Biotyping coagulase negative staphylococci

0095-1137/88/101950-07$02.00/0

Copyright(O 1988,American Society for Microbiology

Biotyping

Coagulase-Negative Staphylococci

G. ANN HÉBERT,* ROBERT C.COOKSEY, NANCYE C. CLARK, BERTHA C. HILL, WILLIAM R. JARVIS, ANDCLYDE THORNSBERRY

Hospital Infections Program, Center for Infectious Diseases, Centers forDiseaseControl, Atlanta, Georgia30333

Received 14 April 1988/Accepted 28June1988

The biochemical profiles obtained with Staph-Ident (Analytab Products, Plainview, N.Y.) panels were combined with' the results ofadherence and synergistic hemolysistests todefine biotypesamong1,064 clinical isolates representing eight species of coagulase-negative staphylococci. The 672 isolates of Staphylococcus

epidermidis were aligned in 69of144 potential biotypes in our scheme because of 18 different biochemical profilesand the eight physiologic subtypes. Isolates ofmost other species werein fewer biotypes because of

moreuniformadherence andsynergistic hemolysisdatà,aswellasfewer biochemical profiles. Sinceadherence andsynergistic hemolysis may prove tobe related tovirulence and pathogenicity, biotyping with thesetest results would help evaluate the reliability of adherence and synergistic hemolysisas possible indices ofthe clinical significance ofsome ofthese organisms. When the antimicrobialsusceptibility and plasmid profiles

obtainedontwoclustersofS.epidermidisisolateswerecomparedwith thebiotyping results,oneclusterwasnot furtherdifferentiated by plasmid profiles, butwasby antimicrobial profiles;the other cluster with only two

biotypes was further divided into five distinct types by plasmid profiles but was not separated at all by

antimicrobial profiles.

The natural habitats of Staphylococcus epidermidis and several other species of coagulase-negative staphylococci

include the skin and nares (11); a clinical specimen could,

therefore,contain both contaminants and pathogens of these

species. If a contaminant is mistakenly identified as a patho-gen, i.e., the source of theisolate was the skin and not an

infectiveprocess,the patientwill probably receive unneeded

antimicrobial therapy. When coagulase-negative

staph-ylococci are isolated from a specimen, particularly one

obtainedby skinpuncture,theirsignificance is often doubted and twoquestionsare oftenasked. Is the isolate apotential pathogen, and is it identical to skin isolates? Furthermore,

thequestion of whether strains areidentical may be raised when more than one isolate of coagulase-negative

staphylo-cocci is obtained froma specimen, particularly from blood

cultures. These considerations have become more critical

since coagulase-negative staphylococci have become oneof themost frequent causesof nosocomial infections(9).

Onewayto

approach

the

question

ofwhether strainsare

identical is to use a biotyping scheme. The biochemical

profiles

obtained with the

Staph-Ident

system

(Analytab

Products, Plainview, N.Y.) have been combined with anti-biograms, slime production, and phage types to identify strainsinepidemiologic investigations; plasmid banding pat-ternshavebeenused todifferentiate strains ofS.epidermidis (17) andtoenhance epidemiologic studies (16, 18). Onetest

that has been prominently proposedas atool formeasuring

theclinical significanceofcoagulase-negative staphylococci (3, 4, 10) is slime productionoradherence (2), buttestsfor

exotoxinproduction orsynergistic hemolysis (8) have also beensuggested. If thesecharacteristics areconsidered tobe indicators of pathogenicity, the biotyping scheme that

in-cludedthemcouldserve adual purpose: totype theisolates andtodetermine theirclinical significance.

In a companion report (7), we presented the results of

biochemical studies, adherence tests, and synergistic

he-molysis tests of a large collection of coagulase-negative

* Correspondingauthor.

staphylococci. We reexamined those data to see whether they could be usedto differentiatestrainsforepidemiologic studies. The result was to combine data from several test

systems into a biotyping scheme. In this report, we define

our biotyping scheme, show its application to our culture

collection,anddiscussitsusewithplasmidandantimicrobial

profiles on two smallclusters of S. epidermidisisolates.

