Evaluation of the Minitek system for identification of nonfermentative and nonenteric fermentative Gram negative bacteria

p.

0095-1137/80/10-0509/08$02.00/0 Vol.12, No. 4

Evaluation of the Minitek System for

Identification

of

Nonfermentative and Nonenteric

Fermentative

Gram-Negative

Bacteria

BRENTCHESTER"*AND TIMOTHY J. CLEARY'

Laboratory Service, VeteransAdministrationHospital,'andDepartments ofPathology and

Microbiology,"',

University

of

Miami

School

of

Medicine,

Miami,

Florida 33125

The Minitek

identification

system

(MT) was compared with a conventional

testing battery for the characterization

of 735 isolates whichincluded 57 species

and

groups

of

nonfermentative

(NF) and nonenteric fermentative (NEF)

gram-negative bacteria. The

MT

correctly

identified 585 of 616 NF (94.96%) and 115 of 119

NEF

(96.65%) bacteria and 700 of 735 strains

(95.24%) overall.

A total of 31

NF and NEF

(4.22%) bacteria were misidentified, and no identification was

determined for

four strains (0.69%). All strains of Acinetobacter anitratus,

Pseu-domonas maltophilia, P. fluorescens, and P. putida and

all

but one

strain

of P.

aeruginosa

were correctly

identified.

The most frequently

misidentified

taxa were

CDC group

Va-1,

P.

pickettii (Va-2),

P.

mendocina,

and

Moraxella

urethralis

(M-4).

Supplemental

tests were

needed for the

complete identification of

214

strains

(29.11%).

An average

of

1.54

supplemental

tests were

used with each of

these strains.

A

total of

134

strains

(18.23%)

had

their identification delayed by

1

day due

to

supplemental testing.

We recommend

the

use

of the 42°C growth

test

with the MT. When used in accord with the

manufacturer' instructions

and with

the MT

code

book,

the

MT

was

found

to

be

a

valuable

system for the identification

of

a

wide

variety

of

common

and

infrequently

encountered NF and NEF bacteria.

Although

most

clinical isolates of

gram-nega-tive

bacteriaare

enterics,

nonfermentative

(NF)

and nonenteric fermentative

(NEF)

bacteria

are

encountered

daily

in

most

laboratories.

At one

of

our

laboratories,

perhaps

asa

consequence of

the

large

number of chronic and debilitated

pa-tients, NF and NEF bacteria

account

for

more

than 10%

of

all

gram-negative

isolates.

The

identification

of

NF

and NEF bacteria

has been

a

continuing problem for

most

labora-tories.

Although conventional methodology

de-signed

to

deal with this problem has been

avail-able for

many years,

it

has

nevertheless

failed

to

gain

widespread

acceptance.

Recently,

a variety of

commercial,

packaged

systems for

the

identi-fication of these

two

bacterial

groups

have

be-come

available

to

the

microbiologist. These

sys-tems offer an attractive alternative to

conven-tional

methodology through such features

as

rapid (24

to 48

h) and

computer-assisted

identi-fication, convenience,

easeof use, and cost-effec-tiveness.

This

study

is

the

first

to evaluate one such system, the Minitek

(MT;

BBL

Microbiology

Systems,

Cockeysviile,

Md.)

with

strict adher-ancetothe

manufacturer's

instructions and

with

the aid of a

computerized

identification

code

book.

Although three previous studies of the MT

system

for

the identification of NF and NEF

bacteria have been published

(2, 15; M. Slifkin

and

G. R.

Pouchet,

Abstr.

Annu.

Meet.

Am.

Soc.

Microbiol. 1977,

C88,

p.

50),

none

had the

benefit

of the

code book

nor

several

recent

modifications

of the MT

methodology.

Importantly, this study

represents

the

most

comprehensive evaluation,

to

date, of

any

com-mercial

packaged

system

designed for the

iden-tification of NF and

NEF

bacteria.

