A Dichotomous Key for the Identification of Miscellaneous Gram-Negative Bacteria

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Tech Com:

Microbiology

A Dichotomous Key for the

Identification of Miscellaneous

Gram-Negative Bacteria

by Jim Romeo, MT(ASCP)SM

In recent years the development of rapid identifica-tion systems along w i t h the availability of various new media have greatly simplified the identification of c o m m o n clinically significant bacteria. The identi-fication of certain less c o m m o n clinical isolates still

poses problems for many laboratories.1 The isolation

of u n c o m m o n bacteria, some currently classified only by letters and numbers, provides a challenge and sometimes frustration for the clinical microbi-ologist faced w i t h the task of identifying t h e m .

Many clinical laboratories in the United States now use the Center for Disease Control (CDC) bacterial

identification key2 or one of its various

modifica-tions.1'3-4 After the Gram stain and g r o w t h

charac-teristics on MacConkey agar are evaluated, and the oxidase and OF glucose reactions are d e t e r m i n e d , the choices for identification of an organism are re-duced to a g r o u p of f r o m one to 22 organisms. Various tables and written descriptions of the organisms direct one to the additional substrates or tests needed for final identification, a process that can be very t i m e - c o n s u m i n g and occasionally misleading.

To simplify this process, a supplemental dichoto-mous key was developed and has been used in our laboratory for the past t w o years. This key was

de-veloped using the data presented in the CDC key2

and crosschecked w i t h other references.1 , 5 , f iThe

con-fidence level for the branch points in the key is 90% or greater, except where indicated otherwise. It is not suggested that this key should replace tables currently in use or that the number of tests required for final identification of a particular isolate should necessarily be reduced. This key usually leads one t o a rapid preliminary identification and facilitates selec-tion of culture media for further testing and confirm-atory reactions.

Materials and Methods

The Center for Disease Control recommends the f o l l o w i n g media and tests for the identification of Jim Romeo, MT(ASCP)SM, is with the Laboratory of the King Faisal Specialist Hospital and Research Centre in Riyadh, Saudi Arabia.

organisms included in this key. The reader is referred to the references cited for more detailed descriptions

and formulas.1'2

The oxidase test is performed by adding one or t w o drops of 0.5% aqueous tetramethyl-p-phenyl-enediamine dihydrochloride to colonies on a culture plate. The Kovacs oxidase method is also satisfactory. MacConkey agar slants are used to detect g r o w t h (+) or no g r o w t h ( - ) .

Three different substrates are used to determine carbohydrate reactions for glucose, xylose, man-n i t o l , lactose, sucrose, maltose aman-nd levulose.

Semi-solid OF medium of King1 w i t h phenol red indicator

and 1 % carbohydrate is used for oxidative bacteria. Liquid peptone basal medium with Andrade's indi-cator is recommended for fermentative bacteria, and cystine tryptophan agar medium (CTA) is used for Neisseria. In the d i c h o t o m o u s key, OF or CTA pre-cedes the name of the carbohydrate where these substrates are used. Fermentative substrates may be assumed when a carbohydrate is named w i t h o u t a prefix.

Other Media and Tests

1. Blood agar plate (heart infusion agar w i t h 5%

defibrinated rabbit b l o o d at 35°C in 3% C 02) .

2. Catalase test p e r f o r m e d by f l o o d i n g t r y p t o n e -glucose-yeast extract (TGY) agar slants w i t h 3% hydrogen peroxide.

3. Christensen's urea agar slant. 4. Esculin agar.

5. Leifson flagella stain t o determine n u m b e r and arrangement of flagella.

6. Loeffler media for pigment p r o d u c t i o n of certain organisms as indicated in the key.

7. L-lysine decarboxylase, ninhydrin (Carlquist nin-hydrin base) is recommended for Pseudomonas cepacia.

8. Methyl red-Voges-Proskauer m e d i u m .

9. Moeller decarboxylase broths for lysine and o r n i t h i n e decarboxylase and arginine d i h y d r o -lase.

0007/5027/79/0900/547 $01.10 © A m e r i c a n Society of Clinical Pathologists

547

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Table 1—Organisms Listed in the CDC Key (August of Bergey's CDC Key Achromobacter species Achromobacter xylosoxidans Alcaligenes denitrificans Alcaligenes odorans HB-1 {Eikenella corrodens) Neisseria catarrhalis Neisseria fiava Neisseria perflava Pasteurella gallinarum Vibrio extorquens Vibrio fetus "Related Vibrios"

