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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
Applied Veterinary Bacteriology and Mycology:
Identification of aerobic and facultative
anaerobic bacteria
Chapter 9: Miscellaneous Gram-negative, nonfermentative bacilli
Author: Dr. M.M. Henton and Dr. J.A. Picard
Licensed under a Creative Commons Attribution license.
TABLE OF CONTENTS
INTRODUCTION ...........................................................................................................................................3
Table 9.1: Identifying characteristics of several genera of non-fermentative bacilli ......................3
Table 9.2: Reactions of some Gram-negative, glucose non-fermenting bacteria..........................4
Bordetella and Alcaligenes spp. ................................................................................................................4
Table 9.3: Differential characteristics of the genus Bordetella and other morphologically and
physiologically similar genera ........................................................................................................6
Table 9.4: Differential characteristics for Gram-negative, non-fermentative bacteria with
phenotypic characteristics similar to those of Bordetella and Alcaligenes species .......................6
Table 9.5: Differential characteristics of B. bronchiseptica, B. avium, Alcaligenes spp., and
CDC groups IVc and Oligella. All are motile (except B. parapertussis), oxidase (except B.
avium and B. parapertussis) – and catalase-positive, and grow on MacConkey agar) .................7
Riemerella Anatipestifer .............................................................................................................................7
Moraxella spp. ..............................................................................................................................................7
Table 9.6: Summary of the disease and sites of isolation of some the Moraxella species ...........8
Table 9.7: Differentiation of the Moraxella/Brahamnella species ..................................................8
Pseudomonas spp. and other related genera ..........................................................................................9
Table 9.8: Pathogenicity and the normal habitat of Pseudomonas/Burkholderia species of
veterinary importance.....................................................................................................................9
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
Table 9.9: Main characteristics of the pathogenic Pseudomonas/Burkholderia species .............10
Table 9.10: Identification of common members of the Pseudomonaceae (all are motile) ...........12
Shewenella .................................................................................................................................................13
Table 9.11: Identification of Shewenella species (all are Gram-negative curved or straight
rods, catalase and oxidase positive, motile and grow in 3% NaCl. Negative for arginine
dehydrolase, lysine or ornithine decarboxylase and indole production) ......................................14
Flavobacterium and related bacteria ......................................................................................................14
APPENDIX 1 ...............................................................................................................................................15
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
INTRODUCTION
Many Gram-negative bacteria fall into this group and include: Alcaligenes spp., Acinetobacter spp.,
Bordetella spp, Branhamella spp., Flavobacterium spp., Francisella tularensis, Moraxella spp., Neisseria
spp., Pseudomonas spp., and Burkholderia spp. Very few isolates are clinically significant, with most
being isolated as normal commensal flora or as sample contaminants. Bacteria that are potentially
pathogenic include Bordetella bronchiseptica, Pseudomonas aeruginosa, Burkholderia mallei,
Burkholderia pseudomallei and Moraxella bovis. These bacteria are particularly difficult to identify as they
show few biochemical reactions, and commercial tests such as the API 20 NE are recommended.
Although dealt with in a separate chapter, note that Brucella spp. are similar to this group. Bacteria of
veterinary significance will be discussed under their respective headings. Tables 9.1, 9.2 and 9.3 list the
characteristics of some of these bacteria. As they are a diverse and difficult group to identify, an
identification key is included to assist in grouping them.
Table 9.1: Identifying characteristics of several genera of non-fermentative bacilli
Genus
Metabolism
Motility
Oxidase
Growth on
MacConkey
agar
Acinetobacter
Oxidative or
nonsaccharolytic
Non-motile
Negative
Good growth
except for
some strains
of A. lwoffii
Alcaligenes
Oxidative
Motile by means
of peritrichous
flagella
Positive
Good growth
Nonsaccharolytic
Motile by
peritrichous
flagella
Positive
Good growth
Oxidative
Variable. Those
species motile
aro peritrichous
flagella are B.
bronchiseptica,
B. avium, and B.
hinzii)
Most are
positive except
B.
parapertussis
is negative
and B. avium
is variable.
