In vitro method to differentiate isolates of type III Streptococcus agalactiae from symptomatic and asymptomatic patients

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In

Vitro Method To

Differentiate Isolates of

Type

III

Streptococcus

agalactiae from

Symptomatic and Asymptomatic Patients

JOHN J. MAURERAND STEPHEN J. MATTINGLY*

Department of Microbiology, University of Texas Health Science CenteratSan Antonio, SanAntonio, Texas78284 Received8September 1987/Accepted14December1987

Streptococcus agalactiae (groupBstreptococci) isolates from infected infants have been demonstratedtohave three- to fourfold or higher levels of cell-associated lipoteichoic acid than isolates from asymptomatically

colonized infants, suggestingarolefor this cell surface polymer in the relative virulence of these organisms. The

presentstudyindicates that symptomatic isolates oftypeIIIgroupB streptococcicanbereadily differentiated

from asymptomatic strains by theirresponse tovarious levelsof phosphate in achemically defined medium

(FMC). Both classesof isolates had the same doubling time (TD of 30to35 min) in FMC containing 65 mM sodium phosphate. However, levels of phosphate >125 mM distinguished the two classes of strains. Asymptomatic strains pregrown in 65 mM phosphate to the stationary phase rapidly initiated growth at elevatedphosphate levels, whilesymptomatic strains initiated growth only afteraprolongedincubation period

(>400 min). These results suggest that the physiological growth response of clinical isolates of group B

streptococci to phosphate canserve as a diagnostic aid in screening potentially virulent strains inpregnant

women andnewborn infants.

The virulence determinants of Streptococcus agalactiae (group B streptococci) have been the subject of recent studies in several laboratories. The type-specific antigens (TSA)arewell recognized asimportant antiphagocytic

fac-tors, andantibodies raised against these type-specific poly-saccharides(Ia, lb, II, III,and IV)aswellastheIbc protein

have been showntobeprotective in several animal models (7). An additional potential virulence determinant, the lipo-teichoic (LTA) ofgroup B streptococci, has been shownto mediatespecific adherence ofgroupBstreptococcitohuman fetalcelllines,incontrast tomuch less efficientadherenceto adult cells (13). Isolates (stationary phase) from infected infants had LTA chainsof 30to35glycerolphosphate units, while asymptomatic isolates (from colonized infants) had LTA chains of 10 to 12 units (14). Symptomatic isolates required eightfold-higher levels ofpurified LTA todisplace theseorganisms from human fetal lung cells compared with asymptomatic isolates, demonstrating thegreatly enhanced binding capacity of symptomatic type III strains (14). In view of the net negative charge contributed by the TSA (sialic acid) and LTA (phosphate), the present study was

initiatedto determine whether the ionic compositionof the growth medium would be useful in differentiating sympto-matic andasymptomatic isolates oftype IIIgroupB strep-tococci, which presumably would have widely varying chargesontheircell surfaces. Since the level of phosphate in

the medium has been shownto affect synthesis of both the TSA(16) and LTA (12), its influenceonthegrowthresponse

ofsymptomatic and asymptomatic strains oftypeIIIgroupB streptococciwasexamined.

MATERIALSANDMETHODS

Bacterialstrains, media,andgrowthconditions. S. agalac-tiae (group B streptococci) strains used in this study have been described elsewhere (5, 10). Group B streptococcus isolates were cultured on5% sheep bloodagarplates (BBL

Microbiology Systems, Cockeysville, Md.)at37°C for 16to 24 hbefore eachexperiment. A chemically defined medium

* Correspondingauthor.

