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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Applied Veterinary Bacteriology and Mycology:
Identification of aerobic and facultative
anaerobic bacteria
Chapter 10: Regular and coryneform non-sporing Grampositive rods
Author: Dr. J.A. Picard
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
Table 10.1: Differences between Listeria monocytogenes and Erysipelothrix rhusiopathiae ......2
Listeria ..........................................................................................................................................................2
Table 10.2: Differentiation of Listeria species ...............................................................................3
Erysipelothrix Rhusiopathiae .....................................................................................................................5
Corynebacterium species ...........................................................................................................................7
Table 10.3: Distinguishing features of corynebacteria of veterinary importance ..........................8
Table 10.4: Identification characteristics of regular, non-sporing, Gram-positive rods ................11
Table 10.5: Biochemical and morphological characteristics of genera of Gram-positive rods
isolated from animals. ..................................................................................................................12
Table 10.5 continued: Biochemical and morphological characteristics of genera of Grampositive rods isolated from animals ..............................................................................................14
Table 10.6: Diseases associated with pathogenic coryneforms and Rhodococcus equi.16Use of laboratory an
Rhodococcus equi ....................................................................................................................................16
Trueperella (previously Arcanobacterium) pyogenes ...........................................................................17
Brochothrix thermasphactum ..................................................................................................................18
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
INTRODUCTION
Gram-positive rods are divided into two groups based on morphology and the guanine-cytosine (G+C)
content of the DNA. Those with a high G+C content and irregular cell shape are included in the
actinomyces group and are discussed in a different chapter, with Trueperella (Arcanobacterium)
pyogenes, Corynebacterium and Rhodococcus equi being dealt with in this chapter. Those with a low
G+C content and with irregular staining include the genera Bacillus, Clostridium, Listeria,
Erysipelothrix and Lactobacillus. The latter three will be discussed in this chapter.
Listeria monocytogenes and Erysipelothrix rhusiopathiae are important animal pathogens and
Lactobacillus spp. are common commensals of the gastrointestinal tract and mucous membranes of
animals. All these agents have a similar morphology and must be distinguished by culture morphology
and various identification tests. Refer to Table 10.1
Table 10.1: Differences between Listeria monocytogenes and Erysipelothrix
rhusiopathiae
Characteristic
Disease
Morphology
Gelatine stab after two weeks at 22°C
Motility
Blood agar
Growth at 4°C
Aesculin hydrolysis
Catalase production
MR and VP reactions
Pathogenicity
Erysipelothrix rhusiopathiae
Septicaemia, diamond skin disease,
arthritis and endocarditis in pigs. In
other animals and humans skin and joint
lesions
Smooth colony: short rods
Rough colony: filaments
Brush growth
Non-motile
α-haemolysis
Kills pigeons but not guinea pigs
Listeria monocytogenes
Septicaemia, abortions and
meningoencephalitis in most animals
(rare disease in South Africa)
Thick rods
Umbrella-like growth
Motile
β-haemolysis
+
+
+
+
Kills guinea pigs but not pigeons
LISTERIA
Listeria species are medium-sized (0.5-2μm in length), Gram-positive rods that are non-acid-fast and
are motile at 22°C. Although they are facultative anaerobes, their growth is enhanced by 5 – 10% CO2.
They are catalase-positive, oxidase-negative, hydrolyse aesculin, tolerate 10% salt (NaCl) and will not
grow on MacConkey agar.
They are widely spread in nature and have been isolated from both sick and healthy animals as well
as from the soil, water, silage and sewage.
Only some strains of L. monocytogens (more common) and L. ivanovii have been associated with
disease. Pathogenic strains of L. monocytogenes can cause meningoencephalitis, septicaemia and
abortions in primarily domestic ruminants, but also other animals and humans (zoonosis). Sheep and
goats may sometimes harbour the bacteria without showing clinical signs. When such animals are
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
stressed they may then begin to shed the bacteria and may become sick. The main source of infection
in ruminants has been the feeding of poor quality (alkaline) silage.
Occasionally L. monocytogenes is shed in the milk of infected goats, cows and ewes. In humans
disease is often associated with the intake of contaminated dairy products, such as improperly
pasteurised milk, poultry meat and coleslaw.
LABORATORY DIAGNOSIS
Specimens
Septicaemia: material from lesions in the liver, kidney and spleen.
Meningoencephalitis: spinal fluid, brainstem, and tissue from several sites on the medulla oblongata.
Abortion: Placenta (cotyledons), foetal abomasal contents and/or uterine discharges.
