Enzymatic characterization of some oral and nonoral gram negative bacteria with the API ZYM system

0095-1137/81/090288-07$02.00/0

Enzymatic

Characterization of

Some Oral

and Nonoral

Gram-Negative Bacteria with the API ZYM System

J0RGEN SLOTS

Departmentof Oral Biology and Periodontal Disease Clinical ResearchCenter,SchoolofDentistry, State

University ofNew York atBuffalo,Buffalo, New York 14226

Received 12February1981/Accepted 24April1981

The API ZYM system (Analytab Products, Plainview, N.Y.), containing 19 chromogenic substrates, was utilized semiquantitatively to detect extracellular acid and alkalinephosphatases, aminopeptidases,proteases,esterase-lipase,

phos-phoamidase, andglycosidasesin128oral and nonoral isolates ofblack-pigmented Bacteroides, Actinobacillus, Haemophilus aphrophilus, Capnocytophaga, Fu-sobacteriumnucleatum, Wolinella recta, and Veillonellaparvula. Inthe

black-pigmentedBacteroides group oforganisms,astrongtrypsinreactionwaspresent inBacteroidesgingivalis (oralspecies)butnotinBacteroidesasaccharolyticus

(nonoral species). Bacteroidesmelaninogenicus subsp.melaninogenicus, in

con-trast to Bacteroides melaninogenicus subsp. intermedius, exhibited strong

N-acetyl-f8-glucosaminidase

activity.H. aphrophilus produced

/8-galactosidase

and

a-glucosidase, but thecloselyrelatedActinobacillus

actinomycetemcomitans

did not. Capnocytophaga was distinct with respect to strong aminopeptidase

reac-tions. Thisstudyshowed thatawide rangeof enzymes which have thepotential

of causing tissue injury and inflammation can be elaborated from major oral

gram-negative species. Also, the API ZYM system appears to be a valuable

adjunctto traditional biochemicaltestingin

identifying

oral

gram-negative

spe-cies.

Gram-negative

organisms

appear to

play

a central role in the

etiology

and

pathogenesis

of

periodontal disease and other oral diseases of humans. These bacteriaarepresent

only

inlow numbers inhealthy

gingival

sites,

but

they

com-prisemorethan 75% of the cultivable microflora in most advanced

periodontitis

lesions

(12).

Gram-negative species

arealso

frequent

isolates from teeth with acute

pulpitis

and

periapical

osteitis (17) and from tonsillar infections (4). Enzymes released

by

these

organisms

have been

implicated in the soft tissue destruction

taking

placeinthese diseases.

In our

experience,

five

species

of

gram-nega-tiveanaerobicbacteriaandtwo

species

of

gram-negative

facultatively

anaerobic bacteria

consti-tute

approximately

75% of the cultivable gram-negative microflora of most odontogenic infec-tions inhumans. The anaerobicspeciesare: Bac-teroidesgingivalis, previouslyoralBacteroides

asaccharolyticus(3); Bacteroides melaninogen-icus subspecies; Fusobacterium nucleatum; Wolinella recta (a newspecies, proposed by A. C. R. Tanner and S. S. Socransky [personal

communication], which has the following key characteristics: strictly anaerobic, predomi-nantlystraight-sided cells, size of0.5 by 2 to 4

ym, with rounded ends, motile, stimulated by formate andfumarate, asaccharolytic, benzidine

positive, catalasenegative, oxidase negative, and sensitive to dyes and antibiotics); and Veillo-nellaparvula. The two species of facultatively anaerobicgram-negative bacteria are

Capnocy-tophagaspecies, previously Bacteroides ochra-ceus (16), and Actinobacillus actinomycetem-comitans.Procedures currently used for identi-fication of these species are time consuming and

costly,involvingthe preparation and inoculation ofseveral test media and often the use of ex-tended incubation periods. Furthermore, certain oral gram-negative species grow poorly in com-monly used broth media for biochemical testing, and some gram-negative isolates from the oral cavity cannotreadily be classified by the con-ventionalphenotypic tests. From many points of view, therefore, it is desirable to develop rapid

and reliable methods of identification of oral gram-negative species.

