them-selves under the conditions of selective transfer.As a consequence, in the selective transfer of thecolonial types, we have adhered as closely as pos-sible to the originally described characteristics inpicking colonies for transfer. The original strain(F62) colonial types, which have been carried asseparate entities for 3.5 years, are morphologicallyvery similar to colonial types obtained from sam-ples that were frozen or lyophilized in 1963. Thetwo colonial types, Ti and T2, obtained frompatients are characterized by their glistening con-vexity, dark-brown to black coloration, and smallsize (0.5 mm and 0.4 mm, respectively; Fig. 1).These characteristics are apparent only when ob-served on a transparent medium with the use ofdiffuse, angled light transmitted through themedium from below the plate. The colonial typeT2 differs from Ti in having a slight internalgranularity, a very marked definition of colonialedge, a friable consistency, and a greater ability toreflect light. The last characteristic, which is ob-served with a combination of transmitted andedge lighting, is consistent with T2 colonies havinga thicker surface film of reflective material than TIcolonies. In Fig. 1, several points are of interest asthey relate to colonial structure. Evidence of the

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KELLOGG ET AL.

distinctions between Ti and T2 colonies can beseen in three aspects of the colonies: (i) the nar-row crescent of refracted light (caused mainly bytransmitted light from below the plate) at theupper edge of the T2 colony as compared with thebroad crescent on the Ti colony; (ii) the brighthighlight on the left side of the T2 colony (fromedge lighting from second light source at side);and (iii) the sharply defined lower edge of the T2colony. The T3 colony, large and granular, posses-ses an inner ring of lighter material which can beseen as a narrow light band running from left toright approximately halfway between the bottomedge and the center of the colony. Small irregularareas of granularity similar to that of the T3colony can be seen in the colorless T4 colony. Thelighting used in photographing these colonies wasestablished in advance as optimal for depiction ofcolonial characteristics; then, without further ad-justment, the individual colonies were photo-graphed. Figure 2 illustrates- the structural and:

|::::i:. :..|:. :: ..F :.. .:.|.E:t: :::.E:.:.: :......|:::::.::: :.:: :.:F....'t.: : ::. ::

:. .:[:.: . ::

IT1

elevational characteristics of the four colonialtypes in a cross-section view.Both Ti and T2 colonies were observed to

acquire a surface roughness and a variation ofcolonial border which tends toward an inter-mediate colony type that we designated T21.Progeny of T21 colonies are predominantly colo-nial type TI,with the rest colonial type T2. Theappearance of such colonies indicated some typeof variation in the cultivation medium and servedas an index to the reproducibility of our culturalconditions. Several hundred isolations of N.gonorrhoeae from both males and females furthersubstantiated the relationship of these types withthe disease state and the relative consistency of themorphological characteristics from strain tostrain.The two colonial types associated with nonse-

lectively transferred laboratory strains of N.gonorrhoeae T3 and T4 are characterized by theirnearly flat elevation, minimal coloration, and

T:2.....~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~~~~~~...........}....jg..........is~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...=7.e. ......,.,XXE.:....................: iX 11 ** -_gU:...........................................

.4011| L: .s |@k~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...E.: .......FIG. 1. Colonial types ofN. gonorrhoeae. Combined illumination: tranismitted light from below the plate with a

ground-glass diffuser anid tilted mirror, but no condensin2g system; edge lighting from second light source at side at15° above flat plane. X 58.

.71

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VIRULENT N. GONORRHOEAE IN VITRO

Tl T2 T3 T4

FIG. 2. Cross sections of colonies of N. gonorrhoeae.

larger size (1.0 to 1.2 mm). The T3 colonial typeis distinguished from the T4 type by an internallight-brown granularity.

