INSTRUCTIONS FOR USE – BBL™ Enterotube II

IA-273176.01 Rev.: Jan. 2007

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BD™ BBL™ Enterotube™ II INTENDED USE BBL Enterotube II is a ready-to-use identification system for the identification of Enterobacteriaceae and a variety of other oxidase negative Gram negative rods. PRINCIPLES AND EXPLANATION OF THE PROCEDURE Microbiological method. Enterobacteriaceae play a major role as infectious agents. This group of bacteria utilizes carbohydrates principally by fermentation and by oxidation. They are catalase positive and nearly all of them are oxidase negative [It has been proposed recently to include Plesiomonas shigelloides into the Enterobacteriaceae based on 16S rRNA sequencing and because it contains the enterobacterial common antigen.1,2 Previously, this organism which is is oxidase positive, was included into the family Vibrionaceae]. The identification of Enterobacteriaceae is based on biochemical reactions as described by Ewing and by Farmer. For details, consult the references.1-11 While a huge variety of genera and species has been described over the years, the organisms that are isolated from clinical specimens belong to 20 to 25 species that are well-known for many years.1

BBL Enterotube II is a self-contained, compartmented plastic tube containing twelve different media that allow the determination of 15 biochemical reactions (glucose, gas production from glucose, lysine decarboxylase, ornithine decarboxylase, hydrogen sulfide (H2S), indole, adonitol, lactose, arabinose, sorbitol, Voges-Proskauer (VP), dulcitol, phenylalanine deaminase (PA), urea and citrate). The enclosed inoculating wire allows inoculation of all compartments in one step from one or a few single colonies of an isolate. The resulting combination of reactions, together with the Interpretation Guide (codebook), allow identification of clinically significant Enterobacteriaceae. The Interpretation Guide (codebook) for BBL Enterotube II was constructed using the percentage data contained in the references for the biochemical tests performed by BBL Enterotube II.3-11 The Results Pad and Color Reaction Chart permit a rapid check of the positive reactions obtained with BBL Enterotube II. The checked positive numbers are totaled, and the composite number is then located in the Interpretation Guide to identify the organisms. Where two or more organisms are listed, the confirmatory tests required to further identify them are also given. The following flow diagram demonstrates how the oxidase test may used to differentiate members of the family Enterobacteriaceae from nonfermentative, oxidase positive or negative, and from fermentative, oxidase positive Gram negative bacteria and when BBL Enterotube II or BBL Oxi/Ferm™ Tube II are used:

Pure culture on nonselective medium

↓ Oxidase test

positive negative ↓ ↓ Inoculate BBL Oxi/Ferm Tube II Inoculate BBL Enterotube II ↓ ↓

Anaerobic glucose Glucose Positive Negative Positive Negative*

Aeromonas spp., Plesiomonas shigelloides, Vibrio spp.

Achromobacter spp., Alcaligenes spp., Bordetella bronchiseptica, Moraxella spp., Pasteurella spp., etc.

Enterobacteriaceae Acinetobacter spp., Stenotrophomonas maltophilia, etc.

* These species may be identified with the BBL Enterotube II system, but use of BBL Oxi/Ferm Tube II may be necessary for confirmation. REAGENTS BBL Enterotube II Substrates, other active ingredients, and coloration of the uninoculated media:

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• Medium 1 (Glucose): Glucose (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. The medium is covered with wax to provide anaerobic conditions and to allow detection of gas formation. Uninoculated: red.

• Medium 2 (Lysine): Lysine (10.0 g/l) contained in an appropriate base medium, with bromcresol purple as a pH indicator. The medium is covered with wax to provide anaerobic conditions. Uninoculated: yellow.

• Medium 3 (Ornithine): Ornithine (10.0 g/l) contained in an appropriate base medium, with bromcresol purple as a pH indicator. The medium is covered with wax to provide anaerobic conditions. Uninoculated: yellow.

• Medium 4 (H2S/Indole): Sodium thiosulfate (4.7 g/l) , ferric ammonium citrate (0.6 g/l) , and tryptophan (1.2 g/l) in an appropriate base medium. Uninoculated: Beige to light amber.

