Page 1

3.18

Schemes for Identification of Aerobic Bacteria

3.18.1

Identification of Gram-Positive Bacteria [Updated March 2007]

I.

PRINCIPLE

Unlike gram-negative rods, it can be very difficult to sort out the identification of gram-positive cocci and rods. Many kits for staphylococcal identification have proved to be less sensitive than desired, and new DNA studies indicate that we have misidentified many streptococci. Gram-positive rods have been difficult to identify because there are hundreds of named species and thousands of genotypes or biochemical variants found in the environment and the normal microbiota of the human body, including skin, mucosal membranes, oropharynx, and genitourinary and gastrointestinal tracts. Thus, it is not within the scope of this handbook to identify all isolates, but to detect and iden-

tify the known pathogenic microorganisms in the human biosphere and to limit other identifications to those bacteria that are involved in disease from invasively collected specimens. The figures and tables that follow are designed to rapidly determine the agents of infection and to provide guidance for when to perform a kit identification or pursue other microorganisms. The charts may not include all the options at each step, since the observance of a colony morphology may lead to performing a test but the lack of that morphology may suggest doing a different test. The figures are designed to do the minimum to arrive at an excellent identi-

II.

Gram-positive rods or cocci as determined by Gram stain. 䊓 NOTE: Because of the lack of a unique colony morphology of many grampositive microorganisms, the Gram stain must be performed on all isolates. Colonies of Streptococcus and Aerococcus may appear similar, but these organisms can be differentiated by the arrangement of the cells in the Gram stain. Group B streptococci and Listeria colonies may also have a similar appearance. Gram-variable microorganisms are considered to be gram positive. When in doubt, the Gram reaction enzymatic test (procedure 3.17.20) may be helpful.

MICROORGANISMS TESTED

III. MEDIA, REAGENTS, AND SUPPLIES

䊓 NOTE: See individual tests in procedure 3.17 for methods for use of tests. To the extent that tests are available in kits, it is not necessary to stock the separate tests. A. Media 1. Bile-esculin (procedure 3.17.5) 2. Glucans (optional) (procedure 3.17.19) 3. H2S production medium (procedure 3.17.22) 4. Human blood agar for Gardnerella

fication, without doing further tests unless the morphology warrants them. For species identification of viridans group streptococci, enterococci, and corynebacteria, commercial kits are easier to perform than standard tube biochemical tests (see Tables 3.16–1, 3.16–3, and 3.16–4) but are still limited in their accuracy. No system available can do as good a job as cell wall analysis and DNA studies, both of which are beyond the scope of most laboratories and beyond reasonable cost. For identification of other aerobic gram-positive rods in the actinomycete group, refer to section 6 of this handbook; for further information on Actinomyces spp., refer to section 4 and reference 5.

5. Lipophilism test (procedure 3.17.28) 6. Ornithine and arginine decarboxylase (procedure 3.17.15) 7. 6.5% NaCl broth (procedure 3.17.43) 8. Broth for motility (procedure 3.17.31) 9. Lecithinase agar (procedure 3.17.27) B. Reagents and supplies 1. Catalase (3% H2O2) (procedure 3.17.10)

3.18.1.1


3.18.1.2

III. MEDIA, REAGENTS, AND SUPPLIES (continued)

IV.

PROCEDURE

V. REPORTING AND INTERPRETATION OF RESULTS

Aerobic Bacteriology 2. Polymyxin B (300 U), novobiocin (5 g), bacitracin (0.4 U), and vancomycin disks (procedure 3.17.4) 3. CAMP test (procedure 3.17.8) 4. Coagulase by rabbit plasma and (optionally) agglutination (procedures 3.17.13 and 3.17.14) 5. Spot bile reagent (procedure 3.17.6) 6. Hippurate (optional) (procedure 3.17.21) 7. Leucine aminopeptidase (LAP) (procedure 3.17.26) 8. Optochin disks (procedure 3.17.38)

9. Streptococcal grouping antisera (section 11) 10. Pyrrolidonyl-b-naphthylamide (PYR) (procedure 3.17.41) 11. Urea test (procedure 3.17.48) 12. Kits for identification of enterococci and viridans group streptococcus gram-positive and grampositive rod identifications (corynebacterium and anaerobe kits) (Tables 3.16–1, 3.16–3, and 3.16–4)

A. Observe colony morphology on BAP and CHOC if growth is lacking on BAP. B. Perform catalase and Gram stain from BAP or CHOC. 䊓 NOTE: To avoid misidentifications, do not skip this step. C. Use Fig. 3.18.1–1 if the organism is a catalase-positive, gram-positive coccus. D. Use Fig. 3.18.1–2 if organism is a beta-hemolytic, catalase-negative, grampositive coccus. E. Use Fig. 3.18.1–3 if organism is a catalase-negative, gram-positive coccus that is not hemolytic (except those with characteristic group B streptococcus morphology) and either is PYR negative or does not grow on BAP, except around staphylococci. F. Proceed to Fig. 3.18.1–4 if the catalase-negative, gram-positive coccus is PYR positive and not identified from the other figures. G. For gram-positive rods, proceed to Fig. 3.18.1–5. 1. If isolate is catalase positive a. Perform CAMP test (and test for lipophilism, if microorganism demonstrates small colonies or poor growth at 24 h). b. Check for motility by wet mount from young growth of either broth or agar cultures. 2. If isolate is catalase negative, follow Fig. 3.18.1–5 based on the anatomical site of isolation.