It must be emphasized that Ti and T2 colonialtypes can be maintained only by selective transferof individual colonies having the typical mor-phology as observed through the dissecting mi-croscope. All strains of N. gonorrhoeae studiedunder conditions of nonselective transfer demon-strate a conversion from colonial types Ti and/orT2 to colonial types T3 and/or T4. The conver-sion can be prevented only by selective transfer orstorage. Unsuitable media complicated the main-tenance of colonial types Ti and T2 by increasingthe proportion of T3 and T4 colonial types foundat each successive nonselective transfer. Stabiliza-tion of a strain as colonial type TI or T2 has notbeen possible on any medium employed; however,with our particular medium, there was a selectionof colonial type Ti for greater stability. For ex-ample, when colonial type Ti cells of a represent-ative group of eight primary isolate strains werespread on agar-medium plates and the types of theresulting colonies were recorded, the results wereas seen in Fig. 3. After four selective in vitro trans-fers of colonial type Ti cells, three strains were

well adapted and produced progeny, 98 to 99%of which were TI colonies. Three strains producedprogeny with only 50 to 65% Ti colonies; twostrains produced 30% and 5% Ti colonies, re-spectively. Five selective passages later, all eightstrains produced 98 to 99% colonial type Tiprogeny. A more complex medium, such ashemolyzed whole rabbit blood-agar medium(5%), which supports excellent growth of N.gonorrhoeae, had no effect upon the assumptionof stability, nor did a synthetic medium of simplecomposition. The synthetic medium, which sup-ported sparse but equivalent growth in terms ofnumbers of clones of each of the four colonialtypes, did demonstrate a reduced rate of appear-ance of colonial types T3 and T4. This reductioncould have been the result of the restricted amountof growth which allowed less opportunity forexpressing a genetic change. Materials allowingcolonial types T3 and T4 to make superior growthwere absent, as indicated by the equivalent colonydiameters of the four colonial types on the syn-thetic medium. All primary isolates grew on thesynthetic medium.

After the first studies of colonial variation inN. gonorrhoeae (12), the apparent stepwise colo-nial step TI to T4 selection in vitro could bereadily reversed, another characteristic of subse-quently isolated strains. However, after 15 monthsin vitro, strain F62 colonial type T4 was back-selected from T4 colonial morphology to T3 andthen T2 morphology only. Colonial type Ti

T1*

4 91 4 91 1 4 19 1.4 9 1 1 4 19 4i919E 1 T 2 1 1 3 1 1 4 T 5 6-8

N. GONORRHOEAE STRAINS

ElTI

T2

T3, T4

Passagt- Number

Strain Identification |

r FIG. 3. Stabilization ofcolonial type in N. gonorrhoeae strains. Each passage in vitro was selectivefor Ti colonialmorphology and was spread-inoculated for examination ofpercentage of individual colony types present.

100

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. '...-..-...--. ....--. .. ,..-- I

VOL. 96, 1968 599

KELLOGG ET AL.

clones were not detected in the back-selected T2line, even though they were detected in the T2 linewhich had been carried as T2 for 15 months. Ap-parently, the Ti colonial characteristics were re-tained in the T2 inheritance with occasional ex-pression, but were lost from the T4 inheritanceafter continued selection in vitro. Long termstorage (1 month or longer) of the colonial typeshas been successful, either frozen in glycerol-broth at -40 C or lyophilized. For routinemanagement of active stocks for periods of lessthan one month or more than one weekend, weused 20% glycerol-G C Base Medium-broth and-20 C storage. Some glycerol-broth (-20 C)stored suspensions were thawed, sampled, and re-frozen at least six times, and, although there was adecline in viable cells each time, there was noproblem in obtaining adequate growth in 18 hr onagar-medium.

