Vol. 11, No. 6JOURNAL OF CLINICAL MICROBIOLOGY, June 1980, p. 694-7020095-1 137/80/06-0694/09$02.00/0

Collaborative Investigation of the AutoMicrobic SystemEnterobacteriaceae Biochemical Card

HENRY D. ISENBERG,`* THOMAS L. GAVAN,2 PETER B. SMITH,3 ALEX SONNENWIRTH,4W. TAYLOR,5 W. J. MARTIN,6 DWANE RHODEN,3 AND ALBERT BALOWS3

Long Island Jewish-Hillside Medical Center, New Hyde Park, New York 110421; Cleveland Clinic andFoundation, Cleveland, Ohio 441063; Center for Disease Control, Atlanta, Georgia 303333; Jewish Hospitalof St. Louis, St. Louis, Missouri 631104; St. Mary's Hospital, Chicago, Illinois 606225; and UCLA Clinical

Center, Los Angeles, California 900246

The Enterobacteriaceae biochemical card was used in six separate laboratoriesto identify 170 representatives of Enterobacteriaceae. The AutoMicrobic System(Vitek Systems, Inc.) performed with an accuracy of 97.8% as compared with98.1% by the standard method selected and 97.6% by a commercially preparedmanual system approach. During this time, 5,450 clinical isolates belonging toEnterobacteriaceae were analyzed. Compared with the routine methods used inthe various laboratories, the AutoMicrobic System identified 96.4% correctly.

The commercially produced systems or kitapproaches to the biochemical recognition ofclinically significant bacteria and yeasts haveprovided standardized, easy-to-use reagents tothe staffs of many clinical laboratories whoheretofore could not offer the advantages ofsuchservice (4) to clinicians in their institutions orcommunities. Compared with other clinical lab-oratory disciplines, the clinical microbiology ser-vice has remained labor-consuming despite theproliferation of these kits or systems, all ofwhichinvolve manual manipulation and individualevaluation and interpretation. The Auto-Microbic System (AMS; Vitek Systems, Inc.,subsidiary of McDonnell-Douglas, Hazelwood,Mo.), a fully automated approach to the enu-meration and recognition of selected bacteria inclinical urine specimens and certain antibio-grams of machine-sequestered organisms (1, 5,8, 9), has been expanded in its application toclinical microbiology by the introduction of adisposable card designed to identify members ofEnterobacteriaceae. A feasibility study of thisapproach has been reported (M. C. Meyer, J. J.Underwood, R. Wilkinson, and L. V. Woods,Abstr. Annu. Meet. Am. Soc. Microbiol., C142,p. 333, 1979; H. D. Isenberg, J. Scherer, and S.Freedman, Abstr. Annu. Meet. Am. Soc. Micro-biol., C 143, p. 334, 1979), and a carefully designedcollaborative evaluation of this fully automatedapproach has been conducted simultaneouslywith the application of the Enterobacteriaceaebiochemical card (EBC) to the identification ofmembers of the family isolated from any clinicalspecimen. This report is a summary of theselatter investigations.

MATERIALS AND METHODS

Bacteria used. For the collaborative challenge tothe AMS, 170 members of the family Enterobacteri-aceae, in numbers indicated in Table 1, were selectedby the collaborating laboratory of the Center for Dis-ease Control (CDC), coded, and shipped to each of theparticipants.At the same time, during and after this period of

the collaborative investigation, any member of thefamily Enterobacteriaceae isolated in the laboratoriesof the participants was also examined.

Materials used. All microbiological reagents used,other than the material required for the automatedsystem, were supplied to all participants by a singlecommercial media manufacturer. These included theprimary isolation agars, the various substrates for thestandard manual method of determining the biochem-ical reactions of all bacteria, and those reagents re-quired for detecting final reactions. All the materialswere prepared for all laboratories at the same timeand represenited the same lot of media and chemicals.Two of the laboratories (H.D.I. and T.L.G.) exam-

ined the bacteria used in the collaborative study withthe API system (Analytab Products, Inc., Plainview,N.Y.), referred to subsequently as the routine method.Since this product is a frequently used systems ap-proach to the identification of Enterobacteriaceae, noattempt was made to standardize the lot.

Inoculum preparation. The challenge bacteriaused in the collaborative study were subcultured onthe blood agar and MacConkey agar provided for thestudy. After 18 to 24 h of incubation, several colonieswere selected from blood agar and suspended in 1.8 mlof 0.5% NaCl until McFarland no. 1 standard densitywas obtained.