1974) which Appear under a Different Name in the Eighth Edition Manual of Determinative Bacteriology

Bergey's Manual Alcaligenes species Alcaligenes species Alcaligenes faecalis Alcaligenes faecalis Bacteroides corrodens Branhamella catarrhalis Neisseria subflava Neisseria subflava Pasteurella multocida Methylamonas species Campylobacter fetus

Campylobacter fetus ss. jejuni

10. Motility test by the hanging drop m e t h o d . 11. Motility m e d i u m .

12. Nitrate medium (infusion base incubated 48 hours).

13. Nitrate medium (peptone base incubated 48 hours).

14. Nitrite reduction b r o t h .

15. Nutrient gelatin to determine gelatin lique-faction.

16. Phenylalanine deaminase agar.

17. Pseudomonas agars for detection of pyocyanin and fluorescein (A and B media of King).

18. Simmon's citrate agar.

19. SS agar.

20. Triple sugar iron agar slant (TSI).

21. 2% tryptone broth for indole p r o d u c t i o n (incu-bate 48 hours and extract w i t h xylol before addi-t i o n of Ehrlich-Boehme reagenaddi-t).

22. Tryptone-glucose-yeast extract (TGY) agar slants are incubated at 25°C, 35°C and 42°C to deter-mine optimal growth temperatures. (Thioglycol-late broth is used for Campylobacter species; i.e., Vibrio fetus.)

It is suggested that the preliminary w o r k u p of an organism to be identified include an evaluation o f : 1) Gram stain reaction and m o r p h o l o g y , 2) TSI (triple sugar iron agar) reaction, 3) OF glucose reaction, 4)

Table II—Organisms Listed as Nomina Incertae Sedis in the Eighth Edition of Bergey's Manual

of Determinative Bacteriology

Actinobacillus actinomycetem —comitans Actinobacillus suis Comamonas terrigena Moraxella kingii Neisseria lactamicus Pasteurella anatipestifer Pseudomonas denitrificans Pseudomonas putrefaciens

oxidase reaction, and 5) growth on MacConkey agar. After making these preliminary assessments, w h i c h may be enlarged upon at the discretion of the user, other substrates or tests may be prudently selected f r o m the dichotomous key. Final confirmation of bac-terial identification may require further testing.

It should be emphasized that w i t h this g r o u p of organisms, many of the observed reactions may be weak or delayed for several days. Therefore it is often necessary to hold the test media and observe reac-tions daily for a week and occasionally longer. The OF reactions in particular may be slow, sometimes adding t o the difficulty in making an i d e n t i f i c a t i o n ; for exam pie, an organism that appears to be a glucose oxidizer initially may actually be a "weak glucose

f e r m e n t e r . " Recent revisions of the CDC key2 have

discounted the use of the term "weak f e r m e n t e r s , " and the organisms previously included under this heading are now classed as " o x i d i z e r s . " W h i l e this change has the advantage of simplifying the inter-pretation of OF glucose reactions, the suggestion of using fermentative substrates has been eliminated. Whichever way this reaction is interpreted, the tech-nologist is required to select fermentative substrates at the appropriate times. The term "weak f e r m e n t e r " is used in the key presented here w i t h a f o o t n o t e indicating that the user should consider this g r o u p whenever a positive reaction is observed only in the open tube of the OF glucose test.

Nomenclature of the Bacteria in the Key

The wide spectrum of bacteria included in this key encompasses many familiar and well-established or-ganisms of clinical significance. It also includes a number of species and groups w h i c h have been clas-sified only recently, and the taxonomic position of many of these organisms is still being debated. All of the organisms included have been isolated f r o m human sources and are considered to be potentially opportunistic pathogens. Some of the groups have 5 4 8 LABORATORY MEDICINE • VOL. 10. NO. 9. SEPTEMBER 1979

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Table III —List of CDC Groups of Unclassified Gram-Negative Bacilli

Ve 1 Pseudomonas-I ike bacteria. Possibly members of the genus Pseudomonas or possibly of the genus Ve 2 Xanthomonas

Va Ilk type 1 Ilk type 2

Vb-2 Pseudomonas-like bacteria resembling Pseudomonas stutzeri except for a positive arginine reaction Vb-3

HB-1 Eikenella corrodens (Bacteroides corrodens)

HB-5 A group of fermentative microorganisms with uniform biochemical characteristics IVe Resemble Alcaligenes species in some respects