TheAPI ZYM(Analytab Products Inc.,

Plain-view, N.Y.) system isasemiquantitative micro-method which allowsrapiddetermination of19

enzymatic reactions. The API ZYM system has been reportedtobe useful in the identification of oralstreptococci (7), oralactinomycetes (9), 288

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and nonoral gram-negative anaerobic rods (5, 18). This paper reports on the enzymatic char-acterization of common oral andcloselyrelated nonoralgram-negative speciesand on theability of the API ZYM systemtoidentifythese orga-nisms.

MATERIALS AND METHODS

Bacterial strains. Thestudy strains,thesitesfrom

whichtheywereisolated,and thesourcesfrom which

theywerereceivedarepresented in Table1.

Classifi-cation of these strainswascarriedoutby established

procedures(6).

TheAPI ZYMtests.The API ZYM system

con-sists ofaseries ofmicrocupules containing dehydrated

chromogenic enzyme substrate, detector reagent A

[tris(hydroxymethyl)aminomethane, 2.5 g; 37% HCl,

1.1 ml;laurylsulfate,1 g; anddistilled water, 10ml],

detector reagent B(fast blueBB,35mg; and

2-meth-oxyethanol, 10 ml), incubation tray and cover, and

color chart. The enzymestrip assays for alkaline

phos-phatase, butyrate esterase, caprylate esterase-lipase,

myristatelipase,leucine aminopeptidase, valine

ami-nopeptidase, cystineaminopeptidase, trypsin,

chymo-trypsin, acidphosphatase, phosphoamidase,

a-galac-tosidase, 1B-galactosidase, /3-glucuronidase,

a-glucosi-dase, 83-glucosidase, N-acetyl-,8-glucosaminidase,

a-mannosidase, and a-fucosidase. The API ZYM system

was purchased from Analytab Products, Plainview,

N.Y.

The API ZYM strips were inoculated, incubated,

andread accordingtothe manufacturer's directions.

Becausepreliminary studies showed no important

dif-ferences between API ZYM results when the assay

wasperformedaerobicallyoranaerobically,our

enzy-matictestingwascarried out under aerobic conditions.

Organisms wereobtainedfrom2-to 4-day anaerobic

(85% N2, 10%H2, 5% CO2) cultures on Trypticase soy

agar(BBL Microbiology Systems) supplemented with

10% sheepblood, 5,ugofhemin per ml, and 0.2 ,ug of

menadione per ml.Bacterialcells were emulsified in

0.85% NaCl indistilledwater toa turbidity between a

McFarlandno. 5and no. 6 standard. Eachmicrocupule

ofthe API ZYM tray was inoculated with 0.05 ml of

the standardized bacterial suspension. After aerobic

incubation in the dark for 4 h at370C, 0.025 ml each

of reagents A and Bwasaddedtoeachmicrocupule,

and thecolor reactions which developed within 5 min

weregraded from0 to 5by using the API ZYM color

reaction chart. Tests given grade 0 were regarded as

negative, grades1and 2 were regarded as weak

posi-tivereactions, and grades 3 through 5 were regarded

as moderate to strong positive reactions. Most test

strainswereexaminedatleast twice on separate days,

utilizingdifferentsubcultures.

RESULTS

The 128 isolates evaluated in this studyand the results of their enzymatic reactions in the API ZYMsystemarelisted in Table2.

Theblack-pigmentedBacteroidesspeciesand

subspecies tested varied

considerably

in enzy-matic activity.B.gingivalis possessedastrong

trypsin-like activity. In contrast, the human

strains ofB.asaccharolyticus, B.

melaninogen-icussubsp. intermedius, and B.

melaninogeni-cus subsp. melaninogenicus were negative for trypsin. Variable trypsin activity was found among the nonhuman strains of B.

melanino-genicus subsp. levii,B. macacae,and the cata-lase-positive beagle dog bacteroides. All strains of B. melaninogenicus subsp. melaninogenicus andmoststrains of B. melaninogenicus subsp.

intermedius were positive for a-glucosidase, whereas the asaccharolytic Bacteroidesstrains weredevoid ofa-glucosidase activity. Also,weak a-fucosidaseactivity was detected only in strains

ofB. melaninogenicus subsp.melaninogenicus and B. melaninogenicus subsp. intermedius. A

strong

N-acetyl-,f-glucosaminidase

reactionwas

present in B. melaninogenicussubsp. melanin-ogenicus butwas notfound inB. melaninogen-icussusbp. intermedius.