Direct microscopy
Histopathology examination (of formalin fixed specimens) is more useful as Gram-positive rods will be
seen in association with lesions characteristic of listeriosis.
Isolation
Suspected material is inoculated on blood and MacConkey agar to rule out any contaminants.
Selective media containing antibiotics or blood agar containing 0.5% potassium tellurite to inhibit many
Gram-negative bacteria can be used. Examples of selective media are PALCAM (polymixin-acriflavinlithium chloride-ceftazidime-esculin-mannitol) agar where grey-green colonies form and Oxford (lithium
chloride-phenylethanol-moxalactam) agar where black colonies surrounded by a black halo form.
In the visceral form of the disease sample material can be directly inoculated onto agar, but in the
neural form of the disease nervous tissue should be placed in a broth and the cold enrichment
technique followed. The cold enrichment technique is a more sensitive method for recovering Listeria
species. Small pieces of brain tissue are homogenized and a 10% suspension is made in nutrient
broth. The suspension is placed in a refrigerator at 4°C and sub-cultured weekly onto blood agar for
up to 12 weeks before being considered negative.
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-
+/(+)
-
(+)
-
-
-
-
+ (wide
zone)
+
-
-
-
-
-
-
-
-
-
+
L. ivanovii
L. welshimeri
L. seeligeri
L. innocua
(+)
L. denitrificans
CAMP-test (S. aureus)
CAMP-test (R. equi)
Acid production from:
L-Arabinose
-
L. murrayi
β-haemolysis
+/-
L. grayi
Irregular rods with uneven staining
L. monocytogenes
Characteristics
Table 10.2: Differentiation of Listeria species
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Dextrin
Galactose
Glycogen
Lactose
D-Lyxose
Mannitol
Melezitose
Melibiose
α-Methyl-D-glucoside
α-Methyl-D-mannoside
L-Rhamnose
Sorbitol
Soluble starch
Sucrose
D-xylose
Voges-Proskauer
Hydrolysis of
Cellulose
Hippurate
Starch
Lecithinase
Phosphatase
Reduction of NO3 to NO2
Pathogenicity for mice
d
d
d
d
+
+
+
-/+
+
+
d
+
+
d
d
d
+
-
-
-/+
-
+
d
+
+
+
+
d
+
+
d
+
d
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
d
+
+
-
+
+
+
d
d
d
d
+
+
+
+
+
+
+
+
+
1/2a,1/2
1/2a,1/2
1/2b,4c,
5
Specific
b, 1/2c,
b,1/2c,U
6a,6b,U
3a, 3b,
S,4b,4d,
S
Serotypes
3c, 4a,
6b
4ab, 4b,
4c, 4d,
4e, 7
L. ivanovii subsp. londoniensis is ribose negative, it is also positive to N-acetyl-β-D-mannosamine, whereas L. ivanovii is
negative.
-
+
+
+
+
+
+?
Cultural and staining characteristics
Small round transparent colonies appear on the blood agar after 24 hours of incubation. They become
greyish-white with a diameter of 0.5-2 mm after 48 hours of incubation. Listeria monocytogenes and
the non-pathogenic L. seelegeri produce a narrow zone whereas L. ivanovii produces a wide zone of
β-haemolysis. Sometimes haemolysis can only be noted under the colonies. Smooth and rough colony
forms may be seen.
Gram-stain reveals the presence of Gram-positive rods. Primary cultures are often pleomorphic and
may be confused with Corynebacterium and Streptococcus species. The organisms easily decolourize
and may be mistaken for Gram-negative rods.
Identification
Listeria species can be differentiated from other related genera e.g. from motile Corynebacterium spp.
by aerobic acid production from glucose, inability to hydrolyse urea and positive VP reaction; from
Streptococcus spp. by its catalase positive reaction; from Lactobacillus spp. by it being motile and
catalase positive; from Kurthia spp. by being a facultative anaerobe and its aesculin hydrolysis.
All the Listeria species hydrolyse aesculin. L. monocytogenes has a typical tumbling motility in a 2 to 4
hour broth culture incubated at 25°C when examined by the hanging drop method. When grown on
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
semi-solid motility medium or gelatine they have a characteristic umbrella-shaped growth on the
subsurface.
Modified CAMP tests using Staphylocccus aureus and Rhodococcus equi help distinguish the βhaemolytic Listeria species. (Table 10.2)
Other tests that can be used to distinguish the different Listeria species include acid production from
carbohydrates (Table 10.2), API-Listeria test, phage typing and DNA fingerprinting.
