by robert i. and wesleyw. spinkdm5migu4zj3pb.cloudfront.net/manuscripts/103000/103041/... · 2014....

THE INFLUENCE OF ANTIBIOTICS ONTHE ORIGIN OF SMALLCOLONIES (G VARIANTS) OF MICROCOCCUS

PYOGENESVAR. AUREUS'

By ROBERTI. WISE AND WESLEYW. SPINK

(From the Department of Medicine, University of Minnesota Hospitals, and Medical School,Minneapolis, Minn.)

(Submitted for publication June 7, 1954; accepted August 5, 1954)

In attempting to select out penicillin-resistantcolonies of staphylococcus from sensitive culturesby the method of Kirby (1), small Gcolonies wereobtained. When isolated on plates of agar, thecolonies in some instances appeared so minute,that growth was detected only with the aid ofmagnification. A study of this phenomenon waspursued further with many cultures of staphylo-coccus and with different antibiotics, and minutecolonies were again isolated. The small coloniesreverted to the normal larger colonies when cul-tured in the absence of antibiotics. Since theminute colonies originated under the influence ofantibiotics commonly used in the therapy ofstaphylococcal diseases, the possibility presenteditself that these colonies might also appear in pa-tients while undergoing treatment with antibiotics.

This report is concerned first, with a brief sum-mary of the literature on G colonies; second, ex-periments relating to the in vitro conditions underwhich the minute colonies appeared with the dif-ferent antibiotics; third, changes in virulence andresistance to the antibiotics displayed by theparent colonies, small colonies, and the revertedlarge colonies; fourth, the sensitivity to bacterio-phage of parent colonies, G colonies and revertedcolonies; and, lastly, the isolation of minute col-onies from human patients with staphylococcaldisease.

REVIEW OF LITERATURE

Small colony variants have been described formany species of bacteria. Most of the variantsreported by investigators were obtained from cul-tures submitted to unfavorable metabolic influ-ences, such as chemicals, antibiotics, and aging.In some instances, isolation was from patients

1 Presented at the Forty-fifth Annual Meeting of theAmerican Society for Clinical Investigation, AtlanticCity, N. J., May 4, 1953.

following treatment with antibiotics. Interest insmall colonies was stimulated in 1931 by Hadley,Delves, and Klimek (2), who isolated very smallcolonies of Shigella from broth cultures containinglithium chloride, and reported that they passedthrough bacteriological filters, and suggested thatthey were gonidial mutants and part of a normallife cycle. Prior to their report, other workers(3-8) had noted similar types of colonies.

The method of Hadley, which incorporates se-lective inhibitory substances in bacteriologicalmedia, has been widely used for the isolation ofG colonies from cultures of different bacterial spe-cies. These substances include lithium chloride,barium chloride, 2-methyl-1,4-naphthoquinone,and sterile filtrates of bacterial cultures (9-23).Appearance of G colonies in cultures of filtrates of"transitional" colonies of Salmonella, which ap-peared on MacConkey agar, has also been re-ported (24). Small colonies of the G type havebeen isolated, without the aid of added inhibitorysubstances, from old cultures of bacteria and,yeasts (12, 25-37).

The selection of G colonies with antibioticsin vitro was first described by Schnitzer, Camagni,and Buck (20) when they observed small coloniesin areas where growth of staphylococci had beeninhibited by a crude extract of Penicillium no-tatum. These minute colonies reverted to normalcolonial forms when cultivated in the absence ofthe antibiotic. Similar observations were reportedby others (38, 39) who used more purified prepa-rations of penicillin. Barbour (40) noted thatstreptomycin caused the appearance of G formsduring the development of resistance in five strainsof Staphylococcus pyogenes.

The primary isolation of G cultures from ani-mal or human sources is of considerable clinicalinterest, for little is known concerning the pos-sible role of such bacterial variants in disease.

1611

ROBERT I. WISE AND WESLEYW. SPINK

Edwards (41 ) encountered "dwarf" colonies ofShigella equerulis, an organism associated with adisease of horses, in 25 per cent of post mortemexaminations. Bliss and Long (42) observedalmost invisible colonies of hemolytic strepto-cocci which were isolated from throats of patientswith upper respiratory infections. Small coloniesof gonococci were isolated by Morton and Shoe-maker (43) from 3 of 135 cases of gonorrhea.It was not known whether these three cases hadreceived treatment. Morris, Sellers, and Brown(44) isolated from blood, feces, urine, and sputumsmall colony variants of Eberthella typhosa from10 of many patients between 1934 and 1940. Halland Spink (45) isolated streptomycin-resistantand streptomycin-dependent small colonies ofBrucella abortus from a patient with subacute bac-terial endocarditis following treatment with strep-tomycin. Voureka (46) obtained small coloniesfrom urine of patients with urinary-tract infec-tions caused by Pseudomonas and coliform organ-isms following treatment with chloramphenicol.Very small colonies of staphylococcus have beenisolated in pure culture from closed abscesses ofthree patients. In the case reported by Hale (47)small colonies were isolated from the abscess, aswell as from the patient's nose. One of two pa-tients reported by Sherris (48) had never re-ceived antibiotics; the other had been treated withpenicillin.

