Enterobacteriaceae Identification BERT F

Home , Enterobacter, Enterobacteriaceae, Proteus vulgaris, Shigella

Evaluation of the AutoSCAN-3 and Sceptor Systems for Enterobacteriaceae Identification BERT F. WOOLFREY,* RICHARD T. LALLY, AND CHARLES 0. QUALL Clinical Microbiology Section, Department ofAnatomic and Clinical Pathology, St. Paul-Ramsey Medical Center, St. Paul, Minnesota 55101 Received 4 October 1982/Accepted 21 January 1983

To evaluate the accuracy and cost effectiveness of the AutoSCAN-3 (Micro- Scan Systems of America, Sacramento, Calif.) and Sceptor (BBL Microbiology Systems, Cockeysville, Md.) systems for identification of members of the Enterobacteriaceae, we performed parallel tests on 678 stock cultures of well- characterized clinical isolates of Enterobacteriaceae. Automated results by AutoSCAN-3 correctly identified 95.1% at the genus level and 94.9% at the species level. However, 15 of 42 Shigella isolates were misidentified as members of other genera. In contrast to the automated results, visual interpretation of panels produced 97.9% agreement at the genus level, missing only three Shigella isolates. Sceptor correctly identified 96.8% at the genus level and 93.4% at the species level. Of 42 Shigella isolates, 3 were missed and were designated as Salmonella spp. Although all Salmonella spp. were correctly identified, six other isolates were misidentified as Salmonella spp. Test costs were found to be comparable for each system, with the cost per test increasing markedly with fewer than 10 to 15 tests performed per day.

AutoSCAN-3 (MicroScan Systems of Ameri- were then also tested by the API 20E system as a ca, Sacramento, Calif.) and Sceptor (BBL Mi- check on the original characterization of the stock crobiology Systems, Cockeysville, Md.) are isolates. Tables were designed to evaluate and compare the results. During the course of the study, semiautomated systems that provide microbial technical time requirements for performing Auto- identification and minimum inhibitory concen- SCAN and Sceptor tests were determined. These were tration determination. AutoSCAN-3 (Auto- used in conjunction with material costs for an analysis SCAN) uses AutoSCAN instrumentation to read of cost effectiveness. and interpret MicroScan panels which are sup- Microorganisms. A total of 678 clinical isolates of plied frozen. Sceptor provides instrumentation Enterobacteriaceae representing 11 genera (141 Esch- which automatically hydrates and inoculates de- erichia spp., 134 Klebsiella spp., 86 Enterobacter hydrated Sceptor panels. MicroScan and Scep- spp., 89 Serratia spp., 41 Salmonella spp., 42 Shigella tor panels differ slightly in their choice of bio- spp., 27 Citrobacter spp., 101 Proteus spp., 8 Provi- dencia spp., 8 Morganella spp., and 1 Hafnia spp.) chemicals and antimicrobial agents. The present were selected for the study. All had been used for a investigation evaluates the accuracy and cost previous study (2) and had been well characterized by effectiveness of these two systems for identify- the API 20E system and classical biochemical tests. ing members of the Enterobacteriaceae. Isolates were retrieved from frozen stock cultures by sampling each with a hot wire loop and streaking to a MATERIALS AND METHODS sheep blood agar plate which was incubated in air at Experimental design. Microbial identifications were 35C for 24 h. From each blood agar plate, the center performed in parallel by AutoSCAN with MicroScan of a single well-isolated colony was used as a common Gram-Negative Combo Panels and by Sceptor with growth source to initiate the appropriate inoculum Gram-Negative MIC/id Panels for 678 stock cultures suspension required by each system. of clinical isolates which had been previously well AutoSCAN system. AutoSCAN provides instrumen- characterized. Isolates with any discrepancy among tation for automated reading and interpretation of the AutoSCAN, Sceptor, and the original stock cul- MicroScan panels. MicroScan panels are supplied ture identifications were retested by AutoSCAN and frozen and use the following 24 tests for identification Sceptor in parallel with the API 20E system (Analytab of members of the Enterobacteriaceae: fermentation Products, Plainview, N.Y.) as follows. Subculture of adonitol, arabinose, glucose, inositol, melibiose, purity plates, originating from the initial AutoSCAN raffinose, rhamnose, sorbitol, and sucrose; urease; and Sceptor identification tests, were interchanged H2S production; indole production; decarboxylation and processed for identification by the other system. of lysine and ornithine; arginine dihydrolase; trypto- Subcultures derived from the original stock isolate phan deaminase; esculin hydrolysis; Voges-Pros- 807 808 WOOLFREY, LALLY, AND QUALL J. CLIN. MICROBIOL. TABLE 1. Comparison of AutoSCAN identification with referee designations for 678 isolates of Enterobacteriaceae AutoSCAN identification and no. (%) Entero- Entero- Entero- Ece-Kieb- Kleb- Klebsiella Kleb- Entero- bacter bacter Referee identification (total) Escher- rhino- siella bactEbacter~~~~~~~~agglom- aero- gergov- coli Pniepneu-oxytoca scleromatis ozaenae cloacae erans genes iae Escherichia coli (141) 136 (96) 1 Klebsiella pneumoniae (91) 90 (99) Klebsiella oxytoca (42) 1 41 (98) Klebsiella rhinoscleromatis (1) 0 Enterobacter cloacae (53) 50 (94) Enterobacter agglomerans (3) 1 2 (67) Enterobacter aerogenes (29) 29 Enterobacter gergoviae (1) 1 Serratia marcescens (88) Serratia liquefaciens (1) Salmonella spp. (41) Shigella spp. (42) 6 (15) 3 (7) 1 (2) Citrobacterfreundii (18) Citrobacter diversus (9) Proteus mirabilis (91) Proteus vulgaris (10) Providencia stuartii (2) Providencia rettgeri (6) Morganella morganii (8) Hafnia alvei (1)

