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Rapid Automated Identification of Gram-Positive and Gram-Negative Bacteria in the Phoenixtm System J As presented at the 99th General Meeting of the American Society for Microbiology, May 1999. Rapid Automated Identification of Gram-Positive and Gram-Negative Bacteria in the PhoenixTM System J. SALOMON, T. DUNK, C. YU, J. POLLITT, AND J. REUBEN BD Biosciences • 7 Loveton Circle • Sparks, MD, USA 21152 ABSTRACT I Feasibility of rapid and reliable identification of over 225 taxa that included gram-negative and gram-positive species was evaluated in the PhoenixTM Automated Microbiology System (BD Biosciences, Sparks, MD). The PHOENIX Phoenix system is intended for the rapid identification of clinically relevant aerobic bacteria without supplemental INTRODUCTION tests. Seventy-five glucose-fermenting and 50 glucose- Accurate and rapid identification of clinically relevant gram- positive and gram-negative bacteria is becoming increasingly more nonfermenting gram-negative species and isolates from important in infectious disease therapy. Bacterial identification with Staphylococcus, Streptococcus, Enterococcus, commercially available, miniaturized, automated and manual systems has been on the rise for decades. More recently, miniaturized Corynebacterium and Bacillus species were included in identification systems have successfully employed a combination of this evaluation. Additionally, a total of 74 isolates from biochemical and enzymatic substrates to identify bacteria to the species level. This study was conducted to determine the feasibility of Campylobacteraceae that included Campylobacter, rapid identification of aerobic, gram-positive and gram-negative bacteria in the Phoenix Automated Microbiology System (BD Helicobacter and Arcobacter species were also tested in Biosciences, Sparks, MD). Identification in the Phoenix system is this new system. All test strains were previously identified based on phenotypic profiles of bacterial reactivity in the presence of substrates that are kinetically monitored. Observed reproducibility of by currently recommended reference methods. substrate reactivity as well as discrimination of species-specific biochemical reactivity in the Phoenix system is described. The Phoenix system utilizes a total of 45 biochemical and METHODS enzymatic substrates with a variety of colorimetric and Two Phoenix Identification panels, one for gram-positive bacteria fluorescent indicators. All strains were tested with a and one for gram-negative bacilli were formulated, manufactured bacterial inoculum concentration equivalent to a 0.5 and tested with the Phoenix Incubation/Reader module. Each panel contained a total of 45 dried substrates including 16 fluorogenic, McFarland Standard. Kinetic readings in the instrument 14 fermentation, 8 carbon source, 5 chromogenic, and 2 miscellaneous substrates (Table 1). at 15-minute intervals were evaluated for detectable Approximately 1250 gram-negative isolates (from 125 species) biochemical reactivity. Reproducible reactions with and over 1000 gram-positive isolates (from 100 species) were included in this evaluation (Table 2). All test isolates were referenced significant levels of discrimination were observed among all using recommended classical and conventional methods. classes of substrates starting at 2-3 hours. Pseudomonas, Each isolate was subcultured twice to ensure viability and purity. Trypticase® Soy Agar with 5% sheep blood (TSA) and Columbia Comamonas, Oligella species as well as Campylobacter Blood Agar with 5% sheep blood (CBA) were used for all gram- positive isolates, while TSA and MacConkey Agar (MAC) were used and Helicobacter species that are generally less reactive for gram-negative strains to include reactivity from different media. showed adequate reactivity for differentiation. The Phoenix test panels were inoculated with an organism concentration approximately equivalent to 1.5x108 CFU/ml, in Phoenix system demonstrates an excellent capability buffered saline and placed in the Phoenix instrument for incubation for providing a rapid and reliable means for bacterial and automated reading. Kinetic measurements of colorimetric and fluorescent signals were collected every 20 minutes and normalized. identification in the automated clinical laboratory. Based on biochemical reactivity and resulting signals in the visible and UV spectra, substrate specific metrics were selected and preliminary algorithms were applied to each of the five different substrate classes. Probability of substrate specific reactivity was generated with 3h data for each of the taxa to determine the discriminatory ability of the system. Table 1. Table 2. List of Substrates Tested Gram-Positive Target Taxa in the Phoenix System GRAM-NEGATIVES GRAM-POSITIVES Actinomyces pyogenes Leuconostoc mesenteroides subsp. cremoris