Determination of pathogenicity
As many strains of L. monocytogenes and L. ivanovii can be non-pathogenic, laboratory animals can
be used to assess the virulence of this agent.
1.
Anton test: inoculation into the conjunctiva of a rabbit or guinea pig. L. monocytogenes will cause
2.
a purulent keratoconjunctivitis within 24 to 36 hours after inoculation.
Intraperitoneal inoculation of mice with a broth culture of either L. monocytogenes or L. ivanovii. If
virulent, death will occur within 5 days with necrotic lesions present in the liver.
3.
4.
Inoculation of the chorioallantoic membrane of embryonated hen’s eggs.
Cell culture cytotoxicity using the human intestinal cell line Caco-2.
Serotyping
Strains of Listeria species can be divided into serotypes on the basis of somatic (O) and flagellar (H)
antigens.
Antimicrobial susceptibility
In vitro the organism is sensitive to penicillin, ampicillin, gentamycin, potentiated sulphonamides,
erythromycin, tetracycline and chloramphenicol. Since aminoglycosides enhance the activity of
penicillin, this combination is recommended in the therapy of disease. In human isolates resistance
has been found to develop to chloramphenicol, erythromycin and tetracycline. Cephalosporins are
ineffective.
ERYSIPELOTHRIX RHUSIOPATHIAE
Rhusos= red; Pathos = pain; Thrix = hair (describes the disease in pigs)
Organisms in this group of bacteria are slender, Gram-positive, non-motile bacteria. They generally do
not grow on MacConkey agar. There are both smooth and rough variants. The smooth colonies are
often present in peracute and acute disease, and the rough forms predominate in chronic disease.
The organisms grow with a test tube brush appearance in a gelatine stab.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Pigs carry these organisms on the tonsils and in the intestines. They are present in the soil and pig
manure and will survive for prolonged periods in an alkaline environment. It has also been isolated
from the slime layer on the skin of fish and from free-living avian species that died from septicaemia.
It causes swine erysipelas and can manifest as a septicaemia, a skin form known as “diamond skin
disease”, chronic arthritis and valvular endocarditis (heart valve disease). In sheep, cattle and horses
the disease may manifest as a polyarthritis. A severe septicaemia disease often occurs in turkeys.
There have been a few reports of valvular endocarditis in dogs. Humans mainly develop a localized
skin infection known as erysipeloid that usually involves the hands and fingers. Rarely systemic
infection occurs.
Direct microscopy
Gram stained smears made from the blood of the heart will reveal the presence of large numbers of
slender Gram-positive rods in acute disease and long filaments in chronic disease. The filaments stain
poorly with Gram’s stain.
LABORATORY DIAGNOSIS
Specimens:
Liver, spleen, kidney, heart and synovial tissue/fluid can be used. Recovery of the organism from skin
lesions and chronic forms of the disease can be difficult.
Isolation Procedures:
Suspected erysipelas material is inoculated onto blood agar. Blood agar containing sodium azide
(1:1000) or crystal violet (0,01%) may be added to suppress contaminants. Inoculated plates are put in
a candle jar or 10% CO2 in order to accelerate growth and incubated at 37°C for 24 – 48 hours.
Cultural and staining characteristics
Non-haemolytic pin-point colonies are seen after 24 hours of incubation. After 48 hours a zone of
greenish haemolysis appears just under the colonies. On further incubation a zone of clear (β)
haemolysis may develop. Smooth colony types (S) are often small (0,1-1,5 mm in diameter) and
round, whereas rough (R) colonies are larger, flatter, and more opaque with irregular borders. This
colony variation is best seen after 48 hours of incubation.
Gram stain of S-colonies reveals the presence of slender Gram-positive rods indistinguishable from L.
monocytogenes, whereas R-colonies reveal filaments of varying length.
Identification
Biochemically, they are catalase-negative, aesculin-negative and H2S positive (Table 10.1).
After stab inoculation of TSI agar and an incubation period of 24 hours at 37°C, a thin black line
indicating H2S production is produced.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Rough colony forms give a “bottle-brush” type of growth in nutrient gelatine after 5 days of incubation
at room temperature.
Carbohydrate tests should be carried out in peptone water with added sterile serum (0.5 – 1%) or in
nutrient broth plus the test carbohydrate with phenol red as an acid indicator. Although the
fermentation pattern is variable, they usually ferment lactose, glucose, levulose and dextrin.
