Evaluation of the Enteric Analyzer, an instrument to aid in the identification of Enterobacteriaceae

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Vol. 2, No. 3 Printed in U.S.A.

Evaluation of the Enteric Analyzer,

an

Instrument

to

Aid

in

the

Identification of Enterobacteriaceae

DON J. BRENNER* ANDALBERT BALOWS Centerfor Disease Control,Atlanta, Georgia 30333

Received for publication 4 June 1975

This study evaluated the Enteric Analyzer, aninstrument designed to

iden-tifyEnterobacteriaceae, with data obtained fromthe Modified r/b Enteric

Differ-entialSystem, otherrapididentificationsystems, orconventionalidentification

systems. It is programmed for 19 reactions with data obtained from Center for

Disease Control publications. The instrument is very simple to use. Typical

strains from 25 of 28specieswereunequivocallyidentified. Withthe otherthree

species, twochoicesweregiven. Aswitch allows theusertoidentifyallspecies

possibilities where agiven biochemical patternoccurs inmore than 1%of the

strains or morethan 10%of the strains. The instrument is useful both in the

clinical laboratoryand forteachingpurposes.

Thesignificantincreasesinnosocomial

infec-tions, inmultiple drug-resistant bacteria, and

inbacteriathatarebiochemically atypicaldue

tothe presence ofplasmid-mediatedmetabolic

genes are but some of the reasons why more

laboratories tend to speciate

Enterobacteri-aceae. The policy in individual laboratories

withregardtoidentificationatthespecies level

iscontingent upon cost, time,andspace

require-ments, aswell asthe number of personnel and

their level ofcompetence.

A numberof kitsorsystemsare now

commer-cially available for the rapid identification of Enterobacteriaceae. These systems have been

evaluated in a number of laboratories (11-16)

and also have been foundtobepracticalfroma

cost-accounting point of view. By properly

us-ingthesesystemsandproperlyinterpreting re-sults, trained personnel arebetter ableto

spe-ciateorganisms. Theuseofthesesystemshelps

to eliminate errors and variations in results

caused by using different media, reagents,

tests,ordifferencesinqualitycontrol. The

abil-ity to compare results obtained from different

laboratories should now be substantially in-creased.

Withtheavailabilityofthesesystems, a

ma-jorremainingproblemishow besttoapply the

data to speciation. Various approaches have

been recommendedincludingrelying on the

ex-pertise of the bacteriologist, providing flow

sheets for identification, and providing either

biochemical profile analysis keys or a

com-puteranalysis of biochemical data.

DiagnosticReasearch,Inc., hasdeveloped an

instrument called the Enteric Analyzer. This

instrumentisprogrammed according to

percent-23

agespublished byEwingetal. (2-9)toidentify Enterobacteriaceae basedon 18biochemical

re-actionsand motility.

As seen inFig. 1, the keyboardhas switches

for each reaction. Each switch has apositive, neutral, and negative position. Above the

key-boardare 28 red lights, each correspondingto

anenteric speciesorgroupofspecies. As

posi-tive ornegative reactions areentered, the red

lights are extinguished as species are

elimi-nated. Also on the keyboard is a switch with

two positions labeled 90 and 99%. In the 90%

mode a species is eliminated when the test

strain exhibits apositive or negative reaction

seen in less than 10% of the strains of that

species. In the 99% mode, a species is

elimi-nated only when a given reaction is seen in less

than 1%of its strains.

TheEnteric Analyzer isdesigned for use with

the Modified r/b Enteric Differential System. The manufacturer states that it can also be

used with any otherrapid identificationsystem

or a conventional identification system which

includes the reactions covered by the

instru-ment.Inthepresentstudythis instrument was

evaluated with data obtained from approxi-mately 1,000 gram-negative, oxidase-negative,

fermentative strains that hadbeenpreviously

identified as Enterobacteriaceae by

conven-tional biochemical methods.

MATERIALS AND METHODS

Bacteria.Thespecies used inthis study are listed

in Table 1, along with the corresponding

designa-tions ontheEntericAnalyzer.Theywere pure cul-tures isolated from clinical specimens including

stools, sputa, urines,blood, and wounds.

