COLONIAL MORPHOLOGY AND VIRULENCE OF PASTEURELLA PESTIS'

DANIEL M. EISLER, GAYLE KUBIK, AND HERBERT PRESTON

The Naval Biological Laboratory, School of Public Health, University of California, Berkeley, California

Received for publication February 3, 1958

The relationship of the colonial morphology ofPasteurella pestis to its virulence has beencontroversial. Gotschlich (1900), Markl (1914),Petrie (1929), Burgess (1930), Otten (1936),Bhatnagar (1940) and Jawetz and Meyer (1943)described numerous colonial types for whichinterchangeable descriptions are lacking. Mostagreed upon an inconstant hereditary trans-mission of colonial characteristics in formsdiffering from the usual smooth, pearly typecommon to many strains of P. pestis.On the other hand, Pirie (1929), Rachinsky

(1930), Bessonowa and Lenskaja (1931), Watsand Puduval (1940) and Crumpton and Davies(1956) describe more or less stable smooth orrough colonial morphology in various strains.

Inability to correlate virulence with colonialmorphology has been common. Thus, Markl(1914), Pirie (1929), Burgess (1930), Otten(1936), Wats and Puduval (1940), Jawetz andMeyer (1943) and Burrows and Bacon (1954)found avirulent and virulent rough and smoothcolonial types in numerous strains. An extremeopinion was held by Bessonowa and Lenskaja(1931) who considered all virulent strains to berough. Pollitzer (1954), however, considers itprobable that the smooth type is characteristicof virulent freshly isolated strains.

This communication describes a simple methodwhich emphasizes the differences between smoothand nonsmooth colonies of P. pestis. It is ob-served that the hereditary transmission ofcolonial characters is not as unstable as has beenthought. The resultant definition of smooth andnonsmooth forms has permitted reconsiderationof the relation of colonial morphology to the

I This work was supported by the Office of NavalResearch. The opinions contained in this reportare not to be construed as reflecting the views ofthe Navy Department or the Naval Service atlarge. (Article 1252 U. S. Navy Regulations,1948.) Reproduction in whole or in part is per-mitted for any purpose of the United States Gov-ernment.

taxonomy, virulence, immunology, and bio-chemistry of P. pestis.

MATERIALS AND METHODS

Selection of smooth and nonsmooth forms of P.pestis. Representative smooth and nonsmoothcolonies were recovered from stock strains bydilution of slant (blood agar base (BAB, Difco)containing 3 per cent whole human blood)growth in 1 per cent peptone water and theinoculation of aliquots thereof upon thick (40to 50 ml) blood agar base plates. Colonies(optimally 25 to 125 per plate) were incubatedat 28 C for 2 days followed by 6 to 8 days storageat room temperature. Their numbers andmorphology were observed. One strain (138) ofP. pestis provided by Dr. Alcor Brown of theLaboratories, Department of Health, State ofCalifornia, was analyzed directly from materialaspirated from a bubo in a human being.A similar technique was employed to study

the progeny of selected smooth and nonsmoothcolonies grown in heart infusion broth (HIB,Difco) and replated upon the surfaces of bloodagar base plates. In some cases, where nonsmoothstrains were characterized by a high frequencyof dissociation to smooth, the isolated colonieswere picked off the agar, suspended in 1 per centpeptone water, heart infusion broth or saline andused directly in various tests. In such casessmooth colonies arising from the same parentstock were similarly chosen for study.A specific bacteriophage, developed by Gun-

nison et al. (1951) was used as an aid in establish-ing the purity of P. pestis recovered both frombroth and agar cultures and from organs ofinfected mice. The bacteriophage was propagatedon a stock strain (139L) of P. pestis grown inheart infusion broth. Portions, of the Seitzfiltered bacteriophage cultures were tested forsterility by incubation for 48 to 72 hr in heartinfusion broth.