B.
parapertussis
and B.
bronchiseptica
Bordetella
Flavobacterium
Oxidative
(some strains
are slow
fermenters)
Non-motile
Positive
Poor or
negative
Moraxella
Oxidative or
nonsaccharolytic
Non-motile
Positive
Scant or
negative
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Additional characteristics
Special growth factors are not
required. Acid production from
glucose is weak (A. baumannii) or
lacking (A. lwoffii). Cells appear
coccoid in Gram-stained
preparations. Most strains are
penicillin resistant.
Obligate aerobe. Glucose may be
oxidized slowly (5 days); xylose is
oxidized rapidly (24 hours)
Strict aerobe, although some
strains utilize nitrate instead of
oxygen as the final electron
acceptor.
B. bronchiseptica rapidly splits
urea (within 4 hours)
Supplemental nitrogen and Bcomplex vitamins required for
growth of many strains. Yellow
pigment often produced. No
denitrification of nitrates. Growth
optimal at 30°C. All species are
resistant to polymyxin B. Most
species are weak indole positive
(F. odoratum is indole negative).
Most strains are fastidious in
growth requirements, some
requiring serum supplement. Strict
aerobes. May appear as coccobacilli on Gram’s stain. Highly
susceptible to penicillin.
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
Pigment
Penicillin susceptibility
Gelatinase
Indole
Urease
Nitrate reduction
Oxidase
Catalase
Motility
Growth on MacConkey
agar
Haemolysis
Acid from glucose
Table 9.2: Reactions of some Gram-negative, glucose non-fermenting bacteria
Alcaligenes faecalis
A. xylosidans
A. denitrificans
A. piechaudii
Acinetobacter
calcoaceticus
A. lwoffii
Branhamella catarrhalis
v
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
v
-
-
-
R
R
v
R
-
+
-
+
-
+
-
-
v
-
-
R
-
-
+
-
-
+
+
+
(+)
-
-
-
v
S
-
Moraxella caviae
-
-
-
+
+
+
-
-
-
S
-
M. cuniculi
M. bovis
M. ovis
Neisseria canis
N. flavescens
N. sicca
N. lactamica
N. denitrificans
N. mucosa
N. weaveri
N. elongata
Flavobacterium
meningosepticum
F. indologenes
F. odoratum
F. multovorum
Weeksella zoohelcum
CDC group EF-4
O
O
O
O
-

(w)

()
v
v
-
v
v
-
+
(+)
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
(+)
+
+
+
-
-
(+)
+
-
S
S
S
S
S
S
S
S
S
S
(S)
Yellow
Yellow
v (Yellow)
Yellow
v(Yellow)
v(Yellow)
-
O
()
+
-
+
+
-
-
+
+
R
v(Yellow)
O
O
(O)
v
()
()
v()
-
v
+
+
v
-
+
+
+
+
+
+
+
+
+
+
v
+
(-)
+
+
+
-
+
+
-
(+)
+
+
-
R
R
R
S
*
Yellow
v(Yellow)
Yellow
Yellow/tan
O =oxidative, - = unreactive, (O)= most strains oxidative, + = positive reaction, (+) = most strains positive,
(-) = most strains negative, v = variable, S = susceptible, R = resistant, * = data unavailable,
 = beta-haemolysis,  = alpha-haemolysis, () = most strains.
BORDETELLA AND ALCALIGENES SPP.
These are small, motile, non-fermentative, Gram-negative rods that are catalase- and oxidase positive.
Bordetella bronchiseptica is the cause of respiratory disease in mammals and B. avium the cause of
turkey coryza and respiratory disease in poultry. Alcaligenes species are saprophytes present in the
intestinal tracts of vertebrates. They may be opportunistic invaders and are difficult to distinguish from
Bordetella. Bordetella parapertussis has been reported in sheep in Europe, and B. hinzii is not known to
be pathogenic.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
Specimens
Specimens for bacterial isolation include nasal swabs, tracheal washings and pneumonic lungs. If nasal
swabs are to be taken from animals where the nasal orifice is small, flexible swabs designed for human
infants should be used.
Direct microscopy
As Bordetella is a small Gram-negative cocco-bacillus, smears made directly from specimens are not very
useful.
Isolation
They can all be isolated on blood agar. A selective medium described by Smith and Baskerville (See
Appendix 1) can be used for contaminated lung samples. Bordetella bronchiseptica and B. avium forms
blue, convex, smooth colonies 1 – 2 mm in diameter after 48 – 72 hours of incubation at 37°C.