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236 BRENNER AND BALOWS

4cWmr4mZUrAoUa Wy

S s QtmCl_Am ,t \AS

Oll

,~~

FIG. 1. TheEntericAnalyzer. The keyboardcontains anOnlOff switch,90% mode or 99% mode, and 19

switchesfor enteringreactions. The reaction switches haveapositive, neutral, and negative position. The

display has28boxeseach with aspecies nameandaredlightemittingdiode.

Evaluation. The data used to evaluate the En-tericAnalyzer hadpreviously been obtained by

con-ventionalbiochemicaltests(1-10)onapproximately

1,000 gram-negative, oxidase-negative, fermenta-tive strains. Representatives of all of the genera and/or species covered by the instrument were

in-cluded. In most cases all reactions coded by the instrument were used. These were phenylalanine

deaminase, hydrogen sulfide production,gas produc-tion fromglucose, lysine decarboxylase, lactose fer-mentation,indole production, ornithine decarboxyl-ase,motility, citrateutilization, rhamnose fermenta-tion,deoxyribonuclease, raffinose fermentation,

sor-bitol fermentation, arabinose fermentation,

malo-nateutilization,urease,inositol fermentation, adon-itol fermentation, and esculin utilization. All

reac-tions were read at 24and 48 h. Any reaction that became positive after 48 h was deemed negative.

Specificcommentsusually applytostrains exhibit-ing only one atypical reaction. An attempt was

made to include strains exhibiting each atypical reaction occurring in from 1 to 10% of the total

strainsofagiven species.

RESULTS

Use of the Enteric Analyzer. The Enteric

Analyzer isdesigned forusewiththe Modified

r/b EntericDifferential System. The

manufac-turer states that it can also be used with any

other rapid identification system or

conven-tionalidentification system. The instrument is

extremely simple to operate. The instruction

bookletisclearly and concisely written.

Exam-plesareincludedtohelp theuserlearnto

oper-atetheinstrument, whichcanbeaccomplished

within 15 min. With practice, the data canbe

easily entered and the result obtainedin 30 s to

1 min.

When only one species isindicatedno

inter-pretation of results is necessary. When all

spe-cies areruledout inboth the90and99%modes,

the culture can be presumptively reported as

unidentified. In these cases the users are

di-rected tovarious publications listed in the

in-J. CLIN. MICROBIOL.

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ENTERIC ANALYZER 237 TABLE 1. Species studied and Enteric Analyzer

designations

Species Enteric Analyzer designa-_tion'

Arizona hinshawii Arizona sp. (typical)

Citrobacter diversus

C.freundii

Edwardsiella tarda Enterobacter aerogenes

E.agglomerans

E.cloacae

E.hafniae

Escherichia coli Klebsiella ozaenae

K.pneumoniae

K. rhinoscleromatis

Proteus mirabilis

P. morganii

P.rettgeri

P. vulgaris

Providencia

alcalifa-ciens

P.stuartii

Salmonella cholerae- Salmonella sp. (typical)

suis

S. enteritidis Salmonella sp. (typical)

S. enteritidis serparaty- Salmonellaparatyphi A

phiA

S.typhi

Serratia rubidaea

Shigella boydii Shigella sp.(typical)

S.dysenteriae Shigella sp.(typical)

S.flexneri Shigella sp. (typical)

S.sonnei

Yersinia enterocolitica

Y.pseudotuberculosis

aOnly listed when differentfrom designation in

the lefthand column.

structionmanualfor

guidance

inthe

identifica-tion. When more than one

species

possibility

exists in either the 90 or 99% mode the user

mustchoose betweenthe

possibilities

ordecide

what additional tests are

required

because: (i) thereisnoindicationas towhich of the choices

has a greater probability ofbeing the correct

one; and(ii) there are no

guidelines

for which

additional tests, if any, should becarriedout.

Identification of typical strains. For

pur-posesofevaluation,a

typical

strainexhibitsthe

reactionshownbymorethan 50% of all strains

included in the Center for Disease Control

ta-bles for the19 tests

programmed

inthe Enteric

Analyzer(1-10). Ideally, inthe90%

mode,

the

EntericAnalyzershould

unequivocally

identify

every typical strain.