MATERIALS AND METHODS

Cultures andgrowth conditions. Allof the isolates

exam-ined forthis studywerepartofalargercollectionof strains described in the companion report (7). A total of 1,064 of

those clinical isolates (7) were included in this biotyping study; these included 672 S. epidermidis, 133 S.

haemnoly-ticus, 103 S.hominis,48 S.simulans,38 S.saprophyticus, 40 S. warneri, 15 S. capitis, and 15 S.

xylosus.

Fiveof the S.

epidermidis isolates were from the cardiac-care wing of

hospitalA;four ofthefivecamefrom pumpblood collected

in theoperating room fromheart-lung bypassmachines, and

thefifthcame fromablood culture taken 6days

postopera-tionfromoneofthefourpatients. SevenotherS.

epidermi-disisolates were frommultiple blood cultures taken froma

single patientwho hadpneumoniaanda cardiacmurmur at

hospitalB. Alloftheculturesweregrownon

Trypticase

soy agarcontaining 5% defibrinated sheep blood (TSA II;BBL

Microbiology

Systems, Cockeysville, Md.)

and were incu-batedaerobically for 18to 24h at 35°C.

Staph-Ident system. AlU of the isolatesweretested with the

Staph-Identsystemas

reported

in the

companion

report

(7).

This panel included tests for the

production

of alkaline

phosphatase,

P-glucosidase,

P-glucuronidase,

and

,-galacto-sidase;for the utilization of

urea`and

arginine;

and foracid

productionfrom mannose, mannitol,

trehalose,

and salicin. The panelswere inoculated andprocessed

according

to the

procedures recommended by the manufacturer. The result-ing biochemical profiles were expressed as

four-digit

num-bers.

Adherence.All of the isolatesweretestedfor adherenceto

glass culture tubesasdescribedbyChristensenetal.

(2);

the

1950

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details of the

qualitative procedure

weused werediscussed in the

companion

report

(7).

Briefly,

after the cells had grown

overnight

ina

glass

tubeof

broth,

theturbid brothwas

poured

out; the tubes were then rinsed once, filled to the

original

broth level with safranin tostain for30

min,

rinsed

twice,

and turned

upside

down to drain and

dry.

The dry tubes were observed

by

transmitted

light

for evidenceofa

stained

film;

and the reactions were recorded as

negative,

weakly positive,

moderately positive,

or

strongly positive.

Controls includedthereference strains of Christensenetal.

(3);

control strains ATCC 35983

(RP12)

and ATCC 35984

(RP62A)

were

strongly

positive,

and strain ATCC 35982

(SP2)

was

negative

in the adherence tube test.

Synergistic

hemolysis.

All of the isolates were tested for

synergistic hemolysis

asdescribed

previously

(8).

Astrainof S. intermedus

(AB148)

was streaked downthe centerofa

TSA

II

plate.

The test strains were streaked

perpendicular

to, but not

touching,

thecenter inoculum. The

plates

were

incubated

aerobically

at

35°C

for18to20h and heldatroom

temperature

for4to6

h;

thereactions werereadat

approx-imately

24 h. Azoneof

complete hemolysis

(where

thetest

strain was

growing),

within the zone of

incomplete

he-molysis

caused

by

the

beta-lysin

from S. intermedius

growth,

was a

positive

test.

Plasmid

profiles.

Twelve of the S.

epidermidis

isolates

were examined

by plasmid

DNA

electrophoresis.