Clinical isolates, grown on each laboratory's partic-ular blood agar, MacConkey agar, or eosin methyleneblue agar, were used. The inoculum was prepared asdescribed. Before transfer into the single-barreled card

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TABLE 1. Collaborative comparison ofAMS with standard and routine manual methodsNo. of strains No. identified correctly"

Bacterium' submitted/ Total no. testedlaboratory AMS Standard Routine'

Arizona hinshawii (Salmo-nella arizonae)

Citrobacter amalonaticus(intermedius)

C. diversus (intermedius)C. freundiiEdwardsiella tardaEnterobacter aerogenesE. agglomerans (Erwinia

herbicola)E. cloacaeE. gergoviae (E. cloacae)Escherichia coliHafnia alveiKlebsiella ozaenaeK. pneumoniaeK. rhinoscleromatisMorganella morganii (Pro-

teus morganii)Proteus mirabilisP. vulgarisProvidencia alcalifaciens

(Proteus inconstans)P. rettgeri (Proteus rettgeri)P. stuartii (Proteus rettgeri)Salmonella sp.S. typhiSerratia liquefaciensS. marcescensS. rubidaea (Bacterium rub-

idaeum)Shigella dysenteriaeS. sonneiShigella sp. (S. boydii and S.

flexneri)Yersinia enterocoliticaNot members of Enterobac-

teriaceaedTotal

2 12 11 (91.7) 12 (100) 4 (100)

2 12

272106

101

1152

1125

1022

52

274252

2116

210

170

10 (83.3)

12 12 (100)42 38 (90.5)12 12 (100)60 60 (100)36 30 (83.3)

60 54 (90.0)6 6 (100)

66 66 (100)30 30 (100)12 12 (100)66 66 (100)12 12 (100)30 30 (100)

60 60 (100)12 12 (100)12 12 (100)

3012

16224123012

126636

1260

1,020

30 (100)11 (91.7)

162 (100)24 (100)12 (100)30 (100)10 (83.3)

12 (100)66 (100)36 (100)

12 (100)60 (100)

998 (97.8) 1,001 (98.1) 332 (97.6)" Identified by reference laboratory at CDC; the clinical microbiology laboratory at CDC participated in the

collaborative evaluation.Numbers in parentheses indicate percentage of the particular bacterium identified correctly.

' Applied consistently in the laboratories of two participants only.d Consisted of: Pseudomonas aeruginosa, two; P. maltophilia, one; P. cepacia, one; P. stutzeri, one;

Acinetobacter calcoaceticus var. anitratus, one; A. calcoaceticus var. lwoffi, one; Flavobacterium meningosep-ticum, one; group CDC 11 K 1, one; group CDC VE 1, one.

sample injector assembly, the suspension of bacteriawas agitated on a Vortex mixer for 15 to 30 s. TheEBC was affixed to the single-barreled card sampleinjector after it had been properly marked for com-

puter recognition, and the contents of the injectorwere introduced into the EBC by the vacuum assem-

bly furnished with the instrument. The operator in-spected the card to verify that it was properly filled.The card was then placed into the reader/incubatorfor analysis. Members of Enterobacteriaceae are iden-tified within 8 h, with all reactions recorded on a

printout. Identification of each organism is accompa-

nied by a probability number for the accuracy of thisidentification. The computer also provides the nextclosest possible identification. The differences in prob-abilities usually are extremely large.EBC. The EBC (Fig. 1) is a 30-compartment card

containing 26 dried biochemical broths and a growthcontrol broth. Three empty wells are for addition ofother substrates in the future. The card contains 14carbohydrate substrates, 2 decarboxylases, 1 deami-nase, and 1 dihydrolase, in addition to materials suchas urea, citrate, malonate, and a system for detectingH2S. The carbohydrate base, with the exception of the

3 (25)

12 (100)42 (100)12 (100)60 (100)34 (94.4)

59 (98.3)4 (66.6)

66 (100)30 (100)12 (100)66 (100)11 (91.7)30 (100)

60 (100)12 (100)12 (100)

30 (100)10 (83.3)

162 (100)24 (100)12 (100)30 (100)12 (100)

12 (100)65 (98.5)35 (97.2)

12 (100)60 (100)

0 (0)

4 (100)14 (100)4 (100)20 (100)12 (100)

20 (100)2 (100)

22 (100)10 (100)4 (100)

22 (100)4 (100)10 (100)

20 (100)4 (100)4 (100)

10 (100)3 (75)

53 (98.1)8 (100)4 (100)10 (100)3 (75.0)

4 (100)22 (100)11 (91.6)

4 (100)20 (100)

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FIG. 1. EBC card. (A) Full-length guide ledge forproper orientation of EBC in incubator/reader; (B)bubble traps; (C) number entry blocks for identifyingspecimen, using up to seven numerals; (D) guidenotches for internal instrument manipulation; (E)distribution channels connecting each compartmentto inoculum injection port; (F) injection port for in-oculum introduction. The substrates are distributedas designated: (1) DP300; (2) blank; (3) growth controlmedium; (4) blank; (5) p-coumaric acid; (6) indoxyl-,B-D-glucoside; (7) urea; (8) citrate; (9) malonate; (10)tryptophan; (11) raffinose; (12) rhamnose; (13) mal-tose; (14) sorbitol; (15) melibiose; (16) mannitol; (17)xylose; (18) sucrose; (19) inositol; (20) adonitol; (21)blank; (22) substrate for H2S detection; (23) ONPG;(24) lactose; (25) arabinose; (26) glucose; (27) argi-nine; (28) lysine; (29) base control for decarboxylasesand dihydrolase; (30) ornithine.

