Vol. 43, No. 1INFECTION AND IMMUNITY, Jan. 1984, p. 217-2230019-9567/84/010217-07$02.00/0Copyright ©3 1984, American Society for Microbiology

Isolation and Characterization of Bordetella pertussis PhenotypeVariants Capable of Growing on Nutrient Agar: Comparison with

Phases III and IVMARK S. PEPPLERt* AND MERRY E. SCHRUMPF

Laboratory of Microbial Struicture and Fu(nction, Rocky Moi(ntain Laboratories, Hamilton, Montania 59840

Received 25 May 1983/Accepted 7 October 1983

Unsupplemented nutrient agar (NA) was used to select spontaneous phenotype variants (PVs) ofBordetella pertlussis Tohama I and 3779 which, by their growth on NA, could possibly be consideredequivalent to phase IV in the system of Leslie and Gardner (P. H. Leslie and A. D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu,Kitasato Arch. Exp. Med. 26:121-134, 1953). NA growers (Gna+) were selected from the flat, nonhemoly-tic, non-NA grower (Dom- Hly- Gna-) PV of both strains at a rate of between 10-7 to 10-8 per cell per

generation. When cultured on Bordet-Gengou agar (BGA), more than one colony type was observed instrain 3779; these all retained the Gna+ characteristic during 10 to 30 passages on BGA. Analysis of 1251_surface-labeled whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no majorchanges between the Dom- Hly- Gna+ PV and their Dom- Hly- Gna- PV parents in polypeptide profile(by Coomassie stain), in surface exposure of proteins (by autoradiography), or in lipopolysaccharide profile(by silver stain). Increased resistance to oleic acid, tetracycline, erythromycin, rifampin, and penicillin G,however, was characteristic for the Dom- Hly- Gna' PV. Five phase IV strains and a phase III B. pertuississtrain had similar antibiotic and oleic acid sensitivity profiles as the Dom- Hly- Gna' isolates and platedwith similar efficiency on NA, despite heterogeneity in BGA colonial morphology and lipopolysaccharideprofile.

Bordetella pertuissis has been recognized to exist in atleast four serological phenotypes when grown on Bordet-Gengou agar (BGA) at 37°C (9, 13). The variation from wild-type fresh isolate to rough or degraded phenotype(s) hasbeen termed phase variation by Leslie and Gardner (13) andKasuga et al. (9) and has been shown to represent a series oflosses in antigenic (9, 13, 18) and virulence-associated (5, 15)properties as well as changes in colonial morphology onBGA (9, 10, 18). More recently, however, the distinction(s)between what has been called phase III and phase IV in thesystem of Leslie and Gardner or phase III and rough phase inthe system of Kasuga et al. has been lost from the literature(15, 21). Contributing to this loss, undoubtedly, is the lack ofreference phenotype variants (PVs), antisera to phase-asso-ciated antigens, and even standardized procedures for uni-form growth conditions among different investigators (15).This current lack of reference material created a dilemmawhen one of us (M.S.P.) reported on the isolation of variantsof B. pertussis which had a flat, nonhemolytic (Dom- Hly-)colonial morphology on BGA distinct from the domed,hemolytic (Dom' Hly+) colonial morphology of wild-type B.pertussis (16). It was suggested that the Dom- Hly- PV wasakin to the phase III prototype strain, Sakairi, of Kasuga etal., partly because the colonial morphology of both Sakairiand the Dom- Hly- PV was smooth and entire. Thismorphology was distinct from that reported for the roughphase types of Kasuga et al. which were irregular in outlinewith a rough surface texture. In addition, the Dom- Hly-PV did not grow on nutrient agar (NA) (Gna-) even atinocula of 108 CFU per petri plate, distinguishing them fromLeslie and Gardner's phase IV organisms (1, 3). As wereport here, the relationships among these PVs are more

* Corresponding author.t Present address: Department of Medical Microbiology, Univer-

sity of Alberta, Edmonton, Alberta, Canada T6G 2H7.

complex than previously anticipated but they are easilydefinable.We investigated the properties of phase III and phase IV

by two main strategies. First, if growth on NA is a propertyof phase IV (1, 3), then NA should select for NA growers(Gna+) from a population of NA nongrowers (Gna-). Byemploying a Luria and Delbruck fluctuation test, the rate ofGna- to Gna+ variation could be estimated, and isogenicsets of Gna- and Gna+ organisms could be compared.

Second, we wanted to survey extant phase IV organismsand apply nonserological criteria such as antibiotic sensitiv-ity (5), sensitivity to oleic acid (16), and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ofwhole-cell lysates (16) to distinguish these organisms fromother PVs.Although we show that our Dom- Hly- Gna PVs exhibit

colonial heterogeneity, they were all consistently more resis-tant to several antibiotics and oleic acid than their Dom-Hly- Gna- parents. They share this characteristic resistancewith B. pertuissis organisms sent to us as phase III or phaseIV. Of particular note was the observation in phase IVcultures of two distinct lipopolysaccharide (LPS) profiles bySDS-PAGE and silver stain. The relationships among theDom- Hly- Gna+ PV and phase III and phase IV arediscussed.