Serotyping
This is usually only done in reference laboratories. Twenty-two serovars have been identified on the
basis of somatic antigens. Serovars 1 and 2 account for 70 – 80% of all isolates. Serovar 7 is now
known as E. tonsillarum, an avirulent commensal in the oropharynx of pigs.
CORYNEBACTERIUM SPECIES
Coryne = club shaped.
Members of this genus are Gram-positive, small pleomorphic rods (about 0.5 μm in diameter). They
are non-spore forming, not acid-fast, catalase positive and oxidase negative. The majority are nonmotile except for the plant pathogens. These bacteria are frequently arranged in parallel “palisades” or
at sharp right-angles to each other “Chinese letters”. Corynemycolic and corynemycolinic acids are
present in the cell walls. Many have metachromic granules (high-energy phosphate stores) in their
protoplasm, which is particularly noticeable in the type species Corynebacterium diphtheriae, the
cause of diptheria in humans.
Members of this genus and related genera belong to the actinomycetes group, which includes
Corynebacterium, Actinomyces, Arcanobacterium, Brevibacterium, Mycobacterium, Rhodococcus,
Nocardia, Bifidobacterium and many others.
Many occur widely in nature and those isolated from animals are often found as commensals on the
skin and mucous membranes. More than 30 Corynebacterium species have been identified, of which
about a third are parasitic on humans and animals and are often found in clinical samples. Only a few,
however, cause disease.
7|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Pigment
Haemolysis
Catalase
O/F
Lipophilic
Gelatin hydrolysis
Urease
Bile aesculin
Nitrate
ALP
Acid phosphatase
Acid production
Glucose
Lactose
Maltose
Mannitol
Ribose
Sucrose
D-xylose
8|Page
lemon
+
F
+
+
+
-
β
+
F
-
C. lipophiloflavum
C. pilosum
C. renale
C. cystitidis
C. mastitidis
C. pseudotuberculosis
C. bovis
C. amycolatum
C. argentoratense
C. auris
C. coyleae
C. jeikeium
C. striatum
C. camporealensis
Corynebacterium
capitovis
Characteristic
Table 10.3: Distinguishing features of corynebacteria of veterinary importance
F
-
F
-
O
+
F
-
O
-
F
-
F
-
+
O
+
O
+
+
F
-
lemon
+
F
-
yellow
+
F
-
O
+
-
-
-
-
-
-
-
+
+
V
+
+
+
+
+
-
+
+
+
+
+
+
+
v
+
+
V
v
+
+
-
+
+
+
v
+
+
+
-
+
+
+
+
-
+
+
+
-
-
-
v
-
-
-
v
-
+
v
-
-
-
-
-
v
-
+
-
-
-
v
-
v
v
-
-
-
-
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
LABORATORY DIAGNOSIS
Specimens
Pus or exudates from recent abscesses or suppurative lesions, or swabs from the edge of abscesses.
Mid-stream urine for the isolation of members of the “C. renale” group.
Direct microscopy
Pleomorphic rods are usually observed within the cytoplasm of neutrophils or free in pus. They may be
confused with streptococci, staphylococci, enterococci and Listeria species.
Isolation
For routine isolation, blood and MacConkey agars can be used. The inoculated plates are incubated at
37°C for 24 – 48 hours. If no contaminants are suspected, the specimen can also be inoculated into a
serum-containing or brain heart infusion broth. A selective medium containing fosfomycin, nalidixic
acid and the culture supernatant of Rhodococcus equi has been developed for the culture of
Corynebacterium pseudotuberculosis and is known as FNR medium.
Identification
Colony morphology
A rapid visual identification of certain similar Gram-positive rods is available in Table 10.3.
C. bovis: small, white, dry, non-haemolytic colonies that tend to appear in the wells of plates
inoculated with milk, as it is a lipophilic organism.
C. pseudotuberculosis: small, white and dry colonies, which after 48-72 hours of incubation will be
surrounded by a narrow zone of β-haemolysis. After several days of incubation the colonies can reach
3 mm in diameter and appear dry, crumbly and cream in colour.
C. kutscheri: resembles the colonies of C. pseudotuberculosis. Occasional strains may be βhaemolytic.
C. renale, C. pilosum and C. cystitidis: very small, non-haemolytic. With time colonies of C. renale
become cream to light yellow, C. pilosum yellow and C. cystitidis remains translucent to white in
appearance.
