JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1980, p. 271-2780095-1137/80/08-0271/08$02.00/0

Vol. 12, No. 2

Four Methods for Identification of Gram-NegativeNonfermenting Rods: Organisms More Commonly

Encountered in Clinical SpecimensPETER C. APPELBAUM,* JUDY STAVITZ, MICHAEL S. BENTZ, AND LARRY C. VON KUSTERDepartment ofPathology (Clinical Microbiology), Hershey Medical Center, Hershey, Pennsylvania 17033

Four commercial kits, Oxi/Ferm (OF), API 20E (AP), Minitek (MT; BBLMicrobiology Systems), and Flow N/F (NF), were evaluated, without additionaltests, for identification of 258 gram-negative nonfermentative rods. OF and MTwere read after 48 h of incubation, and AP and NF were read after both 24 and48 h of incubation, respectively. Overall, OF correctly identified 51% of strains,with 46% as part (but not first) of a spectrum of identifications (SI), and 3%incorrect species identification. MT yielded 85% correct identification, with 15%SI. Of 126 glucose-positive strains, or those with 23 positive AP reactions after 24h, 60% were correctly identified, with 40% SI; incubation for an additional 24 hraised the rate of correct identification to 99%, with 1% SI. A total of 132 strainsyielded <3 positive AP reactions after 24 h and were identified after 48 h only; ofthese, 82% were correctly identified, with 17% SI and 1% incorrect speciesidentification. NF correctly identified 79% of cultures after 24 h, with 21% SI;corresponding figures after an additional 24 h of incubation were 80% and 20%,respectively. Ail four commercial methods show promise; OF is easiest to inocu-late, but requires extra tests for optimal identification. AP reliably identifies themajority of clinically important nonfermenters, with fairly good species identifi-cation of saccharolytic strains after 24 h. MT yields reliable identification of mostnonfermenters and has the advantage of flexibility. NF is easy to inoculate, yieldssatisfactory identification rates, and may be read after 24 h of incubation.

Gram-negative nonfermenting aerobic rodsare becoming increasingly implicated in humandisease, especially of nosocomial origin (11, 17,20). The complex physicochemical properties ofthese organisms necessitate a battery of conven-tional tests for their precise identification. Inaddition, much confusion still exists concerningthe taxonomic status of many of these orga-nisms. Lack of personnel or other resources pre-vents many clinical microbiology laboratoriesfrom identifying most of these organisms byconventional means. Therefore, identificationhas often been neglected, especially when anti-microbial sensitivity results are available. A1-though such data may be adequate for patienttreatment, it does not yield satisfactory epide-miological information. As interest in these or-ganisms has increased, a number of computer-ized rapid identification systems have been de-veloped and marketed (1, 2, 6, 7, 9, 10, 12-15, 18,19, 21).The smaller microbiology laboratory is in

need of a rapid, reliable method of nonfermenteridentification. The purpose of this study was tocompare the ability of four commercial kits-Oxi/Ferm (OF; Roche Diagnostics, Div. Hoff-

mann-La Roche, Inc., Nutley, N.J.), API 20E(AP; Analytab Products, Inc., Plainview, N.Y.),Minitek (MT; BBL Microbiology Systems,Cockeysville, Md.), and Flow N/F system (NF;Flow Laboratories, Roslyn, N.Y.)-to identifycommonly encountered, clinically significantgram-negative nonfermenting rods, without theaid of supplementary conventional tests.

(Part of this work was presented at the 80thannual meeting of the American Society forMicrobiology [P. C. Appelbaum, J. Stavitz, M.S. Bentz, and L. C. von Kuster, Abstr. Annu.Meet. Am. Soc. Microbiol. 1980, C190, p. 306].)

MATERIALS AND METHODSBacteria. A total of 258 commonly encountered,

clinically significant nonfermenters were tested; 84%of these were kindly provided by Gerald Gilardi (Hos-pital for Joint Diseases and Medical Center, NewYork, N.Y.), and the remaining 16% were provided bycommercial companies (Analytab Products, Flow Lab-oratories, Roche Diagnostics). Strains tested are listedin Tables 1 to 5. Cultures from G. L. Gilardi and FlowLaboratories were identified by methods previouslydescribed (5). Cultures from Roche and AnalytabProducts were originally obtained from G. L. Gilardi,the Center for Disease Control, and the firms' respec-

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272 APPELBAUM ET AL.

tive reference laboratories. Stock cultures stored inlitmus milk at -30°C were transferred and subculturedtwice onto 5% sheep blood agar plates before inocula-tion of kits. Oxidase testing was with 1.0% tetra-methyl-p-phenylenediamine dihydrochloride (MarionScientific Corp., Kansas City, Mo.). Incubation was at35°C, unless otherwise stated. Cultures used to inoc-ulate kits were checked for purity and viability byinoculation of Trypticase soy agar plates (BBL).