Virulence tests. The Namru strain of whitemice (Garber and Hauth, 1950) used in these

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Figure 1. Smooth forms of Pasteurella pestis strain 53Figure 2. Nonsmooth forms of Pasteurella pestis strain 53

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MORPHOLOGY AND VIRULENCE OF P. PESTIS

experiments is susceptible to infection with 2 to5 virulent P. pestis. Subcutaneous LD5o valueswere derived by correlating percentage mortalityand estimated dose per animal (EDA) data uponthe Linearized Mortality Grid devised by Gold-berg et al. (1954). Usually 4 groups of 8 miceeach were injected with 0.1 ml amounts ofappropriate decimal serial concentrations oforganisms.

RESULTS

Figures 1 and 2 show typical smooth and non-

smooth colonies obtained from P. pestis strain53. Slight variations of these forms occur in otherstrains. The forms may be differentially describedby the scheme designed by Buck (1944). By hisclassification the smooth is IV (convex), X(entire) and XXI (opaque) and XXIII (finelygranular) and has a pearly lustre. The non-

smooth form is V (convex papillate), XIII(undulate) and XXI (opaque). In daylight ithas a dull, ground glass appearance.

Table 1 describes the colonial morphology andvirulence for mice of parent smooth and non-

smooth stocks subcultured upon thick blood agar

base after 24 hr growth in heart infusion brothat 28 C. The results parallel those obtained by

suspending portions of the stock slant growthsin peptone water or heart infusion broth andsubculturing upon blood agar base with no

intervening period of incubation. The datapresented for 5 strains are typical of 31; limita-tion of space does not permit presentation of alldata.Smooth forms of subculture maintained their

characteristic morphology more readily thandid several nonsmooth strains. However, byrepeated selection in vitro the latter could bestabilized. Virulent nonsmooth colonies whenhomogeneously suspended in 1 per cent peptonewater and injected into mice were recoverable inpure culture from the spleen. No difficulty was

met in maintaining true breeding, nonsmoothcolonies of strain Poona by selective passage inmice, one passage only being required to establisha pure culture of this type. Pure smooth formswere similarly recovered. The results indicatethat both smooth and nonsmooth variants are

entities capable of survival during mouse passage.

Further evidence supporting the view thatsmooth and nonsmooth variants of P. pestis are

equally capable of survival may be obtainedfrom in vitro generation time studies. Deter-minations by the method of Wilson and Miles

BLE 1*The colonial morphology and mouse virulence of Pasteurella pestis variants

grown on thick blood agar base medium

Nonsmooth Smooth Mortality Data

Strain No.tColony count Per cent Colony count Per cent No. of organismst condence interval

31 Pt 0 0 126 100 >370St 0 0 106 100 >308

37 P 144 29 347 71 12 4.5 to 25NSt 54 98 1 2 3.2 1.4 to 6.5S 0 0 57 100 >98

53 P 39 80 10 20 27 11 to 60NS 67 98 1 2 12 4.5 to 25S 0 0 317 100 1.2 0.48 to 2.6

59 P 710 100 2 0.3 15 6 to 31NS 969 99 5 0.5 5 2 to 10S 0 0 291 100 2,400 1400 to 7000

137 P 16 2 798 98 1.4 0.87 to 2.8NS 109 100 0 0 25 10 to 55S 0 0 597 100 1.5 0.6 to 3.2

* This table presents typical data for 5 of 31 strains observed.t P = parent (original stock); S = smooth variant; NS = nonsmooth variant.t Expressed as number of organisms per LD60 following subcutaneous injection of Namru mice.

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TABLE 2Virulence and source of parent, nonsmooth, and smooth stock cultures of Pasteurella pestis

P.pesi aet Non- Smoh Year of LoStraineNo. r smooth Smooth Ion ation of Isolation SourceNo.moh.slto

WyomingNevadaIdahoOregonNew MexicoColoradoLos Angeles, Calif.Yreka, Calif.New MexicoJaffa, IsraelJaffa, IsraelSan Francisco, Calif.

Yreka, Calif.TunisiaArmyOregon

NevadaOregonArmy 53New Mexico

IndiaJaffa, IsraelHaifa, IsraelSiamHawaiiTexasG.Hooper Foundation,San Francisco, Calif.

