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SNP MICROBIOLOGY
The Commonsense Bug Workbook
The Commonsense Bug Workbook
A realistic & practical approach to the identification of
difficult & medically important bacteria
Source: The Commonsense Bug Workbook.doc
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Table of Contents:
Preface:............................................................................................................................................. 3
General: ............................................................................................................................................ 5
Identification Methods: ...................................................................................................................... 5
Characteristics: ............................................................................................................................... 10
Microscopic Appearance:................................................................................................................ 10
Culture Appearance: ....................................................................................................................... 11
Motility: ............................................................................................................................................ 11
Biochemical tests: ........................................................................................................................... 11
Growth Requirements of Value:...................................................................................................... 12
Safety Considerations:.................................................................................................................... 12
Identification of Listeria species ...................................................................................................... 13
Identification of Corynebacterium species and other non-sporing Gram positive rods .................. 16
Identification of the main clinically significant Corynebacterium species ....................................... 19
Identification of other Non-sporing Gram positive rods .................................................................. 21
Identification of Streptococcus species, Enterococcus species and similar organisms ................. 25
Identification of Staphylococcus species, Micrococcus species and Stomatococcus species....... 36
Identification of Non-sporing, Non-branching Anaerobes............................................................... 40
Identification of Anaerobic Cocci..................................................................................................... 49
Identification of Clostridium species ............................................................................................... 52
Identification of Anaerobic Gram positive non-sporing rods ........................................................... 56
Identification of Vibrio & related species ......................................................................................... 58
Identification of Campylobacter and related species ...................................................................... 63
Identification of Helicobacter species ............................................................................................. 66
Identification of Haemophilus species and the HACEK group of organisms.................................. 67
Identification of Pasteurella Species ............................................................................................... 72
Identification of Moraxella species and Morphologically similar species ........................................ 76
References...................................................................................................................................... 80
Source: The Commonsense Bug Workbook.doc
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Preface:
This workbook has been formulated from textbooks, journals and the testing of a large number of
clinical isolates collected over many years. It is intended to be a guide only, a ‘hint’ book, and is
best used in combination with relevant textbooks and bench method manuals. It is meant to be a
useful tool for microbiology staff, which due to factors such as cost constraints, large workload,
lack of expertise, lack of staff or geographical location, do not have the tools of a large reference
to identify difficult organisms.
The aim of this workbook is to provide a “commonsense” guide to the identification of
unusual/difficult organisms encountered in the clinical microbiology laboratory.
Identification of bacteria in the clinical microbiology laboratory is usually based on phenotypic
characteristics. Experience with different specimen types, and knowledge of the bacteria involved
in infections associated with these sites is valuable.
“Brain Storming” amongst peers when encountered with a difficult organism is of great value,
providing different ideas and solutions. Bacteria often vary in their reactions and morphology as
stated in textbooks, even within a genus, e.g. with differing basic biochemical reactions such as
oxidase.
Until potentially dangerous organisms have been excluded, e.g. Brucella, all testing on unknown,
unusual/difficult organisms should be performed in a biological safety cabinet (BSC).
Questions that should be asked prior to identifying an organism in the clinical microbiology
laboratory are:
•
Do I need to identify this organism?
Ø
Ø
Is it clinically relevant or significant?
Is it the cause of the disease?
If the answer to these questions is ‘yes’ (ascertained by clinical aspects), then the next question
is:
•
How far (to what level) do I identify an organism?
Ø
Clinical significance
• clinical data
• specimen site/type
• isolation from multiple sites or specimens
• culture purity and amount of growth
• leucocyte response
• presence or absence of normal regional flora.
Ø
Costs versus Clinical Relevance
Ø
Expertise and staffing levels
Ø
Available testing facilities (i.e. BSC, reagents and identification systems).
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SNP MICROBIOLOGY
•
The Commonsense Bug Workbook
Groups/organisms not included in this workbook:
Ø
Enterobacteriace
There are many systems available for the identification of Enterobacteriace,
which are easy to use, accurate and have a rapid turn-around time.
Ensure, whichever system is used, that it is maintained and quality controlled as
per manufacturers instruction, and that regular updates of the database are
performed.
The Enterobacteriace consists of a very large number of bacteria, which other
than a few “weirdo’s” can be defined as:
“Gram negative bacilli, that are facultative anaerobes, oxidase negative, catalase
positive, grow on MacConkey agar, ferment glucose and reduce nitrate to nitrite*”
*Nitrate negative species include some members of the genus Citrobacter,
Panteoa (Enterobacter) agglomerans, Yersinia and Photorhabdus.
Ø
Legionella species
Few laboratories culture for Legionella species.
Identification is usually
performed by a reference laboratory. Other pathology tests are used for detection
i.e. urine antigen test, serology.
Ø
Bordetella species
Culturing is being replaced by PCR.
Ø
Bacillus species
Refer to separate texts eg Manual of Clinical Microbiology
Ø
Mycobacterium or Nocardia species
Presumptive identification can and should be performed by most clinical
laboratories. Definitive and species identification is usually performed by a
reference laboratory.
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
General:
Identification Methods:
There are three main approaches to identification, particularly of unusual/difficult organisms. The
ability to perform each method will vary between laboratories.
1. The first relies heavily on expertise, the “Guru Factor”. Likely identification is based on
clinical data, growth on different media and under different atmospheric conditions. A
small number of tests are then performed to support the identification.
2. The second is putting the organism through a large number of phenotypic tests such as
those found in commercial systems. The choice of the identification system will vary in
different laboratories and be dependent on the success in preliminary tests. The reaction
data is examined and compared to test results or machine databases in order to provide
identification. Knowledge of the organisms present in the identification system databases
is critical. This dictates which system to use and also how to the interpret results.
Commercial systems should always be used according to manufacturer’s instructions.
3. The third method is a detailed conventional approach. Primary fundamental tests such as
Gram stain, oxidase and catalase tests are performed and results of these tests lead to
secondary/tertiary tests being perform. As more tests are performed, the likely identity of
an organism is revealed. This method is usually laborious, costly and slow as tests can
only be performed when results of the previous tests are available. Setting up a barrage
of tests blindly is impracticable. Although this method can be very effective and does not
rely heavily on expertise, incorrect results at any point may force the investigation down
the wrong path. The result is a wrong identification, and a waste of valuable time and
resources.
The best method is a combination of all three methods, resulting in a cost effective and efficient
identification process, which is within the scope of the average clinical microbiology laboratory.
Any identification result, irrespective of method or confidence, should always be supported by
clinical details, antibiograms (when available), other laboratory results and textbook descriptions,
to reduce the risk of misidentification.
Note:
•
Always use a fresh restreak from nonselective media (where possible) for testing.
•
Always perform and examine purity restreak before accepting any identification.
•
Always use the recommended inoculum.
•
Incubate all tests under the correct conditions and for recommended testing times.
•
Run test controls particularly on infrequently used tests.
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The Commonsense Bug Workbook
Characteristics:
When identifying bacteria, examine all available test results. In this workbook, certain
characteristics have been selected for the purpose of identification.
The first step is the performance of a few simple tests listed below, which will usually allow for
primary provisional placement into a bacterial group:
•
•
•
•
Morphology (Gram, wet preparation)
Atmospheric requirement (growth in the presence/absence of oxygen)
Growth performance of different agar types
Catalase and oxidase tests
Microscopic Appearance:
Gram stain and/or wet preparation provide information on shape, arrangement, organism size
and presence or absence of inclusions e.g. spores. The Gram stain also usually allows
division into Gram positive or Gram negative groups.
*Hint:
• Perform from young cultures
• From non-selective media
• Away from antibiotics
• From broth cultures when possible.
Terms valuable in microscopic preparations:
•
Staining – even, irregular, bipolar, beaded, barred
•
Shape – coccoid, coccobacilli (short rods), long rods, filamentous, curve rods,
spiral forms.
•
Motility – darting, wobbling, tumbling
•
Spores – spherical, oval, centered, sub terminal, terminal, cause bulging,
presence outside cell.
•
Capsule – presence/absence
•
Size – length and width
•
Shape of sides and ends – pointed ends, parallel sides, bulging sides
•
Arrangement – singly, in pairs, chairs, tetrads, Chinese letter shapes
•
Irregular forms – variation in shape and size, branched, fusiform or swollen
forms.
•
Pleomorphism – variation in shape e.g. filaments and coccobacilary forms.
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Culture Appearance:
Colonies of a bacteria growing on specific media under certain specific conditions are
described by size, shape, consistency and pigment production. This in many instances is
critical, as it provides one of the primary steps in the identification process.
Most good textbooks will provide you with further information and detailed characteristics.
Presence/absence and type of haemolysis may also be very valuable in bacterial
identification.
Hint: When checking for pigment production observe both old and new cultures and
look for pigment on swab after touching individual colonies.
Motility:
Many bacteria are observed to be motile and move from one position to another when
suspended in fluid. True motility must not be confused with Brownian movement (vibration
caused by molecular bombardment) or convection currents.
Microscopic examination may indicate whether a motile organism has polar flagellae, shown
by a darting, zigzag movement or peritrichate flagellae, which cause a less vigorous and
more vibratory movement.
Some bacteria may be motile at different temperatures e.g. motile at ambient temperature but
not at 37ºC, or vice versa.
Hint: To exclude Brownian motion, check for upstream swimming – phase
microscopy is an advantage. A negative hanging drop or wet preparation may not
mean the organism is non-motile
Biochemical tests:
Numerous biochemical tests may be used for the identification of bacteria. Some such as
catalase and oxidase are rapid and easy to perform and may be used for preliminary
differentiation purposes. The fermentation of glucose may also be used to distinguish
between groups of organisms.