Escherichia coli and Klebsiella species form yellow colonies and Alcaligenes and Pseudomonas species
green colonies. For isolates from dogs and rabbits, the medium should be prepared without gentamicin.
After 24 hours of incubation on sheep and horse blood agar, B. bronchiseptica forms small, convex,
smooth colonies that may be beta-haemolytic. The colonies of B .avium are similar, but non-haemolytic.
Phase modulation occurs in both species and is thought to be due to loss of a capsule-like structure on
subculture. Phase I colonies are convex and shiny, phase II are larger circular and convex with a smooth
surface and phase III are large, flat, and granular with irregular edges. The colonies on MacConkey agar
are small, pale with a pinkish hue and amber discolouration of the underlying medium. Bordetella colonies
tend to be small after 24 hours, enlarging greatly at 48 hours, whereas Pseudomonas and Alcaligenes
colonies are large from the start.
Biochemical identification
Differential characteristics for these genera are in Tables 9.3, 9.4 and 9.5.
All will grow on MacConkey agar, are catalase- and oxidase-positive and produce an alkaline slant with an
alkaline or no reaction in the butt of a TSI slant. Bordetella bronchiseptica, B. avium and Alcaligenes
species are motile. The API rapid NFT will identify Bordetella bronchiseptica and Alcaligenes. Bordetella
avium is not listed, but will assimilate adipate but not caprate, while A. faecalis assimilates caprate but not
adipate. The API20E system will also identify Bordetella species. However, the CDC group may also be
confused with Bordetella and Alcaligenes.
Haemagglutination test
Bordetella bronchiseptica possesses a haemagglutinin that will haemagglutinate washed sheep red blood
cells. A young 24-hour culture should be used, as older cultures tend to lose their haemagglutinating
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
ability. Two colonies of a suspected B. bronchiseptica culture are suspended in a drop of physiological
saline on a slide. An equal volume of a 3% suspension of washed sheep red cells is added and mixed. To
check for auto-agglutination, controls should include a suspension of colonies without red blood cells and
a suspension of red blood cells alone. B. bronchiseptica will auto-agglutinate the red cells within 1-2
minutes.
Serology
Tube agglutination, micro-agglutination and ELISA procedures have been developed for B. avium and B.
bronchiseptica.
Detection of dermo-necrotic toxin of Bordetella bronchiseptica
Bordetella bronchiseptica produces an intracellular, heat-labile toxin that is lethal when injected
intraperitoneally into mice, and produces necrosis when inoculated intradermally into guinea pigs.
Table 9.3: Differential characteristics of the genus Bordetella and other
morphologically and physiologically similar genera
Characteristics
Strictly parasitic
Saprophytic
Localise on respiratory cilia
Strictly aerobic
Growth requirements:
Thiamine
Nicotinamide
X and/or V factor
Ferments carbohydrates
Nitrate reduction
Litmus milk/ alkaline
Oxidation of amino acids
Tetrazolium reduction
Bordetella
+
+
+
Alcaligenes
+
+
Brucella
+
(+)
Haemophilus
+
-
Riemerella
+
(+)
+
d
+
+
+
d
+
+
-
+
+
+
d
+
+
+
+
+
d
Table 9.4: Differential characteristics for Gram-negative, non-fermentative bacteria
with phenotypic characteristics similar to those of Bordetella and Alcaligenes
species
Bacterial
genera
Bordetella
Alcaligenes
Pseudomonas/
Burkholderia
Flavobacterium
Moraxella
Eikenella
Acinetobacter
Glucose
oxidation
d
Oxidase
+
+
+
+
Growth on
MacConkey
+
+
+
+
Rods/
cocci
R
R
Type of
flagella
peritrichous
peritrichous
Motility
d
+
d
+
+*
R
polar
d
d
+
+
+
+
(+)
+
-
d
d
d
-
R
C
R
C
-
* Burkholderia mallei is non-motile
6|Page
Catalase
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
Table 9.5: Differential characteristics of B. bronchiseptica, B. avium, Alcaligenes
spp., and CDC groups IVc and Oligella. All are motile (except B. parapertussis),
oxidase (except B. avium and B. parapertussis) – and catalase-positive, and grow
on MacConkey agar)
Bacteria genera
B. bronchiseptica
B. avium
B. parapertussis
B. hinzii
A. faecalis
A. xylosoxydans
A. denitrificans
A. piechaudii
CDC group IVc-2
O. ureolytica
Urease
+
+
d
+
+
Nitrate
reduction
+
+
+
+
+
Oxidation of
Glucose
+
-
Simmon’s citrate
Malonate
+
+
+
+
+
+
d
-
+
+
+
+
+
+
+
+
RIEMERELLA ANATIPESTIFER
Riemerella anatipestifer (previously designated Pasteurella anatipestifer) causes septicaemia and
respiratory disease in poultry, especially ducks. It is catalase- and oxidase-positive, and does not grow on
MacConkey agar. It prefers microaerophilic conditions. It is variable for urease, negative for nitrate and is
usually gelatine positive. Acid production from sugars is normally negative, except glucose and maltose
that are positive.