The

plas-mids of the five isolates from

hospital

Awere extracted

by

the

procedure

of

Goering

and Ruff

(6)

and

separated by

vertical

electrophoresis

in an agarose

gel

(0.85%

in

Tris-acetate

buffer)

for 12 h at35 mA. The seven isolates from

hospital

B were tested

by

the

procedure

of Takahashi and

Nagano (19),

in which crude cellular

lysates

were

electro-phoresed

in a horizontal agarose

gel

(0.7%

in Tris-acetate

buffer)

for4 hat 55 mA. Eachofthe

gels

was stainedwith

ethidium bromideto

develope

the

bands, destained,

and then

photographed using

transilluminated

shortwaveUV

light.

Antimicrobial

profiles.

The 12 S.

epidermidis

isolates from

hospitals

Aand Bweretestedfor

susceptibility

to a setof13

antimicrobial agents

by

agar disk diffusion as described

previously (14).

The antibiotic disks

(BBL)

usedwere

peni-cillin

(10 U),

oxacillin

(1

,ug),

methicillin

(5

,ug),

amoxicillin-clavulanate

(20/10

,ug),

cephalothin

(30 ,ug),

erythromycin

(15 ,ug),

clindamycin (2 ,ug), chloramphenicol

(30 ,ug),

tetra-cycline

(30 ,ug), sulfamethoxazole-trimethoprim (23.75/1.25

,ug),

rifampin

(5 ,ug), vancomycin (30 ,ug),

and

gentamicin

(10

itg).

P-Lactamase

test. The same 12 isolates were also

exam-ined for

P-lactamase

production

by

a

rapid chromogenic

cephalosporin

test

(15,

20).

A

500-,ug/ml

solution of

nitro-cefin

(Glaxo, Ltd., Greenford,

Middlesex, England)

was

prepared by dissolving

20mgof nitrocefinin 2mlof

dimethyl

sulfoxide and

diluting

the mixture to 40 ml with fresh

phosphate

buffer

(pH

7),

whichwasmade

by mixing

39.2 ml

of1/15 M

monopotassium phosphate

with60.8mlof 1/15M

disodium

phosphate.

A

loopful

of18-to24-h

growth

onTSA

II

wasusedtoprepareadense

ceil suspension

intwodrops

ofnitrocefin solutionin the well ofamicrodilutionplate. All inoculatedwellsweresealed with

strips

ofcellophanetape to prevent

evaporation.

The mixture was incubated at room

temperature

and observed fora color change immediately,

after 15

min,

and after 1 h. The reaction was recorded as

negative (yellow),

weakly positive (dark

orange),orpositive

(red)

after 1 h. When an isolate tested negative, it was retested after induction with methicillin by repeating the

nitrocefintestwitha

loopful

of the 18-to24-hgrowth around the methicillin disk

(5 ,ug).

Known

,-lactamase-positive

and

TABLE 1. SummaryofStaph-Ident, adherence,andsynergistic

hemolysis results usedforbiotyping

Staph-Ident %positivefor: profiles

Organism(no.)

No. No. Adherence Synergistic seen unique tube test hemolysis

S.epidermidis (672) 18 2 83 73

S. haemolyticus (133) 17 6 42 99

S. hominis (103) 5 1 56 52

S.simulans(48) 10 3 71 96

S. saprophyticus (38) 9 4 71 0

S. warneri(40) 13 7 5 63

S.capitis(15) 5 3 7 93

S.xylosus(15) 12 10 73 53

,-lactamase-negative strains of S. aureus were included as

controls.

Independent testing and dataanalysis.Theplasmidstudies weredoneby twoofus,and theantimicrobialsusceptibility

studies weredone byanother one ofusindependentof any otheranalyses.Alldata except theplasmidand antimicrobial results were stored and analyzed with computer software

(dBASE III PLUS; Ashton-Tate, Torrance, Calif.) and a

portable computer (COMPAQ) upgraded to 640 kilobytes

(7).