MATERIALS AND METHODS

Bacteria. The 735 bacteria tested contained rep-resentativesof57speciesandgroupsofNF and NEF bacteria (Table 1). Of the total bact ria, 306strains were reference cultures, primarily of the less fre-quently encountered members of thesetwo bacterial groups(courtesy of R.E.Weaver,

Special

Bacteriology

Section,Center for Disease

Control, Atlanta,

Ga. and G. L.Gilardi, Hospitalfor Joint Diseases and Medical Center, New York City, N.Y.). The remainder, 429 strains, were fresh clinical isolates (not transferred

more thantwo times) from themicrobiology labora-tories ofsevenhospitals.With theexception of Pseu-domonasmallei and Moraxellalacunata,each of the 56speciesand groupsforwhich the MTsystem makes

anidentification claimwastested. One isolateof Vi-brio vulnificus (6) (Vibriospecies, lactose

positive),

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TABLE 1. NF and NEFgram-negative bacteria examined

Taxon

Acinetobacter anitratus A.lwoffi

Achromobacter species (Vd) A.xylosoxidans Aeromonashydrophila Alcaligenesdenitrificans A.faecalis A. odorans Actinobacilluslignieresii Bordetella bronchiseptica B.parapertussis Cardiobacterium hominis Chromobacterium violaceum CDCgroup EF-4 1If

IIj

IIk-2 IVc-2 IVe Va-1 Ve-1 Ve-2

Flavobacterium odoratum(M-4f) Flavobacterium speciesIIb F.meningosepticum Haemophilusaphrophilus Moraxella bovis M.nonliquefaciens M.osloensis M.phenylpyruvica M.urethralis(M-4) Pasteurellahaemolytica P.multocida P.pneumotropica P.ureae Plesiomonasshigelloides Pseudomonas acidovorans P.alcaligenes P.aeruginosa Pyocyanogenic Apyocyanogenic Mucoid P. cepacia P.diminuta P.fluorescens P.maltophilia P.mendocina (Vb-2) P.paucimobilis(IIk-1) P.pickettii (Va-2) P.pseudoalcaligenes P.pseudomallei P.putida P.putrefaciens P.stutzeri(Vb-1,Vb-3) P. testosterone P. vesicularis Vibrioalginolyticus V.cholerae V.parahaemolyticus V. vulnificus Fresh clinical isolates 36 il 16 18 10

o

6 20 O 2 1 4 O 3 O 4 2 O

o

1

o

19 il 7 2

o

O 2

o

O

o

4 O

o

1 3 1 74 19 6 7 7 16 29 1 1

o

12

o

27 8 18 3

o

4 1 5 1 Refer-ence strains 15 3 18 3 5 2 5 2 2 8 3 2 2 1 7 9 7 4 8 8 10 9 7 1 6 1 1 3 O 8 4 5 8 3 7 8 9 6 i 3

o

3 4 8 4 4 7 6 8 2 6 8 5 5 10 6 4 7 O the results.

Conventional methodology. All strains were identifiedusingcriteria andproceduresrecommended by the American Society for Microbiology (8), the CenterforDiseaseControl (14),andG. L. Gilardi(7). In addition, amodified silverstain forflagella (16), a semi-quantitative catalasetest(3), andadistilled wa-terimmobilizationtestfor vibrios (4) wereemployed. MTsystemfor NF and NEFbacteria. The MT systemfor theidentificationofNF and NEFbacteria consists ofpaperdisksimpregnated with appropriate biochemicals:dextrose,maltose, sucrose,xylose,urea, citrate,nitrate(reduction anddenitrification), phenyl-alanine, ornithine, arginine, lysine, o-nitrophenyl-,f-D-galactopyranoside (ONPG), and starch. The sus-pendingbroth serves assubstrate for the indole test. The disks aredispensed intoaplastic plate containing wells. Into each well containing a disk, 0.05 ml of organismsuspendedinbroth(MT enteric and nonfer-menterbroth,BBLMicrobiologySystems) is pipetted. Intothe wellcontaining the arginine disk, 0.10 ml of inoculumispipetted. For indoledetection, 0.15 ml of inoculum isplaced intoanempty well. After overlay-ing withoilthose diskscontaining dextrose (a second dextrosedisk isnotoverlaid),urea,ornithine, arginine, andlysine, theplates are incubated inahumidorat 35°C for48h(ONPG andurea arereadat24h). After the addition ofappropriatereagentsforindole,nitrate reduction and denitrification, phenylalanine deami-nation,andstarchhydrolysis, the observed reactions areinterpretedwith tables and acode book provided by the manufacturer.