CAMP test
The CAMP tests are quick presumptive tests for C. pseudotuberculosis, C. renale and R. equi. Bovine
or sheep blood agar should be used for the test. The results are as follows:
Staphylococcal β-haemolysin
C. pseudotuberculosis
Inhibition
C. renale
Enhancement
R. equi
Enhancement
9|Page
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
A. pyogenes
Enhancement
There is also enhancement (synergism) between the R. equi “equi factors” and the haemolysin of C.
pseudotuberculosis.
Biochemical tests
The definitive identification of the corynebacteria is based upon biochemical tests (See tables 10.4
and 10.5). APICoryne can also be used to identify these bacteria.
10 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Table 10.4: Identification characteristics of regular, non-sporing, Gram-positive rods
Characteristics
Cell morphology
Multi cellular rods
(trichomes)
Diameter of rods
Motile
Catalase reaction
Growth at 35°C
H2S production
Acid from glucose
Major fermentation
products
Normal habitat
11 | P a g e
Catalase-negative
Facultative anaerobe
Lactobacillus
Erysipelothrix
Usually straight rods,
Sender rods to
sometimes
filaments
coccobacilli
Catalase-positive
Facultative anaerobe
Brochothrix
Listeria
Sender rods to
filaments
Short rods often short
chains and filaments
Strict aerobic
Kurthia
Regular rods in
chains, cocci in old
cultures
Caryophanon
Renibacterium
Short rods in chains
Short rods often in
pairs
-
-
-
-
-
+
-
0.5-1.6
-/(+)
-/ (+)
+
+
Mainly lactate (some
may also give acetate,
ethanol and CO2)
0.2-0.5
+
+
+
0.6-0.8
+ (media dependent)
+
0.4-0.5
+ (25°C)
+
+
+
0.7-0.9
+
+
+
-/ (+)
+
1.4-3.2
+
+
0.3-1.0
+
Lactate
Mainly lactate
Lactate
No acid
No acid
No acid
Widespread in
fermentable materials,
rarely pathogenic
Widespread
pathogen in
vertebrates
Meat products, nonpathogenic
Widespread in
decaying material,
may be a vertebrate
pathogen
Faeces of farm
animals, meat
products, nonpathogenic
Cow dung, nonpathogenic
Pathogen in salmonid
fish
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 10: Regular and coryneform non-sporing Gram-positive rods
All
G
H
H
G
M
Ch
G
D
P
RC
M
F
Ch
G
A
P
PG
H
S
PC
H
FM
A
A
MF
A
A
A
M
H
F
A
H
H
A
Turicella
Streptomyces
Sanguibacter
Rothia
Rhodococcus
Renibacterium
Propioniferax
Proprionibacterium
Oerskovia (Callatomonas
turbata)
Nocardia
Mycobacterium
Microbacterium
Listeria
Lactobacillus
Kurthia
Exguobacterium
Erysipelothrix
Dermatophilus
Dermabacter
C. aquaticum
Curtobacterium
Corynebacterium (plant
pathogens)
Corynebacterium
Cellulomas
Caseobacter
Caryophanon
Brochothrix
Brevibacterium
Aureobacterium
Arthrobacter
Arcanobacterium
Arachnia (P. propionicus)
Agromyces
Actinomyces
Characteristic
Table 10.5: Biochemical and morphological characteristics of genera of Gram-positive rods isolated from animals.