RESULTS

AsummaryoftheStaph-Ident, adherence,and

synergistic

hemolysis test results is shown in Table 1. The isolates in each ofthese

eight species

ofcoagulase-negative staphylo-coccigaveatleast fivedifferentprofiles withtheStaph-Ident

system, and within each species some ofthe profiles were

unique. Thiswas moststrikingamongisolatesof S.xylosus, because those 15 isolates gave 12 different biochemical

profiles, and only2of thoseprofileswereproduced bymore than 1 isolate. Because the distribution of several

biochem-ical

profiles

within eachspecies seemedagood basis foran

initial separation into biotypes, the biochemical profiles obtained with the Staph-Identsystemfor each specieswere

listed in the order of frequency of occurrence and were

assigned

letter codes forbiotyping (leavingoutOand Qto

avoid confusion).

Inthe adherence test, 42 to83% oftheisolatesinsix of the

eight species

were positive. In addition, the

adherence-positive

isolates were graded as strong,moderate, orweak

(7). Amongthe 747adherence-positive isolatesin these eight

species, 29% were strongly positive, 55% were moderately

positive,and16%wereweakly positive.Most (200 of 214) of the strongly positive isolates amongthese species were S.

epidermidis; however, among the 556 adherence-positive S.

epidermidis isolates,

36%werestrong,50%weremoderate,

and 14% were weak. These adherence test results suggest

that thistestcanbe used for the subtyping of isolates.

The synergistic hemolysistestresults showed homogene-itywithin four of thesespecies;none of the S. saprophyticus were positive;and nearly all the S. haemolyticus (99%), S. simulans(96%),and S. capitis(93%)isolates were positive.

Amongthe other four species, however, 52 to 73% of the

isolateswerepositive. With this range of values, synergistic

hemolysiscould also be a toolforsubtyping isolates.

There-fore,

thephysiologic subtypesforbiotypingwere defined by

the results obtained with the adherence and synergistic

hemolysistests(Table 2). The subtypes 1 and 2 were further

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TABLE 2. Definition of physiologic subtypesfor biotyping coagulase-negative staphylococci

Resultof:

Subtype

code Adherence tube test hemolysis

la Positive (strong) Positive

lb Positive(moderate) Positive

lc Positive(weak) Positive

2a Positive (strong) Negative

2b Positive(moderate) Negative

2c Positive (weak) Negative

3 Negative Positive

4 Negative Negative

divided into classes by the strong, moderate, and weak levels of positivity obtained in adherence tests.

The 144 biotypes and subtypes of S. epiderinidis that resulted from these definitions are shown in Table 3. These 672 isolates gave 18 different Staph-Ident profiles, but 493 (73%) ofthe isolateswere inonly 2 profiles: 3040 (51%) and 3000(22%).The 18 profiles included twoof the phosphatase-negative codes forS. hominis: 2040 (5.6%) and 2000(2.4%). Most(633 of 672) of the S. epidermidis gave oneofonly six biochemical profiles. The data for S. epidermidis reflect the highpercentage ofstrains that were positive for both phys-iologictests,regardlessof their biochemical profile, because 60% (401 of 672) of the isolates weresubtype 1; however, the adherence classes separated each of the large groups in biotypes A through F into three much smaller groups. Isolates were seen in each of the eight possible subtypes of

biotypes A, B, and C; seven subtypes of biotype D; six subtypes ofbiotype E; andfive subtypesofbiotype F.In the biotypes G through R that contained more than one isolate, thesubtypesgave manyuniquecodes that representedsingle isolates.

The many biotypes and subtypes among the other seven species of coagulase-negative staphylococci are shown in Table 4. The Staph-Ident profiles obtained with the 103 isolates ofS. hominiswerecontained in fiveprofiles,but 100 (97%) werein oneofthreeprofiles: 2000 (36%), 2400 (31%), and 2040 (30%). The data on S. hominis showed a nearly

even distribution ofstrains among the subtypes in the three

major biotypes. As expected, only twosubtypesper species

were useful in separating the isolates within most of the otherspecies. Most ofthe isolates of S.haemolyticus andS. simulans werein subtypes 1and 3, because isolates inthese speciesarerarelynegative in synergistic hemolysis tests. All

ofthe isolates ofS.saprophyticus were in subtypes 2 and 4,

because isolates of this species do not exhibit synergistic

hemolysis; and most of the isolates of S. warneri were in

subtypes 3 and 4, because adherence isseldom, ifever, seen

among isolates of this species. The data on adherence

classesarenotshownfor theseseven species;butthey were

veryuseful inseparating thesubtype 1 isolates ofS.hominis, S. haemolyticius, andS. simulans and thesubtype 2 isolates of S. hominis.