compartment containing o-nitrophenyl-D-galactopy-ranoside (ONPG), consists of (per liter): 2 g of proteosepeptone no. 3 (Difco Laboratories, Detroit, Mich.); 0.5g of K2HPO1; 0.5 g of sodium deoxycholate; and 0.2 gof reduced aniline blue as indicator. Adonitol, arabi-nose, glucose, maltose, mannitol, sucrose, and xyloseare present in concentrations of 10 g/liter; inositol,lactose, melibiose, raffinose, rhamnose, and sorbitolare present at 5 g/liter.

The ONPG base consists of (per liter): proteosepeptone no. 3, 0.2 g; beef extract (Difco), 2 g; ONPG,0.1 g; i-propyl-D-thiogalactopyranoside, 0.1 g; and re-duced aniline blue as indicator, 0.2 g.The base for the decarboxylases and arginine dihy-

drolase consists of the following (per liter): Thiotone(BBL Microbiology Systems, Cockeysville, Md.), 2 g;glucose, 1 g; pyridoxyl 5-phosphate, 0.005 g; and bro-mothymol blue as indicator, 0.1 g. The amino acids areadded at a level of 1%. The tryptophan deaminase isdissolved in a base consisting of (per liter): NaCl, 2 g;Trypticase (BBL), 1 g; tryptophan, 5 g; and ferriccitrate as indicator, 10 g.

Citrate medium has the following composition (perliter): Thiotone (BBL), 0.5 g; sodium citrate, 2 g;MgSO4.7H20, 0.2 g; (NH4)2HPO4, 1 g; bromothymolblue as indicator, 0.2 g. Malonate is prepared as follows(per liter): Thiotone, 0.5 g; (NH4)2SO4, 0.5 g; malonicacid, 4 g; glucose, 0.2 g; bromothymol blue as indicator,0.2 g. Urea medium contains (per liter): urea, 20 g;K2HPO4, 2 g; proteose peptone no. 3 (Difco), 1 g;sodium thioglycolate, 1 g; bromothymol blue as indi-cator, 0.2 g. To assess H2S generation, the followingsubstrate is provided, containing (per liter): Thiotone(BBL), 2 g; beef extract (Difco), 2 g; glucose, 0.5 g;sodium thiosulfate, 0.5 g; L-cystine, 0.1 g; ferric citrateas indicator, 0.3 g.

Three novel substrates useful in the division ofEnterobacteriaceae were incorporated into EBC. In-doxyl-D-glucoside (plant indican) in a concentration of1 g/liter is suspended in proteose peptone no. 3 (Difco)(1 g/liter) and K2HPO4 (1 g/liter). p-Coumaric acid issuspended in a medium containing (per liter): cou-maric acid, 1 g; K2HPO4, 0.5 g; Trypticase (BBL), 2 g;glucose, 10 g. Reduced aniline blue, 0.02 g/liter, is theindicator added. Irgasan (DP300), a broad-spectrumbacteriostat (CIBA-GEIGY, Nutley, N.J.), is sus-pended in medium containing (per liter): Thiotone(BBL), 3 g; sodium deoxycholate, 4 g; glucose, 10 g;DP300, 10 ml of 1% stock. Reduced aniline blue, 0.2 g/liter, serves as indicator.Standard biochemical test method. All cultures

submitted simultaneously to the collaborating labo-ratories for analysis were tested by the standard pro-cedure advocated by CDC as outlined in Edwards andEwing (3). As mentioned previously, all media wereprepared in one commercial laboratory from the samelot and shipped to all participants at the same time.The following tests and media were included in thisanalysis: tests for oxidase, triple sugar iron agar, mo-tility medium, methyl red-Voges-Proskauer broth, in-dole broth, citrate medium, Christensen urea, decar-boxylase media for lysine and ornithine, arginine di-hydrolase, phenylalanine deaminase, malonate, gela-tin, deoxyribonucleic acid hydrolysis, and the pre-scribed media for carbohydrate fermentation contain-ing arabinose, adonitol, inositol, sorbitol, raffinose, andrhamnose. This approach measured 23 characteristicsof the microorganisms.

