An Identification Scheme for Rapidly and Aerobically Growing Gram-positive Rods

Zbl. Bakt. 284, 246-254 (1996) © Gustav Fischer Verlag, Stuttgart· Jena . New York

An Identification Scheme for Rapidly and Aerobically Growing Gram-positive Rods ALEXANDER VON GRAEVENITZ and GUIDO FUNKE Department of Medical Microbiology, University of Zurich, Switzerland Received March 4, 1996 . Accepted March 25,1996

Summary An identification scheme for aerobically growing Gram-positive rods (genera Actinomyces, Arcanobacterium, Aureobacterium, Bacillus, Brevibacterium, Cellulomonas, Corynebacterium, Dermabacter, Erysipelothrix, Gardnerella, Lactobacillus, Listeria, Microbacterium, Oerskovia, Propionibacterium, Rhodococcus, Rothia, Turicella, as well as unnamed CDC groups, Clostridium tertium, and Mycobacterium fortuitumlchelonae) is presented. It is derived from the Hollis-Weaver scheme and uses catalase, oxidative/fermentative carbohydrate metabolism and motility as primary reactions. Tests for lipophilism, nitrate reduction, urease, esculin hydrolysis, the CAMP reaction, acid formation from five carbohydrates, as well as for some facultative reactions should lead to a correct diagnosis based on information available at the end of 1995.

The identification of aerobically growing Gram-positive rods (GPR) in a routine diagnostic laboratory presents several problems. Some laboratories still consider most of these bacteria as contaminants, others are content with characterizing even strains with suggestive clinical significance as "diphtheroids" (but may perform susceptibility tests), again others use miniaturized identification schemes which, however, may have a limited scope. In the past decade, a few laboratories have shown that the identification of Grampositive rods, at least from material that is normally sterile, is a worthwhile undertaking (30). Such specimens may contain strains of taxa that have always been thought to be of pathogenic significance (e. g., Actinomyces, Arcanobacterium, Listeria, some Corynebacterium spp., Clostridium tertium) or have come to be regarded as potentially significant, such as Brevibacterium, Propionibacterium, Lactobacillus, and Rothia (30). The complete spectrum can at present only be covered by traditional tests. Hollis and Weaver (18) were the first to outline an identification scheme which was later augDedicated to Prof. Dr. H. Brandis on the occasion of his 80 th birthday.

Identification of Gram-positive Rods

247

mented (17). It uses a rather large number of tests and has, in part, been superseded by new developments in taxonomy and additional species found in specimens from humans (see Tables). Here we present an identification scheme which is based on the Hollis-Weaver schemes and which will, of course, also have a limited life span. Its decision tree relies on three key reactions: catalase, the type of carbohydrate metabolism (i. e., oxidative vs. fermentative), and motility. It has been tried on numerous reference strains. The primary isolation medium is blood agar, possibly containing selective agents such as colistin plus nalidixic acid. A reminder: the Gram stain in these bacteria may, particularly in older cultures, be negative so that the use of a confirmatory test (e. g., a negative L-alanine aminopeptidase test [Merck AG] [2]) may be advisable. The catalase test is done in a traditional way using liquid media that do not contain eukaryotic cells. It has to be noted that most lactobacilli growing on blood-containing media may form a heme catalase (31). The test for fermentativelnonfermentative carbohydrate metabolism is best done on Kligler's or Triple Sugar Iron Agar with a drop of serum or Tween 80 added to make lipophilic Corynebacterium species grow (a control tube without serum or Tween 80 could be used for comparison). Nonfermentative, nonoxidative species show no change in the butt and no change or alkalinization of the slant while fermentative species show an acid (yellow) butt and a slant that is alkaline or shows no change. Nonfermentative oxidative species may colour the slant yellow but will not grow in the butt (which may, however, become yellow after prolonged incubation due to diffusion of Table 1. Genera and species of original CDC taxa (17, 18)

CDC Group ANF-l ANF-3 A-I, A-2 A-3 A-4 A-5 B-1, B-3 D-2 E F-2 G-l, G-2 1-1 1-2

JK

1 2 3,5 6

C, Corynebacterium. A, Actinomyces. C suggestive. d D, Dermabacter.