Physiological characteristics of colonial types.The factors responsible for virulence are probablyinherent in some aspect of the cellular physiologyor antigenic mosaic which is reflected in the grosscharacteristic of colonial morphology. None ofthe characteristics previously associated withvirulence in other microorganisms has been de-tected in N. gonorrhoeae cells. No evidence hasbeen found for the presence of hemolysins, coagu-lases, or fibrinolysins. The toxic characteristics ofthe gonococcus are present in approximatelyequivalent concentrations in the cells of all fourcolonial types as assayed in CFW mice. No evi-dence of infection was observed in any of the miceduring postmortem examinations. No distinctcapsules were detected on any of the cells of thefour colonial types, regardless of their source (pri-mary isolates or laboratory strains) or cultivationmedium. Cells of colonial type T2 exhibit a singu-lar ability to grow throughout agar-medium shaketubes-a characteristic not shared by cells of theother three colonial types. The possible signifi-cance of this characteristic in terms of virulenceis not known.An outstanding requirement of both virulent

and avirulent cells was for the presence of ferricions in our medium. In Fig. 4, the logs of the con-centrations of ferric ions (in micrograms per cent)are plotted against the relative increase in colonialdiameters over the controls. Stimulation began at5 to 10 g per cent and increased to a maximumat approximately 280 Ag per cent where the colo-nial size had doubled that of the controls. No fur-ther increase in colonial size was observed beyond1,400 ,ug per cent, and the only alteration of colo-nial morphology was an increase in the darknessof colonial coloration. The types of anions accom-panying the ferric ions had no effecton the results.

X 2.04

V

zu0

° 1.0

1.0 2.0 3.0 4.0

LOG OF CONCENTRATIONS IN MICROGRAMS/100 ml.

FIG. 4. Growth stimulation by iron ions. Growth onunsupplemented G C Base Medium equals 1.0. Glu-cose/ferric curve represents the effect of increasingglucose concentrations in the presence of a maximalstimulation level offerric ions.

Glucose alone was ineffective, but with the ferricions produced an additive effect. A ratio of glu-cose to ferric ions of less than one depressed thecolony size to a level intermediate between thatobtained with either additive alone. Hemin andferrous gluconate were not stimulatory. Alumi-num ions were nearly as effective as ferric ions,but manganese and magnesium ions were notstimulatory in our medium. The ferric ions couldbe incorporated into the medium before auto-claving without reducing the stimulation.

Virulence studies with N. gonorrhoeae colonialtypes. After 440 selective transfers in vitro over aperiod of 17 months, cells of strain F62 Ti and T2colonies were found to be virulent for four malevolunteers. Strain F62 T3 and T4 colonies werefound avirulent at 17 months. There were severaldifferences in the course of events in the3e men ascompared to those of the previous study (12).Specimens were obtained from each man each dayafter infection and were tested by direct and de-layed FA techniques (22) and cultural procedures.

Inoculation with cells of colonial types T3 andT4 resulted in either a watery discharge or none atall. Exudates and urethral scrapings from thesevolunteers contained rare FA-positive diplococciof poor morphology both intra- and extracellularto polymorphonuclear leukocytes at 24 hr postin-oculation. Many polymorphonuclear leukocytescontaining 2+ to 3 + FA-positive granules wereseen, as well as occasional histiocytes and epithe-lial cells. No FA-positive diplococci were seen in

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specimens taken at 48 hr postinoculation. Thenumber of polymorphonuclear leukocytes in theexudates varied among the volunteers from few tomany, and the FA-positive granules varied from1+ to 3 + in intensity. Each 24-hr period postin-oculation saw a decreasing number of polymor-phonuclear leukocytes with decreasing FA-stain-ing intensity of the granules in the exudates. Thevolunteers acquired a considerable tenderness ofthe inguinal lymph nodes starting about 24 to 36hr postinoculation and lasting 3 to 4 days, withprogressively decreasing severity. N. gonorrhoeaewas not recovered in cultures from these volun-teers at 24 hr postinoculation, or at any subse-quent time.

Inoculation with cells of colonial types Tl or T2resulted in the formation of moderate amounts ofpurulent exudate. These exudates contained manyFA-positive diplococci intra- and extracellularto polymorphonuclear leukocytes. Histiocytes,epithelial cells, and many polymorphonuclearleukocytes, containing 1+ FA-positive granules,were observed. By 72 hr post inoculation, the pro-portions of intra- and extracellular FA-positivediplococci in relation to polymorphonuclear leu-kocytes had begun to shift to more extracellularthan intracellular. Between 96 hr and 120 hr, theshift was nearly complete and only rare intracellu-lar diplococci were seen. By 168 hr and 192 hr,there were varying numbers of extracellular di-plococci among the volunteers, and many poly-morphonuclear leukocytes which contained 3+

100

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>.) 75 - Nc% 4.