Quality control. The reaction of the EBC wascontrolled with a series of six bacteria. These orga-nisms were: Proteus mirabilis (ATCC 7002), whichreacts positively with glucose, ornithine, xylose, H2S,urea, citrate, tryptophan (tryptophan deaminase), andp-coumaric acid; Citrobacter freundii (ATCC 6750),

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which is positive for glucose, ONPG, lactose, arabi-nose, mannitol, xylose, rhamnose, maltose, melibiose,H2S, citrate, DP300, and coumaric acid; Shigella flex-neri (ATCC 12661), which is positive for glucose andmannitol; Pseudomonas aeruginosa (ATCC 27315),which is positive for arginine, citrate, malonate, andp-coumaric acid; Klebsiella pneumoniae (ATCC13883), which is positive for glucose, lysine, ONPG,lactose, arabinose, mannitol, xylose, sucrose, inositol,adonitol, raffinose, rhamnose, maltose, sorbitol, meli-biose, plant indican, citrate, and malonate; and Enter-obacter cloacae (ATCC 23355), which is positive forglucose, arginine, ornithine, ONPG, arabinose, man-nitol, xylose, sucrose, raffinose, rhamnose, maltose,sorbitol, melibiose, plant indican, citrate, and malo-nate.

RESULTSTable 1 summarizes the collaborative compar-

ison of the AMS with standard and routinemanual methods, using a total of 170 represent-atives of Enterobacteriaceae. Since there weresix participants, the total numbers of each straintested (column 3, Table 1) are the number ofstrains that should have been identified cor-rectly by all participants. The results indicatethat 22 of 29 species analyzed by the AMS wereidentified correctly by all of the automatedequipment in the six laboratories. The manualstandard method identified all of 21 of theseEnterobacteriaceae species, whereas the routinelaboratory method (API) identified 24 of thespecies without error. Of course, it must be em-phasized that only two of the participating lab-oratories used the so-called routine method con-sistently; therefore, the opportunities for correctand erroneous identification were reduced three-fold. Correct identification of the species sub-mitted as unknown samples to each laboratorydepended on the number of opportunities eachlaboratory had to identify representatives of aspecific species. These opportunities varied from1 to as many as 27 in the case of the varioussalmonellae included. Percentages listed in thetable reflect the total number of opportunitiesto identify the species submitted as unknowns.Thus, if two representatives of a species weresubmitted, there were 12 opportunities for thevarious methods to identify that particular or-ganism. The total number of strains tested (col-umn 3, Table 1) is the basis for determining thepercentage identified correctly. The table doesnot reflect the performance of individual labo-ratories. The overall correct identification per-fornance by the AMS in the six laboratories wasas follows: A, 94.3%; B, 99%; C, 96.8%; D, 98%; E,97.5%; and F, 96.2%. Their performance with theotheir mn-thods was of the same order of magni-tude.

Discrepancies in AMS identifications of thecollaborative study organisms are listed in Table

2. This table permits review of the reproducibil-ity of the AMS in various laboratories in termsof correct as well as incorrect identification. Itfurther permits an evaluation of the reasons forsome of the discrepancies observed. The AMSin each of the laboratories participating in thiscollaborative evaluation of unknown represent-atives of Enterobacteriaceae was equipped witha tape-recording device which permitted a cen-tralized computer to judge the results obtainedin 6, 8, and 10 h. This device was helpful inestablishing analyses of discrepant reactions.Thus, the one Arizona hinshawii was identifiedby one AMS as Hafnia alvei in 8 h. The reactionresponsible for this mistake was negative citrateutilization. A 10-h reading of this culture by thesame AMS identified the organism correctly.The additional 2 h of incubation allowed theArizona to express citrate utilization. The fail-ure to react properly in 8 h may reflect one oftwo possibilities: (i) the reaction may have beenslow in this particular EBC card, because sub-strate availability or enzyme expression washindered, or (ii) since all otherAMS instrumentsidentified the organism correctly, the inoculummay have been prepared incorrectly.

All reactions necessary to identify this orga-nism as A. hinshawii were positive except forthe citrate reaction. This result favored identi-fication as H. alvei, with a probability of 49% ascompared with 32% for A. hinshawii. When suchclose correspondence between identificationprobabilities is observed at low levels, one ques-tions the adequacy of the reactions for identify-ing either organism. With a positive citrate re-action, the probability of identification for A.hinshawii becomes 96%, whereas that for H.alvei approaches 0%.

Citrobacter amalonaticus was misidentifiedby two machines. Different strains of the orga-nism were involved. In one instance the orga-nism was identified as a C. freundii in 8 h andcorrectly in 10. Indoxyl-D-glucosidase was notexpressed in the shorter incubation period, thusleading to the incorrect identification as C.freundii, with a probability of 62% as comparedwith 36% for C. amalonaticus. The indoxyl-D-glucosidase is positive in 90% of C. amalonaticusand negative in 99% of the C. freundii. Oncemore, a slow reaction or an inadequately denseinoculum may explain the incorrect reaction inthe 8-h period. The second AMS identified adifferent culture of C. amalonaticus as C. di-versus. The identification was based on malo-nate utilization, which is 99% negative for C.amalonaticus. The reaction profile elicited iden-tified this bacterium as a C. diversus with aprobability of 94%. Comparing these reactionswith the other five AMS results indicates that

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TABLE 2. AMS identification discrepancies in collaborative study bacteria v