The general characteristics of G colonies, whichhave been described for many species of bacteriaare similar in most instances but there have beensome reports at variance with each other. Thereported characteristics may be summarizedbriefly as follows: Colonies are translucent, some-times opaque, do not produce pigment and varyin size from 0.02 to 1 mm. (9, 12, 18, 24, 27, 40).Broth cultures exhibit slight turbidity. Stainedspecimens have revealed microorganisms whichwere described as normal and uniform, or irregu-lar and small. The germination period has beendescribed as 1 hour and 45 minutes to 3 hoursand 50 minutes for the G organisms, as comparedto 36 minutes for the parent organisms (27).Hemolytic, coagulating and fermentative activityis decreased. The size of the colonies is in-creased by cultivation in an environment of CO2or by the use of thiamine, or dextrose and blood

in the media (12, 17, 47). Bacteriophage sus-ceptibility between parent cultures and their Gcultures is reported to be different, the same, ordecreased in case of the G colony (9, 11, 21, 24,40). Cross agglutination with antisera producedwith the parent cultures and with the cultures ofG colonies is reported to occur in most cases (6,9, 12, 15, 26, 27, 30, 34, 41, 45). However, someinvestigators (2, 3) report no cross agglutinationand others (11, 24) state that antisera preparedagainst G cultures do not agglutinate parent cul-tures, whereas antisera prepared against parentcultures agglutinate both parent and G cultures.Most investigators (2, 9, 12, 41) have reportedthe G cultures to be nonvirulent; others (18, 24,26, 32) report a decrease in virulence; whereasHadley and Carapetian (15) found an increasein virulence. Heat resistance of G colonies is in-creased or decreased (9, 21, 22). The rate of re-duction of methylene blue is decreased (27, 31).There is a decreased catophoretic velocity (27,31). The most controversial property of the Gorganisms is the question of their passage throughbacteriological filters. They have been reportedby some (2, 9, 15, 24, 25, 28) to be filterable, butothers (12, 14, 16, 21, 26, 27, 30, 31, 33) havenot been able to demonstrate this characteristic.

The nature and function of G colonies are notknown. Hadley, Delves, and Klimek (2) believedthem to be part of a normal life cycle. Youmans(13) considered them to be bacteria with a tem-porary loss of inactivity of certain enzyme func-tions and a lowered rate of metabolism, broughton by an unfavorable environment. A similarview was expressed by Weinberg (17), whothought that they were biochemically deficientvariants. Lindegren (49) stated that the normalG-type transformation resembled very closely atransgenation (point mutation, gene mutation)which he believed to serve as an adaptive device.

MATERIALS ANDMETHODS

For the selection of G colonies of staphylococci withantibiotics, a method was employed similar to that usedby Kirby (1) in studying the action of penicillin onstaphylococci. Large inocula of a broth culture of staphy-lococci, which had been incubated for 18 hours, wereadded to each of several Klett-Summerson colorimetertubes containing 5 ml. of tryptose phosphate broth 2 and

2 Difco Laboratories, Inc., Detroit, Michigan.

1612

ANTIBIOTICS AND G COLONIES OF STAPHYLOCOCCUS

0 35

105, --/I00

5

06 2 1 24303642484 6 8 10 12 14hours Time days

FIG. 1. INFLUENCE OF VARYING CONCENTRATIONSOF PENICILLIN UPON BROTHCULTURESOF STAPHYLOCOCCUS

Lysis of the organisms occurred with all the concentrations, but G colonies ap-peared only in the tube with lowest concentration of penicillin01 unit per ml.

different concentrations of antibiotics. The antibioticsused were penicillin, streptomycin, chloramphenicol, baci-tracin, carbomycin, and oxytetracyc'ine in concentrationsof 0.1, 1.0, 10.0, and 100.0 units or micrograms per ml.,and erythromycin in concentrations of 0.25, 2.5, 5.0, 10,20, 40, and 80 micrograms per ml. The bacterial inocu-lum added to each tube was that amount necessary toproduce an increase of 25 units in the turbidity of broth,as measured by a Klett-Summerson photoelectric color-imeter, and varied from 0.3 to 1.0 ml. in all the experi-ments, but a constant amount was used with each anti-biotic. A series of control tubes were prepared to de-termine the rate of growth in the absence of antibiotics,to indicate the possibility of contamiration of uninocu-lated broth containing antibiotics, and to serve as a con-trol for turbidimetric readings. All tubes were incu-bated at 370 C. The turbidity of each tube was deter-mined at frequent intervals for 14 days. Every two daysa portion of the contents of each tube was subculturedto plates of trypticase soy agar.3 After incubation theagar plates were examined with the aid of a stereoscopicmicroscope and selected small colonies were subculturedin broth for 24 hours. Reverted large colonies (RV)were obtained from all G cultures. All cultures weremaintained on agar plates in the refrigerator and inbroth-at minus 100 C.

