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 Phoenix System
TM
J. SALOMON, T. DUNK, C. YU, J. POLLITT, AND J. REUBEN
BD Biosciences • 7 Loveton Circle • Sparks, MD, USA 21152
ABSTRACT
■ 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
P H O E N I X
Phoenix system is intended for the rapid identification of
clinically relevant aerobic bacteria without supplemental
tests. Seventy-five glucose-fermenting and 50 glucosenonfermenting gram-negative species and isolates from
Staphylococcus, Streptococcus, Enterococcus,
Corynebacterium and Bacillus species were included in
this evaluation. Additionally, a total of 74 isolates from
Campylobacteraceae that included Campylobacter,
Helicobacter and Arcobacter species were also tested in
this new system. All test strains were previously identified
by currently recommended reference methods.
The Phoenix system utilizes a total of 45 biochemical and
enzymatic substrates with a variety of colorimetric and
fluorescent indicators. All strains were tested with a
bacterial inoculum concentration equivalent to a 0.5
McFarland Standard. Kinetic readings in the instrument
at 15-minute intervals were evaluated for detectable
biochemical reactivity. Reproducible reactions with
significant levels of discrimination were observed among all
classes of substrates starting at 2-3 hours. Pseudomonas,
Comamonas, Oligella species as well as Campylobacter
and Helicobacter species that are generally less reactive
showed adequate reactivity for differentiation. The
Phoenix system demonstrates an excellent capability
for providing a rapid and reliable means for bacterial
identification in the automated clinical laboratory.
INTRODUCTION
■ Accurate and rapid identification of clinically relevant grampositive and gram-negative bacteria is becoming increasingly more
important in infectious disease therapy. Bacterial identification with
commercially available, miniaturized, automated and manual systems
has been on the rise for decades. More recently, miniaturized
identification systems have successfully employed a combination of
biochemical and enzymatic substrates to identify bacteria to the
species level. This study was conducted to determine the feasibility of
rapid identification of aerobic, gram-positive and gram-negative
bacteria in the Phoenix Automated Microbiology System (BD
Biosciences, Sparks, MD). Identification in the Phoenix system is
based on phenotypic profiles of bacterial reactivity in the presence of
substrates that are kinetically monitored. Observed reproducibility of
substrate reactivity as well as discrimination of species-specific
biochemical reactivity in the Phoenix system is described.
METHODS
■ Two Phoenix Identification panels, one for gram-positive bacteria
and one for gram-negative bacilli were formulated, manufactured
and tested with the Phoenix Incubation/Reader module. Each panel
contained a total of 45 dried substrates including 16 fluorogenic,
14 fermentation, 8 carbon source, 5 chromogenic, and 2
miscellaneous substrates (Table 1).
Approximately 1250 gram-negative isolates (from 125 species)
and over 1000 gram-positive isolates (from 100 species) were
included in this evaluation (Table 2). All test isolates were referenced
using recommended classical and conventional methods.
Each isolate was subcultured twice to ensure viability and purity.
Trypticase® Soy Agar with 5% sheep blood (TSA) and Columbia
Blood Agar with 5% sheep blood (CBA) were used for all grampositive isolates, while TSA and MacConkey Agar (MAC) were used
for gram-negative strains to include reactivity from different media.
Phoenix test panels were inoculated with an organism
concentration approximately equivalent to 1.5x10 8 CFU/ml, in
buffered saline and placed in the Phoenix instrument for incubation
and automated reading. Kinetic measurements of colorimetric and
fluorescent signals were collected every 20 minutes and normalized.