• Medium 5 (Adonitol): Adonitol (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

• Medium 6 (Lactose): Lactose (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

• Medium 7 (Arabinose): Arabinose (20.0 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

• Medium 8 (Sorbitol): Sorbitol (20 g/l) contained in an appropriate base medium, with cresol red as a pH indicator. Uninoculated: red.

• Medium 9 (Voges-Proskauer): Glucose (20.0 g/l) contained in an appropriate base medium. Uninoculated: colorless to light amber.

• Medium 10 (Dulcitol/PA): Dulcitol (20.0 g/l) , phenylalanine (7.0 g/l), and ferric ammonium citrate (0.5 g/l) contained in an appropriate base medium, with bromthymol blue as a pH indicator. Uninoculated: green.

• Medium 11 (Urea): Urea (20.0 g/l) contained in an appropriate base medium, with phenol red as a pH indicator. Uninoculated: beige to light amber.

• Medium 12 (Citrate): Sodium citrate (2.0 g/l) contained in an appropriate base medium, with bromthymol blue as a pH indicator. Uninoculated: green.

PRECAUTIONS

. For professional use only Do not use tubes if they show evidence of microbial contamination, discoloration, wax detachment from the respective media, drying, cracking or other signs of deterioration. Any of the following conditions may interfere with the accuracy of BBL Enterotube II: dehydration or liquefaction, lifting of wax from media surfaces, any change of media colors from those indicated under “Uninoculated” in the REAGENTS section, and any indication of growth on media surfaces. The following precautions pertain to the individual compartments indicated: • Glucose: The wax overlay in this compartment produces the degree of anaerobiosis necessary to

allow only the true fermentation reaction characteristic of all Enterobacteriaceae. If this medium remains red after inoculation and incubation, the strain does not belong to the Enterobacteriaceae. Examples of oxidase-negative non-Enterobacteriaceae that fail to ferment glucose under anaerobic conditions in BBL Enterotube II are glucose negative Acinetobacter species. Although these organisms can be differentiated from Enterobacteriaceae using BBL Enterotube II (in the Interpretation Guide, use the database for the oxidase negative nonfermenters), BBL Oxi/Ferm Tube II may be required for their further identification.

• Phenylalanine (PA): The medium may become a deeper shade of green after incubation. This should be considered negative.

Warning: When preparing and handling indole and VP reagents, observe appropriate handling instructions and Risk and Safety Phrases. Consult GENERAL INSTRUCTIONS FOR USE document for aseptic handling procedures, biohazards, and disposal of used product. STORAGE AND SHELF LIFE On receipt, store BBL Enterotube II in the dark at 2 to 8° C, in the original box until just prior to use. Avoid freezing and overheating. The tubes may be inoculated up to the expiration date and incubated for the recommended incubation times. Tubes from opened packages can be used up to the expiration date. Opened tubes must be used immediately.

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USER QUALITY CONTROL Inoculate BBL Enterotube II with the strains mentioned below. For inoculation, incubation, and reading, proceed as described in Test Procedure.