E

05o

0

25

FA-positive granules. The extracellular diplococcihad a poor morphological appearance whichcould be described as "moth eaten." The consider-able tenderness of the inguinal lymph nodes seenin the volunteers who received colonial types T3and T4 was not seen in the volunteers who re-ceived colonial types TI and T2.An interesting situation was observed when the

daily isolates from the infected volunteers wereexamined for their relative percentages of T1 andT2 colonial types. Colonial types T3 and T4 wereso rare that they were not included in the deter-minations. In Fig. 5, the percentages of type Tland T2 colonies per isolate are shown for each daypostinoculation. When the inoculum was 100%Tl cells, successive daily isolates showed a pro-gressive change in the composition of the popula-tion to 90% T2 colonies. A similar change wasseen when the inoculum was colonial type T2; i.e.,altered to 90% colonial type Tl. The number ofcolonies per sample was approximately the samefor each volunteer, as was the exudate content(number of polymorphonuclear leukocytes, intra-and extracellular FA-positive diplococci, andtissue cells). The rate of colonial change was aboutthe same among volunteers who received colonialtype Tl cells. However, the rate of colonial changeshowed more variation with the individual menwho had received colonial type T2 cells (Fig. 6).By the 2nd day, the colonial types from two of thevolunteers had altered to greater than 70% colo-nial type Tl. The exudate from one volunteer did

I 0

*m ~* TI Inoculum*~~m T2 Inoculum

l0

25 0

50 E#' S

_ ~~~~~~~~~~~~~759L

2 3 4 5Post Inoculation (Days)

FiG. 5. Alteration ofcolony type in exudates. Average percentage of Ti and T2 colonies found on successive daysafter inoculation, considering total number of Ti and T2 colonies to equal 100%.

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KELLOGG ET AL.

INDIVIDUAL ALTERATION CURVES - T2 INOCULUM

0

I-)

u

o

0

0

U

Sa-

P

c0

2 3 i 5Post Inoculation (Days)

FIG. 6. Ilidividual alteration curves-T2 inioculum. Variationi in response offour voluniteers to similar inocula interms of change of coloniy type in daily exudate samples to alternate virulenit type, considerinig total Ti and T2 col-onies to equal 100%.

not begin to show colonial type alteration untilthe 4th day. Although the colonial changes oc-curred at different rates among the volunteers, thechange seen in the FA pattern did not coincidewith the colonial changes.Animals other than man, such as mice, ham-

sters, rabbits, and chimpanzees, could not be in-fected with colonial types TI and T2 of N. gonor-rhoeae. Different routes of inoculation were usedon mice and chimpanzees without effect, exceptthat in some circumstances a rise in antibody titerwas demonstrated.FA and colonial patterns after treatment. There

was a progressive decline in the numbers of extra-cellular FA-positive diplococci with time aftertreatment. No alteration was observed in the in-tensity of FA staining of the individual diplococcias the result of treatment. FA-positive diplococciwere rarely observed at 6 to 12 hr after treatment,the length of time depending on the volunteer. Nocorrelation was found between the rates of colo-nial type change and the subsequent posttreat-ment decline of FA-positive diplococci in exu-dates. The rates of decline of the number of FA-positive diplococci in exudates corresponded tothe rates of declining numbers of viable cells foreach volunteer. Viable diplococci were obtainedfrom exudates of each volunteer during approxi-mately the same period posttreatment as FA-posi-

tive diplococci could be detected extracellular topolymorphonuclear leukocytes. The number ofcolonies isolated from the exudates remained ap-proximately constant for each volunteer for 3 hrafter treatment and then began to decline. Withsome variance among volunteers, the colonialtypes after treatment were the same as pretreat-ment, until 4 to 6 hr after treatment, when theybecame untypable and alike in morphologicalappearance. No change in colonial type was ob-served during the treatment period up to the timethey became untypable. This was expected sincethere was no difference reported between the colo-nial types of this strain in their susceptibility totetracycline, penicillin, chloramphenicol, erythro-mycin, and oleandomycin (Thayer, personal coin-munication) .

