3.18.1.

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3.18
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3.18.1 Identication of Gram-Positive
Bacteria
[Updated March 2007]
I. PRINCIPLE
Unlike gram-negative rods, it can be very
difcult to sort out the identication of
gram-positive cocci and rods. Many kits
for staphylococcal identication have
proved to be less sensitive than desired,
and new DNA studies indicate that we
have misidentied many streptococci.
Gram-positive rods have been difcult to
identify because there are hundreds of
named species and thousands of genotypes
or biochemical variants found in the en-
vironment and the normal microbiota of
the human body, including skin, mucosal
membranes, oropharynx, and genitouri-
nary 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 microorgan-
isms in the human biosphere and to limit
other identications to those bacteria that
are involved in disease from invasively
collected specimens. The gures and ta-
bles that follow are designed to rapidly de-
termine the agents of infection and to pro-
vide guidance for when to perform a kit
identication or pursue other microorgan-
isms.
The charts may not include all the op-
tions at each step, since the observance of
a colony morphology may lead to per-
forming a test but the lack of that mor-
phology may suggest doing a different
test. The gures are designed to do the
minimum to arrive at an excellent identi-
cation, without doing further tests unless
the morphology warrants them. For spe-
cies identication of viridans group strep-
tococci, enterococci, and corynebacteria,
commercial kits are easier to perform than
standard tube biochemical tests (see Ta-
bles 3.161, 3.163, and 3.164) 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 identi-
cation 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.
II. MICROORGANISMS TESTED
Gram-positive rods or cocci as determined by Gram stain.
NOTE: Because of the lack of a unique colony morphology of many gram-
positive 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 strepto-
cocci and Listeria colonies may also have a similar appearance. Gram-variable
microorganisms are considered to be gram positive. When in doubt, the Gram re-
action enzymatic test (procedure 3.17.20) may be helpful.
III. MEDIA, REAGENTS, AND
SUPPLIES
NOTE: See individual tests in proce-
dure 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. H
2
S production medium(procedure
3.17.22)
4. Human blood agar for Gardnerella
5. Lipophilism test (procedure
3.17.28)
6. Ornithine and arginine decarbox-
ylase (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% H
2
O
2
) (procedure
3.17.10)
2. Polymyxin B (300 U), novobiocin
(5 g), bacitracin (0.4 U), and van-
comycin disks (procedure 3.17.4)
3. CAMP test (procedure 3.17.8)
4. Coagulase by rabbit plasma and
(optionally) agglutination (proce-
dures 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)
3.18.1.2 Aerobic Bacteriology
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 identication of entero-
cocci and viridans group strepto-
coccus gram-positive and gram-
positive rod identications
(corynebacterium and anaerobe
kits) (Tables 3.161, 3.163, and
3.164)
III. MEDIA, REAGENTS, AND
SUPPLIES (continued)
IV. PROCEDURE
A. Observe colony morphology on BAP and CHOC if growth is lacking on BAP.
B. Perform catalase and Gram stain from BAP or CHOC.
NOTE: To avoid misidentications, do not skip this step.
C. Use Fig. 3.18.11 if the organism is a catalase-positive, gram-positive coccus.
D. Use Fig. 3.18.12 if organism is a beta-hemolytic, catalase-negative, gram-
positive coccus.
E. Use Fig. 3.18.13 if organism is a catalase-negative, gram-positive coccus that
is not hemolytic (except those with characteristic group B streptococcus mor-
phology) and either is PYR negative or does not grow on BAP, except around
staphylococci.
F. Proceed to Fig. 3.18.14 if the catalase-negative, gram-positive coccus is PYR
positive and not identied from the other gures.
G. For gram-positive rods, proceed to Fig. 3.18.15.
1. If isolate is catalase positive
a. Perform CAMP test (and test for lipophilism, if microorganism demon-
strates 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. 3.18.15 based on the anatomical
site of isolation.
V. REPORTING AND
INTERPRETATION OF RESULTS
A. Follow tables and kit identications to report genus and species as appropriate
without delay.
B. Use commercial kits for identication of Enterococcus faecalis and Enterococ-
cus faecium, but perform motility testing on vancomycin-intermediate or -re-
sistant E. faecium.
Identication of Gram-Positive Bacteria 3.18.1.3
Figure 3.18.11 Flowchart for identication of catalase-positive, gram-positive cocci.
Figure 3.18.12 Flowchart for identication of catalase-negative, gram-positive cocci,
either beta-hemolytic or nonhemolytic, with morphology of group B streptococci.
3.18.1.4 Aerobic Bacteriology
Figure 3.18.13 Flowchart for identication 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 identication problematic.
B. Some staphylococci are catalase-negative. See the aminolevulinic acid (ALA)
test for options.
C. Streptococci are increasingly difcult to identify to the species level, even with
commercial kits. The LAP test is important to at least conrm the genus of
streptococci or enterococci.
D. Gram-positive rods are most difcult to identify. Every laboratorian should be
able to recognize Listeria monocytogenes, Erysipelothrix rhusiopathiae, Bacil-
lus cereus, Arcanobacterium haemolyticum, and Gardnerella vaginalis and be
able to presumptively recognize Bacillus anthracis and Corynebacterium
diphtheriae. Some other Corynebacterium species are identied using a com-
mercial kit. For other gram-positive rods of signicance, a reference laboratory
is usually needed.
Identication of Gram-Positive Bacteria 3.18.1.5
Figure 3.18.14 Flowchart for identication of PYR-positive, catalase-negative, gram-
positive cocci. R, resistant; S, susceptible; VRE, vancomycin-resistant enterococci; MGP,
methyl--D-glucopyranoside.
3.18.1.6 Aerobic Bacteriology
Figure 3.18.15 Guide to distinguish genera and signicant species of gram-positive rods.
VP, Voges-Proskauer. (Refer to section 6 for aerobic actinomycetes.)
Identication of Gram-Positive Bacteria 3.18.1.7
Table 3.18.11 Key biochemical reactions of the common and/or signicant gram-positive cocci that are catalase positive with large
white to yellow colonies
a
Organism(s) Selected characteristic(s) Slide coag SAG Tube coag
Bacit
(0.04 U)
Poly B
(300 U)
PYR
b
Rothia mucilaginosa Adherent, sticky S R V
Micrococcus group
c
Often yellow S S V
S. aureus VP V R R
S. intermedius (dogs) VP V V R S
Staphylococcus delphini
(dolphins)
VP
NA
R NA NA
Staphylococcus hyicus (pigs) VP NA V R R
S. lugdunensis Ornithine V V R R
S. schleiferi Ornithine V