EF-4 Unnamed fermentative bacteria

M-3 Bacteria that resemble members of the genus Moraxella M-4

M-4f M-5 M-6

lie Nonmotile organisms which demonstrate weak indole production. Similarities to Flavobacterium species lie

llf llh in iij

TM-1 Unnamed group of organisms which have been isolated frequently on Thayer-Martin media inoculated with pharyngeal swabs

not been named and are assigned l e t t e r - n u m b e r symbols at C D C . The n o m e n c l a t u r e used in this key corresponds to that used in the CDC key.2

Tables I, II and III are p r o v i d e d to clarify the no-menclature used in the d i c h o t o m o u s key. Table I lists those organisms w h i c h appear in the eighth edi-t i o n of Bergey's Manual of Deedi-terminaedi-tive Bacedi-teriol-

Bacteriol-ogy* under a new name. The taxonomic p o s i t i o n of

some of these is still contested by the CDC g r o u p .1 A c h r o m o b a c t e r , for example, is not recognized in

Bergey's Manual; rather, it is i n c l u d e d in the

discussion of Alcaligenes species. At CDC t h e genus A c h r o mobacter is defined as oxidasepositive p e r i t r i -chously flagellated gram-negative rods w h i c h are strictly aerobic and attack carbohydrates oxidatively. Alcaligenes species have similar characteristics but are nonsaccharolytic. Similarly the CDC g r o u p sug-gests that the microaerophilic bacteria f o r m e r l y designated HB-1 and later named Eikenella corrodens should be recognized as a separate genus f r o m the strict anaerobe Bacteroides corrodens.

Table IV lists the abbreviations used in Figs. 1-12. Legends for Figs. 1-12 appear on page 553.

Use of the Key

Fig. 1 shows the reactions of f o u r major groups of organisms as divided by the use of the OF glucose

test, g r o w t h on MacConkey agar and the oxidase reaction. N u m b e r s in parentheses indicate t h e a d d i -tional figures in this article w h i c h must be c o n s u l t e d to differentiate the organisms i n c l u d e d in these f o u r

Table I V

-+

-ARG beta cd db dc ig lr LYS ORN PD srps sy tr

-Abbreviations Used in the Identification Keys (Figs. 1-12)

—90% or more of strains positive —90% or more of strains negative —arginine

—beta hemolysis around isolated colonies —coccoid, diplococcoid and plump bacillary

forms

—diplobacilli, some cocci in chains —diplococci

—lavender-green coloration under heavy growth (proteolysis)

— large rods, cell diameter >0.9 /x

— lysine —ornithine

— phenylalanine deaminase

—short to medium length pale-staining rods — slight yellow or tan

—tiny coccoid rods TSI Reactions—triple sugar iron agar

N/N K/A K/N vlg

—neutral slant and butt —alkaline slant, acid butt —alkaline slant, neutral butt —very light growth

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+ —90% or more of strains positive —90% or more of strains negative ARG —arginine

beta —beta hemolysis around isolated colonies cd —coccoid, diplococcoid and plump bacillary

forms

db —diplobacilli, some cocci in chains dc —diplococci

Ig —lavender-green coloration under heavy growth (proteolysis)

lr —large rods, cell diameter aO.9 (JL LYS —lysine

ORN —ornithine PD —phenylalanine deaminase

srps —short to medium length pale-staining rods sy —slight yellow or tan

tr —tiny coccoid rods TSI Reactions—triple sugar iron agar

N N —neutral slant and butt K/A —alkaline slant, acid butt K/N —alkaline slant, neutral butt vlg —very light growth

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• O X I D I Z E R S MacCONKEY + O X I D A S E + (2) (2) (2) O X I D A S E + (3) N O N O X I D I Z E R S MacCONKEY +

I I

O X I D A S E + - O X I D A S E + (4) (5) (6) (V) F E R M E N T E R S MacCONKEY • W E A K F E R M E N T E R S MacCONKEY + - O X I D A S E + - O X I D A S E + O X I D A S E + - O X I D A S E + (8) (9) HB-5 & Enterobacteriaceae (10) I l k type 1 (11) None (12) Fig. 1

* W E A K F E R M E N T E R S are classed as O X I D I Z E R S in recent publications of the CDC key. Organisms which give a positive reaction in only the open tube of OF glucose media may actually belong in the weak fermenter group. Refer t o the discussion in the accompanying text.