After 720 selective transfers in vitro over a pe-riod of 35 months, the cells of strain F62 TI colo-nies were still virulent for male volunteers. In eachcase, N. gonorrhoeae was reisolated from eachman each postinoculation day and characterizedin the same manner as after 17 months' in vitrocultivation. Two major differences were seen be-tween the results with the 17- and 35-monthinocula. First, there were more symptoms with35-month cell inoculum than 17-month cell inocu-lum where symptoms were mild to nonexistent,although there were moderate amounts of puru-

602 J. BACTERIOL.

VIRULENT N. GONORRHOEAE IN VITRO

lent exudate. The observation was considered sig-nificant, even though the physicians attending thesubjects were not the same at 17 months (A.L.S.)and at 35 months (I.R.C.). With 35-month cells,6 of 10 volunteers had a discharge within 24 hr,accompanied by a burning sensation. By 4 dayspostinoculation, these six men had all developed atender lymph node or testes, or both, and one hada tender epididymis. The remaining four mendeveloped discharges ranging from scant andwatery to profuse and bloody by 48 hr postinocu-lation. No correlation was observed between thevolunteers' response to inoculation and their pasthistory of infection. Second, the daily isolate colo-nial type was neither colonial type Ti nor T2, butresembled a cross between the two morphologies.This morphology had been seen previously duringroutine passaging and had been designated colo-nial type T21. Progeny of these T21 colonies werepredominantly TI in colonial morphology.Serologically, most of these volunteers becamereactive by the microprecipitin test within 24 hrpostinoculation and remained reactive for periodsranging from 50 days to 100 + days postinocula-tion (Fig. 7). Such reactivity was not stable uponstorage of the sera at -40 C, and the sera becamenonreactive within a few months, in contradistinc-tion to sera obtained from clinic patients thatretained reactivity for several years.

125

100

>- 75a

0

0" 50

2

25

DISCUSSION

A significant result of these studies is the dem-onstration of a close relationship between colonialmorphology of N. gonorrhoeae and its virulencefor humans. Since the human is the only knownmammal which can be infected by N. gonorrhoeae,the ability to identify the virulent cells and studytheir characteristics in vitro should expedite thedevelopment of serological tests and immunizingagents for this peculiarly human disease.Another conclusion to be drawn from these

results is that the genetic bases of colonial mor-phology and virulence in cells of colonial types Tiand T2 are closely related in the bacterial genome.There were many opportunities for a dissociationof these two characteristics during the 720 selec-tive transfers since the original isolation. Manytransient variations in colonial morphology withinthe described colonial characteristics were ob-served, which apparently resulted from environ-mental factors such as medium alterations, gasstate variability, agar-medium surface moisture,and others. Occasionally, variants of Ti mor-phology were detected that would perpetuatethemselves, indicating that a genetic change hadoccurred. The continued appearance of T3 and T4colonies in the Ti and T2 lines after many selec-tive in vitro transfers is probably the result of agenetic instability in the Ti cells rather than a se-lection for a coexistent cell type. Evidence for this

untested

0 reactive

non-reactive

53Z C ......

3

2 3 4 5 6 7

Individual Human Subjects8 9 10

FIG. 7. Precipitini test reactivities.