•
Catalase: Hydrogen peroxide is formed by some bacteria as an oxidative end
product of the aerobic breakdown of sugars and, if allowed to accumulate, is highly
toxic. The catalase enzyme breaks down hydrogen peroxide to water and gaseous
oxygen.
•
Oxidase: The oxidase test is used to detect an intracellular cytochrome oxidase
enzyme system. This system is usually present only in aerobic organisms, which are
capable of utilizing oxygen as the final hydrogen acceptor.
•
Fermentation of glucose: Some aerobic organisms metabolise glucose oxidatively
(i.e. oxygen is the ultimate hydrogen acceptor). Other organisms ferment glucose
and the hydrogen acceptor is then another element such as sulphur.
Source: The Commonsense Bug Workbook.doc
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The Commonsense Bug Workbook
Growth Requirements of Value:
•
Atmosphere
Ø
Ø
Ø
Ø
Ø
Strict aerobes → grow only in the presence of O2
Strict anaerobes → grow only in the absence of O2
Facultative → organisms from aerobic and anaerobic
Microaerophilic → grow best in reduced O2 atmosphere
(addition of 5-10% CO2enhances growth)
Capnophilic → organisms require CO2 for growth
Hint: Use the same media, same inoculum and the same incubation times
•
Temperature
Ø
Ø
Ø
Psychrophilic → grow at ↓ temperatures
Mesophiles → grow at temperatures between 10-45ºC (optimal 30-40ºC)
Thermophiles → will not grow or grow poorly at 37ºC (optimal 40-60ºC)
Hint: Most clinically significant organisms are mesophiles.
•
Nutrition
Ø
Ø
Ø
Ø
The ability to grow on ordinary nutrient agar.
Growth performance on blood or chocolate agar.
Growth on MacConkey agar.
Requirements for specific factors such as charcoal, X factor (haemin) or V
factor (NAD), lipids.
Safety Considerations:
•
Laboratory gowns
recommendations.
and
gloves
•
Laboratory methods that give rise to infectious aerosols must be conducted in an
appropriate biological safety cabinet particularly on unknown bacterial isolates.
•
Laboratories must comply with transport regulation of biological agents, if
transportations of bacteria are required.
Source: The Commonsense Bug Workbook.doc
should
be
used
as
dictated
by
current
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The Commonsense Bug Workbook
Identification of Listeria species
Target Organisms
•
•
•
Listeria monocytogenes
Listeria ivanovii
Listeria seeligeri
Taxonomy
•
There are currently seven species in the genus Listeria.
Characteristics
•
Members of the genus are facultative anaerobes.
•
They are non-sporing, non-acid fast and do not possess a capsule.
•
Listeria species are motile by peritichous flagella when grown at < 30ºC and display a
characteristic “tumbling” motility. Best viewed using phase microscopy.
•
They are catalase positive, oxidase negative and ferment carbohydrates.
Microscopic appearance
•
Gram positive rods approximately 0.5 x 0.5-3µm with rounded ends, occurring singly
or sometimes in pairs and may resemble “coryneforms’ or diplococci.
•
They are non-sporing, non-branching and non-capsulated
Identification
•
Colonies on blood agar or Listeria selective agar are identified by colonial
appearance, Gram stain, catalase production and tumbling motility at ambient
temperature. .
•
All identification tests should ideally be performed from non-selective agar.
•
If confirmation of identification is required, isolates should be sent to a Reference
Laboratory.
Primary isolation media
•
Blood agar incubated in 5-10% CO2 at 35-37ºC for 16-48 hours.
•
Listeria selective agar incubated in air at 35-37 ºC for 40-48 hours
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Colonial appearance
•
All Listeria species produce non-pigmented colonies on blood agar, which have a
characteristic ground glass appearance.
•
On Listeria selective agar, Listeria species appear as black colonies with a
surrounding black zone produced by hydrolysis of the aesculin.
•
The appearance of L.monocytogenes, L.ivanovii, and L.seeligeri is described below.
L.monocytogenes
Blood agar: Colonies are 0.5-1.5mm in diameter,
smooth, translucent and emulsifiable, with zones of hazy
β-haemolysis. Colonies resemble Group B Streptococci.
Non-haemolytic colonies occasionally occur
L.ivanovii
Blood agar: colonies are similar to L.monocytogenes
but develop larger zones of complete haemolysis with
outer zones of partial haemolysis.
L.seeligeri
Blood agar: zones of β-haemolysis are produced.
Test Procedures
•
Motility test
All Listeria species exhibit tumbling motility at ambient temperatures (<30ºC) but not
at 37ºC
•
Commercial identification kit
•
Summary table of results
Species
Catalase
Tumbling
motility at
<30ºC
Tumbling
motility at
37ºC
L.monocytogenes
+
+
-
L.ivanovii
+
+
-
L.seeligeri
+
+
-
Source: The Commonsense Bug Workbook.doc
Commercial
identification kit
Refer to
manufacturer’s
Instructions
Refer to
manufacturer’s
Instructions
Refer to
manufacturer’s
Instructions
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The Commonsense Bug Workbook
Flowchart – Identification of Listeria species
Clinical specimens
Primary isolation plate (blood agar)
Blood agar – non-pigmented translucent
haemolytic colonies*
Gram stain
Gram positive rods
Catalase
Positive
Negative
Tumbling motility
(Hanging drop, ambient temperature <30º
Positive
Negative
All Listeria
species
Not Listeria
species
Not Listeria species
Commercial identification
kit
If required, save pure isolate on a blood or nutrient agar slope for referral to a reference laboratory.
*Non-haemolytic strains of L.monocytogenes occur occasionally
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The Commonsense Bug Workbook
Identification of Corynebacterium species and other
non-sporing Gram positive rods
Target Organisms:
Corynebacterium species reported to have caused human infection:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Corynebacterium accolens
Corynebacterium afermentans (two
subspecies)
Corynebacterium minutissimum
Corynebacterium mucifaciens
Corynebacterium propinquum
Corynebacterium pseudodiphtheriticum
Corynebacterium pseudotuberculosis
Corynebacterium coyleae
Corynebacterium diphtheriae
Corynebacterium striatum
Corynebacterium sundsvallense
Corynebacterium thomssenii
Corynebacterium ulcerans
Corynebacterium jeikeium
Corynebacterium lipophiloflavum
•
Corynebacterium macginleyi
•
Corynebacterium matruchotii
•
•
•
•
•
•
•
•
•
•
•
•
Corynebacterium amycolatum
Corynebacterium aquaticum
Corynebacterium argentoratense
Corynebacterium auris
Corynebacterium confusum
Corynebacterium riegelii
Corynebacterium seminale
Corynebacterium durum
Corynebacterium falsenii
Corynebacterium glucuronolyticum
Corynebacterium imitans
Corynebacterium urealyticum
Note: Other Corynebacterium species may be clinically significant
Species morphologically similar to Listeria and Corynebacterium species known to
have caused human infection:
•
•
•
•
•
•
•
•
Arcanobacterium species
Aureobacterium species
Bifidobacterium species
Brevibacterium species
Cellulomonas species
Dermabacter hominis
Erysipelothrix rhusiopathiae
Lactobacillus species
•
•
•
•
•
•
•
Microbacterium species
Mycobacterium species (MOTT)
Oerskovia species
Propionibacterium species
Rhodococcus species
Rothia dentocariosa
Turicella otitidis
Taxonomy
•
The organisms classified as non-sporing Gram-positive rods are very diverse not only
morphologically, but also metabolically and structurally.
Source: The Commonsense Bug Workbook.doc
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The Commonsense Bug Workbook
Microscopic appearance
•
Gram-positive rods, microscopic appearance varies with the species.
Identification
•
Colonies on blood agar are identified by colonial appearance, Gram stain, catalaseproduction and motility.
•
Identification is confirmed by further biochemical tests and/or referral to a Reference
Laboratory.
•
All identification tests should ideally be performed from non-selective agar.
Identification can be performed from Tween containing agar.
Primary isolation media
•
Blood agar incubated in 5-10% CO2 at 35-37°C for 16-48h
Colonial appearance
Corynebacterium species
Characteristics of growth on horse blood agar after
aerobic incubation at 35-37°C for 16-48h in CO2
C.accolens
White, dry colonies. 0.5-1mm after 24h. Sticks to agar
C.afermentans subspecies
afermentans
Non-haemolytic, white. 1-2mm after 24h
C.afermentans subspecies
lipophilum
Grey, glassy colonies
C.amycolatum
White/grey dry colonies. 1-1.5mm after 24h
C.aquaticum
Non-haemolytic, yellow convex colonies after 24h
C.argentoratense
Cream non-haemolytic colonies. 2mm after 24h
C.auris
Non-haemolytic dry colonies become
prolonged incubation. 1-2mm after 48h
C.confusum
White glistening, convex creamy colonies. Up to 1.5mm
after 48h
C.coyleae
White, slightly glistening, sticky colonies with entire edges
after 24h in CO2
C.diphtheriae
White, “Staph-like” colonies (may be Staph latex pos)
C. durum
Non-haemolytic, small convoluted beige colonies with
irregular edges after 48h
Source: The Commonsense Bug Workbook.doc
yellow
after
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Corynebacterium species
Characteristics of growth on blood agar after aerobic
incubation at 35-37°C for 16-48h
C. falsenii
White glistening smooth colonies with entire edge. 2mm
after 24h. Exhibit yellow pigment >72h
C. glucuronolyticum
Non-haemolytic white/yellow convex colonies. 1-1.5mm
after 24h
C. imitans
White/grey, glistening, creamy colony with entire edge. 12mm after 24h
C. jeikeium
Non-haemolytic grey/white. Entire, low, convex. Good
growth on blood agar at 42ºC, poor growth at 22ºC.
Confluent on Tween containing media.