MATERIALS AND METHODS

Organisms and media. B. pertuissis 3779 and Tohama Iwere obtained from J. J. Munoz, Rocky Mountain Labora-tories, Hamilton, Mont., and have been described in detailpreviously (16). Strains 10901, 11089, and 10902 were ob-tained from the National Collection of Type Cultures, Lon-don, England. Strain 10901 was formerly called 364, a phaseIV of Leslie and Gardner (13), and 11089 and 10902 were

previously called L84, phases I and IV, respectively. For

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simplicity in making comparisons, we refer to 11089 as L84,phase I, and 10902 as L84, phase IV. Strain Sakairi, theprototype phase III organism in the system of Kasuga et al.(9), was obtained from K. Sekiya (Kitasato University,Tokyo, Japan). Strain L51, phase IV, was obtained fromC. R. Manclark (National Center for Drugs and Biologics,Bethesda, Md.), and strain 11615, a prototype phase IV B.pertiussis was obtained from the American Type CultureCollection, Rockville, Md. Strain D3148, phase I and phaseIV, were the gift of J. M. Dolby (Clinical Research Centre,Harrow, England).

All strains were stored at -70°C in modified Greavessolution (7). For routine growth, cultures were maintainedon BGA as previously described (16).NA (Difco Laboratories, Detroit, Mich.) was used in the

selection of B. pertulssis Gna+ isolates, and Stainer-Scholteagar (SSA) was used to select Dom- Hly- Gna- PVs fromwild-type Dom' Hly+ Gna- PVs as previously described(16).SDS-PAGE. The discontinuous Tris-glycine buffer system

of Laemmli (12) was used in 12.8% (wt/vol) total acrylamideslab gels as previously described (6). Low-molecular-weightprotein standards (Bio-Rad Laboratories, Richmond, Calif.)were used as a reference for gels with nonradiolabeledsamples. For radiolabeled samples, "4C-molecular weightmarkers were used (Amersham Corp., Arlington Heights,Ill.). Gels were fixed in 7% acetic acid-25% isopropanol andstained for LPS with silver by the method of Hitchcock andBrown (8) or for proteins by 0.2% Coomassie brilliant blueR-250 in fixing solution.

lodination of whole organisms. 1,3,4,6-tetrachloro-3ox,6a,diphenylglycouril (lodogen; Pierce Chemical Co., Rockford,Ill.) was used to label surface proteins of whole B. pertiussisorganisms with 1-51 as detailed elsewhere (16). Autoradio-graphs were also made as previously described (16).

Efficiency of plating. Viable counts of B. pertuissis on

duplicate NA plates were compared to their viable counts on

triplicate BGA plates and expressed as a percentage (assum-ing 100% for BGA). Counts were made after 10 days ofincubation at 37°C in a humidified incubator.Frequency of variation, estimation of mutation rate, and

selection of Gna+ mutants. Dom' Hly' Gna- and Dom-Hly- Gna- PVs of B. pertlussis were grown for 3 days on

BGA and harvested into TGS, and 50 [l of suspensioncontaining 1 x 108 to 3 x 108 CFU was plated onto 40 9-cmNA plates. A sample of the inoculum was diluted in TGS to

calculate CFU on BGA. NA plates were incubated for up to

28 days and observed for growth of individual colonies. Thefrequency of Gna' organisms within a population of Gna-organisms was calculated as the number of Gna+ isolatesdivided by the total number of organisms plated.

Mutation rates for the Gna+ phenotypes were estimatedby using a modified version of the fluctuation test of Luriaand Delbruck (14). Single colonies of BGA-grown organismswere inoculated into 10 ml of Stainer-Scholte broth (SSB) (20)

(pH 7.5) in a 25-cm2 tissue culture flask. The organisms were

grown statically at 37°C in a humidified incubator for 4 days.The organisms were then adjusted to 0.12 absorbance at 540nm in test tubes (13 by 100 mm) and diluted 10-5 in SSB.

Replicate 150-pl samples of the 10-5 dilution were distribut-ed to all 96 wells of a flat-bottomed microtiter tray andincubated at 37°C as before. Fifty-microliter samples of the

10-5 diluted organisms were also plated on BGA for viablecounts of the microtiter tray inoculum (1.0 x 103 to 1.5 x 103CFU per well). The microtiter cultures were grown for 7

days, at which time 40-[.l samples were taken from each well

and inoculated onto NA, eight samples per 9-cm plate. Fourwells from the 7-day growth were pooled and plated forviable count on BGA. NA plates were incubated at 37°C for15 days, colonies were counted, and the mutation rates wereestimated. The mutation rate, i.e., number of mutants perorganism per generation, was calculated by the formula ofBraun (4).