The electrophoretograms obtained during the plasmid

studies are shown as Fig. 1 and 2. The profile in lane 1 on

each gel andall similarprofiles on thatgel within that set of isolates were given the code letter A; the next profile

encountered was designated B, and so on, until all profiles

for agiven set ofisolates were assigned a code.

Therewerethree different plasmid profilesamongthefive

isolatesfrom hospital A(Fig. 1). Isolates 1 and 3withprofile A contained one large plasmid band and several smaller bands that were not seenin the otherthree isolates. Profile B of isolate 2 also contained several bands, but itwas unique amongthesefive isolates. Although theprofiles of isolates 4 and 5 were slightlydifferent, each wasdesignatedaseparate subtype ofprofile C because of thepossibleloss in isolate 4 of the large plasmid seenin isolate 5.

There werefive differentplasmid profilesamongtheseven

isolates from hospital B (Fig. 2). Isolates 1, 2, and 4 had identical banding patterns; but the other four isolates had profiles differentfrom these and from each other.

The disk-diffusionantimicrobial susceptibility profiles ob-tained with the five isolates from hospital A are shown in Table 5. Theprofile of isolate 1, and anysimilarprofileinthis setof fiveisolates, wasassignedthecode letter A; thenext

profileencountered wasassignedthe letterB,andsoonuntil all profiles were assigned codes. Isolates 1, 2, and 3 were

resistant to six or seven of theantibiotics tested. Isolates 1

and 3 of profile A were identical, including their mixed

sulfamethoxazole-trimethoprimtestresults;eachproduceda

zone of susceptibility with this disk, but both zones

con-tained several discrete colonies that indicated some resis-tance to this drug. Isolate 2 was the only one that was

resistant to tetracycline and gentamicin and was assigned

profile B. Isolates 4 and 5 were very susceptible and quite

different from the other three in this set; isolate 4 was

1-lactamase positive

and resistant to

penicillin only,

but isolate 5 was

1-lactamase

negative and susceptibleto all of

the antibiotics tested, so they were assigned to separate profiles (C and D, respectively). The susceptibility datafor

theisolatesfrom hospitalBare notshown, becausethey all

had the same antimicrobial profile. All seven isolates from

hospital B were

P-lactamase

positive and resistant to

peni-cillin but were susceptible totheother 12antibiotics tested. The combined data on the two clusters ofS. epidermidis

isolatesare shown in Table 6. Thetestresults thatproduced

the biotype codes are included in Table 6 to illustrate the conversion of data to code. There were three different

biotypes among the five isolates fromhospitalA. Isolates1,

TABLE 3. Biotypesand subtypesof 672 S. epidermidisisolates Biotype Staph-Ident No.with No. of isolates in each subtype

code profile profile la lb lc 2a 2b 2c 3 4

A 3040 342 63 106 34 24 42 11 45 17

B 3000 151 25 48 15 19 19 2 17 6

C 7040 65 16 25 4 2 5 1 10 2

D 2040 38 10 8 4 1 6 1 8

E 7000 21 6 5 3 5 1 1

F 2000 16 6 1 5 2 2

G 1000 7 2 2 1 2

H 7100 5 5

I 3100 5 3 1 1

J 6040 5 1 1 1 1 1

K 1040 4 2 1 1

L 2100 3 3

M 6000 2 1 1

N 3140 2 1 1

P 0000 2 1 1

R 1100 2 1 1

S 0040 1 1

T 5000 1 1

Total 141 199 61 59 77 19 89 27

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TABLE 4. Biotypes andsubtypes of coagulase-negative staphylococciotherthan S. epidermidis