A. Follow tables and kit identifications to report genus and species as appropriate without delay. B. Use commercial kits for identification of Enterococcus faecalis and Enterococcus faecium, but perform motility testing on vancomycin-intermediate or -resistant E. faecium.


Identification of Gram-Positive Bacteria

3.18.1.3

Figure 3.18.1–1 Flowchart for identification of catalase-positive, gram-positive cocci.

Figure 3.18.1–2 Flowchart for identification of catalase-negative, gram-positive cocci, either beta-hemolytic or nonhemolytic, with morphology of group B streptococci.


3.18.1.4

Aerobic Bacteriology

Figure 3.18.1–3 Flowchart for identification of gram-positive, catalase-negative, cocci that are not beta-hemolytic. R, resistant; S, susceptible.

VI.

LIMITATIONS

A. A number of gram-positive cocci are either coagulase or agglutination positive but are not Staphylococcus aureus, making identification problematic. B. Some staphylococci are catalase-negative. See the aminolevulinic acid (ALA) test for options. C. Streptococci are increasingly difficult to identify to the species level, even with commercial kits. The LAP test is important to at least confirm the genus of streptococci or enterococci. D. Gram-positive rods are most difficult to identify. Every laboratorian should be able to recognize Listeria monocytogenes, Erysipelothrix rhusiopathiae, Bacillus cereus, Arcanobacterium haemolyticum, and Gardnerella vaginalis and be able to presumptively recognize Bacillus anthracis and Corynebacterium diphtheriae. Some other Corynebacterium species are identified using a commercial kit. For other gram-positive rods of significance, a reference laboratory is usually needed.


Identification of Gram-Positive Bacteria

3.18.1.5

Figure 3.18.1–4 Flowchart for identification of PYR-positive, catalase-negative, grampositive cocci. R, resistant; S, susceptible; VRE, vancomycin-resistant enterococci; MGP, methyl-�-D-glucopyranoside.


3.18.1.6

Aerobic Bacteriology

Figure 3.18.1–5 Guide to distinguish genera and significant species of gram-positive rods. VP, Voges-Proskauer. (Refer to section 6 for aerobic actinomycetes.)


Identification of Gram-Positive Bacteria

3.18.1.7

Table 3.18.1–1 Key biochemical reactions of the common and/or significant gram-positive cocci that are catalase positive with large white to yellow coloniesa Organism(s) Rothia mucilaginosa Micrococcus groupc S. aureus S. intermedius (dogs) Staphylococcus delphini (dolphins) Staphylococcus hyicus (pigs) S. lugdunensis S. schleiferi S. saprophyticus Staphylococcus epidermidis S. haemolyticus Staphylococcus caprae Other coagulase-negative staphylococci

Selected characteristic(s) Adherent, sticky Often yellow VP Ⳮ VP ⳮ VP ⳮ VP ⳮ Ornithine Ⳮ Ornithine ⳮ Urine; novo R; nonhemolytic Nonhemolytic Urease ⳮ, VP Ⳮ, DNase ⳮ Urease Ⳮ, DNase Ⳮ Novo V, urease V; nonhemolytic or delayed hemolysis

Slide coag

SAG

Tube coag

V V

Ⳮ V

Ⳮ Ⳮ

NA

ⳮ V Ⳮ ⳮ ⳮ ⳮ ⳮ

NA V V V ⳮ ⳮ ⳮ

V ⳮ ⳮⳭ ⳮ ⳮ ⳮ ⳮ

V

Bacit (0.04 U)

Poly B (300 U)

PYRb

S S R R R

R S R S NA

V V ⳮ Ⳮ NA

R R R R R R R R

R R S S R S S S

ⳮ Ⳮ Ⳮ ⳮ ⳮ Ⳮ Ⳮ V

Symbols and abbreviations: Ⳮ, greater than 90% of strains positive in 48 h; ⳮ, greater than 90% of strains negative; V, results are between 90 and 10% positive; ⳮⳭ, most strains are negative but rare positive strains exist; NA, not applicable or available; R, resistant; S, susceptible; Bacit, bacitracin; Poly B, polymyxin B; coag, coagulase; SAG, staphylococcal protein A or clumping factor agglutination; VP, Voges-Proskauer; Novo, novobiocin. Data are from references 1, 6, 12, 15, 16, 19, 23, and 30. Catalase can be weak for Rothia. b PYR data are for broth test. Weak positive results with S. aureus ATCC 29213 and ATCC 25923 occur with the disk test, suggesting that this test is unreliable to separate S. aureus from S. intermedius (M. York, personal communication). c Includes related taxa. The genus Micrococcus has been divided into additional genera, including Kytococcus and Kocuria. a