Antibiotic and oleic acid sensitivity testing. SSB cultures inflat-bottomed microtiter trays were used in determining theminimum inhibitory concentration (MIC) of five antibioticsand oleic acid for B. pertiissis PVs. Concentrated stocksolutions (2,000x) of the antibiotics and oleic acid weremade the day of the test as follows: penicillin G potassium(Eli Lilly & Co., Indianapolis, Ind.), 100 mg/ml in distilledwater; erythromycin (Sigma Chemical Co., St. Louis, Mo.),4 mg/ml in dimethyl sulfoxide; polymyxin B sulfate (Sigma),10 mg/ml in distilled water, rifampin (Sigma), 20 mg/ml indimethyl sulfoxide; tetracycline hydrochloride (Sigma), 40mg/ml in distilled water; and oleic acid (Fisher ScientificCo., Fairlawn, N.J.), 200 mM in ethanol. Stock solutionswere diluted 10-3 in SSB, distributed into the first row of themicrotiter trays in duplicate, and further serially twofolddiluted in 50-1L1 volumes of SSB. Organisms were harvestedfrom BGA after 3 days of growth and suspended in SSB to aconcentration of 4 x 107 to 10 x 107 CFU/ml. Fifty microli-ters of suspension was added per well, and the microtitertrays were then incubated statically at 37°C in a humidifiedincubator for 4 days. Turbidity associated with organismgrowth was observed with dark ground illumination througha Bausch & Lomb Stereo Zoom 4 (Bausch & Lomb,Rochester, N.Y.). The growth in control wells was assigneda value of 4+, and the test wells were scored on a scale from0 to 4+. The MIC was the lowest concentration of antibioticor oleic acid to reduce turbidity to <2+.

RESULTSSelection of Gna+ phenotype. A total of between 10") and

101 1 CFU (based on BGA) of Dom Hly Gna- and Dom-Hly- Gna- PVs from strains Tohama I and 3779 were platedon NA. After 15 days at 37°C, only the NA plates inoculatedwith Dom- Hly- Gna- PVs from both strains showedcolonial growth. The frequency of Gna colonies within theDom- Hly- Gna- population was 7.4 x 10- (1(14/1.9 x 10'0)for strain 3779 and 3.8 x 10-1 (2/5.2 x 10'°) for strainTohama I. No growth was observed on the plates inoculatedwith the Dom+ Hiyt Gna- PV from either strain, even after28 days of incubation.Two representative Gna+ colonies (INA1, 1NA2) selected

from 3779, PV Dom- Hly- Gna-, and two Gnat coloniesselected from Tohama I, PV Dom- Hly- Gna- (2NA18,2NA19), were cloned onto NA for further study.

Rate of mutation. Further attempts to select Gnat organ-isms simply by plating large inocula on NA (as done above)gave erratic results. From the low frequency describedabove, it was likely that the irregular isolation of Gna+organisms reflected differences in the time at which sponta-neous mutants arose within the population of the inoculum.A type of fluctuation test was therefore employed to deter-mine the rate of mutation and to implement a reliable meansof selecting Gna+ variants from other strains.The Dom- Hly- Gna- PV of strains Tohama I and 3779

were tested first. Of 96 wells inoculated, 14 were positive forGna+ in Tohama I, and 18 were positive in 3779. The actualnumber of Gna+ mutants (based on colony counts on NA)ranged from 1 to 61 per 40-pl sample and followed noparticular pattern relative to the wells of origin. The muta-

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FIG. 1. Colonial morphology of B. pertutssis Dom - Hly Gna+grown on NA. Representative isolates from strains 3779 (iNAl) andTohama I (2NA18) after 10 days of growth at 37°C and viewed bysubstage lighting with a silvered mirror for opacity and texture. Bar.1.0 mm.

tion rates were calculated as 3.4 x 10-8 for Tohama I and 8.4x 10-8 for 3779. We were unable to obtain Gna' mutantsfrom the Dom' Hly+ Gna- PV from any of four strainstested. With the method described here, the fluctuation testcould detect rates no lower than 1.8 x 10- "'. Thus, it followsthat if Gna+ mutants arise from Dom' Hly' Gna- PVparents, they occur at spontaneous rates of less than 1.8 x10-1(. Gnat mutants selected by the fluctuation techniquewere identical, in the comparisons that follow, to Gna+organisms obtained earlier by direct plating of large inoculaon NA.