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-NEGATIVES
GRAM-POSITIVES
L-TRYPTOPHAN-AMC
L-PROLINE-AMC
L-ARGININE-AMC
L-PHENYLALANINE-AMC
L-LEUCINE-AMC
L-ORNITHINE-AMC
L-SERINE-AMC
GLYCINE-AMC
L-ALANINE-AMC
L-LYSINE-AMC
4MU-BD-GLUCOSIDE
4MU-BD-GALACTOSIDE
4MU-BD-MANNOSIDE
4MU-AD-GLUCOSIDE
4MU-AD-GALACTOSIDE
4MU-BD-GLUCURONIDE
L-LYSINE-AMC
L-ALANINE-AMC
GLYCINE-AMC
L-VALINE-AMC
L-HISTIDINE-AMC
L-TYROSINE-AMC
L-ISOLEUCINE-AMC
L-SERINE-AMC
L-PROLINE-AMC
L-METHIONINE-AMC
4MU-BD-CELLOBIOSIDE
4MU-BD-GLUCOSIDE
4MU-PHOSPHATE
4MU-BD-FUCOSIDE
4MU-BD-GALACTOSIDE
4MU-AD-GLUCOSIDE
D-ARABITOL
D-GLUCOSE
D-SORBITOL
L-ARABINOSE
L-RHAMNOSE
D-FRUCTOSE
D-LACTOSE
MALTOSE
D-MANNITOL
D-TREHALOSE
D-RAFFINOSE
D-SUCROSE
D-XYLOSE
METHYL-BD-GLUCOSIDE
DEXTRIN
D-GLUCOSE
D-SORBITOL
L-ARABINOSE
D-MANNITOL
D-FRUCTOSE
D-TREHALOSE
CELLOBIOSE
SALICIN
MALTOSE
GLYCEROL
D-GALACTOSE
D-XYLOSE
D-SUCROSE
PNP-AD-GALACTOSIDE
PNP-BD-CELLOBIOSIDE
PNP-N-ACETYL-D-GLUCOSAMINE
ONP-BD-GLUCOSIDE
L-METHIONINE-P-NITROANILIDE
PNP-BD-GLUCOSIDE
PNP-BD-CELLOBIOSIDE
PNP-BD-GALACTOSIDE
PNP-PHOSPHATE
L-ALANINE-P-NITROANILIDE
MALONATE
TARTRATE
ACETATE
GALACTURONIC ACID
CITRATE
GLUCONIC ACID
METHYL-GLUTARIC ACID
PHENYLPROPIOLIC ACID
METHYL-ADIPIC ACID
GLUCONIC ACID
THYMIDINE
TROPIC ACID
HYDROXYBENZOIC ACID
PENTAERYTHRITOL
LAMINARIN
SEDOHEPTULOSE
UREA
ORNITHINE
UREA
ARGININE
Gram-Positive Target Taxa in the Phoenix System
Actinomyces pyogenes
Aerococcus urinae
Aerococcus viridans
Arcanobacterium haemolyticum
Bacillus brevis
Bacillus cereus
Bacillus licheniformis
Bacillus subtilis
Bacillus thuringiensis
Corynebacterium diphtheriae
Corynebacterium jeikeium
Corynebacterium pseudodiphtheriticum
Corynebacterium pseudotuberculosis
Corynebacterium striatum
Corynebacterium urealyticum
Corynebacterium xerosis
Enterococcus avium
Enterococcus casseliflavus
Enterococcus durans
Enterococcus faecalis
Enterococcus faecium
Enterococcus gallinarum
Enterococcus hirae
Enterococcus raffinosus
Erysipelothrix rhusiopathiae
Gardnerella vaginalis
Gemella haemolysans
Gemella morbillorum
Kocuria kristinae
Kocuria rosea
Kocuria varians
Kytococcus sedentarius
Lactococcus lactis ssp. cremoris
Lactococcus lactis ssp. hordniae
Lactococcus lactis ssp. lactis
Leuconostoc mesenteroides subsp. cremoris
Leuconostoc mesenteroides subsp. mesenteroides
Leuconostoc pseudomesenteroides
Listeria monocytogenes
Micrococcus luteus
Micrococcus lylae
Oerskovia turbata
Oerskovia xanthineolytica
Pediococcus acidilactici
Pediococcus damnosus
Pediococcus pentosaceus
Rhodococcus equi
Rothia dentocariosa
Staphylococcus aureus
Staphylococcus auricularis
Staphylococcus capitis ssp ureolyticus
Staphylococcus capitis ssp. capitis
Staphylococcus caprae
Staphylococcus carnosus
Staphylococcus caseolyticus
Staphylococcus chromogenes
Staphylococcus cohnii ssp urealyticum
Staphylococcus cohnii ssp. cohnii
Staphylococcus epidermidis
Staphylococcus equorum
Staphylococcus gallinarum
Staphylococcus haemolyticus
Staphylococcus hominis
Staphylococcus hyicus
Staphylococcus intermedius
Staphylococcus kloosii
Staphylococcus lentus
Staphylococcus lugdunensis