StandardBacterium AMS identification No. AMS Reaction discrepancy' 10-h reading and/or Explanation

routine

Arizona hinshawii Hafnia alvei 1/6 CIT- A. hinshawii Correct Slow reactionCitrobacter ama- C. freundii 1/6 PLI- C. amalonaticus Incorrect Slow reaction

lonaticusC. amalonaticus C. diversus 1/6 MAL+ C. diversus Incorrect See textC. freundii Escherichia coli 1/6 CIT- C. freundii Correct Slow reactionC. freundii Enterobacter ag- 1/6 H2S- C. freundii Correct Slow reaction

glomeransC. freundii Yersinia enter- 2/6 PLI+, CIT- C. freundii Correct See text

ocoliticaEnterobacter ag- Y. enterocolitica 2/6 ONP-, XYL-, Y. enterocolitica Incorrect See textglomerans DP3-

ONP- E. agglomerans Correct Slow reactionE. agglomerans Y. enterocolitica 3/6 CIT- E. agglomerans Correct Slow reactionE. cloacae Y. enterocolitica 6/6 ARG-, LAC-, Y. enterocolitica Correct See text

RAF-, MEL-,CIT-

Providencia stuar- E. agglomerans 1/6 LAC+, ARA+, E. agglomerans Incorrect Clerical errortii MAN+, RHA+,

SOR+Serratia rubidaea Y. enterocolitica 2/6 LYS-, RHA-, CIT- S. rubidaea Correct Slow reaction

'Abbreviations: CIT, citrate; PLI, plant indican; MAL, malonate; ONP, ONPG; XYL, xylose; DP3, irgasan; ARG, argininedihydrolase; LAC, lactose; RAF, raffinose; MEL, melibiose; ARA, arabinose; MAN, mannitol; RHA, rhamnose; SOR, sorbitol.

all reactions were identical except for the malo-nate utilization. The only adequate explanationfor this incorrect singular reaction is an improperthreshold adjustment arising from the paucityof C. amalonaticus for inclusion in the database.There were four misidentifications of C. freun-

dii involving four AMS results and three differ-ent strains of the bacterium. In one machine C.freundii was called Escherichia coli in 8 h butcorrectly as C. freundii in 10 h (Table 2), againsuggesting a slow reaction. The substrates in-volved in this misidentification were negative 8-h utilizations of citrate and DP300. Citrateshould be positive in more than 90% of reactionswith C. freundii. The chance of encountering a

citrate-negative representative is approximately10%. Therefore, the reaction battery led to thedesignation ofE. coli, characteristically negativefor citrate at a 99% level. Citrate was utilizedafter 10 h of incubation, suggesting once againan inoculum or enzyme effect. In another in-stance, the identification in the 8-h period of C.freundii as Enterobacter agglomerans wasbased on a series of reactions requiring a positiveH2S for correct identification of this particularC. freundii. Although in the previous case C.freundii belonged to the 21% which do not gen-erate H2S, the failure to do so with this partic-ular representative indicated the probability ofa C. freundii at a level of only 29% and an E.agglomerans with a probability of 70%. The 10-h reading elicited the proper reaction, suggestingonce again inoculum or slow enzyme effects.

In two of the six AMS, one strain of C. freundii

was identified as Yersinia enterocolitica on thebasis oftwo reaction discrepancies: false-positiveplant indican and false-negative citrate. In 10 hthe correct citrate was obtained. Indoxyl-D-glu-coside is negative for C. freundii with a fre-quency of 99%, indicating the need for thresholdadjustment.Two of the six representatives of E. agglom-

erans showed discrepancies in the AMS analy-sis. The first of these was identified in one of thelaboratories as Y. enterocolitica even at the 10-h incubation period. The standard and routinemethods also identified the orlg'nism incor-rectly. Three incorrect reactions led to the mis-identification. Therefore, the probability of iden-tifying this organism as an E. agglomerans be-came very small, whereas Y. entetocolitica wascorrect at the level of 97%. FailurS- of the stan-dard and routine methods to identify this partic-ular bacterium correctly strongly suggests a cler-ical error in the numbering of this particularculture. In another laboratory, the organism wasidentified as a Y. enterocolitica on the basis ofa negative ONPG in 8 h; it was identified as a Y.enterocolitica at 51% and as an E. agglomeransat 31% probability levels. The belatedly positiveONPG led to the correct identification with aprobability of 90%.The second strain of E. agglomerans was

identified in three laboratories as Y. enterocolit-ica on the basis of a negative citrate. In 10 h,this reaction was corrected in the three AMS,and the organism was identified properly. Again,either an inoculum, card, or threshold effectcould explain the delayed diagnoses. Of course,