Twenty-five

of the 28

spe-ciesareidentifiedinthe 90% mode. The

excep-tions are as follows: (i) Enterobacter cloacae

cannotbedistinguished from Citrobacter

freun-dii. The addition of the methyl red and Voges-Proskauer tests would allow differentiation of

these species. (ii) Yersiniapseudotuberculosis

cannotbe distinguished from Enterobacter

ag-glomerans. The addition of Voges-Proskauer,

sucrose, and motility tests at 22 C would allow

differentiation of these species. (iii) Shigella

sonnei is not distinguished from Shigella sp.

(typical). S. sonnei is ornithine positive,

whereas strains of other shigellae are 98% or

more ornithine negative. Therefore, S. sonnei

canbedistinguished from other shigellae

with-out any additional tests. This apears to be a

programming error.

Identification of atypical strains. Any

strainof a speciesexhibiting a reaction shown

by less than 10% of the total strains of that species is eliminated in the 90% mode. If the

reaction occurs in less than 1% of the strains,

the atypical strain is also eliminated at 99%. Of particular interest are atypical strains that ex-hibit reactions shared by 1 to 10% of the total

strainsof a species.Theseareidentified at 99%,

but achoiceoftenmustbe made between two or

more possibilities. As shown in Table 2, this

type of strain is encountered in almost every

species. The instrument gives no indication of

which possibility is more likely. Furthermore,

astrainthat exhibitsoneatypical reaction

(oc-curringin 1 to 10%ofstrainsin agiven species)

is notdistinguishedfrom a strain exhibiting as

many as six or more atypical reactions.

Ineach case, additional reactions are

neces-sary tochoosebetweenpossible species. Some of

these reactions areshown inTable 3. In some

cases these are commonly used tests such as

methyl red, Voges-Proskauer, KCN, arginine, and maltose. In other instances the reactions

such as corn oil, gelatin, a-methyl glucoside,

mucate, and tartrate are less commonly used.

Possible programming errors. There are

several caseswhereastrain isnot identified in

the90%modeorthe 99% mode even though the

atypical reaction occurs in more than 1 or 10%

ofthe total strains (Table 4). There are also

instances when a strain exhibiting a reaction shared by less than 10% of the total strains is identified in the 90% mode (Table 4). These

reactions are most likely theresult of errors in

programming the Enteric Analyzer.

Delayed reactions. Rapid identification

sys-tems are usually designed to be read between

18and 24 h. Conventionalsystems may be read

anywhere from 18 h to two weeks or longer. There is no mention of delayed reactions in the instruction manual. Some examples of

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TABLE 2. Identification of atypical strains of enteric bacteria by use of the Enteric Analyzer in the99%mode

Species Reactiona Choices

Klebsiellaozaenae K.pneumoniae

Enterobacteraerogenes

E.cloacae

E.hafniae

Anaerogenic E.

agglom-erans

Serratiamarcescens

S. liquefaciens Salmonella typhi Salmonella sp. (typical)

Shigella sonnei Shigella sp. (typical) Escherichiacoli

Providencia

alcalifa-ciens

P. alcalifaciens P. stuartii

Proteusmirabilis P. vulgaris

orn+(2%) mal+(6%) ado-(1%) gas-(3.5%) lys-(3%) lac-(2%) ind+(6%) cit-(2%)

mal-(7.5%)

ure-(5.5%)

ino-(2%) lys-(l%)

mot-(3%) orn-(4%)

mot-(5.5%) raf-(3%) sor-(5.5%)

ure+(3%) esc+(6%) mot-(7%)

ado+(3%)

mot-(1%) cit-(l%)

mot-(7%) esc-(5%)

H2S-(6%) H2S-(8%) lys-(5%) rha-(10%) raf+(3%) sor-(6%) lac+(2%)

gas+(2%)

gas-(8%)

lac-(8%) ind-(1.5%) ado-(6%) phe-(3%)

mot-(4%)

cit-(2%)

ado-(5%)

phe-(6%) lac-(4%) cit-(4%) raf+(6%)

sor+(4%)

ara+(5%) ado+(4%) ino-(2.5%)

orn-(1%) H2S-(5%)