TABLE 1-Continued AutoSCAN identification and no. (%) Serra- Citro Citro- Entero- Serratia tia Serratia Salmo- Shigella bacter eSerratia Lctr Referee identification (total) sakazakii mensas odor- lique rubidea nella spp freundiifreudii diver- sakazakii cens ifer faciens sus Escherichia coli (141) 1 Klebsiella pneumoniae (91) 1 Klebsiella oxytoca (42) Klebsiella rhinoscleromatis (1) Enterobacter cloacae (53) 2 (4) Enterobacter agglomerans (3) Enterobacter aerogenes (29) Enterobacter gergoviae (1) Serratia marcescens (88) 88 Serratia liquefaciens (1) 1 Salmonella spp. (41) 40 (98) Shigella spp. (42) 5 (12) 27 (64) Citrobacterfreundii (18) 18 Citrobacter diversus (9) 9 Proteus mirabilis (91) Proteus vulgaris (10) Providencia stuartii (2) Providencia rettgeri (6) Morganella morganii (8) Hafnia alvei (1)

kauer; utilization of citrate, malonate, and tartrate; 0- each microwell by light transmission to detect bacteri- nitrophenyl-4-D-galactopyranoside; and growth in the al growth or color change. The electronic information presence of cephalothin (8 ,ug/ml) and colistin (4 so generated is automatically analyzed by a micro- ,uglml). After inoculation and incubation, panels are processor program which determines the most proba- inserted into the AutoSCAN reader, which examines ble microorganism identifications and likelihood per- VOL. 17, 1983 EVALUATION OF AUTOSCAN AND SCEPTOR 809 TABLE 1-Continued

AutoSCAN identification and no. (%)

