Ability of the MicroScan Rapid Gram-Negative ID Type 3 Panel To Identify Nonenteric Glucose-Fermenting and Nonfermenting Gram-Negative Bacilli

The MicroScan Rapid Neg ID3 panel is designed for the identificationof Enterobacteriaceae and nonenteric glucose-fermenting andnonfermenting gram-negative bacilli. We evaluated this panelfor its ability to identify gram-negative non-Enterobacteriaceaebacteria. A total of 134 strains, representing 26 genera and42 species, were taken from storage at -70^oC, passaged threetimes before testing, and inoculated into the panels accordingto the manufacturer's directions before being inserted intoa Walk/Away 96 instrument loaded with version 22.28 software.At the end of the initial 2.5-h incubation period, 89 isolates(66.4%) were correctly identified at a probability level of $>=$ 85%. After additional testing recommended by the manufacturerwas completed, another 11 isolates (8.2%) were correctly identifiedat probability levels of $>=$ 85%. Twenty-five (18.7%) isolates werecorrectly identified after additional testing, but the probabilitylevels were less than 85%. Two isolates were unidentified, andseven (5.2%) were incorrectly identified. The seven misidentifiedstrains were not concentrated in any one genus. With an accuracyapproaching 75%, this product may be used for the identificationof the commonly isolated non-Enterobacteriaceae bacteria butmay present problems in identification of other non-glucose-fermentinggram-negative bacilli.

INTRODUCTION

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Introduction

Commercial systems for bacterial identification have been availablefor approximately 30 years. The first manual systems were readvisually but were gradually replaced by those more advancedin their substrates. Eventually automated systems that oftenincorporate substrates that cannot be read visually came onthe market. Of the original systems, only the API 20E (bioMérieux,Inc., Durham, N.C.) remains available, while some MicroScanpanels (Dade Behring, Inc., MicroScan Inc., West Sacramento,Calif.) and Vitek cards (bioMérieux, Inc.) have madethe leap in technology, incorporating many enzymatic substratesthat necessitate electronic reading of the tests. Other systemsoffer a combination of the pH-based and enzymatic tests.

Evaluations of these systems for their ability to identify membersof the family Enterobacteriaceae correctly are common, whilereports of their accuracy in the identification of nonentericglucose-fermenting and nonfermenting gram-negative bacilli arenot as prevalent. Shelly et al. (6) and Visser et al. (9) recentlyevaluated rapid panels, while Shelly et al. (6) looked specificallyat the ability of conventional panels to identify members ofthe genus Burkholderia. van Pelt et al. (8) evaluated both conventionaland rapid panels for their accuracy in identifying Burkholderiaspecies.

MATERIALS AND METHODS

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Materials and Methods

Isolates. A total of 134 strains of biochemically typical and atypicalglucose-fermenting and nonfermenting nonenteric gram-negativebacilli from the stock culture collection of the Centers forDisease Control and Prevention were used in the study (Table1). The collection included both oxidase-negative and oxidase-positivestrains.

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TABLE 1. Strains tested in Rapid Neg ID3 panel

Reference method. Each isolate identification was compared with that obtainedby using conventional biochemical tests as performed at theCenters for Disease Control and Prevention (1-3, 10). Commercialmedia were used whenever possible.

Additional tests. Additional tests necessary for the completion of an identificationby the instrument were performed according to the manufacturer'sinstructions. These may include any of 42 commonly used biochemicaltests plus staining for flagella. The tests that were performedin this study were urea; motility; arginine dihydrolase; fermentationof oxidation-fermentation (OF) glucose, OF maltose, OF mannitol,or OF xylose; oxidase; nitrate reduction; and lecithinase productionon egg yolk agar (at 25°C), as well as flagellum staining.

System. A MicroScan Walk/Away 96 instrument (Dade Behring, Inc., MicroScanInc.) was used in the evaluation with the Rapid Neg ID3 panel.The software version was 22.28. The Rapid Neg ID3 panel is designedfor the identification to species level of rapidly growing aerobicand facultatively anaerobic non-glucose-fermenting and non-Enterobacteriaceaeglucose-fermenting gram-negative bacilli. The time to completionof the identification was 2.5 h.

Taxonomy. Organism identifications are given as listed in the MicroScandatabase even though some taxonomic changes may have occurredsince the database was compiled.