Appearance, growth, and stability of Gna+ isolates. Colo-nies of the Gna+ isolates on NA were generally flat andentire, with colony surfaces of different smoothness. Colonysurfaces became rougher as the cultures aged at 37°C. TheGna+ from Tohama I, for example, appeared to have arougher colonial surface than the Gna+ from 3779 (Fig. 1).Both opaque and transparent forms (based on obliquelytransmitted light using a silvered substage mirror) werenoted early in growth (not shown). Colonies became moreuniformly opaque as the cultures aged. The cellular morphol-ogy of the organisms was principally coccobacillary in thecolonies grown on NA, even after 10 days of growth at 37°C.Organisms grown on NA, harvested, and washed once in

TGS showed efficiencies of plating on subsequent transfer tofresh NA plates of 3.3 and 16.0% for 3779 Gna+ isolateslNAl and 1NA2, respectively, and 44.3 and 29.2% forTohama I Gna+ isolates INA18 and 2NA19, respectively.When transferred from NA to BGA, the Gna+ organisms

from strain 3779 formed two major colony types. Both wereflat and nonhemolytic (Dom- Hly-), but one was distinctlymore marbled or milky (Mil) than the other (Mil-) (Fig. 2).The Mil + colonial variant was composed of long gram-negative rods and chains of rods (Fig. 2, bottom) andrepresented 98.0% of the colonies plated onto BGA fromiNAl but only 9.2% of the colonies from 1NA2. Mil+colonial variant plated with an efficiency of 40.6% whentransferred to NA from BGA, but reverted to the Mil-colonial variant at a frequency of 1.6 x 10-1 when furtherpassaged on BGA.

In contrast, the Mil- colonial variants were composed ofgram-negative coccobacilli, were phenotypically stable uponserial BGA passage, and retained a plating efficiency of.23.2% on NA, even after 30 BGA passages.Gna+ isolates from Tohama I formed only Dom- Hly-

Mil- colony types on BGA and were identical in appearanceto the Mil+ type of 3779 Gna+ isolates. In addition, Mil-variants of Dom- Hly- Gna+ isolates from both Tohama I

and 3779 possessed BGA colonial phenotypes virtually indis-tinguishable from the Dom- Hly- Gna- parents from whichthey were derived.

Neither MilW nor Mil- variants of Dom- Hly- Gna+ PVsfrom either strain agglutinated spontaneously in TGS or0.85% NaCl, even after standing for 24 h at ambient tempera-ture. Unless otherwise stated, the studies that follow wereconducted on the more stable Mil- variant of the Dom-Hly- Gnat PV.SDS-PAGE of Dom- Hly- Gna+ isolates. SDS-PAGE of

[1251]Iodogen-labeled viable organisms was used to comparethe Dom- Hly- Gnat PV to the Dom' Hly+ Gna- or Dom-Hly- Gna- PV of the same strain for differences in (i) LPSby silver stain, (ii) polypeptide profile by Coomassie stain-ing, or (iii) surface labeling profile by autoradiography.

Figure 3A shows that LPS profiles are qualitatively thesame for all three PVs of each strain and look similarbetween strains Tohama I and 3779 as well. All organismspossess a dark-stained a band and a lightly stained b band,although the Dom' Hly+ Gna- PV of both strains (lanes aand e) appear to have quantitatively less of the b band thando the Dom- Hly- Gna- or Dom- Hly- Gna+ PVs (17).LPS profiles from the Dom- Hly- Gna' PV were essentiallythe same whether the organisms were grown on BGA or NA.The silver-stained bands of high apparent molecular weight,including the 200,000-dalton molecular weight marker in Fig.3A, appeared on occasions when the stain was overdevel-oped. These bands may represent LPS associated withprotein in the whole-cell lysate. However, they do notappear to represent LPS by itself (17).

In Fig. 3B, no obvious difference can be seen in Coomas-sie-polypeptide profiles between Dom- Hly- Gna' andDom- Hly- Gna- PVs. The differences between Dom'Hlyt Gna- and Dom- Hly- Gna- PVs are consistent withthose reported before (16). Dom- Hly- Gna organismsgrown on NA or BGA gave equivalent polypeptide profiles.

[ 151]Iodogen labeling of Dom- Hly- Gna' organismsgrown on BGA gave a pattern similar to Dom- Hly- Gna-organisms (also grown on BGA [Fig. 3C]). Dom- Hly-

3779 DomrHly Gna+

FIG. 2. Colonial morphology and cell morphology (insets) of thetwo Dom- Hly- Gna+ phenotypes isolated from strain 3779 (1NA2)after 4 days of growth on BGA at 37°C. Milky (Mil+) and nonmilky(Mil-) colonies grown on the same plate were viewed by reflectedlight from a universal illuminator as previously described (16). Forcolonies, the white bar = 1.0 mm; for organisms. the black bar = 5.0,um.