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we must also consider that the small differencesin the rate of citrate transport and utilization ascarbon source of a particular strain may play asignificant role in the delays noted.One strain of Enterobacter cloacae was misi-

dentified by all AMS instruments as a Y. enter-ocolitica through differences in five reactions.These were: negative arginine dihydrolase, fail-ure to ferment lactose, raffinose, and melibiose,and inability to utilize citrate. Usually, E. cloa-cae in the AMS produces arginine dihydrolaseat a frequency of 99%, ferments lactose at 76%,raffinose at 90%, and melibiose at 95%, and uti-lizes citrate at a 98% level. All of these reactions,with the possible exception of lactose fermenta-tion, are significant for identifying E. cloacae.Arginine is probably the pivotal reaction, sincethe other reactions are shared by several speciesand genera. Thus, these reactions might have fitan exceptional S. liquefaciens. The negative re-actions in the five substrates listed argue verystrongly for a Y. enterocolitica despite the pos-itive urease production usually encountered.This discrepancy in all of the instruments couldhave been due to an improperly labeled culture,but both the standard and routine methods iden-tified the bacterium properly. In an attempt tounderstand these discrepancies, one participanttested the same culture at a different time witha different lot of EBC. Correct reactions wereelicited, suggesting that the particular lot ofEBC used in the collaborative study was inade-quate for identifying this particular E. cloacae,requiring substrate or threshold adjustments.The error in identifying Providencia stuartii

by one AMS suggests a clerical error. Therewere five reactions incompatible with the iden-tification of P. stuartii, unchanged in 10 h andconfirmed by routine and standard methodswhich also identified the organism as an E.agglomerans.One strain of Serratia rubidaea was identified

as a Y. enterocolitica by two of the AMS in the8-h reading. The misidentification was based onnegative reactions for lysine, raffinose, and cit-rate. The 10-h reading in both laboratories re-versed these negative reactions and identifiedthe culture properly. The correct identificationwas also made by the manual methods applied.Once more, a slow reaction possibly resultingfrom an inadequate inoculum density may havebeen involved.While the collaborative analysis of the un-

known cultures was in progress, 5,450 represent-atives of the family Enterobacteriaceae wereisolated in the participating laboratories fromclinical specimens. These were analyzed by theroutine methods used in each of the laboratoriesand with the EBC. For the purposes of this

particular evaluation, we make the tacit assump-tion that the manual identification of these or-ganisms was correct. Identifications by the AMSof these cultures were in agreement with themanual method in 5,255 instances (96.4%). Atthe same time, 932 such clinical isolates weretested in one laboratory by the standard methodused in the collaborative study. Of these, 894(96%) were correctly identified by the AMS.Among the Enterobacteriaceae encountered inthe clinical specimens were organisms such asKlebsiella oxytoca and Providencia stuartii,urease positive. The computer memory of theAMS also has the capability of identifying Y.ruckeri, Y. pestis, and Y. pseudotuberculosis.However, identification of the first two of theseyersiniae must await fine tuning of the instru-ments to accommodate the temperature limita-tions of Y. ruckeri and the incubation time re-quirement of Y. pestis. Because of the differentmethods used for identification, the unequal dis-tribution of organisms tested in the various lab-oratories, and the distribution of representativesencountered in the clinical setting, presentationof a detailed analysis of these clinical sampleswould serve no purpose.

DISCUSSIONThe capability of the AMS to identify Enter-

obacteriaceae at the same level as the classicaland at least one of the manual systems ap-proaches has been demonstrated in this study.This analysis pertains to clinical specimens aswell as to the bacteria especially selected for thecollaborative evaluation. The bacteria chosenchallenge the limits of identification in all sys-tems. Reproducibility from instrument to instru-ment was excellent.

It is, however, the reaction file which is thecritical part of the EBC processing by the AMScomputer. The EBC reactions only characterizethe unknown specimen; they cannot identify thebacterium. Experimental testing of many repre-sentatives of Enterobacteriaceae in the AMSwas required to extract a statistical expressionof the percentages (of each species) of positiveand negative reactions for each substrate in theEBC for each identification. This is especiallyimportant because the environment of the AMSdiffers from the conditions that govern the man-ually prepared standard and the commerciallymanufactured systems. The passage of atmos-pheric gases, the small volume within each re-action chamber or well, the shorter incubationtime, and the inoculum density contribute tothese differences and require a special AMSidentification file. Each species has the sameinitial probability. No biochemical substrate is

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given more weight regardless of its importanceto delineate a species. No species receives addi-tional advantages because of the frequency withwhich it occurs in clinical samples. The fre-quency with which each species acts on eachsubstrate influences the conditional probabilityconsideration of the computer. Before compar-ing the reactions obtained for a specific organismand attempting identification, the AMS per-forms several preliminary checks. Thus, the glu-cose reaction is screened to ascertain that theorganism belongs to Enterobacteriaceae. If theglucose reaction is not positive, the control wellis checked to establish that adequate prolifera-tion has taken place. When the growth controlwell is positive but glucose is negative, the ureaseand ornithine decarboxylase compartments arechecked. If these are positive, the computer as-sumes the glucose to be positive but reactingslowly. If glucose, urease, and ornithine are neg-ative while the growth control well is positive,the computer will report that this particularorganism is not a member of Enterobacteria-ceae. The probability computation performedby the computer follows the usual Baysian cal-culation.The EBC card contains three novel substrates