IndonesiaPoona, Western IndiaCorona, CaliforniaJaffa, Israel

Lice from guinea pigGuinea pig tissueGuinea pig tissueGuinea pig tissueFleas from guinea pigFleas from guinea pigRatHumanFleas from guinea pigUnknownUnknownIndian rat

HumanHuman buboUnknownFleas from mouse

Fleas from guinea pigHumanUnknownHuman

HumanUnknownRatUnknownHuman lungFleas from guinea pigUnknown

Human, bloodHumanHuman buboUnknown

* A = avirulent; V = virulent.t Strain 51: 2 of 4 smooth and 1 of 4 nonsmooth colonies were found aviruilent in Namru mice.

(1946), and based on viable cell numbers, were

found to be 68 min for parent, smooth, andnonsmooth forms of strain 139L.

In table 2 the 31 strains are categorized as tocolonial morphology and virulence. In additionsome epidemiological data have been listed foreach strain. All strains proving completelysmooth (12 of 31) were also avirulent for Namrustrain mice over a dose range of from 1 to 4 x

100 to 1 to 4 X 102 cells, the range within whichvirulent strains are lethal. In 4 other strainsonly the nonsmooth colonial types were virulent,

the smooth avirulent. In 4 strains the smooth andnonsmooth types were both avirulent. Finally in11 strains both smooth and nonsmooth formswere virulent.

Sugar fermentation tests performed by con-

ventional methods showed biochemical dif-ferences between the two forms. Table 3 presentsthe differential fermentation reactions after 21days incubation of P. pestis strain 139L andvariants, parent, smooth, and nonsmooth, and,for comparison, the avirulent morphologicallysmooth strains EV 76 and A 1122. No dif-

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MORPHOLOGY AND VIRULENCE OF P. PESTIS11

TABLE 3

Selected carbohydrate reactions of Pasteurella pestisstrains A 1122, EV 76, 139L, 189L smooth, and

139L nonsmooth*

Strains of Pasteurella pestis

Carbohydrate 139LA 1122 EV 76 139L S139L Non-miloot smooth

Lactose ....... Alkt Act Alk Ac AlkXylose ....... Alk Alk Alk Ac AlkTrehalose ..... Alk Ac Alk Ac AlkArabinose...... Alk Ac Alk Ac Alk

* Final reading after 21 days incubation inphenol-red broth base (Difco) containing 0.1 percent of indicated carbohydrates.

t Alkaline reaction.t Acid reaction.

ferences were observed as a result of growth inthe following media: phenol-red broth base(Difco) plus glucose, sucrose, maltose, mannite,i-inositol, dulcitol, melibiose, galactose, i,rham-nose, raffinose, levulose, D-sorbitol, inulin andD(-)ribose. Subcultures of all carbohydratebroths 3 weeks after inoculation resulted inrecovery of viable organisms in all cases but thatof glucose. Other smooth and nonsmooth strainshave shown differences in their fermentationreactions, which, however, were variable fromstrain to strain and would not serve as a basisfor classification without further study.With the aid of bacteriophage, carbohydrate

fermentations and the characteristic pathologicchanges occurring during animal infection it wasnot difficult to establish the identity of virulentnonsmooth strains as P. pestis. The specificity ofthe smooth avirulent form might, however, bequestioned.

Bacteriophage tests and inability to utilizerhamnose (D'Aunoy, 1923; Bessonowa, 1930;Jawetz, 1940) partially proved the identity ofthe smooth avirulent strains. Further, separateexperiments were performed to prove the im-munological specificity of avirulent smoothvariants of P. pestis, strains 139L, EV 76, and A1122. A high rate of protection was afforded allvaccinated mice against challenge with virulentstrain 139L (2.4 x 106 to 2.4 X 108 viableorganisms per dose) which killed nominally 100per cent of the control mice.