C. kroppenstedtii
Non-haemolytic,
convex colonies
C. lipophiloflavum
Requires lipid for growth. Non-haemolytic <0.2mm on
blood agar. With addition of Tween colonies are 1mm and
yellow
C. macginleyi
Requires lipid for growth. Non-haemolytic, 1mm after 48h
without lipid. With Tween red/beige colonies, 2-4mm
C. matruchotii
Flat colonies. Older cultures are circular, convex, rough
and may be irregular
C. minutissimum
Shiny, moist, convex and circular. Entire edges. 1-1.5mm
after 24h
C. mucifaciens
Yellow glistening, very mucoid colonies. 1-1.5mm after
24h in CO2
C. propinquum
Non-haemolytic, matted surface. 1-2mm after 24h
C. pseudodiphtheriticum
White/cream butyrous colonies
C. pseudotuberculosis
White, dry, sometimes
Mycobacterium colony.
C. riegelii
White, glistening, convex colonies. Entire edges. Up to
1.5mm after 48h
C. seminale
Non-haemolytic grey/beige non-mucoid colonies. Slight
growth at 24h. Abundant growth at 48h
C. striatum
Non-haemolytic white, moist, smooth colonies. >2mm
after 24h
C. sundsvallense
Non-haemolytic, buff/slightly yellow, opaque, shiny
colonies. Adhere to the surface of the agar after 48-72h
C. thomssenii
Slightly glistens, smooth, convex colonies. Very small after
24h. Molar tooth-like after 96h. Adhere to surface of the
agar
non-pigmented,
small,
“cheesy”,
smooth
may
look
and
like
C. ulcerans
C. urealyticum
Non-haemolytic, white, smooth and convex. Pinpoint
colonies after 48h in CO2. Grey/white colonies on CLED.
Confluent growth on Tween containing media
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Identification of the main clinically significant
Corynebacterium species
Target Organisms:
C.diphtheriae
C.ulcerans
C.pseudodiphtheriticum
C.pseudotuberculosis
C.jeikeium
C.urealyticum
C.macginleyi
•
•
•
•
•
•
•
Corynebacterium species which are potentially toxigenic
Corynebacterium diphtheriae var belfanti
Corynebacterium diphtheriae var gravis
Corynebacterium diphtheriae var intermedius
Corynebacterium diphtheriae var mitis
Corynebacterium pseudotuberculosis
•
•
•
•
•
•
•
•
•
•
Corynebacterium ulcerans
Corynebacterium pseudodiphtheriticum
Corynebacterium ureolyticum
Corynebacterium jeikeium
Corynebacterium ulcerans
Primary Separation:
Table 1: (Non-lipid requiring)
C.diphtheriae*
C.ulcerans
C.pseudodiphtheriticum
C.pseudotuberculosis
Nitrate
+/+
v
Urea
+
+
+
Glucose
+
+
+
Sucrose
v
*C.diphtheriae var belfanti is the only nitrate negative biotype.
Table 2: (Lipid requiring)
#
C.jeikeium
#
C.urealyticum
C.macginleyi
Nitrate*
+
Urea*
+
-
Glucose*
+
+
Sucrose*
+
Resistant
+
+
-
*Hint: A heavy inoculum is required and a few drops of sterile serum or Tween 80
needs to be added to the CTA sugar tubes.
#
Resistant to most antibiotics except vancomycin.
Confirmation using a commercial system e.g. API Coryne should be used on all provisionally
identified clinically significant Corynebacterium isolates.
To test for lipid requirement, restreak on media containing Tween 80.
(Nalidixic acid Tween agar) is recommended.
Source: The Commonsense Bug Workbook.doc
The use of NAT
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The Commonsense Bug Workbook
Primary screening of non-lipophilic Corynebacterium species
* Further identification if regarded as significant, perform a commercial identification or refer to
a reference laboratory.
**Possible C.diphtheriae isolate, confirm using a commercial system ie API Coryne & forward
to a reference laboratory for toxin testing.
Consult Pathologist/Clinician urgently for clinical assessment of patient.
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Identification of other Non-sporing Gram positive rods
Other non-sporing Gram positive
rods
Characteristics of growth on blood agar after
aerobic incubation at 35-37°C for 16-48h
Arcanobacterium species
β-haemolysis, which may not be present after
24h.Haemolysis of A. bernardiae is variable
Aureobacterium species
Non-haemolytic, yellow pigment
Bifidobacterium species
Colonies are low, greyish-brown, ovoid with a
brown opaque centre and a translucent crenated
edge
Brevibacterium species
Non-haemolytic, may turn yellow to green after 48h
Cellulomonas species
Non-haemolytic, yellow- or orange-pigmented
Dermabacter hominis
Non-haemolytic, small grey/white convex colonies
Erysipelothrix rhusiopathiae
At 48h 2 distinct colony types appear: a small
smooth form, 0.3-1.5mm, transparent, convex and
circular with entire edges. The large rough form is
flatter, more opaque, with a matt surface and an
irregular edge. Both exhibit α-haemolysis. Look like
a viridans Streptococcus.
Colonies are small and often α-haemolytic on blood
after 48h. Sweet smell better growth anaerobically.
Lactobacillus species
Microbacterium species
May produce a yellow or orange pigment
Mycobacterium species (MOTT)
Rough or smooth colonies. May produce
yellow/orange pigment when grown either in the
light or the dark.
Oerskovia species
Most strains produce a yellow pigment
Propionibacterium species
Small colonies after 48h, grow better anaerobically.
Rhodococcus species
Colonies may be rough, smooth or mucoid and may
be pigmented, cream, beige, yellow, orange or red
Rothia dentocariosa
Non-haemolytic, creamy, dry, crumbly or mucoid
Turicella otitidis
Non-haemolytic, convex, whitish, creamy
Test procedures
•
Catalase activity test (reactions vary).
•
Motility test - performed at 37°C and <30°C for all organisms (reactions vary).
•
Commercial identification kit
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Characteristics
Arcanobacterium species
•
Arcanobacterium pyogenes (formerly Corynebacterium or Actinomyces pyogenes) is
a Gram-positive rod, which may show branching. Colonies on blood agar produce
sharp zones of β-haemolysis after 48 hours incubation. A. pyogenes is facultatively
anaerobic, non-motile, and catalase-negative. Differentiation between A. pyogenes
and A.haemolyticum may prove difficult but they may be distinguished by fermentation
of α-mannose, pyrazinamidase and gelatin tests. They are both mupirocin resistant.
•
Arcanobacterium haemolyticum (formerly Corynebacterium haemolyticum) is a Grampositive rod. Colonies on blood agar after 48 hours produce zones of β-haemolysis
and are similar in appearance to A.pyogenes. A.haemolyticum is non-motile,
facultatively anaerobic and, unlike Corynebacterium species, it is catalase-negative.
•
Arcanobacterium bernardiae formerly (Actinomyces bernardiae) exhibits variable
haemolysis.
Aureobacterium species
•
Aureobacterium species are Gram-positive, irregular, short rods and are catalasepositive. They are obligate aerobes, which produce acid from carbohydrates by
oxidation rather than by fermentation. Strains may be vancomycin resistant and can
be distinguished from C.aquaticum by casein and gelatin hydrolysis.
Bifidobacterium species
•
Bifidobacterium species vary in shape and may be curved, clubbed or branched rods
or occasionally coccoid, Gram-positive forms, 0.5-1.3 x 1.5-8µm. Cells often stain
irregularly. Growth is anaerobic but some species can grow in air enriched with 10%
CO2. Bifidobacterium species are non-sporing, non-acid fast and non-motile.
Bifidobacterium species ferment carbohydrates and are catalase negative.
Brevibacterium species
•
Brevibacterium species are Gram-positive rods, which that show a marked rod-coccus
cycle. On fresh subculture, cells appear as bacilli but become coccal in older cultures.
Colonies on blood agar are non-haemolytic and may turn a yellow to green colour
after 48 hours incubation. Brevibacterium species are non-motile, aerobic, ureasenegative and catalase-positive.
Cellulomonas species
•
Cellulomonas species produce yellow or orange-pigmented colonies. They are
catalase-positive and some species are motile. Cellulomonas species differ from
Oerskovia species as they lack hyphal growth.
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Dermabacter hominis
•
Dermabacter species are very short Gram-positive rods that may be mis-interpreted
as cocci. Dermabacter hominis, currently the only member of the genus, is nonhaemolytic, non-motile and catalase-positive. Dermabacter species are fermentative
and produce acid from glucose, lactose, sucrose and maltose. They hydrolyse
aesculin and do not reduce nitrate or produce pyrazinamidase or DNase.
Erysipelothrix rhusiopathiae
•
E.rhusiopathiae is a Gram-positive rod, which produces α-haemolysis on blood agar.
It is facultatively anaerobic, non-motile and catalase-negative. Erysipelothrix species
can be distinguished from Lactobacillus species by its ability to produce H2S in a triple
sugar iron agar slant (may need ≥48hrs, a heavy inoculum is required).
Gardnerella vaginalis
•
Gardnerella vaginalis is a pleomorphic, Gram-variable rod. It is facultatively anaerobic
and non-motile. G.vaginalis is non-sporing, non-encapsulated and both oxidase and
catalase-negative but hippurate positive.
Lactobacillus species
•
Lactobacillus species are long Gram-positive rods. Colonies are small and often αhaemolytic on blood agar after 48 hours. They are facultatively anaerobic, rarely
motile and catalase-negative.
•
Lactobacillus species is may not always be Vancomycin resistant.
Microbacterium species
•
Microbacterium species are Gram-positive slender rods. They may produce a yellow
or orange pigment. The optimum growth temperature is 30°C. Microbacterium species
are aerobic and some species are motile. All species are catalase-positive.
Mycobacterium species
•
Mycobacterium species other than Mycobacterium tuberculosis (MOTT) may be
isolated on primary culture within 48 hours. Refer to the Reference Laboratory.