3.18.1.8

Aerobic Bacteriology

Table 3.18.1–2 Separation of the common groups of viridans group streptococci (PYR-negative, LAP-positive, 6.5% NaCl-negative cocci in chains)a Group

Species

VP

ARG

MAN

SOR

Esculin

Mutans

S. mutans, S. sobrinus, S. ratti

V

Ⳮ, puddles and droplets

␣, b. Sometimes dry and adherent. S. sobrinus can be sorbitol negative; S. ratti is arginine positive.

Salivarius

S. salivarius, S. vestibularis

Ⳮ, mucoid, firm

␣, c. S. vestibularis is ␣, VP and esculin variable, and glucan negative

Bovis

I

Ⳮ, watery, spreads

II/1 II/2

Ⳮ Ⳮ

ⳮ ⳮ

ⳮ ⳮ

ⳮ ⳮ

Ⳮ Ⳮ

Ⳮ, watery, spreads ⳮ

c. Bile-esculin positive (3). Taxonomic revisions suggest that human S. bovis I isolates be renamed as S. gallolyticus, and biotypes II/2 and II/1 have proposed species names S. pasteurianus and S. infantarius, respectively (3, 4, 10, 27)

Anginosus (“S. milleri”)

S. anginosus, S. constellatus, S. intermedius

ⳮⳭ

Ⳮⳮ

␣, b, c. S. constellatus is divided into subspecies constellatus and pharyngis. All species are esculin positive, except that S. constellatus subsp. constellatus is esculin variable (28).

Mitis

S. sanguis, S. parasanguis, S. gordonii, S. cristatus

V

Ⳮⳮ

V, hard, adheres to agar

S. mitis, S. oralis

ⳮⳭ

V

␣. S. cristatus may be arginine and esculin negative. S. parasanguis may be esculin negative. S. sanguis may be sorbitol positive. ␣. Can be pencillin resistant. S. oralis can be esculin positive.

a

Glucans

Hemolysis and comments

Abbreviations: ARG, hydrolysis of arginine; MAN, acid production from mannitol; SOR, acid production from sorbitol; Ⳮⳮ, most strains positive but rare negative strains exist. See Table 3.18.1–1, footnote a for other abbreviations and symbols. Strains do not always produce glucans; test is only useful if positive. Commercial kits for identification of streptococci are helpful to resolve variable reactions. Data are extrapolated from reference 35. Also see references 7, 10, 25, and 28.


