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Evaluation of the Titertek NF system for identification of gram negative nonfermentative and oxidase positive fermentative bacteria

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0095-1137/89/061201-05$02.00/0

Copyright 1989, American Society forMicrobiology

Evaluation of the Titertek-NF System for Identification

of

Gram-Negative Nonfermentative and Oxidase-Positive

Fermentative Bacteria

PETER KAMPFER* AND WOLFGANG DOTT

FachgebietHygiene, Technische Univ'ersitatBerlin, Ainrurnerstrasse 32, D-1000Berlin 65, FederalRepublic of Germrany Received 28 July1988/Accepted 22 February 1989

TheTitertek-NF (TT-NF)system (Flow LaboratoriesGmbH, Meckenheim, Federal Republic of Germany)

was evaluated for the identification of 1,289 strains of gram-negative, nonfermentative bacteria and some

gram-negative, oxidase-positive bacteria. The oxidasetestwasalsoperformed. Identificationswereclassifiedas

correct, notidentified (twoor moretaxa possible and identification scoreof<80%; supplementary testsfor furthering the identication were not performed), and incorrect. Correct identification results were further

subdivided by thecorrectlevelof speciesorbiotype identificationas -98% (categorya),90to97% (category b), and 80to89% (categoryc). When compared withconventional identification results,the TT-NF system

correctly identified 90.3% ofstrains(1,164 of 1,289strains), with 72.5% (935 strains) belongingtocategorya,

14.7% of strains (189 strains) belongingto categoryb, and3.1% of strains (40 strains) belongingto category c. Among the remaining strains, 104 (8.1%) were notidentified and 14 (1.1%) were misidentified, and the system failed to generateidentificationresultsfor 7 strains (0.5%). Reactions within the TT-NFsystemwere

reproducible, withanestimated probability oferroneoustest results of 0.2%. Although gram-negative, nonfermentative bacteria

in-creasingly are becoming implicated in humandisease, iden-tification of these organisms has often been neglected in clinicalmicrobiology laboratories, especiallywhen antibiotic susceptibility data are available (2). A battery of

conven-tional biochemical tests is needed for their precise identifi-cation of the taxonomically heterogeneous group of

gram-negative, nonfermentative bacteria (13-15, 27). In recent

years, several commercial kits have been developed, some

of which areforthe identification ofmembersofthefamily

Enterobacteriaceae and commonly encountered nonfer-mentersandothers of whicharefor the specific identification

ofgram-negative nonfermentative bacteria (8).

Duringa3-year study,ascreeningwasperformedinorder to find miniaturized biochemical tests that would make possible an extensive species differentiation within these

bacterial genera (20, 21). As a result, in cooperation with

Flow Laboratories GmbH (Meckenheim, Federal Republic ofGermany), the Titertek-NF (TT-NF) system was

devel-oped; itprovides for thecomputer-aided identification of 57 different species. This study was undertaken to determine the accuracy, suitability, and reproducibility ofthe TT-NF systemforuse in clinical microbiology laboratories.

MATERIALS AND METHODS

Bacteria. A total of 1,289 strains of nonfermentative, gram-negative bacteria and fermentative, oxidase-positive organismsweretested. Of these,621wereobtained from the

National Collection ofType Cultures, Central PublicHealth Laboratory (Colindale, London, United Kingdom) (123

ref-erence strainsand 498 field strains). Theywereidentifiedby

the methods of the National Collection of Type Cultures (18). An additional 668strains were stock cultures obtained

from the Institute of Medical Microbiology, University of Heidelberg, Heidelberg, FederalRepublicofGermany; Cen-tre Hospitalier St. Nicolas, Sarrebourg, France; and the

*Correspondingauthor.

Culture Collection of the University Gôteborg, Gôteborg, Sweden, and were identified by conventional methods (13) or the API 20NE system (Analytab Products, Plainview, N.Y.) (12). Before testing,all isolates weregrown on sheep blood agar (Oxoid, Ltd., Hampshire, United Kingdom) for

24 hat30°C (strains ofthegeneraMoraxellaandPasteurella

and Bordetella bronchiseptica isolates weregrown for48 h at 30°C). Oxidase testing was carried out by using oxidase test strips (E. Merck AG, Darmstadt, Federal Republic of Germany).Toassesspurity,cultureswerechecked through-outthe study by colony morphology.