MT code book. The MT NF and NEF bacteria code bookcontains the MT numerical identification system. Basedon the reactionsof a testisolate with the 16 MTsubstrates, aswell asthe indophenol oxi-dase reaction and theability of the isolatetogrow on MacConkeyagarwithin48h,asix-digit profilenumber isgenerated. When listed in the codebook,theprofile number is accompanied by threepossible identifica-tions. The firstselection identification has thehighest confidence value (100 isperfect), followed by the sec-ond and thirdselection. Also listed foreachselection is its biotype validity value (1 is a perfect "fit"). Supplementaltests arefrequentlylistedtoaid in the differentiation of the threeselections. Whenaprofile numberisnotlisted in the codebook, atoll-freecail is madetotheBBLTechnicalService Departmentto obtainanyinformation in themastercodepertaining to theprofilenumber inquestion.

Criteria for correct identifications. In this study, a correct identification as "system only" was one inwhichthe firstselectionhad aconfidencevalue of90% or greater andmatchedtheidentification ob-tained withconventional methodology.

An identification was scored as "correct with the MT systemandsupplementaltests" when the conven-tional identification matchedone ofthe three selec-tions,noneof which hadaconfidencevalue of at least 90%,sothatsupplemental testingwasneeded.

Correct identifications obtained from the BBL TechnicalServiceDepartment were "correct with tel-ephone assistance."

notincluded in the MT coding system,wasalso ex-amined. Nosingle taxonrepresented morethan 14% ofthe total examined, to prevent anyone group or

speciesfromassertingadisproportionateinfluenceon

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MINITEK FOR NONFERMENTERS AND FERMENTERS 511

RESULTS

The MTsystemcorrectly

identified

700of 735

(95.24%)

strains of NF and NEF

bacteria.

Thirty-one (4.22%)were

misidentified,

andfour

(0.69%)werenotgivenaMT

identification

(Ta-ble2).

NF bacteria.

Of

the 616 strains of NF bacte-ria tested the MT system correctly

identified

585

(94.96%),

misidentified 28

(4.55%),

and had

noidentification for 3 (0.49%) isolates (Table 3).

Among

the 585 strainscorrectly

identified,

371

(63.42%)

were identified by the MT system alone, 203

(34.70%)

requiredone or more

supple-mental tests, and 11

(1.88%)

needed the aid of the MT telephone assistance service. A 100%

accuracyof

identification

levelwasreached for

30 of the 40NFbacterialtaxa

examined,

includ-ing strains of P. aeruginosa, A. anitratus, P.

maltophilia,

P.putida, and P.

fluorescens,

but with the exception of one pyocyanogenic P.

aeruginosa isolate.

Among

the 28 misidentifi-cations, 16

(57.10%)

wereassigned tothewrong

genusandtheremaining12(42.90%)were

incor-rect atthe specieslevel. The poorest

perform-anceof the MT systemwasseenwith P.pickettii

(Va-2), CDC groupVa-1,P. mendocina

(Vb-2),

and M. urethralis (M-4). Due to a code book

misprint,

five of six strains ofCDCgroup Va-1

were misidentified as A. xylosoxidans or P.

pseudoalcaligenes.

Four of sixstrains of P. pick-ettii (Va-2) were misidentified as A. xylosoxi-dans. Only two of five P. mendocina

(Vb-2)

isolates were correctly

identified,

although this

taxon is notincluded inthe code book.

All

four strainsofM. urethralis(M-4)were

misidentified

(Table 5).

NEF bacteria.

Of

the 119 strains of NEF bacteria examined, the MT system correctly identified 115

(96.65%),

misidentified 3

(2.52%),

and hadnoidentification for1(0.86%) (Table 4). Among the 115 strains correctly

identified,

103

(90.19%)

were identified by the MT system

alone,

10

(8.25%)

required one or more

supple-mental tests, and 2

(1.65%)

needed telephone assistance. Thirteen of the 17 NEFspeciesand

groupshadallstrainscorrectlyidentified(Table

4). One Pasteurella haemolytica isolate was

misidentified

asP. multocida,one strain of

Vi-brio

alginolyticus

was incorrectly identifiedas

Aeromonas

hydrophila,

anda

strain of V.

vul-nificus,

notincluded in the MT codingsystem,

wasmisidentifiedas V.cholerae (Table5).

Supplemental

testing.