3 BAltimore Biological Products Laboratory, Baltimore,Md.

RESULTS

Thirty individual strains of coagulase-positivestaphylococci were studied. All strains weresusceptible to low concentrations of all the anti-biotics. The number of strains tested with eachantibiotic was as follows: penicillin, 20; erythro-mycin, 10; carbomycin, 9; bacitracin, 9; strepto-mycin, 13; oxytetracycline, 8; and chlorampheni-col, 8. Although many small colonies were se-lected for subculture, the majority yielded largecultures after 24 hours of incubation. However,fifteen stable G cultures were obtained. The num-ber of G cultures produced with each antibioticwas as follows: penicillin, 6; erythromycin, 2;carbomycin, 3; and bacitracin, 4. None was iso-lated in the presence of streptomycin and chloram-phenicol. Color changes which occurred withoxytetracycline precluded adequate determinationsof turbidity, and no G colonies were obtained withthis antibiotic. In most instances, G colonieswere isolated after the parent culture had beenlysed by the antibiotic, and were present in onlyone concentration of the antibiotic.

The pattern of changes of turbidity of the cul-

1613


0I40 -f / -f /

i1 / // 0

20o I? N'oo .

-I

E120

NI G - ~~~~~~colonies

40 0 units per mnl.

-1.0

20 ~ ~ ~ ~- 100

0 1 2 3 4 5 6 7 8 9 10 11Time in doys

FIG. 2. INFLUENCE OF VARYING CONCENTRATIONSOF BACITRACIN UPON BROTHCULTURESOF STAPHYLOCOCCUS

Note that lysis of the organisms occurred only with the highest concentration ofbacitracin-100 units per ml. G colonies were isolated only from the tube dis-playing lysis.

4 5Time in days

FIG. 3. INFLUENCE OF VARYING CONCENTRATIONSOF ERYTHROMYCINUPONBROTHCULTURESOF STAPHYLOCOCCUS

Lysis of the culture was followed by the appearance of G colonies in the tubehaving a concentration of 5 mcg. per ml.

1614


tures was characteristic, though different for eachantibiotic. With penicillin (Figure 1) the curves-of turbidity showed lysis occurring in all tubes.In agreement with Kirby (1), this took place morecompletely in the presence of the lower concentra-tions of antibiotic. After an initial clearing ofthe broth because of the lysis of the staphylococci,turbidity began to increase on the eighth day inthe broth containing 0.1 unit per ml. The sub-culture from this tube showed hundreds of G col-onies.

In the presence of bacitracin (Figure 2) rapidgrowth of the parent culture occurred in the lowerconcentrations, whereas lysis took place in thetube containing 100 units per ml. followed by theappearance of G cultures in this tube on the sev-enth day.

In the case of erythromycin (Figure 3) the bac-terial turbidity increased for one day with partiallysis occurring over a period of five to seven days,when small colony variants began to appear.

With carbomycin (Figure 4) G colonies wereobtained on the ninth day in the tube containing10 micrograms per ml.

Since it had been demonstrated that G coloniescould be selected out with different antibiotics,comparative studies were made with organismsfrom large colonies, G colonies and reverted largecolonies. These investigations included a studyof the general characteristics, susceptibility to bac-teriophage, sensitivity to the antibiotics, produc-tion of penicillinase and virulence for small ani-mals. Because reversion of the G colonies tolarge colonies occurred readily, carefully con-trolled measures had to be employed to detectwhether Gcolonies or reverted large colonies werebeing used in the initial inoculum. This was doneby inoculating the surface of agar plates with aportion of the broth culture used as the initialinoculum. Thus, after an incubation period of 24hours, the composition of the culture used at thebeginning of an experiment could be ascertained.Similarly, since reversions could occur during theperiod of observation both in vitro and in vivo,subcultures of the contents of tubes and materialfrom animals were made at the conclusion of theexperiment. Only in this way could significantobservations be interpreted.

Time in days

FIG. 4. INFLUENCE OF VARYING CONCENTRATIONSOF CARBOMYCINUPON BROTHCULTURESOF STAPHYLOCOCCUS

Lysis of the organisms in a concentration of 10 mcg. per ml. was followed bythe appearance of G colonies.