RefereeRefreeidntiictioidentification (ota)(total) Proteus Proteus Provi- denciaProv'i- Morga-nella Hafnia Arizona Yersinia Rare mirabiliSmraili vlgaisdencial!ulgaris stuartii rett- inorganii avial'ei SPs spp.P geri Escherichia coli (141) 2 (1) Klebsiella pneumoniae (91) Klebsiella oxytoca (42) Klebsiella rhinoscleromatis (1) 1 Enterobacter cloacae (53) 1 Enterobacter agglomerans (3) Enterobacter aerogenes (29) Enterobacter gergoviae (1) Serratia marcescens (88) Serratia liquefaciens (1) Salmonella spp. (41) 1 Shigella spp. (42) Citrobacterfreundii (18) Citrobacter diversus (9) Proteus mirabilis (91) 90 (99) Proteus vulgaris (10) 10 Providencia stuartii (2) 2 Prov'idencia rettgeri (6) 6 Morganella morganii (8) 8 Hafnia alvei (1) 0 (0)

centages. These results, along with comments or interpreted visually without prior knowledge of the instructions such as requests for visual interpretation automated results. A seven-digit code number was of some or all of the microwell reactions, are displayed constructed, and the microorganism identification was on a cathode ray tube. After review and entry of any found by using the MicroScan Biotype Codebook. needed information relative to visual interpretation of Data so generated were used to compare the results of plates, the results are then printed as a single most automated versus visual interpretation of MicroScan probable microorganism identification. panels. Approximately 50 to 60 isolates, including Inoculum suspensions were prepared by transfer- quality control isolates as suggested by the manufac- ring microorganisms from a common growth source turer, were processed daily for identification. colony, as described above, to a tube containing 0.5 ml Sceptor system. The Sceptor system provides an of brain heart infusion broth. This was incubated for 4 instrument for simultaneous rehydration and inocula- h in air at 35°C, at which time 0.05 ml was transferred tion of desiccated panels and an instrument for visual with a calibrated pipettor (Medical Laboratory Auto- interpretation. For identification of members of the mation, Mount Vernon, N.Y.) to a tube containing 25 Enterobacteriaceae, 21 biochemical tests are used ml of inoculum water with 0.02% Tween 80 supplied which included: fermentation of adonitol, arabinose, by the manufacturer and then to a purity plate. After dextrose, inositol, mannitol, melibiose, rhamnose, multiple inversions, the contents of the tube were sorbitol, sucrose, and xylose; H2S production; indole poured into a disposable plastic tray whose cover production; tryptophan deaminase; esculin hydrolysis; served as a device to inoculate a previously thawed urease; O-nitrophenyl-,3-D-galactopyranoside; utiliza- panel. This procedure was said by the manufacturer to tion of citrate and malonate; arginine dihydrolase; and transfer 0.05 ml of inoculum to each microwell, pro- decarboxylation of lysine and ornithine. Each inocu- viding a final inoculum suspension of 105 CFU/ml. lum suspension was prepared in 5 ml of tryptic soy Arginine, glucose. H2S, lysine, ornithine, and urease broth from a single colony on the common blood agar wells were overlaid with mineral oil, and plates were plate growth source. After 3 to 5 h of incubation in air then incubated in stacks of five for 18 h in air at 35°C. at 35°C, each broth suspension was adjusted with After incubation, reagents were added to the indole, tryptic soy broth to the density of a 0.5 McFarland tryptophan deaminase, and Voges-Proskauer wells. standard. With a Sceptor disposable loop, 10 ,.1 of All panels were interpreted within 90 min after remov- each suspension was transferred to a tube containing al from incubation beginning 10 min after the addition 10 ml of Sceptor Gram-Negative MIC/id Broth. After of reagents and within 20 min after all reagents were preparation of a purity plate with the disposable loop added. Profile numbers resulting in a "very rare and immediately after multiple inversions, the con- biotype" response by the AutoSCAN reader were tents of the tube were transferred to a disposable called to an AutoSCAN computer telephone service. Preparation Station Reservoir for the simultaneous Immediately upon completion of the AutoSCAN iden- hydration and inoculation of a panel by the Sceptor tification for each panel, the reactions were also Automated Preparation Station. A sterile mineral oil 810 WOOLFREY, LALLY, AND QUALL J. CLIN. MICROBIOL. TABLE 2. Comparison of Sceptor identification with referee designations for 678 isolates of Enterobacteriaceae Sceptor identification and no. (%) Kleeb- Klebsiel- Kleb- Entero- Entero- Entero- Entero- ReedtitiEscher-c ieu- siellala rhino- siella Enter bacter bacter bacter bacter ichia coli pneu- orytocascleroma- ozae- cloacae agglo- aeroge- gergo- saka- moniae oytoca tis nae merans nes viae zakii Escherichia coli (141) 136 (96) Klebsiella pneumoniae (91) 86 (95) 1 1 2 (2) Klebsiella oxytoca (42) 1 35 (83) 1 Klebsiella rhinoscleromatis (1) 1 Enterobacter cloacae (53) 45 (85) 3 (6) Enterobacter agglomerans (3) 3 Enterobacter aerogenes (29) 2 (7) 27 (93) Enterobacter gergoviae (1) 1 Serratia marcescens (88) Serratia liquefaciens (1) Salmonella spp. (41) Shigella spp. (42) Citrobacterfreundii (18) Citrobacter diversus (9) Citrobacter amalonaticus Proteus mirabilis (91) Proteus vulgaris (10) Providencia stuartii (2) Providencia rettgeri (6) Morganella morganii (8) Hafnia alvei (1)