a

b

New name

References

Co" afermentans, C. auris, Turicella

8,11,25 24 28 9 6,9

C. propinquum

Oerskovia spp. Cellulomonas spp. Microbacterium sp. Cellulomonas sp. Microbacterium sp. Brevibacterium sp. C. urea!ytieum A." radingae, A. turicensis C. amyco!atum( CDC Group G C. striatum' C. amywlatum( C.jeikeium A.neuri A. bernardiae 0: 1hominis C. aew/ens

6 15 21 32 1,3 27 3 1 19 13

10 12 23

Vh V

V

+

V

+

C. xerosis

C. striatum C. minutissimum C.argentoratense C. glucuronolyticum

C. matruchotii

A A A A

A A

A A A A A A

A

A A A A

A

AI A A A

Glu

V A A V

A A

A A A A

A

A

A

V V

A

V

Suc

A V

A A

A A A A

V

A

V

A

A

A A A V

C

Mal

V A A V

V

Xyl

+ +

A A

V V

+ +

+

Lys+o Orn+ P Lac-r Lac-r grey colonies AP_s PYZ-

Lac+ r

I

f3-glucuronidase+ Whip handle (Gram)

PYZL reverse Glycogen A reverse 01129 Ri Dry colonies Yellow pigment, a-glucosidase+ V tyrosine+ 0/129 si

CAMP Others

V

V

V

Mann

23 27 18 18,27

18 17

18 18 18 13,17 13,17 12,17

18

1,3,20 33 26 4

7

18 18 18 1, 3, 7, 33

References

f

A, acid; g PYZ, pyrazinamidase; h V, variable; i 0/129 R , resistant to the vibriocidal compound 0/129; 0/129 s, susceptible to the vibriocidal compound 0/129; (any zone); k P, Propionibacterium; I esculin anaerobic+ in P. avidum, - in P. granulosum; m A, Actinomyces; n D, Dermabacter, ° Iv~. Iv~ine clec::Irhoxvl::lse, P om. ornithine decarhoxvlase: q R. Rothia, r Lac. lactose: S Ao-. alkaline ohosohatase.

* GPR, Gram-positive rods; a C, Corynebacterium; b var. intermedius is lipophilic; +, positive; d var. belfanti is nitrate-negative; e -, negative;

+

+ + V V

C. accolens C. macginleyi CDC Group F-l CDC Group G

+ + + +

+ V

+ V

R. q dentocariosa CDC Group 4

V +

V

V + +

V

V

Esc

P. acnes A. m viscosus A. neuii subsp. neuii A. neuii subsp. anitratus D.nhominis

p. k avidumlgranulosum

+ + V

V

-e

+c, d

C. a diphtheriae C. ulcerans c. pseudotuberculosis C. amycolatum

-b

Ur

Lipo- N0 3 phil ism

Species

Table 2. Catalase-positive, fermentative, nonmotile GPR *

...

~

:::

=

>Tj

0

0-

:::

~

~

CJ ... ...-< ... 2. F

;<:

?--

00

.j:>.

N

C, Corynebacterium. T, Turicella. C B, Bacillus. d M, Mycobacterium.

a

b

I

e

+

V

V

V

V

Esc

A

A

A

A V

V

V

Glu

A

A

V

Suc

V

A

A

V

V

Mal

V

V

V

Xyl

A

V

V

V

Mann

V

+

+

V +

Fructose-

Long rods Casein+, Gelatinase+, Methanethiol + Motile, oxidase+ Acid-fastness Salmon-colored colonies Yellow colonies, some motile Gelatinase-, Casein-, motile

Slight adherence to agar

CAMP Others

18 18

25

14

14

18 18

18

5, 15

11

24 18

25 8

References

R, Rhodococcus. Some sucrose-negative strains of C. minutissimum may exhibit the same biochemical reactions (maltose being +), even appearing nonfermentative but never lipophilic or fructose-negative.

V

"C. " aquaticum

+ +

V

Aureobacterium sp.

+

V V

V V

C. afermentans subsp. lipophilum C. jeikeiumf C. urealyticum

V

+

Ur

V

V

+ +

Lipo- N0 3 philism

B. C sphaericus, B. brevis M. d fortuitum chelonae R.e equi

C. propinquum C. pseudodiphtheriticum T. h otitidis Brevibacterium sp.

C." afermentans subsp. afermentans C. auris

Species

Table 3. Catalase-positive, nonfermentative GPR

~

\0

N



0 0-

i'C

(l)

::t. <:

~.

-00

a

o· ::l 0 ..... CJ .., III

~

(")

'"'

a.