DISCUSSION

From the evidence provided by this study it isnot surprising that there is a disparity of opinionon the relationship of virulence and colonialmorphology of P. pestis. P. pestis can no longerbe assumed to be homogeneous in colonialmorphology. The demonstration of smooth andnonsmooth forms upon agar plates inoculatedwith adequately separated cells of parent stockis not necessarily explained as a newly arisingmutation since the forms may have coexistedeven in the natural state, as was indicated by thedirect isolation from material aspirated from aplague bubo of both smooth and nonsmoothvirulent types. The question of mutation inrespect to the smooth z nonsmooth dissociationis another matter and will be treated in anothercommunication.

While smooth forms breed true, with rare dis-sociation to the nonsmooth type, the nonsmoothform when isolated and subcultured varies inits ability to maintain its morphology. Somestrains of P. pestis give rise to pure line non-smooth forms while the selected nonsmoothcolonies of others are characterized by a highrate of dissociation to smooth. These observa-tions are at variance with those of Jawetz (1940)who states that no true breeding colony formshad ever been obtained in plague.With the reservation that these observations

have been limited to the study of a stock culturecollection, these results broadly suggest thefollowing: no virulent parent stock is entirelycomposed of smooth constituents; virulent parentstrains may contain virulent or avirulent smoothforms, but generally possess virulent nonsmoothcomponents; no avirulent parent is entirelycomposed of nonsmooth elements; many aviru-lent strains are completely smooth; no stockparent strain, virulent or avirulent, is free ofsmooth forms. The data further suggest thatavirulence in a strain originally composed ofvirulent smooth and nonsmooth constituentsmay first have become an attribute of themajority of the smooth elements, secondarily ofthe nonsmooth as well as of the smooth, andthat persistence of virulence is greater in thenonsmooth even though this form may be theminority population in the composite parentstrain. It appears that the wild population of P.pestis is a complex of the smooth and nonsmoothboth of which may be virulent.

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Since no geographical or biological sourcespeculiar to smooth or nonsmooth morphologicaltypes were found it is concluded that the mixedmorphological-virulence characteristics are as-sociated with strains of P. pestis irrespective ofplace of origin.

SUMMARY

Of 31 strains of Pasteurella pestis culturedupon thick blood agar base plates for 6 to 8 days,19 revealed the presence of smooth and non-smooth colonies. The remainder containedsmooth colonies only. The two types were alsoderived from material aspirated directly from ahuman plague bubo.The smooth forms for the most part retained

their characteristic morphology throughout sub-culture. The nonsmooth strains varied in homo-geneity under such conditions. On the otherhand pure cultures of both forms were maintainedon agar plates inoculated with splenic materialfrom mice infected with either smooth or non-smooth strain Poona.The two forms (whether virulent or avirulent)

were shown to be true P. pestis by pathogenicity,bacteriophage, immunological, and some commonbiochemical reactions. In addition to morpholog-ical differences, other differences were demon-strated by fermentation reactions.

Parent, smooth, and nonsmooth variantswere classified in relation to their mouse viru-lence. While most avirulent strains were entirelysmooth, some contained avirulent nonsmoothforms. Virulent strains all contained nonsmoothelements, and also possessed smooth forms whichVaried in virulence from strain to strain. Allstrains contained smooth forms.Both morphological types of P. pestis have

been isolated from parent strains collected fromwidely scattered places and hosts, indicatingworld wide distribution of strains composed ofboth smooth and nonsmooth variants.

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nose als Differenzierungsnaihrboden fur B.pestis und B. pseudotuberculosis rod. Pfeif-feri. Zentr. Bakteriol. Parasitenk. Abt. I.Orig., 119, 32-35.

BESSONOWA, A. AND LENSKAJA, G. 1931 Bouil-lontrubende Varietaten des B. pestis. Unter-suchungen uber die Dissociation des B. pestis.

Zentr. Bakteriol. Parasitenk. Abt. I. Orig.,119, 430-443.

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GUNNISON, J. B., LARSON, A., AND LAZARUS,A. S. 1951 Rapid differentiation betweenPasteurella pestis and Pasteurella pseudotuber-culosis by action of bacteriophage. J. Infec-tious Diseases, 88, 254-255.

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