All test isolates of the Actinobacillus-Hae-mophilus group of organisms exhibited strong alkaline and acid phosphatase reactions.

Hae-mophilus aphrophilus differed fromA.

actino-mycetemcomitans byproducingfl-galactosidase anda-glucosidase. Furthermore, Actinobacillus

equuli, Actinobacillus lignieresii, and Actino-bacillus suispossessed/3-galactosidase activity and Actinobacillusseminiswaspositive for

fl-glucuronidase, characteristics which permitted differentiation between thesespecies and A.

ac-tinomycetemcomitans. Strong esterase and es-terase-lipase activities were foundinA. equuli and A. suis but not in A.

lignieresii

and A.

seminis.

The Capnocytophaga strains tested consis-tently produced strong alkaline phosphatase, acid phosphatase, leucine aminopeptidase,

va-line aminopeptidase, cystine aminopeptidase, anda-glucosidase reactions. The study strains of

F.nucleatum andW. rectagaveeithernegative

or weakpositivereactions in all of the enzyme tests performed. V. parvula produced strong

acid phosphatase and phosphoamidase; other-wise,theseorganisms weregenerally inactive.

Study on reproducibility of the API ZYM system revealed that strains of Actinobacillus

species,H.aphrophilus,F.nucleatum, W.recta, and V.parvula exhibitedlittle or no variation betweentestings. On the other hand, strains of

black-pigmented Bacteroides and Capnocyto-phaga varied in many enzyme tests between negative and weak positive reactions or between weakpositive and strong positive reactions; how-ever, variation between anegative and a strong

positivereactionwasonlyobserved foralkaline phosphataseand 8-glucosidase in two B.

mela-ninogenicussubsp.melaninogenicus strains, for 14,

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TABLE 1. Sources and sites of isolation of the strains useda

Organism and strain(siteof isolation) Source Bacteroides gingivalis

1021, 1112, 1312, 1432, 2561, 4A2-22,4A5-25, 5A1-3 (subgingival plaque)

Ki10, 381 (subgingival plaque)

Bacteroides asaccharolyticus ATCC27067(leg wound)

207-72(peritonealabscess)

B536(feces)

Umbilicus,amnioticfluid

In4(unknown)

18477(unknown)

Bacteroidesmelaninogenicus subsp. intermedius

20-3, 24, 2481, 2465,L5A2-7, 6122, 6303, 6306, 6317, 6619, 5A1-6

(subgingival plaque)

532-70A(cervical swab)

Bacteroides melaninogenicussubsp. melaninogenicus

L4A4-1 4A2-24, 4A3-11, 5A3-33, 6207,6509, 7229 (subgingival plaque)

ATCC25845(sputum), ATCC 15930(gingival crevice) VPI 9085 (gingival crevice)

Bacteroidesmelaninogenicus subsp.levii

1815, 1818(subgingival plaque)

VPI 12466 (bovine mastitis), VPI 10450 (calfrumen)

JP2(cattle horn abscess)

Bacteroidesmacacae

7511L-11A, MD1-17(monkey subgingivalplaque)

ATCC33141(monkey subgingivalplaque)

Bacteroidesmelaninogenicus, catalase-positive

434R-1,435R-5, 436R-2,437L-5, 441L-2, 442R-3, 443AL-2, 443R-2, 448R-3

(beagle dog subgingivalplaque)

889.8(beagledogsubgingivalplaque)

Actinobacillus actinomycetemcomitans

1, 4, 14, 34,39, 64,68, 73, 88,100(subgingivalplaque, representativesof10

biotypes)

ATCC29522 (mandibular abscess), ATCC29523(blood), ATCC29524

(chest aspirate)

NCTC9709(abscess), NCTC9710(abscess)

Y4(subgingival plaque)

