Evaluation of the modified Micro ID system for identification of Enterobacteriaceae

CLINICAL 0095-1137/79/10-0454/05$02.00/0

Evaluation

of the

Modified

Micro-ID

System

for Identification

of Enterobacteriaceae

WILLIAM J.BUESCHING,' DWANE L.RHODEN,2 ANN 0. ESAIAS,2 PETER B.SMITH,2 AND

JOHN A. WASHINGTON II'*

Sectionof ClinicalMicrobiology, Mayo Clinic andMayo Foundation, Rochester, Minnesota 55901,1 and

Center for DiseaseControl, Atlanta,Georgia 303332

Receivedforpublication30July1979

Micro-IDis asystemdesignedtoidentifythe Enterobacteriaceae by utilizing reagent-impregnated disks for 15 biochemical tests. Since its initial evaluations, the system hasundergonemodification in formulation and in its computerdata

base. In a dual-centerevaluation,306 isolates of Enterobacteriaceae were tested:

145 commonandtypicalisolates at the MayoClinic and 161 unusual oratypical isolates at the Center for Disease Control. Each laboratory also exchanged 50

cultures

to test the

system's

reproducibility.

Micro-ID

correctly

identified 142

(98%) of the common clinical isolates and 123 (76%) ofthe unusual or atypical organisms. However, in this latter group, three species tested were not in the

system's data base. When these

organisms

weredeleted from the

analysis,

138of

146(95%) of the unusual oratypical isolateswerecorrectlyidentified.Analysisof

the100isolatesidentified in

duplicate

revealed 93%

reproducibility

of genus and species identification and 62%

reproducibility

of octal code numbers. Of the 31

strains with the sameidentification butdifferent code numbers, 74% differed in only one biochemicaltest.

In many

laboratories,

a

majority

of the work loadinvolves the isolation and identification of members of the family Enterobacteriaceae.

These

organisms

areoften the causative agents

ofsevere and

life-threatening infections.

Thus,

therapid and accurate identification of enteric

pathogens by

the clinical

microbiology

labora-toryis ofsome

importance.

A newbiochemical identificationkit, the Micro-ID system (General

Diagnostics,

Morris

Plains, N.J.), provides

iden-tification of the Enterobacteriaceae 4 h after inoculation. This system utilizes reagent-im-pregnated filter paper disks for 15 biochemical

tests.A

five-digit

octal code number is generated

from each setofbiochemical reactions, and an identification is derived fromacodebook. Since its initial evaluation (1), the system has

under-goneextensive modification. The 15biochemical

tests arethe same, but selected changes in

sub-strate/indicator ratios have been made. Further,

thesystem's computer data base has been mod-ified. It was the purpose of this work to deter-mine the abilityofthe modified Micro-ID

sys-tem to identify both common and unusual

iso-lates of Enterobacteriaceae and to obtain an estimate of the reproducibility of the system.

MATERIALS AND METHODS

Culturestested. A total of 306 stock cultures

rep-resenting 34 species of Enterobacteriaceae were

tested: 145 commonandtypicalisolatesattheMayo

Clinic and 161 atypical or unusual isolates at the

Centerfor DiseaseControl.Culturesweremaintained

at roomtemperature ontrypticsoy agarslants

over-laidwithsterile mineraloilorintrypticsoy agarstabs.

Totestthesystem's reproducibility, eachlaboratory

exchanged50cultures. Thus,100isolateswere

identi-fied induplicate.Allorganismswereassigneda

num-ber, and the identity ofno isolate was known until

testingwascomplete.

Identificationmethods. Thestockculturestested

attheMayoClinic hadbeen previously identified by

meansofclassicaltests(3,5) and theAPI20E system

(Analytab Products Inc.). The API 20E strips were

inoculated, incubated,and readaccordingtothe

man-ufacturer's instructions. Sincetheperformanceofthis

systemhas beenwellcharacterized(2,6, 7,8),further evaluation in this study was not attempted. Those

isolatestested at theCenterfor Disease Control had

beenpreviously identified by using conventional bio-chemical tubedmedia (4, 6). The nomenclature used in thisstudywasbasedonthetaxonomic systems of Edwards andEwing (4), Ewing and Martin (6), and Brenneretal. (2).