TT-NF system. The TT-NF system consists of 32 dehy-drated substrates in a standard microplate format (28 test and 4 control media). The system includes tests for indole production; esculin hydrolysis;urease;arginine dihydrolase; ornithine decarboxylase; fermentation of glucose and

su-crose; assimilation of D-glucose, mannose, maltose,

N-acetyl-D-glucosamine, mannitol, gluconate,

P-hydroxybu-tyrate, DL-lactate, adipate, suberate, malate, phenylacetate, and histidine; and hydrolysis tests for

o-nitrophenyl-p-D-galactopyranoside, p-nitrophenylphosphorylcholine, 2-de-oxythymidine-5'-p-nitrophenylphosphate, L-proline-p-nitro-anilide, p-nitrophenyl-a-D-maltoside, p-nitrophenyl-N-acetyl-,-D-glucosaminide, and bis-p-nitrophenylphosphate. Each microplate contains three test sets for the identification of three different isolates. Inoculation of the test kit was as

follows. Each well was filled with 100 ,ul of a bacterial

suspensioninadefined minimalbasal medium withadensity

equivalent to a 0.1 McFarland standard. Test wells and

control wells ofesculin,esculincontrol, arginine dihydrolase, ornithinedecarboxylase, urease,glucoseandsucrose fermen-tation, and the fermentation controlwere overlaid with 50,ul

of sterile mineral oil. After inoculation, the microplate was

sealed withasterileplasticsealer.Followingincubationfor 24 hat30°C,50 plof thedimethylaminocinnamaldehyde reagent was added to the indole test wells, and then the test plates

were read with a photometer (Multiscan MCC 340; Flow Laboratories) connected to a personal computer

(Desk-1201

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pro286; Compaq, Munich, Federal Republic of Germany). Different tests were read at different wavelengths, and the resulting adsorbance values werecoded inbinary data. The

resultingtestprofileswere compared with aprobability

ma-trix, accordingtothe methodsofnumerical identification (39). This probability matrixwasconstructed by themethods given

in the literature (17, 24), but with upperand lower limits of

0.999 and 0.001, respectively. The evaluation results were

classifiedby the differentrangesof likelihoods(identification

scores), which are given as percentages throughout this

report.

The reproducibility of the TT-NFsystemwasdetermined

by testing 50 randomly chosen isolates on 3 differentdays.

Theresultingtestprofileswerecompared, andanestimate of

the probability ofan erroneoustestresultwascalculated by

methods described elsewhere (19).

RESULTS

The results of the evaluation of the TT-NF system are

summarized in Table 1. Of 1,289 strains tested, 935 (72.5%)

were correctly identified with an identification score of

-98%. For 189 strains (14.2%), the correct identification

scores werebetween 90 and 97%, while for 40 strains (3.1%),

thecorrectidentification scores were between 80 and 89%.

Foratotal of 104 strains (8.1%), the identification score was

below 80%, and hence, these strains were not identified correctly. To further the identification of 79 strains, oneto

fouradditionaltests were necessary; these appeared in the

formofpercentpositivetestresults onthecomputerscreen

incasesof low discrimination (additionaltestsrecommended by Flow Laboratories for the TT-NF system were nitrogen

formationfrom nitrate, nitrite formation from nitrate, motil-ity, growth on salmonella-shigella agar,growth in the pres-enceofcetrimide, growthon MacConkey agar, andgrowth

at42°C).

Incorrect identifications were obtained for 14 strains

(1.1%) (Table 2). Forseven strains (0.5%) no identification

could be obtained. Investigations on the reproducibility of

the TT-NF systemprovided the following results. A total of 50 strains were chosen at random, and 28 tests were

per-formed oneach strain, in triplicate, foratotal of4,200test

results. Of these 4,200 test results, there were 7 errors (a single negative result in one of the triplicate tests was

classified as an error). The corrected error rate was

calcu-latedasfollows:

C = 1/2(1- V/1-T4E/N)

where E is the number of errors observed, and N is the

number oftest results, as described previously (19). The

correctederrorratefor theTT-NF systemwascalculatedto

be0.2%, witha95% confidence interval of 0.7to 1.4%. The reproducibility of individualtests is summarized inTable3.