Actinobacillusequuli

ATCC19392(blood offoal) Actinobacilluslignieresii

ATCC19393(bovine lesion)

Actinobacillus seminis ATCC15768(ovine semen)

Actinobacillussuis

ATCC15557(bloodofswine)

Haemophilusaphrophilus

ATCC13252(unknown),ATCC19415(endocarditis)

NCTC5906(endocarditis),NCTC5907 (endocarditis),NCTC5908

(endocarditis)

Own isolates

S. S.Socranskyb

ATCCC

D. W. Lambe,Jr.d

V. L. Suttere K.H.Wicherf

H. R.Inghamr

G.L.Lombardh

Own isolates

D. W. Lambe, Jr.

Own isolates ATCC J.M.Hardie'

Own isolates

L.V.Holdeman'

J. M. Hardie

Own isolates ATCC

Own isolates

K.S.Kornmank

Ownisolates

ATCC

NCTC'

S.S.Socransky

ATCC

ATCC

ATCC

ATCC

ATCC NCTC

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TABLE 1-Continued

Organism and strain (site ofisolation) Source

Capnocytophaga

2482,BA2-17,BA2-33, 3M-15,3M-16, EK2-15,EK3-21,L7A4, L8A3,

C4295, C6100(subgingival plaque)

4, 27, 2010(subgingival plaque)

Fusobacterium nucleatum

2431, 2491,MG4M,MG7M-3,KRY-14M(subgingivalplaque)

D28J-15,D31A-24,D33A-5,E2D-3A,E2D-20(oral cavity)

Wolinella recta

1225, 2130,2141, 3101,3109,3112, 3214, 3409,L4A2-1,25M-14(subgingival

plaque)

Veillonellaparvula

LlA1-8,L5A1-7, L5A3-3,MG7M-7, 3A1-9, 4A1-8, 5A2-2, 2112, 3109, 4211,

7116, 7130(subgingivalplaque)

aAll strainswereisolated from humans

unless

otherwisedesignated.

bForsyth DentalCenter, Boston,Mass.

eATCC, AmericanTypeCulture

Collection, Rockville,

Md.

d EastTennessee StateUniversity,JohnsonCity.

'Wadsworth Veterans

Administration

Hospital,LosAngeles,

Calif.

fErieCounty Health Center,Buffalo,N.Y.

gGeneralHospital, Newcastle-upon-Tyne,England.

hCenters for DiseaseControl, Atlanta,Ga.

'London

HospitalMedicalCollege, London, England.

VirginiaPolytechnic Institute,Blacksburg.

kUniversityofConnecticut, Farmington.

'NCTC,TheNational Collection ofType Cultures, London,England.

alkalinephosphataseandtrypsininoneB.

ma-cacae strain, and for esterase and

N-acetyl-,B-glucosaminidase in two Capnocytophaga strains.It should also be noted that saline which

hadbeen flooded on the surface of asterile blood

agarplatecould occasionally produce weak

re-actionsof alkalinephosphatase,esterase,

ester-ase-lipase,

and

phosphoamidase.

DISCUSSION

Despite numerous reports on phenotypic characteristicsoforalgram-negative species, lit-tle is known about the

specific

enzymatic activ-itiesof these organisms. Such information may be useful with respecttotaxonomy and

evalua-tionof

pathogenicity.

B.

gingivalis

and B.

asaccharolyticus,

al-though genetically markedly different (3), can

presently onlybe differentiated on the basis of

serological

assays(11), directhemagglutination of sheep erythrocytes (13), or production of

phenylacetic acidinglucose broth cultures (8). In this study, a strong trypsin reaction of B.

gingivalis

was shown also to

distinguish

this

species fromB. asaccharolyticus.

Discriminat-ing between B. melaninogenicus subsp.

inter-medius and B. melaninogenicus subsp.

mela-Own isolates

S. S. Socransky

Own isolates

L. V.Holdeman

Own isolates

Own isolates

ninogenicuscanbedifficult whenusing

conven-tionalbiochemical tests. Thestrong

N-acetyl-f8-glucosaminidase

reaction which waspresent inalltest strains of B. melaninogenicus subsp.

melaninogenicus, but not in any strains ofB.

melaninogenicus subsp. internedius,may prove tobeavaluable differential phenotypic charac-ter.The finding that a-glucosidase activity was

absent inasaccharolytic Bacteroidesspecies and present in B.melaninogenicus subsp. interme-dius andB.melaninogenicussubsp.

melanino-genicus

agreeswiththe

capability

of these or-ganisms offermenting maltose (6).