Micro-ID system. The Micro-ID system consists ofamolded styrene tray containing 15 reaction wells

and a hinged cover. The first five wells contain a

substratedisk andanindicatordisk; the remaining 10 wells contain asingle combination substrate/indicator disk. The 15 biochemical tests used by the system include: Voges-Proskauer, nitrate, phenylalanine

de-aminase, hydrogensulfide(H2S), indole, ornithine

de-carboxylase, lysine dede-carboxylase,malonate, urea,

es-454

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culin,o-nitrophenyl-fi-D-galactopyranoside,arabinose,

adonitol,inositol, and sorbitol. Organisms tested were

taken fromMacConkeyoreosin-methylene blue agar

platesafter overnight incubation at 35°C. Each

orga-nism wassuspended in isotonic saline to a turbidity of

a no. 2McFarland standard. Each Micro-ID strip was

labeled, and 0.2 ml of the organism suspension was pipetted into each inoculation well at the top of the

unit.The cover wasclosed, and the strip was placed in

anuprightposition and gently tapped, ensuring that

theorganism suspension was incontact with all

sub-strate disks. Each strip wasincubated in an upright

position at35°C for4 hinanatmosphere withoutCO2.

Afterincubation, two drops (0.1 ml) of 20% KOH

wereadded to theinoculationwellofthe

Voges-Pros-kauer testchamber, and the strip was set upright to

allow

the KOH to flow down into the test solution.

The strip was thenrotated about 90° so that the upper

indicatordisks in the first fivewells became wet. The

strip wasagain set upright and the reactions were read

accordingtothecolorguideprovidedwith thekit. The

reactions wererecorded on data sheets, and the

five-digit octal number was calculated. Each organism was identified by using the Micro-ID identification man-ual, edition 09178. When identification discrepancies occurred, the organism was tested again with the Micro-ID system. With occasional isolates, the

bio-chemical reaction patternswereinsufficienttoprovide

aspecies identification. In such cases, additional

bio-chemical tests,assuggestedintheMicro-ID

identifi-cation manual, were performed withconventional

bio-chemical media. Organisms identified as species of

Salnonella orShigella were confirmed by serology

(4), assuggestedin theidentification manual.

Certainbiochemicaltestresults asdeterminedby

Micro-ID and other systems differ because of

differ-encesinformulation(1). Since the revised Micro-ID

computer data basewasdeveloped independentlyof

any othersystem, it is consistent within itself.

There-fore,atest-by-testcomparisonof individual reactions

wasnotattempted in this study. This evaluation was

designedtodetermine the overall accuracy of

identi-fication andreproducibility of the Micro-IDsystem.

RESULTS

Overall identification.

A

total of

306

iso-lates

of

Enterobacteriaceae

were

tested:

145

commonand

typical

isolatesattheMayo Clinic and 161

atypical

orunusualisolates at the

Cen-ter

for Disease

Control. The Micro-ID

system

correctly identified

142

(97.9%)

of the

common

clinical isolates and

123

(76.4%)

of the

atypical

or

unusual

organisms

(Table 1).

Of

these

atypi-cal

or

unusual

isolates,

the

biochemical reaction

patterns

of three

species,

Citrobacter

amalona-ticus

(malonate-

and

H2S-negative

C.

freundii),

Enterobacter

gergoviae

(sp.

nov.;

2),

and

Pec-tobacterium

(Erwinia-like),

were not included

in

the

system's

data base. When these

organisms

were

deleted

from

the

analysis,

138 of 146

(94.5%) of

the

unusual

organisms

were

correctly

identified. Deletion of

these

organisms

from the

analysis also increased

theoverall

identification

rate

from

86.6 to

96.2%. Thirty-two

of the

iso-lates

tested

were

identified only

to the genus

level, and additional biochemical

testswere

re-quired for identification

tospecies level.

The

performance

of

Micro-ID was

comparable

to

that of

API 20E for the

identification

of

common

clinical isolates (Table

2). The

former

system

correctly identified

97.9% of the orga-nisms

tested, the latter

system

correctly

identi-fied

99.3%.

Of

the 161 unusual or atypical

orga-nisms

tested,

154

(95.6%)

were

correctly

identi-fied with conventional

biochemical

media as

compared

to 123

(77%) with the Micro-ID

sys-TABLE 1. Summaryof organism identification by

Micro-ID

No.requiring No. No.cor- additional

Organismclassification tested recta biochemical

testsa

Commonandtypical 145 142(97.9) 6 (4.1)

Unusual andatypical 161 123(76.4) 26(16.1)

Total . .... 306 265(86.6) 32(10.4)

Common andtypical 145 142(97.9) 6 (4.1)

Unusual and

atypical-revised.

146 138(94.5) 25(17.1)

Total.291 280(96.2) 31(10.6)

aNumbers inparenthesesindicate percentages.

bAnalysisexcludingCitrobacter

amnalonaticus,

En-terobactergergoviae,andPectobacterium.