which aid in evaluating and determining thespecies that comprise the family Enterobacteri-aceae. Two of these substrates, which act byinterfering with glucose fermentation, are DP300and p-coumaric acid. The organisms capable offermenting glucose despite the presence ofDP300 are Yersinia sp., Citrobacter sp., Serra-tia sp., Morganella (Proteus) morganii, Provi-dencia (Proteus) rettgeri, and Providenciastuartii, including the urea-positive variant. Theorganisms that can ferment glucose in the pres-ence of p-coumaric acid are Yersinia sp., Citro-bacter sp., Serratia sp., Arizona hinshawii, Sal-monella sp., Escherichia coli, and most speciesof Shigella. The indoxyl-f-D-glucoside is brokendown by certain bacteria into indigo blue by aspecific f8-glucosidase. Organisms which can pro-duce this enzyme are the various species of thegenus Serratia; the various Enterobacter speciesincluding E. gergoviae; the various Klebsiellaspecies including K. oxytoca; Citrobacter diver-sus; and some Providencia rettgeri. The level ofpositive reaction for the last group is better than90% for the organisms listed, whereas more than80% of the genera listed for the DP300 and p-coumaric acid reactions are positive in ferment-ing glucose in the presence of these compounds.In identifying the various Enterobacteriaceaespecies, these positive reactions are helpful ad-juncts for their automated recognition, espe-cially in the absence of certain classical reactions

often performed, such as the test for indole. TheEBC does contain a well with ordinary nutrientbroth which can serve as a ready reservoir forthis test and as a source of antigen for immu-nological corroboration.The discrepancies observed provide the op-

portunity to discuss the advantages inherent inthis particular automated approach and proposemeans to improve the AMS further. In additionto its labor-saving aspects, the AMS reduces thechances for human error. One of the contributingfactors is, of course, the computer, which canhandle the numerous reactions and search itsmemory rapidly and accurately (7). Also, thecomputer's software can be updated continu-ously to accommodate changes in reaction se-quences for newly established species or signifi-cant biotypes. It could also be instructed tocritically scrutinize results which indicate a closeprobability of identification between organisms.Under those circumstances, the computer couldinstruct the instrument to prolong incubationfor 1 or 2 h or to resolve difficulties which mightarise from inadequate or improper inoculumpreparation or result from an inherent, slowlyexpressed enzyme sequence manifested in a par-ticular organism. The computer also possessesthe a bility to judge the purity of the culture.analyzed and could be instructed to evaluate aseries of reactions which imply an impure cul-ture. Also, it is possible to update computerinstructions to request additional tests not onlywhen immunological confirmation of identifica-tion is desirable, but also in those rare instanceswhere additional biochemical tests should beperformed to confirm the identification. To thisend, the EBC cGntains a growth control com-partment which can be entered, and the contentscan be used for immunological and additionalbiochemical tests.The computer memory has the capability of

dividing the genera Salmonella and the genusShigella in various "species" or serotypes. Thisgrouping of the salmonellae would seem unnec-essary in identifying a clinical isolate, since reli-ance on immunological confirmation at least intogroups is so well ingrained into clinical labora-tory personnel that reliance solely on biochemi-cal reactions might be deemed unacceptable.Only Salmonella typhi has therefore been se-questered from the remaining salmonellae. Thefrequency with which different shigellae are en-countered in the clinical laboratory is recognizedin the instructions to the AMS computer. Thus,Shigella dysenteriae and S. sonnei can be iden-tified by their biochemical reactions, but thecomputer continues to advise immunologicalconfirmation. S. boydii and S. flexneri are

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grouped together, for the same reasons as thesalmonellae. In all instances, confirmation byimmunological analysis is recommended on thecomputer printout.Some of the discrepancies observed which

were ascribed to inoculum density can also becorrected by fine tuning the threshold setting inthe computer for a particular well in the EBC.Each ofthese wells has a distinct point or thresh-old which discriminates between positive andnegative reactions. This particular decision de-pends on the amount of light transmitted andrecognized by the several detecting elementswhich scan the well at hourly intervals, as wellas the ability of the detectors to recognize sig-nificant color changes. There are a number ofreactions which may require a high thresholdsetting to accommodate equivocal areas. How-ever, the computer linked to the detectors couldbe i istructed to direct the perusal of other di-agnos-dc reaction compartments, to evaluate theequivocal threshold reading by interpolating theslope of the growth curve to a longer incubationtime, and to decide on the identity of the partic-ular isolate. Additionally, the contents in eachEBC well can be fine tuned. Experience withmany biotypes of the species may require ad-justment in certain substrate concentrations orbasal media to ease the recognition process. Forexample, slow-reacing citrate utilization and ar-ginine dihydrolase production can be adjustedin this fashion.The 6-, 8-, and 10-h computer readings taken

during the evaluation of the collaboratively an-alyzed cultures were intended to confirm theoptimal incubation time commensurate with ac-curacy and the environment provided by theEBC. The shorter time period, compared withthe standard and most commercially availablesystems, is an added advantage to the decisionprowess of the AMS. Although it is possible todetermine the presence of some bacterial en-

zymes very rapidly, the detection of others re-

quires proliferation and synthesis, at times in-volving sequences of enzymes bearing on thefinal reaction. The excellent performance of theinstrument indicates the suitability of the 8-hincubation. Adjustments for the majority of theinoculum-dependent discrepancies can be incor-porated into the computer memory.