A R G I N I N E I l k type 1 Pseudomonas mallei C A T A L A S E Moraxella kingii M O T I L I T Y

Brucella species Vibrio ex torquens (1 F L A G E L L A ) (PINK PIGMENT) M O T I L I T Y F L A G E L L A S T A I N or O F M A N N I T O L + L Y S I N E ' Acinetobacter calcoaceticus var. anitratum Pseudomonas A R G I N I N E cepacia or ESCULIN Pseudomonas cepacia Ve2 Ve1 *L-lysine decarboxylase, ninhydrin is recommended 1

Fig. 2

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peritrichous

F L A G E L L A

STAIN polar

OF MALTOSE FLUORESCENT PIGMENT +

"Achromobacter sp. Type 1 'Achromobacter sp. Type 2 GROWTH A T 42°C + G E L A T I N Pseudomonas putida Pseudomonas aeruginosa ( U S U A L L Y Pseudomonas P Y O C Y A N I N + ) fluorescens ( P Y O C Y A N I N - ) A R G I N I N E L Y S I N E " OF MALTOSE SS Pseudomonas cepacia (ONPG +) G E L A T I N Pseudomonas stutzeri Vb-2 ( G E L A T I N & OF LACTOSE N E G A T I V E ) G E L A T I N ' -& OF LACTOSE Vb-3 OF SUCROSE + Pseudomonas vesicularis Pseudomonas pseudomallei Va Ilk type 2 Refer t o Table I.

*L-lysine decarboxylase, ninhydrin is recommended.

Fig. 3

groups. The user of the key is cautioned to recog-nize that organisms classified as weak fermenters may appear initially to be oxidizers. Also, in certain instances it may be necessary to enrich the OF basal media by adding t w o drops of rabbit serum t o each tube of media to enhance the g r o w t h of fastidious organisms.

Pseudomonas mallei and Ilk type 1 are the only oxidative gram-negative rods w h i c h characteristically do not grow on MacConkey agar and are oxidase negative. As shown in Fig. 2a, they may be differen-tiated by the arginine dihydrolase test. Ilk type 1 colonies exhibit a deep yellow pigment.

Oxidizers w h i c h grow on MacConkey agar and are oxidase negative can be differentiated first by motil-ity (Fig. 2c). N o n m o t i l e strains of Pseudomonas cepacia may be differentiated from/4c/nefo6acferca/-coaceticus by flagella stains and OF mannitol reac-tions. Acinetobacter is characteristically pleomor-phic on Cram stain, while Pseudomonas species show straight or slightly curved rods. Lysine decar-boxylase separates P. cepacia f r o m the Ve g r o u p .

Oxidizers w h i c h grow on MacConkey agar and are oxidase positive (Fig. 3) include Pseudomonas spe-cies, Pseudomonas-like bacteria and Achromobacter species. Flagella stains are very useful and are some-times required for final identification of this g r o u p of

Oxidizers w h i c h do not grow on MacConkey agar and are oxidase positive (Fig. 2b) can be easily dif-ferentiated by Gram stain characteristics, by colonial pigmentation and by the motility and catalase tests. Brucella species are small gram-negative rods, usu-ally coccobacillary, and produce small nonpig-mented colonies. Vibrio extorquens (Methylamonas) morphologically are curved or branched, and the colonies have a pink pigment due to the presence of carotenoid. Bordetella parapertussis G R A M S T A I N cd Ir Acinetobacter caicoaceticus var. Iwofii (Mima polymorpha) Bacillus species Fig. 4

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O l O l to I-

>

CD

o

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URE A CATALAS E BET A HEMOLYSI S P D *HB-1 (Eikenella corrodens) Moraxella bo vis NITRAT E + _ MOTILIT Y + Neisseria d c GRA M STAI N d b 1 flavescens | I GELATI N Neisseria catarrhal is Moraxella phenylpyruvica Vibrio extorquens Bacillu s specie s GELATI N P D + INDOL E + Moraxella ***Pasteurella phenylpyruvica anatipestifer GRA M STAI N + I r GELATI N GRA M STAI N d b d c srp s Neisseria flavescens GELATI N WIT H ADDE D SERU M + *HB-1 (Eikenella corrodens) Bacillus antfiracis MOTILIT Y Bacillu s specie s tr GRA M STAI N d b "Vibrio extorquens Brucell a specie s Moraxella osloensis HEMOLYSI S lq bet a NITRATE* * + Moraxella lacunata Moraxella osleonsis Moraxella nonliquefaciens NITRAT E ll f d b GRA M STAI N * * 'Pasteurella anatipestifer Moraxella lacunata Moraxella bovis Bacillus cereus * Refe r t o Tabl e I . * * M. osloensis i s 28 % nitrat e positive . ** P. anatipestifer i s show n a s ure a positiv e i n th e CD C ke y an d ure a negativ e i n Bergey's Manual. Fig. 5 by guest on September 2, 2016 http://labmed.oxfordjournals.org/ Downloaded from