Identification of organisms. Identification wasclassified as correct, part (but not first) of a spectrumof identifications (SI), or incorrect. Where only genusidentification was possible with a specific system, thiswas regarded as correct.OF system. Procedures for inoculation and incu-

bation were as described previously (2, 6, 7, 10, 12-14,18). No supplemental tests were done. Where defini-tive code book identification was not possible withoutsupplemental tests, identification was as SI; in suchcases, no statistical information is available in the OFcode book at this time.AP system. Strips, OF, and motility media were

inoculated and incubated according to standard prac-tice (2, 5, 9, 12, 13, 19, 20). Reagents were added after48 h in all cases. If glucose positivity or -3 positivereactions (excepting oxidase) was found after 24 h,tryptophan deaminase, Voges-Proskauer, and indolereactions were extrapolated to 24 h, and organismswere identified from 24-h, as well as 48-h, results,respectively. Where organisms yielded <3 positive re-actions after 24 h, results were encoded after 48 h.Indole, Voges-Proskauer, and tryptophan deaminasereactions which are positive after 48 h are uniformlypositive after 24 h (Karla M. Tomfohrde, personalcommunication). The above method of reagent addi-tion, although differing from that used in clinical lab-oratories, was used to generate as much data as pos-sible for the AP system. Interpretation of code num-bers was according to the manufacturer's instructions.Where identification was as "good likelihood but lowselectivity" with a spectrum of possibilities, supple-mental tests were not done, and the statistically mostprobable identification was used for organism identi-fication.MT system. A dense homogenous suspension of

organisms in MT nonfermenter broth (MacFarlandno. 5) was used to inoculate MacConkey plates andthe following range of MT disks: two of dextrosewithout nitrate, and one each of maltose, sucrose, D-xylose, arginine dihydrolase, ornithine and lysine de-carboxylase, urease, o-nitrophenyl-,f-D-galactopyran-oside (ONPG), nitrate reductase, starch, phenylala-nine deaminase, citrate. For indole production, theinoculum was placed in an empty well. In addition,gelatin disks (Key Scientific Products, Inc., Los An-geles, Calif.), esculin, mannitol, and malonate (notcommercially available in the MT panel) were inocu-lated. Reagents were added, and tests were interpretedwith the aid of a code book, according to the manufac-turer's instructions. The firm's computer facilitieswere consulted for organisms whose code did notappear in the book. Where supplemental tests wererequired for precise identification of a range of possi-bilities, the organism with the highest confidence value

and lowest biotype validity was selected as correct.NF system. Two-tube screens and Uni-N/F-Tek

wheels were inoculated according to the manufac-turer's recommendations (1, 21). Pseudomonasaeruginosa strains were initially inoculated into thetwo-tube screen; only organisms which failed to keyout with the latter were inoculated onto the wheel.Strains of Pseudomonas fluorescens and Pseudomo-nasputida were inoculated into both tubes and wheelsirrespective of whether positive identification was pos-sible with screen tubes only; this was to generate moredata for the NF system. Oxidase-negative strains wereinoculated onto the wheel only. 42P screen tubes wereincubated at 42°C, and GNF screens plus wheels wereincubated at 35°C. Interpretation of reactions wasaccording to the manufacturer's recommendations,utilizing a computer code book (1, 21). Numbers whichdid not appear in the code book were referred to thefirm's computer facilities. Where the code book re-vealed a spectrum of more than one organism as thepossible species, identification was with the organismof greatest statistical likelihood, without performanceof supplemental tests.

RESULTSBiochemical reactions of strains in the four

systems are available from the authors on re-quest. In general, most tests gave expected re-actions. OF nitrogen (N2) gas production wasoften difficult to interpret, owing to accumula-tion of bubbles under the wax overlay. Citrateutilization tests also presented difficulty withOF, owing to equivocal greenish-blue color pro-duction, which could have been interpreted aseither positive or negative. Nitrate reactionswith AP were satisfactory only if the methodrecommended by the manufacturer was strictlyadhered to: if powdered instead of granular zincwas used, or if strips were not left open for atleast 10 to 15 min after zinc addition, equivocalreactions resulted. Motility was the most diffi-cult test to interpret in AP. In many cases, onlya few motile outgrowths from the primary inoc-ulum were found, necessitating interpretation byan experienced technologist for optimal results.Positive AP indole reactions were often weakand took 1 to 2 min to develop satisfactorily.MT nitrate reductase tests gave rapid results,but inaccuracies occurred, especially false-nega-tive tests for N2 gas production. The sensitivityof Key gelatin disks, as presently available inthe MT system, was low. NF gave rise to prob-lems in interpretation of N2 gas and indole pro-duction. N2 gas interpretation difficulties arosewhen scanty, small bubbles were produced onthe butt surface beneath the constriction; thesewere read as positive, whereas bubbles or cracksalong the inoculation stab line were taken asnegative. The Flow indole method was difficultto interpret: when a red color did develop, this

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IDENTIFICATION OF GRAM-NEGATIVE NONFERMENTERS 273

was often weak and difficult to see.