(l)

0:

+

V

Cellulomonas Sp.h. c

Microbacterium sp. b

c

b

V V

Ur

V

+

+ +/(-) +

Esc

+

+

+ + +

Glu

May also be nonmotile on primary isolation or at 37°C. May be nonmotile. C. fermentans is catalase-negative.

V V

Listeria spp. Bacillus spp. Oerskovia spp.b

a

NO]

Species

Table 4. Catalase-positive, fermentative, motile" CPR

+

+

V V +

Suc

+

+

+ V +

Mal

V

V

V

Vb V

Vb V + +

Mann

Xyl + V reverse or-

CAMP

Most cellulase+, most yellow Yellow or orange

Motile at 22 0C: Straight rods

Others

6

9

18 18 18,28

References

Vo

tv

(1)

:-;-

::l

C

'T1

o

'"::l0-

J"

;:;.

::l

< (1)

(1)

'"

:<

?-..,o

o

V

+

+

Ur

+ V

_/(+)d

-1+

+ + V

Esc

+ + + + + + + + +/NC I

+ + +

Glu

+/NC

+ V + + V VINC

+ + V

Mal

V + + + + + +(-)

V

V + +

Sue

a

A. israelii: f3-galactosidase+, a-glucosidase+. Some strains do grow aerobically. A. odontolyticus: f3-galactosidase-, a-glucosidase-. b A, Actinomyces. c After ~5 days. d ( ), weak reaction only. e P, Propionibacterium. I NC, no change; ND, no data.

A.pyogenes A. bernardiae A. radingae/turicensis Arcanobacterium haemolyticum p. e propionicum Clostridium tertium Lactobacillus spp. Erysipelothrix rhusiopathiae Gardnerella vaginalis

V V

Actinomyces israeliid A." naeslundii A. odontolyticusd

+

NO)

Species

Table 5. Catalase-negative, nonmotile GPR

VlNC

+ V

+

+

V V V

Xyl

-INC

+ + V

-/(+)

V

V

Mann

reverse NDf

HzS+

Gas, motile

18 18 18 18 18 18

18

18 18 18

References

10 f3-hemolysis-/( +) 32

Brownish pigment' f3-hemolysis+

CAMP Others

V,

N

'"

P-

~

~

'"s:

...:,

S o

~

o .....

::l

o

::t.

I'l

()

S. :tl

0: <>

252

A. v. Graevenitz, and G. Funke

acid). OfF media can, of course, also be used to indicate the type of carbohydrate metabolism but several taxa will not grow on them. For the acidification of individual carbohydrates, we have, therefore, used CTA medium (Becton Dickinson) with 1 % of the substrate. CTA sugars can also be used for observation whether an organism has a fermentative (acid production at the bottom) or an oxidative (acid production at the surface) metabolism. The motility test is, for most strains, confirmatory only. It is best done with a hanging drop at either 22 °C, 30°C, or 3JOe. Of the other tests, those for nitrate reduction, Christensen's urease, esculin hydrolysis, lysine and ornithine decarboxylase, deoxyribonuclease (DNase), casein and gelatin hydrolysis, and CAMP factor (using the ~-hemolysin-producing strain S. au reus ATCC 25923) are traditional ones (16, 22). Cellulase activity can be tested by degradation of copy paper (9). Enzymes such as a-glucosidase or ~-glucuronidase may be detected in miniaturized systems such as API Coryne or API Zym (API bioMerieux, La Balme-les-Grottes, France; the former also contains the sugars used in our system). Lipophilism is best detected by growth enhancement on a blood agar plate containing 0.1 to I % Tween 80 (25). Incubation of all media is for 24-48 h, i. e., until changes in the carbohydrate media are observed. The system outlined will have to be supplemented by further traditional tests (to be found in the references), and possibly by analysis of the end products of glucose metabolism, analysis of cellular fatty acids, and cell wall analysis but only in the rare cases when it does not clearly differentiate between the taxa included in the tables.