overlay was added to the arginine, H2S, lysine, orni- andria, Va.). A workload unit value of 6.0, as recom- thine, and urease wells before incubation at 35°C in air mended by the College of American Pathologists, was for 18 h. Immediately after incubation, Kovac reagent used for API 20E identification (3). Time related to and ferric chloride were added to the indole and production and use of MIC-2000 panels had been tryptophan wells, respectively, and panels were inter- previously determined by us to be 9.0 units. Labor preted within a time period similar to that described costs were determined by using the average hourly above for AutoSCAN. Reactions were visually inter- wage of $13.20 for technologists performing similar preted and recorded with the Sceptor Reader/Re- tests. Materiel cost for each system, including trans- corder. From the panel reactions, seven-digit profile portation cost to maintain a 3-month inventory, was numbers were constructed for which a current Sceptor determined on the basis of current charges for all ID Taxonomy Guide provided microbial identification. materials used from the point of preparation of initial Profile numbers not in the Taxonomy Guide were inoculum suspensions to the final printed results. called to the computer telephone service. Quality control costs for commercial systems were API 20E system. The API 20E system was inoculat- based upon daily testing of those organisms and re- ed and interpreted as described previously (2). Profile agents which the manufacturer indicated were neces- numbers were interpreted with the current API 20E sary to assure accuracy of the test system. AutoSCAN Analytical Profile Index and, when necessary, the API required five microorganisms to be tested daily, and 20E computer telephone service. Sceptor required six. The API 20E system required Cost comparison. After thorough familiarization and one API 20E reagent QC strip daily. Quality control experience with both AutoSCAN and Sceptor instru- for MIC-2000 plates conformed to that recommended ments and procedures, a 4-day period was used to by the National Committee for Clinical Laboratory record the time required for all aspects of AutoSCAN Standards (1). Instrumentation costs were based on and Sceptor identification tests, including quality con- current list prices and were allocated on a straight-line, trol, from preparation of the initial broth inoculum 5-year depreciation scheme which included mainte- suspensions to the final identification results. Purchas- nance. ing and inventory times were not included. Approxi- mately 50 to 60 isolates were processed each day RESULTS during the study. As a base of reference for cost comparison, a workload unit value was determined for Table 1 compares the identifications provided microorganism identification and minimum inhibitory by AutoSCAN with the referee identifications of concentration testing for the combined use of API 20E the 678 isolates. There was 95.1% agreement at and microdilution panels produced by us, using the the genus level and 94.9% at the species level. A MIC-2000 system (Dynatech Laboratories, Inc., Alex- majority of the 33 genus disagreements involved VOL. 17, 1983 EVALUATION OF AUTOSCAN AND SCEPTOR 811