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_

cd GRAM STAIN Acinetobacter calcoaceticus var. Iwoffi (gram-negative, coccoid, some rod forms, some chains)

OF MALTOSE tr

"1

Bordetella parapertussis (gram-negative, short rods) + Pseudomonas maltophilia LYS+ ARGO R N -Fig. 6

organisms. A flagella stain should be p e r f o r m e d on any organism w h i c h has the characteristics of this g r o u p and fails to produce fluorescein (fluorescent pigment). Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas putida, w h e n g r o w n on appropriate media, are the only organisms in the key that produce a fluorescent pigment w h e n viewed under ultraviolet light. O f these three organisms, P. aeruginosa is the most frequent isolate b u t , since P. fluorescens and P. putida are occasionally isolated f r o m clinical specimens, confirmatory tests should be p e r f o r m e d . Demonstration of the presence of pyocyanin may be used to c o n f i r m the identification of P. aeruginosa; but, when this pigment is absent, P. fluorescens and P. putida are possibilities. As shown in Fig. 3, the best tests to differentiate the three organisms are g r o w t h at 42°C and gelatin lique-faction. Susceptibility patterns are also helpful in recognizing these organisms. P. aeruginosa generally is susceptible to carbenicillin and resistant to kana-mycin and tetracycline. This pattern generally is

re-versed w i t h P. putida and P. fluorescens.5

The three organisms listed in Fig. 4 are easily dif-ferentiated by colony and Gram stain m o r p h o l o g y . Acinetobacter species are c o c c o i d , diplococcoid and p l u m p bacillary forms, and occasionally long thick filaments w i t h large swollen areas are seen. Typically, rods (1.0-1.5 /xm by 1.5-2.5 ^im) are seen d u r i n g the logarithmic stage of g r o w t h . Brucella species are tiny coccobacilli (0.2-0.3 /xm by 0.5-1.0 /xm) occur-ring singly, in pairs or rarely in short chains. Bacillus species are large straight rods (0.3-2.2 /xm by 1.2-7.0 /xm). These organisms are usually gram-positive dur-ing early g r o w t h stages, but are frequently gramnegative in older cultures. Bacillus species are i n -cluded here and elsewhere in the key because they are occasionally isolated (usually as contaminants) in the clinical laboratory and may stain gram-negative.

Fig. 5 includes a g r o u p of organisms difficult t o identify, requiring careful observations of Gram stain m o r p h o l o g y and hemolytic reactions on b l o o d agar

Legends for Figures

Fig. 1.

Four major groups of gram-negative organisms as divided by OF glucose reactions. Numbers in parentheses refer to the figure numbers of the identification keys.

Fig. 2.

Identification keys for organisms with the following character-istics: oxidizers that do not grow on MacConkey agar and are oxidase negative (Fig. 2a); oxidizers that do not grow on MacConkey agar and are oxidase positive (Fig. 2b); and oxi-dizers that grow on MacConkey agar and are oxidase negative (Fig. 2c).

Fig. 3

Identification key for organisms that are oxidizers, grow on MacConkey agar and are oxidase positive.

Fig. 4

Identification key for organisms that are nonoxidizers, do not grow on MacConkey agar and are oxidase negative.

Fig. 5

Identification key for organisms that are nonoxidizers, do not grow on MacConkey agar and are oxidase positive.

Fig. 6

Identification key for organisms that are nonoxidizers, grow on MacConkey agar and are oxidase negative.

Fig. 7

Identification key for organisms that are nonoxidizers, grow on MacConkey agar and are oxidase positive.

Fig. 8

Identification key for organisms that are fermenters, do not grow on MacConkey agar and are oxidase negative.

Fig. 9

Identification key for organisms that are fermenters, do not grow on MacConkey agar and are oxidase positive.

Fig. 10

Identification key for organisms that are fermenters, grow on MacConkey agar and are oxidase positive.

Fig. 11

Identification key for organisms that are weak fermenters, do not grow on MacConkey agar and are oxidase positive.

Fig. 12

Identification key for organisms that are weak fermenters, grow on MacConkey agar and are oxidase positive.