ⳮ ⳮ ⳮ ⳮ ⳮ Ⳮⳮ Ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ

ⳮ ⳮ Ⳮ

ⳮ Ⳮⳮ Ⳮⳮ ⳮ

ⳮ ⳮ

ⳮ ⳮ ⳮ

E. avium E. raffinosus Vagococcus fluvialis

E. faecium E. gallinarum E. casseliflavus E. mundtii

E. faecalisd Lactococcus garvieae

E. durans E. hirae E. dispar

ⳮ ⳮ Ⳮ

ⳮ ⳮ

ⳮ Ⳮ Ⳮ ⳮ

V V Ⳮ

MGP

Ⳮ Ⳮ Ⳮ

Ⳮⳮ Ⳮ

Ⳮ Ⳮⳮ Ⳮⳮ Ⳮ

ⳮ ⳮ ⳮ

Arginine dihydrolase

ⳮ ⳮ ⳮ

ⳮ ⳮ

Ⳮ Ⳮ Ⳮ Ⳮ

Ⳮ Ⳮ ⳮ

Arabinose

ⳮ ⳮ ⳮ

Ⳮⳮ Ⳮ

Ⳮⳮ Ⳮ Ⳮ Ⳮ

Ⳮ Ⳮ Ⳮ

Mannitol

Ⳮ Ⳮ Ⳮ

Ⳮd Ⳮ

Ⳮ Ⳮ Ⳮ Ⳮ

Ⳮ Ⳮ ⳮ

Lactose

ⳮ Ⳮ Ⳮ

ⳮ ⳮ

V Ⳮ D Ⳮ

ⳮ Ⳮ ⳮ

Raffinose

Ⳮ Ⳮ Ⳮ

Ⳮ Ⳮ

Ⳮ Ⳮ V Ⳮ

Ⳮ Ⳮ Ⳮ

Ribose

ⳮ ⳮ ⳮ

Ⳮ ⳮ

V D V V

Ⳮ Ⳮ Ⳮ

Sorbitol

Ⳮ Ⳮ ⳮ

Ⳮ ⳮ

Ⳮ Ⳮ Ⳮ Ⳮ

Ⳮ Ⳮ ⳮ

45⬚C growth

S S S

V S

V R R S

S V S

Vancomycin

a

All species grow well on BAP and in 6.5% NaCl and are PYR, bile-esculin, and LAP positive. PYR-negative species E. cecorum, E. columbae, and E. saccharolyticus are not included and have not been isolated from humans. PYR-positive strains E. malodoratus, E. pseudoavium, E. asini, and E. sulfureus (H2SⳭ) also are not listed since they have not been isolated from humans. E. gilvus and E. pallens have been described to occur in humans but are extremely rare. Abbreviations: MGP, methyl-␣-D-glucopyranoside; D, different reactions in references. See footnote a to Tables 3.18.1–1 and 3.18.1–2 for other abbreviations and symbols. Table adapted from references 11, 26, and 33. Also see references 20, 21, and 32. b Motility is done in 0.5 ml of BHI or TSB incubated at 30⬚C for 2 h. c Pigment (yellow) is observed by swabbing a blood agar plate incubated at 35⬚C in 5% CO2 for 24 to 48 h and observing swab for bright yellow color (Ⳮ). d Lactose-negative asaccharolytic E. faecalis exists.

Pigmentc

Motilityb

Organism

Table 3.18.1–3 Common species of enterococci and related PYR-positive cocci in chainsa

Identification of Gram-Positive Bacteria 3.18.1.9


3.18.1.10

Aerobic Bacteriology

Table 3.18.1–4a Biochemical reactions of PYR-positive, catalase-negative or weakly positive, gram-positive cocci (excluding Streptococcus pyogenes) Phenotypic characteristicsa Genus or species Enterococccus (some motile) Lactococcus Vagococcus (motile) Abiotrophia/Granulicatella Globicatella Dolosicoccus Aerococcus viridans Helcococcus kunzii Gemella Facklamia (hippurate Ⳮ) Alloiococcus otitis Ignavigranum (hippurate ⳮ) Rothia mucilaginosa Dolosigranulum a

Gram stain

CAT

LAP

NaCl

10⬚C

45⬚C

Colony on BAP

Hemolysis

Bile-esculin

CH CH CH CH CH CH CL/T CL/T CL/T/CH CL/CH CL/T CL/CH CL CL/T

ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ, W ⳮ ⳮ ⳮ W, Ⳮ ⳮ ⳮ, W, Ⳮ ⳮ

Ⳮ Ⳮ Ⳮ Ⳮ ⳮ ⳮ ⳮ ⳮ V Ⳮ Ⳮ Ⳮ Ⳮ Ⳮ

Ⳮ V Ⳮ ⳮ Ⳮ ⳮ Ⳮ V ⳮ Ⳮ Ⳮ Ⳮ ⳮ Ⳮ

Ⳮ Ⳮ Ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ NA ⳮ

Ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ NA ⳮ

Large Large Large Satellite Small Small Large Tiny Tiny, 48 h to grow Small Tiny, 72 h to grow Satellite (V) or small Sticky Small

␣, c, b ␣, c ␣, c ␣, c ␣ ␣ ␣ c ␣, c c ␣ c c c

Ⳮ Ⳮ Ⳮ ⳮ V NA V ⳮ ⳮ ⳮ NA ⳮ V NA

CAT, catalase production; NaCl, growth in broth containing 6.5% NaCl; 10⬚C and 45⬚C, growth at 10 and 45⬚C, respectively (for the latter, use campylobacter incubator if heat block not available). Abbreviations for cell arrangement in Gram stain: CL, clusters; T, tetrads; CH, chains; W, weak. Large colonies are approximately 1 mm; small colonies are about the size of viridans group streptococci. See Table 3.18.1–1, footnote a, for other abbreviations and symbols. Tables adapted from references 9, 17, 22, and 25. Also see reference 6.

Table 3.18.1–4b Biochemical reactions of PYR-negative, catalase-negative gram-positive cocci Phenotypic characteristicsa Genus or species Leuconostoc Weissella confusa Pediococcus Streptococcus Aerococcus urinae a

Gram stain

LAP

NaCl

Van

Arginine

45⬚C

MRS

CH, rods CH, rods CL/T CH CL/T

ⳮ ⳮ Ⳮ Ⳮ Ⳮ

V V V ⳮ Ⳮ

R R R S S

ⳮ Ⳮ

ⳮ Ⳮ V V ⳮ

Ⳮ Ⳮ ⳮ ⳮ ⳮ

Van, vancomycin; MRS, gas production in MRS broth. See footnote a to Table 3.18.1–4a and Table 3.18.1 for other abbreviations.