686

(FMC) was prepared as described previously (15) with 65

mM sodium phosphate serving as the standard phosphate

concentration andwas always used within 24 h of

prepara-tion.Growthwas monitoredasoutlined in previous studies

(12). The cell density of each culture inoculumwasadjusted

to 13 to 15.0 ,ug of cellular dry weight per ml, which

correspondedto adjusted optical density units (15) of 30to 35asdeterminedonaspectrophotometer (Junior model 35;

The Perkin-Elmer Corp., Oak Brook, Ill.). One adjusted optical density unit is equivalentto 0.43 ,ug of cellular dry weight per ml (11). When the cultures reached the

mid-exponential phase (approximately 170 ,ug of cellular dry weightperml), the pH was routinely monitored and

main-tainedat 7.0 by periodic addition of 2 N NaOH. Cultures

wereconsidered tobe in the stationaryphase when the cell density failedtoincreaseexponentially withinanintervalof

30to60 min.

Effect ofphosphateongrowth ofsymptomaticand

asymp-tomatic isolates ofgroupB streptococci. Stationary cultures

were used to inoculate 10 ml ofFMC containing elevated sodiumphosphate levels (100to200mM). Theinitial inocu-lum was modified to 25 adjusted optical density units, and growthwas monitored to determineany differencesamong

isolates in regard to length of lag phase, growth rate (TD), andcell yield. Experiments examining the effect of growth phase on reinitiation ofgrowth in200mM phosphate were

performed by the procedure outlined above. Strains were

pregrown in standard FMC, and samples were obtained at various times throughout growth and immediately chilledin

an ice bath. After all samples were obtained, the cultures were inoculated into FMC (initial inoculum, 25 adjusted optical density units) containing 200 mM phosphate and growthwas monitored asdescribed above. Subculturingof

symptomatic isolate GBS122 and asymptomatic strain GBS182 in 200 mM phosphate was performed by initially

growing strainstothestationary phase in standard FMCand using1-mlsamplestoinoculateanadditional 9 ml of 200 mM phosphate medium whichwasincubated for24 h before each

passage. This procedure was repeatedfor seven passages. Aftereach passage, the subcultured strain was isolated on

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sheep blood plates and stocks were made and stored at -700C.

Extraction,purification, and quantitation of TSA and LTA. TheTSAwasprepared by lysing whole cells with

mutanoly-sin (Sigma Chemical Co., St. Louis, Mo.) as previously

described (16) except the 40% sucrose in the lysing buffer

wasomitted. Cell suspensionswereincubatedat370C (30to 60 min) until there was a >95% decrease in the initial cell

density. The lysate was incubatedat37°C for anadditional

hour in thepresenceof bovine DNase II (Sigma) and RNase

I-A (Sigma) (1 mg/ml for both nucleases). The lysate was

dialyzed (for 18to24 hat40C) three timesagainst deionized waterwith dialysis tubing withanexclusion limit of >3,500

molecular weight (American Scientific Products, McGraw Park, 111.). Sialic acid was released by hydrolyzing the

antigen with 1 N HClat840Cfor 15 min andquantitated by theAminoff method (1). Extraction, purification, and

quan-tificationof LTA has been detailed inprevious work (9, 12).

RESULTS

Effect of phosphate levels on growth of symptomatic and

asymptomatic or avirulent strains of group B streptococci. Studies were initially conducted to determine the effect of

various phosphate levelsonthe growthresponseof selected

isolates, since previous reports indicated that altering the phosphate level markedly influenced the amount of cell surface and extracellular TSA (16) and LTA (12) synthe-sized. Two strains (GBS122 and D136C) were selected as

representative of symptomatic and asymptomatic or aviru-lent isolates (16). Regrowth of stationary-phase cells in sodiumphosphate levelsgreaterthan 125 mMreadily distin-guished GBS122 from D136C (Fig. 1 and 2). However, the mostpronounced differencewas seenwith200 mM sodium

phosphate, in which the lag time was even moreapparent, with thesymptomatic strain GBS122 exhibitingalongerlag

time in the highphosphate than D136C. The low cell yield

100-0

o6

1o00

3005

40.10soM0

ca

O05M

0O2OM

20'0

300 400 500 600

Time

(min)

FIG. 1. Effectofphosphatelevelsonthegrowthof S.agalactiae

GBS122 (symptomatic infant).Theculturewasgrowntothe

station-aryphase in standard FMC andusedto inoculate 10 ml of FMC containing elevated sodiumphosphate (100to200mM).