ORGANISMS

Glu

cose

Gas

Lysi

ne

Orn

ithin

e

H2S

Indo

le

Ado

nito

l

Lact

ose

Ara

bino

se

Sorb

itol

VP

Dul

cito

l

Phen

yl-

alan

ine

Ure

a

Citr

ate

Acceptable biocodes

Proteus mirabilis ATCC™ 12453

+ +/- - + + - - - - - - - + + +/- 66007 / 66006 / 46007

Proteus vulgaris ATCC 6380

+/(+) +/- - - + + - - - - - - + + + 63007 / 43007

Providencia alcali-faciens ATCC 9886

+ +/- - - - + + - - - - - + - -/+ 61404 / 41405 / 41404 / 61405

Serratia marcescens ATCC 13880

+ +/- + + - - +/- - - + + - - - + 74461 / 54461 / 74061 / 54061

Escherichia coli ATCC 25922

+ +/- + + - + - + + + - - - - - 75340 / 55340

Klebsiella pneumoniae ATCC 27736

+ +/- + - - - + + + + + + - -/+ + 50771 / 70771 / 70773 / 50773

Pseudomonas aeruginosa ATCC 27853

- - - - - - - - -/+ - - - - -/(+) + *

* P. aeruginosa is oxidase positive and is, therefore, not included in the BBL Enterotube II databases. However, this strain is useful as a control organism to detect negative reactions in the glucose and gas compartments. += positive; − = negative; (+) weakly positive; +/- = positive or (rarely) negative; +/(+) = positive or (rarely) weakly positive; -/+= negative or (rarely) positive. PROCEDURE Materials Provided BBL Enterotube II. Microbiologically controlled. Results Pad and Color Reaction Chart for BBL Enterotube II. Materials Not Provided BBL Enterotube II Interpretation Guide (codebook), document no. CM-273176.CE, which is divided into three databases: (1) Oxidase negative nonfermenters, (2) Enterobacteriaceae – method without VP, and (3) Enterobacteriaceae – method with VP. Kovacs‘ indole reagent (Indole Dropper Reagent, 50 ampoules, cat. no. 261185) and Voges-Proskauer reagents (Voges-Proskauer A Dropper Reagent, 50 ampoules, cat. no. 261192; Voges-Proskauer B Dropper Reagent, cat. no. 261193) reagents are available from BD. Materials and reagents to perform the supplemental tests as mentioned in the BBL Enterotube II Interpretation Guide. BBL Oxi/Ferm II identification system (cat. no. 212116) for the identification of non-Enterobacteriaceae. Ancillary culture media, reagents and laboratory equipment as described. Kovacs‘ reagent may be prepared in the laboratory as follows: Amyl or Isoamyl Alcohol 75 ml p-Dimethylaminobenzaldehyde 5 g Hydrochloric acid, concentrated 25 ml Dissolve p-dimethylaminobenzaldehyde (the reagent should be light yellow in color) in alcohol and then slowly add the acid. The prepared reagent should have a straw yellow color and be stored in a brown bottle under refrigeration for maximum stability. Any reagent having a dark brown color should not be used. Voges-Proskauer reagents may be prepared as follows: 5% Alpha-naphthol solution alpha-Naphthol 5 g Ethanol (ethyl alcohol), absolute 100 ml

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20% Potassium hydroxide solution Water 100 ml Potassium hydroxide (pellets) 20 g Creatine 300 mg After preparation, Voges-Proskauer reagents are stable in tightly closed bottles for two weeks at 2 to 8° C in the dark. Specimen Types BBL Enterotube II is a biochemical identification system and must not be used directly with clinical specimens. Use isolates from appropriate media (see Test Procedure). BBL Enterotube II may be used to identify aerobic, facultatively anaerobic Gram negative rods (Enterobacteriaceae) isolated from any specimen. Test Procedure For the inoculation of BBL Enterotube II, growth from MacConkey (MAC), Eosin Methylene Blue (EMB), Salmonella Shigella (SS) or Hektoen Enteric (HE) agars, or from nonselective blood agar media can be used. The culture used for the inoculation should be at least 18 hours old, but should generally not be older than 48 hours. Make sure that the isolate that shall be identified with BBL Enterotube II is a pure culture of a Gram negative rod. BBL Enterotube II should be used only with oxidase-negative isolates since oxidase-positive organisms (non-Enterobacteriaceae) require the use of BBL Oxi/Ferm Tube II for their identification. 1. Prepare one sheet from the coding pad for the isolate by entering patient name, specimen number,

and date. Note: It may be decided by the user if the databases with or without VP are used. Depending on this decision, use the frontside or the backside of the coding pad. If the database without VP is chosen, it may be necessary to perform the VP reaction as a supplemental test for selected organisms.

2. Take one BBL Enterotube II tube and enter patient name, specimen number, and date on the label. 3. Remove both caps. The tip of the inoculating wire is under the white cap. Do not flame wire. 4. Pick a well isolated colony directly with the tip of the BBL Enterotube II inoculating wire (Figure 1). A

visible amount of inoculum should be seen at the tip and the side of the wire. Avoid touching agar with wire. Utilize one or more BBL Enterotube II to pick additional colonies as experience dictates.

5. Inoculate BBL Enterotube II by first twisting wire, then withdrawing wire through all twelve compartments applying a turning motion (Figure 2).

6. Reinsert wire (without sterilizing) into BBL Enterotube II, using a turning motion through all 12 compartments, until the notch on the wire is aligned with the opening of the tube (Figure 3). The tip of the wire should be seen in the citrate compartment. Break wire at notch by bending. The portion of the wire remaining in the tube maintains anaerobic conditions necessary for true fermentation of glucose, production of as and decarboxylation of lysine and ornithine.