Definitions. "Correct at 2.5 h" means that the identification is correctto genus and species level at the end of the initial 2.5-h incubationperiod at a probability level of at least 85%, which is thelower limit of "acceptability" as defined by the software. "Correctwith additional tests" means that the identification is correctto genus and species level after additional tests requestedby the software were performed. "No ID" means that the softwarewas unable to identify the organism at the end of the incubationtime and a report of "No ID" was generated. "Error" means thatthe identification was incorrect at either the genus or thespecies level when that identification was included in the databasefor this panel.

Strains used in the study were removed from -70°C storageand plated on tryptic soy agar with 5% sheep blood (TSAII; BDBiosciences, Sparks, Md.). Incubation was at 30°C over theweekend (2.5 days). The strains were then transferred once moreon sheep blood agar before being transferred the third timeonto MacConkey's agar (MacConkey II; BD Biosciences). If anorganism did not grow on MacConkey's agar, the third transferwas made to sheep blood agar. These last two incubations wereat 35°C for 24 h each.

Rapid Neg ID3 panels were removed from the refrigerator andallowed to warm to room temperature before being inoculatedaccording to the manufacturer's directions and loaded into theMicroScan Walk/Away 96 instrument.

If an identification was in error, the strain was transferredagain onto the appropriate agar and incubated, and panels wererepeated in duplicate. If two of the three answers agreed withthe reference identification, the answer was considered correct.

RESULTS AND DISCUSSION

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Results and Discussion

We tested 134 strains that represented 26 genera and 42 differentspecies. Results of this testing are shown in Table 1. At theend of the initial 2.5-h incubation period, 89 (66.4%) of theidentifications were correct at a level of $>=$ 85%. Of the 36 strainsthat were correct after additional testing was completed, theprobability levels ranged from 3.4 to 99.9%. Eleven (8.2%) strainsgave identifications with probability levels of 85% or greater,while 25 (18.7%) strains gave identifications with probabilitylevels of less than 85% and should be classified as uninterpretable.When the probability is in the lower range, the laboratorianhas to make a decision based on colonial morphology, the sourceof the isolate, and antimicrobial susceptibility data as towhether the identification could be correct. If the likelihoodis very low, an alternative system of identification shouldbe used.

Of the remaining nine strains, seven were misidentified andtwo were unidentified. Table 2 lists the misidentified strains,along with the probability level of the answers that were given.Of the two unidentified strains, one was an isolate of Bergeyellazoohelcum that was negative for arginine. We suspect that thelack of identification was due to the fact that the databasematrix has the arginine reaction for this organism set at 90%positive. The printout had this test listed as atypical. Theother isolate that the instrument could not identify was Comamonastestosteroni.

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TABLE 2. Strains misidentified by the MicroScan Rapid Neg ID3 panel

As stated earlier, if an initial identification was in error,the strain was transferred again onto the appropriate agar andthe panels were repeated in duplicate. Table 3 lists the 20isolates that were repeated and the number of identificationsthat were correct on repeat along with the two that gave "NoID" on repeat. Of the 13 identifications that were correct,8 were of $>=$ 85% probability. We have unpublished data from previousstudies that show that many of the initial incorrect identificationsare related to the number of passages of an isolate upon removalfrom -70°C storage and that, upon continued passage, anidentification will correct itself.

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TABLE 3. Isolates that gave initial erroneous identifications

Table 4 lists other studies that evaluated either conventionalor rapid MicroScan panels for the identification of glucosenonfermenters.

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TABLE 4. Other studies that evaluated MicroScan panels

The study by Shelly et al. (6) looked specifically at the identificationof Burkholderia cepacia and showed a 15% (three misidentifiedisolates) error rate with the Rapid Neg ID panel. Two of thethree misidentified isolates were reported as Alcaligenes xylosoxidans,and one was reported as Burkholderia gladioli.

Visser et al. (9) studied 41 strains, including Pseudomonasaeruginosa, Acinetobacter calcoaceticus (baumannii), and Xanthomonas(Stenotrophomonas) maltophilia. Only one strain, of A. calcoaceticus,was misidentified, for an accuracy rate of 97.0%.

In an earlier study of ours (4), 36 isolates of P. aeruginosa,Stenotrophomonas maltophilia, Acinetobacter baumannii, and Acinetobacterlwoffi were correctly identified 81% of the time.

The overall accuracy of the Rapid Neg ID3 panel for the identificationof a challenge set of non-Enterobacteriaceae bacteria afteradditional testing was 74.6% with an additional 20.2% of theisolates having results that either were not able to be interpretedor were unidentified.

FOOTNOTES

* Corresponding author. Mailing address: Mailstop C16, Atlanta, GA 30333. Phone: (404) 639-2316. Fax: (404) 639-3822. E-mail: cmo1{at}cdc.gov.

REFERENCES

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