TABLE 2. Comparisonof identifications by

Micro-ID andAPI 20E

No. No.

conrect

No. correct

Organism

tested by API

20E"

bymicro-ID'

Citrobacter diversus 9 9

(100)

9(100)

Citrobacter

freundii

9 9(100) 9(100)

Enterobacter

aero-genes ... 13 13(100) 12

(92.3)

Enterobacter

cloa-cae. 12 12(100) 12(100)

Escherichiacoli ... 15 15(100) 15(100) Klebsiella

pneumo-niae ... 9 9

(100)

9

(100)

Klebsiella

oxytoca

(K.pneumoniae) 4 3(75) 4

(100)

Morganella

mor-ganii .. 12 12 (100) 11(91.7)

Proteus mirabilis 12 12

(100)

12(100)

Proteusvulgaris ... 13 13(100) 12(92.3)

Salmonella ... 13 13(100) 13(100)

Salmonella typhi 1 1(100) 1 (100)

Serratiamarcescens 12 12(100) 12(100)

Shigellasonnei .... 3 3(100) 3(100)

Shigella

species

8 8(100) 8(100)

Avg .. (99.3) (97.9)

aNumbersin

parentheses

indicatepercentages.

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tem.

However,

deletion of the

species

not in-cluded in the Micro-ID data base resulted in correct identification of 94.5% of theseorganisms

(Table

3).

Misidentifications.

A list of

organisms

mis-identified

by

the Micro-IDsystem is

presented

inTable 4. Identificationerrorsinvolveda vari-ety oforganisms with no single genus or species

predominating.

In several

instances,

identifica-tion

errors

appeared

tobe duetothe insensitiv-ity of certain of the Micro-ID biochemical tests.

For

example,

two

of three

strains of Citrobacter

freundii

(H2S

negative)

were

incorrectly

identi-fied

as

Citrobacter diversus

(C. intermedius).

The main source of error

appeared

to be the

inability

of Micro-IDtodetect the

production

of

acid

from

adonitol

by C. divetsus.

Further,

two

strains

of

Serratia (Enterobacter)

liquefaciens

TABLE 3. Rateofcorrectidentificationsby

Micro-ID

No. No. correct by

Organism

tested Micro-ID'

Arizona hinshawii(Salmonella

arizonae) ... 16 15(93.8)

Citrobacteramalonaticusb .... 5 0(0)

Citrobacterfreundii(H2S

nega-tive) ... 3 1 (33.3)

Edwardsiella tarda ... 10 10(100)

Enterobacteragglomerans

(Er-winia) ... 6 6(100)

-b

Enterobactergergoviae ... 5 0(0)

Escherichia coli (H2S positive) 5 4(80)

E. coli (atypical) ... 5 4(80)

Hafnia alvei ... ... 15 15 (100)

Klebsiella ozaenae ... 5 5(100)

Klebsiella rhinoscleromatis ... 5 3(60)

Pectobacterium.

5 1(25)c

Providenciaalcalifaciens

(Pro-teusinconstans) ... 6 6(100)

Providenciarettgeri (Proteus

rettgeri) ... 13 11(84.6)

Providencia stuartii(Proteus

inconstans) ... 11 10 (91)

Salmonella cholerae-suis ... 4 3 (75)

Salmonella paratyphiA ... 4 2 (50)

Salmonella(atypical) . ... 5 5(100)

Serratia liquefaciens ... 11 8 (72.7)

Serratia rubidaea ... 4 3 (75)

Yersinia enterocolitica ... 10 7 (70)

Yersiniapseudotuberculosis .. 4 3 (75)

Avg (77)

Avg excluding C. amalonaticus,

E.gergoviae, and

Pectobac-teriumrm... (94.5)

aNumbersinparentheses indicate percentages.

bBiochemical reaction patterns for this organism

arenotincludedin theMicro-ID data base.

'Fouradditionalstrains classified as Enterobacter

agglomerans.

wereincorrectly identified as Serratia marces-cens because of false-negative arabinose reac-tions. The small number of strains tested within

each

species,

however, precluded an

in-depth

analysis

ofidentification errors.

TABLE 4. Errors in identification by the Micro-ID system

Incorrectly identified

Organism(no.) as:

Citrobacter

amalon-aticusa(5) ...

Citrobacterfreundii

(H2Snegative) (2)

Edwardsiella tarda (1) ...

Enterobacter

aero-genes (1) ...

Enterobacter

gergo-viaea (3) ...

Enterobacter

gergo-viaea (2)

Escherichia coli(1)

E.coli(1) ...

Klebsiella

rhino-scleromatis(1) ...

K.rhinoscleromatis

(1) ..

Morganella

mor-ganii (1) ...