CL1O 0.125M 7Wg0M/ .200M

ID 0.095M | /

Cu

10.

0 100 200 300 400

500

600 Time

(min)

FIG. 2. Effect ofphosphatelevelsonthegrowthof S.agalactiae D136C(aviruent).

after16 hofincubationin 200 mM

phosphate

didnotappear tobe the

consequence

of

growth

inhibition sincethe symp-tomatic strain GBS122 reached a final cellular

dry weight

similar to that of the avirulent strain D136C after a 36-h incubation

(Table 1).

Atconcentrations of

phosphate

greater than 200

mM,

the

yield

wasreducedafter 36 hofincubation

(Table 1).

Differenceswerealsonoted within the 225 to250 mM

phosphate

range,withtheavirulentstrainD136C

grow-ing

to a

higher

cell

density.

However,

growth

wasreduced 10-to30-foldwhen

compared

with thatof cultures grown in 200 mM

phosphate

aftera36-h incubation. Differenceswere also noted between GBS122 and D136C when

stationary-phase

cells were grown in medium

containing

various amounts ofNaCl

(50

to 200

mM). However,

after 16 h of incubation in 200 mM

NaCl,

both strains had reached the

stationary

phase

(data

not

shown),

in

contrast'to previous

results above

demonstrating

the

inability

of GBS122 to

TABLE 1. Effect ofphosphateonthe cellyieldsof S.agalactiae GBS122(symptomatic)andaviruent D136C

Phosphate levelin Totalcellyield

Strain medium (mM) wt])after36 h

GBS122 200 5.58l

225 0.19

250 0.23

300 0.16

D136C 200 5.93b

225 0.66

250 0.68

300 0.13

aTotalcellyield(milligramsofcellulardry weight) after20 hofincubation

was0.50.

bFinalcellyield after20hof incubation.

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initiate growth during this same period, suggesting that the prolongedlagperiodisnot entirelyattributableto a nonspe-cific salteffect.

Effect of growth phase on reinitiation ofgrowth in FMC containing 200 mM phosphate. Differences have previously been observed in theamount ofLTA expressed by clinical isolates (12). Symptomaticisolates produce three-to fourfold more LTA thanasymptomatic isolates. Thisdistinctionwas observedonlywhen cells were grown to the late exponential or early stationary phase (12). A study was conducted to determine whether the growth response to high phosphate was a function of the growth phase for some of the clinical isolates. Symptomatic and asymptomatic strains were pre-grown in standard FMC to various points in the growth curve and examined for a growth response in 200 mM phosphateby theprocedures discussed above. An asympto-matic strain, GBS182, was used in place ofthe avirulent strain since ithad beenpreviouslyshown to have a short lag time in 200 mM phosphate (data not shown) and would be morerepresentative ofsomeoftheclinical isolatesthan the Lancefield typingstrain D136C. Theresponseto high phos-phatelevels bytheasymptomaticstrain GBS182(Fig. 3) was thesameforeach growth phase. However, the symptomatic isolate GBS122 exhibited a lag time when cells were pre-grown tothe lateexponential or earlystationary phase but hadgrowthkinetics similartothoseof GBS182 whengrown to the earlyormid-exponential phase(Fig. 4).