Identification of Gram-Positive Bacteria

3.18.1.11

Table 3.18.1–5 Catalase-negative, gram-positive rods that can grow aerobicallya Organism(s)

H2S Vancomycin Hemolysis Hippurate Motility Nitrate Esculin

Weissella spp. Erysipelothrix rhusiopathiae

ⳮ Ⳮ

R R

Alpha Alpha

ⳮ ⳮ

ⳮ ⳮ

NA ⳮ

Ⳮ ⳮ

Lactobacillus spp.

R

Alpha

V

V

Arcanobacterium haemolyticum

S

Beta

Arcanobacterium pyogenes

S

Beta

Arcanobacterium bernardiae

S

V

V

Gardnerella vaginalis Bifidobacterium spp.

S

Ⳮⳮ

NA

S

Actinomyces israelii

S

NA

Actinomyces spp.

S

V

V

V

Aerotolerant Clostridium

S

NA

V

Ⳮⳮ

a

Gram stain morphology Small, short GPR; gas in MRS broth (22) Has two cell forms; the long-chaining form can be confused with Lactobacillus, and the short form can be confused with Actinomyces or even Enterococcus (PYR positive) Most lactobacilli are long regular-chaining rods. Some are C shaped. Branching, which can be rudimentary; reverse-CAMP positive, lecithinase positive, gelatin negative Branching, which can be rudimentary; reverse-CAMP negative, lecithinase negative, gelatin positive Reverse-CAMP negative, gelatin negative; does not branch; not beta-hemolytic on human blood. Kits can misidentify as Gardnerella. SPS sensitive; beta-hemolytic on human blood Some are aerotolerant; can look like Actinomyces or Gardnerella; not beta-hemolytic on human blood Branching, which can be rudimentary; anaerobic kits will identify; urease negative Not all species show branching; A. naeslundii and others are urease positive. Some colonies of A. meyerii-odontolyticus group turn red after 1 wk. Forms spores; medium to large regular rod; grows slowly compared to Bacillus; anaerobic kits will identify

Data from references 2, 5, 8, and 24. Once G. vaginalis, Arcanobacterium, Weissella, and E. rhusiopathiae are ruled out, either call “Anaerobic grampositive rod” or do anaerobic identification kit. GPR, gram-positive rods; SPS, sodium polyanethol sulfonate. See footnote a to Tables 3.18.1–1 and 3.18.1– 2 for other abbreviations and symbols. See Table 3.18.1–9 for catalase-positive Actinomyces.


3.18.1.12

Aerobic Bacteriology

Table 3.18.1–6 Catalase-positive, usually yellow- or pink-pigmented gram-positive rodsa Organism(s)

Motility Fermentation Nitrate Urease Esculin Gelatin Glucose

Cellulosimicrobium/Cellulomonas (Oerskovia) spp. Microbacterium (Aureobacterium) spp. Exiguobacterium acetylicum Leifsonia aquatica Corynebacterium falsenii Corynebacterium lipophiloflavum Corynebacterium mucifaciens Rhodococcus equi

a

Comment

Ⳮ/V

Ⳮⳮ

V

V

V

Ⳮⳮ

V

V

V

Golden yellow to orange

V

V

V

V

W

V

W

V

NA

W

Previously called “Corynebacterium aquaticum” Yellow after 72 h

W

Lipophilic, rarely isolated

V

Slightly to deep yellow mucoid colonies, CAMP negative Mucoid in 48 h, usually pink after 4–7 days, can be yellowish, CAMP positive. Can be acid fast. Important pathogen.

Subsurface or surface hyphae/pseudohyphae may be present.

If motile and yellow or positive for esculin and/or gelatin, report as “Motile coryneform, not Corynebacterium spp.” If of clinical significance, use kit (e.g., Coryne API or RapID Coryne) or send to reference laboratory. Other yellow Corynebacterium spp. (C. aurimucosum, C. falsenii, and C. sanguinis) are rarely isolated or associated with human disease. Data are from references 13, 14, 29, and 31; also see section 6. See footnote a to Tables 3.18.1–1, 3.18.1– 2, and 3.18.1–3 for abbreviations and symbols.

Table 3.18.1–7 Large, regular catalase-positive, gram-positive rods that usually produce spores and are usually motilea Organism(s) B. anthracis B. cereus Bacillus thuringiensis Bacillus mycoides Bacillus megaterium Other Bacillus spp. and related groupsc