Pectobacterium a

(1)

.... Proteusvulgaris

(1)

... Providenciarettgeri (1) ...

P.

rettgeri

(1) ...

Salmonella

cholerae-suis (1) ...

Salmonella

paraty-phiA (1) ...

S.:paratyphiA (1) ..

Salmonella typhi (1)

Serratia liquefaciens

(2) ...

S. liquefaciens (1)

Serratia rubidaea (1) ...

Yersinia enterocolit-ica (1) ...

Y.enterocolitica

(2) ...

Yersinia

pseudotu-berculosis (1) ...

Citrobacter diversus C. diversus Salmonella chol-erae-suis Serratia liquefa-ciens Klebsiella pneumo-niae Hafnia alvei Citrobacterfreundii Serratia marcescens Klebsiella ozaenae Enterobacter ag-glomerans Proteus rettgeri Yersinia pestis Proteus mirabilis

Notonfile

Providencia alcali-faciens Citrobacter diversus Escherichia coli Hafnia alvei H. alvei Serratia marcescens Serratia rubidaea Enterobacter ag-glomerans Escherichia coli Yersinia pseudotu-berculosis Yersinia pestis

aBiochemical reaction patterns for this organism

are notincludedin the Micro-ID data base.

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Reproducibility. To

test

the

reproducibility

of the Micro-ID

system, each laboratory

ex-changed

50 cultures. Thus, both laboratories

independently

identified 100isolates. Of the 100

isolates tested in

duplicate,

93were identified as the same genus and species. Sixty-two of the

organisms

produced

identical profile numbers.

Of the

38

organisms

with different profile

num-bers, 69% varied

in

one biochemical

test per

Micro-IDprofile, 21% varied in two, 5% varied inthree, 0% varied in four, and 5% varied in five. In 31

isolates,

the same identification was

ob-tained but

with differing

profile numbers. Of

these,

23

(74%)

differed

in only one of the 15

biochemical

tests, seven (23%) showed variation

in twobiochemical tests, and one (3%) exhibited

discrepancies in three biochemical

reactions.

All

50common

clinical isolates

were correctly

iden-tified by both laboratories,

whereas 7 of the 50

unusual

or

atypical organisms

were identified as

a

different

genus or

species

or both.

Of

these

seven

strains, three showed

a

single variable

biochemical reaction,

one varied in two

reac-tions,

one

differed

in

three

reactions, and two

showed variation

in

five

biochemical tests. These

discrepancies

in

identification

were:Escherichia

coli

versus

C. diversus (2);

E.

coli

versus

S.

marcescens

(1); Salmonella paratyphi

A versus

E.

coli

(1);

Salmonella typhi

versus

Hafnia

alvei

(1);S.

liquefaciens

versus

S.

marcescens (1); and

S.

marcescens versus

Serratia rubidaea

(sp. nov.;

reference

6) (1).

DISCUSSION

The

Micro-ID

system

utilizes

reagent-impreg-nated

filter

paper

disks

to

provide

a

4-h

identi-fication of the Enterobacteriaceae.

Inoculation

of the

unit

withan

organism-saline suspension

is

convenient. Inoculation does

not

require

the

use

of sterile saline

or

sterile

pipettes

or

pipette

tips; however, the

use

of saline

preparations

containing

preservatives,

such

as

sodium

azide,

must

be

avoided. Since stock cultures

were

used

in this

evaluation,

we

could

not

determine

the

frequency

with which the

required

0.5

Mc-Farland inoculum

density could

be

achieved

without

overnight incubation

in a

clinical

set-ting.

A

previous

report

(1)

indicated

that

suffi-cient

growth

to

bring

the

inoculum

to

the

re-quired

turbidity

was achieved from the initial

isolation

plate

for 74% of the

clinical

isolates

tested.

The

performance

of the Micro-ID systemwas

comparable

tothose of the API 20E and

classical

tubed

media.

As

expected,

unusual

or

atypical

organisms

presented

the greatest

challenge

for

the system.

Of this

group

of

organisms,

three

species tested

in this

study,

C.

amalonaticus,

E.

gergoviae, and Pectobacterium,

were not

in-cluded in the

Micro-ID data base

and thuscould

not

be identified by the

system.

It

was noted in

a

previous

study

(1)

that several

misidentifica-tions were due to "book errors" in the identifi-cation

manual.

These appear to have been

elim-inated in the current edition of the manual. The

original identification

manual was based on the

data of Edwards and

Ewing

(4).

The

current

edition

of

the

manual

is based on tests of nu-merous

Enterobacteriaceae

with Micro-ID.