K. ozaenae, E.aerogenes, S. liquefaciens K. ozaenae, E. aerogenes, E.agglomerans K. ozaenae, E.aerogenes, E. agglomerans

K.pneumoniae, K. ozaenae, E.aerogenes, S. rubidaea K. pneumoniae, K. ozaenae, E.aerogenes, E. cloacae,E.

agglomerans S. rubidaea K.ozaenae, E. aerogenes K. pneumoniae, S. rubidaea

K.pneumoniae, K. ozaenae, E.aerogenes, S. rubidaea K. pneumoniae, K. ozaenae, E.aerogenes, S.rubidaea

K.pneumoniae, K. ozaenae, E.aerogenes, S.rubidaea

E. aerogenes, E. cloacae E.aerogenes, K. ozaenae

E.cloacae, E. aerogenes, E. agglomerans, C.freundii, S. rubidaea

E.cloacae, E. aerogenes, K. ozaenae, C.freundii

E. cloacae,E.aerogenes,C.freundii

E. cloacae, E.aerogenes, C. freundii

E.hafniae,E. aerogenes

E.hafniae,E.aerogenes

E.hafniae,E.aerogenes, K. ozaenae

E.agglomerans,E.cloacae, E. aerogenes

S. marcescens, S.liquefaciens S. marcescens, S.liquefaciens

S. liquefaciens,K.ozaenae

S. liquefaciens, Salmonellasp. (typical)

S. typhi, Salmonellasp. (typical)

Salmonellasp.(typical),E.aerogenes,S.liquefaciens

Salmonellasp. (typical), C.freundii

Salmonella sp. (typical),Arizona

S.sonnei, C.freundii,E.coli,K.ozaenae, E.aerogenes,

S.liquefaciens

Shigella sp. (typical), Salmonella sp. (typical),E.

ag-glomerans

E.coli, S. liquefaciens

E.coli,S. liquefaciens

E.coli.E.aerogenes,S.liquefaciens

E.coli, S.liquefaciens

P.alcalifaciens,E.agglomerans

P.alcalifaciens,E. agglomerans

P.alcalifaciens,P. vulgaris, P.mirabilis,E.

agglomer-ans

P.stuartii,E.agglomerans

P.stuartii, E.agglomerans

P.stuartii,P.alcalifaciens,P.mirabilis,P.vulgaris, E.

agglomerans

P.mirabilis,P.vulgaris

P.vulgaris, P.rettgeri

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TABLE 2-Continued

Species Reactiona Choices

P. rettgeri phe-(2%) P.rettgeri, E. agglomerans

lac+(9%) P.rettgeri, E. agglomerans mot-(6%) P.rettgeri, E. agglomerans cit-(4%) P.rettgeri, E. agglomerans

raf'(9.5%) P.rettgeri, E. agglomerans

ino-(7%) P.rettgeri, E. agglomerans

P. morganii mal'(5%) P. morganii, P. mirabilis

ure-(2%) P. morganii, P. mirabilis

phe-(5%) P.morganii, S. liquefaciens

orn-(3%) P. morganii, P. mirabilis, P. rettgeri, P. vulgaris, E.

agglomerans

Yersinia enterocolitica gas-(1.3%) K.ozaenae, S. liquefaciens

rha'(1.3%) C.freundii, K. ozaenae, E.aerogenes,S.liquefaciens

DNase+(3%) Y. enterocolitica, K. ozaenae, S.liquefaciens

raf'(7%) Y. enterocolitica,K.ozaenae, S.liquefaciens

aThe numbers in parentheses are the percentages of strains of a given species exhibiting the listed

reaction.