A. actinomycetemcomitans is designated "species incertae sedis"inBergey's Manual of

DeterminativeBacteriology,8thed. (1). Uncer-tainty especially exists in the taxonomic

rela-tionship between various actinobacilli species and H. aphrophilus, organisms which do not require X (hemin) or V (nicotinamide adenine

dinucleotide) factors for growth. Many API ZYM testsonlyexhibited weak activity for

Ac-tinobacillusand

Haemophilus;

however, the re-actions werereproducible and therefore

poten-tially useful in taxonomy. H. aphrophilus

pro-duced,8f-galactosidase

anda-glucosidase, but the

closely related A. actinomycetemcomitans did

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J. CLIN. MICROBIOL.

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293 not.Thepositive

,B-galactosidase

reaction in H.

aphrophilusagrees with this

organism's

ability

to ferment

lactose,

a key character in identifi-cation of H.

aphrophilus (10).

On the basis of

B-galactosidase,

a-glucosidase, 8i-glucuronidase,

andesterase-lipase

reactions,

theothertest

Ac-tinobacillus

species

could also be identified. The API ZYM systemdidnotdiscriminate between the 10 biotypes of A. actinomycetemcomitans described

by

Slotsetal. (14).

The

Capnocytophaga

genus exhibits great

variance in

morphological

and

physiological

characteristics, and the species

singularity

of

these

organisms

isstillnotclear

(16).

The API

ZYMpattern obtained for

Capnocytophaga

was distinct with respect to strong leucine, valine, and

cystine

aminopeptidase

reactions.Other

en-zymatic reactions varied in

activity

between strains of

Capnocytophaga,

butnoobvious

cor-relation was demonstrated between API ZYM testsand key

phenotypic

characteristics. Also,

variability

in

activity

between

repeated

testings

of

Capnocytophaga

strains limited the

taxon-omical value ofmany enzymetests.

The absence of glycosidase reactions in all strains ofF.

nucleatum,

W. recta, and V. par-vulawasconsistent with the

asaccharolytic

na-tureof these

organisms.

Inrespecttotaxonomy and

typing

of these three

species,

theAPIZYM system

appeared

tohave limited value.

The widerange of enzymeswhichthisstudy showedcanbeelaborated from oral gram-nega-tivespeciesare

capable

of

acting

on avarietyof

host

macromolecules,

such as constituents of

connective

tissue,

complement

components,and

immunoglobulins.

It is not

clear, however,

to

whatextentthese enzymesareinvolvedin

bio-logical

eventsinvivo. Little is known about the actual concentration of freeenzymes inthe oral

cavity

(2). Human

polymorphonuclear

granulo-cytesandother hostcells

furthernore produce

many oftheenzymes whichcan bedetectedin

bacteria (15), and the relative

importance

of

bacterialenzymes and host-derivedenzymes in

the

inflammatory

processisunknown. The effect ofspecific antibodiesandtheplasma inhibitors

alpha1-antitrypsin, alpha1-antichymotrypsin,

and

alpha2-macroglobulin

in the inactivation and elimination of bacterialenzymesalso awaits

furtherstudy.

In summary, the results presented in this

study indicate that enzyme activities of oral

gram-negative isolatescanbedetectedbyusing the API ZYM system. The method has the

potentialtodistinguishbetweenmajor oral bac-terial genera, and it appears to be useful in

differentiatingB.gingivalisand B. asaccharo-lyticus,B.

melaninogenicus

subsp. intermedius

andB. melaninogenicus subsp.

melaninogeni-cus, andH.aphrophilus andvarious

Actinoba-cillus

species.

ACKNOWLEDGMENTS

This work wassupported by Public Health Service grant DE-04898 from the National Institute of Dental Research.

The technical assistance of P. M. Lobbins is highly appre-ciated.

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