603VOL. 96, 1968

KELLOGG ET AL.

hypothesis is as follows. (i) The appearance of T3and T4 cells in Ti or T2 colonies is a discreteevent, as seen morphologically by the formationof "pie segments" of T3 and T4 cells in Ti or T2colonies; (ii) the frequency of their occurrence is aconstant for each strain; (iii) their rate of occur-rence was not affected by the nutritional characterof the medium; and (iv) Tl colonies could not beback-selected from T4 colony lines which hadbeen propagated in vitro for 2 years. In spite ofthese genotypic and phenotypic fluctuations,virulence and colonial morphology in Ti and T2colonies remained inseparable.A relationship between virulence and colonial

morphology has been observed in other genera ofbacteria (1, 2, 7, 14); however, in most cases, thishas been either an association of colonial mor-phology with a degree of virulence or an associa-.tion of these two characteristics in which eithercould vary independently of the other. In thelatter category also fall several characteristicsassociated with virulence, such as cell surfacestructure, enzymes, and toxins. Of these character-istics, only the first can be associated exclusivelywith either virulence or colonial morphology inN. gonorrhoeae. The cells of virulent colonies arerough in saline, a property which may be mani-fested colonially by their increased convexity.Differences in cell surface structure have been re-garded as reflecting variation in antigenic charac-ter (1). Differences have been observed betweenvirulent and avirulent cells of N. gonorrhoeae bymeans of direct and indirect FA procedures, geldiffusion studies, and serum sorptions which maybe associated with their antigenic structure (3, 11).

Antigenic differences between virulent andavirulent cells may be qualitative or quantitativein character; however, present evidence seems tosupport a quantitative distinction between viru-lent and avirulent cells. "Natural" antibodieshave been demonstrated in persons uninfectedwith N. gonorrhoeae (3). These antibodies mayresult from experiences with other members of thegenus Neisseria or members of other genera (3, 4,17; 0. Grados and W. H. Ewing, Bacterial Proc.,1965, p. 53). The host does recognize the virulentcell, as seen by the development of antibodiesdetectable by both the indirect FA (4) and pre-cipitin (16) procedures. Both virulent and aviru-lent N. gonorrhoeae cells possess heat-labile sur-face antigens which are related to their specificrather than common relationships (5) and whosepresence on the cell surface depresses reactivitiesof the common antigens. It is possible, therefore,that the specific antigens interfere with recogni-tion of the virulent cell. On the other hand, thevirulent cells are engulfed by host phagocytic cells,

as seen by the FA examination of exudate smears.Since the toxin levels of virulent and avirulentcells are approximately equivalent and the nodesdid not become tender, it may be that the rate ofremoval of virulent cells from the urethra to theregional lymph node is less than with avirulentcells. This could be a result of an inability of thephagocytic cells to degrade the virulent gonococcias effectively as the avirulent gonococci. This hasbeen noted with naturally acquired gonorrhealinfections (15). A recent review of phagocytosis(10) indicates that, with a few exceptions in theMycobacterium, Brucella, and Salmonella, mostbacteria are rapidly degraded after phagocytosis.Another author (18) feels that these exceptionscould be explained by a lack of opsonization be-fore ingestion. The elevated levels of antibodiesseen in sera of the volunteers (I. R. Cohen, D. S.Kellogg, Jr., and L. C. Norins, in preparation)and in sera of clinic patients without parallel dis-appearance of virulent cells would seem to indi-cate that any possible opsonins might be specific,not general, in character.The stability of the association of virulence with

an easily identifiable colonial characteristic andantigenic distinctiveness in N. gonorrhoeae shouldprovide a useful tool with which to study someaspects of virulence as it is related to phagocytosisin a disease peculiar to humans. The high inci-dence of gonorrhea, coupled with an increasingnumber of penicillin-resistant strains, highlightsthe importance of understanding the basic char-acteristics of the infection so as to devise moreeffective detection procedures, and to acquire theknowledge requisite for inducing partial or com-plete immunity.

ACKNOWLEDGMENTS

We thank Wilbur E. Deacon and M. BrittainMoore, Jr., for their support and encouragement dur-ing the 2 years required for these studies, and CharlesCravens for his conscientious and able technical as-sistance.

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p. 26-36. Symp. Am. Assoc. Advan. Sci. TheWilliams & Wilkins Co., Baltimore.