No.of isolatesineach Organism(no.)and Staph-Ident of the following

biotypecode profile subtypes:

1 2 3 4

S.hominis (103) A

B C D E

S.haemolyticus (133) A B C D E F G H I J K

S.simulans(48) A B

c

D E F G

S.saprophyticus (38) A

B

c

D E

S. warneri(40) A B C D E F

S.capitis (15) A

B

S.xylosus(15) A B 2000 2400 2040 0400 0040 0660 0460 4440 4640 0640 4400 4460 0060 4040 4060 4660 Unique' 2461 2041 2061 2441 2741 2661 3061 Unique 2001 2401 6001 2040 2000 Unique 6400 6460 6660 6640 6600 6440 Unique 0040 0140 Unique 6021 6621 Unique 15 10 8 6 9 8 1 1 7 9 13 5 4 4 3 2 3 1 1 3 6 9 7 5 3 2 2 15 4 6 2 2 4 8 9 9 9 5 1 25 14 1 3 6

s

4 5 4 3 2

FIG. 1. Agarose gelelectrophoresis ofplasmidDNA extracted from the S. epidermidis isolates received fromhospitalA. Lanes 1 through 5,isolates1through 5,respectively; lettersAthrough Care the plasmid profile codes. After extraction (6), the lysates were electrophoresedin0.85%agarosefor12 h at 35 mA.

1 1

3, 4,

and 5 had the same

Staph-Ident

profile

but could be

3 separatedinto twosubtypes bytheadherence and synergis-tic hemolysistestresults. Isolate 2 gave astrongpositive in theadherence test, and theStaph-Identprofile of2000shows 4 2 that it was negative in the testfor alkalinephosphataseand

1 did not utilize arginine. The biotype, plasmid, and

antimi-2 crobial data

agreed

thatisolates 1 and 3 were identical and thatisolates4and 5weresomewhatsimilar,but that isolate

2 2 was not like either of these

pairs.

Isolates

1, 3, 4,

and 5

2 werethefourobtained frompump

blood;

isolate 2wasfrom

theblood culture ofa

patient

who had beenonthepumpthat yielded isolate 1.

ThecombineddataontheisolatesfromhospitalBarealso 1 shown inTable 6.Allwerestrongly positive in the adherence

4 test, but therewere twodistinctbiotypes among these seven

2

2 A A B A C D E

1 2 3 4 5 6 7

1-'1-V I y-,--Ir -

IolE-1 1 8 2

4 4 3 1 2 2 1 2 2 6 1 1 7 3 3 1

1 1 1

1 1

5 3 1 1

FIG. 2. Agarosegel electrophoresis of plasmid DNA extracted fromthe S.epidermidis isolates received from hospital B. Lanes 1 through 7, isolates 1 through 7,respectively; letters A through Eare the plasmid profile codes. After extraction (19), the lysates were

electrophoresed in 0.7%agarosefor 4 hat55 mA.

A B

1 2

A Cl C2

3 4 5

aUnique indicatesaStaph-Ident biochemicalprofile produced by only one

isolate.

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TABLE 5. Disk-diffusionaantimicrobial susceptibility profileson theclusterof S. epidermidis isolates from hospital A

Antimicrobial profiles ofisolatesb: Antibiotic

1 2 3 4 5

Penicillin R R R R S

Oxacillin R R R S S

Methicillin R R R S S

Amoxicillin-clavulanate R S R S S

Erythromycin R R R S S

Clindamycin R R R S S

Tetracycline S R S S S

Sulfa-trimethoprimc S/Rd S S/R S S

Gentamicin S R S S S

,-Lactamasetestresulted + + + +

Profile code A B A C D

a National Committee for Clinical Laboratory Standards, standard M2-A3 (14).

b All five isolates were susceptible to cephalothin, chloramphenicol, rifam-pin, and vancomycin; R, resistant;S, susceptible.