Staphylococcus saprophyticus
Staphylococcus schleiferi ssp coagulans
Staphylococcus schleiferi ssp schleiferi
Staphylococcus sciuri
Staphylococcus simulans
Staphylococcus warneri
Staphylococcus xylosus
Stomatococcus mucilaginosus
Streptococcus acidominimus
Streptococcus agalactiae
Streptococcus anginosus
Streptococcus bovis
Streptococcus constellatus
Streptococcus cricetus
Streptococcus crista
Streptococcus downei
Streptococcus equi subsp zooepidemicus
Streptococcus equi subsp. equi
Streptococcus equinus
Streptococcus equisimilis
Streptococcus gordonii
Streptococcus group G
Streptococcus intermedius
Streptococcus mitis
Streptococcus mutans
Streptococcus oralis
Streptococcus parasanguis
Streptococcus pneumoniae
Streptococcus porcinus
Streptococcus pyogenes
Streptococcus salivarius
Streptococcus sanguis
Streptococcus sobrinus
Streptococcus uberis
Streptococcus vestibularis
Table 3.
Gram-Negative Target Taxa in the Phoenix System
Acinetobacter baumanii
Enterobacter amnigenus
Photobacterium damsela
Tatumella ptyseos
Acinetobacter haemolyticus
Enterobacter asburiae
Plesiomonas shigelloides
Vibrio alginolyticus
Acinetobacter lwoffi
Enterobacter cancerogenus (taylorae)
Proteus mirabilis
Vibrio cholerae
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 vulneris
Pseudomonas flourescens
Yersinia frederiksenii
Brevundimonas vesicularis
Ewingella americana
Pseudomonas mendocina
Yersinia intermedia
Burkholderia cepacia
Flavimonas oryzihabitans
Pseudomonas pseudoalcaligenes
Yersinia kristensenii
Burkholderia gladioli
Flavobacterium odoratum
Pseudomonas putida
Yersinia pseudotuberculosis
Burkholderia pickettii
Hafnia alvei
Pseudomonas stutzeri
Yersinia ruckeri
CDC EF-4b
Klebsiella ornithinolytica
Salmonella arizonae
Yokenella regensburgei
CDC EO-2
Klebsiella oxytoca
Salmonella choleraesuis
CDC group IVc-2
Klebsiella pneumoniae ssp ozaenae
Salmonella paratyphi A
Cedecea davisae
Klebsiella pneumoniae ssp rhinoscleromatis
Salmonella species
Cedecea lapagei
Klebsiella pneumoniae ssp. pneumoniae
Salmonella typhi
Cedecea neteri
Kluyvera species
Serratia ficaria
Chryseobacterium gleum
Leclercia adecarboxylata
Serratia fonticola
Chryseobacterium indologenes
Leminorella species
Serratia liquefaciens
Chryseobacterium meningosepticum
Moellerella wisconsensis
Serratia marcescens
Chryseomonas luteola
Moraxella species
Serratia odorifera
Citrobacter amalonaticus
Morganella morganii
Serratia plymuthica
Citrobacter freundii
Ochrobactrum anthropi
Serratia rubidaea
Citrobacter koseri
Oligella ureolytica
Shewanella putrefaciens
Comamonas acidovorans
Oligella urethralis
Shigella sonnei
Comamonas testosteroni
Pantoea agglomerans
Shigella species
Edwardsiella tarda
Pasteurella aerogenes
Sphingobacterium multivorum
Eikenella corrodens
Pasteurella haemolytica
Sphingobacterium spiritivorum
Empedobacter brevis
Pasteurella multocida
Sphingomonas paucimobilis
Enterobacter aerogenes
Pasteurella pneumotropica
Stenotrophomonas maltophilia
Graph 1. Three-hour Discrimination of Nonfermenters in Phoenix System
Graph 2. Three-hour Discrimination of Enterics in Phoenix System
30
0.8