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Oerskovia species
•
Oerskovia species are Gram-positive branching rods. They form a mycelium, an
extensively branching substrate hyphae that breaks up to form rod-shaped or coccoidrod elements. Most strains produce a yellow pigment. They are facultatively
anaerobic, motile and catalase-positive.
Propionibacterium species
•
Propionibacterium species are Gram-positive pleomorphic rods (short “Y” forms).
Strains generally grow better anaerobically, particularly on primary isolation,
producing small colonies after 48 hours. Propionibacterium species are facultatively
anaerobic and are non-motile. They are catalase-positive except Propionibacterium
propionicum (formerly known as Arachnia propionica), which is catalase negative.
•
The indole test for P.acnes, if not positive directly from colonies, is best performed on
a broth culture (TSB), after overnight incubation.
Hint: A sheen around colonies, is usually present due to propionic acid production.
Rhodococcus species
•
Rhodococcus species usually stain Gram-positive and cells form as cocci or short
rods which grow in length and may form an extensively branched vegetative mycelium
which may fragment. They are usually partially acid-fast. Colonies may be rough,
smooth or mucoid and are colourless, cream, beige, yellow, orange or red. Moisture
and extended incubation enhances pigment production.
Rothia dentocariosa
•
R. dentocariosa is a Gram-positive irregular rod and may show branching in young
cultures. In older broth cultures cells may be coccoid, which distinguishes them from
Actinomyces species. It grows well on simple media and colonies may be creamy,
dry, crumbly or mucoid. Rothia species are facultatively anaerobic, non-motile,
catalase positive and ferment carbohydrates.
Turicella otitidis
•
This genus comprises a single species, Turicella otitidis. Microscopically it resembles
a coryneform but has longer cells. It may be distinguished by colonial morphology
from C.afermentans and C.auris. T.otitidis colonies are convex, whitish, creamy and
non-haemolytic compared with the flat, grey-white and non haemolytic colonies of
C.afermentans and the convex, dry, adherent, yellowish colonies of C.auris. T.otitidis
is non-fermentative and occurs either alone or with Gram-negative rods. Strains
exhibit a strong CAMP reaction and it is catalase-positive. T.otitidis may be
misidentified, often as Corynebacterium species, by some commercial identification
systems .
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Identification of Streptococcus species, Enterococcus
species and similar organisms
Target organisms
•
Streptococcus species reported to have caused human infection
•
Streptococci possessing Lancefield group antigens A-G
Group A
Streptococcus pyogenes (Streptococcus anginosus and
Streptococcus constellatus may cross react with the Lancefield
group A antigen
Group B
Streptococcus agalactiae
Group C
Streptococcus dysgalactiae subspecies equisimilis,
Streptococcus equi subspecies equi, Streptococcus equi
subspecies zooepidemicus (Streptococcus anginosus and
Streptococcus the Lancefield group C antigen)
Group D
Enterococcus species (see below)
Streptococcus bovis
Streptococcus gallolyticus
Group F
Streptococcus anginosus
Streptococcus constellatus
Group G
Group G streptococci (Streptococcus constellatus may cross
react with the Lancefield group G antigen,
Other streptococci
Streptococcus pneumoniae
Streptococcus suis
Streptococcus
anginosus/milleri group
Streptococcus anginosus
Streptococcus constellatus
Streptococcus intermedius
Streptococcus mutans group
Streptococcus mutans
Streptococcus sobrinus
Streptococcus sanguis group
Streptococcus cristatus
Streptococcus gordonii
Streptococcus snaguinis
Streptococcus parasanguinis
Streptococcus mitis group
Streptococcus mitis
Streptococcus oralis
Streptococcus salivarius group
Streptococcus salivarius
Streptococcus vestibularis
Nutritionally variant
streptococci
Abiotrophia adjacens
Abiotrophia defective
Abiotrophia elegans
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Enterococcus species reported to have caused human infections
•
•
•
•
•
•
•
•
Enterococcus faecalis
Enterococcus faecium
Enterococcus casseliflavus
Enterococcus dispar
Enterococcus durans
Enterococcus flavescens
Enterococcus gallinarum
Enterococcus raffinosus
Other genera reported to have caused human infections
•
•
•
•
•
•
Aerococcus species
Gemella species
Helcococcus species
Lactococcus species
Leuconostoc species
Pediococcus species
Microscopic appearance
•
Gram stain
Ø
Streptococcus, Enterococcus and Lactococcus species are Gram-positive,
round or ovoid cells occurring in pairs, short or long chains or sometimes in
clusters
Ø
Streptococcus pneumoniae are Gram-positive, lanceolate cells occurring in
pairs, often with a visible capsule
Ø
Aerococcus, Pediococcus and Helcococcus species are Gram-positive cocci
in clusters or tetrads
Ø
Gemella and Leuconostoc are Gram-positive cocci occurring in pairs, clusters
and short chains (Gemella may be easily decolourised)
Primary isolation media
•
Blood agar incubated in 5-10% CO2 at 35-37ºC for 16-48h
•
Staph/Strep agar incubated in air at 35-37ºC for 16-48h
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Flowchart: Identification of Streptococcus & Enterococcus
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Colonial appearance
Organism “group”
Haemolysis
Characteristics of growth on blood agar after
incubation at 35-37oC for 16-24h
β-haemolytic Streptococci
β
Approximately 0.5mm, entire edged, may have a
dry appearance, colonies may be difficult to pick off
the plate
“viridans” Streptococcus
α or non
Colonies are 0.5 - 1.0mm, entire edged
Enterococcus species
α,β or non
Colonies are larger than those of streptococci,
usually 1-2mm, with a wet appearance. Haemolysis
is variable
S. pneumoniae
α
Colonies are 1-2mm and may appear as
“draughtsman” colonies. After anaerobic incubation
colonies may be larger and mucoid
“S. anginosus”
α,β or non
Colonies are small (≤ 0.5mm), haemolysis is
variable. Some strains have a white “heaped” up
colony
NVS
α or non
Colonies are small (≤ 0.5mm), require pyridoxal for
growth
Aerococcus species
α
Resemble “viridans” Streptococci
Gemella species
α or non
Resemble “viridans” Streptococci
Helcococcus species
non
Colonies are 0.5 - 1.0mm, entire edged
Lactococcus species
α or non
Resemble Enterococcus
Leuconostoc species
α or non
Colonies are 0.5 - 1.0mm, entire edged
Pediococcus species
α or non
Resemble “viridans” streptococci
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Summary of test results
Possess Lancefield
grouping antigen
(Commercial kit)
Optochin
sensitivity
Catalase
Bile Aesculin
hydrolysis
PYR
Group B, C, F and G
+
R
-
-
-
S. pneumoniae
–
S
-
-
D
Group D
+
R
-
+
-
Enterococci
+
R
V
+
+
S. bovis
+
R
-
+
-
Aerococcus species
-
R
V
V
D
Group A
+
R
-
-
+
S. anginosus group
V
R
-
V
-
“viridans” streptococci
-
V
-
Gemella species
R
W
-
+
Helcococcus species
R
-
-
+
Leuconostoc species
R
D
D
+
-
Pediococcus species
CR
R
-
V= variable, R=resistant , S=sensitive, CR=cross reacts, D=6-84% strains,
W=weak positive reaction
Characteristics
Streptococcus pyogenes (Lancefield group A)
•
Streptococcus pyogenes is a Gram positive coccus occurring in chains. After 18-24h
incubation at 35-37ºC on blood agar colonies are approximately 0.5mm, domed, with
an entire edge. Some strains may produce mucoid colonies. Haemolysis is best
observed by growing the culture under anaerobic conditions because the
haemolysins are more stable in the absence of oxygen.
•
Lancefield group A streptococci will not grow on media containing bile.
•
Low concentration bacitracin susceptibility has been used for screening purposes but
is unreliable. Resistance to benzyl penicillin has not been reported.
•
The pyrrolidonyl arylamidase (PYR-aminopeptidase) (PYR) test is positive for Group
A streptococci and negative for most other groupable streptococci, although some
human strains of groups C and G may be positive. Enterococcus are also PYR
positive.
•
Minute colony forms of the S. anginosus group may cross react with Lancefield
groups A C and G antibodies and may grow on media containing bile.
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Streptococcus agalactiae (Lancefield group B)
•
Streptococcus agalactiae are Gram positive coccus, occurring in chains. After 18-24h
incubation at 35-37ºC, colonies tend to be slightly larger than other streptococci
(approximately 1mm) and have a less distinct zone of β-haemolysis. Some strains
may be non-haemolytic.
•
Lancefield group B Streptococci will grow on media containing bile.
•
Islam’s medium, to detect orange pigment production, may be useful for primary
isolation and presumptive identification, but is not recommended in this workbook.
Streptococcus dysgalactiae subspecies equisimilis (Lancefield grps A,C,G and L)
Streptococcus equi subspecies zooepidemicus (Lancefield group C) and Lancefield
group G Streptococcus
•
Microscopically these species are Gram positive cocci, occurring in chains. Large
colony forms of Lancefield groups C and G Streptococcus (0.5mm) produce similar
colonies to Group A Streptococcus.
•
Lancefield groups C and G streptococci will not grow on media containing bile.
•
Minute colony forms of the S.anginosus group (formerly the S.milleri group) can cross
react with the Lancefield groups A C and G antibodies and may grow on media
containing bile.
Enterococcus species, Streptococcus bovis group (Lancefield group D)
•
The genus Enterococcus and organisms of the S.bovis group possess Lancefield
group D antigen. Microscopically the organisms are Gram positive cocci, spherical or
ovoid in shape (0.6-2.5µm), usually occurring in pairs or short chains in broth culture.