Slightly

more than

half

of

the

species andgroups(29 of57)required no

supplemental testing

forcorrectidentification with

the MT

system.

Of

the28taxacontaining strains which required supplemental testing,an

average of 1.54 tests per strain were needed.

However,

the majority of strains belonging to

these

28taxa(56.77%) requirednosupplemental tests

(Table

6).

DISCUSSION

The MT system was used to identify 735 isolates representing 57 taxa of NF and NEF

bacteria.

This study is the mostcomprehensive evaluation,todate, of the ability ofacommercial system to

identify members of these

two

bacte-rial groups. Overall, the MT system correctly

identified

95.4% ofthese

isolates

(Table 2) and reached a slightly higher level with the NEF

bacteria,

96.65%, than with the NF

bacteria,

94.96%(Tables 3 and 4). The performance of the MT system is especially

impressive

in view of the variety,number, and

distribution

of

bacteria

involved.

Previous

studies (1, 2, 5, 7, 10-13, 15) which used

commercial

systemsfor the

identi-fication of NF and NEFbacteriawerelimited in

the number oftaxastudied. Inthepresentstudy

notaxoncomprisesmorethan 14% of the total.

Theabove evaluationswereunbalanced interms of bacterial

distribution,

with P.aeruginosa iso-lates

accounting

forasmuchas75% ofthe total

examined. The identification rates reported in the above studiesranged from 43to95.3%(1,2, 5,7, 10-13, 15).

Itwassignificant that only 1 isolate of 176 P.

aeruginosa, P.

fluorescens,

P.putida, P. mal-tophilia, and A. anitratus was misidentified

(Table 3). In a recent MT system

evaluation,

only 2 of 137 isolates of thesesamefivegroups

were misidentified (2). The fact that these five

speciesare the most common NF bacteria

en-countered insignificant clinical situationsis

re-flectedinthe 1979 blood culture statistics from TABLE 2. Overallidentification ability ofthe MT systemforNF and NEFgram-negativebacteria