H
Source*
PB
BF
PB
PC
RC
S
R@
M
PR
R
C
S
P
P
P
P
BF
FR
RC
R
R
P
P
PB
BF
P
R
R
P
FB
Shape§
D
+C
(+)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
D
+
+
+
+
+
(D)
+
+
+
+
+
Aerobic growth
O
Anaerobic growth
+
(+)
+
+
-
-
-
+
-
+
+
+d
-
-
+
+
-
+
+
-
-
-
+
+
-
-
+
-
37°C
+
+
+
+
D
D
-d
-
+
(+)
+
+
D
+
+
+
+
+
+
+
+
+
+
+
-
D
+
+
Adherence
D
+
+
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
D-
+
+
-
-
D+
-
+
Aerial hyphae
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
D-
+
-
-
-
D-
-
+
Microcols filament
D
+
+
-
-
-
-
-
-
-
-
d
-
-
+#
-
-
-
-
-
-
-
+
-
-
D
A
+
Pigment¥
DR
(Y)
-
-
D
Y
DY
-
-/P
Y
DY
YB
DY
Y
-
D/
-
-
DY
+
YR
Y
YR
YR
-
(mycelium)
12 | P a g e
Y
-
G
W
Y
+
W
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Y
Haemolysis
D
D
+
-
Oxidase
D
-
-
Catalase
D
-
-
-
+-
-
-
-
-
D
+
-/+
-
-
-
+
+
+
+
+
-
-
-
-
d
-
+
+
+
+
-
-
-
-
-
D
-
(+)
-
D
D
D
D
-
D
-
-
D
+
+
+
(+)
-
-
-
-
-
-
-
+
-
+
-
+
+-
+
+
+
D
-
-
-
-
-
D
-
(+)
-
-
+S
-
+/-
-
+2
+
D
-
D
-
-
-
-
-
+
-
-
(+)
5
-
+
+
-
-
+
+
+
+
-
-
+
+
-
-
-
D
-
D
D
-
-
D
-
-
-
D+
+
+
+/-
+
-
D
+
D+
D
+/-
+
D
-
D
+
-
D
V
-
-
+
+
+
Acid fast (milk)
(+)
-
-
-
-
-
D
-
-
+
-
D
-
+
-
+
Motility
+
3w
+
+
-
(+)
+/-
+
-
(+)
+
+(-
+
(+)
-
+
+
+
(+)
-
-
+
Glucose acid
)
Glucose gas
-
-
(+)
-
-
Nitrate
+/-
-
+
D
D
Urea
D
-
-
D
D
-
D
-
-
-/+
PG
+
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
+
D
-
D
-/+
D
D
(+)
V
-
-
-
V
-
-
D
-
-
-
-
+
-
-
-
-
-
D
D
+
-
+
+
-
V
-
-
-
D
+
+/-
-
V
+
-
+
-
D
+
+
+
+
-
-
-
D
-/+
-
-
+
V
D
-
D
-
+
-
Gelatine
§
Aesculin
D
TSI
D
H2S (LA)
D
13 | P a g e
+
D
D
+
D
D
D
-
-
-
-
D
D
D
+
-
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Hippurate
-
-
-
Indole
-
+
-
D
D
-
-
D
-
-
-
-
D
-
-
+
D
+
-
-
-
-
-
D
-
-
D
D
-
DNase
D
-
+
+
D
+
Casein
+/-
-
-
+
D
+
Phosphatase
-
Arginine
D
Sabouraud’s agar
-
ONPG
-
14 | P a g e
-
-
-
-
-
-
+
-
-
+
+/-
D
+
+
-
+
-
-
+
D
-
D
-
+
-
-
D
D
+
D
D
(+)
+
-
-
-
D
-
+
D
-
D
-
D
+
-
+
D
+
D
+
D
-
-
+
+
-
D
+
D
+
V
-
-
+
-
-
D
-
+
+
-
-
Turicella
Streptomyces
D
+
+
Sanguibacter
Rothia
Rhodococcus
Renibacterium
Propioniferax
Proprionibacterium
Oeskovia (Callatomonas turbata)
-
-
D
Nocardia
Mycobacterium
Microbacterium
Listeria
Lactobacillus
Kurthia
+
Exguobacterium
+
Erysipelothrix
+
C. aquaticum
-
D
Curtobacterium
-
Corynebacterium (plant pathogens)
+
Corynebacterium
Caryophanon
-
-
-
D
-
Dermatophilus
D
Dermabacter
-/+
Cellulomas
D
Caseobacter
Starch hydrolysis
Brochothrix
Brevibacterium
Aureobacterium
Arthrobacter
Arcanobacterium
Arachnia (P. propionicus)
Agromyces
Actinomyces
Characteristic
Table 10.5 continued: Biochemical and morphological characteristics of genera of Gram-positive rods isolated from animals
-
+
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria  Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Penicillin
S
S
Filter paper
trypsin
-
-
-
-
-
Volatile fatty acids
-
+
-
-
-
-
S
-
S
-
-
+
-
-
-
-
-
S
-
-
-
-
S
S
-
-
-
-
-
S
R
-
-
-
-
-
-
R
-
S
S
S
D
-
-
-
-
+
-
-
-
A: all; C: poultry, P: pigs, S: sheep; F: faeces; M: meat; Ch: cheese; M: milk; G: soil; P: plants; D: skin, H: humans §: PG: poor growth,
R: rods, FR: filamentous rods, BF: branching filaments, P: pleomorphic; RC: cocco-bacilli, R@: long rods in loops, MR: multi celled rods, Y: yellow; P: pink; DY: dull yellow, #: only in co2,
15 | P a g e
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Table 10.6: Diseases associated with pathogenic coryneforms and Rhodococcus
equi.