After 18-24h incubation at 35-37ºC on blood agar colonies are 1-2mm and may be α,
β or non-haemolytic on horse blood agar. Most species will grow on nutrient agar at
45ºC. A few will grow at 50ºC, at pH 9.6 and in 6.5% NaCl. They can also survive
60ºC for 30 minutes.
•
Lancefield group D streptococci will grow on media containing bile and may be
differentiated from other streptococci by rapid hydrolysis of aesculin in the presence
of 40% bile.
•
Enterococcus species are also heat resistant (60oC/30mins) and PYR-positive which
differentiates them from S.bovis and S.gallolyticus.
•
There are six species included in the S.bovis group: S. bovis, S.equinus,
S.gallolyticus (formerly S.bovis biotype I), S.infantarius (formerly S.bovis biotype II),
S.pasteurianus (formerly S.bovis biotype II/2) and S.lutetensis.
•
Microscopically these species are Gram-positive cocci, occurring in chains after 1824h incubation at 35-37ºC in CO2 or anaerobically.
•
Colonies are usually non-haemolytic on blood agar and 1-2mm in diameter.
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•
Members of the S.bovis group may be misidentified as Enterococcus species as
many strains share the group D antigen. It is important to identify S.bovis group
organisms from clinical material especially in cases of bacteraemia, as S.gallolyticus
and S.pasteurianus are associated with chronic bowel disease particularly
adenocarcinoma of the colon. S.bovis group may be differentiated from Enterococcus
species by a negative reaction in both PYR and arginine tests, whereas Enterococcus
is usually positive for both.
•
Enterococcus species are facultative anaerobes. Two species within the genus are
motile, Enterococcus cassiflavus and Enterococcus gallinarum. Enterococcus are
oxidase negative and ferment carbohydrates. Most species are catalase negative, but
some strains produce a pseudocatalase.
•
Most Enterococcus possess the group D antigen although some strains may cross
react with Lancefield group G antiserum.
•
E. faecalis are very rarely resistant to ampicillin.
Streptococcus anginosus, Streptococcus constellatus, Streptococcus intermedius
(formerly the Streptococcus milleri group)
•
Microscopically these species are Gram positive cocci, occurring in chains. Colonies
on blood agar are small (≤0.5mm) and may exhibit α, β or no haemolysis after 16-24h
at 35-37ºC. Incubation conditions may be of some value for the presumptive
identification of the S.anginosus group as growth is enhanced by a low oxygen
tension and raised CO2 levels.
•
Organisms of this group may possess the Lancefield group A, C, F or G antigen and
in some instances may be ungroupable. S.intermedius is non-haemolytic and
possesses no group antigen. S.constellatus may express group C, F or N and
S.anginosus group A, C, F, G or N antigens. Streptococci in this group will grow on
media containing bile although they are not salt tolerant.
•
Resistance to sulphonamides and bacitracin may be used as screening tests for
organisms of the S.anginosus group.
Streptococcus pneumoniae
•
Streptococcus pneumoniae are Gram positive typically lanceolate cells occurring in
pairs, which may be capsular. Colonies are 1-2mm, α-haemolytic and may appear as
‘draughtsman' colonies due to autolysis of the organisms after incubation in 5-10%
CO2 at 35-37oC for 16-24h. Under anaerobic conditions colonies may appear larger
and more mucoid.
•
Pneumococci are usually sensitive to Optochin (ethylhydrocupreine hydrochloride),
which enables rapid identification of the organism, but resistance may occur.
Pneumococci are also soluble in bile salts solution.
•
Pneumococci may also be identified by serological methods. The ‘Quellung reaction'
(capsular swelling) may be used microscopically to identify specific types of
pneumococci. Commercial agglutination tests are also available for the rapid
detection of pneumococcal antigen.
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Streptococcus suis
•
S.suis is β-haemolytic on horse blood agar, Optochin resistant and PYR-negative.
They are commonly associated with the Lancefield groups R, S and T. S.suis I is
associated with group S and S.suis II with group R. They do not grow in 6.5% NaCl
broth. Some strains are able to grow in the presence of 40% bile and all are able to
hydrolyse aesculin.
Other streptococci (“viridans” streptococci)
•
These species are Gram-positive cocci occurring in chains, which are
indistinguishable from β-haemolytic streptococci. Colonies are 0.5-1.0mm and may
be α or non-haemolytic on blood agar after anaerobic incubation at 35-37ºC in CO2 for
16-24h.
•
Generally these streptococci would not require further identification, other than as α
or non-haemolytic streptococcus, when isolated from sites where they are considered
normal flora. Identification of streptococci in cases of suspected endocarditis has
some value in the confirmation of the diagnosis and for epidemiological purposes.
Some species of streptococci, eg Streptococcus sanguis and Streptococcus oralis
(formerly mitior), may account for up to 80% of all streptococcal endocarditis cases.
Nutritionally Variant Streptococci (NVS)
•
NVS have now been reclassified as Abiotrophia adiacens, Abiotrophia elegans and
Abiotrophia defectiva. NVS require media supplemented with either pyridoxal or
cysteine for growth.
•
NVS should be suspected when Gram positive cocci resembling streptococci are
seen in positive blood cultures, which subsequently fail to grow on subculture. Repeat
subculture of suspect broth should include a blood agar plate with a Staphylococcus
aureus streak which is examined for satellitism of NVS around the staphylococcus.
Alternatively, media may be supplemented with 10mg/L pyridoxal hydrochloride.
Aerococcus species
•
Aerococci resemble “viridans” streptococci on culture but differ microscopically by
characteristically occurring as tetrads or clusters, similar to staphylococci. Sometimes
a weak catalase or pseudocatalase reaction is produced. Some strains of Aerococcus
viridans are bile aesculin-positive and PYR-positive. Aerococcus urinae is bile
aesculin-negative, PYR-negative.
•
In some commercial identification systems A.viridans may be mis-identified as
Helcococcus kunzii.
Gemella species
•
There are four species: Gemella haemolysans, Gemella morbillorum (formerly
Streptococcus morbillorum), Gemella bergeriae and Gemella sanguinis. These
bacteria easily decolourise on Gram staining, occurring as Gram negative cocci in
pairs, tetrads, clusters or short chains. Gemella species are α or non-haemolytic on
blood agar and resemble colonies of viridans streptococci.
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Helcococcus kunzii
•
Helcococcus kunzii, similar to Aerococcus species, has recently been described.
H.kunzii produces tiny grey, slightly α-haemolytic colonies; growth is stimulated by the
addition of serum or Tween 80 to the basal medium.
•
In some commercial identification systems A.viridans may be mis-identified as
Helcococcus kunzii.
Lactococcus species
•
Lactococcus species are physiologically similar to Enterococcus. They are α or nonhaemolytic, Gram positive cocci which occur singly, in pairs or chains. They are bile
aesculin positive, but do not possess group D antigen.
Leuconostoc species
•
Leuconostoc species are Gram positive lenticular cocci occurring in pairs and chains
and are characteristically vancomycin resistant. They may be confused with the
Enterococcus because most Leuconostoc species are bile aesculin positive and
some cross-react with the group D antisera.
Pediococcus species
•
Pediococcus species may resemble viridans streptococci on culture, but
microscopically they are similar to staphylococci. They are Gram positive cocci
appearing in pairs, clusters and tetrads and are vancomycin resistant. They may be
confused with Enterococcus because they are bile aesculin-positive and cross-react
with the Group D antisera.
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Flowchart: Non-β-haemolytic Gram positive cocci
Non-beta-hemolytic catalase-negative gram-positive-cocci
with “streptococcal” Gram stain
(coccobacilli in pairs and chains)
Vancomycin
S
R
Leuconostoc
LAP
+
Globicatella
PYR
+
Streptococcus
Lactococcus
Satelliting
behavior
+
Abiotrophia
(formerly nutritionally
variant streptococci)
Enterococcus
Lactococcus
Enterococci grow at both 10 and 45oC.
Lactococci grow at 10oC but not at 45oC.
Streptococci may grow at 45oC but not at 10oC.
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Flowchart: Non-beta haemolytic, catalase negative Gram positive cocci with
“Staphylococcal” Gram stain (cocci in clusters, tetrads, pairs)
Non-beta-hemolytic catalase-negative Gram-positive cocci with
“staphylococcal” Gramstain (cocci in clusters, tetrads, pairs)a
PYR
+
Vancomycin
LAP
+
R
S
Pedicoccus
Esculin
hydrolysis
+
-
Stomatococcus
Gemellab
Stomatococcus
Aerococcus urinae
6.5%
NaCl
6.5% NaCl
+
+
-
Aerococcus
urinae
Stomatococcus
-
Aerococcus viridans
(microaerophilic)
Esculin
hydrolysis
Helcoccus (facultative)
+
-
Helcoccus
Gemellab
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Identification of Staphylococcus species,
Micrococcus species and Stomatococcus species
Target Organisms
Staphylococcus species reported to have caused human infection
•
•
Staphylococcus
Staphylococcus
aureus
aureus
•
•
•
•
•
•
•
•
•
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
epidermidis
capitis
capitis
hominis
hominis
haemolyticus
lugdunensis
saccharolyticus
warneri
•
•
•
•
Staphylococcus
Staphylococcus
Staphylococcus
saprophyticus
cohnii
cohnii
•
•
•
•
•
•
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
Staphylococcus
caprae
hyicus
intermedius
schleiferi
schleiferi
simulans
subspecies
aureus
anaerobius
capitis
ureolyticus
hominis
novobiosepticus
cohnii
ureolyticus
S.epidermidis group
S. saprophyticus group
coagulans
schleiferi
Other species reported to have caused human infection
•
•
Micrococcus luteus
Stomatococcus mucilaginosus
Staphylococcus species
Introduction
•
The staphylococci most frequently associated with human infection are S. aureus,
S.epidermidis and S.saprophyticus. Other Staphylococcus species may also be
associated with human infection.