Staphylococcus schleiferi ssp schleiferi Aerococcus urinae Leuconostoc mesenteroides subsp. mesenteroides Staphylococcus sciuri L-TRYPTOPHAN-AMC L-LYSINE-AMC Aerococcus viridans Leuconostoc pseudomesenteroides Staphylococcus simulans L-PROLINE-AMC L-ALANINE-AMC Arcanobacterium haemolyticum Listeria monocytogenes Staphylococcus warneri L-ARGININE-AMC GLYCINE-AMC Bacillus brevis Micrococcus luteus Staphylococcus xylosus L-PHENYLALANINE-AMC L-VALINE-AMC Bacillus cereus Micrococcus lylae Stomatococcus mucilaginosus L-LEUCINE-AMC L-HISTIDINE-AMC Bacillus licheniformis Oerskovia turbata Streptococcus acidominimus L-ORNITHINE-AMC L-TYROSINE-AMC Bacillus subtilis Oerskovia xanthineolytica Streptococcus agalactiae L-SERINE-AMC L-ISOLEUCINE-AMC Bacillus thuringiensis Pediococcus acidilactici Streptococcus anginosus GLYCINE-AMC L-SERINE-AMC Corynebacterium diphtheriae Pediococcus damnosus Streptococcus bovis L-ALANINE-AMC L-PROLINE-AMC Corynebacterium jeikeium Pediococcus pentosaceus Streptococcus constellatus L-LYSINE-AMC L-METHIONINE-AMC Corynebacterium pseudodiphtheriticum Rhodococcus equi Streptococcus cricetus 4MU-BD-GLUCOSIDE 4MU-BD-CELLOBIOSIDE Corynebacterium pseudotuberculosis Rothia dentocariosa Streptococcus crista 4MU-BD-GALACTOSIDE 4MU-BD-GLUCOSIDE Corynebacterium striatum Staphylococcus aureus Streptococcus downei 4MU-BD-MANNOSIDE 4MU-PHOSPHATE 4MU-AD-GLUCOSIDE 4MU-BD-FUCOSIDE Corynebacterium urealyticum Staphylococcus auricularis Streptococcus equi subsp zooepidemicus 4MU-AD-GALACTOSIDE 4MU-BD-GALACTOSIDE Corynebacterium xerosis Staphylococcus capitis ssp ureolyticus Streptococcus equi subsp. equi 4MU-BD-GLUCURONIDE 4MU-AD-GLUCOSIDE Enterococcus avium Staphylococcus capitis ssp. capitis Streptococcus equinus Enterococcus casseliflavus Staphylococcus caprae Streptococcus equisimilis D-ARABITOL DEXTRIN Enterococcus durans Staphylococcus carnosus Streptococcus gordonii D-GLUCOSE D-GLUCOSE Enterococcus faecalis Staphylococcus caseolyticus Streptococcus group G D-SORBITOL D-SORBITOL Enterococcus faecium Staphylococcus chromogenes Streptococcus intermedius L-ARABINOSE L-ARABINOSE Enterococcus gallinarum Staphylococcus cohnii ssp urealyticum Streptococcus mitis L-RHAMNOSE D-MANNITOL Enterococcus hirae Staphylococcus cohnii ssp. cohnii Streptococcus mutans D-FRUCTOSE D-FRUCTOSE Enterococcus raffinosus Staphylococcus epidermidis Streptococcus oralis D-LACTOSE D-TREHALOSE Erysipelothrix rhusiopathiae Staphylococcus equorum Streptococcus parasanguis MALTOSE CELLOBIOSE Gardnerella vaginalis Staphylococcus gallinarum Streptococcus pneumoniae D-MANNITOL SALICIN Gemella haemolysans Staphylococcus haemolyticus Streptococcus porcinus D-TREHALOSE MALTOSE Gemella morbillorum Staphylococcus hominis Streptococcus pyogenes D-RAFFINOSE GLYCEROL Kocuria kristinae Staphylococcus hyicus Streptococcus salivarius D-SUCROSE D-GALACTOSE Kocuria rosea Staphylococcus intermedius Streptococcus sanguis D-XYLOSE D-XYLOSE Kocuria varians Staphylococcus kloosii Streptococcus sobrinus METHYL-BD-GLUCOSIDE D-SUCROSE Kytococcus sedentarius Staphylococcus lentus Streptococcus uberis PNP-AD-GALACTOSIDE PNP-BD-GLUCOSIDE Lactococcus lactis ssp. cremoris Staphylococcus lugdunensis Streptococcus vestibularis PNP-BD-CELLOBIOSIDE PNP-BD-CELLOBIOSIDE Lactococcus lactis ssp. hordniae Staphylococcus saprophyticus PNP-N-ACETYL-D-GLUCOSAMINE PNP-BD-GALACTOSIDE Lactococcus lactis ssp. lactis Staphylococcus schleiferi ssp coagulans ONP-BD-GLUCOSIDE PNP-PHOSPHATE L-METHIONINE-P-NITROANILIDE L-ALANINE-P-NITROANILIDE MALONATE METHYL-ADIPIC ACID TARTRATE GLUCONIC ACID Table 3. ACETATE THYMIDINE GALACTURONIC ACID TROPIC ACID CITRATE HYDROXYBENZOIC ACID GLUCONIC ACID PENTAERYTHRITOL Gram-Negative Target Taxa in the Phoenix System METHYL-GLUTARIC ACID LAMINARIN PHENYLPROPIOLIC ACID SEDOHEPTULOSE Acinetobacter baumanii Enterobacter amnigenus Photobacterium damsela Tatumella ptyseos Acinetobacter haemolyticus Enterobacter asburiae Plesiomonas shigelloides Vibrio alginolyticus UREA UREA Acinetobacter lwoffi Enterobacter cancerogenus (taylorae) Proteus mirabilis Vibrio cholerae ORNITHINE ARGININE Actinobacillus ureae Enterobacter cloacae Proteus penneri Vibrio fluvialis Aeromonas caviae Enterobacter gergoviae Proteus vulgaris Vibrio hollisae Aeromonas hydrophila Enterobacter hormaechei Providencia alcalifaciens Vibrio metschnikovii Aeromonas veronii biovar sobria Enterobacter intermedium Providencia rettgeri Vibrio mimicus Agrobacterium radiobacter Enterobacter sakazakii Providencia rustigianii Vibrio parahaemolyticus Alcaligenes xylosoxidans ssp xylosoxidans Escherichia coli Providencia stuartii Vibrio vulnificus Bergeyella zoohelcum Escherichia fergusonii Pseudomonas aeruginosa Weeksella virosa Bordetella bronchiseptica Escherichia hermannii Pseudomonas alcaligenes Yersinia enterocolitica Brevundimonas diminuta Escherichia
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