7. With the broken off part of the wire, punch holes through the foil covering the air inlets of the last eight compartments (adonitol, lactose, arabinose, sorbitol, Voges-Proskauer, dulcitol/PA, urea and citrate) in order to support aerobic growth in these compartments (Figure 4). Replace both caps.

8. Incubate at 35 to 37° C for 18 to 24 hours with BBL Enterotube II lying on its flat surface or in an upright position. Allow for air circulation between incubated tubes.

9. Interpret and record all reactions with exception of indole and Voges-Proskauer. (For complete instructions on how to read results of BBL Enterotube II, see Results section.) All other tests must be read before the indole and Voges-Proskauer tests are performed as the reagents added for these tests may alter the remainder of the BBL Enterotube II reactions.

Indole and Voges-Proskauer Reagent Addition: 1. Place BBL Enterotube II horizontally on the bench or vertically in a rack with glucose compartment

pointing downward. By using a needle or the broken-off end of a BBL Enterotube II inoculating wire or by melting with a hot inoculating wire, punch or melt a hole of about 3-4 mm in diameter in the plastic film above the Indole/H2S and VP compartments.

2. To perform the indole test: Add 3 - 4 drops of Kovacs‘ reagent (cat. no. 261185 or laboratory

prepared; see section Materials Not provided) to the H2S/indole compartment. Allow reagent to contact the surface of the medium. A positive test is indicated by development of a red color in the added reagent within 10 sec.

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3. To perform the Voges-Proskauer test: This test is mandatory if the database with VP is chosen. It may also be necessary to perform this test if needed as a confirmatory test when using the database without VP. When using the laboratory prepared reagents (see section Materials Not Provided), add two drops of 20% potassium hydroxide solution containing 0.3% creatine and three drops of 5% alpha-naphthol in absolute ethyl alcohol. A positive test is indicated by the development of a red color within 20 min. Do not wait longer than 20 min before reading the results! When using Dropper Reagents, add 3 to 5 drops of Voges-Proskauer A reagent (cat. no. 261192) and 1 to 2 drops of Voges-Proskauer B reagent (cat. no. 261193). A positive reaction is indicated by the development of a cherry-red color within 5 to 15 min. A copper to brownish discoloration is read as negative. Do not wait longer than 15 min before reading the results!

Results Follow the instructions given below to identify the isolate. Information on the appearance of positive and negative reactions is provided in Table 1. Note: If no color change, or an orange color, is observed in the glucose compartment and growth is evident by color changes in other compartments, the organism is not a member of the family Enterobacteriaceae. (See PRECAUTIONS section). 1. After 18 to 24 hours of incubation, interpret all reactions. With the exception of indole and VP, read the

reactions in a sequential fashion by comparing the colors of the media in the tube after incubation with those given in the color scheme on the cover of the coding pad and (eventually) with an uninoculated BBL Enterotube II which must be brought to room temperature first (Figure 5). All reactions which should be positive may be of equal, greater or lesser intensity than the colors indicated in the Color Reaction Chart.

2. Indicate each positive test result by circling the number appearing below the appropriate compartment on the Results Pad (Figure 6).

3. Finally, perform the indole and VP tests. If positive, circle the appropriate numbers on the prepared sheet. The VP test is mandatory only if the database with VP is used.

4. Add circled numbers in the bracketed section and enter the sum in the space provided below the arrow (Figure 6).

5. Locate the five digit number in the Interpretation Guide (codebook) and find the best answer(s) in the column entitled “ID Value“. Make sure to use the right database [(1) Oxidase negative nonfermenters, (2) Enterobacteriaceae – method without VP, and (3) Entero-bacteriaceae – method with VP]. In the example given in Figure 6, the identification as Klebsiella pneumoniae is obtained.