(heart infusion agar containing 5% defibrinated rab-bit b l o o d is recommended). Moraxella species are typically short, p l u m p rods (1.0-1.5 /xm by 1.5-2.5 /xm) approaching coccoid shape, usually in pairs or short chains, and p l e o m o r p h i c forms are often seen. Neisseria species are spherical cells seen in pairs or masses with flattened adjacent sides. The Gram stain m o r p h o l o g y of Brucella species and Bacillus

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MOTILIT Y P D or URE A CITRAT E GELATI N + 'CATALAS E M-6 'SLIGH T YELLO W PIGMEN T M-4 (rarel y Moraxella + osloensis) Moraxella phenylpyruvlca M-4 f Moraxella osloensis o r M-3 1-2 pola r flagell a microaerophili c M-5 1-2 pola r flagell a FLAGELL A STAI N pola r tuf t wit h mor e peritrichou s flagell a tha n 2 flagell a GROWT H a t 42 ° C + H 2 S(TSI ) + URE A * Vibrio fetus (Campylobacte r species ) "Relate d Vibrios " **GELATI N + Pseudomonas putrefaciens O F XYLOS E + + Comamonas terrigena S S + O F LEVULOS E + Pseudomonas diminuta Pseudomonas alcaligenes o r *Pseudomonas denitrificans N0 3 -*ga s *Achromobacter IV e Bordetella N0 2 -^ga s + xylosoxidans bronchiseptica Pseudomonas pseud oalcaligenes F R UIT Y * Alcaligenes ODO R + denitrificans Ili a N0 3 — N0 2 MlbN0 3 —N0 2 +ga s Alcaligenes faecalis * Alcaligenes odorans Refe r t o Tabl e I . * * Confidenc e leve l o f branchpoin t les s tha n 90% . by guest on September 2, 2016 http://labmed.oxfordjournals.org/ Downloaded from

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NITRATE Haemophilus vaginalis (Corynebacterium vaginale) (INDOLE & C A T A L A S E - , NG or N/N T! TSI A/A) Haemophilus aphrophilus (CATALASE - or weak+)

species was previously described. Pasteurella anati-pestifer appears in Sergey's Manual" under species incertae sedis and is " p r o b a b l y a Moraxella species."

Nonoxidizers that grow on MacConkey agar and are oxidase negative (Fig. 6) can be differentiated by OF maltose reaction and Gram stain m o r p h o l o g y as described.

The large group of nonoxidizers in Fig. 7 is first divided by the motility test into a subgroup of Moraxella species and a larger heterogenous group of motile organisms possessing flagella. Motility tests c o m m o n l y employed for the identification of the Enterobacteriaceae are not satisfactory. The reader should refer to references cited for more detailed discussion on motility testing.

Comamonas terrigena is the only organism in Fig. 7 w h i c h possesses polar tufts of three or more fla-gella; this is the single most important identifying characteristic of this organism. The organisms w i t h peritrichous flagella are differentiated by urease pro-d u c t i o n , OF-xylose reaction anpro-d the repro-duction of nitrates and nitrites. The Pseudomonas species in-cluded in Fig. 7 are characterized by one to t w o polar flagella. Pseudomonas putrefaciens is easily recog-nized by the production of H_,S on TSI agar. The other four species are not easily separated. Positive gelatin liquefaction separates Pseudomonas dimin-uta, but only about 75% exhibit this characteristic. Pseudomonas denitrificans is listed in Bergey's

Manual1'' as species incertae sedis and is not

de-scribed. There is no clear differentiation of this or-ganism f r o m Pseudomonas alcaligenes by the reac-tions used in this key.

The Campylobacter species are unique compared to the other organisms included in Fig. 7 because of their microaerophilic properties. Best g r o w t h is obtained in a reduced oxygen atmosphere.

Occa-+

INDOLE + A / A o r K / A HB-5 [ (TSI A/A, CATALASE - ) Actinobacillus actinomycetem<omitans or Bacillus species (Both CATALASE +) 8

sionally some strains grow slightly under aerobic conditions. Campylobacter species grow well in t h i o -glycollate b r o t h , and tests for g r o w t h at 42°C should be performed in this m e d i u m .

Nitrate reduction, indole, TSI and catalase reac-tions differentiate the four fermenters in Fig. 8.

Ac-cording to Bergey's Manual,B Haemophilus vaginalis

does not belong in the genus Haemophilus but its taxonomic position is not yet settled. Because it is widely identified by this name, it still appears in the manual.