1615


FIG. 5. SHOWINGCOMPARATIVESIZE OF G COLONIES AND NORMAL-SIZED LARGECOLONIES OF STAPHYLOCOCCI

Small colonies in the presence of larger re-verted colonies of normal size are demonstratedin Figure 5. The Gcolonies were translucent andnonpigmented, having a diameter of less than 0.1mm., and were seen with difficulty except by re-flected light or with magnification. They could beeasily overlooked. Whencultivated in the absenceof antibiotics, they reverted to colonies of normalappearance. The reversion proceeded at slow orrapid rates; some cultures reverted almost com-pletely within 24 hours, while others remainedstable for periods of a month or more. The rateof reversion was characteristic of the strain.

Contrary to the report of Youmans and Delves(19), G colonies could readily be produced fromreverted cultures, and with much more successthan they could be obtained from normal parentcultures. The method used for procuring G cul-tures (G2) from reverted cultures (RV) was thesame as that employed for determining sensitivityof bacterial cultures to antibiotics with the tube-dilution method (50). This consisted of adding0.5 ml. of a 1-100 dilution of an 18-hour brothculture of the reverted strain to test tubes con-taining 0.5 ml. of twofold serial dilutions of the

antibiotic. After incubation of 24 hours, sub-cultures from the tubes containing the lowestconcentration of antibiotic in which no bacterialgrowth was visible showed the presence of G col-onies (G2) in most instances.

The stained cells of G cultures of staphylococciwere usually indistinguishable from those of theparent cultures in regard to uniformity in size,shape, and tinctorial properties. Some G strainsshowed pleomorphism with large swollen cells.The G cultures did not show any evidence ofgrowth in Gladstone's synthetic medium, whereasthis medium did support growth of the parent cul-tures (51). The G colonies were nonhemolytic.Although coagulase activity could not be demon-strated by the tube method in the usual threehours, some strains showed slight coagulase ac-tivity at 18 hours. It is possible that the coagu-lase activity at the end of this longer period wasdue to reverted cells. Penicillinase could not bedemonstrated. G cultures which were adaptedto grow in concentrations of penicillin up to 2000units per ml. did not manifest penicillinase activity.With reversion of the adapted penicillin-resistantG strains to the large colony forms there was a

1616


TABLE I

Penicillin-sensitivity of reverted cultures obtained fromadapted penicillin-resistant G cultures

StrainNo.

28283236

1A9

Units of penicillin per ml.inhibiting growth

G Revertedcultures cultures

125 <3.9500 0.5125 1.9

15.6 1.915.6 0.125

loss of the acquired resistance (Table I). The Gcultures were susceptible to the bactericidal ac-tion of human defibrinated whole blood, whereasthe parent and reverted strains were resistant tothis action. This was observed using a methoddescribed by Spink and Vivino (52). The cul-tures were incubated in blood for 24 hours at 370C. and tested for viability. The results are shownin Table II. With reversion there was a return

TABLE II

Bactericidal action of human defibrinated whole blood onparent, G, and reverted cultures

Bactericidal testtdilution of organisms

Strain*No. 102 103 104 106 106

Parent 79 + + + + 0G 79 0 0 0 0 0RV 79 + + + + 0

* All cultures were of equal turbidity.t + = growth.

to the original properties of the parent strainwith respect to hemolysin, coagulase, pigmentation,and characteristics of growth.

Bacteriophage typing was carried out with thecolonial variants to eliminate the possibility thatthe G or reverted cultures were contaminants, and

also, to compare their susceptibility to bacterio-phage action.4 Table III shows the results of typ-ing the parent (P), small colony variant (G) andreverted strains (RV) of strain 79, which hadbeen treated with penicillin, and strain 12, whichhad been treated with erythromycin. Of 11 setsof cultures studied (a total of 33 strains), threesets (9 strains) were nontypeable. Eight setsconsisting of 24 strains were typeable. Most ofthe G cultures showed no lysis with the dilutedbacteriophages which were effective for the pa-rent and reverted strains, but required undilutedbacteriophage for the demonstration of lysis. Itis significant that sensitivity to at least one bac-teriophage was retained by the G form, and thatreversion was accompanied by a return to a lyticpattern comparable or identical with that of theparent strain. Thus, in the process of comparingthe variants, there was no evidence of contamina-tion of the cultures.

In an attempt to obtain more information con-

cerning the selective action of penicillin for Gcolonies, a comparison of the penicillin-sensitivityof the related variants was made using the tube-dilution technique. Fifteen strains of G cultureswere isolated from one parent and compared totheir 15 reverted strains and duplicate culturesof the parent. Results showed growth of theparent strain to be inhibited by 0.06 units per

ml.; the G cultures were inhibited in a range from0.1 to 0.5 units per ml. and the reverted culturesfrom 0.06 to 0.12. This indicates that the G cul-tures were two to eightfold more resistant thanthe parent strain, and on reversion there was a

return to a sensitivity similar to that of the parent,

4 Bacteriophage studies were performed with the co-

operation of Dr. John Blair, Hospital for Joint Diseases,New York.