Figure 1 Figure 2 Figure 3

Figure 4 Figure 5

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(A)

(B)

Figure 6: Results Pad from BBL Enterotube II (A = database with VP; B = database without VP) Table 1: Appearance of negative and positive reactions in BBL Enterotube II

REACTIONS Reagents Negative Positive

REMARKS

COMPARTMENT 1 Glucose red (to

orange) yellow (to golden)

Glucose (GLU): The end products of bacterial fermentation of glucose are either acid, or acid and gas. The shift in pH due to the production of acid is indicated by change in the color of the indicator in the medium from red (alkaline) to yellow (acidic). Any degree of yellow should be interpreted as a positive reaction; orange should be considered negative.

Gas production

wax not lifted

wax lifted Gas production (GAS): This is evidenced by definite and complete separation of the wax overlay from the surface of the glucose medium, but not by bubbles in the medium. Since the amount of gas produced by different bacteria varies, the amount of separation between medium and overlay will also vary with the strain being tested.

COMPARTMENT 2 Lysine Yellow purple Lysine decarboxylase (LYS): Bacterial decarboxylation of lysine, which

results in the formation of the alkaline end product cadaverine, is indicated by a change in the color of the indicator in the medium from pale yellow (acidic) to purple (alkaline). The medium remains yellow if decarboxylation of lysine does not occur.

COMPARTMENT 3 Ornithine Yellow purple Ornithine decarboxylase (ORN): Decarboxylation of ornithine, which

results in the formation of the alkaline end product putrescine, is indicated by a change in the color of the indicator in the medium from pale yellow (acidic) to purple (alkaline). The medium remains yellow if decarboxylation of ornithine does not occur.

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REACTIONS Reagents Negative Positive

REMARKS

COMPARTMENT 4 H2S beige to

light amber black Hydrogen sulfide production (H2S): Hydrogen sulfide is produced by

bacteria capable of reducing sulfur-containing compounds, such as peptones and sodium thiosulfate present in the medium. The hydrogen sulfide reacts with the iron salts also present in the medium to form a black precipitate of ferric sulfide usually along the line of inoculation. Some Providencia strains may produce a diffuse brown coloration in this medium, however, this should not be confused with true H2S production as indicated by the presence of black color. Note: The black precipitate may fade or revert back to negative if BBL Enterotube II is read after 24 h incubation.

Indole formation

colorless pink-red Indole formation (IND): The production of indole from the metabolism of tryptophan by the bacterial enzyme tryptophanase is detected by the development of a pink to red color after the addition of Kovacs‘ indole reagent, which is added into the compartment after 18 to 24 h incubation of the tube (see Test Procedure section).

COMPARTMENTS 5,6,7,8 Adonitol, Lactose, Arabinose, Sorbitol

red (to orange)

yellow (to golden)

Adonitol (ADON), Lactose (LAC), Arabinose (ARAB), Sorbitol (SORB): Bacterial fermentation of adonitol, lactose, arabinose, and sorbitol result in the formation of acidic end products, is indicated by a change in color of the indicator present in the medium from red (alkaline) to yellow (acidic). Any sign of yellow should be interpreted as a positive reaction; orange should be considered negative.

COMPARTMENT 9 Voges-Proskauer

colorless to light amber

red Voges-Proskauer (VP): Acetylmethylcarbinol (acetoin) is an intermediate in the production of butylene glycol from glucose fermentation. The production of acetoin is detected after the addition of the VP reagents after incubation (see Test Procedure section). The presence of acetoin is indicated by the development of a red color within 5 to 20 min.

COMPARTMENT 10 Dulcitol green yellow (to

golden) Dulcitol (DUL): Bacterial fermentation of dulcitol, which results in the formation of acidic end products, is indicated by a change in color of the indicator present in the medium from green (alkaline) to yellow or pale yellow (acidic).

PA any shade of green

black-smoky-gray

Phenylalanine deaminase (PA): This test detects the formation of pyruvic acid from the deamination of phenylalanine. The pyruvic acid formed reacts with a ferric salt present in the medium to produce a characteristic black to smoky gray color. Ferric chloride need not to be added since the medium already contains an iron salt.

COMPARTMENT 11 Urea beige to

light amber light pink to pink

Urea (UREA): Urease, an enzyme possessed by various microorganisms, hydrolyzes urea to ammonia causing the color of the indicator in the medium to shift from yellow (acidic) to red-purple (alkaline). The urease test is strongly positive for Proteus species and may be evident as early as 4 to 6 h after incubation; it is weakly positive (light pink color) after 18 to 24 h incubation for Klebsiella and Enterobacter species.