TABLE 3. Additional testsof value inidentifyingatypical strainsofenteric bacteria

Species Reactions

Klebsiellaozaenaefrom K.rhinoscleromatis

K.ozaenaefrom K.pneumoniae

K.ozaenaefrom Enterobacteraerogenes

K. ozaenaefrom E.agglomerans

K.pneumoniae from E.aerogenes K.pneumoniae from E. cloacae K.pneumoniaefrom E.agglomerans K.pneumoniae from Serratia rubidaea E.aerogenes fromE. cloacae

E.cloacae from Citrobacterfreundii E.hafniae fromE.aerogenes E.hafniae fromK.ozaenae

E.agglomerans fromE.aerogenes E.agglomerans fromE.cloacae E.agglomerans from S. rubidaea E.agglomerans from C.freundii

S. marcescensfromS. liquefaciens

S.liquefaciens from K.ozaenae

S.liquefaciens from Salmonella sp. (typical) S.rubidaea from K.ozaenae

Salmonellatyphi fromSalmonellasp. (typical) Salmonella sp.(typical)fromArizona hinshawii Salmonella sp. (typical)fromC.freundii Salmonella sp. (typical)from E.aerogenes C.freundiifrom E.aerogenes

Edwardsiellatardafrom S. liquefaciens E.tarda fromSalmonellasp. (typical) Shigella sonnei fromEscherichiacoli S.sonnei fromSalmonella sp.(typical)

S.sonneifromC.freundii S.sonneifrom K.ozaenae

S.sonnei fromE.aerogenes

S.sonnei fromS. liquefaciens

Shigellasp.(typical)fromSalmonellasp.(typical) Shigellasp. (typical)fromE.agglomerans Shigellasp. (typical) fromE.coli

Shigellasp. (typical)fromK.ozaenae

Sucrose, starch,a-methylglucoside

Methyl red, Voges-Proskauer

Methylred,Voges-Proskauer

a-Methyl glucoside, gelatin Gelatin

Arginine, gelatin

Tartrate, a-methylglucoside

Cornoil, gelatin

Arginine,gelatin

Methyl red, sucrose, salicin

Voges-Proskauer, salicin

Methyl red,Voges-Proskauer, KCN,tartrate

Methyl red, arginine,a-methyl glucoside

Mucate, cornoil,tartrate

Methyl red, Voges-Proskauer, arginine

Methyl red, Voges-Proskauer, xylose

Voges-Proskauer,xylose

KCN, sucrose,gelatin

Methyl red, Voges-Proskauer, gelatin,cornoil

Dulcitol, mucate, sodiumacetate

Gelatin, dulcitol,tartrate

KCN, tartrate, 13-glactosidase

Methyl red, Voges-Proskauer, KCN, sucrose

Methyl red, Voges-Proskauer KCN, sucrose,xylose, mannitol

Arginine, dulcitol,xylose, mannitol

Dulcitol, salicin, mucate

Dulcitol, xylose, mucate

KCN, xylose, mucate

KCN, salicin

Methyl red, Voges-Proskauer, KCN

KCN, gelatin, cornoil

Xylose, maltose, tartrate,mucate

Voges-Proskauer, sucrose, xylose

Xylose, mucate, tartrate

Methyl red, Voges-Proskauer, salicin

ENTERIC ANALYZER

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TABLE 3-Continued

Species Reactions

Shigellasp. (typical) from K. rhinoscleromatis

Shigellasp. (typical) from Proteus morganii

Shigella sp. (typical) from Providencia

alcalifa-ciens

Shigellasp. (typical) from P. stuartii

E.coli from S.liquefaciens

E.coli from Arizonahinshawii

E.colifrom E. aerogenes

E.coli from S. rubidaea

E.coli fromE.agglomerans

P.alcalifaciens from P. vulgaris

P.alcalifaciens from P. mirabilis

P. alcalifaciens from E. agglomerans P.stuartii from P. alcalifaciens P.stuartiifrom P. mirabilis P. stuartii from P. vulgaris P.stuartii from E. agglomerans P. mirabilis from P. vulgaris P. vulgaris from P. rettgeri P.rettgeri from E. agglomerans P. morganii from P. rettgeri P. morganii from P. mirabilis P. morganii from P. vulgaris P. morganii from S.liquefaciens