TABLE III

Comparative results of typing variants of two strains of staphylococcus with bacteriophages *

Source of culture Bacteriophages producing lysis

Parent-A 79 7 42C 42D 53 55 52AG colonies-G 79 42DReverted colonies-RV 79 7 42D 47B 47C 53 55 52A

Parent-12 E 42B 47CG colonies-G 12E 42BReverted colonies-RV 12E 42B 47C

* The parent culture of strain 79 had been treated with penicillin, and that of strain 12 with erythromycin.

1617


which confirms the observations of others (20,38). On the other hand, Hale (21) reported thatthe G colony was four times more sensitive topenicillin than the parent strain. Sensitivity ofrelated variants in which the G culture had beenselected with penicillin showed no significant dif-ferences in sensitivity to chlortetracycline. Incomparing related variants in similar studieswith erythromycin, carbomycin, and bacitracin,there was observed a two to threefold increase inresistance of the Gcultures over the parent strains;however, on reversion a decrease in resistancecould not be adequately demonstrated since, asindicated previously, the reverted strains pro-duced many G colonies when placed in the anti-biotics.

Comparative virulence studies with related vari-ants were carried out by injecting the strains intra-dermally into rabbits and noting the skin reac-tions, and intravenously into mice and observingthe lethal effect (53). For these studies all cul-tures were incubated 24 hours in tryptose phos-phate broth. To standardize the inocula for in-jection, the parent and reverted cultures werediluted with sterile broth to a degree of turbidityequal to the turbidity of the Gculture. The num-ber of bacteria could not be determined in the

22

20

18 S

16 's \CP.14 *4>

1 2 - *A

I 04

0)n8E

z-

G cultures since subsurface colonies could not bereadily seen with the plate method. The inoculumof the diluted parent and reverted cultures, how-ever, contained from 200,000,000 to 1,200,000,000cells per ml. All intradermal and intravenous in-jections consisted of 0.25 ml.

In the rabbit the sites of intradermal injectionsof the G cultures were just discernible after 48hours. But at the sites of injections of the parentand reverted strains, there were edema, erythema,and pustule formation, and after four to six daysareas of necrosis appeared. The reverted strainsproduced necrotic reactions in the skin that wereof similar or greater severity than the reactionproduced by the related parent strains.

The lethal results following the intravenous in-oculation of mice are charted in Figure 6. Twentywhite mice of the same age, weight, and geneticstrain were injected with each culture of the re-lated variants. At the end of 120 hours there was100 per cent survival of mice receiving the G cul-ture. These mice continued in apparent goodhealth for two weeks, at which time they weresacrificed and G cultures were isolated from theirkidneys. At the end of 24 hours the fatality ratesof the "reverted" and "parent" groups were 90per cent and 25 per cent, respectively. All mice

Time in hours

FIG. 6. SURVIVAL CURVES OF MICE FOLLOWING THE INTRAVENOUS INJECTIONOF PARENT, G AND REVERTEDCULTURESOF A STRAIN OF STAPHYLOCOCCUS

1618


receiving the reverted strain were dead in 72hours and all receiving the parent strain weredead in 120 hours. Cultures of the spleens, livers,and kidneys of mice which had been injected withthe parent and reverted strains revealed staphylo-cocci in great numbers. The outstanding featuresof these studies were first, the increased virulencewhich occurred with reversion of the avirulentsmall-colony variants, and second, the continuedexistence of the G cultures in the kidneys of ap-parently normal mice.

The presence of G colonies of staphylococci inhuman material was investigated by examiningwith magnification the original cultures of ap-proximately 500 specimens of blood, exudate, urine,and other body fluids. Ten cultures of G col-onies were isolated from eight patients, as shownin Table IV. Seven of the patients were being

TABLE IV

Isolation of G colonies of staphylococcus fromhuman patients

No. of Treatment receivedculture Source of G colonies by patients

1 Fluid from chest cavity Penicillin2 Urine Gantrisin3 Throat Penicillin

Streptomycin4 Throat Penicillin

Streptomycin5 Urine Chlortetracycline6 Urine Gantrisin

Chlortetracycline7 Blood Penicillin8 Urine None

treated with an antibiotic or sulfonamide at thetime the isolates were made. When the first eightcultures were examined, there was no knowledgeof the identity of the specimens or the history ofthe patient. It is interesting that cultures 3 and4 were from the same patient. Gram stains of Gcultures 1 to 8 inclusive revealed Gram-positivecocci. These cultures gradually reverted to col-onies of normal size. The characteristics of cul-tures 9 and 10 differed from the others and were