Correct identifications Misidentifications

Group

~~~~~~System

Tl No

iden-Group Strains System with sup- Tele Wrong Wrong tification

phone

as-tested only plemental sistance genus species

tests

NF 616 371 203 11 16 12 3

NEF 119 101 12 2 1 2 1

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TABLE 3. Ability of the MT systemto

identify

NFgram-negativebacteria

Correctidentifications Misidentifica-tions

Taxon System Tele- No

iden-Strains System with phone Wrong Wrong tification tested only

mental

assist- genus species

test ance

Acinetobacter anitratus 51 36 15 0 0 0 0

A.lwoffi 14 14 0 0 0 0 0

Achromobacterspecies(Vd) 34 30 1 1 2 0 0

A.xylosoxidans 21 12 9 0 0 0 0

Alcaligenesdenitrificans 2 0 0 0 0 0 0

A.faecalis il 0 il 0 0 0 0

A. odorans 22 0 22 0 0 0 0

Bordetella bronchiseptica 10 10 0 0 0 0 0

B.parapertussis 4 4 0 0 0 0 0

CDC group

IIf 10 6 0 2 0 2 0

IIj 9 7 2 0 0 0 0

IIk-2 il il 0 0 0 0 0

IVc-2 6 0 6 0 0 0 0

IVe 8 3 4 0 1 0 0

Va-i

8 0 3 0 2 3 0

Ve-1 il 9 2 0 0 0 0

Ve-2 9 2 7 0 0 0 0

Flavobacterium odoratum(M-4f) 26 22 0 2 2 0 0

Moraxellabovis 1 0 1 0 0 0 0

M.nonliquefaciens 3 0 3 0 0 0 0

M. osloensis 2 0 2 0 0 0 0

M.phenylpyruvica 8 2 4 1 0 0 1

M.urethralis (M-4) 4 0 0 0 3 1 0

Pseudomonas acidovorans 12 0 12 0 0 0 0

P.alcaligenes 7 0 7 0 0 0 0

P.aeruginosa

Pyocyanogenic 75 64 10 0 0 1 0

Apyocyanogenic 22 16 4 0 0 0 2

Mucoid 6 3 3 0 0 0 0

P.cepacia 10 10 0 0 0 0 0

P. diminuta il il 0 0 0 0 0

P.fluorescens 24 0 24 0 0 0 0

P.maltophilia 33 32 1 0 0 0 0

P.mendocina (Vb-2) 5 0 0 1 2 2 0

P.paucimobilis (IIk-1) 8 6 2 0 0 0 0

P.pickettii(Va-2) 6 0 1 1 4 0 0

P.pseudoalcaligenes 20 12 7 1 0 0 0

P.pseudomallei 2 2 0 0 0 0 0

P.putida 33 O 33 O O O O

P.putrefaciens 16 15 0 1 0 0 0

P.stutzeri(Vb-1, Vb-3) 23 21 0 1 0 1 0

P.testosteroni 8 0 8 0 0 0 0

P.vesicularis 10 10 0 0 0 0 0

one of our laboratories (T.J.C.). It was

found

M. urethralis (M-4) (4 isolates) (Table 5). The

that,

of130NF isolates from blood cultures of remaining 14 misidentifications were scattered 130

patients,

115

belonged

tothese five species amongeight taxa of NF bacteria and three spe-of NF bacteria. cies of NEF bacteria. Each of five isolates of Among the 31 isolates misidentified, 17 be- CDC group

Va-i

was misidentified due to an

longed

tooneof fourtaxaof NF bacteria: CDC MT coding error which programmed positive group

Va-i

(5

isolates);

P.

pickettii

(Va-2) (4

maltose

reactions

for members of this group.

isolates);

P.

mendocina

(Vb-2) (4 isolates); and These bacteria areuniformly negative for

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MINITEK FOR NONFERMENTERS AND FERMENTERS 513 TABLE 4. Ability of the MT system to

identify

NEF gram-negative bacteria

Correctidentifications

Misidentifica-tions

Taxon System Tele- No

iden-Strains System _supple-with hone Wrong Wrong tification tested only mental assist- genus species

tests ance

Aeromonashydrophila 15 14 0 1 0 0 0

Actinobacillus lignieresii 2 2 0 0 0 0 0

Cardiobacteriumhominis 3 3 0 0 0 0 0

Chromobacteriumviolaceum 6 6 0 0 0 0 0

CDC group EF-4 1 1 0 0 0 0 0

Flavobacterium

species

IIb 12 10 2 0 0 0 0

F. meningosepticum 13 9 3 1 0 0 0

Haemophilus aphrophilus 3 3 0 0 0 0 0

Pasteurellahaemolytica 5 4 0 0 0 1 0

P.multocida 12 6 5 0 0 0 1

P.pneumotropica 3 3 0 0 0 0 0

P. ureae 7 7 0 0 0 0 0

Plesiomonasshigelloides 9 9 0 0 0 0 0

Vibrio alginolyticus 10 9 0 0 1 0 0

V. cholerae 5 5 0 0 0 0 0

V.parahaemolyticus 12 12 0 0 0 0 0

V. vulnificus 1 0 0 0 0 1 0

tose

utilization

in

the MT

system. Each of four

isolates of P. pickettii (Va-2) was misidentified

asA.

xylosoxidans. Both of

these bacteria gen-erate

the

same

profile

number, but the MT

system

is

programmed

to

select A. xylosoxidans

as

the first

choice

(confidence

value greater than

90%)

in

this

situation. To avoid