P. morganrii from E. agglomerans

Yersiniaenterocolitica from K. ozaenae

Y.enterocolitica from S.liquefaciens

Y.enterocolitica from C. freundii Y. enterocolitica from E. aerogenes

Y.enterocolitica from Salmonella sp. (typical) Y. enterocolitica from Shigella sp. (typical) Y.enterocolitica from E. coli

Y.pseudotuberculosis from E. agglomerans Y.pseudotuberculosis from K. ozaenae

Y.pseudotuberculosis from E. aerogenes

Y.pseudotuberculosis from P. rettgeri

Methyl red, Voges-Proskauer, sucrose

KCN,tartrate

KCN, tartrate

KCN, gelatin,mucate

Gelatin, tartrate

Methyl red, Voges-Proskauer, KCN

Methylred, Voges-Proskauer, arginine, tartrate

Sucrose, maltose, xylose, gelatin, corn oil Gelatin, corn oil, xylose

Voges-Proskauer, tartrate,xylose, maltose

Trehalose, sodiumacetate

Gelatin, cornoil, xylose

Gelatin, sucrose, cornoil, xylose

Voges-Proskauer, maltose, xylose, tartrate

Sucrose, maltose, salicin

Sucrose, maltose, xylose

Voges-Proskauer, maltose, xylose, gelatin Mannitol, glycerol, erythritol

Xylose,gelatin, corn oil

Sucrose, gelatin, corn oil, maltose

Sucrose, xylose, salicin, gelatin

Voges-Proskauer, gelatin, maltose, tartrate

Motility (22C), KCN, corn oil

Motility(22 C), KCN, gelatin

Motility(22 C), KCN, sucrose

Motility (22 C), KCN, methyl red,

Voges-Pros-kauer

Motility (22C), arginine, sucrose, dulcitol

Motility (22C), corn oil, cellobiose

Motility(22 C), corn oil, mucate, cellobiose

Motility(22 C), Voges-Proskauer, sucrose

Motility(22 C), KCN, salicin,cellobiose

Motility (22C), methylred, Voges-Proskauer

Motility(22C), KCN, mannitol, maltose

mon,delayed reactions that will result in mis-identification onthe Enteric Analyzer are

lac-tose orcitratefor Enterobacterhafniae, lactose

orraffinoseforS.

sonnei,

esculin for Citrobac-terdiversus, and sorbitol for Providencia

stuar-tii. Theusershouldbecautioned that reactions

turning positive after 24h areprogrammedas

negative inthe EntericAnalyzer.

Flexibility and service. The instruction

manual statesthat allrepairs andadjustments

must be made by the manufacturer. We have

hadnomalfunctionduring7months ofuseand

thereforecannot commentonthetimeinvolved

or cost ofrepairs. Since the usercannot make

adjustments, any updating in percentages,

changes in reactions, or in recognized species

will have to be carried out by the

manufac-turer.

DISCUSSION

TheModified r/b Enteric Differential System

is considered to be "highly satisfactory as a

system for identification ofEnterobacteriaceae"

(12). The Enteric Analyzeris designed foruse

with this system; however, it is programmed

with data obtained from conventional

biochemi-cal tests performed at the Center for Disease

Control. This datawasobtained at 24and48h

and should be perfectly compatible with the

dataprogrammed inthe instrumentand valid

for purposes of evaluation. Some variation in

testresults amongvarious rapid identification

systemsand between these systems and

conven-tional biochemical methods is to be expected.

We have not yet attempted to evaluate the

EntericAnalyzer with data obtained from any

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ENTERIC ANALYZER 241

TABLE 4. Possibleprogramming errors in the

present Enteric Analyzer

Enteric

Analyzer Species Reactiona identification

90% 99%

mode mode

Klebsiella ozaenae raf+(12%)

K.pneumoniae lac-(2%)

Enterobacter hafniae lac+(<l%) +

cit+(<l%) +

rafl(<l%) +

glu-(<1%) +

ara-(4%)

-Serratia marcescens lac+(2%)

-raf+(2%)

-S. rubidaea esc-(10%)

Arizona hinshawii ara-(2%)

-Salmonella sp. (typi- lac+(1%)

-cal)

ind-(1%)

-rha-(10%)

Citrobacter freundii cit-(10%)