obtained under the following circumstances: Pa-tient C. C. was a 77 year old male with cystitiscaused by Staphylococcus aureus, which appearedto be present in pure culture. An attempt was

then made to study the production of G coloniesin vivo in this patient. He was treated with

erythromycin, 250 mg. every six hours. Urinewas collected daily for culture, and the plateswere examined carefully for G colonies with theaid of magnification. There was a gradual elimi-nation of normal staphylococcal colonies over aperiod of four days. On the fifth day there weretwo normal colonies and many microcolonieswhich could be seen only with magnification.These small colonies were transferred to brothand to agar. Stained smears of these colonies re-vealed Gram-positive pleomorphic rods withcocco-bacillary and coccoid forms similar to thosedescribed by Voureka (46), which she had iso-lated from urine during therapy with chloram-phenicol. These colonies remained stable throughmany subcultures and did not revert to large col-onies.

DISCUSSION

It has long been known that bacteria producesmall colony variants and that these forms can berecovered more readily when the cultures aresubjected to unfavorable environments. In thepresent studies, G cultures have appeared underin vitro experimental conditions, and in patientsfollowing exposure of bacteria to antibacterialagents. Under in vitro conditions, there existsan optimum concentration of antibiotic for re-covering G colonies. The G forms are two toeight times more resistant than are the parentcells. Therefore, the slow growing G variantscan be isolated most readily with concentrationsof antibiotic that eliminate or inhibit growth ofthe normal forms, and permit the G forms to re-produce. This optimum concentration falls withina narrow range. If the concentration is reduced,the normal forms reproduce rapidly and overgrowthe less active G forms. If the concentration ofantibiotic is too great, there is inhibition or elimi-nation of the normal parent cells, as well as theG forms.

The G cultures are devoid of hemolysin andcoagulase activity; there is an increase in nutri-tional requirements; and biochemical activity isdiminished. The G variants have a loss of viru-lence and remain viable in animal tissues withoutproducing signs of infection. Reversion to cul-tures of large colonies in vivo has not been proved.The small forms remain stable in the presence ofantibiotics in concentrations that do not destroy

1619

2ROBERTI. WISE AND WESLEYW. SPINK

them. When cultivated in the absence of antibi-otics, G colonies revert to large colonies, and thereis a restoration of the reproduction of hemolysinand coagulase. The reverted form is as virulentas the original parent strain, and in some cases ismore virulent. The reverted strain is similar butnot identical to the parent, because the revertedstrain may display increased virulence, and G col-onies are obtained from reverted cultures morereadily than they can be selected from the parent.Bacteriophage typing of related strains provedtheir genetic relationship and eliminated the pos-sibility that these cultures were contaminants.

Small colony variants have been isolated fromhuman sources, in which the patients with oneexception gave a history of therapy with antibi-otics or sulfonamides. It is not unlikely that op-timum concentrations of antibiotics for the sur-vival of G colonies may occur in human tissuesduring therapy of staphylococcal infections, andunder these conditions avirulent small-colony vari-ants may be selected out, and remain undetected.Following cessation of therapy, reversion to avirulent form may occur, and relapse of the infec-tion may ensue. Careful bacteriological studiesshould include a search for G colonies subse-quent to apparently successful treatment in pa-tients with staphylococcal disease. Further in-vestigations are now under way to determine howlong the G variants will remain in tissues and un-der what conditions reversion to the large virulentcolonies will occur.

CONCLUSIONS

1. Small colony variants (G variants) ofstaphylococci were selected out with the aid ofantibiotics.

2. The characteristics of the G variants werestudied and they are as follows: Compared to theparent strain, they have less hemolytic activityand produce less coagulase. The nutritional re-quirements are more demanding. The G culturesare less virulent, and remain viable in tissues ofapparently normal animals. The G cultures weretwo to eightfold more resistant to the antibioticsthan the parent cultures.

3. Reversion of G cultures to large coloniestook place when cultivated in the absence of anti-

biotics. The characteristics of the reverted formswere the same as the parent cells, except the viru-lence of the reverted forms was increased in someinstances.

4. Bacteriophage typing of the parent cells, andrelated G forms and reverted large colonies,showed a genetic relationship, which ruled outthe possibilities of contamination.

5. Cultures of G colonies were obtained fromhuman sources following treatment with anti-bacterial drugs.

REFERENCES

1. Kirby, W. M. M., Bacteriostatic and lytic actions ofpenicillin on sensitive and resistant staphylococci.J. Clin. Invest., 1945, 24, 165.

2. Hadley, P., Delves, E., and Klimek, J., The filter-able forms of bacteria. I. A filterable stage inthe life history of the Shiga dysentery bacillus.J. Infect. Dis., 1931, 48, 1.