0.7
25
Absorbance
Fluorescence
0.6
20
15
0.5
0.4
0.3
10
0.2
5
0.1
0
L-Tryptophan-AMC
Methyl-B-D-Glucoside
Graph 3. Reactivity of Bacillus and Corynebacterium in Phoenix System (3H)
Graph 4. Three-hour Discrimination of Streptococci in Phoenix System
0.8
1.2
1.1
0.7
1.0
0.6
Absorbance
Absorbance
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.4
0.2
0.3
0.2
0.5
0.1
BACILIC
BACIBRE
BACICER
BACITHU
BACISUB
CORPSD
CORJEI
CORSTR
CORPST
STRAGA
STREQUZ
STRINR
STRPYO
STRANG
STRBOVI
STRVES
STRCON
STRACI
STRBOVII
STREQUE
STRPNE
STRUBE
CORXER
CORURE
Hydroxybenzoic Acid
Graph 5. Three-hour Discrimination of Staphylococci in Phoenix System
Sucrose
Graph 6. Three-hour Discrimination of Enterococci in Phoenix System
18
2.0
1.9
16
1.8
1.7
Fluorescence
14
Absorbance
1.6
1.5
1.4
1.3
1.2
1.1
12
10
8
6
1.0
4
0.9
0.8
2
STAEP1
STAHAE
STAGAL
STALEN
STASAP
STASIM
STAXYL
STAAUE
STAHOM
STAHYI
STALUG
STASCI
STAWAR
PNP-BD-Glucoside
RESULTS
■ A review of individual substrate graphs, as well as the
3 hour probability tables, showed good overall substrate
reactivity with each of the different substrate classes
exhibiting discriminatory power. For gram-negatives, the
Enterobacteriaceae reacted with all substrate classes and
indicated possible differentiation in 2-3 hours.
Nonfermenters displayed similar results with most of the
substrate classes in a 2-4 hour time frame. For grampositives, the three major genera — Staphylococcus,
Streptococcus, and Enterococcus, all exhibited differential
reactivity in 2-4 hours, while Corynebacterium and
Bacillus also demonstrated separation potential in a
similar time frame (see graphs).
Replicate QC testing of five panel lots revealed
satisfactory reproducibility results for the distinct
substrate classes and preliminary stability studies indicate
acceptable results over time. In addition, substrate
robustness in both systems allows for enhanced separation
through flexible threshold selection capabilities, which can
be individually customized.
ENTCAVI
ENTCCAS
ENTCDUR
ENTCFAA
ENTCFAI
ENTCGAL
ENTCHIR
ENTCRAF
Tyrosine-AMC
CONCLUSION
■ Phoenix Automated Microbiology System has the
ability to identify over 125 gram-negative and over
100 gram-positive taxa to the species level.
■ The system uses a combination of growth-based and
enzymatic-based substrates. Multiple substrate classes
utilizing both colorimetric and fluorometric detection
provide robustness in differentiating the various
organism groups.
■ The system is capable of identifying a wide variety
of clinically relevant bacterial pathogens in a very short
incubation time.
■ Kinetic monitoring allows the system to apply different
metrics (such as rate and acceleration) to bacterial reactivity enhancing the level of discrimination between species.
■ User efficiency is created by obviating the need for
add-on reagents or off-line testing.
YERENT
SHISON
SERMAR
SALSPE
PROVSTU
PROTMIR
PROTVUL
KLEPNEP
MORGMOR
HAFALV
KLEOXY
ESCOOL
ENTBCLO
ENTBAER
CITFRE
EDWTAR
PSELUT
CDCEF4b
EIKCOR
SHEPUT
WEEVIR
ALCXYL
EMPHBRE
SPHMPAU
PSEAER
STEMAL
MYRODA
BURCEP
SPHEMUL
BORBROS
CHRBMEN
0.0
LR650