DISCUSSION

The TT-NF system was easy to set up and posed no

problems of interpretation. Indole tests were sometimes

difficulttoreadphotometrically, because somestrains of the

Alcaligenes faecalis-A. odoransgroupand alsosome

Pseul-domonas acidovorans strains produced a cherry red color

after the wells werefilled with the indolereagent (4-dimeth-ylaminocinnamaldehyde). This caused higher adsorbance valuesand, hence, false-positive results. These nonspecific reactionswereconsidered in theprobability values of Alcali-genesfaecalis-A. odorans and Psezidomonas alcaligenes in the data base.

The reproducibility ofthekit tests was

excellent,

withan

estimated probability of erroneous results of 0.2%. This value is low when compared with probabilities of 2 to 4% found in other studies (19),

probably

because thetests that wereperformedare well established in our

laboratory.

Seventeen

species

of the genus Pseudomonas were in-cluded inour study. Most strains were identified correctly

with an identification score of

-90%.

For the 5 of 18 Pseudomonas

pseudoalcaligenes

strains and the 8 of 24

Pseudomonastestosteroni strains

tested, only

identification scores of <80% could be obtained. The differentiation

amonginactive strains of the

species

Pseudomonas alcali-genes,Pseudomonas

pseudoalcaligenes,

and Pseudomonas testosteroni was not

satisfactory,

as has

already

been

de-scribed for other test systems,

especially

the API 20NE

system

(2, 23, 26).

Strains ofChryseomonas luteola

(CDC

group Ve-1)were

correctly

identified,

whereas for two of the five strains of

Flavimonasoryzihabitans (CDC groupVe-2),

higher

identi-fication scoresof -80%were

prevented.

Within the genus

Alcaligenes,

Alcaligenes faecalis

and

Alcaligenes

odorans were

grouped together

(Alcaligenes

odorans isalater synonymfor

Alcaligenes faecalis [22]).

Five taxa ofthe genus Moraxella were tested. The two

species

Moraxella

nonliquefaciens

and Moraxella lacunata

are not

easily separated

on routine biochemical tests and were therefore combined into a

single

taxon. Of the 14

strains of these two

species,

13 achieved identification scores of

only

68.7%. For 8 strains of the 12 Moraxella

phenylpyruvica

strains,

ascoreof<80%was obtained. For

the 11strains of

Oligella

urethralis

(Moraxella

urethralis),

10

identification scores were only at a level of77.1%. Within the genera

Flavobacterium, Pasteurella,

and

Vibrio,

asfor

the

species

Aeromonas

hydrophila,

Plesiomonas

shigelloides,

and Chromobacterium

violaceum,

identifica-tion in most cases were correct, with scores of >90%. Strains identified with alower accuracy (scores from80 to

90%)

inmost casesshowed

atypical

testpatterns. ForVibrio

vulnificus, percentages

should be

verified,

with the inclusion ofmorereference

organisms

in the database needed.

Previous

investigations

onthe accuracy of

commercially

available systems for the identification of nonfermenters produced various results (2, 8, 23, 26). Identification of gram-negative

fermenting

and

nonfermenting

bacteria

by

probabilisticmethods has beendescribed in theliterature(6,

16, 18), and most

commercially

available test kits for the

identification ofvarious bacteriaarebasedonthese methods

(40). Most evaluation resultsof

commercially

available kits

reported

difficulties in

identifying

weak oxidativeorinactive

strains. The combination of carbon substrate assimilation

testswith enzymetestsin the API 20NEsystemhasoffered

the

highest

accuracysofar for identificationtothe

species

or

biogroup

level

(2).

Carbon source assimilation tests and

carbon substrate alkalinization testshave beenused earlier for the differentiation of

representatives

of the families Pseiidomonadaceae(3, 4, 25, 28, 30, 31, 33-35, 37,

38)

and

Alcaligenaceae

(29, 32) and the genus Flavobacterium

(7).

Miniaturization of these methods (9-11, 20, 21) has made their

application possible

in

commercially

availabletestkits.