Edwardsiella tarda glu-(%)

-ind-(1.5%)

-Escherichia coli ino+(1%)

-Providencia stuartii mal+(1%)

-adoN(4%) +

Proteus mirabilis raf'(1%)

-P.rettgeri sor+(1%)

-mal(1%)

-Yersiniaenterocoli- glu-(%)

-tica

rha+(1%)

-aThenumbers inparentheses are the percentages

of strains ofa given species exhibiting the listed reaction.

rapidsystem. However,previous reports detail our evaluation of the r/b system (12, 16). On balance, the EntericAnalyzer isavery useful adjunct for rapidconversionof biochemical test

resultsto identificationat an acceptablelevel.

Whenmorethanonespeciesfitsthe biochemi-cal profile, the user must have aconsiderable degreeof competence to choose between them.

There isnoway

provided

toassesseither

abso-luteorrelative

probabilities

of

possible species.

For instance, aKlebsiella pneumoniae strain

that is citrate negative (2% ofK.

pneumoniae

arecitratenegative) islistedinthe99%mode.

Thesameistruefora

hypothetical

strainofK. pneumoniae that does not

produce

gas from glucose(3.5%ofstrains), is citratenegative (2% ofstrains),lysine negative (3%ofstrains),indol positive (6% ofstrains), ureasenegative (5.5% of strains), and inositol

negative

(2% ofstrains).

Assuming that these traitsare not

linked,

the

probability ofthe second strain is about10-8.

In most cases additional reactions will

re-solve the instances where more than one

spe-cies ispossible. The reactionsfelt to be useful

arelisted in Table3. Aflow sheetdiagramfor

someof theseadditionalreactionsinthe identi-fication ofenteric bacteria is giveninreference

12. Taxonomy of Enterobacteriaceae is a

dy-namicfield.Until 2or 3years ago H2S-positive

Escherichia coli were notrecognized. Nowthey aremorefrequently isolated.Newer species

des-ignations includeSerratia rubidaea, E.

agglom-erans, and C. diversus. The instrument

cor-rectly identified these, but will have to be

pe-riodicallyupdated as new groups are recognized

and changes occur in reaction frequency. For

instance,urease-positive E. coli, KCN-positive

E.coli,Pectobacterium, andErwinia mayhave

tobe includedinthe future. Yersinia pestis is not included. The instrument has no built-in

flexibility. This may become a serious

draw-back ifupdatingcannotbe carriedoutquickly

andatlow cost.

The presence of the Enteric Analyzer has stimulated our personnel to compare, check,

anddiscuss their results. Used either

individ-ually or by groups the instrument is an ex-tremely useful teaching and reference tool for both studentsandtechnicians.The effectofany

given reaction or groupofreactions onagenus

orspecies canrapidly be determined. Atypical or hypothetical strains can be entered at will and thendiscussed. Againit mustbenotedthat not all useful tests are programmed, andthat other sources must be consulted for problem organisms.

The following recommendations are madein anefforttoincreasetheoverall effectivenessof

the Enteric Analyzer. (i) The nomenclature

usedisthatrecommended byEwing (2-10). For

consistency, Arizona sp. (typical) should be

changedtoArizonahinshawii, andSalmonella paratyphi A should be changed to Salmonella enteritidis ser

paratyphi

A.

(ii)

Klebsiella rhi-noscleromatis is extremely rare. Perhaps this

species should be combined with Klebsiella

ozaenae. Klebsiellae would then occupy two

slots: K. pneumoniae and Klebsiella sp. (iii)