Diam of cell usually above 1 lm

Motilityb

Beta-hemolysis

Lecithinase

Penicillin

Large colony at 24 h

Sticky, tenacious colony at 24 h

Ⳮ Ⳮ Ⳮ Ⳮ Ⳮ ⳮ

ⳮ Ⳮ Ⳮ, V ⳮ Ⳮ, V Ⳮ, V

ⳮ Ⳮ Ⳮ W V ⳮⳭ

Ⳮ Ⳮ Ⳮ Ⳮ ⳮ ⳮ

Sb R R R S V

Ⳮ Ⳮ Ⳮ Ⳮ Ⳮ V

Ⳮ ⳮ ⳮ ⳮ ⳮ V

B. cereus, B. thuringiensis (insect pathogen used in horticulture), B. mycoides (rhizoids or hairy projections in agar), and B. anthracis are included in the B. cereus group. If organism is motile with spores, penicillin resistant, hemolytic, with cells greater than 1 lm in diameter, and/or lecithinase positive, report as “Bacillus cereus group, not B. anthracis.” Otherwise, if organism is motile, with spores, but nonhemolytic and/or lecithinase negative, report as “Bacillus, not B. anthracis or B. cereus.” Data are from references 18 and 34. Spores can be induced by growing on urea, bile-esculin agar, or an agar plate with vancomycin disk or at 45⬚C. Spores can be proved by heating broth culture to 80⬚C for 10 min and subculturing to BAP. Viable colonies indicate that spores survived the heating. See Table 3.18.1–1, footnote a, for other abbreviations and symbols. b Submit any nonmotile, spore-forming strain to designated higher reference laboratory to rule out B. anthracis, regardless of the penicillin susceptibility. c Kurthia (diameter, 0.8 to 1.2 lm) organisms are motile and nonhemolytic and do not produce spores (13). a


Identification of Gram-Positive Bacteria

3.18.1.13

Table 3.18.1–8 Urease-positive Corynebacterium spp. of clinical importancea Organism

Nitrate

Urease

Pyrazinamidase

Glucose

Sucrose

Lipophilism

CAMP reaction

C. glucuronolyticum C. pseudotuberculosisb C. ulceransb C. pseudodiphtheriticumc C. riegeliid C. urealyticume C. amycolatum CDC group F1

V V ⳮ ⳮ ⳮ ⳮ V V

V Ⳮ Ⳮ Ⳮ Ⳮ Ⳮ V Ⳮ

Ⳮ ⳮ ⳮ Ⳮ V Ⳮ Ⳮ Ⳮ

Ⳮ Ⳮ Ⳮ ⳮ ⳮ ⳮ Ⳮ Ⳮ

Ⳮ V ⳮ ⳮ ⳮ ⳮ V Ⳮ

ⳮ ⳮ ⳮ ⳮ ⳮ Ⳮ ⳮ Ⳮ

Ⳮ Reverse Ⳮ Reverse Ⳮ ⳮ ⳮ ⳮ ⳮ ⳮ

Data are from references 13 and 14. Other urease-positive or -variable species of less clinical significance include C. durum, C. falsenii, C. singulare, C. sundsvallense, and C. thomssenii, which are CAMP and reverse-CAMP negative, are not lypophilic, and ferment glucose. See Table 3.18.1–1, footnote a, for abbreviations and symbols. b Submit to reference laboratory for diphtheria toxin testing. C. pseudotuberculosis is associated with sheep handlers. c Respiratory pathogen; not able to acidify maltose, ribose, or trehalose. d C. riegelii is rarely isolated but has been found in urine and other body sites (13). It is able to acidify maltose. e Urinary pathogen; multiresistant to antimicrobials. a

Table 3.18.1–9 Catalase-positive, urease-negative, gram-positive rods, excluding Corynebacterium spp. and yellow- or pink-pigmented rodsa Gram stain or colony appearance

Organism(s)

Fermentation

Nitrate

Esculin

Gelatin

Glucose

CAMP

Actinomyces neuii Actinomyces viscosus Propionibacterium avidum/granulosum Turicella otitidis

Ⳮ Ⳮ Ⳮ

V Ⳮ ⳮ

ⳮ ⳮ V

ⳮ NA V

Ⳮ Ⳮ Ⳮ

Ⳮ ⳮ Ⳮ

Nonhemolytic, slight branching

Brevibacterium spp. Dermabacter hominis

ⳮ Ⳮ

V ⳮ

ⳮ Ⳮ

Ⳮ Ⳮ

ⳮ Ⳮ

ⳮ ⳮ

Rothia spp.

V

Large rod, branching; ear pathogen CAMP-positive C. auris and C. afermentans have similar reactions Some yellowish, distinct odor Coccoid rods, distinct odor Lysine Ⳮ, arginine ⳮ, ornithine Ⳮ Some branching, some black pigmented; if sticky, refer to Tables 3.18.1–1 and 3.18.1–4a for R. mucilaginosa (previously Stomatococcus mucilaginosus) (6).

a

Beta-hemolytic, branching

Usually irregular rods. Identify only if clinically significant, but all can be pathogens (13, 14). The important tests are fermentation (use Andrade’s base or cysteine Trypticase agar), CAMP, and Gram stain, with careful reading of Gram stain morphology. Coryneform identification kits can be helpful. For abbreviations and symbols, see Table 3.18.1–1, footnote a.