this error,

bac-teria

identified

by the MT system as A.

xylosox-idans

should be

shown

to

demonstrate

a

rapidly

positive catalase

test,

whereas

a

delayed

positive

reaction would

indicate further testing with

P.

pickettii

as a

possible

identification

(3).

In a recent

evaluation

of the MT system,

93.4% of the NF

bacteria tested

were

correctly

identified

(2). This result

strongly

supports the

94.96%

accuracy rate

obtained

in

the

present

study.

In

another

published

report

(15)

using the

MT system

for

the identification of

NF and NEF

bacteria, 92.6% of the isolates

tested were

iden-tified

to

the

genus

level and 88.3%

were correct at

the

species

level. These lower identification

rates, in

comparison with

our

95.2%

rate, can

be

attributed

to

three factors.

First,

at

the

time of the

prior

evaluation,

the MT

coding

systemwas not available.

Secondly,

certain

improvements

in the MT system

methodology,

e.g., increased

inoculum

size for

arginine,

were not

utiized.

Finally,

the author did not use the manufac-turer's recommended

battery

of

substrate

disks but chose to delete

starch,

ONPG,

phenylala-nine,

and sucrosewhile

adding

lactose and

man-nitol.

Also, only

the nitrate

reduction

reaction

but

not

denitrification results

were

recorded.

Had the MT code

book, improved methodology,

and the recommended disks been used in the

prior

evaluation,

12

of the

27

misidentifications

would have been avoided. The

corresponding

increase

in correct

identification

would have

been 95.23%

at

the

genus

level and 93.48%

at

the

species level.

It

was necessary to use the BBL Technical

Service

Department's telephone assistance

with

only

13

(1.8%) of the isolates

examined.This low usage was

in marked

contrast to

the

25

and 36%

rates for

telephone-assisted identifications

re-ported for another commercial

system (5, 10).

Of the

57taxa, 29

contained

no

strains

requir-ing

supplemental

tests

and

were

identified by

the basic

MT system.

Among the

remaining

28 groups

and

species,

an average

of

1.54

supple-mental

tests were

needed for those strains

re-quiring

additional

testing.

When

same-day

sup-plemental

tests

such

as

catalase,

pigmentation,

and

motility

are

discounted,

only

134

(18.23%)

of the

735

isolates in

the

study

had their

identi-fications

delayed,

usually by

1

day

(Table 6).

Inview of the need for

testing

for

growth

at

420C

to

differentiate

apyocyanogenic

strains of P.

aeruginosa,

especially

denitrification-nega-tive

isolates,

from P.

fluorescens

andP.

putida,

we

recommend

the routine use ofthis test in

addition

to the MT

system

to avoid an undue

delay

in

identification.

A

similar

suggestion

was

proposed

ina recent

evaluation

of the MT sys-12,1980

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TABLE 5. MTsystemmisidentificationsofNF and NEFgram-negative bacteria

Correctidentification MTidentification MT pro-_{file no.} MTdiscrepancies" Achromobacter species (Vd) Pseudomonas pseudoalcaligenes

Achromobacterspecies (Vd) Alcaligenesdentrificans A.denitrificans CDCgroupI1f

CDCgroupIIf

CDCgroupIVe

Flavobacterium odoratum (M-4f) F.odoratum (M-4f)

Moraxella urethralis(M-4) M.urethralis(M-4) M.urethralis (M-4) M.urethralis (M-4) Pasteurellahaemolytica Pseudomonasaeruginosa Pseudomonasmendocina(Vb-2) P.mendocina(Vb-2)

P. mendocina (Vb-2)

P.mendocina(Vb-2) P.pickettii(Va-2) P.pickettii(Va-2) P.pickettii(Va-2) P.pickettii (Va-2) CDCgroupVa-1

CDCgroupVa-1

CDCgroupVa-1

CDCgroupVa-1

CDCgroupVa-i

Pseudomonasstutzeri(Vb-1, Vb-3)

Vibrioalginolyticus

Vibriovulnificus

Moraxella phenylpyruvica Alcaligenes faecalis A.faecalis

CDCgroupIIj

CDCgroupIIj

M. phenylpyruvica P.paucimobilis (IIk-1)

P.paucimobilis (IIk-1)