Its biochemical reactions are listed in Table 9.3.
MORAXELLA SPP.
Moraxella are short, plump Gram-negative rods (1–1,5 x 1,5-2,5 µm) found characteristically in pairs.
Some strains approach a completely coccoid shape. They are strict aerobes, oxidative-, oxidase- and
catalase-positive, non-motile and do not attack carbohydrates. Although they grow on non-enriched
media, their growth is enhanced by the addition of blood or serum. The optimal temperature for growth is
33 – 35°C. Most Psychrobacter phenylpyruvica strains will grow on MacConkey agar, but M. bovis and M.
lacunata will not.
A summary of the differential characteristics is included in Table 9.7.
LABORATORY DIAGNOSIS
Moraxella bovis
Specimens
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
A swab of the lachrymal secretions is taken from the deep canthus of the eye. Ideally BTA plates should
be inoculated immediately after collection. If this is not possible, each swab is placed in about 1– 2 ml of
sterile distilled water, to prevent desiccation, and the specimens taken to the laboratory within two hours
after collection.
Table 9.6: Summary of the disease and sites of isolation of some the Moraxella
species
Species
Host
Cattle
Horses
Many animal species
M. lacunata
Humans
Psychrobacter
phenylpyruvica
Disease
Conjunctiva or nasopharynx of
cattle older than 2 years
M. bovis
M. ovis
Natural habitat
Sheep
Sheep & cattle
Pigs
Goats
Infectious bovine keratoconjunctivitis
Conjunctivitis
Opportunistic pathogen: septicaemia,
abortions
Mucous membranes of
animals
Conjunctivitis
Not pathogenic
Mucous membranes
Urogenital tract
Urogenital tract
Intestinal tract
Pathogenicity for animals is unknown
Table 9.7: Differentiation of the Moraxella/Brahamnella species
Characteristic
Beta-haemolysis (blood)
Growth on MacConkey agar
Oxidase
Catalase
Nitrate reduction
Urease
DNAse
Peptonisation of milk
Gelatinase or Loeffler serum
slope
M. bovis
+ (not
equine
strains)
+
(+)
+
(+)
B. ovis
M. lacunata
Other Moraxella
P. phenylpyruvica
+
-
-
-
+
+
+
(+)
+
-
+
+
+
-
(+)
+
+
(+)
+
+
d
+
+
d
d
(-)
+
-
-
Direct microscopy
Gram-stained smears show Gram-negative short, plump diplobacilli. A fluorescent antibody technique will
demonstrate and identify M. bovis if sufficient bacterial cells are present.
Isolation
Inoculate BTA and incubate at 35°C for 48–72 hours. The inoculation of the MacConkey plate is useful to
gauge the degree of contamination by other Gram-negative bacteria, as M. bovis does not grow on this
agar.
Colony identification
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
After 48 hours of incubation, small greyish-white colonies, surrounded by a narrow zone of betahaemolysis are seen. They look similar to streptococci. New isolates are often pilated and erode the agar,
sinking into it. Colonial growth autoagglutinates when suspended in saline. Some colonies become nonhaemolytic. Equine strains of M. bovis are non-haemolytic. So is M. lacunata and P. phenylpyruvica.
Some strains of P. phenylpyruvica will grow on MacConkey agar. Moraxella bovis colonies enlarge
markedly after 3 days of growth.