Taxonomy
•
More than thirty species of staphylococci have been recognised, most of which are
found only in lower mammals. The coagulase positive staphylococci are S. aureus,
S.intermedius, S.hyicus and S.schleiferi. The coagulase negative staphylococci
(CNS) can be divided into six major groups but the species found on humans are
located within two groups.
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Characteristics
•
Staphylococcus species are Gram positive, non-motile, non-sporing cocci occurring
singly, in pairs and in irregular clusters, size may be variable.
•
Colonies are opaque and may be white or cream and are occasionally yellow or
orange. The optimum growth temperature is 30-37°C.
•
They are facultative anaerobes and have a fermentative metabolism.
•
Staphylococcus species are usually catalase-positive and oxidase-negative. Nitrate is
often reduced to nitrite. Some species are susceptible to lysis by lysostaphin but not
by lysozyme and are usually able to grow in 10% sodium chloride. Some species
produce extracellular toxins. Staphylococci may be identified by the production of
deoxyribonuclease (Dnase) and/or a heat-stable DNase (thermostable nuclease).
Coagulase-positive Staphylococcus species
Staphylococcus aureus
•
Staphylococcus aureus is a primary pathogen, which may be associated with severe
infection. It is important to distinguish it from the opportunistic coagulase negative
Staphylococci. In routine laboratory practice, the production of coagulase is frequently
used as the sole criterion to distinguish S.aureus from other Staphylococci; as other
coagulase positive Staphylococci have been found only occasionally in human
infection or carriage. The production of coagulase and thermostable nuclease by
these staphylococci may lead to their misidentification as S.aureus.
•
S. aureus produces virulence factors such as protein A, capsular polysaccharides; α
toxin and some strains produce toxic shock syndrome -1 toxin (TSST-1) or other
toxins. Multiresistance to antibiotics may be associated with methicillin resistant
strains. It is thermostable nuclease-positive.
Staphylococcus aureus subspecies anaerobius
•
S.aureus subspecies anaerobius is rarely isolated from clinical specimens. It grows
poorly aerobically and growth may be CO2 dependent. It is slide coagulase negative
and thermonuclease negative. It may be catalase negative. Strains may be identified
by better growth anaerobically and they may give a positive coagulase test result.
However, because growth may be poor the coagulase result may be negative and
suspected isolates should be referred to a Reference Laboratory.
Other coagulase positive Staphylococcus species
•
S.hyicus may be coagulase-positive (11-89% of strains) and thermostable nucleasepositive. S.intermedius is coagulase-positive and thermostable nuclease-positive.
S.schleiferi subspecies coagulans is coagulase-positive and thermostable nuclease
positive, and S.schleiferi subspecies schleiferi is coagulase-negative and
thermostable nuclease-positive.
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Coagulase negative Staphylococcus species
•
The CNS group are opportunistic pathogens, which lack many of the virulence factors
associated with S.aureus. There are more than 30 species of CNS. S.epidermidis and
S.saprophyticus are the species most often associated with infection but S.capitis,
S.cohnii, S.haemolyticus, S.hominis, S.lugdenensis, S.schleiferi subspecies schleiferi,
S.simulans and S.warneri have also been implicated.
•
Many of these species are also thermostable nuclease-negative. Multi-resistance is
associated with some strains of S.epidermidis. It is thermostable nuclease-negative.
S.haemolyticus is often multi-resistant and frequently demonstrates reduced
susceptibility to teicoplanin. S.saprophyticus is novobiocin resistant. S.pasteuri can
be phenotypically distinguished from all of the other novobiocin-susceptible
staphylococci except S.warneri, from which it can only be differentiated by
genotyping.
•
S.saccharolyticus was previously known as Peptococcus saccharolyticus.
Micrococcus species
•
Micrococcus species are strictly aerobic. Micrococcus luteus produces yellow
colonies. Cells are large Gram positive cocci arranged in tetrads. Micrococci may be
distinguished from staphylococci by a modified oxidase test. Staphylococcus species,
with the exception of S.sciuri, S.lentus and S.vutulus are oxidase-negative and
Micrococcus species are oxidase positive.
Stomatococcus species
•
Stomatococcus species are weakly catalase-positive. Growth is facultatively
anaerobic. The species associated with infection is S.mucilaginosus previously
known a Micrococcus mucilaginosus or Staphylococcus salivarius.
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Presumptive identification of Staphylococcus species
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Identification of Non-sporing, Non-branching
Anaerobes
Introduction
•
This section describes the characterisation of non-sporing, non-branching anaerobic
bacteria. Identification of this group of organisms to species level is difficult without
the use of techniques such as gas-liquid chromatography (GLC) or PCR methods.
Because of the large number of species, it is difficult to provide genus definitions and
a single biochemical table to differentiate all species. In the routine laboratory,
susceptibility to metronidazole is frequently regarded as sufficient indicator of an
anaerobe being present in a clinical specimen. However, this approach is
fundamentally flawed because if anaerobes are by definition sensitive to
metronidazole, no metronidazole resistant anaerobes will ever be detected. An
increasing number of metronidazole resistant Bacteroides fragilis are being recorded
and these organisms will be missed by such an approach. Most clinical microbiology
laboratories regard a report of “mixed anaerobes” as adequate in many instances .
Taxonomy
•
Anaerobic Gram-negative rods
The taxonomy of the anaerobic bacteria is in a state of continuous change with the
addition of new species and reclassification of old species. An example occurs in the
genus Bacteroides, most of the saccharolytic pigmented species are now included in
the genus Prevotella and the asaccharolytic species have been assigned to the
genus Porphyromonas. There are more than 20 genera of anaerobic Gram-negative
rods. The commonest human isolates are Bacteroides, Fusobacterium,
Porphyromonas and Prevotella.
•
Anaerobic Gram-negative cocci
Three genera are included in the anaerobic Gram-negative cocci, but only one Veillonella - is found in clinical material. There are seven species of Veillonella, of
which Veillonella parvula is the most commonly isolated species from human
specimens.
•
Anaerobic Gram-positive cocci
The classification of the anaerobic Gram-positive cocci is continually changing with
the addition of new species and renaming old species. There are six genera of
anaerobic Gram-positive cocci which may be isolated from humans. They are:
Peptostreptococcus (12 species with three proposed new species), Peptococcus (one
species), Atopobium (one species), Coprococcus (three species), Ruminococcus (10
species) and Sarcina (three species). The majority of human isolates are
Peptostreptococcus species.
•
Anaerobic Gram-positive rods
The only genus of non-sporing, non-branching anaerobic Gram-positive rods of
clinical importance is Eubacterium. There are 25 members of the genus. The genus is
currently ill-defined and further taxonomic work is likely to sub-divide it. Eubacterium
lentum has been reclassified as Eggerthella. E.lenta and is the species most
frequently associated with human infections.
Note: Some clostridia do not produce spores in vitro (eg.C.perfringens and
C.ramosum)
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Flowchart: Identification of Anaerobes
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Presumptive identification of Non-sporing, Non-branching Anaerobes
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Characteristics of anaerobic Gram-negative rods
•
Bacteroides species
Bacteroides species are rod shaped of variable size. Many are pleomorphic and
show terminal or central swellings, vacuoles or filaments. They are bile resistant and
carbohydrates are fermented. Bacteroides fragilis is the most commonly isolated
species and is often β-lactamase positive.
•
Fusobacterium species
Fusobacterium species are rods which may be spindle-shaped (Fusobacterium
nucleatum) or pleomorphic (Fusobacterium necrophorum) These two species are the
most commonly isolated from human clinical material. F.necrophorum is a cause of
serious infections (necrobacillosis or Lemierre’s disease) commonly diagnosed in
young adults and it can be recognised by production of indole and lipase on egg yolk
agar. Fusobacterium species grown on Fastidious Anaerobe Agar containing blood
may fluoresce yellow green (chartreuse) when exposed to long wave (365 nm)
ultraviolet light. This phenomenon is medium-dependent.
•
Porphyromonas species
The genus Porphyromonas includes asaccharolytic, catalase-negative species of
human and animal origin. They are short rods 0.5-0.8 x 1.0-3.0 µm. Bile sensitive.
Most Porphyromonas species isolated from humans are catalase-negative whilst
those from animals are catalase-positive. Porphyromonas species may fluoresce
brick red when exposed to long wave (365 nm) ultraviolet light and may produce a
pigment (buff to tan to black) when grown on blood containing media due to porphyrin
production. This phenomenon is medium dependent & Bile sensitive
•
Prevotella species
The genus Prevotella is composed of mainly saccharolytic, pigmented or nonpigmented species previously classified as Bacteroides. They are usually
pleomorphic. Young cultures of Prevotella species may fluoresce brick red when
exposed to long wave (365 nm) ultraviolet light and this may fade to a tan or black
pigment when grown on blood containing media for extended periods.
Characteristics of anaerobic Gram-negative cocci
•
Veillonella species
Veillonella species are small asaccharolytic cocci, measuring only 0.5 µm in diameter.
They are the only Gram-negative anaerobic cocci which are isolated from human
clinical material and are rarely found in pure culture. Veillonella species also fluoresce
red on exposure to ultraviolet light (365 nm), but this is medium dependent and may
fade in a few minutes, on exposure to oxygen. Some species produce catalase.
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Characteristics of anaerobic Gram-positive rods
•
Eubacterium species
Eubacterium species are irregularly sized rods 0.2-2.0 x 0.3-10.0 µm, varying in
shape from coccoid to long rods. They may be arranged singly, in pairs or chains.
They are not frequently encountered in clinical specimens. Eubacterium lentum (now
reclassified as Eggerthella lenta) is the commonest species, usually occurring in
mixed cultures.