Alcaligenes/Pseudomonas Pseudomonasdiminuta Moraxellaatlantae (M-3) Alcaligenes/Pseudomonas Pasteurellamultocida P. pseudoalcaligenes Achromobacter species(Vd) P.aeruginosa

Achromobacter species(Vd)

P.aeruginosa

Achromobacterxylosoxidans A.xylosoxidans

A. xylosoxidans A.xylosoxidans A. xylosoxidans

P.pseudoalcaligenes

P.pseudoalcaligenes

A.xylosoxidans

P.pseudoalcaligenes

P. aeruginosa

Aeromonashydrophila

Vibriocholerae

a-,Negative;+,positive; Dex, dextrose; Malt, maltose;

Suc,

sucrose; Xyl, xylose.

644022 Urea-, denitrifica-

tion-604222 Denitrification-600021 Denitrification-600021 Denitrification-474042 Dex+,Malt',Suc+,

Xyl+

474042 Dex+,Malt',Suc+, Xyl+

600223 Phenylalanine+ 674202 Dex+,Malt',Suc+,

Xyl+

674202 Dex+,Malt', Suc+, Xyl+

600001 Phenylalanine-600002 Citrate-600000 Phenylalanine-600001 Phenylalanine-574420 Ornithine+ 642021 Xylose-644013 Arginine-646011 Not in code book 646013 Misprint in code

book,correct identificationvia telephone 646011 Not in code book 604021

Urea-604021 Urea-604021 Urea-604021

Urea-604021 Code book mis-print 604023 Codebook

mis-print 604023 Code book

mis-print 604001 Codebook

mis-print 604021 Code book

mis-print 646211 Urea+,starch+

772161 Notin code book duetoarginine+, ONPG+ 770560 Not in code book

tem (2). Another

helpful

addition to the MT systemwould be anacetamide disk.Acetamide utilization can be usedto provide the differen-tiation described inregard tothe 42°C growth test.In

addition,

anacetamide disk would negate

supplemental

testing nowrequired for the MT identification of P.acidovorans. The acetamide disk would alsoserve to

distinguish

A.

xylosox-idans

(positive)

from P.

pickettii

(Va-2)

(nega-tive)

and therefore

strengthen

a weak identifi-cation

capabiity

ofthe MT system (Table 5).

The importance of

adhering

strictly to the MT

procedure,

asisthecasewith any commer-cial system, cannot be overemphasized. This adherance

applied particularly

totheuseof MT reagents andtocolor

interpretations

of the sub-stratedisks. Itis also importanttoconsider the

morphology

of the testisolate in relationtothe MTidentificationoffered.Inour

study,

theMT system was found to be a valuable system for the identification of awide variety of common andinfrequentlyencountered NFand NEF

bac-514

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MINITEK FOR NONFERMENTERS AND FERMENTERS 515

TABLE 6. Taxaof NF and NEF gram-negative bacteria requiring supplemental tests with the MT system

Strains No. of

Strin requiring tests

Taxon

tested

supple- Alternative identification Supplementaltests Noo

mentary

strain

tests

Acinetobacter anitratus 51 15 CDC group Ve-1, Ve-2

Achromobacterspecies(Vd) A.xylosoxidans

Alcaligenesfaecalis

A.odorans

CDC groupIIj

CDC group IVc-2 CDC group IVe CDC groupVa-i

CDC group Ve-1

CDC group Ve-2

FlavobacteriumspeciesIIb F.meningosepticum Moraxella bovis M.nonliquefaciens M.osloensis M.phenylpyruvica Pasteurella multocida Pseudomonasacidovorans P.alcaligenes P.aeruginosa Pyocyanogenic Apocyanogenic Mucoid P.fluorescens P.maltophilia P.paucimobilis P.pickettii (Va-2) P.pseudoalcaligenes

P.putida P.testosteroni

34 1 Pseudomonaspaucimobilis 21 9 P.pickettii (Va-2)

il 1 Alcaligenes, Pseudomonas acidovorans,P. testos-teroni, P. alcaligenes, P.pseudoalcaligenes 22 22 Alcaligenes, P.acidovorans,

P. testosteroni, P. alcalig-enes, P. pseudoal-caligenes

9 2 Cardiobacterium hominis, Pasteurella multocida 6 6 CDC group IVe

8 4 Moraxella phenylpyruvica 8 3 P.pseudoalcaligenes,A.

xylosoxidans

11 2 A. anitratus, P. pauci-mobilis

9 7 A.anitratus

12 13 1 3 2 8 12 12 7 12 3 1 3 2 4 5 12 C.hominis

FlavobacteriumspeciesIIb M.osloensis

M.osloensis,M.lacunata M.bovis,M.lacunata CDCgroupIVe C.hominis

Alcaligenes, P. testosteroni

7 Alcaligenes, P. pseudo-alcaligenes