3. Jacobsen, K. A., Mitteilungen uiber einen variablenTyphusstamm (Bacterium typhi mutabile), sowiejiber eine eigentumliche hemmende Wirkung desgewohnlichen Agar, verursacht durch Autokla-vierung. Centralb. f. Bakt. 1910, Abt. 1, 56, 208.

4. Eisenberg, P., Untersuchungen jiber die Variabilititder Bakterien. IV. Ueber den Variationskreis desB. prodigiosum und B. violaceum. Centralb. f.Bakt., 1914, Abt. 1, 73, 449 and 466.

5. Baerthlein, K., Ueber bakterielle Variabililat, ins-besondere sogenannte Bakterienmutationen: Cen-tralb. f. Bakt., 1918, 1, 81, 369.

6. Fiirth, J., Variationsversuche mit Paratyphus :S.(Weil). Ztschr. f. Immunititsforsch u. exper.Therap., 1922, 35, 155.

7. Fiirth, J., Rezeptorenanalyse und Variationsversuchemit B. Paratyphus Aertryck. Ztschr. f. Im-munitatsforsch u. exper. Therap., 1922, 35, 162.

8. Friedberger, E., Ueber Bacterienvirus (kryptanti-genes Virus) nach Versuchen mit Typhusvirus.Ztschr. f. Immunitatsforsch u. exper. Therap., 1927,53, 339.

9. Hoffstadt, R. E., and Youmans, G. P., Staphylococcusaureus: Dissociation and its relation to infectionand to immunity. J. Infect. Dis., 1932, 51, 216.

10. Hoffstadt, R. E., and Youmans, G. P., The geneticsignificance of the dissociants of Staphylococcusaureus. J. Bact., 1934, 27, 551.

11. Hoffstadt, R. E., Youmans, G. P., and Clark, W.,Antigenic structure of Staphylococcus aureus andits variants. J. Bact., 1934, 27, 97.

12. Swingle, E. L., Studies on small colony variants ofStaphylococcus aureus. Proc. Soc. Exper. Biol.& Med., 1934, 31, 981.

1620


13. Youmans, G. P., Production of small-colony variantsof Staphylococcus aureus. Proc. Soc. Exper. Biol.& Med., 1937, 36, 94.

14. Rettger, L. F., and Gillespie, H. B., Bacterial varia-tion, with special reference to pleomorphism andfilterability. J. Bact., 1933, 26, 289.

15. Hadley, P., and Carapetian, H., A study of theinfective qualities possessed by G phase culturesof Bacillus typhosus. J. Bact., 1933, 25, 94.

16. Raney, M. E., and Kopeloff, N., Dissociation and fil-tration studies with L. acidophilus. J. Bact., 1934,27, 45.

17. Weinberg, E. D., Vitamin requirements of dwarfcolony variants of bacteria. J. Infect. Dis., 1950,87, 299.

18. Duff, D. C. B., Dissociation in Bacillus salmonicida,with special reference to the appearance of a Gform of culture. J. Bact., 1937, 34, 49.

19. Youmans, G. P., and Delves, E., The effect of in-organic salts on the production of small colonyvariants by Staphylococcus aureus. J. Bact., 1942,44, 127.

20. Schnitzer, R. J., Camagni, L. J., and Buck, M., Resist-ance of small colony variants (G-forms) of a

staphylococcus toward the bacteriostatic activityof penicillin. Proc. Soc. Exper. Biol. & Med.,1943, 53, 75.

21. Hale, J. H., Studies on staphylococcus mutation:Characteristics of the "G" (gonidial) variant andfactors concerned in its production. Brit. J. Ex-per. Path., 1947, 28, 202.

22. Colwell, C. A., Small colony variants of Escherichiacoli. J. Bact., 1946, 52, 417.

23. Hoffstadt, R. E., and Almaden, P., Dissociation ofStaphylococcus aureus. III. Relation of bacterio-phage to the dissociation of Staphylococcus aureus.

J. Infect. Dis., 1934, 54, 253.24. Haddow, A., Small-colony variation in B. paratypho-

sus B (Tidy) and other bacteria, with special ref-erence to the G type of Hadley. J. Infect. Dis.,1938, 63, 129.

25. Novak, M. V., and Henrici, A. T., Pleomorphic or-

ganism showing relationships between staphylo-cocci and actinomycetes. J. Infect. Dis., 1933, 52,253.

26. Swingle, E. L., Studies on small colony variants ofStaphylococcus aureus. J. Bact., 1935, 29, 467.

27. Chinn, B. D., Characteristics of small colony variantsof Shigella paradysenteriae Sonne and Staphylo-coccus aureus. Proc. Soc. Exper. Biol. & Med.,1936, 34, 237.