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220 PEPPLER AND SCHRUMPF

A. B. C.SILVER STAIN COOMASSIE BLUE of A. AUTORADIOGRAPH of B.

3779 Tohama 1 3779 Tohama 1 3779 Tohama

MW MW MW

a b c'c d'd e f g' g h'h a b c'c d'd e f gsg h'h a b c'c d' d e f g'g h'h

on

Awl

.-.AL

,_S_m :low t...q._ ".+

-200,< 1 0 0-92.5

'\ 46

-30

'4

_ -14.3

FIG. 3. SDS-PAGE of whole-cell lysates from the three major phenotypes of strains 3779 and Tohama I vectorially labeled with 1251 by

lodogen. (A) Silver-stained gel by the Ag-LPS method of Hitchcock alnd Brown (8). Principal LPS bands (l and b are noted by arrowheads. (B)Coomassie brilliant blue R-250 counterstaining of silver-stained gel in A. (C) Autoradiograph of the silver- and Coomassie-stained gel picturedin (B) after drying it onto paper. c', d'" g', and h' indicate lysates of organisms grown on NA; a. b, c, d, etc., indicate lysates of organismsgrown on BGA. PVs from 3779: a, Dom' Hly+ Gna ; b. Dom Hly Gna; c' and c, Dom Hly Gna' isolate 1NA1; d' and d. Dom HlyGna' isolate 1NA2. PVs from Tohama 1: e. Dom' Hly+ Gna ; f, Dom- Hly- Gna; g' and g. Dom Hly Gna+ isolate 2NA18; h' and h,Dom- Hly Gna+ isolate 2NA19.

Gnat grown on NA were labeled differently than when theywere grown on BGA. Most notably, the polypeptide ofapparent molecular weight 18,000 (arrow) labeled muchmore weakly in the NA-grown organisms than in the BGA-grown organisms, with an exception being the organisms inlane d. Although the intensities of labeling varied for individ-ual preparations, the overall profiles were consistent fromtest to test.

Antibiotic and oleic acid sensitivity profiles of Dom- Hly-Gna+ isolates. All PVs from 3779 and Tohama I were testedagainst five antibiotics and oleic acid to determine theirMICs. The data presented in Table 1 are from a repre-sentative experiment. Although the MICs are not absolute,the trends in sensitivity for the different PVs were reproduc-ible. As seen in Table 1, Dom- Hly- Gna' organisms are

generally greater than or equal to fourfold more resistant torifampin, tetracycline, penicillin G, erythromycin, and oleicacid than are their Dom- Hly- Gna- PV parents. Inaddition, the Dom- Hly- Gna- organisms form a group withMIC profiles intermediate between Dom' Hly' Gna- andDom- Hly- Gnat PVs.Comparison of Dom- Hly- Gna+ isolates with phase III and

phase IV organisms: efficiency of plating. Six strains inputative phase III or phase IV were obtained from sources

listed above and tested for efficiency of plating on NA,colonial morphology on BGA, and cellular morphology.Table 2 summarizes the findings. As the data suggest, phase

III and phase IV could be described as Dom- Hly- GnatPVs, although colonial heterogeneity was noted in each ofthe strains. Representative examples of this heterogeneity inboth colonial morphology and cellular morphology are

shown by the Mil and Mil- variants of strains L51 and11615 in Fig. 4. All the colony types described were stablefor at least five passages, except the Mil' variant of L84,which was lost after three subcultures. None of the phase IIIor phase IV organisms spontaneously agglutinated in saline.Their efficiency of plating on NA ranged from 22 to 100%,with the exception of 11615 M-. which plated with an

efficiency of 0.02% (Table 2).SDS-PAGE of phase III and phase IV organisms. Figure 5A

shows differences among the SDS-PAGE LPS profiles fromvarious strains in phase IV, but similarity in the LPS profilesfrom colonial variants of phase IV within a strain. Morespecifically, the Dom- Hly- Gna+ PVs of 10901, both Mil'and Mil- subtypes, D3148 (phase IV), Mil+ subtype, and11615, Milt subtype, all have LPS bands which migratecloser to the dye front than do the LPSs of the other strains.The other colonial subtypes of Dom- Hly- Gna PVs fromstrains 11615 and D3148 (phase IV) also had this faster-migrating LPS (not shown). In contrast, the LPSs of L51(phase IV), Mil-, L84 (phase IV), Mil' and Mil-, and D3148(phase I) are similar to the LPS seen in all three Tohama IPVs. Sakairi (not shown) also had an LPS profile identical tothat of Tohama I. Of all the strains tested, only D3148

TABLE 1. Antibiotic and oleic acid sensitivities of isogenic B. peritssis PVs in SSB

MIC' (,ug/ml)

Strain PV OleicPenG Eryth PxB Rif Tet acid

(pM)

3779 Dom' Hly+ Gna- 0.8 0.03 0.63 0.63 0.63 6.33779 Dom-- Hly-- Gna- 12.5 0.25 1.25 2.5 2.5 25.03779 (INA1) Dom-- Hly- Gna+ >50.0 1.0 2.5 10.0 10.0 >100.0Tohama I Dom' Hly+ Gna- <0.8 <0.03 0.63 <0.16 0.63 3.1Tohama I Dom- Hly Gna 6.3 0.5 2.5 2.5 2.5 25.0Tohama I (2NA18) Dom - Hly Gna+ 50.0 2.0 2.5 5.0 10.0 >100.0

" PenG, Penicillin G Eryth, erythromycin; PxB, polymyxin B; Rif, rifampin; Tet, tetracycline.