75 10 P.fluorescens/P.putida 22 4 P.fluorescens/P. putida 6 3 P.fluorescens/P. putida 24 24 P.aeruginosa,P.putida 33 1 Pseudomonas cepacia

8 2 Pasteurellahaemolytica 6 1 A.xylosoxidans,P.

pseudoalcaligenes 20 7 Pseudomonasputrefaciens,

Alcaligenes,P. alcalig-enes

33 33 P.aeruginosa,P. fluores-cens

8 8 Alcaligenes,P.acidovorans

Mannitol oryellow pigment Mannitol 42°C or catalase Flagella stain, denitrification, fruity odor Fruity odor Catalase, gelatin Rapid urea Motility

Flagella stain or cat-alase, denitrifica-tion

Mannitol oryellow pigment Mannitol oryellow

pigment Catalase Mannitol Beta-hemolysis Gelatin,Loeffler's Beta-hemolysis, Loeffler's Motility Catalase Flagella stain, acetamide Flagella stain, fructose 420C 420C 420C 42°C, lecithinase Deoxyribonuclease Mannitol

420Corcatalase, denitrification Gelatin, flagella

stain, fructose

42°C, lecithinase

Flagella stain, acet-amide

teria. Itwasaccurateandeasyto use, provided-rapididentificationswithminimalsupplemental testing,andwascost-effective.

LITERATURE CITED

1. Barnishan, J.,andL W.Ayers. 1979.Rapid

identifi-cation of nonfermentative gram-negative rodsby the

Corning N/F system.J. Clin.Microbiol.9:239-243.

2. Burdash,N.M.,E.R.Bannister,J.P.Manos,andM.

E.West. 1980. Acomparisonof fourcommercial

sys-temsfor the identification of nonfermentative bacilli.

Am. J.Clin. Pathol.73:564-569.

3. Chester,B.1979.Semiquantitativecatalase testas anaid

in theidentification ofoxidative andnonsaccharolytic

gram-negativebacteria.J. Clin. Microbiol. 10:525-528.

1 1 i 2.27 1 2 1 1 1.66 i i i i 1 2 2 1 1 2 2 1 1 1 1.83 1 i 2 1.57 1.90 2

VOL.12,1980

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4. Chester, B., andE.G. Poulos.1980.Rapid, presumptive identification of vibriosby immobilization in distilled water. J.Clin.Microbiol.11:543-545.

5. Dowda, H. 1977. Evaluation oftwo rapid methods for identification of commonly encountered nonfermenting oroxidase-positive,gram-negative rods. J. Clin. Micro-biol.6:605-609.

6. Farmer, J. J. III.1979.Vibrio ("Beneckea") vulnificus, thebacterium associated withsepsis,septicemia, and the sea. Lancetii:903.

7. Gilardi, G. L. 1980. Identification of nonfermentative gram-negative bacteria.Hospital for Joint Diseases and Medical Center, New YorkCity.

8. Lennette,E.H.,E.H.Spaulding, and J. P. Truant (ed.). 1974. Manual of clinical microbiology, 2nd ed. AmericanSociety for Microbiology, Washington, D.C. 9. Nadler,H., H. George, and J. Barr. 1979. Accuracy and

reproducibility of the Oxi/Ferm system in identifying a select groupofunusualgram-negativebacilli. J. Clin. Microbiol. 9:180-185.

10. Oberhofer, T.1979.Comparison of the API-20E and Oxi/ Ferm systemsinidentification ofnonfermentative and oxidase-positive fermentative bacteria. J. Clin. Micro-biol. 9:220-226.

11. Oberhofer, T. R., J. W. Rowen, G. F. Cunningham, andJ.W.Higbee.1977.Evaluation of theOxi/Ferm

tube system with selected gram-negative bacteria. J. Clin. Microbiol. 6:559-566.

12. Shayegani,M., A.M.Lee, and D. M.McGlynn. 1978. Evaluation of theOxi/Ferm tube system for identifica-tionofnonfermentativegram-negative bacteria. J. Clin. Microbiol. 7:533-538.

13. Shayegani, M., P. G. Maupin,and D. M. McGlynn. 1978.Evaluation of the API-20E system for identifica-tion of nonfermentativegram-negativebacteria. J. Clin. Microbiol. 7:539-545.

14. Weaver, R.E.,H. W.Tatum,andD. G.Hollis.1972.

Theidentification of unusualpathogenicgram-negative bacteria(Elizabeth O. King). Center for Disease Con-trol, Atlanta, Ga.

15.Wellstood-Neusse,S. 1979.Comparison of thé Minitek system with conventional methods for identification of nonfermentative and oxidase-positive fermentative gram-negativebacilli. J. Clin. Microbiol. 9:511-516. 16. West, M., N. M. Burdash, and F. Freimuth. 1977.

Simplified silver-plating stain for flagella. J. Clin. Mi-crobiol.6:414-419.

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