3.18.1.14

Aerobic Bacteriology

Table 3.18.1–10 Urease-negative Corynebacterium spp. of clinical importancea Organism(s)

Nitrate Urease Pyrazinamidase

Alkaline Glucose Maltose Sucrose Lipophilism CAMP phosphatase

C. accolens CDC group Gb C. jeikeiumb C. afermentans

Ⳮ V ⳮ ⳮ

ⳮ ⳮ ⳮ ⳮ

V Ⳮ Ⳮ V

ⳮ Ⳮ Ⳮ Ⳮ

Ⳮ Ⳮ Ⳮ ⳮ

ⳮ V V ⳮ

V V ⳮ ⳮ

Ⳮ Ⳮ Ⳮ V

ⳮ ⳮ ⳮ V

C. macginleyi

C. diphtheriaec

C. propinquum C. amycolatumb

Ⳮ V

ⳮ V

V Ⳮ

V Ⳮ

ⳮ Ⳮ

ⳮ V

ⳮ V

ⳮ ⳮ

ⳮ ⳮ

C. minutissimum

V

C. striatum C. xerosis

Ⳮ V

ⳮ ⳮ

Ⳮ Ⳮ

Ⳮ Ⳮ

Ⳮ Ⳮ

ⳮ Ⳮ

V Ⳮ

ⳮ ⳮ

V ⳮ

Comment(s)

Fructose positive Fructose negative One subspecies is lipophilic. Found in eye specimens. C. diphtheriae/belfanti is nitrate negative; C. diphtheriae/intermedius is lipophilic. Dry colony, O/129 R, a common species of human resident microbiota; can be misidentified as C. xerosis. O/129 S, DNase positive, PYR positive O/129 S Creamy colony, O/129 S, LAP positive

Other species that are rare are not listed (see references 13 and 14), including some CAMP test-positive species. Some Corynebacterium organisms have black-pigmented colonies. For identification of species in this table, the combination of CAMP test, lipophilism, O/129 disk, and commercial kits for corynebacteria should be used if identification is clinically important. For abbreviations and symbols, see footnote a to Tables 3.18.1–1 and 3.18.1–9. b Multiresistant to antimicrobials. c Submit to reference laboratory for diphtheria toxin testing. a

REFERENCES

1. Bannerman, T. 2003. Staphylococcus, Micrococcus, and other catalase-positive cocci that grow aerobically, p. 384–404. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C. 2. Bille, J., J. Rocourt, and B. Swaminathan. 2003. Listeria and Erysipelothrix, p. 461–471. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C. 3. Chuard, C., and L. B. Reller. 1998. Bile-esculin test for presumptive identification of enterococci and streptococci: effects of bile concentration, inoculation technique, and incubation time. J. Clin. Microbiol. 36:1135– 1136. 4. Clarridge, J. E., III, J. S. M. Attorri, Q. Zhang, and J. Bartell. 2001. 16S ribosomal DNA sequence analysis distinguishes biotypes of Streptococcus bovis: Streptococcus bovis biotype II/2 is a separate genospecies and the predominant clinical isolate in adult males. J. Clin. Microbiol. 39:1549–1552.

5. Clarridge, J. E., III, and Q. Zhang. 2002. Genotypic diversity of clinical Actinomyces species: phenotype, source, and disease correlation among genospecies. J. Clin. Microbiol. 40:3442–3448. 6. Collins, M. D., R. A. Hutson, V. Baverud, and E. Falsen. 2000. Characterization of a Rothia-like organism from a mouse: description of Rothia nasimurium sp. nov. and reclassification of Stomatococcus mucilaginosus as Rothia mucilaginosa comb. nov. Int. J. Syst. Evol. Microbiol. 50:1247–1251. 7. Coykendall, A. L. 1989. Classification and identification of the viridans streptococci. Clin. Microbiol. Rev. 2:315–328. 8. Dunbar, S. A., and J. E. Clarridge III. 2000. Potential errors in the recognition of Erysipelothrix rhusiopathiae. J. Clin. Microbiol. 38:1302–1304. 9. Facklam, R., and J. A. Elliott. 1995. Identification, classification, and clinical relevance of catalase-negative, gram-positive cocci, excluding the streptococci and enterococci. Clin. Microbiol. Rev. 8:479–495.