Growthresponse of clinical isolates of group B streptococci in FMC containing 200 mM phosphate. Various clinical isolates were examined to determine whether there was a correlation between the growth response (short orlong lag time)inhighphosphateandthe sourceof the isolate

(symp-2000

1000

1o20 0

Tim

(mw*in)

10- . , . Nfi. . .5 0

c

410

200

300

700

800 Time~~~Tm

(min)

1001

FIG. 3. Effect ofgrowth phaseonthereinitiation ofgrowthofS. agalactiae GBS182 (asymptomatic) in FMC containing 200 mM phosphate. Cells were pregrown in standard FMC, and samples

were'obtained

atvarious times throughoutgrowth (see inset)and immediatelychilled inanice bath. After allsampleswereobtained,

cultureswereinoculated into FMCcontaining200 mMphosphate. Symbols correspond to, the different points in the growth curve

(inset) at which samples were taken andused to inoculate FMC containing200 mMphosphate.

CL)

1000-'n

100- Tin(m)

N

<9~~~~~~~~~~~~~~C

G>100'00~~~~~~~~~~~~0200 300 700 800 9001000

Time

(min)

FIG. 4. Effect ofgrowth phase on the reinitiation of GBS122 (virulent) in FMC containing 200 mM phosphate. Experimental conditionswerethesame asthosedescribed in thelegendto,Fig. 3. tomaticor

asymptomatic).

Thestrainswerepregrowntothe

stationary phase

in standard

FMC,

inoculatedinto200mM

phosphate,

and incubated at 37°C for 16 h. The total cell

yield

ofthe

asymptomatic

isolates was 30-fold

higher

than thatof the

symptomatic

isolates after 16 h ofincubation in 200 mM

phosphate

(Table 2).

When the total cellular

dry

weight

ofthe various clinical

isolates

was

compared

with that ofthe

prototype

avirulent strain

D136C,

there was a

statistically

significant

difference

(P

<<

0.005)

between D136C and the

symptomatic

isolates examined in Table

2,

while there were no differences

(P

> 0.

100)

seen in the

asymptomatic

isolates. After sixpassages in200 mM

phos-phate,

a variant of GBS122 was obtained that

responded

with a shorter

lag

time in

high-phosphate

conditions

(see

below).

When the total cellular

dry

weight

ofthis

variant,

VP6,

was

compared

with that of

D136C,

there was no

statistically significant

difference

(P

>

0.05).

Analysis

ofTSA andLTAlevelsof

symptomatic, avirulent,

and

phosphate-adapted

mutants of group B

streptococci.

Mutantswere selected with

greatly

shortened

lag

times

by

subculturing

in FMC

containing

200 mM

phosphate.

After three passages in

high phosphate,

a variant ofthe virulent strainwasselected thathada

fivefold-higher

cell

yield

than the

parental

strain after

growth

in 200 mM

phosphate (400

min).

Subculturing

ofthe

asymptomatic

isolate selected fora strain that hadatwofoldincreasein cell

yi

eld

compared

with the wild type after seven passages in the

high-phosphate

medium

(Table 3).

The amount ofTSA was

analyzed

for each of thesubculturedstrainstodetermine whether

passag-ing

had any effect on TSAlevels. Cells weregrown tothe

stationary phase

under standardconditionsand

analyzed

for TSA.

Passaging

apparently

hadnoeffecton the TSAlevels of thesubculturedstrains

(Table 4).

However,

when

parental

strain GBS122wasgrownunderdifferent

phosphate

levels,

theamount of TSAdecreased

linearly

with

increasing

con-centrations of

phosphate (Fig. 5).

The

phosphate

concentra-tion

appeared

tohavelittle effectonthe

expression

of TSA in- the avirulent strain

D136C,

as

previously reported (16).