Characteristics of anaerobic Gram-positive cocci associated with
human infection
•
Peptostreptococcus species
Peptostreptococcus species vary in size from 0.3 to 2.0µm and can be arranged in
chains, pairs, tetrads or clumps, although the majority are present either as clumps or
chains. Some species are aerotolerant. Some species are asaccharolytic and others
are strongly saccharolytic.
•
Peptococcus species
The genus Peptococcus now contains only one species, Peptococcus niger.
Typically, cells are 0.3 to 1.3µm in diameter arranged singly, in pairs or clumps and it
grows very slowly. Black pigment is produced after five days incubation but is lost on
subculture.
Other Gram-positive cocci associated with human infection
•
•
•
Atopobium parvula
Coprococcus species
Ruminococcus species
Principles of anaerobic identification
•
Colonies are usually isolated on fastidious anaerobe agar (or equivalent) or blood
agar incubated anaerobically and may be characterised by colonial morphology,
Gram’s stain reaction and are sensitive to metronidazole. Some species may require
longer than 48 hours incubation to grow. Identification tends to be undertaken only in
exceptional situations if clinically indicated. Further identification tests include
fluorescence under long wave UV light (365 nm), pigment production, bile tolerance,
glucose fermentation and lecithinase and lipase activity on egg yolk agar.
Classification of many anaerobes to species or even genus level requires additional
biochemical tests, metabolic end product analysis by GLC or molecular techniques.
Identification may be undertaken using commercial kits. For reliable results it is
important to ensure purity by subculture on non-selective agar before attempting
identification.
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Anaerobic Gram negative rods
Target Organisms
Bacteroides fragilis group reported to have caused human infection
•
•
•
•
•
•
•
•
•
•
B. fragilis
B. caccae
B. distasonis
B. eggerthii
B. merdae
B. ovatus
B. stercoralis
B. thetaiotaomicron
B. uniformis
B. vulgatus
Bacteroides species (taxonomic position uncertain) reported to have caused human
infection
•
•
•
•
•
•
•
•
B. capillosus
B. coagulans
B. forsythus
B. putredinis
B. pyogenes
B. splanchnicus
B. tectum
B. ureolyticus
Fusobacterium species reported to have caused human infection
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
F. alocis
F. gonidiafromis
F. mortiferum
F. naviforme
F. necrogenes
F. necrophorum
F. necrophorum subspecies funduliforme
F. necrophorum subspecies necrophorum
F. nucleatum
F. nucleatum subspecies fusiforme
F. nucleatum subspecies nucleatum
F. nucleatum subspecies polymorphum
F. nucleatum subspecies vincentii
F. periodonticum
F. russii
F. sulci
F. ulcerans
F. varium
Porphyromonas species reported to have caused human infection
•
•
•
•
P. asaccharolytica
P. catoniae
P. endodontalis
P. gingivalis
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Other Gram-negative anaerobic rods associated with human infections
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Anaerobiospirillum species
Anaerorhabdus species
Bilophila species
Catonella species
Centipeda species
Dialister species
Hallella species
Johnsonella species
Leptotrichia species
Mitsuokella species
Oribaculum species
Selenomonas species
Succinivibrio species
Sutterella wadsworthia
Prevotella species reported to have caused human infection
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
P. bivia
P. buccae
P. corporis*
P. dentalis
P. denticola*
P. disiens
P. enoeca
P. heparinolytica
P. intermedia*
P. loescheii*
P. melaninogenica*
P. nigrescens*
P. oris
P. tannerae*
Pigmented species
Other genera of Gram-negative rods reported to have caused human infection
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Anaerobiospirillum succiniproducens
Anaerobiospirillum thomasii
Anaerorhabdus furcosus
Bilophila wadsworthia
Catonella morbi
Centipeda periodontii
Dialister pneumosintes
Hallella seregens
Johnsonella ignava
Leptotrichia buccalis
Sneathia sanguinegens
Mitsuokella multiacida
Selenomonas artemidis
Selenomonas dianae
Selenomonas flueggei
Selenomonas infelix
Selenomonas noxia
Selenomonas sputigena
Succinivibrio dextrinosolvens
Sutterella wadsworthia
Other species may be associated with human disease
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Flowchart: Anaerobic Gram Negative Bacilli
Anaerobic Gram negative bacilli
Growth on BBE
Yes - Growth
(Bile tolerant)
Positive
+ Black
(Esculin Pos)
1. Va = R (Bacteroides fragilis group)
K=R
C=R
No Black
(Esculin Neg)
2. Va = R (Bacteroides group)
K=R
C = S/R
No Growth
(Bile sensitive)
Negative
Zone size > 10mm = Susceptible
3. Va = R
K=S
C=S
Presumptive
Fusobacterium
Mortiferium/varium gp
1. Va=S
K=R
C=R
Porphyromonas sp.
(Pigmenters, most indole POS)
2. Va = R
K = R/S
C = R/S
Pigment
Bile sensitive
Prevotella
species
No Pigment
UREA
Positive - B.ureolyticus
(Pitting or flat or cols)
3. Va = R
K=S
C=S
Commercial
identification kit
eg sterile site
Negative
Positive
Spot Indole
Fusobacterium nucleatum (pointed ends)
Fusobacterium necrophorum (rounded ends,pleomorphic)
Other Fusobacterium species
Negative
Commercial Identification Kit
(if required - eg Sterile site)
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Microscopic appearance
Gram stain
•
Bacteroides, Porphyromonas and Prevotella species are small, Gram-negative rods
of variable length.
•
Fusobacterium species are Gram-negative rods, highly variable in length and width
and may have pointed ends. F.nucleatum is a slim filamentous rod usually with
pointed ends and is indole positive. F.necrophorum also produces indole and lipase
on egg yolk agar.
Primary isolation media
•
Fastidious anaerobe agar or equivalent, (with or without neomycin – (some organisms
may be inhibited by neomycin) 40-48h incubation anaerobically at 35-37°C. Note: some
species may require longer incubation.
insert diagram from p 39 (Specimen inoculated on to appropriate media) of manual
Colonial appearance
Genus
Characteristics of growth on blood anaerobe agar after
incubation anaerobically at 35-37°C
Bacteroides
Colonies are 1-3 mm diameter, circular, low convex, smooth, semi-opaque
grey and are often moist or even mucoid. Mostly non-haemolytic and
resistant to an ox-bile disc.
Fusobacterium
Colonial appearance is variable, but most are 1-3mm diameter, with an
irregular or dentate edge. They vary from translucent to granular and
opaque, F.necrophorum may be beta-haemolytic.
Porphyromonas
1.0 mm diameter after 48 h incubation, smooth, shiny and grey. Dark
brown or black pigment develops after 3-7 days. Growth may be
enhanced by “satellitism” around colonies of other organisms e.g.
staphylococci
Prevotella
Colonies are similar to those of Bacteroides, except some
species are pigmented (may be pale brown to black). Most
pigmented species are haemolytic
Colonial appearance varies with the other genera of Gram negative rods
Identification of Commonly Isolated Fusobacterium Species
Species
Cellular Morphology
Indole
Bile
Growth
Lipase
Esculin
hydrolysis
F.nucleatum
Slender, pointed ends
+
-
-
-
F.mortiforum
Bizarre,round bodies
-
+
-
+
F.necrophorum
Large, pleomorphic
+
-/+
+
-
V
+
-
-
F.varium
Hint: Do not heat fix Gram of F.necrophorum, as bizarre shape morphology may not be seen.
Greening of agar on oxygen exposure commonly occurs. This due to H2O2 production.
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Identification of Anaerobic Cocci
Target Organisms
Peptostreptococcus species reported to have caused human infection
•
•
•
•
•
•
•
•
•
•
•
•
•
•
P. anaerobius
P. asaccharolyticus
P. hydrogenalis
P. harei
P. indolicus
P. ivorii
P. lacrimalis
P. lactolyticus
P. magnus
P. micros
P. octavius
P. prevotii
P. tetradius
P. vaginalis
Peptococcus species reported to have caused human infection
•
P. niger
Other genera of Gram-positive cocci reported to have caused human infection
•
•
•
•
•
•
•
•
•
Atopobium parvulum
Ruminococcus hansenii
Ruminococcus productus
Sarcina albus
Sarcina pasteurii
Sarcina ventriculi
Coprococcus eutactus
Coprococcus comes
Coprococcus catus
Veillonella species reported to have caused human infection
•
•
•
V. parvula
V. atypica
V. dispar
Other species may be associated with human disease
Microscopic appearance
Gram stain
• Peptostreptococcus and Peptococcus are Gram-positive cocci arranged in chains, pairs,
tetrads or clumps. Veillonella are small Gram-negative cocci arranged in clumps.
Primary isolation media
•
Fastidious anaerobe agar or equivalent (with or without neomycin) 40-48h incubation
anaerobically at 35-37°C. Note: some species may require longer incubation.
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Flowchart: Anaerobic Cocci
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Colonial appearance
Genus
Characteristics of growth on fastidious anaerobe agar
after incubation anaerobically at 35-37°C
Peptostreptococcus
magnus
Small colonies (<1.0mm), often with variation in size and colour.
Colonies may be convex and whitish and flatter and translucent on the
same plate.
Peptostreptococcus
anaerobius
Colonies 1-2mm in diameter, grey with slightly raised off-white centres,
sensitive to SPS (liquoid) disc.
Peptostreptococcus
asaccharolyticus
Colonies 1-2mm in diameter, glistening, low convex usually whitish to
lemon-yellow.
Peptostreptococcus
micros
Small colonies (<1.0mm), typically white (but sometimes grey),
glistening and domed, sometimes surrounded by a yellow-brown halo
up to 2mm wide.
Peptococcus species
Small colonies (<1.0mm), raised, grey, becoming dark brown/black.
Veillonella species
Small colonies (<1.0mm) after 48 hours incubation. May fluoresce red
under long wavelength UV light (365nm).