28. Colien, F. E., A study of microbic variation in a

yellow pigment-producing coccus. J. Bact., 1935,30, 301.

29. Dienst, R. B., A study of some of the factors pro-

moting dissociation of Bacterium dysenteriae Sonne.J. Bact., 1933, 26, 489.

30. Koser, S. A., and Dienst, R. B., Small colpny vari-ants or G forms of Eberthella dysenteriae Sonne.J. Infect. Dis., 1934, 54, 131.

31. Chinn, B. D., Characteristics of small colony variantswith special reference to Shigella paradysenteriaeSonne. J. Infect. Dis., 1936, 59, 137.

32. Roe, A. F., Dissociation of Cl. welchii. J. Bact., 1934,27, 46.

33. Kopeloff, N., Dissociation and filtration of Lactobacil-lus acidophilus. J. Infect. Dis., 1934, 55, 368.

34. Raven, C., Dissociation of the gonococcus. J. In-fect. Dis., 1934, 55, 328.

35. Raven, C., Dissociation of the gonococcus. Proc.Soc. Exper. Biol. & Med., 1934, 31, 899.

36. Fabian, F. W., and McCullough, N. B., Dissociationin yeasts. J. Bact., 1934, 27, 43.

37. Huddleson, I. F., and Baltzer, B., The characteristicsand dissociation pattern of type G (micro-colonytype) of Brucella abortus in Studies in Brucellosis,III. A series of five papers. East Lansing, Michi-gan State College, Agr. Exp. Sta., Department ofBacteriology and Public Health, 1952, p. 64.

38. Youmans, G. P., Williston, E. H., and Simon, M.,Production of small colony variants of Staphlococ-cus aureus by the action of penicillin. Proc. Soc.Exper. Biol. & Med., 1945, 58, 56.

39. Eriksen, K., Studies on induced resistance to peni-cillin in staphylococci. Acta path. et microbial.Scandinav., 1946, 23, 284.

40. Barbour, R. G. H., Small colony variants ("G" forms)produced by Staph. pyogenes during the develop-ment of resistance to streptomycin. Australian J.Exper. Biol. & M. Sc., 1950, 28, 411.

41. Edwards, P. R., Studies on Shigella equirulis. III.The occurrence of dwarf colony variants. Ken-tucky Agr. Exp. Sta. Bull., 1931, No. 320, 320.

42. Bliss, E. A., and Long, P. H., Studies on minutehemolytic streptococci. I. The cultural character-istics of minute hemolytic streptococci. J. Bact.,1934, 27, 105.

43. Morton, H. E., and Shoemaker, J., The identificationof Neisseria gonorrhoeae by means of bacterialvariation and the detection of small colony formsin clinical material. J. Bact., 1945, 50, 585.

44. Morris, J. F., Sellers, T. F., and Brown, A. W., Theprimary isolation of small colony strains of Eber-thella typhosa from blood, feces, urine, and sputum.J. Infect. Dis., 1941, 68, 117.

45. Hall, W. H., and Spink, W. W., In vitro sensitivityof Brucella to streptomycin: Development of re-sistance during streptomycin treatment. Proc. Soc.Exper. Biol. & Med., 1947, 64, 403.

46. Voureka, A., Bacterial variants in patients treatedwith chloramphenicol. Lancet, 1951, i, 27.

47. Hale, J. H., Studies on staphylococcus mutation:A naturally occurring "G" gonidial variant and its

1621


carbon dioxide requirements. Brit. J. Exper.Path., 1951, 32, 307.

48. Sherris, J. C., Two small colony variants of Staph.aureus isolated in pure culture from closed infectedlesions and their carbon dioxide requirements.J. Clin. Path., 1952, 5, 354.

49. Lindegren, C. C., Genetical studies of bacteria. III.Origin of G-type colonies by transgenation. Zen-tralbl. f. Bakt., II, 1935, 93, 389.

50. Waisbren, B. A., Carr, C., and Dunnette, J., Thetube dilution method of determining bacterial sen-

sitivity to antibiotics. Am. J. Clin. Path., 1951,21, 884.

51. Gladstone, G. P., The nutrition of Staphylococcusaureus: nitrogen requirements. Brit. J. Exper.Path., 1937, 18, 322.

52. Spink, W. W., and Vivino, J. J., The coagulase testfor staphylococci and its correlation with the re-

sistance of the organisms to the bactericidal ac-

tion of human blood. J. Clin. Invest., 1942, 21, 353.53. Gorrill, R. H., Experimental staphylococcal infec-

tions in mice. Brit. J. Exper. Path., 1951, 32, 151.

1622

by robert i. and wesleyw. spinkdm5migu4zj3pb.cloudfront.net/manuscripts/103000/103041/... · 2014....

Documents