R S
S. saprophyticus Urine; novo R; nonhemolytic V R S
Staphylococcus epidermidis Nonhemolytic R R
S. haemolyticus Urease , VP , DNase R S
Staphylococcus caprae Urease , DNase R S
Other coagulase-negative
staphylococci
Novo V, urease V; nonhemo-
lytic or delayed hemolysis
V
R S V
a
Symbols and abbreviations: , greater than 90% of strains positive in 48 h; , greater than 90% of strains negative; V, results are between 90 and 10%
positive;

, 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.
3.18.1.8 Aerobic Bacteriology
Table 3.18.12 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 Glucans Hemolysis and comments
Mutans S. mutans, S. sobri-
nus, S. ratti
V , puddles and
droplets
, b. Sometimes dry and adher-
ent. S. sobrinus can be sorbi-
tol negative; S. ratti is argi-
nine positive.
Salivarius S. salivarius,
S. vestibularis
, mucoid, rm , c. S. vestibularis is , VP
and esculin variable, and glu-
can negative
Bovis I
II/1
II/2

, watery, spreads
, watery, spreads

c. Bile-esculin positive (3). Tax-

onomic 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. infan-
tarius, respectively (3, 4, 10,
27)
Anginosus
(S. milleri)
S. anginosus, S.
constellatus, S.
intermedius

, b, c. S. constellatus is di-
vided into subspecies constel-
latus and pharyngis. All spe-
cies are esculin positive,
except that S. constellatus
subsp. constellatus is esculin
variable (28).
Mitis S. sanguis, S. para-
sanguis, S. gor-
donii, S. crista-
tus
S. mitis, S. oralis

V, hard, adheres to
agar
V
. S. cristatus may be arginine
and esculin negative. S. para-
sanguis may be esculin nega-
tive. S. sanguis may be sorbi-
tol positive.
. Can be pencillin resistant. S.
oralis can be esculin positive.
a
Abbreviations: ARG, hydrolysis of arginine; MAN, acid production from mannitol; SOR, acid production from sorbitol;