TABLE 2-Continued Pro- Provi- Provi- Serratia Serra-ti Serratia Serra- SalmF Citro- Citro- Citro- Proteus Morga- Haf- marce- iu- tuia nella Shigella bacter bacter bacter mir- teus dencia dencia Morga- Hiaf rubi- spp. freun- diver- amal vul- stu- rett- nel scenssnarce-iodori-fera faciens dea spp. dii sus naticus Proabilis garis artEi geri morganii alveima 1 1 3 (2) 1 5 (12) 1 ~~~2(4) 2 (4)

73 (83) 10 (11) 5 (6) 1 41 3 (7) 39 (93) 18 9 90 (99) 10 2 6 8

5 Escherichia coli, 6 Enterobacter cloacae, and the visually generated identifications agreed 15 Shigella spp. AutoSCAN misidentified the 15 with the referee identification, including 12 of Shigella spp. isolates as 9 Klebsiella spp., 5 the Shigella spp. which had been misidentified Salmonella spp., and 1 Enterobacter sp., owing, by AutoSCAN. Total genus level agreement for in the majority of cases, to false-positive inter- Shigella spp. with the reference identification pretation of carbohydrate reactions. A total of was thus 97.9% for visual interpretation, com- 37 isolates were designated as very rare biotype, pared with 64% for AutoSCAN interpretation. requiring visual interpretation ofthe entire plate, Among the nine remaining discrepancies, only construction of a profile number, and use of the six of the AutoSCAN identifications agreed with current MicroScan Biotype Codebook for Aero- the reference designation, including three S. bic Gram-Negative Bacilli, which in turn cor- marcescens and one each of E. cloacae, Klebsi- rectly identified 27 isolates as Serratia marces- ella pneumoniae, and Shigella spp. In the re- cens and one as Proteus mirabilis. The maining three instances, both visual and auto- remaining nine profile numbers had no corre- mated results disagreed with the reference sponding biotypes in the codebook and were designation, including one each of E. coli, Kleb- called to the telephone computer service. Of the siella rhinoscleromatis, and Shigella spp. microorganisms, four were correctly identified, Table 2 summarizes the Sceptor identifica- including three E. cloacae and one S. marces- tions for the 678 isolates. There was 96.8% cens. After interchange of subculture purity genus agreement and 93.4% species agreement. plates to retest discrepant AutoSCAN results, There were 22 discrepancies at the genus level 22 of the misidentifications, including 10 of the which included 5 E. coli, 5 Klebsiella oxytoca, misidentified Shigella spp., were identified cor- and 5 E. cloacae. Of the 42 stock Shigella spp. rectly. isolates, 3 were misidentified as Salmonella spp. All 678 MicroScan plates were also interpret- Of the 23 species discrepancies, 15 occurred ed visually without the use of the AutoSCAN with the genus Serratia, all involving misidentifi- reader and without knowledge of the Auto- cation of S. marcescens as other Serratia spp. SCAN-generated identification. A total of 28 owing to misinterpretation of carbohydrate reac- identification discrepancies, all at the genus lev- tions. On 55 occasions, the profile number gen- el, were found between automated and visually erated from the Sceptor panel was not listed in generated results. Of the 28 discrepancies, 19 of the Sceptor ID Taxonomy Guide and required 812 WOOLFREY, LALLY, AND QUALL J. CLIN. MICROBIOL. use of the telephone computer service, which 000% 00 & o oW 00 ' or- provided 32 additional correct identifications. Of %C a) 11D r -X en a the 73 correct S. marcescens identifications by .0 Sceptor, 11 were provided by the telephone 0 computer service. After interchange of subculture purity plates to retest discrepant Sceptor r lqe0% r- ri results, 27 of the misidentifications, including all 0 t N O O 00 0 e o -t r-- 1. 'I 00 rl- 00 en 0a 15 S. marcescens, were identified correctly. 0 An 88.1% species level agreement was found for AutoSCAN, Sceptor, and referee identifica- CO tions for the 678 isolates. On two occasions, 0 Nb 0 o m00r^ each involving identification of E. cloacae, both CO A0 0 £0 00 00 u 0 AutoSCAN and Sceptor disagreed with the ref- CO eree designation. AutoSCAN required visual a) plate interpretation resulting in a profile number C) CO which was not in the code book, whereas Scep- 0X.. tor identified one isolate as Enterobacter saka- 00 m m O 0 0 0 zakii and the other as a Salmonella sp. In all ' N esN >.a) t- C\ . 0. other instances in which one system disagreed .E with the referee designation, the other was in agreement. 0_ .D 0 6 0 Table 3 summarizes the costs for performing 0% 00 t M r4,. r4 "t CO 00o v) a- t- ON fi t 0. combined identification and susceptibility tests CO I" 0;06 , c Wf~f -4 by AutoSCAN and Sceptor in comparison with CZ m costs associated with the API 20E system for a) '4- 0 C u identification combined with the MIC-2000 sys- 0 tem for susceptibility. For all three systems, a 0 0 0 T IC C1 as C4" t b en decrease in cost per test occurred as the number - W W b W 000 O 0 cn . . of tests performed per day increased. The initial 'T0-4 .- 0 CO marked decrease in cost per test was largely the 0^ result of distribution of quality control costs. 0. E .U a) After 10 to 15 tests per day, quality control costs 0 became less significant, and total test costs for a- C- " ") " - W) It tn en W en t t No 0000 t c aNs all three systems showed only gradual declines.