Also, it may be possible to use the growthcontrol well as control for adequate inoculumdensity. This specific compartment can bescreened to determine the level of growth beforeidentification is made. If a preset threshold isnot reached in 8 h, as might be expected withslower-growing organisms or inadequately denseinocula, the AMS computer can be instructed to

prolong the incubation period before reaching adecision. Since 7 of the 11 discrepancies observedwere the result of potential inoculum inadequa-cies, slowly elaborated enzyme sequences, orslowly synthesized enzyme combinations de-tected phenotypically, the advantages of thiscontrol procedure are evident. Although the re-producibility from instrument to instrument wasexcellent, fine tuning of individual instrumentsmay be another means of overcoming any dis-crepant reactions.The family Enterobacteriaceae has received

much attention by investigators attempting tobring some order to microbiological taxonomy.The initial, outstanding contributions of Ed-wards and Ewing (3) have been expanded bynumerical taxonomy and deoxyribonucleic acidanalysis (2) so that interspecies differences arenow determined by phenotypic expressions ofspecific genomes. However, since numerical tax-onomy and deoxyribonucleic acid analysis arebeyond the capabilities of clinical microbiologylaboratories, biochemical tests which reflect thespecies genome must be chosen and used forbacterial species identification. In the future, itmay well be shown that a number of the reac-tions incorporated into the EBC and, for thatmatter, into the manual systems approaches orthe standard method are superfluous. Adjust-ments certainly can be made in all of thesesystems to refine and possibly reduce the testingrequired to arrive at meaningful microbiologicaldiagnoses.The EBC is an important addition to the

utility of the AMS in the clinical microbiologylaboratory, expanding the application of auto-mation to another important time- and labor-consuming activity. The recent preliminary re-port on AMS general susceptibility and minimalinhibitory concentration capabilities (6) indi-cates even further progress in the application ofautomation and computer technologies by mi-crobiology laboratories charged with the resp,Ui.sibility of providing accurate and rapid servicefor the benefit of patients.

ACKNOWLEDGMENTSWe gratefully acknowledge the important contributions of

the many individuals working in our laboratories. Withouttheir efforts and devotion, this study could not have beenconducted.

LITERATURE CITED1. Aldridge, C., P. W. Jones, S. Gibson, J. Lanham, M.

Meyer, R. Vannest, and R. Charles. 1977. Automatedmicrobiological detection/identification system. J. Clin.Microbiol. 6:406-413.

2. Brenner, D. J. 1978. Characterization and clinical iden-tification of Enterobacteriaceae by DNA hybridization.Prog. Clin. Pathol. 7:71-117.

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3. Edwards, P. R., and W. H. Ewing. 1972. Identificationof Enterobacteriaceae, 3rd ed. Burgess Publishing Co.,Minneapolis.

4. Isenberg, H. D. 1976. Biochemical "rapid identification"of Enterobacteriaceae, p. 41-49. In J. E. Prier, J. Bar-tola, and H. Friedman (ed.), Modern methods in medi-cal microbiology. University Park Press, Baltimore.

5. Isenberg, H. D., T. L. Gavan, A. Sonnenwirth, W. I.Taylor, and J. A. Washington II. 1979. Clinical lab-oratory evaluation of automated microbial detection/identification system in analysis of clinical urine speci-mens. J. Clin. Microbiol. 10:226-230.

6. Isenberg, H. D., and J. Sampson-Scherer. 1980. Clin-ical laboratory feasibility study of antibiotic suscepti-bility determined by the Auto Microbic System, p. 526-

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528. In J. D. Nelson and C. Grassi (ed.), Current chem-otherapy and infectious disease. American Society forMicrobiology, Washington, D.C.

7. MacLowry, J. D., E. A. Robertson, and R. J. Elin.1978. The place of the computer in diagnostic medicalbacteriology. Proc. Clin. Pathol. 7:49-70.

8. Smith, P. B., T. L. Gavan, H. D. Isenberg, A. Sonnen-wirth, W. I. Taylor, J. A. Washington II, and A.Balows. 1978. Multi-laboratory evaluation of an auto-mated microbial detection/identification system. J.Clin. Microbiol. 8:657-666.

9. Sonnenwirth, A. C. 1977. Preprototype of an automatedmicrobial detection and identification system: a devel-opmental investigation. J. Clin. Microbiol. 6:400-405.

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