Only the NF system offered species identifi-cation of most strains. With the exception ofMoraxella phenylpyruvica, Moraxella strainswere identifiable to genus only by OF and MT;AP offers only genus identification of moraxel-las. OF and AP identify Alcaligenes strains togenus only, whereas MT identifies these orga-

nisms as Alcaligenes/Pseudomonas species.Acinetobacter calcoaceticus biotypes haemoly-ticus and alcaligenes were identified as biotypesanitratus and lwoffi, respectively, by all foursystems. The differentiation of A. calcoaceticusinto four biotypes on the grounds of sugar oxi-dation, hemolysis, and proteolysis (4) is not rec-

ognized by all taxonomists (G. L. Gilardi, per-

sonal communication). Differentiation betweenMoraxella urethralis and Moraxella osloensisstrains may be difficult (8); therefore, when theFlow system (the only method which offeredspecies identification of moraxellas) included M.osloensis in the SI for M. urethralis strains, thiswas taken as correct group identification. MTand NF systems did not differentiate between P.fluorescens and P. putida strains. Flavobacter-ium meningosepticum strains were identified togenus only with OF, and to genus (two strains)or species (seven strains) with AP.

Identification ofnonfermenter strains with thevarious systems is listed in Tables 1 to 5. OFcorrectly identified 51% of strains (mainly P.aeruginosa,, Pseudomonas maltophilia, A. cal-coaceticus oiotype alcaligenes, Alcaligenesodorans, F. meningosepticum) without supple-mental tests; 46% of remaining organisms re-

quired additional tests, not included in OF, forprecise identification, and 3% of strains wereincorrectly identified. Of 126 strains with posi-tive AP glucose reactions or 23 positive reac-tions after 24 h, 60% were correctly identified(mainly P. maltophilia, A. calcoaceticus bio-types anitratus and haemolyticus, Pseudomo-nas cepacia), with 40% requiring additionaltests. Incubation for an additional 24 h raisedthe rate of correct identification to 99%, with 1%SI. Ail P. aeruginosa strains keyed as SI (mainlyP. fluorescens group) after 24 h and requiredOF, motiity, nitrate reduction, and MacConkeytests for precise identification. A total of 132strains yielded <3 positive AP reactions after 24h and were identified after 48 h only; of these,82% were correctly identified, with 17% SI(mainly P. fluorescens, Alcaligenes species) and1% incorrect identification. Of 77 organismsidentified as the first likelihood of a "good like-lihood but low selectivity" range, 61% yieldedthe correct result, with 39% SI. MT correctlyidentified 85% of strains, with 15% SI; the latter

TABLE 1. Identification of nonfermenters with OF

Correct One of a IncorrectOrganism identifica- spectrum identc

tion' of identifi- tidentiacations'P. aeruginosa (52)Y 5e __dP. fluorescens (11) _ ilP. putida (7) - 7 -

P. maltophilia (34) 28 1 5A. calcoaceticus biotype 2 24 -

anitratus (26)A. calcoaceticus biotype 7 9

lwoffi (16)A. calcoaceticus biotype 5 10

haemolyticus' (15)A. calcoaceticus biotype 14 - -

alcaligenesf (14)A. faecalisg (12) - 12 -

A. odoransg (14) 13 1 -

A. denitrificansg (12) 3 9 -

M. osléensis- (12) - 7 -

M. phenylpyruvica (2) - 1 1M. urethralisg (4) 2 2 -

M. nonliquefaciensg (11) - 11 -

F. meningosepticumg (9) 7 1 1P. cepacia (12) - 1il 1

a Includes free tests (reactions on Trypticase soy agar,hemolysis).

b Extra tests necessary for correct identification.C Number of strains.d- None.' Identified as biotype anitratus.f Identified as biotype lwoffi.g Organisms identifiable to genus only with this system.

TABLE 2. AP results of nonfernenters with >3positive reactions after 24 ha

Correct identi- Oneofafiction spectrum ofOrganism identifica-

Time No. tons(h)

P. aeruginosa (48) 24 _b 4848 48 -

P. fluorescens (1) 24 - 148 - 1

P. putida (1) 24 - 148 1 -

P. maltophilia (23) 24 23 -

48 23 -A. calcoaceticus biotype 24 25 -

anitratus (25) 48 25 -

A. calcoaceticus biotype 24 15 -

haemolyticusc (15) 48 15 -

F. meningosepticum (2) 24 2d -

48 2d -

P. cepacia (11) 24 10 148 il -

a There were no incorrect identifications.-_ None.