The

application

of

qualitative

enzyme testswith

chromoge-nic substrates improved the accuracy of differentiation of

glucose-nonfermenting,

gram-negative,

and

oxidase-posi-tive,

glucose-fermenting

bacteria(1,

5).

The TT-NF system is a combination of miniaturized classical biochemical tests, carbon substrate assimilation tests, and growth-dependent

qualitative

enzyme tests with

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TABLE 1. Results of the evaluation of the TT-NFsystem

No.of strainscorrectly identified No.of strains No.of No.of

No.of with an identification score of: with an strains strains

Species strainstested identification incorrectly not

>98% 90-97% 80-89% scoreof <80%" identified identified' Pseudomonas aeruginosa Pseudomonasfluorescens Pseudomonasputida Pseludomonaspseudomallei Pseudomonas cepacia Pseudomonas pickettii Pseudomonasstutzeri Pseudomonas mendocina Pseudomonasalcaligenes Pseudomonas pseudoalcaligenes Pseudomonas acidovorans Pseudomonas testosteroni Pseudomonas diminuta Pseudomonas vesicularis Pseudomonas maltophilia Pseudomonas paucimobilis Shewanellaputrefaciens

Chryseomonas luteola (CDCgroupVe-1) Flavimonas oryzihabitans(CDCgroupVe-2) Alcaligenesfaecalis-A. ordorans

Alcaligenesxylosoxidans subsp. denitrificans Alicaligens xylosoxidans subsp. xylosoxidans Ochrobactrumanthropi (Achromobacter

groupV-d)

Bordetellabronchiseptica Agrobacterium tumefaciens

Moraxellanonliquefaciens-M. lacunata Moraxellaosloensis

Moraxella phenylpyruvica Moraxellaatlantae

Oligella urethralis (Moraxellaurethralis) Acinetobacter calcoaceticus Acinetobacterlwoffii Flavobacterium meningosepticum Flavobacterium odoraltun Flavobacterium breve Flavobacterium multivorum Flavobacteriumthalpophilum Flavobacteriumspiritivorum Flavobacterium species group IIb

Weeksellazoohelcum (CDCgroup Ilj) Pasteurellaaerogenes

Pasteurella multocida

Pasteurellahaemolytica type A Pasteurella haemolytica type T Pasteurella pneumotropica Pasteurellaureae Aeromonashydrophila Plesiomnonas shigelloides Vibrioalginolyticus Vibrioparahaemolyticus Vibriovulnificus Vibriocholerae Vibriomimicus Vibriometschnikovii Vibriofurnissii Vibriofluvialis Chromnobacterium violaceuni 113 72 61 29 42 19 22 22 8 18 44 24 24 25 57 20 17 6 5 54 13 il 23 28 30 14 10 12 s il 26 25 25 21 18 42 7 31 28 14 1 34 2 2 3 4 28 23 22 18 10 23 s 12 10 6 10 106 58 42 27 39 15 20 4 38 21 20 47 16 17 6 3 50 2 10 19 6 6 6 3 20 7 9 3 15 1 3 7 1 4 25 26 9 4 4 12 il 20 3 25 15 3 9 7 il 22 s 29 22 1 14 32 2 25 17 20 14 2 19 2 12 7 3 8 i

s

s

2 1 1 1 i 1 1 2 2 1 2 6 1 1 3 8 i 2 1 2 1

s

2 8 1 2 1 2 2 4 1 1 13 1 8 1 10 1 2 2 1 3 2 1 3 1 i 1 2 1 1 1 1 1 1 1 1 2 2 2 4 1 1 2 2 2 3 1 1 i i 2 2 i 1 1 4 1 1 2 1 1 1 1 1 1 1

1,289(100) 935(72.5) 189(14.7) 40(3.1) 104(8.1) 14(1.1) 7(0.5) Total (percent)

"Lowdiscriminationresults(identificationresults of strainsgroupedin this category showedtwoor more taxapossible [amongthemthecorrecttaxonl),but

theyfailed to reach the identification level (scores of.80%);acorrectidentificationcanbe madebyperformingthesuggestedsupplementarytests todifferentiate

between themostlikelytaxasuggested.

bStrains reached theidentificationlevel (scoresof.80%),buttothe incorrecttaxon.