Biochemical reactions
Non-fermentative, non-motile, indole-negative and all are sensitive to penicillin. M. bovis will slowly pit a
Loeffler serum slope and will grow on 5% saline medium. Litmus or Crossley milk inoculated with M. bovis
becomes alkaline (blue) = peptonisation, with three zones: a blue upperlayer, a soft blue curd in the centre
and the bottom white. This helps distinguish it from other related species. Moraxella species have been
differentiated by the analysis of cellular fatty acid.
Animal inoculation
The inoculation of virulent, haemolytic and pilated strains of M. bovis intraperitoneally into guinea-pigs or
mice results in a fatal infection.
PSEUDOMONAS SPP. AND OTHER RELATED GENERA
A meaningful number of taxonomic changes have recently been made. This group includes
Pseudomonas, Burkholderia, Stenotrophomas, Shewenella and others. Pseudomonas spp. are medium
sized (0,5–1,0 x 1,5–5μm), Gram-negative rods. They are strict aerobes, oxidative, catalase- and oxidasepositive, and motile by one or several polar flagella. Burkholderia mallei is the only one that is non-motile.
Many produce soluble pigments and most will grow on MacConkey agar.
Diseases caused by Pseudomonas species in animals are shown in Table 9.8 and main differential
characteristics are in Tables 9.9 and 9.10.
Table 9.8: Pathogenicity and the normal habitat of Pseudomonas/Burkholderia
species of veterinary importance
Agent
Natural habitat
B. mallei
nasopharynx of
carrier horses
B. pseudomallei
soil & water
P. aeruginosa
(Bacillus of green
pus)
soil & water
9|Page
Disease in animals
Glanders or farcy in horses
Acute septicaemic disease in man.
Melioidosis or pseudoglanders in most
mammals. Septicemic disease, affects also
the joints and lymph nodes
Opportunistic pathogen:
Cattle: mastitis, endometritis, abscesses,
enteritis & arthritis.
Sheep & goats: mastitis, pneumonia, “green
wool”.
Horses: metritis, respiratory infections &
Notes
Zoonosis. Must use a
biosafety cabinet when
handling this bacterium.
Zoonosis. Must use a
biosafety cabinet when
handling this bacterium.
Polymicrobial
resistance common
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
mastitis.
Dogs & cats: otitis externa, cystitis,
endocarditis, dermatitis, wound infections &
conjunctivitis.
Reptiles: necrotic stomatitis & other necrotic
lesions.
S. maltophilia, B.
cepacia, P. putida, P.
fluorescens, P.
stutzeri, Shewanella
soil & water
Rare opportunistic pathogens. P. fluorescens
causes food spoilage and lesions in reptiles &
fish
Sample contaminants
Table 9.9: Main characteristics of the pathogenic Pseudomonas/Burkholderia
species
Characteristic
P. aeruginosa
Pigment produced
++
Odour
Growth on MacConkey
Growth at 5°C
Growth at 42°C
Oxidation of:
Glucose
Lactose
Arginine dehydrolase
Reduction of nitrate to
nitrite
Reduction of nitrate to
N2 gas
Motility
“fruity’ grape-like
+
+
B. pseudomallei
but colonies become orange
to cream
Putrid, becoming earthy
+
+
B. mallei
but colonies are
yellow to brown
-
B. cepacia
+
+
+
+
+
+
(+)
+
+
-
+
+
+
d
v
+
-
d
+
+
-
+
Yellow
Sweet
+
d
LABORATORY DIAGNOSIS
Specimens
Swabs or tissue samples, taken from various sites with lesions, for culture and antimicrobial sensitivity
test.
Direct microscopy
Seen as a medium sized Gram-negative rod in exudate smears. Not characteristic. A fluorescent antibody
test can be used to detect B. mallei and B. pseudomallei
Isolation
Pseudomonas is non-fastidious and will grow on most bacteriological media. The growth of B. mallei is
enhanced by 1% glycerol. A selective medium for B. mallei can be made by adding 1 000 units of
polymyxin B, 1250 units of bacitracin and 0,25mg actidione to 100 ml of trypticase soy broth. Incubate
aerobically at 37°C for 24-48 hours. Some of the saprophytic Pseudomonas species such as P.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
fluorescens will grow poorly if not at all at 37°C, and they need to be incubated at room temperature.
Pseudomonas will grow in a candle jar, but prefer normal atmosphere.