C Identified as biotype anitratus.d One organism identified to species, the other to

genus only.

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274 APPELBAUM ET AL.

TABLE 3. AP results at 48 h of nonfermenters with<3 positive reactions after 24 h

Correct One of a IncorrectOrganism identifica- spectrum identifica-

tion cation tioncahions

P. aeruginosa (4) 4a -bP. fluorescens (10) 1 9P. putida (6) 6P. maltophilia (11) 11iA. calcoaceticus biotype la

anitratus (1)A. calcoaceticus biotype 16

lwoffi (16)A. calcoaceticus biotype 14

alcaligenesd (14)

A. faecalis' (12) 8 4A. odorans' (14) 7 7A. denitrificans' (12) 10 1 1M. osloensis' (7) 7M. phenylpyruvicae (2) 1 1M urethralis' (4) 3 1M. nonliquefaciens' (11) 1i1F. meningosepticum (7) 71 _P. cepacia (1) 1

a All keyed out as SI after 24 h.b_, None.

All keyed out as P. maltophilia after 24 h.d Identifiable as biotype lwoffi.' Identifiable to genus only with this system.fSix correct species identifications, one identification to

genus only.

included P. aeruginosa, A. calcoaceticus bio-types anitratus and lwoffi, F. meningosepticum.False-negative tests for N2 gas production ac-counted for problems with P. aeruginosa; all 12A. calcoaceticus biotype anitratus and F. men-

ingosepticum strains identified as SI keyed outcorrectly when results of mannitol-esculin testswere used. Positive tests for N2 gas-urease were

responsible for SI of A. calcoaceticus biotypelwoffi strains. The Flow two-tube screeningmethod identified 98% of P. aeruginosa, 18% ofP. fluorescens, and 45% of P. putida strains after24 h; all remaining organisms in these threegroups were correctly identified on addition ofthe wheel. The Flow method identified 79% ofcultures after 24 h, with 21% SI; correspondingfigures after an additional 24-h incubation were80% and 20%, respectively. Organisms identifiedas SI were mainly P. maltophilia, Alcaligenesfaecalis, and Moraxella strains.

Extra tests most commonly needed for correctidentification of SI strains are listed in Table 6.As can be seen, some members of all organismgroups required a variety of additional tests topinpoint a correct identification from an SI spec-

trum. Incorrect identifications are presented inTable 7. Eight strains were misidentified by OF;false-positive citrate tests were responsible foridentifying three strains of P. maltophilia as A.

TABLE 4. Identification of nonfermenters with MT'

Correct OneofaOrganism identifica- spectrum

tion of identifi-cations

P. aeruginosa (52) 35 17P. fluorescens (11) lob 1P. putida (7) 7b _éP. maltophilia (34) 34A. calcoaceticus biotype 18 8d

anitratus (26)A. calcoaceticus biotype 10 6

lwoffi (16)A. calcoaceticus biotype 15 -

haemolyticuse (15)A. calcoaceticus biotype 14

alcaligenesf (14)A. faecalis5 (12) il 1A. odoransg (14) 14A. denitrificansg (12) 12M. osloensish (7) 7M. phenylpyruvica (2) ih 1M. urethralish (4) 3 1M. nonliquefaciensh (11) il -

F. meningosepticum (9) 5 4dP. cepacia (12) 12 -

a There were no incorrect identifications.b Identified as P. fluorescens/P. putida with this

system.c-, None.d Eight strains of biotype anitratus and four of F.

meningosepticum required mannitol/esculin (not in-cluded in regular panel) for correct identification.

e Identified as biotype anitratus.f Identified as biotype lwoffi.Identifiable as Alcaligenes/Pseudo>#onas species

in this system.h Identifiable to genus only with this system.

calcoaceticus biotype lwoffi and two strains asP. cepacia. A false-positive citrate test was alsoresponsible for one F. meningosepticum strainnot being coded. One M. phenylpyruvica strainwas misidentified as group 4E Alcaligenes-like,due to a false-positive aLkaline dextrose andrapid urease tests. One strain of P. cepacia wasmisidentified as Pseudomonas species/A. fae-calis/M-4 Moraxella-like, due to a false-nega-tive aerobic dextrose reaction. The one incorrectidentification by AP of an Alcaligenes denitri-ficans strain as Pseudomonas species other/Moraxella/Pasteurella was due to a false-neg-ative reaction in API motility medium.