References 1. Barreau, c., E Bimet, M. Kiredjian, N. Rouillon, and C. Bizet: Comparative chemotaxonomic studies of mycolic acid-free coryneform bacteria of human origin. J. Clin. Microbial. 31 (1993) 2085-2090 2. Carlone, G. M., M.]. Valadez, and M.J. Pickett: Methods for distinguishing Grampositive from Gram-negative bacteria. J. Clin. Microbial. 16 (1983) 1157-1159 3. de Briel, D., F. Couderc, P. Riegel, F.Jehl, and R. Minck: High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms. J. Clin. Microbiol. 30 (1992) 1407-1417 4. Funke, C., K. A. Bernard, C. Bucher, C. E. Pfyffer, and M. D. Collins: Corynebacterium glucurono/yticum sp. nov. isolated from male patients with genitourinary infections. Med. Microbiol. Lett. 4 (1995) 204-215 5. Funke, C. and A. Car/otti: Differentiation of Brevibacterium spp. encountered in clinical specimens. J. Clin. Microbiol. 32 (1994) 1729-1732 6. Funke, C., E. Fa/sen, and C. Barreau: Primary identification of Microbacterium spp. encountered in clinical specimens as CDC coryneform group A-4 and A-5 bacteria. J. Clin. Microbiol. 33 (1995) 188-192 7. Funke, C., P. A. Lawson, K. A. Bernard, and M. D. Collins: Most Corynebacterium xerosis strains identified in the routine clinical laboratory correspond to Corynebacterium amyco/atum. J. Chn. Microbiol. 34 (1996) 1124-1128 8. Funke, G., P. A. Lawson, and M. D. Collins: Heterogeneity within Centers for Disease Control and Prevention coryneform group ANF-1-like bacteria and description of Corynebacterium auris sp. nov. Int.]. Syst. Bacteriol. 45 (1995) 735-739 9. Funke, G., C. Pascual Ramos, and M. D. Collins: Identification of some clinical strains of CDC coryneform group A-3 and group A-4 bacteria as Cellulomonas species and pro po-

Identification of Gram-positive Rods

253

sal of Cellulomonas hominis sp. nov. for some group A-3 strains.]. Clin. Microbiol. 33 (1995) 2091-2097 10. Funke, G., C. Pascual Ramos, J. Fernandez-Garayzaba/, N. Weiss, and M. D. Collins: Description of human-derived Centers for Disease Control coryneform group 2 bacteria as Actinomyces bernardiae sp. nov. Int. J. Syst. Bacteriol. 45 (1995) 57-60 11. Funke, G., S. Stubbs, M. Altwegg, A. Carlotti, and M. D. Collins: Turicella otitidis gen. nov. sp. nov., a coryneform bacterium isolated from patients with otitis media. Int. J. Syst. Bacteriol. 44 (1994) 270-273 12. Funke, G., S. Stubbs, G. E. Pfyffer, M. Marchiani, and M. D. Collins: Characteristics of CDC group 3 and group 5 coryneform bacteria isolated from clinical specimens and assignment to the genus Dermabacter. J. Chn. Microbiol. 32 (1994) 1223-1228 13. Funke, G., S. Stubbs, A. uon Graeuenitz, and M. D. Collins: Assignment of humanderived CDC group 1 coryneform bacteria and CDC group I-like coryneform bacteria to the genus Actinomyces as Actinomyces neuii subsp. neuii sp. nov., subsp. nov., and Actinomyces neuii subsp. anitrdtus subsp. nov. Int. J. Syst. Bacterial. 44 (1994) 167-