Colony morphology
P. aeruginosa.
Large greyish-blue spreading colonies (3 – 4mm) on BTA. It has a characteristic grape-like odour due to
aminoacetophenone. Most strains will produce beta-haemolysis on BTA. The bacterium is non-lactose
fermenting and produces various shades of blue to green pigments (pyocyanin) on MacConkey agar. Red
colonies are seen on brilliant green and XLD agars, no H2S is produced.
B. pseudomallei.
Colony growth varies from smooth to mucoid to rough with a dull wrinkled corrugated surface. Aged
colonies develop a yellow-tinge. The growth has a characteristic musty odour. Partial and later complete
haemolysis is seen on sheep BTA. It is lactose-positive on MacConkey agar (B. mallei is negative) but
doesn’t grow on deoxylate or Salmonella-Shigella agar.
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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
Table 9.10: Identification of common members of the Pseudomonaceae (all are motile)
Chryseomonas luteola
RNA GROUP III
Conamonas acidovoras*
C. terrigena
C. testosteroni
Flavimonas oryzihabitans
RNA GROUP I
Fluorescent group
Pseudomonas
aeruginosa*
P. fluorescens
P. putida
Stutzeri group
P. stutzeri
P. mendocina
CDC group Vb3
Alcaligenes group
P. alcaligenes
P. pseudoalcaligenes
P. species group I
RNA GROUP II
Pseudomallei group
Burkholderia pseudomallei
B. mallei§
B. cepacia
B. gladioli
B. picketti
RNA GROUP IV
Diminuta group
P. dimuta
P. vesicularis
P. paucimobilis
Shewenella putrifaciens
RNA GROUP IV
Xanthomonas maltophilia
Oxidase
-
Pyoverdin
-
Glucose
+
Maltose
+
Lactose
-
Mannitol
+
Arginine
v
Lysine
-
Nitrite
v
Nitrate
-
Urea
v
ONPG
+
DNase
-
Aesculin
+
Polymixin
S
+
+
+
-
-
+
+
-
+
-
-
+
+
+
-
-
v
-
-
NA
NA
-
v
S
S
S
+
+
+
V
-
+
+
-
+
V
V
-
-
-
S
+
+
+
+
+
+
V
V
-
+
-
+
+
-
V
-
-
V
v
-
-
-
S
S
+
+
+
-
+
+
+
+
+
-
V
+
+
+
-
+
+
+
V
V
V
-
-
-
-
S
S
S
+
+
+
-
-
V
-
-
-
V
V
-
V
+
+
-
V
-
-
-
-
s
S
S
+
-
+
+
+
+
+
-
+
+
v
-
-
-
R
W
+
-
+
+
+
+
v
+
v
+
+
v
-
+
-
V
V
v
v
V
+
+
V
+
-
-
v
-
R
R
R
+
+
+
+
-
V
+
+
V
+
V
+
-
-
-
-
+
-
-
V
+
-
V
+
+
+
-
V
S
S
S
-
-
+
++
+
-
-
+
v
-
-
+
+
+
S
* Acetamide positive § non-motile
12 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
B. mallei.
Growth is slower than that of P. aeruginosa, with smaller colonies at 24-48 hours (1 – 2mm in
diameter). Initially the colonies are smooth and white to cream, as they age they become yellow. B.
mallei cannot grow on MacConkey agar.
The opportunist, B. cepacia may be confused with both B. mallei and B. pseudomallei.
Immunological tests
Melioidosis: Complement-fixation and indirect haemagglutination tests can be used to detect
antibodies to B. pseudomallei. Diagnosis is however, based upon isolation and identification of the
bacterium.
Glanders: Complement-fixation, indirect haemagglutination and counter immunoelectrophoresis tests
are used in the diagnosis of glanders. False positive reactions may be obtained in areas that are
endemic for melioidosis, due to cross reactivity.
The mallein test that demonstrates hypersensitivity to B. mallei infection is used to detect carrier
horses. Mallein is a glycoprotein extracted from the bacterium. Either subcutaneous injection or
instillation into the conjunctival sac will result in a localised swelling in positive animals.
Animal inoculation
The Shwartzman phenomenon is seen in male guinea-pigs inoculated intraperitoneally with infective
material containing either B. pseudomallei or B. mallei. A localised peritonitis and purulent
inflammation of the testes develop in 2 – 3 days.