DISCUSSIONThis study attempted to evaluate the relative

ability of each of four commercially availablesystems to identify a group of commonly en-countered, clinically significant gram-negativenonfermenters. The potential of these organisms

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IDENTIFICATION OF GRAM-NEGATIVE NONFERMENTERS 275

TABLE 5. Identification of nonfermenters with NF'Correct identi- One of a

fiction spectrum ofOrganism identifica-Time No. tions

_____________________ (h) ________

P. aeruginosa (52)

P. fluorescensc (11)

P. PutidaC (7)

P. maltophilia (34)

A. calcoaceticus biotypeanitratus (26)

A. calcoaceticus biotypelwoffi (16)

A. calcoaceticus biotypehaemolyticusd (15)

A. calcoaceticus biotypealcaligenes' (14)

A. faecalis (12)

A. odorans (14)

A. denitrificans (12)

M. osloensis (7)

M. phenylpyruvica (2)

M. urethralisf (4)

M. nonliquefaciens (11)

M. meningosepticum (9)

P. cepacia (12)

24482448244824482448244824482448244824482448244824482448244824482448

5252il

il77

23212626161515151414

68610

66991212

a There were no incorrect identifications.b_ None.

C Identified as P. fluorescens/P. putida with thissystem.

d Identified as biotype anitratus.e Ideittified as biotype lwoffi.f Does not key out to species with this system (see

text).

for causing nosocomial infection, as well as theirspecific antibiotic susceptibiity spectrum (11,17, 20), makes accurate identification manda-tory.The OF system, without supplemental tests,

correctly identified (to genus or species) 98% ofP. aeruginosa, 82% of P. maltophilia, 100% ofA. calcoaceticus biotype alcaligenes, 93% of A.odorans, and 78% of F. meningosepticumstrains. Other isolates were identified with vary-ing degrees of accuracy (Table 1), and mostrequired additional tests for more precise iden-tification (Table 7). Previous workers (2, 6, 7, 10,12-14, 18) have reported the ability of OF to

identify most commonly encountered nonfer-menters, ifrecommended supplemental tests areperformed. If these tests are not carried out,accuracy of OF drops (2, 6, 13, 18). Difficultiesin interpretation of N2 gas reactions have beendescribed before (10). Conflicting reports on theaccuracy of the OF citrate test have appeared inthe literature (2, 6, 7, 10, 12-14). In our hands,citrate utilization reactions were difficult to in-terpret and responsible for mis-identification offive P. maltophilia strains, as well as non-iden-tification of one strain of F. meningosepticum(code number 0251). Advantages of OF includeease of inoculation and the infrequent need toconsult the manufacturer's computer facilitieswith numbers not present in the code book. Inour opinion, the value of OF, especially in thesmaller laboratory without facilities for manyrecommended conventional tests, would be im-proved by inclusion of more reactions (e.g., man-nitol, lactose, maltose, deoxyribonuclease, escu-lin, ONPG) in the tube, with correspondinglylonger code numbers and a more comprehensivecode book. When a spectrum of identificationsis encountered, statistical data on the likelihoodof each would also be valuable. Improvement ofexisting N2 gas and citrate tests would furtherenhance the value of the OF system.

Identification by AP of saccharolytic nonfer-menters after 24 h was satisfactory for the ma-jority of strains, with the exception of P. aerugi-nosa (Table 2). Ail 48 strains of the latter orga-nism were identified as SI (mainly P. fluorescensgroup) after 24 h, with precise species identifi-cation only after additional expanded AP tests.Gelatin negativity of 10 out of 11 P. fluorescensstrains in AP tests precluded accurate identifi-cation of this organism group. Problems in in-terpretation of the AP motility test were respon-sible for SI of Alcaligenes strains and for theone incorrect AP identification (Table 7); im-provement in sensitivity of the latter test wouldenhance the capabiity of the AP system toidentify nonfermenters. Reports in the literatureon the accuracy of AP nonfermenter identifica-tion have varied; most workers agree that oxi-dase-negative bacteria, such as P. maltophiliaand A. calcoaceticus biotypes, are reliably iden-tified by this system (2, 6, 9, 12, 13, 19, 21), butproblems have been encountered in the speciesidentification of P. aeruginosa (6, 9, 13, 21). Inthe light of these findings as well as our own, itseems that the expanded 48-h AP system maybe necessary for differentiation of P. aeruginosafrom other saccharolytic oxidase-positive non-fermenters. Problems in AP identification of P.cepacia reported by others (2, 6, 9, 21) were notencountered in our study. A practical difficulty

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TABLE 6. Extra tests most commonly needed for correct identification (SI)Organism OF AP MT NF