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TABLE 2. Properties of B. pertussis strains designated phase III or phase IV% Efficien-

Strain Phase Colonial phenotypes on BGA' cy of plating Cell morphology from BGAbon NA

10901 IV Dom- Hly- Scs+ Ece+ Mil- 97.0 CoccobacilliDom- Hly- Scs+ Ece+ Mil+ 74.0 Short rods

L84 IV Dom- Hly- Scs+ Ece+ Mil- 71.0 Coccobacilli, some in chainsDom- Hly- Scs+ Ece+ Mil+ NDC Coccobacilli and short rods

L51 IV Dom- Hly- Scs+ Ece+ Mil- 100.0 CoccobacilliDom- Hly- Scs+ Ece+ Mil+ 96.0 Long rods and coccobacilli

11615 IV Dom- Hly- Scs+ Ece+ Mil- 0.02 Short rodsDom- Hly- Scs+ Ece+ Mil+ 22.0 Coccobacilli and short rods

D3148 IV Dom- Hly- Scs+ Ece+ Mil- 92.0 Coccobacilli and short rodsDom- Hly- Scs+ Ece+ Mil+ 57.0 Coccobacilli and short rods

Sakairi III Dom- Hly- Scs+ Ece+ Mil- 68.0 Coccobacillia Appearance on BGA after 3 to 5 days of growth at 37°C: Dom-, flat, not domed; Hly-, nonhemolytic; Scs+, smooth, moist colony

surface; Ece+, entire, nonconvoluted colony edge; Mil+, milky appearance and watery consistency; Mil-, not milky, indistinguishable fromDom- Hly- Gna- phenotype on BGA.

b Gram stain on 3-day-old cultures suspended in TGS, air dried, and methanol fixed.' ND, Not determined.

L51 11615Mile Mil-

.k.* A-> Vi $ 3 b9 * {f ; **

-J ,

_ -.

FIG. 4. Colony morphology and cell morphology of representative colonial variants of phase IV from strains L51 and 11615 grown for 4days on BGA at 37°C. Mil+, milky; Mil-, nonmilky. For colonies, the white bar = 1.0 mm; for cells, the black bar = 5.0 ,um.

-0,~~-t 0)

cn OD 0)- 0 -j T

a) IIVIMd MrFMM

oo

EL

't 0)

mw _ LOu I IV I -- - mwa b c _ >- Mil MilMil MiIMiI

-'.V.A

moo.

FIG. 5. SDS-PAGE of whole-cell lysates from the three major phenotypes of strain Tohama I and various strains in phase IV, or bothphase IV and phase I, as indicated. All organisms were grown on BGA for 3 days. (A) silver stain as in Fig. 3; (B) Coomassie blue counterstainof (A).

Eco toA ro -

0 cD(0Cr-

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TABLE 3. Antibiotic and oleic acid sensitivities of B. pertussisstrains designated phase III or phase IV (Mil- subtypes)'

MIC'7 (Lg/rnl)

Strain OleicPenG Eryth PxB Rif Tet acid

(1±M)

10901 6.3 0.25 1.25 0.63 10.0 >100.0L84 >50.0 >2.0 1.25 >10.0 >10.0 >100.0L51 >50.0 >2.0 1.25 >10.0 >20.0 >100.0D3148 >50.0 2.0 2.5 >10.0 20.0 >100.0Sakairi >50.0 2.0 2.5 10.0 20.0 >100.0

a See Table 2.b For abbreviations, see footnote a of Table 1.

possessed both LPS profiles, one type in phase I and theother in phase IV. Coomassie poststaining of the silver-stained gel (Fig. 5B) shows polypeptide profiles for all phaseIVs that are typical of B. peri-tssis of the Dom- Hly- Gna-PV (16).

Antibiotic and oleic acid sensitivity profiles of phase III andphase IV organisms. The Mil- variants of Dom- Hly- Gna+PVs from the strains listed in Table 2 were tested forsensitivity toward the same five antibiotics and oleic acid asthe prototype strains and PVs in Table 1.

Table 3 shows similarity among the phase IV or phase IIIstrains tested, with the exception of 10901, which appears tohave a greater sensitivity toward penicillin G, erythromycin,and rifampin. Nevertheless, 10901 and the other five strainshave tetracycline and oleic acid resistances similar to theDom- Hly- Gna+ PVs isolated from 3779 and Tohama I(Table 1) regardless of the LPS type they possess (Fig. 5).Strain 11615, both Mil+ and Mil- variants, failed to grow inSSB and could not be tested in this assay.