COMPARTMENT 12 Citrate green blue Citrate (CIT): This test detects those organisms which are capable of

utilizing citrate, in the form of its sodium salt, as the sole source of carbon. Organisms capable of utilizing citrate produce alkaline metabolites which change in the color of the indicator from green (acidic) to deep blue (alkaline). Any degree of blue should be considered positive. NOTE: Certain microorganisms will not always produce the ideal „strong“ positive color change. Lighter shades of the same basic color should also be considered positive.

PERFORMANCE CHARACTERISTICS AND LIMITATIONS OF THE PROCEDURE BBL Enterotube II is designed specifically to identify Gram negative rods which are oxidase-negative and should only be used to differentiate members of the Enterobacteriaceae family. A limited number of oxidase negative nonfermenters can also be identified with the system, but the use of BBL Oxi/Ferm Tube II is recommended for these organisms.

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Performance Characteristics BBL Enterotube II has been evaluated in several external studies. Published data indicate an accuracy of identification of 87 to 99%.12-14

Reproducibility: A total of ten organisms were tested in duplicate over three days by three independent laboratories using the BBL Enterotube II. Three (30%) of the organisms showed identical biocodes with every replicate at each lab tested. Five (50%) of the organisms showed slight variations in biocodes but gave identical organisms identification with each replicate. Two (20%) of the organisms showed slight variations in biocodes with identical identification with two of 18 replicates for these organisms resulting in unidentifiable biocodes. Over all replicates, agreement in identification was 98.9%. A study of 247 previously identified strains (including known ATCC cultures) were analyzed using the BBL Enterotube II Identification System. On initial inoculation, nine organisms showed biochemical discrepants. Upon reinoculation, eight of these discrepants were resolved. In five cases the resolved result was equivalent to the reference method initial result. In three cases the resolved test was equivalent to the BBL Enterotube II initial result. In one case the resolved result was different than either initial result. The discrepant biochemical test was resolved by use of a third method which gave a similar result as the BBL Enterotube II. The study samples are summarized in Table 2. Provided is a listing of the genus, species, number of strains tested and the percent of organisms giving positive biochemical reactions. Limitations of the Procedure BBL Enterotube II is designed for the taxa provided. Taxa other than those listed in Table 3 are not intended for use in this system. BBL Enterotube II biocodes cannot be used to establish phenotypic identity between isolates from the same or different specimens. Biochemical results obtained with the BBL Enterotube II may differ from other methods and published material. Serological testing is required for confirming Salmonella and Shigella spp. Isolates of these organims should be sent to appropriate reference laboratories for complete serotyping and epidemiological surveillance. Identification of Gram negative bacteria should be made with the consideration of additional characteristics such as source of specimen, history of the patient, colonial and microscopic morphology, serology and antimicrobial susceptibility patterns. Identification of rare isolates should be repeated or additional testing performed to verify the identification of such organisms. Some strains of organisms may exhibit atypical biochemical reactions due to unusual nutritional requirements or mutations and may be difficult to identify. Some organisms may require longer than 24 hours incubation for proper identification. Although it has been recently proposed to include Plesiomonas shigelloides into the family Enterobacteriaceae, this species is not included in the BBL Enterotube II databases.1,2 Instead, this organism must be tested using the BBL Oxi/Ferm Tube II system and database. Table 2: Results of the performance evaluation

% of strains reacting positive

Organisms

No.

str

ains

te

sted

GLU

GA

S

LYS

OR

N

H2S

IND

AD

O

LAC

AR

A

SOR

DU

L

PA

UR

E

CIT

ACINETOBACTER A. anitratus 1 0 0 0 0 0 0 0 0 100 0 0 0 0 0 A. Iwoffii* 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 BURKHOLDERIA B. cepacia 1 0 0 0 0 0 0 0 0 0 0 0 0 0 100 CEDECEA C, davisae 1 100 0 0 100 0 0 0 0 0 0 0 0 0 100 C. lapagei* 1 100 0 0 0 0 0 0 100 0 0 0 0 0 100 CITROBACTER C: amalonaticus 1 100 100 0 100 0 100 0 0 100 100 0 0 100 100 C: freundii 4 100 100 0 0 100 0 0 50 100 100 0 0 50 50 ESCHERICHIA E: coli 83 100 92 100 78 0 99 0 88 99 100 1 0 0 0 ENTERIC GROUPS Enteric Group 60* 2 100 100 0 100 0 0 0 0 100 0 0 0 0 0