P. aeruginosa _b - 42°C; CET; ACE (P.fluorescens group)Y

P. fluorescens/P. 42'C; GEL; PB; AMP Conventional GEL (P.putida (P. fluorescens group) fluorescens group)

A. calcoaceticus MOT (P. cepacia) - MANN; ESC (CDC VE- -biotype anitratus 1, 2)

A. calcoaceticus MOT (P. maltophilia) - MOT (B. parapertussis;biotype lwoffi Alcaligenes!

Pseudomonas)

Alcaligenes species MOT; NAL; PEN; CET; ACE; SS; FLA; MOT ( )" MOT; NIT; PAD; SS;MAC; ANG; FLA; BB 6.5% NaCI; N2 from FLA ( >"nitrite ( >d

Moraxella species PEN; MOT; MAC; SS; PEN; TSI ( )d MOT ( )" NIT; MOT; PAD;ANG; FLA ( )d FLA ( )d

P. maltophilia - - MOT (P.paucimobilis)

F. meningosepticum MOT; ONPG; SUC; - MANN; ESC; CATLYS (Vibrio- (Flavobacterium IIB)Aeromonas)

P. cepacia PB; CAR; KAN; AMP; 10% LAC; PB (CDCMOT (P. fluorescens; VE-1; P. maltophilia;P. stutzeri; CDC VE- P. paucimobilis)2; P. pickettii;Achromobacter)

42°C, Growth at 42°C; MOT, motility; CET, growth on cetrimide; ACE, growth on acetamide; MAC, growth on MacConkeyagar; SS, growth on salmonella-shigella agar; 6.5% NaCi, growth in 6.5% NaCI; NIT, nitrate reduction; PAD, phenylalaninedeamination; GEL, gelatin liquefaction; FLA, flagellar staining; ANG, anaerobic growth; BB, barred appearance on Gram stainfrom broth; TSI, growth on triple sugar iron agar; ONPG, ONPG breakdown; SUC, sucrose oxidation; 10% LAC, utilization of10% lactose; MANN, mannitol oxidation; ESC, esculin hydrolysis; CAT, catalase production; LYS, lysine decarboxylaseproduction; PEN, penicillin G sensitivity; AMP, ampicillin sensitivity; PB, polymixin B sensitivity; CAR, carbenicillin sensitivity;KAN, kanamycin sensitivity; NAL, nalidixic acid sensitivity.

t.. None.Organisms in parentheses represent other SI possibilities.

d Other asaccharolytic/weakly saccharolytic pseudomonads; Pasteurella spp.

with the AP system was the large number (ap-proximately 25%) of profile numbers which didnot appear in the code book, necessitatinglengthy delays in identification because of theneed for computer assistance. Enlargement ofthe existing code book so as to include morenonfermenter profile numbers would help ob-viate this problem. The relatively high (61%)rate of correct identifications when the first like-lihood of a "good likelihood but low selectivity"SI was used as the organism's identification re-flects a possible use for this system in smallerlaboratories with fewer facilities.Few published reports on the use of MT for

nonfermenter identification could be found. Slif-kin and Pouchet (M. Sliflin and G. R. Pouchet,Abstr. Annu. Meet. Am. Soc. Microbiol. 1977,C88, p. 50), in a preliminary study, found thesystem to be satisfactory when supplementedwith motility testing, in the identification of a

wide variety of nonfermenters. In our hands theMT system yielded satisfactory results for mostorganism groups. Recourse to manufacturer'scomputer facilities was low approximatelyy 5%).Problems with SI of P. aeruginosa strains maybe obviated by modifications in the nitrate re-ductase test; also, addition of mannitol and es-culin disks to the existing panel would improveidentification of A. calcoaceticus biotype ani-tratus and F. meningosepticum, as well as CDCVE-1, CDC VE-2, and Flavobacterium groupIIB (P. C. A., unpublished data). If inclusion ofthe gelatin test in the routine MT panel is en-visaged, increased sensitivity is required. Differ-entiation of organisms presently classified asAlcaligenes/Pseudomonas could be effected byinclusion of acetamide, deoxyribonuclease, man-nitol disks, as well as a test for N2 gas productionfrom nitrate.The NF system offered the most accurate

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IDENTIFICATION OF GRAM-NEGATIVE NONFERMENTERS 277

TABLE 7. Major identification differences between OF, AP, MT, and NF methodsOrganisms OF AP MT NF

P. maltophilia (two P. cepacia P. maltophilia P. maltophilia P. paucimobilis; P.strains) maltophilia b

P. maltophilia (three A. calcoaceticus P. maltophilia P. maltophilia P. maltophiliastrains) biotype Iwoffi

A. denitrificans A. faecalis; Pseudomonas other; Alcaligenes/ A. denitrificansAchromobacter Moraxella; Pseudomonasbiotype Ic Pasteurellab