CSulfamethoxazole-trimethoprim (23.75/1.25 ktg).

dThesestrains contained both susceptible and resistant colonies.

e Tested with nitrocefin (seetext).

isolatesbecause of the results of the Staph-Identand

syner-gistic hemolysis tests. The fiveisolates in biotype A2a did not exhibit synergistic hemolysis, but the two isolates in

biotypeFla werepositive for synergistic hemolysis. Both of

thebiotypes amongthese seven isolates were further

sepa-rated by the plasmid profiles; biotype A2a included three

different plasmid profiles (A, B, and E), and biotype Fla

included twoplasmidprofiles (C and D). The antimicrobial profiles for theseisolates were identical and, therefore, did notseparate the isolates at all. These seven isolates were all

from the samepatient.

DISCUSSION

The Staph-Ident system gives goodcorrelation with con-ventional methods (12) for species identification ofclinical isolates ofstaphylococci (7, 13). Thisrapid, 10-test system,

althoughdesigned toidentify species of staphylococci, has

also been usedtobiotype strains withinspecies of

coagulase-negative staphylococci(1, 18).Thebiochemical characteris-tics of these organisms are diverse enough for several

profiles todefine the same species correctly. The

biochem-icalprofiles thencanalso serve asameansofdifferentiating

strains within the same species. In most studies, however,

toofewprofileswere seen perspecies tobe veryhelpful in

epidemiologic studies; therefore, the Staph-Ident system was used with other tests to define subtypes for greater

discrimination. Some of the othertestsusedwere

antimicro-bial susceptibilities, serotyping, phage typing, plasmid

pro-files, and slime productionoradherence (16).

We also used the biochemical profiles of Staph-Ident to

define our biotypes, but we supplemented them with two

physiologic tests, adherence and synergistic hemolysis, to

define four subtypes and then further separated two of the subtypes with three classes each of adherence. Althoughwe

could have separated the other two subtypes with three classes each ofsynergistichemolysis, i.e.,weak,moderate,

orstrong,wefelt that thatdegree ofseparation could best be

usedon asmaller,specific setofstrains withaspecific lot of

media.

Oneof thenewertools fordifferentiatingstrainsor

deter-mining their relatedness is the plasmid profile. The current

methodsarecomplex enough that theyarenotpractical for the routine examination of large groups of strains, norare

they methods that are likely to be used by most clinical laboratories, but the determination ofplasmid profiles may prove tobe valuable in special situations. Perhaps the best

useofthistechnique is the study ofagiven subset of strains

from thesameoutbreakorpatient thatappearidenticalby all

of theothertestsystems.Thetwoclusters of S.epidermidis

isolates examined during this studywereexamplesof this: (i) a set of isolates from a clinic where pump blood was questionedasthesourceofapatient'sinfection and (ii)aset

of isolates fromasingle patient whosephysicianquestioned

whetherall of the isolates werethe same strain. Inthe first

cluster,biotyping alone showed that thepumpblood isolates were not identical to the isolate from the patient, and the plasmid profiles did not give any further differentiation. In the second cluster, however, biotyping defined only two groups of isolates, but the plasmid studies yielded five

distinctprofiles that showed that no more than three ofthe

seven isolates werethesame strain.