3. Cohen, I. R., and L. C. Norins. 1966. Naturalhuman antibodies to gram-negative bacteria:immunoglobulins G, A, and M. Science 152:1257-1259.

4. Cohen, I. R. 1967. Natural and immune humanantibodies reactive with antigens of virulentNeisseria gonorrhoeae: immunoglobulins G, M,and A. J. Bacteriol. 94:141-148.

5. Deacon, W. E., W. L. Peacock, Jr., E. M. Free-

604 J. BACTERIOL.

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6. Dondero, N. C. 1963. Simple and rapid methodfor demonstrating microbial capsules by phase-contrast microscopy. J. Bacteriol. 85:1171-1173.

7. Eigelsbach, H. T., W. Braun, and R. D. Herring.1951. Studies on the variation of Bacteriumtularense. J. Bacteriol. 61:557-569.

8. Gould, R. G., L. W. Kane, and J. H. Mueller.1944. On the growth requirements of Neisseriagonorrhoeae. J. Bacteriol. 47:287-292.

9. Gradwohl, R. B. H. 1963. Clinical laboratorymethods and diagnosis, vol. 1, 6th ed. C. V.Mosby Co., St. Louis.

10. Hirsch, J. G. 1965. Phagocytosis. Ann. Rev.Microbiol. 19:339-350.

11. Kellogg, D. S., Jr., and W. E. Deacon. 1964. Anew rapid immunofluorescent staining tech-nique for identification of Treponema pallidumand Neisseria gonorrhtoeae. Proc. Soc. Exptl.Biol. Med. 115:963-965.

12. Kellogg, D. S., Jr., W. L. Peacock, Jr., W. E.Deacon, L. Brown, and C. I. Pirkle. 1963.Neisseria gonorrhoeae. I. Virulence geneticallylinked to clonal variation. J. Bacteriol. 85:1274-1279.

13. Labzoffsky, N. A., and A. E. Kelen. 1961. Newcomplement fixing antigen for serodiagnosis ofgonorrhea. Can. J. Microbiol. 7:715-723.

14. Page, L. A., R. J. Goodlow, and W. Braun. 1951.The effects of threonine on population changes

and virulence of Salmonella typhimurium. J.Bacteriol. 62:639-647.

15. Pelouze, P. S. 1941. Gonorrhea in the male andfemale, 3rd ed. W. B. Saunders Co., Phila-delphia.

16. Reising, G., and D. S. Kellogg, Jr. 1965. Detec-tion of gonococcal antibody. Proc. Soc. Exptl.Biol. Med. 120:660-663.

17. Reyn, A. 1949. Serological studies on gonococci.I. Technique. Gono-reaction of "normal" rab-bits. Serological relationship between gonococciand pasteurellae. Acta Pathol. Microbiol.Scand. 26:51-70.

18. Rowley, D. 1962. Phagocytosis. Advan. Immunol.2:241-264.

19. Scherp, H. W. 1955. Neisseria and neisserial infec-tions. Ann. Rev. Microbiol. 9:319-334.

20. Thayer, J. D., and J. E. Martin, Jr. 1964. A selec-tive medium for the cultivation of N. gonor-rhoeae and N. meningitidis. Public Health Rept.79 :49-58.

21. U.S. Department of Health, Education, and Wel-fare. 1962 (Revised). Gonococcus-proceduresfor isolation and identification. U.S. PublicHealth Serv. Publ. 499.

22. White, L. A., and D. S. Kellogg, Jr. 1965. Neis-seria gonorrhoeae identitication in direct smearsby a fluorescent antibody-counterstain method.AppI. Microbiol. 13:171-174.

23. White, L. A., and D. S. Kellogg, Jr. 1965. An im-proved fermentation medium for Neisseriagonorrhoeae and other Neisseria. Health Lab.Sci. 2:238-241.

24. Wilson, J. F. 1954. A serological study of Neisseriagonorrhoeae. J. Pathol. Bacteriol. 68:495-510.

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