Bacterial species
C. bovis
C. kutscheri
C. pseudotuberculosis
C. renale, C. pilosum, C.
cystidis
C. renale
“C. ulcerans”
R. equi
Main host(s)
Cattle
Mice, rats (guinea
pigs)
Sheep, goats
Disease(s)
Uncertain
Caseopurulent foci in the liver,
kidneys, lungs and lymph nodes
Caseous lymphadenitis
(Bacterial icterus)
Horses, cattle
Cattle
Ulcerative lymphangitis,
Contagious acne
Pyelonephritis, cystitis
Sheep
Balanoposthitis “Pizzle rot”
Pigs
Cattle
Foals (2–4 months)
Kidney abscesses
Mastitis (rare)
Suppurative bronchopneumonia,
ulcerative colitis
Natural habitat
Teat canal of cows
Mucous membranes of carrier
rodents
Skin, mucous membranes and
gastrointestinal tract of carrier
sheep and the soil of sheep pens
Bovine genital tract, especially in
bulls
Unknown
Unknown
Soil rich in organic material and
faeces of foals
Abscessation
Older horses and other
animals
Gemella morbillorum
Any
These genera are subject to taxonomic changes.
Soil
Opportunistic infections (rare)
Genital tract of humans
Use of laboratory animals
Should one be uncertain as to the identification of C. pseudotuberculosis, guinea pigs can be used as
they develop characteristic lesions.
0,2 ml of a 1:200 dilution of a broth culture is inoculated intramuscularly into the thigh. A swelling
develops in 1 to 3 days, with later enlargement of the inguinal lymph node and suppuration of the local
lesion. Due to spread of the bacteria, it often dies or is euthanized after 3 to 4 days. At necropsy thickwalled, abscesses containing whitish pus are found in or on the liver, spleen and other organs. The
presence of the organism is confirmed by culture. Rabbits and mice can also be used, but develop less
characteristic lesions.
Since the infection can spread between laboratory animals, the inoculated animals must be kept in
isolation for the duration of the test.
RHODOCOCCUS EQUI
These are Gram-positive, aerobic, non-motile, pleomorphic coccobacilli. They grow well on blood agar
forming non-haemolytic, smooth, pinkish, shiny, mucoid colonies. The bacteria commonly appear
coccoid on direct examination of clinical material, but may be rod shaped and rarely have limited
branching.
It is a common cause of bronchopneumonia and ulcerative colitis in 2 to 4 month old foals and rarely in
immunosuppressed adult horses. In other species it causes opportunistic suppurative lesions e.g.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
cervical lymphadenitis in pigs, pyaemia in dogs, ulcerative lymphangitis, abscesses and arthritis in adult
horses. Tuberculosis-like lesions in the lymph nodes of pigs have also been reported.
LABORATORY IDENTIFICATION
Direct microscopy
Gram-stained smears of clinical material reveal the presence of coccoid sometimes rod-shaped and
rarely branched Gram-positive organisms. They may occasionally stain positive (pink) with acid-fast
stains.
Isolation procedures
Clinical material should be streaked onto blood agar and incubated aerobically at 30 to 37°C for 24-48
hours. To isolate it from soil a selective medium is available – refer to Appendix 1.
Cultural characteristics
Within 48 hours, the organisms appear as smooth, mucoid, translucent tear-drop colonies, 3 to 5 mm in
diameter. With age they become larger, mucoid and salmon-pink in colour. Glucose-yeast extract
medium enhances pigment formation.
Identification
They are generally non-reactive in standard tests, are catalase- and urease-positive and do not ferment
carbohydrates.
TRUEPERELLA (PREVIOUSLY ARCANOBACTERIUM) PYOGENES
These are pleomorphic and commonly observed as clusters of Gram-positive, club-shaped, short rods
but may also appear cocco-bacillary or as short, branching rods. They are non-motile and catalasenegative. The organisms are commonly isolated from purulent conditions in sheep, cattle, swine and
occasionally horses. It is often recovered as opportunistic infections from wounds, pneumonia, genital
tract and udder infections.
LABORATORY IDENTIFICATION
Direct Microscopy
Gram-stained smears of pus or mastitis milk samples in T. pyogenes infections usually reveal large
numbers of small, highly pleomorphic, Gram-positive forms. They tend to be a mixture of cocci, rods
and pear-shaped cells. Occasionally short branching forms may be seen.