DISCUSSIONThe following conclusions can be made from the results.B. pertussis organisms capable of growth on unsupple-

mented NA (Gna') can be isolated from the Dom- Hly-Gna- PV at frequencies of between 10-9 and 10-11. Theselow frequencies are markedly different from the higherfrequencies of 5 x 10-5 to 5 x 10-6 reported for theappearance of Dom- Hly- Gna- PVs within a population ofwild-type Dom' Hly' Gna- organisms (16). Furthermore,our Dom- Hly- Gna' isolates occurred at mutation rates ofbetween 10-7 and 10-8 per cell per generation based onfluctuation analysis. These rates are within the range ofspontaneous mutation rates (4), not rates comparable tophase variation (19). Theoretically, there is no reason whyDom- Hly- Gnat PVs should not be isolatable from theDom' Hly+ Gna- PV. The limit of detection in our fluctua-tion test was 1.8 x 10 1o per cell per generation. Increasingthe volumes or numbers of the individual cultures would alsoincrease the sensitivity of the assay. In this way, Alexanderand Redman were able to detect spontaneous streptomycin-resistant mutants of Dom' Hly+ Gna- B. pertiussis at ratesbetween 7 x 10-1 1 and 8 x 10- l per cell per generation (2).Perhaps spontaneous Dom- Hly- Gna+ mutants of Dom+Hly+ Gna- organisms occur at similarly low or lower rates.

Phenotypically the Dom- Hly- Gna+ isolates are hetero-geneous. Different clones from the same strain can showdifferent colonial morphologies on both NA and BGA (e.g.,Mil+ and Mil- variants from 3779 on BGA). Despite thiscolonial heterogeneity, the SDS-PAGE Coomassie-stainedpolypeptide profiles, 125I-surface-labeled autoradiogram pat-

terns (of BGA grown organisms), and silver-stained LPSprofiles are indistinguishable from those of the parent Dom-Hly- Gna- PV. Thus, based on polyacrylamide gel patterns,no overt changes in whole-organism polypeptide composi-tion, surface exposure of polypeptides on whole organisms,or LPS composition were associated with acquisition of theGna' phenotype.The Gna+ phenotype represents a stable mutation in that

Dom- Hly- Gna+ organisms passaged over 30 times onBGA retain an efficiency of plating on NA of 20 to 80%. Inaddition to the high efficiency of plating on NA, the Dom-Hly- Gna+ organisms can be distinguished from their Dom-Hly- Gna- parents by their greater resistances to rifampin,erythromycin, tetracycline, penicillin, and oleic acid. Thus,it appears that a major change has occurred in the Dom-Hly- Gna' organisms which is reflected in the wide range ofantibiotic resistances acquired but is not reflected in anyreadily distinguishable polypeptide or LPS change as visual-ized by SDS-PAGE of whole-cell lysates. Since the whole-cell fatty acid methyl ester profiles of phase I and phase IVB. pertuissis are indistinguishable (5), the change in Gna+organisms may be in membrane phospholipid composition,although this remains to be proven.

Strains of B. pertiussis received from other laboratoriesunder the label phase IV were all Dom- Hly- Gna+ butconsisted of mixed populations of colonial phenotypes onBGA. As with our Dom- Hly- Gna+ organisms, theseintrastrain variations did not affect efficiency of plating onNA (except for 11615, Mil- subtype) or, to any major extent,antibiotic or oleic acid sensitivity. SDS-PAGE profiles ofCoomassie-stained polypeptides or silver-stained LPS werealso unaffected by intrastrain variation among the phase IVs.Notable, however, were the marked interstrain differencesin SDS-PAGE LPS profiles among the phase IV strains.Phase IVs could be broken into two groups based onwhether they possessed the slow-moving wild-type LPS asfound in Tohama I, Dom' Hly+ Gna-, or the variant, faster-migrating LPS, as found in NCTC 10901, Dom- Hly- Gna+.Kasuga et al. described their rough phase organisms as

having heat-stable (100°C, 2 h) 4) antigens, serologicallydistinct from the 0 antigen found in their phases I to III. Inaddition, the rough phase had colonial morphologies charac-teristically different from those of phases I to III. We wereunable to predict LPS type by colonial morphology, al-though we suspect that the 4) antigens are analogous to thefaster-migrating LPS described here.

Regrettably, no rough phase organisms are extant fordirect comparison (K. Sekiya, personal communication).However, the confusion over designations phase III andphase IV may still be resolved by defining the type of LPSpossessed by a Dom- Hly- Gna+ PV (22). For example,NCTC 10901 is an extant phase IV of Leslie and Gardner. Itis a Dom- Hly- Gna+ PV and possessed the variant, faster-migrating LPS. Thus, 10901 could be considered the proto-type of phase IV. Likewise, Sakairi is an extant phase III ofKasuga et al. It is a Dom- Hly- Gna+ PV (11) and possessedthe wild-type LPS. Sakairi could be considered a prototypephase III.Such conjecture on the characteristics of phase III and

phase IV is of historical interest but of questionable valuetoday. First, there is no way to confirm that the phenotypecharacteristics we describe in Sakairi and 10901 were in factpossessed by the organisms described by the original investi-gators nearly 30 or 50 years ago, respectively. Second, theperpetuation of phase designations is a perpetuation ofconfusion and inaccuracy: confusion because of the very