Identification of Gram-Positive Bacteria

REFERENCES (continued)

3.18.1.15 10. Facklam, R. 2002. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin. Microbiol. Rev. 15:613–630. 11. Facklam, R. R., and M. D. Collins. 1989. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J. Clin. Microbiol. 27:731–734. 12. Falk, D., and S. J. Guering. 1983. Differentiation of Staphylococcus and Micrococcus spp. with the Taxo A bacitracin disk. J. Clin. Microbiol. 18:719–721. 13. Funke, G., and K. A. Bernard. 2003. Coryneform gram-positive rods, p. 472–501. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C. 14. Funke, G., A. von Graevenitz, J. E. Clarridge III, and K. Bernard. 1997. Clinical microbiology of coryneform bacteria. Clin. Microbiol. Rev. 10:125–159. 15. He´bert, G. A. 1990. Hemolysins and other characteristics that help differentiate and biotype Staphylococcus lugdunensis and Staphylococcus schleiferi. J. Clin. Microbiol. 28:2425–2431. 16. He´bert, G. A., C. G. Crowder, G. A. Hancock, W. R. Jarvis, and C. Thornsberry. 1988. Characteristics of coagulase-negative staphylococci that help differentiate these species and other members of the family Micrococcaceae. J. Clin. Microbiol. 26:1939–1949. 17. LaClaire, L. L., and R. R. Facklam. 2000. Comparison of three commercial rapid identification systems for the unusual gram-positive cocci Dolosigranulum pigrum, Ignavigranum ruoffiae, and Facklamia species. J. Clin. Microbiol. 38:2037–2042. 18. Logan, N. A., and P. C. B. Turnbull. 2003. Bacillus and other aerobic endospore-forming bacteria, p. 445–460. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C. 19. Mahoudeau, I., X. Delabranche, G. Prevost, H. Monteil, and Y. Piemont. 1997. Frequency of isolation of Staphylococcus intermedius from humans. J. Clin. Microbiol. 35:2153–2154. 20. Manero, A., and A. R. Blanch. 1999. Identification of Enterococcus spp. with a biochemical key. Appl. Environ. Microbiol. 65:4425–4430. 21. Murray, B. E. 1990. The life and times of the Enterococcus. Clin. Microbiol. Rev. 3:46–65. 22. Olano, A., J. Chua, S. Schroeder, A. Minari, M. La Salvia, and G. Hall. 2001. Weissella confusa (basonym: Lactobacillus confusus) bacteremia: a case report. J. Clin. Microbiol. 39:1604–1607. 23. Patel, R., K. E. Piper, M. S. Rouse, J. R. Uhl, F. R. Cockerill III, and J. M. Steckelberg. 2000. Frequency of isolation of Staphylococcus lugdunensis among staphylococcal isolates causing endocarditis: a 20-year experience. J. Clin. Microbiol. 38:4262–4263.

24. Reimer, L. G., and L. B. Reller. 1985. Use of a sodium polyanetholesulfate disk for the identification of Gardnerella vaginalis. J. Clin. Microbiol. 21:146–149. 25. Ruoff, K. L. 2002. Miscellaneous catalase– negative, gram-positive cocci: emerging opportunists. J. Clin. Microbiol. 40:1129–1133. 26. Ruoff, K. L., L. de la Maza, M. J. Murtagh, J. D. Spargo, and M. J. Ferraro. 1990. Species identities of enterococci isolated from clinical specimens. J. Clin. Microbiol. 28:435–437. 27. Ruoff, K. L., S. I. Miller, C. V. Garner, M. J. Ferraro, and S. B. Calderwood. 1989. Bacteremia with Streptococcus bovis and Streptococcus salivarius: clinical correlates of more accurate identification of isolates. J. Clin. Microbiol. 27:305–308. 28. Ruoff, K. L., R. A. Whiley, and D. Beighton. 2003. Streptococcus, p. 405–421. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C. 29. Schumann, P., N. Weiss, and E. Stackebrandt. 2001. Reclassification of Cellulomonas cellulans (Stackebrandt and Keddie 1986) as Cellulosimicrobium cellulans gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 51:1007–1010. 30. Shuttleworth, R., R. J. Behme, A. McNabb, and W. D. Colby. 1997. Human isolates of Staphylococcus caprae: association with bone and joint infections. J. Clin. Microbiol. 35:2537–2541. 31. Takeuchi, M., and K. Hatano. 1998. Union of the genera Microbacterium Orla-Jensen and Aureobacterium Collins et al. in a redefined genus Microbacterium. Int. J. Syst. Bacteriol. 48:739–747. 32. Teixeira, L. M., M. G. Carvalho, V. L. Merquior, A. G. Steigerwalt, D. J. Brenner, and R. R. Facklam. 1997. Phenotypic and genotypic characterization of Vagococcus fluvialis, including strains isolated from human sources. J. Clin. Microbiol. 35:2778–2781. 33. Teixeira, L. M., and R. R. Facklam. 2003. Enterococcus, p. 422–433. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8th ed. ASM Press, Washington, D.C. 34. Turnbull, P. C. B., and J. M. Kramer. 1991. Bacillus, p. 296–303. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 35. Whiley, R. A., and D. Beighton. 1998. Current classification of the oral streptococci. Oral Microbiol. Immunol. 13:195–216.

Identification of gram positive