' Noidentification result is shown(comparisonoftestprofileswith the data basegeneratedidentificationscoresthatweretoosmall).

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TABLE 2. Misidentifications with the TT-NF system Identification with the Conventional identification(no.) TTN

hyte

TT-NFsystern

Pseudoinonasdiminutl (1)...Moraxella atlantae

Pseudoinonas vesicularis(1)...Flat'obh(ateriun

ineningosepli-Pseudotnonaspaucimobilis (1)... Pseudomnonasdiniinuta

Agrobacteriurn rumefaciens(1) ....Flavobacterilm ,nultivorum

Moraxella

nonliquefaciens-M. lacunala (1)...Pasteurellahaeinolvtic-a typeA

Oligellaurethralis (1)...Moraxellaphenvlpvrulvica

Flavobacterinm brei'e(1)...Moraxella

nonliquefaciens-M.lacunata Flavobac-teriun species

groupIIb (1)... Flav'obwterinm eninengosepti-c(un

Aeroinonas hydrophila (1) ...Vibriofurnissii Plesioinonas shigelloides (1)... Pasteurellnllaocida Vibriov'ulnificus (1)...Flai'obal(teriiumi speciesgroup

lIb

Chroinobac'teriun v'iolaceun (1)....Flavobh(ateri-iiii inulti'oro7un AIcaligenes

fjecalis-A. odorans(2)... Akalaigenesxylosox.idans

subsp. denitrificansand

Mor-axelIa

phenvlpyruvica

chromogenic substrates. Hence, this system is designed to offer a relatively specific level of identification compared with that offered by other methods.

The TT-NF system allows, in most cases. the correct

identification (identification scores of >90%) ofcommonly

encountered nonfermenting bacteria and oxidase-positive, fermenting bacteria; and also, those groupsofbacteria that are rarely encountered in the clinical microbiology labora-tory were well separable. Disadvantages of the system included the lack of adequate resolution within the genus Moraxella, the lack of adequate differentiation among non-reactive Pseuidomonas testosteroni, Pseuidomonas alca( li-genes, and Pseudomonas pseudoahlcaligenes species and

Alcaligenes xylosoxidans subsp. denitrifi(ans. Combining these rarer species or genera into groups and identifying

themonthe genus or the group levelonly could improve the percent identification values, as has been suggested for the

PASCO MIC-ID system(36). For the majority of

laborato-ries, collective groupings as Moraxella sp. and

Pseuidomo-nas alcaligenes, Pseudoinonas pseutdoal(ctiligenes, and Pseudomonas testosteroni would be adequate. Vibrio

i'il-nificuiis

isolates also posed problemsfor the TT-NF system.

Certain biotypes are not represented in the data base.

TABLE 3. Reproducibility ofindividual tests

Test' No. of Corrected

errors" error rate(%)' Glucosefermentation 2 1.4(0-2.8)"'

Gluconate assimilation i Histidine assimilation 1

Mannoseassimilation 1 0.7(0-2(.0)

Phenylacetate assimilation 1

Urease 1

Othertests

gave

O

errors

in

the

150

tests

that

were

performed.

b Asinglenegativeresult in oneofthetriplicatetestswasclassified

als

an

error.

Thecorrected error rate isgivenbyC= 1/2 (1 - N. as

de-scribedin the text.

dThe95%confidence intervals aregivenin parentheses.

Despitetheseproblems,the TT-NFsystemprovidesahighly

differentiating approach for the identification of

gram-nega-tive, nonfermenting and gram-negative, oxidase-positive, fermenting bacteria,and thusoffersapracticalalternativeto

already existing identification systems for this

heteroge-neousgroup of

organisms.

ACKNOWLEDGMENTS This studywas supported byFlow Laboratories.

Wethank H. Backes(FlowLaboratories)for invaluablehelpand discussions. Furthermore, we thank Katrin Noack for excellent technical assistanceand H. K. Geiss(Instituteof Medical

Microbi-ology, UniversityofHeidelberg)andG. Barth(CentreHospitalier

St. Nicolas, Sarrebourg, France) for the contributions of test

organisms.

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