M. phenylpyruvica 4E Alcaligenes-like Moraxella spp. CDC IV-E; M. M. nonliquefaciens; M.phenylpyruvica osloensis; P.

testosteroni; A.faecalis; P.alcaligenes; A.denitrificans; M.phenylpyruvica; P.acidovoransb

F. meningosepticum Not coded (0251) F. meningosepticum F. meningosepticum F. meningosepticum

P. cepacia Pseudomonas; A. P. cepacia P. cepacia P. cepaciafaecalis; M-4Moraxella-like'

a Previously identified by conventional methods (see text).b Where a spectrum of possibilities was given, identifications are listed, in descending order of probability, for all systems

except OF.c The spectrum of possibilities with OF is not listed in order of probability (see text).

identification (to species or biotype) of all foursystems. Other advantages ofthis system includeidentification of most P. aeruginosa strains bythe two-tube screen, good identification of moststrains after 24 h (1, 21), and very little need toconsult manufacturer's computer facilities(<1%). Prolongation of the incubation period to48 h increased the correct identification of A.odorans and A. denitrificans strains, but led toa slight decrease in the identification rate of P.maltophilia. In contrast to findings by Warwoodet al. (21), all P. cepacia strains tested werecorrectly identified by NF. Problems in interpre-tation of the NF N2 gas reaction and lack ofprecise identification of asaccharolytic nonfer-menters have been described before (1, 21). Inour opinion, additional tests for phenylalaninedeaminase (possibly in the center of the wheel)and motility/nitrate reductase (possibly as athird combination screen tube) would enhancethe ability of the NF system to identify theAlcaligenes/Moraxella group. More precisepackage instructions on interpretation of N2 gasresults and increased sensitivity of the indoletest would also improve this system.

Utilization of stock organisms supplied by kitmanufacturers could have introduced bias intoresults reported in this study; however, we feelthat the small number of strains from suchsources (16%) as compared to cultures from G.L. Gilardi's laboratory (84%) argues against this

possibility. Additionally, identification of orga-nisms provided by firms did not differ signifi-cantly, when results of the supplier's kit werecompared with those of competitors.The clinical microbiology laboratory at Her-

shey Medical Center currently uses the API 20Esystem for routine nonfermenter identification:in the absence of facilities for conventional tests,it was felt that utilization of well-characterizedstock cultures of clinical isolates would yieldoptimal information, under the circumstances.Edberg et al. (3) have suggested that directcomparison of two commercial kit systems with-out conventional tests merely reflects percent-age agreement figures which represent the sumof innate errors of both; therefore, comparisonof commercial kits would best be made on thebasis oforganism identification, rather than test-by-test comparison. In our study, no attemptwas made to compare results of individual testsin each of the four respective systems; rather,emphasis was placed on accuracy of identifica-tion and shortcomings of individual tests in thesame system.Other methods for nonfermenter identifica-

tion which were not evaluated in this studyinclude the Micro-media system (Micro-media,Inc., Palo Alto, Calif.) and Microscan (Micro-scan, Inc., Ramsey, N.J.). A method which is notcommercially available, but which seems to offerrapid and accurate identification of most nonfer-

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278 APPELBAUM ET AL.

menters from clinical specimens, is the oxidativeattack (OA) system developed by Otto and co-workers (15, 16). If the OA method is marketed,it may prove to be one of the best commercialmethods for identification of this organismgroup.

In summary, all four systems evaluated in thisstudy show promise for identification of com-mon, clinically significant nonfermenters: OF isthe easiest to inoculate, whereas AP has theadvantage of identifying Enterobacteriaceae.The MT system has the advantage of flexibility.The NF method is the only system to identifymost organisms reliably after 24 h and has theadded advantage of identifying most P. aerugi-nosa strains by the two-tube screen only. Allfour systems may be improved with a view touse without additional tests, by addition of newtests or modification of existing ones. In thisway, accurate nonfermenter identification couldbe placed within the reach of smaller laborato-ries which do not possess facilities for extendedconventional testing.

ACKNOWLEDGMENTSSincere thanks go to Gerald Gilardi (Hospital for Joint

Diseases and Medical Center, New York, N.Y.) for provisionof most of the cultures, as well as for invaluable discussionthroughout the course of this study.

This work was supported in part by grants-in-aid fromRoche Diagnostics (Nutley, N.J.), Analytab Products, Inc.(Plainview, N.Y.), Flow Laboratories (Roslyn, N.Y.), and BBLMicrobiology Systems (Cockeysville, Md.). The first threecompanies also provided some of the cultures.

LITERATURE CITED

1. Barnishan, J., and L. W. Ayers. 1979. Rapid identifi-cation of non-fermentative gram-negative rods by theCorning N/F system. J. Clin. Microbiol. 9:239-243.