Antimicrobial sensitivity tests
These need to be carried out with most members of this group, but especially P. aeruginosa as
multiple drug resistance occurs. Resistance is less common to the aminoglycoside group.
SHEWENELLA
Members of this genus are marine bacteria. Only S. putrefaciens and S. algae are important as
opportunists in animals. Both are positive for H2S production on TSI agar, which is diagnostic.
Shewenella algae can grow at 42°C and is haemolytic after 2 days, and S. putrefaciens cannot grow
at 42°C and is not known to be haemolytic. The identification of Shewenella species is in Table 9.11.
13 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
(+)
D
D
(+)
-
+
D
D
D
D
D
-
+
+
+
-
S. woodyi
+
+
+
+
+
+
+
+
-
S. pealeana
25-35
+
(+)
+
+
+
+
S. oneidensis
25-35
+
+
+
(+)
(+)
+
+
+
S. gelidimarina
25-35
S. frigidimarina
-
S. hanedai
-
S. benthica
S. amazonensis
-
S. baltica
S. putrefaciens
Luminescence
Optimal growth
temp
4°C in 24h
35°C
40°C
Nitrate
Gelatinase
Lipase
Haemolysis
H2S
Growth in 0% NaCl
Growth in 6% NaCl
Utilization of
D-Galactose
D-Fructose
Sucrose
Maltose
Lactose
Succinate
Fumarate
Citrate
S. algae
Characteristic
Table 9.11: Identification of Shewenella species (all are Gram-negative curved or
straight rods, catalase and oxidase positive, motile and grow in 3% NaCl.
Negative for arginine dehydrolase, lysine or ornithine decarboxylase and indole
production)
-
-
+
-
-
-
-
-
4-15
25
20-22
15-17
25-35
25-30
25
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
+
+
+
+
+
D
+
(+)
+
+
+
d
+
+
+
-
+
+
+
-
+
+
+
-
+
+
+
-
+
+
+
(+)
+
(+)
-
+
+
+
+
+
+
-
+
-
-
+
-
Flavobacterium and related bacteria
These bacteria are common specimen contaminants that rarely cause disease. The natural habitats of
these bacteria are soil, plants, foodstuffs and water sources. Their taxonomy has not yet been fully
resolved. They are usually penicillin and polymixin B resistant and most species produce yellow
pigments. They are divided into different groups:
Group A:
Saccharolytic, indole positive. Includes F. meningosepticum, F. group IIb and F. breve.
CDC groups IIe, IIh and IIi are similar, but produce no pigment.
Group B:
Asaccharolytic, indole negative. Includes F. odoratum.
Group C:
Saccharolytic, indole negative. Includes F. thalpophilum, F. mizutaii, Sphingobacterium
multivorum and S. spiritivorum.
Group D:
Asacchyrolytic, indole positive. Weeksella.
Since species that are isolated in a clinical setting, i.e. F. meningosepticum, F. group IIb and F.
odoratum, can be resistant to a number of antibiotics i.e. beta-lactams, tetracyclines, amphenicols and
aminoglycosides, it is essential that antimicrobial susceptibility tests be done. As there are usually
14 | P a g e
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria 
Chapter 9: Miscellaneous Gram-negative, non-fermentative bacilli
errors in disk diffusion tests for this group MIC testing is recommended. These bacteria are often
susceptible to the macrolides, potentiated sulphonamides and fluoroquinolones.
APPENDIX 1
Smith-Baskerville Medium for Bordetella bronchiseptica (Smith and Baskerville 1979)
Bacto Peptone
20g
Sodium chloride
5g
Agar
15g
Distilled water
857ml
The basal medium is autoclaved at 121°C for 15 minutes and then cooled to 55°C. The following
supplementary solutions are mixed together and added to the cooled agar medium.
Antimicrobial supplement:
Gentamicin
0,5ug/ml
Penicillin
20ug/ml
Furaltadone
29ug/ml
Carbohydrate supplement:
Glucose (10%)
100ml
Lactose (10%)
100ml
Bromothymol blue solution (filter sterilised)
2% Stock solution
Bromothymol blue
1g
0,1 N NaOH
25ml
Distilled water
475ml
Dilute to 0,2% solution
40ml
15 | P a g e