Both theadherencetestandthesynergistic hemolysistest

have given reproducible results in ourlaboratory whenthe testswere doneexactly asdescribed previously (7, 8). One

oftheproblems encounteredwhenattemptingtouseplasmid

profiles to characterize a set of strains, however, is the

TABLE 6. Combineddataontwoclustersof S. epidermidisisolates

Hospitaland Staph-Ident Adherence Synergistic Biotype Plasmid Antimicrobial

isolateno. profile tubetest hemolysis code profile profile

HospitalA

1 3040 - + A3 A A

3 3040 - + A3 A A

4 3040 + + Alb Ci C

5 3040 + + Alb C2 D

2 2000 + + - F2a B B

HospitalB

1 3040 ++ - A2a A A

2 3040 ++ - A2a A A

4 3040 ++ - A2a A A

3 3040 ++ - A2a B A

7 3040 + + - A2a E A

5 2000 + + + Fla C A

6 2000 ++ + Fla D A

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instability of the individual plasmids. Bacterial strains may

lose antibiotic resistance plasmids and may transfer resis-tance plasmids withinor evenbetween species; inaddition, variations in plasmid extraction methods may alter the

molecularform ofa plasmid, and the conditions of

electro-phoresis can influence the final profile (18). Isolates with

identical plasmid profilesare, therefore, probably clones of

thesame strain; however, isolates with similar, though not

identical, plasmid profilesmayalso be from thesame strain

ifthey have several plasmid bands in common and come

from the same source. A multifaceted approachcould help

overcomethesekinds ofproblems; some methods of

differ-entiating strains are better epidemiologic tools than others, so some combination of methods may be the answer. As

indicated above, plasmid studies may be more usefully

applied to sets of strains from the same source that have

already been shownto be identical by several other

meth-ods.

One of the other methods of differentiating strains is

susceptibility to antibiotics. This is another technique that

maybemosteffective withasubsetof strains fromthesame

outbreak or patient. That was the case with our set of

isolates from pump blood and a patient's blood that were

grouped into three biotypes and had three or fourplasmid

profiles. Only two of these isolates had exactly the same

antimicrobialprofile, but the four isolates frompumpblood

wereeasily placed intotwo similarsetsoftwoisolateseach and theisolate from the patientwasdistinct, although itwas mostlike theisolates inoneof the two sets.This technique didnot,however, differentiate the othersetofsevenisolates that were grouped into twobiotypes and had five separate

plasmid profiles.

Theproblem, then, isnotascarcity oftestsbut howtouse

thebattery oftestsavailable fordifferentiatingthese

organ-isms. The clinician needs a reliable index of significance

when confronted withanisolate of coagulase-negative

staph-ylococci. To establish that indexweneed simple,

reproduc-ible, inexpensive tests that can be done in any clinical

laboratory. Both adherence and synergistic hemolysis are

simple, inexpensive tests thatuse routine media and

over-night incubation, and bothareteststhatcan be done inany

clinical laboratory. In addition, since both adherence and

synergistic hemolysis might be related to virulence and

pathogenicity, theymayhelpto define clinicalsignificance.

It has been suggested that the adherence of some ofthese

organisms tosmooth surfaces enables themto quickly colo-nizeaprosthesis, catheter, orimplant (3). Once established, some of these species can produce one or more cytolytic

toxins thatactsynergisticallywith the toxins ofS.aureus,S.

intermedius, Corynebacterium pseudotuberculosis,

Clos-tridium perfringens, and perhaps others to lyse erythro-cytes(7, 8). Other cytolytic and equally destructive biologic activities may occur in vivo as a result of adherence and

synergism. In a recent review, Gemmell (5) discussed the production of these and other toxins and enzymes by

co-agulase-negative staphylococci and concluded thatwe need more detailed investigations of natural and experimental

infection before we can determine the role (if any) ofthe

many virulencefactors that have been postulated for these

organisms. We suggest, therefore, that abiotyping scheme

that includes tests for adherence and synergistic hemolysis

be applied to collections of isolates with sufficient clinical

datatoevaluate thereliability ofthesetwocharacteristicsas

possible measures ofsignificance. We also suggest that the

system we have proposed for biotyping will be useful but

recognize that this must be confirmed by applying it to a

sufficient andsignificant body of clinical data. ACKNOWLEDGMENT

We thank P. B. Smith for constructive, critical review of the manuscript.

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