, most strains positive but rare

negative strains exist. See Table 3.18.11, footnote a for other abbreviations and symbols. Strains do not always produce glucans; test is only useful if
positive. Commercial kits for identication of streptococci are helpful to resolve variable reactions. Data are extrapolated from reference 35. Also see
references 7, 10, 25, and 28.
Identication of Gram-Positive Bacteria 3.18.1.9
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3.18.1.10 Aerobic Bacteriology
Table 3.18.14a Biochemical reactions of PYR-positive, catalase-negative or weakly positive, gram-positive cocci (excluding
Streptococcus pyogenes)
Phenotypic characteristics
a
Genus or species
Gram stain CAT LAP NaCl 10C 45C Colony on BAP Hemolysis Bile-esculin
Enterococccus (some motile) CH Large , c, b
Lactococcus CH V Large , c
Vagococcus (motile) CH Large , c
Abiotrophia/Granulicatella CH Satellite , c
Globicatella CH Small V
Dolosicoccus CH Small NA
Aerococcus viridans CL/T , W Large V
Helcococcus kunzii CL/T V Tiny c
Gemella CL/T/CH V Tiny, 48 h to grow , c
Facklamia (hippurate ) CL/CH Small c
Alloiococcus otitis CL/T W, Tiny, 72 h to grow NA
Ignavigranum (hippurate ) CL/CH Satellite (V) or small c
Rothia mucilaginosa CL , W, NA NA Sticky c V
Dolosigranulum CL/T Small c NA
a
CAT, catalase production; NaCl, growth in broth containing 6.5% NaCl; 10Cand 45C, growth at 10 and 45C, 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 3.18.11, footnote a, for other abbreviations and symbols.
Tables adapted from references 9, 17, 22, and 25. Also see reference 6.
Table 3.18.14b Biochemical reactions of PYR-negative, catalase-negative gram-positive
cocci
Phenotypic characteristics
a
Genus or species
Gram stain LAP NaCl Van Arginine 45C MRS
Leuconostoc CH, rods V R
Weissella confusa CH, rods V R
Pediococcus CL/T V R V
Streptococcus CH S V
Aerococcus urinae CL/T S
a
Van, vancomycin; MRS, gas production in MRS broth. See footnote a to Table 3.18.14a and Table
3.18.1 for other abbreviations.
Identication of Gram-Positive Bacteria 3.18.1.11
Table 3.18.15 Catalase-negative, gram-positive rods that can grow aerobically
a
Organism(s) H
2
S Vancomycin Hemolysis Hippurate Motility Nitrate Esculin Gram stain morphology
Weissella spp. R Alpha NA Small, short GPR; gas in MRS broth (22)
Erysipelothrix
rhusiopathiae
R Alpha Has two cell forms; the long-chaining
form can be confused with Lactobacil-
lus, and the short form can be confused
with Actinomyces or even Enterococcus
(PYR positive)
Lactobacillus spp. R Alpha V V Most lactobacilli are long regular-chaining
rods. Some are C shaped.
Arcanobacterium
haemolyticum
S Beta Branching, which can be rudimentary; re-
verse-CAMP positive, lecithinase posi-
tive, gelatin negative
Arcanobacterium
pyogenes
S Beta Branching, which can be rudimentary; re-
verse-CAMP negative, lecithinase nega-
tive, gelatin positive
Arcanobacterium
bernardiae
S V V Reverse-CAMP negative, gelatin negative;
does not branch; not beta-hemolytic on
human blood. Kits can misidentify as
Gardnerella.
Gardnerella
vaginalis
S

NA SPS sensitive; beta-hemolytic on human

blood
Bidobacterium
spp.
S Some are aerotolerant; can look like Acti-
nomyces or Gardnerella; not beta-he-
molytic on human blood
Actinomyces
israelii
S NA Branching, which can be rudimentary; an-
aerobic kits will identify; urease nega-
tive
Actinomyces spp. S V V V Not all species show branching; A. naes-
lundii and others are urease positive.
Some colonies of A. meyerii-odontolyti-
cus group turn red after 1 wk.
Aerotolerant Clos-
tridium
S NA V

Forms spores; medium to large regular

rod; grows slowly compared to Bacil-
lus; anaerobic kits will identify
a
Data from references 2, 5, 8, and 24. Once G. vaginalis, Arcanobacterium, Weissella, and E. rhusiopathiae are ruled out, either call Anaerobic gram-
positive rod or do anaerobic identication kit. GPR, gram-positive rods; SPS, sodium polyanethol sulfonate. See footnote a to Tables 3.18.11 and 3.18.1
2 for other abbreviations and symbols. See Table 3.18.19 for catalase-positive Actinomyces.
3.18.1.12 Aerobic Bacteriology
Table 3.18.16 Catalase-positive, usually yellow- or pink-pigmented gram-positive rods
a
Organism(s) Motility Fermentation Nitrate Urease Esculin Gelatin Glucose Comment
Cellulosimicro-
bium/Cellulo-
monas (Oersko-
via) spp.
/V Subsurface or surface hyphae/pseudohy-
phae may be present.
Microbacterium
(Aureobacter-
ium) spp.