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Table 2: continued % of strains reacting positive

Organisms

No.

of

stra

ins

test

ed

GLU

GA

S

LYS

OR

N

H2S

IND

AD

O

LAC

AR

A

SOR

DU

L

PA

UR

E

CIT

ENTEROBACTER E. aerogenes 5 100 100 100 100 0 0 100 80 100 100 0 0 0 100 E. amnigenus bio 1 2 100 0 100 0 0 0 0 100 100 0 0 0 0 100 E. cloacae 10 100 100 10 90 0 0 10 100 100 100 20 0 10 80 EWINGELLA E: americana 1 100 0 0 0 0 0 0 0 0 0 0 0 0 100 HAFNIA H. alvei* 3 100 100 100 100 0 0 0 0 100 0 0 0 0 0 KLEBSIELLA K. oxytoca* 8 100 0 100 0 0 100 100 100 100 100 38 0 100 100 K. ozaenae 4 100 100 100 0 0 100 100 100 100 100 50 0 50 100 K. pneumoniae 20 100 100 100 0 0 0 65 100 100 100 30 0 100 100 KLUYVERA K: ascorbata* 1 100 100 100 100 0 100 0 100 100 0 0 0 0 100 MORGANELLA M: morganii 2 100 100 0 100 0 100 0 0 0 0 0 100 100 50 PROTEUS P. mirabilis* 4 100 75 0 100 100 0 0 0 0 0 0 100 100 100 PROVIDENCIA P: rettgeri* 2 100 0 0 0 0 100 100 0 0 0 0 100 100 100 P: stuartii* 1 100 0 0 0 0 100 0 0 0 0 0 100 0 100 SALMONELLA Salmonella species 41 100 100 100 98 100 0 0 0 100 100 100 0 0 100 Salmonella (ariz) IIIa 5 100 100 100 100 100 0 0 0 100 100 0 0 0 80 SERRATIA S. liquefaciens 3 100 100 100 100 0 0 0 67 100 100 0 100 100 67 S. marcescens* 5 100 20 100 100 80 0 0 0 0 80 0 0 0 100 S. plymuthica 1 100 0 0 0 0 0 0 100 100 100 0 0 0 0 SHIGELLA Shigella srgps A,B,C 10 100 0 0 0 0 20 0 0 20 0 0 0 0 0 S. sonnei 17 100 0 0 100 0 0 0 0 100 0 0 0 0 0 YERSINIA Y. enterocolitica 1 100 0 0 0 0 0 0 0 100 100 0 0 100 0 Y. frederiksenii 2 100 100 0 100 0 100 0 100 100 100 0 0 0 0 Y. ruckeri 2 100 0 50 100 0 0 0 0 0 100 0 0 0 0

* One organism from this group sourced from American Type Culture Collection Table 3: Taxa of Enterobacteriaceae included in the BBL Enterotube II databases Buttiauxella agrestis Hafnia alvei Salmonella serotype Pullorum Cedecea davisae Hafnia alvei biogroup 1 Salmonella (arizonae) IIIa Cedecea lapagei Klebsiella pneumoniae Salmonella (diarizonae) IIIb Cedecea neteri Klebsiella oxytoca Serratia marcescens Cedecea species 3 Raoultella (Klebsiella) ornithinolytica

(group 47) Serratia marcescens biogroup 1

Cedecea species 5 Klebsiella ozaenae Serratia liquefaciens Citrobacter freundii Klebsiella rhinoscleromatis Serratia rubidaea Citrobacter koseri (diversus) Kluyvera ascorbata Serratia odorifera biogroup 1 Citrobacter amalonaticus Kluyvera cryocrescens Serratia odorifera biogroup 2 Citrobacter farmeri Leclercia adecarboxylata Serratia plymuthica Edwardsiella tarda Leminorella sp. Serratia ficaria Edwardsiella tarda biogroup 1 Moellerella wisconsensis Serratia fonticola Edwardsiella hoshinae Morganella morganii Shigella serogroups A, B, C Edwardsiella ictaluri Morganella morganii biogroup 1 Shigella sonnei Enterobacter aerogenes Obesumbacterium proteus biogroup 2 Tatumella ptyseos Enterobacter asburiae Pantoea agglomerans (Enterobacter