Enterobacter agglomerans should perhaps be

subdividedintoaerogenicand anaerogenic

bio-types. These two groups differ inseveral

reac-tions. The two categories would frequently

make E. agglomerans easier to eliminate from

consideration. (iv) The programming error

foundwithtypicalS. sonnei and those apparent

programming errors listed in Table4should be

corrected. (v) Additional tests as outlined in

Table3 areadvisable, particularly methyl red,

Voges-Proskauer, and KCN. In the absence of

VOL. 2, 1975

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http://jcm.asm.org/

(8)

additional tests, the user should be provided with guidelines as to which additional tests are useful in differentiating between pairs of orga-nisms. (vi) It would be extremely beneficial to have some indication of the relative probability

of species. This could beaccomplishedbyeither

having a bright or dim setting for the light next

to each species, by having two or threedifferent

coloredlights next to each species, or by

provid-ing a table of relative probabilities. (vii) There

is no indication given as to the number of

strainsof a species used as the data base. This

canvarysubstantially. For example, the

follow-ingnumbers of strains wereused inthe

refer-encescited: 616 strains of C. freundii and 137

strainsof C. diversus (3); 371 strains of

Salmo-nella (4), only 16 of which are S. typhi, and 16,

S. enteritidis ser paratyphi A (4, 7); 1021

strainsof E. coli (2); and 5166 strains of

shigel-lae (2). The user should be aware of these varia-tions in numbers and in the difference in signifi-cance between data based on 16 strains and 5,000 strains.

ACKNOWLEDGMENTS

We are indebted to V. R. Dowell, Jr., forhis continuing interest in this project, helpful suggestions, and critical reading of the manuscript. We also wish to thank B. R. Davisfor a critical reading of the manuscript.

LITERATURE CITED

1. Darland, G., and B. R. Davis. 1973. Biochemical and serological characterization; hydrogen sulfide produc-ingvariantsofEscherichia coli. Center for Disease Control, Atlanta.

2. Edwards,P. R., andW. H. Ewing. 1972.Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co.,Minneapolis.

3. Ewing, W. H. 1971. Biochemical characterization of Citrobacterfreundii and Citrobacter diversus. Center

for Disease Control, Atlanta.

4. Ewing, W. H. 1972. Isolation and identification of

Sal-monella and Shigella. Center for Disease Control, Atlanta.

5. Ewing, W. H. 1973. Differentiation of

Enterobacteri-aceaeby biochemical reactions. Center for Disease Control,Atlanta.

6. Ewing, W. H. 1973. Biochemical reactions given by Enterobacteriaceae in commonly used tests. Center

for DiseaseControl, Atlanta.

7. Ewing, W. H., andM. M.Ball. 1966. The biochemical reactions of members of the genus Salmonella. Cen-terfor DiseaseControl, Atlanta.

8. Ewing,W.H., and B. R. Davis. 1970.Media andtests

for differentiation of Enterobacteriaceae. Centerfor Disease Control, Atlanta.

9. Ewing, W. H., B. R. Davis, and M. A. Fife, 1972.

Biochemical characterization of Serratia liquefaciens and Serratia rubidaea. Center for Disease Control, Atlanta.

10. Ewing, W. H., andM.A. Fife. 1971.Enterobacter ag-glomerans: the Herbicola-Lathyri bacteria. Center

forDiseaseControl,Atlanta.

11. Goldin, M. 1972. Comparison ofmultiple-test systems for the presumptive identification of Enterobacteri-aceae.Am. J.Med. Technol. 38:288-291.

12. Isenberg,H.D.,P. B.Smith,A.Balows,B. G.Painter, D. L. Rhoden, and K. Tomfohrde. 1974. r/b expan-ders: their use inidentifying routinely and unusually reactingmembers of Enterobacteriaceae.Appl. Micro-biol. 27:575-583.

13. Painter,B.G., andH.G.Isenberg.1973.Clinical labo-ratory experience with the improved Enterotube. Appl. Microbiol. 25:896-899.

14. Rhoden,D. L.,J. M.Tomfohrde, B. P.Smith, andA.

Balows. 1973. Evaluation of the improved Auxotab1

systemfor identifying Enterobacteriaceae. Appl. Mi-crobiol. 26:215-216.

15. Smith, P. B.,J. M.Tomfohrde,D. L. Rhoden, andA.

Balows. 1971. Evaluation of the modified r/b system for identification of Enterobacteriaceae.Appl. Micro-biol.22:928-929.

16. Smith,P. B.,K. M.Tomfohrde,D. L.Rhoden, andA.

Balows. 1972. API system:amultitube micromethod for identification of Enterobacteriaceae. Appl.

Micro-biol. 24:449-452.