Isolation procedures
It grows well on blood agar incubated in 10 percent C0 2 at 37°C and also grows well anaerobically.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Cultural characteristics
Aerobic cultures are dry, white, fine (1mm) colonies at 48 hours, growth not very visible at 24 – 48
hours. At times colonies may appear smooth or mixed rough and smooth. Small zones of fuzzy βhaemolysis are seen even before colony growth is observed. It becomes clearer with time
Identification
Typically Gram-positive, plump pleomorphic rods that are catalase negative. A rapid presumptive test
for T. pyogenes is its ability to pit a Loeffler’s serum slope in 24 – 48 hours (Appendix 1). A. pyogenes
will also give a positive CAMP test with the -haemolysin of Staphylococcus aureus. So too will A. neuii
and some Corynbebacterium species.
BROCHOTHRIX THERMASPHACTUM
It is a spoilage bacterium of meat. It will grow on almost all media, but has a strict temperature
requirement.
1.
Suspend meat in 0.1% peptone water and shake vigorously
2.
Inoculate the fluid onto blood agar and the selective agar (see Appendix 1) and incubate at 22°C
for up to 5 days. Colonies are usually visible in 48 hours.
Identification

Non-haemolytic small white to yellowish colonies

Does not grow at 37 °C (grows best at 22 °C)

Gram-positive regular small rods that often forms chains or clumps

Catalase positive, Oxidase and Indole negative

Thiogel: non-motile, grows throughout the media, gelatine negative


OF: Fermentative, glucose positive
Casein, DNAse, nitrate, H2S and gas negative
REFERENCES
1. Van Netten, P, Perales I, van de Moosdijk, A., Curtis, GWD, & Mossel, DAA, 1989. Liquid and
solid differential media for the detection and enumeration of Listeria monocytogenes and other
Listeria spp. International Journal of Food Microbiology, 8, 299-316.
2. Zhao, H. K., Hiramune, T., Kikuchi, N., Yanagawa, R., Ito, S., Hatta, T., Serikawa, S. & Oe, Y.
1991. Selective medium containing fosfomycin, nalidixic acid, and culture supernatant of
Rhodococcus equi for isolation of Corynebacterium pseudotuberculosis. Journal of Veterinary
Medicine. Series B, 38, 10, 743-748.
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Chapter 10: Regular and coryneform non-sporing Gram-positive rods
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 10: Regular and coryneform non-sporing Gram-positive rods
APPENDIX 1
Glucose-yeast extract medium
Yeast extract
10g
Glucose
10g
Agar
15g
Tap water
1000ml
NANAT Selective medium for the isolation
of Rhodococcus equi.
Base medium
Peptone
20g
Sodium chloride
5g
Yeast extract
5g
Glucose
5g
Sodium hydrogen sulphite
0.2g
Sodium thiosulfate
1.2g
Dipotassium
Loeffler’s serum slant
hydrogen
2g
phosphate
2g
This
medium
is
used
to
isolate
Corynebacterium diptheriae, but also can be
Sodium hydrogen carbonate
300g
Nutrient broth (horse meat broth)
used to demonstrate proteolytic activity of T.
pyogenes.
Agarose
15g
1000ml
70ml
Bovine serum
Infusion
dextrose
broth
2,5g
(dried)
Water
Dissolve by heating and adjust to pH 7.2,
bottle and sterilize at 121°C for 15 min.
Cool to 55°C and add the following 4
Whole egg dried
7.5g
Distilled water
1000ml
antibiotics
to
concentrations
reach
the
following
Final pH: 7.6
Medium preparation
Mix, and dispense in glass heat resistant
bottle. Arrange so that they are in a slant
Novobiocin
25g/ml
actidone [cycloheximide]
40g/ml
potassium tellurite
50g/ml
nalidixic acid
20g/ml
position and autoclave at 120°C for 15
minutes. Properly prepared slants are smooth
and greyish-white in colour.
Test procedure
Brochothrix selective agar
A loopful of the bacterial colony is made in the
Peptone
20g
centre of the slope, taking care not to break
Yeast extract
2g
the surface of the medium. Incubate the
Glycerol
15ml
medium at 37°C for 24 – 48 hours. Pitting of
the slope gives a positive result for T.
pyogenes.
K2HPO4
1g
MgSO4
Agar
1g
3g
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Chapter 10: Regular and coryneform non-sporing Gram-positive rods
Weigh and add to Schott bottle and make up
to 1000 ml using deionised water. The pH
should be 7.0.
Autoclave at 121 °C for 15 minutes
Then add:
Streptomycin sulphate
500 µg/ml
Cycloheximide
Thallium acetate
50 µg/ml
50 µg/ml
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