c) CO CQ u DISCUSSION O- (A) z In general, the performance of AutoSCAN for cU genus- and species-level identification was satis- 00C V400 O a- factory; however, the AutoSCAN-generated

4- _a) identification for Shigella spp. was poor. Accu- 0 S.3 racy for Shigella spp. improved considerably CO * .0. .0. .0 when MicroScan panels were read visually with- 0. out the use of the AutoSCAN reader. In addi- 0. ^N0 M 0 tion, a significant number of panels (33%) had to 0 be interpreted manually for at least one c) microwell reaction due to equivocation by the C) C) 0t 0 AutoSCAN reader, resulting in a considerable U C) increase in manipulation. No single reaction 0 interpretation was requested significantly more cH _0_ 3 often < 0 than others (P 0.05). Both of these _) _ .F.o factors indicate the need for some improvement 00* Y U o * w in the discrimination of the AutoSCAN reader. *O The Sceptor system provides satisfactory re- C1 a) z 0- sults at both the genus and species levels, al- u m N N n u o00 ._ though three Shigella spp. were misidentified as 00 C-4) oi Salmonella spp. One unique advantage of Scep- tor is the use of dehydrated panels which can be stored at room temperature, obviating the ship- ping and storage requirements of frozen panels, VOL. 17, 1983 EVALUATION OF AUTOSCAN AND SCEPTOR 813 as well as the need to thaw before use. Simulta- ACKNOWLEDGMENT neous rehydration and inoculation offers some This work was supported in part by St. Paul-Ramsey assurance against possible evaporation in frozen Hospital Medical Education and Research Foundation grant 8- panels. In addition, the computer telephone ser- 312. vice provides immediate identification informa- LITERATURE CITED 1. National Committee for Clinical Laboratory Standards. tion, whereas the AutoSCAN and API 20E 1979. Proposed standard: PSM-8 for dilution susceptibility computer services require a delay of several tests, p. 27. Villanova, Pa. hours. A reliable device which would aid in 2. Woolfrey, B. F., J. M. K. Fox, and C. 0. Quail. 1981. reducing the time required for individual reading Evaluation of the Repliscan II system for identification of Enterobacteriaceae. J. Clin. Microbiol. 14:408-410. of Sceptor panels and interpretation of biotype 3. Workload Recording Committee. 1982. Laboratory work- code numbers would be a desirable improve- load recording method, p. 34. College of American Patholo- ment. gists, Chicago, Ill.