V V V

V
Exiguobacterium
acetylicum
V V Golden yellow to orange
Leifsonia aquatica V V V V Previously called Corynebacterium aqua-
ticum
Corynebacterium
falsenii
W V W V NA W Yellow after 72 h
Corynebacterium
lipophiloavum
W Lipophilic, rarely isolated
Corynebacterium
mucifaciens
Slightly to deep yellow mucoid colonies,
CAMP negative
Rhodococcus equi V Mucoid in 48 h, usually pink after 47
days, can be yellowish, CAMP positive.
Can be acid fast. Important pathogen.
a
If motile and yellow or positive for esculin and/or gelatin, report as Motile coryneform, not Corynebacterium spp. If of clinical signicance, 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 3.18.11, 3.18.1
2, and 3.18.13 for abbreviations and symbols.
Table 3.18.17 Large, regular catalase-positive, gram-positive rods that usually produce spores and are usually motile
a
Organism(s)
Diam of cell
usually above
1 lm
Motility
b
Beta-hemolysis Lecithinase Penicillin
Large colony
at 24 h
Sticky,
tenacious colony
at 24 h
B. anthracis S
b

B. cereus R
Bacillus thuringiensis , V R
Bacillus mycoides W R
Bacillus megaterium , V V S
Other Bacillus spp. and
related groups
c
, V

V V V
a
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 45C. Spores can be proved by heating broth culture to 80C for 10 min and subculturing to BAP. Viable colonies indicate that
spores survived the heating. See Table 3.18.11, 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).
Identication of Gram-Positive Bacteria 3.18.1.13
Table 3.18.18 Urease-positive Corynebacterium spp. of clinical importance
a
Organism Nitrate Urease Pyrazinamidase Glucose Sucrose Lipophilism CAMP reaction
C. glucuronolyticum V V
C. pseudotuberculosis
b
V V Reverse
C. ulcerans
b
Reverse
C. pseudodiphtheriticum
c

C. riegelii
d
V
C. urealyticum
e

C. amycolatum V V V
CDC group F1 V
a
Data are from references 13 and 14. Other urease-positive or -variable species of less clinical signicance 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 3.18.11, 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.
Table 3.18.19 Catalase-positive, urease-negative, gram-positive rods, excluding Corynebacterium spp. and yellow- or pink-pigmented
rods
a
Organism(s) Fermentation Nitrate Esculin Gelatin Glucose CAMP
Gram stain or colony
appearance
Actinomyces neuii V Nonhemolytic, slight branching
Actinomyces viscosus NA
Propionibacterium av-
idum/granulosum
V V Beta-hemolytic, branching
Turicella otitidis Large rod, branching; ear pathogen
CAMP-positive C. auris and C. af-
ermentans have similar reactions
Brevibacterium spp. V Some yellowish, distinct odor
Dermabacter hominis Coccoid rods, distinct odor
Lysine , arginine , ornithine
Rothia spp. V Some branching, some black pig-
mented; if sticky, refer to Tables
3.18.11 and 3.18.14a for R.
mucilaginosa (previously Stoma-
tococcus mucilaginosus) (6).
a
Usually irregular rods. Identify only if clinically signicant, but all can be pathogens (13, 14). The important tests are fermentation (use Andrades base or
cysteine Trypticase agar), CAMP, and Gram stain, with careful reading of Gram stain morphology. Coryneform identication kits can be helpful. For
abbreviations and symbols, see Table 3.18.11, footnote a.
3.18.1.14 Aerobic Bacteriology
Table 3.18.110 Urease-negative Corynebacterium spp. of clinical importance
a
Organism(s) Nitrate Urease Pyrazinamidase
Alkaline
phosphatase
Glucose Maltose Sucrose Lipophilism CAMP Comment(s)
C. accolens V V
CDC group G
b
V V V Fructose positive
C. jeikeium
b
V Fructose negative
C. afermentans V V V One subspecies is li-
pophilic.
C. macginleyi Found in eye speci-
mens.
C. diphtheriae
c
C. diphtheriae/belfanti
is nitrate negative;
C. diphtheriae/inter-
medius is lipophilic.
C. propinquum V V
C. amycolatum
b
V V V V Dry colony, O/129 R,
a common species
of human resident
microbiota; can be
misidentied as C.
xerosis.
C. minutissimum V O/129 S, DNase posi-
tive, PYR positive
C. striatum V V O/129 S
C. xerosis V Creamy colony, O/129
S, LAP positive
a
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 identication of species in this table, the combination of CAMP test, lipophilism, O/129 disk, and commercial kits for
corynebacteria should be used if identication is clinically important. For abbreviations and symbols, see footnote a to Tables 3.18.11 and 3.18.19.
b
Multiresistant to antimicrobials.
c
Submit to reference laboratory for diphtheria toxin testing.
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