Theamountof LTAwasalso examinedforthevariantofthe

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TABLE 2. Differentiation ofsymptomatic and asymptomatic strainsof type III S. agalactiae by their responseto

elevated (200 mM) phosphate inthegrowth medium

Total cellulardrywt

(mg) after16hof

Strain Source incubationin

mediumcontaining

200 mMphosphate

GBS110 Cerebrospinal fluid (symptomatic) 0.08 ± 0.005"

GBS111 Blood 0.38 ± 0.064a

GBS121 Blood 0.12 ± 0.001l

GBS122 Blood 0.20 ± 0.055a

GBS123 Blood 0.59 ± 0.215a

GBS124 Blood 0.54 ±0.082a

GBS125 Cerebrospinal fluid 0.15 ± 0.031a

GBS149 Blood 0.20 ± 0.068a

GBS150 Blood 0.37 ±0.039a

GBS180 Rectum(asymptomatic) 6.19 ±0.334b

GBS182 Umbilicus 6.30 ±0.188b

GBS184 Rectum 7.58 ±0.590b

GBS188 Umbilicus 4.22 ± 1.372b

GBS207 Umbilicus 4.34 ±0.326e

D136C Prototype(avirulent) 6.35 ±0.778 VP6 High-phosphate-adapted mutant of 7.82 ±0.542c

GBS122 obtainedfromsix

pas-sagesin 200 mMphosphate

p<0.005byunpaired Student'st testwhencomparingthemeancellular

dryweightwith thatof the prototype avirulent D136C.

bp > 0.100 when comparingthe meancellulardry weight with that of D136C.

C p> 0.05 when comparing the mean cellulardry weight withthat of

D136C.

virulent strain and the parental type to determine whether subculturinginhigh phosphate hadanyeffecton LTAlevels. Cells weregrown to the stationary phase in standard FMC with [3H]glyceroltolabelthe LTA(12). The parental strain produced fourfold-higher levels of LTA than the variant (Fig. 6). This difference has also been observed between symptomatic and asymptomatic isolates (12).

DISCUSSION

Theincidence of neonatal group B streptococcal disease has remained nearlyconstantfor almost20yearswithtwoto

TABLE 3. Effect ofpassagingsymptomatic and asymptomatic strains ofS.agalactiae in medium containing200 mM phosphate

andtheirabilitytoreinitiategrowth inhigh-phosphate medium

Totalcellyield(mg[cell dry

Strain wt])after400min of

growth in medium containing

200 mMphosphate

GBS122(symptomaticsource) ... 0.67 GBS122passaged in medium

containing 200mMphosphate

One time... ... 1.54

Threetimes ... 4.44

Fourtimes... 4.92

Six times... ... 4.67

GBS182(asymptomatic source)... 1.94

GBS182 passaged in medium containing200 mM phosphate

Twotimes... ... 2.31

Threetimes... 3.68

Seven times... 4.62

D136C(avirulent strain)... 3.37

TABLE 4. Effect ofpassagingsymptomaticandasymptomatic strains of S.agalactiae inmediumcontaining200mMphosphate

onlevelsofcell-associated TSAexpressedunder low-phosphate(65 mM)growthconditions

,ugof cell-associated

Strain TSA/mg[cell

drywt] GBS122(symptomatic source)... 56.4

GBS122 passagedin mediumcontaining 200 mM phosphate

Onetime... 78.6

Threetimes... 68.9 Fourtimes... 63.3

Six times... 62.1

GBS182 (asymptomaticsource)... 29.1

GBS182 passagedin mediumcontaining 200 mMphosphate

Twotimes... 29.8 Three times... 26.2

Seven times... 23.1

D136C(avirulent strain)... 12.2

threecasesper

1,000

live births andacarrierrateofgroup B

streptococci

in women between 20 and 30%

(2).

A recent

study by Boyer

and Gotoff

(3)

involving intrapartum

treat-mentofcarrierswith

ampicillin significantly

reduced neona-talcolonizationand disease caused

by

group B

streptococci.

However,

with a 23% carrier rate

reported

in their

study,

prevention

would

require

thetreatmentofa

large

percentage of women that would

fall

within their

high-risk

category. Their criteria fortreatment would exclude womenwho are

notincludedin their

high-risk

groupandwho may becarriers of

potentially

virulent strains ofgroup B

streptococci.

There-fore,

it is

imperative

that

diagnostic

methods be

developed

which would aid in the

rapid

differentiationof GBS isolates which have the

potential

to cause disease in the neonate.