In the group of fermenters that are oxidase posi-tive and fail to grow on MacConkey agar (Fig. 9), Neisseria species are easily separated f r o m other or-ganisms in this g r o u p by Gram stain m o r p h o l o g y . Cardiobacterium hominis is separated by a negative catalase test. CTA carbohydrate substrates are used for identification of the Neisseria species. Loeffler slants are useful for demonstrating the y e l l o w pig-ment of the f o l l o w i n g Neisseria species: N. lac-tamicus, N. sicca, N. flava, N. subflava, and N. mucosa.

Pasteurella "gas" is possibly a strain of Pasteurella multocida w h i c h is urea positive and usually pro-duces gas f r o m glucose. The o r n i t h i n e decarboxylase reaction is negative, separating it f r o m Pasteurella pneumotropica according to the CDC key.

Urea, indole, Moeller decarboxylase and carbo-hydrate fermentations speciate the nitrate-positive organisms in Fig. 9.

Fermenters w h i c h grow on MacConkey agar and are oxidase negative (Fig. 1) include the Enterobac-teriaceae and a group of fermentative organisms designated as HB-5. The latter may show light g r o w t h on MacConkey agar and produce acid in the slant and butt on TSI. Indole production by HB-5 is de-monstrable only after extraction w i t h xylol. Catalase,

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NITRAT E CATALAS E URE A Cardiobacterium hominis d c GRA M STAI N tr-c d INDOL E INDOL E - CT A LACTOS E + EF-4 tr-c d GRA M STAI N d c CT A SUCROS E + Neisseria lactamicus CT A LEVULOS E + Neisseria I sicca EF-4 Neisseria subflava 'Neisseria flava SUCROS E MALTOS E + Neisseria mucosa SUCROS E Pasteurella ureae o r Bacillu s Pasteurella specie s "gas" (multocida)

'Pasteurella gallinarum or Bacillu

s HB-5 specie s LYSIN E Pasteurella multocida ORINITHIN E + •Vibrio cholera Pasteurella pneumotropica Refe r t o Tabl e I . '* N. mucosa usuall y produce s a yello w o r sligh t yello w pigmen t o n Loeffle r media . Mucoi d characteristic s var y wit h th e medi a used . 10 'MOTILIT Y URE A LYSIN E Actinobacillus lignieresii TREHALOS E +

I

- ESCULI N GELATI N + ARGININ E SUCROS E Actinobacillus Actinobacillus equui SUIS Providencia species (oxidase — o r wea k + ) SUCROS E + Aeromonas shigelloides Vibrio parahaemoly ticus LEVULOS E + Pasteurella haemoly tica MANNITO L + slo w LACTOS E + EF-4 HB-5 (CATALASE+ ) (CATALAS E o r wea k + ) Vibrio AN T 1,0: 1 alginolyticus I Chromobacterium Aeromonas * * Non-choler a Vibrio violaceum hydrophila Vibri o cholera ' Motil e b y pola r flagell a excep t Chromobacteriu m (pola r an d latera l flagella ) an d Providenc e (peritrichou s flagella) . '* Refe r t o text . by guest on September 2, 2016 http://labmed.oxfordjournals.org/ Downloaded from

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d c GRA M STAI N r(o r deb ) SPECIA L GROWT H REQUIREMENT S CT A DEXTROS E +

I

CTASUCROS E + INDOL E YELLO W PIGMEN T an d - CATALAS E + YELLO W PIGMEN T an d GELATI N + CT A MALTOS E + - NITRAT E + - CT A LACTOS E + Neisseria gonorrhoeae - CT A LEV U LOS E + Neisseria I lac tarnicus GROWT H I N *Neisseria - NUTRIEN T BROT H + flava Neisseria meningitidis Neisseria subflava Neisseria sicca o r "Neisseria pert lava Neisseria mucosa Il k typ e 1 MALTOS E TM-1 PIGMEN T sligh t yello w ver y yello w Flavobacterium Flavobacteriu m meningosepticum sp . li b Moraxella kingii SUCROS E + MALTOS E li e H i ESCULI N + li e ll h Refe r t o Tabl e II . Fig. 11 GLUCOS E LACTOS E SUCROS E MALTOS E + INDOL E an d GELATI N Il k typ e 1 Il k typ e 2 PIGMEN T sligh t yello w ver y yello w Flavobacterium meningosep ticum Flavobacteriu m sp . li b Fig. 12 by guest on September 2, 2016 http://labmed.oxfordjournals.org/ Downloaded from

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urea, citrate, arginine, lysine, o r n i t h i n e and H2S (TSI)

are negative. Few carbohydrates are f e r m e n t e d . Providence species are included w i t h fermenters that grow on MacConkey agar and are oxidase posi-tive (Fig. 10) because members of this species occa-sionally give a weak positive oxidase reaction. Or-ganisms in this group w h i c h may resemble members of the family Enterobacteriaceae are easily separated by their positive oxidase reaction; this suggests the need for routine use of the oxidase test even w h e n isolates appear to be enteric organisms.