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P. PERTUSSIS NUTRIENT AGAR GROWERS 223

reason the present investigation was conducted (see above),and inaccuracy because Dom- Hly- Gna+ mutants do notoccur at phase variation frequencies, whereas Dom- Hly-Gna- variants do. We suggest the use of (i) colonial mor-phology on BGA, (ii) SDS-PAGE/Coomassie- and silver-stained whole-cell profiles, (iii) antibiotic and oleic acidsensitivities, and (iv) plating efficiency on NA as nonsero-logical alternatives for defining PVs of B. pertuissis.

ACKNOWLEDGMENTSThanks to John Foulds (NIADDKD) for helpful discussions, to

the staff of the Laboratory of Microbial Structure and Function forcritiques of the data, to Phyllis Pienta, assistant curator of bacteriol-ogy, ATCC, for special efforts in supplying strain 11615, and toSusan Smaus, Heather Mitchell, Chuck Taylor, and Bob Evans fortheir skill in preparing the manuscript.

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pertuissis that induces bactericidal antibody and its relationshipto protection in mice. J. Gen. Microbiol. 70:371-382.

2. Alexander, H. E., and W. Redman. 1949. Mechanism of einer-gence of resistance to streptomycin of H. pertuissis and H.parapertussis during treatment with this antibiotic. Pediatrics4:461-467.

3. Aprile, M. A. 1972. A reexamination of phase IV Bordete/laperitissis. Can. J. Microbiol. 18:1793-1801.

4. Braun, W. 1965. Bacterial genetics, p. 83. The W. B. SaundersCo., Philadelphia.

5. Dobrogosz, W. J., J. W. Ezzell, W. E. Kloos, and C. R.Manclark. 1979. Physiology of Bo<rdetellai pertiussis, p. 86-93. InC. R. Manclark and J. C. Hill (ed.), International symposium onpertussis. DHEW publication no. (NIH) 79-1830. U.S. Govern-ment Printing Office, Washington, D.C.

6. Field, L. H., and C. D. Parker. 1979. Effects of fatty acids ongrowth of Bordetella pertiussis in defined medium. J. Clin.Microbiol. 9:651-653.

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10. Kasuga, T., Y. Nakase, K. Ukishima, and K. Takatsu. 1954.Studies on Haemnophiliis pertiussis. lII. Some properties of eachphase of H. perttlssis. Kitasato Arch. Exp. Med. 27:37-48.

11. Kumazawa, N. H., and M. Yoshikawa. 1978. Conversion ofBordetella pertutissis to Bordetelal para-apertutissis. J. Hyg. 81:15-23.

12. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

13. Leslie, P. H., and A. D. Gardner. 1931. The phases of HoIetnio-philus pertulssis. J. Hyg. 31:423-434.

14. Luria, S. E., and M. Delbruck. 1943. Mutations of bacteria fromvirus sensitivity to virus resistance. Genetics 28:491-511.

15. Parker, C. D. 1979. The genetics and physiology of Bordetellapertussis, p. 65-69. In C. R. Manclark and J. C. Hill (ed.),International symposium on pertussis. DHEW publication no.(NIH) 79-1830. U.S. Government Printing Office, Washington,D.C.

16. Peppler, M. S. 1982. lsolation and characterization of isogenicpairs of domed hemolytic and flat nonhemolytic colony types ofBordetella pertu.ssis. Infect. Immun. 35:840-851.

17. Peppler, M. S. 1983. Two physically and serologically distinctlipopolysaccharide profiles in strains of Bordetella per tlussis andtheir phenotype variants. Infect. Immun. 43:224-232.

18. Shibley, G. S., and H. Hoelscher. 1934. Studies on whoopingcough. I. Type specific (S) and dissociation (R) forms ofHaeinophiluis pertuissis. J. Exp. Med. 60:403-419.

19. Silverman, M., and M. Simon. 1980. Phase variation geneticanalysis of switching mutants. Cell 19:845-854.

20. Stainer, D. W., and M. J. Scholte. 1971. A simple chemicallydefined medium for the production of phase I Borcdetella pertits-sis. J. Gen. Microbiol. 63:211-220.

21. Wardlaw, A. C., R. Parton, and M. J. Hooker. 1976. Loss ofprotective antigen, histamine-sensitizing factor and envelopepolypeptides in cultural variants of Bordetelka pertiussis. J. Med.Microbiol. 9:89-100.

22. Watanabe, M., and Y. Nakase. 1982. Mutant of Bordetellapertussis which lacks ability to produce filamentous hemaggluti-nin. Infect. Immun. 35:1018-1023.

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