Al-though

it

wasnot

the

purpose of this evaluation to compare the results ofindividual Micro-ID

biochemical

tests withthose of other systems, it

should be

pointed

out that the relative

insensi-tivity

of certain of the

Micro-ID tests (e.g.,

adon-itol and arabinose)

maybe a possible source of

error

in the

system.

Of the

100

isolates identified

in

duplicate,

93%

of

genus

and

species

identifications

and 62% of

profile

numbers were in perfect agreement. Of

the

31

strains with the

same

identification

but

different code

numbers, 74% differed

in

only

one

biochemical

reaction. These

results

are

compa-rable

to

those

reported for the reproducibility of

the

API

20Esystem

(2).

In

several

instances,

the

color reactions of various biochemical

tests were

difficult

to

read

evenwith the

expanded

descrip-tions

provided

by the manufacturer. Some

of

the

testsmay have been

misinterpreted,

thus

con-tributing

tothe

variation

observed.

The use of

a

color chart

with

the

kit

would aid

analysts

in

determining whether

a

given reaction

is

positive

or

negative.

In

conclusion, results

of this

study

indicate

that the Micro-ID

system

compared

favorably

to

other

conventional

systems

for the

identifi-cation

of

Enterobacteriaceae. Since

this isa4-h

system,

it

mayprove

advantageous

in

situations

where

a

"same-day"

identification

is

required.

This

study

was

designed only

to

determine

the

ability of Micro-ID

to

identify

enteric

organisms

and

to

estimate its

reproducibility.

The

perform-ance

of

the system ina

clinical

setting requires

additional evaluation.

ACKNOWLEDGMENT

This workwassupportedinpartbyGeneralDiagnostics,

Division of Warner-LambertCompany, MorrisPlains, N.J. LITERATURE CITED

1. Aldridge,K.E.,B. B.Gardner,S. J.Clark,andJ. M. Matsen. 1978.ComparisonofMicro-ID, API 20E and conventionalmedia systemsinidentificationof Enter-obacteriaceae.J.Clin.Microbiol.7:507-513. 2. Brenner, D.J.,J. J. FarmerIII,F.W.Hickman,M.

A.Asbury, andA.G.Steigerwalt.1977.Taxonomic andnomenclaturechangesinEnterobacteriaceae. Cen-terforDiseaseControl,Atlanta.

3. Butler,D.A.,C.M.Lobregat,andT. L. Gavan. 1975. Reproducibilityof theAnalytab (API20E) system.J.

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458 BUESCHING ET AL.

Clin.Microbiol. 2:322-326.

4. Edwards,P.R., andW. H.Ewing.1972.Identification ofEnterobacteriaceae,3rd ed.BurgessPublishing Co., Minneapolis.

5. Ewing, W. H. 1974. Differentiation of Enterobacteria-ceaeby biochemicalreactions. Center for Disease Con-trol,Atlanta.

6. Ewing,W.H.,and W.J.Martin. 1974. Enterobacteri-aceae, p. 189-221.In E. H.Lennette, E. H.Spaulding, and J. P. Truant(ed.),Manual ofclinicalmicrobiology,

2nd ed. AmericanSocietyforMicrobiology, Washing-ton,D.C.

7. Nord, C.E.,A. A.Lindberg,and A.Dahlback. 1974. Evaluationof five test-kits-API,Auxotab, Enterotube, PathoTec, and R/B-for identification of Enterobac-teriaceae.Med. Microbiol. Immunol.(Berlin) 159:211-220, 1974.

8. Washington, J. A.II. 1976. Laboratory approaches to the identification ofEnterobacteriaceae. Human Pa-thol.7:151-159.

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ERRATUM

Evaluation of the

Modified Micro-ID

System for Identification

of Enterobacteriaceae

WILLIAM J. BUESCHING, DWANE L. RHODEN,ANN 0.ESAIAS,PETER B. SMITH,AND JOHN

A.WASHINGTON II

Sectionof Clinical Microbiology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901, and

Center for Disease Control, Atlanta, Georgia 30333

Volume

10, no. 4. The number of correct

identifications

among

unusual

and

atypical

organisms

"revised" (i.e., excluding Citrobacter amalonaticus, Enterobacter gergoviae, and Pectobacterium)

should be

123

(84.2%),

rather than 138 (94.5%) as stated on p. 454, line 10 of the Abstract; p. 455,

column 1, lines 15-16 of

Results;

p. 455, Table 1; p. 456, column 1, line 3 ofResults; and p. 456, Table 3. The

total

number

of

correctidentifications should, therefore, be 265 (91.1%), rather than 280(96.2%) as stated on p. 455, column 2, line 2 of Results and on p. 455, Table 1.