agglomerans complex) Yersinia enterocolitica

Enterobacter cloacae Proteus mirabilis Yersinia frederiksenii Enterobacter gergoviae Proteus vulgaris Yersinia intermedia Enterobacter sakazakii Proteus penneri Yersinia kristensenii Enterobacter cancerogenus (taylorae) Proteus myxofaciens Yersinia pestis Enterobacter amnigenus biogroup 1 Providencia rettgeri Yersinia pseudotuberculosis Enterobacter amnigenus biogroup 2 Providencia stuartii Yersinia ruckeri Escherichia coli Providencia alcalifaciens Yokenella regensburgeri Escherichia coli (AD), inactive Providencia rustiganii Enteric group 58 Escherichia fergusonii Salmonella species Enteric group 59 Escherichia hermanii Salmonella serotype Typhi Enteric group 60 Escherichia vulneris Salmonella serotype Choleraesuis Escherichia blattae Salmonella serotype Paratyphi A Ewingella americana Salmonella serotype Gallinarum

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REFERENCES 1. Farmer, J.J. III. 2003. Enterobacteriaceae: introduction and identification. In: Murray, P. R., E. J. Baron, J.H.

Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C.

2. Abbott, S.L. 2003. Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and other Enterobacteriaceae. In: Murray, P. R., E. J. Baron, J.H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.). Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C.

3. Farmer, J.J, III., et al. 1985. Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. J. Clin. Microbiol. 21:46-76.

4. Ewing, W.H. 1973. Differentation of Enterobacteriaceae by biochemical reactions, U.S. Department of Health, Education and Welfare, Public Health Service, National Center for Disease Control.

5. Ewing, W.H. 1972. Biochemical characterization of Citrobacter freundii and Citrobacter diversus. U.S. Dept. of Health, Education and Welfare, Public Health Service, National Center for Disease Control.

6. Ewing, W.H., and M.A. Fife. 1971. Enterobacter agglomerans and the Herbicola-Lathyri bacteria. U.S. Dept. of Health, Education and Welfare, Public Health Service, National Center for Disease Control.

7. Darland, G., and B.R. Davis. 1973. Biochemical and serological characterization of hydrogen sulfide producing variants of Escherichia coli. U.S. Dept. of Health, Education and Welfare, Public Health Service, National Center for Disease Control.

8. Ewing, W.H. 1974. Isolation and identification of Salmonella and Shigella. U.S. Dept. of Health, Education and Welfare, Public Health Service, National Center for Disease Control.

9. Ewing, W.H., B.R. Davis, and M.A. Five. 1972. Biochemical characterization of Serratia liquefaciens and Serratia rubidaea. U.S. Dept. of Health, Education and Welfare, Public Health Service, National Center for Disease Control.

10. Brenner, D.J., et al. 1977. Taxonomic and nomenclature changes in Enterobacteriaceae. U.S. Dept. of Health, Education and Welfare, Public Health Service, National Center for Disease Control.:, Oct. 1977.

11. Ewing, W.H. 1986. Edwards and Ewing’s identification of Enterobacteriaceae. Elsevier Science Publishing Co., New York, N.Y.

12. Holmes, B. 1989. Comparative evaluation of the Roche Cobas IDA and Enterotube II systems for identifying members of the family Enterobacteriaceae. J. Clin. Microbiol. 27: 1027-1030.

13. Mastroeni, P. et al. 1983. Comparison of six systems for the identification of Enterobacteriaceae. G. Batteriol. Virol. Immunol. 76: 3-19.

14. Chiaradia, V. et al. 1983. Comparative evaluation of 3 miniaturized systems (API 20E, Enterotube II, Sensititre AP60) commonly used in clinical microbiology laboratories. Quad. Sclavo Diagn. 19: 159-175 [in Italian].

PACKAGING/AVAILABILITY BBL Enterotube II Cat. No. 273176 25 tubes FURTHER INFORMATION For further information please contact your local BD representative.

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