Thus, only

womencolonizedwithvirulentstrains would be

subject

to

intrapartum

treatment with antibiotics.

Inthis report, a

simple

technique

for

differentiating

type III strains

(and

possibly

other

serotypes)

isolated from infected and

asymptomatically

colonized infantsonthebasis

20

015

10

-

5-<'1:

50 100 150 200

Phosphate Concentration

(mM)

FIG. 5. Effect of elevatedphosphate levels (65to200mM)onthe amountof cell surface sialicacid(TSA) (15) of GBS122andD136C. Cellswerepregrown in standardFMCtothemid-exponential phase,

used toinoculate10mlofFMCcontaining elevated phosphatelevels

(65to200mM),andgrown to thestationary phase. Sialic acidwas quantitatedasdescribedinMaterialsandMethods.

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81

4-o'

c>

._

E

C-o

b-'en

o I>

6-

4-

2-I (dLTA)

O S 10 15 20 25

30

35 40 45 50 55 60 65 70

0

c a

o.

Q-c

o-l

fraction

#

FIG. 6. Octyl-Sepharose CL-4B elution profiles of [3H]glycerol-labeled material obtained from 45% aqueous phenol extractions of whole cells ofGBS122 (0) and VP6(O), a high-phosphate-passaged variant of GBS122. A linear gradient of 0 to 60% propan-1-ol in 0.02 M sodium

acetate(pH 4.6)wasapplied to the column, and 4.6-ml fractions were collected and analyzed. Peaks I and II were shown to be deacylated LTA(dLTA) and LTA, respectively.

of thegrowthresponse of the strains in medium containing high levels of phosphate is described. Comparison ofthe variousisolates revealed that symptomatic strains tendedto have a longer lag time in high-phosphate medium. This growth response appears to be related to the levels of LTA expressed byclinical isolates since stationary-phase symp-tomatic isolates produce three-to fourfoldmore LTA than asymptomatic strains. It has been previously shown that increasing levels of phosphate result in decreased TSA production bygroup Bstreptococci (16). The presentstudy

confirmed

this observationathigher levels of phosphate (65 to200 mM). However, when strains were passaged in 200 mMphosphate, thelevelof cell surface-associated TSAwas similartothatof the parentaltypewhencellsweregrown to the stationary phase in standard FMC

containing

65 mM phosphate. However, the phosphate-adapted strain VP6 grown to the stationary phase in standard FMC produced

25%

of the LTA

produced

by the parental type GBS122. These results suggestthat the level ofcell-associated LTA influences the length of the lag phase in high-phosphate medium. This maybe attributedtostimulation of additional LTA synthesis (4), inhibition of cellwallhydrolases by the long-chainLTA(6),or areflection ofdifferences in the level ofprecursorpoolsfor LTA

synthesis

(8).

The markedly different growth responses observed be-tween symptomaticand

asymptomatic

isolates suggest fun-damental geneticand physiological differencesbetween the twoclassesof clinical isolates ofgroup Bstreptococci. This likely is represented bydifferencesinvirulence forneonates, perhaps reflected in their adherence capabilities (12). It is quite possiblethatthistechnique fordifferentiating sympto-matic andasymptomatic strains ofgroup B

streptococci

on

thebasis of theirmarkedly different growthresponse tohigh phosphate could readily be adapted forusein epidemiolog-ical and clinepidemiolog-ical studies. Byidentifying carriers of potentially virulent strains ofgroup B streptococciand selective intra-partum treatment with appropriate antibiotics (3), the inci-denceof infections causedbygroup Bstreptococci might be drastically diminished.

ACKNOWLEDGMENTS

This workwassupported by Public Health Service grant AI 22380 from the National Institutes of Health. J.J.M. was supported by Public Health Servicetraining grant T32AI07271from the National Institutesof Health.

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ou

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