In Fig. 10, " n o n - c h o l e r a v i b r i o " refers t o strains of Vibrio cholera w h i c h belong to antigenic O groups other than the 0 : 1 group to w h i c h cholera vibrios belong." Classic cholera is always associated w i t h strains belonging to serogroup 0 : 1 . Strains belong-ing t o the other groups produce a milder f o r m of the disease.

A g r o u p of Pseudomonas-like organisms desig-nated Ilk type 1 are the only weak fermenters w h i c h are oxidase negative and fail to grow on MacConkey agar (Fig. 1).

Weak fermenters which do not grow on MacConkey agar and are oxidase positive (Fig. 11) include t w o groups of organisms. Neisseria species are differen-tiated f r o m Flavobacterium species and other fer-menters by Gram stain. Neisseria species are gram-negative cocci occurring singly but m o r e often in pairs. O n Gram stain, flavobacteria are long t h i n gram-negative rods, usually w i t h bulbous ends. Ilk type 1 organisms are long rods. Gram stains of Moraxella kingii and TM-1 demonstrate m o r p h o l o g i c characteristics as described earlier for Moraxella spe-cies. Indole p r o d u c t i o n is an important characteristic of Flavobacterium species; however, the reaction is weak and the indole must be extracted w i t h xylol before addition of Ehrlich-Boehme reagent.

The four organisms classed as weak fermenters that grow on MacConkey agar and are oxidase posi-tive (Fig. 12) are differentiated by i n d o l e , gelatin, pigment and carbohydrate fermentation reactions. Ilk type 1 is easily differentiated f r o m Ilk type 2 in that the former fails to ferment any of the four carbo-hydrates shown and the latter ferments all of t h e m .

Discussion

The key presented here contains over 100 species of bacteria w h i c h are subdivided into groups, pro-viding adequate information for presumptive identification, although it is r e c o m m e n d e d in many i n -stances that further confirmatory tests be p e r f o r m e d . The primary advantages of the key are to quickly eliminate certain species f r o m a g r o u p and t o allow the rapid selection of tests to be p e r f o r m e d . Once a preliminary identification is achieved, the micro-biologist can refer to detailed descriptions and select additional tests as needed.

The organisms included in the key f o r m a large heterogenous g r o u p , w i t h the only c o m m o n charac-teristic being their gram-negative staining reaction. Many of the organisms show only subtle differences in biochemical reactions and m o r p h o l o g y . Publica-tions discussing the numbers of organisms received at CDC indicate that many of the groups or species presented have been characterized by studies on fewer than 50 organisms. Obviously, changes in tax-o n tax-o m y may be expected in the f u t u r e , and clinical microbiologists must continue to update their identi-fication schema.

The challenge for the laboratory w o r k e r is to make very careful observations and identify organisms to the fullest extent possible, referring to qualified ref-erence or state laboratories w h e n necessary for

identification of unusual organisms.

References

1. Lennette. E.H., Spaulding, E.H., and Truant. J.P., 1974. Manual of Clinical Microbiology. 2nd edition. Washington. D.C.. American Society for Microbiology.

2. Weaver, R.E., Tatum, H.W.. and Hollis, D.G.. 1975 The Identification of Unusual Pathogenic Gram Negative Bacteria (E.O. King). Atlanta, Georgia, Special Bacteriology Section, Bacteriology Division. Center for Disease Control

3. Wolf, P L , Russell, B., and Shimoda, A., 1975. Practical Clinical Mi-crobiology and Mycology. New York, John Wiley and Sons, pp. 143-150. 4. Frankel, S.. Reitman. S.. and Sonnenwirth. A C 1970 Gradwohl's

Clinical Laboratory Methods and Diagnosis. 7th edition. St. Louis, C.V Mosby, pp 1269-1352

5. Finegold, S.M., Martin, W.J., and Scott, E.G.. 1978. Bailey and Scott's Diagnostic Microbiology. 5th edition. St. Louis. C.V. Mosby.

6. Buchanan, R.E., and Gibbons, N.E.. 1974. Bergey's Manual of Deter-minative Bacteriology. 8th edition. Baltimore, Williams and Wilkins •

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