2. Dowda, H. 1977. Evaluation of two rapid methods foridentification of commonly encountered nonfermentingor oxidase-positive, gram-negative rods. J. Clin. Micro-biol. 6:605-609.

3. Edberg, S. C., D. Clark, M. H. Moore, and J. M.Singer. 1979. Rapid identification of Enterobacteria-ceae from blood cultures with the Micro-ID system. J.Clin. Microbiol. 10:693-697.

4. Gilardi, G. L. 1978. Identification of miscellaneous glu-cose non-fermenting gram-negative bacteria, p. 45-65.In G. L. Gilardi (ed.), Glucose nonfermenting bacteriain clinical microbiology. CRC Press, Inc., West PalmBeach, Fla.

5. Gilardi, G. L. 1980. Identification of glucose nonferment-ing gram-negative bacteria, p. 1-8. Hospital for JointDiseases, New York.

6. Hofherr, L., H. Votava, and D. J. Blazevie. 1978.

Comparison of three methods for identifying nonfer-mentative gram-negative rods. Can. J. Microbiol. 24:1140-1144.

7. Isenberg, H. D., and J. Sampson-Scherer. 1977. Clin-ical laboratory evaluation of a system approach to therecognition of nonfermentative or oxidase-producinggram-negative, rod-shaped bacteria. J. Clin. Microbiol.5:336-340.

8. Lautrop, H. 1974. Genus III. Moraxella, p. 436. In R. E.Buchanan and N. E. Gibbons (ed.), Bergey's manual ofdeterminative bacteriology, 8th ed. The Williams &Wilkins Co., Baltimore.

9. Morris, M. J., V. M. Young, and M. R. Moody. 1978.Evaluation of a multitest system for identification ofsaccharolytic pseudomonads. Am. J. Clin. Pathol. 69:41-47.

10. Nadler, H., H. George, and J. Barr. 1979. Accuracy andreproducibility of the Oxi/Ferm system in identifying aselect group of unusual gram-negative bacilli. J. Clin.Microbiol. 9:180-185.

11. Neu, H. C. 1978. Clinical role of Pseudomonas aerugi-nosa, p. 83-104. In G. L. Gilardi (ed.), Glucose nonfer-menting gram-negative bacteria in clinical microbiol-ogy. CRC Press, Inc., West Palm Beach, Fla.

12. Nord, C.-E., B. Wretlind, and A. Dahlback. 1977. Eval-uation of two test kits-API and Oxi Ferm tube-foridentification of oxidative-fermentative gram-negativerods. Med. Microbiol. Immunol. 163:93-97.

13. Oberhofer, T. R. 1979. Comparison of API 20E and Oxi/Ferm systems in identification of nonfermentative andoxidase-positive fermentative bacteria. J. Clin. Micro-biol. 9:220-226.

14. Oberhofer, T. R., J. W. Rowen, G. F. Cunningham,and J. W. Higbee. 1977. Evaluation of the Oxi/Fermtube system with selected gram-negative bacteria. J.Clin. Microbiol. 6:559-566.

15. Otto, L. A., and U. Blachman. 1979. Nonfermentativebacilli: evaluation of three systems for identification. J.Clin. Microbiol. 10:147-154.

16. Otto, L. A., and M. J. Pickett. 1976. Rapid method foridentification of gram-negative, nonfermentative bacilli.J. Clin. Microbiol. 3:566-575.

17. Rosenthal, S. L. 1978. Clinical role of Acinetobacter andMoraxella, p. 105-117. In G. L. Gilardi (ed.), Glucosenonfermenting gram-negative bacteria in clinical micro-biology. CRC Press, Inc., West Palm Beach, Fla.

18. Shayegani, M., A. M. Lee, and D. M. McGlynn. 1978.Evaluation of the Oxi/Ferm tube system for identifica-tion of nonfermentative gram-negative bacilli. J. Clin.Microbiol. 7:533-538.

19. Shayegani, M., P. S. Maupin, and D. M. McGlynn.1978. Evaluation of the API 20E system for identifica-tion of nonfermentative gram-negative bacteria. J. Clin.Microbiol. 7:539-545.

20. von Graevenitz, A. 1978. Clinical role of infrequentlyencountered nonfermenters, p. 119-153. In G. L. Gilardi(ed.), Glucose nonfermenting gram-negative bacteria inclinical microbiology. CRC Press, Inc., West PalmBeach, Fla.

21. Warwood, N. M., D. J. Blazevie, and L Hofherr. 1979.Comparison of the API 20E and Corning N/F systemsfor identification of nonfermentative gram-negativerods. J. Clin. Microbiol. 10:175-179.

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