171

14. Funke, G., A. UOII Graeuenitz, and N. Weiss: Primary identification of Aureobacterium spp. isolated from clinical specimens as "Corynebacterium aquaticum". J. Clin. Microbial. 32 (1994) 2686-2691 15. Gruner, E., A. C. Steigerwalt, D. G. Hollis, R. S. Weyant, R. E. Weauer, C. W. Moss, M. Daneshuar, .J. M. Brown, and D . .J. Brenner: Human infections caused by Breuibacterium casei, formerlv CDC groups B-1 and B-3. J. Clin. Microbiol. 32 (1994) 15111518 16. Hendrickson, D. A. and M. M. Krenz: Reagents and stains. In: Manual of Clinical Microbiology, 5th edition (A. Balows, W. J. Hausler Jr., K. L. Hermann, H. D. Isenberg, and H..J. Shadomy, eds.), pp. 1289-1314. American Society for Microbiology, Washington, DC (1991) 17. Hollis, D. G.: Conventional approach to the identification of coryneforms. Presented at the 92nd Gen. Meet. Am. Soc. Microbiol., May 26-30, New Orleans, LA 1992 18. Hollis, D. C. and R. E. Weauer: Gram-positive organisms: A guide to identification. Special Bacteriology Section, Centers for Disease Control, Atlanta, GA (1981) 19. Jackman, P..J. H., D. G. Pitcher, S. Pelczynska, and P. Borman: Classification of corynebacteria associated with endocarditis (group JK) as Corynebacterium jeikeium sp. nov. Syst. Appl. Microbiol. 9 (1987) 83-90 20. Martinez-Martinez, L, A. I. Suarez, .J. Winstanley, M. Carmen Ortega, and K. Bernard: Phenotypic characteristics of 31 strains of Corynebacterium striatum isolated from clinical samples. J. Clin, Microhiol. ,)3 (1995) 2458-2461 21. Pitcher, D., A. Soto, F. Soriano, and P. Valero-Guillen: Classification of coryneform bacteria associated with human urinary tract infection (group D2) as Corynebacterium urealyticum sp. nov. Int. J. Syst. Bacteriol. 42 (1992) 178-181 22. Nash, P. and M. M. Krenz: Culture media. In: Manual of Clinical Microbiology, 5th edition (A. Ba/ows, w.J. Hausler Jr., K. L. Hermann, H. D. Isenberg, and H.J. Shadomy, eds.), pp. 1226-1288. American Society for Microbiology, Washington, DC (1991) 23. Neubauer, M.,.J. Sourek, M, Rye. j. Bohacek, M. Mara, and J. Mnukoua. Corynebacterium acwlens sp. nov., a Gram-positive rod exhibiting satellitism, from clinical material. Syst. App!. Microbiol. 14 11991) 46-51 24. Riegel, P., D. de Briel, G. Prcliost, 1-: ]ehl, and H. Monteil: Proposal of Corynebacterium propinquum sp. nov. for CurVllciJaclerium group ANF-3 strains. FEMS Microbiol. Lett. 113 (1993) 229-234 25. Riegel, P., D. de Briel, G. Prcuost, Efehl, H. Monteil, and R. Minck: Taxonomic study of Corynebacterium group ANF-\ strains: proposal of Corynebacterium afermentans sp. nov. containing the subspecies C. afermentans subsp. afermentans subsp. nov. and C. afermentans subsp. lipofJhilum suhsp. nov. Int. J. Syst. Bacteriol. 43 (1993) 287292

254

A. v. Graevenitz, and G. Funke

26. Riegel, P., R. Ruimy, D. de Briel, G. Prevost, RJehl, R Bimet, R. Christen, and H. Montei/. Corynebacterium argentoratense sp. nov. from human throat. Int. J. Syst. Bacteriol. 45 (1995)533-537 27. Riegel, P., R. Ruimy, D. de Briel, G. Prevost, RJehl, R. Christen, and H. Monteil: Genomic diversity and phylogenetic relationships among lipid-requiring diphtheroids from humans and characterization of Corynebacterium macginleyi sp. nov. Int. J. Syst. Bacterio!' 45 (1995) 128-133 28. Sottnek, R 0., J. M. Brown, R. E. Weaver, and G. R Carroll: Recognition of Oerskovia species in the clinical laboratory: characterization of 35 isolates. Int. J. Syst. Bacterio!' 28 (1977) 263-270 29. Taylor, E. and 1. Phillips: The identification of Gardnerella vaginalis. J. Med. Microbio!. 16 (1983) 83-92 30. von Graevenitz, A., V. Punter, E. Gruner, G. E. Pfyffer, and G. Funke: Identification of coryneform and other Gram-positive rods with several methods. APMIS 102 (1994) 381-389 31. Whitten bury, R.: Hydrogen peroxide formation and catalase activity in the lactic acid bacteria. J. Gen. Microbio!. 35 (1964) 12-26 32. Wust,]., S. Stubbs, N. Weiss, G. Funke, and M. D. Collins: Assignment of Actinomyces pyogenes-like (CDC coryneform group E) bacteria to the genus Actinomyces as Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. Lett. App!. Microbio!. 20 (1995) 76-81 33. Zinkernagel, A. S., A. von Graevenitz, and G. Funke: Heterogeneity within Corynebacterium minutissimum strains is explained by misidentified Corynebacterium amycolatum strains. Am. J. Clin. Patho!' (1996) in press

Prof. Dr. Alexander von Graevenitz, Institut fiir Med. Mikrobiologie der Universitiit Ziirich, GloriastraBe 30/32, Postfach, CH-8028 Ziirich, Schweiz