Colonial appearance varies with the other genera
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Identification of Clostridium species
Target Organisms
Clostridium species reported to have caused human disease
Commonly isolated
Rarely isolated
Very rarely isolated
•
C.perfringens
•
C.novyii type A
•
C.tetani
•
C.septicum
•
C.sordellii
•
C.histolyticum
•
C.tertium
•
C.fallax
•
C.difficile
•
C.clostridioforme
•
C.botulinum
Commonly isolated usually “non-pathogenic” clostridia
•
C.sporogenes
•
C.ramosum
•
C.innocuum
•
C.paraputrificum
•
C.cadaveris
Primary isolation media
•
Agar containing blood incubated anaerobically at 35-37oC for 40-48h
Microscopic appearance
Gram stain
•
Gram-positive rods, which may possess a single endospore. Some species may be
Gram-variable
Spore stain
•
Used to determine the shape and position of the spore (phase contrast microscopy is
an alternative option)
•
Gram stain is usually adequate to observe spores.
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Colonial appearance
•
Colonial appearance varies with species.
Organism
Classic Colony & Gram Morphology on Blood agar incubation
at 35ºC for 48 hrs
C.sporogenes
Large (2-6mm), irregularly circular, raised yellowish grey centre and
a “medusa head” periphery
β-haemolytic and adhere to agar
lipase positive
C.sphenoides
Small (1-2mm), non-haemolytic, circular with an entire margin
Usually Gram negative, straight with tapered ends
Spores oval & subterminal & swell the cell
Sporing only occurs after ≥48hrs & involves only small number of
cells. Indole positive
Large, smooth convex colonies, but may be rough with an irregular
edge. Usually have a double zone of haemolysis
Spores rarely seen in vivo, when present are large oval, central or
subterminal and distend the cell
Lecithinase positive
Grey-white, convex, circular colonies with crenated edge, which may
spread. Many are β-haemolytic.
Spores easily, oval, subterminal, often occur as free spores, and
swell the cell slightly. Spores have a thick exosporium.
Lecithinase positive, indole positive, urease positive.
Spreading growth with a narrow zone of β-haemolysis, may spread
over entire plate.
Gram positive in young cultures, but Gram negative in old cultures
Regularly combination of both
Spores oval subterminal and distend the cell with no exosporium.
2-4mm, circular with slightly irregular margins
May look like alpha streptococcus under aerobic conditions with no
spores
Spores are large, oval and terminal and greatly distend the cell.
Large colonies, irregular margins and tend to swarm on moist media
Gram positive in young cultures but become Gram negative after
>24hrs incubation.
Spores round and terminal and distend cell. “spoon” shaped.
Often indole positive.
C.perfringens
C.sordellii
C.septicum
C.tertium
C.tetani
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Identification of Clostridium Species Guidance
Clinical specimens
Primary isolation plate (blood containing agar incubated anaerobically)
Β, or non-haemolytic colonies which may spread
Gram stain
Gram positive rods, which may have a single endospore
(some species may appear Gram-negative)
Further identification
Identify further if clinically indicated
Refer to Identification of Commonly Isolated Clostridia table
Note: it is important to ensure the culture is pure, as the fine spreading growth of some
Clostridium species may mask contamination organisms.
Clostridium septicum
Clostridium sordelli
Clostridium perfringens
Clostridium tertium
Clostridium sphenoides
Clostridium tetani
Clostridium sporogenes
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Identification of Commonly Isolated Clostridia
Species
Spores*
Egg Yolk Agar
Lecthinase
Lipase
Aerobic Growth
Indole
Production
Urease
Comments
C.bifermentans
OS
+
-
-
+
-
Spores don’t swell or are free.
C.perfringens**
OS
+
-
-
-
-
No spores, non-motile, double zone haemolysis,
box car shaped.
C.septicum
OSD
-
-
-
-
-
Swarms on plate.
C.sordelli
OSF
+
-
-
+
+
Spores swell & are free. Colonies are rhizoid
shaped.
C.sporogenes**
OSD
-
+
-
-
-
Spores easily – may be only spores.
OS
-
-
-
-
-
Horse stable smell.
RS/TD
-
-
-
+
-
Few spores, pointed ends, usually stain Gram
negative.
OTD
-
-
+
-
-
Stains Gram positive in young cultures & Gram
negative in old cultures. Aerotolerant, swarms.
C.tetani
RT
-
-
-
+/-
-
Tennis racket shape, may swarm
C.clostridioforme
OS
-
-
-
-/+
-
Usually Gram negative.
C.histolyticum
OS
-
-
+/-
-
-
C.difficile
C.sphenoides
C.tertium**
* O = oval
R = round
F = free
D = distends cell
S = subterminal
T = terminal
**Hints: Clostridium tertium - doesn’t spore aerobically
Clostridium perfringens – haemolysis double zone enhanced by cold
Clostridium sporogenes – ‘weird’ lipase reaction -> less ‘oily’
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Identification of Anaerobic Gram positive non-sporing
rods
Target organisms
Gram positive non-sporing rods reported to have caused human infection
•
•
•
•
Bifidobacterium species
Eubacterium lentum
Propionibacterium species
Actinomyces species
Other species may be associated with human disease
Microscopic appearance
•
Gram stain
Irregular sized Gram positive non-branching rods, may be arranged singly, in pairs or
chains.
Primary isolation media
•
Fastidious anaerobe agar or equivalent (with or without neomycin), 40-48hr
incubation anaerobically at 35-37ºC. For Actinomyces species use Actinomyces
selective agar. Note: some species may require longer incubation.
Colonial appearance
•
Colonies of Eubacterium species are 0.5-2mm diameter, circular, convex and
translucent or slightly opaque, usually sensitive to 5µg metronidazole disc.
Preliminary tests
•
N/A
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The Commonsense Bug Workbook
Presumptive Identification of Anaerobic Gram positive non-sporing rods
Clinical specimen
Primary isolation plate: Fastidious Anaerobe agar or equivalent
with or without neomycin or metronidazole and naladixic acid for
Actinomyces species.
Gram stain
Gram positive rod
Metronidazole 5ug disc senstivity*
Sensitive
May have spore
Large colony
Fast growing
Large rods
Clostridium
Resistant
No spore
Small colony
Slow growing
Small coccobacilli
Actinomyces
Propionibacterium
Bifidobacterium
Lactobacillus
Further identification
Identify further if
clinically indicated
Commercial identification kit
*Rarely, some strains of Clostridium species may be resistant to metronidazole
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Identification of Vibrio & related species
Target Organisms
Vibrio species reported to have caused human disease
•
•
•
•
•
•
•
•
•
•
•
•
•
Vibrio alginolyticus
Vibrio carchariae
Vibrio cholerae
Vibrio cincinnatiensis
Vibrio damsela
Vibrio fluvialis
Vibrio furnissii
Vibrio hollisae
Vibrio metschnikovii
Vibrio mimicus
Vibrio parahaemolyticus
Vibrio vulnificus
Vibrio furnissii
Any species of Vibrio may be found in faeces after the ingestion of seafood or water that
contains them.
Insert vibio flowchart
MICROSCOPIC APPEARANCE
Gram stain
•
Gram negative rods characteristically curved or comma.-shaped. This characteristic
appearance is not always observed when the organism is Gram stained from solid
media, and rarely from TCBS agar.
Primary isolation media
•
•
Blood agar incubated in air at 35-37ºC for 18-24hr
TCBS agar incubated in air at 35-37ºC for 18-24hr
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Flowchart: Identification of Vibrio species
Clinical Specimen
Primary isolation plate
(Blood agar or TCBS)
Blood agar – colonies
2-3mm in diameter (some
colonies haemolytic)
TCBS – yellow/green
2-3 mm diameter colonies
Gram stain
(Characteristically curved or comma-shaped
Gram-negative rods)
Oxidase
(from non-selective media)
Positive
Negative
Vibrio string test
Possible
V.metschnikovii
(check biochemistry
before discarding*)
Negative
Positive
?Aeromonas species
?Shewanella species
?Plesiomonas species
?Vibrio species
Commercial identification kit
(may require NaCl supplementation)
Vibrio species
- confirm using salt tolerance test
- Serological aggs eg V.cholerae
01,0139
Aeromonas species
Shewanella species
*V.metschnikovii produces yellow colonies on TCBS. Oxidase negative
isolates with a different morphological appearance may be discarded
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Colonial Appearance
•
On blood agar colonies are 2-3mm in diameter. Some strains may be haemolytic.
•
After 18-24 hours incubation colonies on TCBS are at least 2mm in diameter and
yellow for sucrose fermenters and green for non-sucrose fermenters.
•
Cultures should be examined quickly after removal from the incubator as the yellow
colouration of the colonies may revert to a green colour when left at room
temperature.
•
Organisms other than Vibrio species grow on TCBS.
Organism
V.cholerae
V.alginolyticus
V.cincinnatiensis
V.damsela
V.carchariae
V.fluvialis
V.furnissii
V.hollisae
V.parahaemolyticus
V.metschnikovii
V.vulnificus
V.mimicus
Aeromonas species
Pseudomonas species
Proteus species
Enterococcus species
Shewanella species
Colour of colonies on TCBS
yellow
yellow
yellow
green
yellow/green
yellow
yellow
green
green
yellow
green
green
yellow
blue/green*
yellow/green*
yellow
blue/green
*The colonies are smaller than those produced by Vibrio species.
Test procedures
Oxidase
Vibrio species are oxidase positive (oxidase tests may give false negative results on
media containing carbohydrates – subculture to nutrient or blood agar before testing).
Sensitivity to pteridine 0129
Most Vibrio species are sensitive with 150 µg but species differ with 10 µg discs
(some strains of V.cholerae 01 and 0139 may be resistant to both disc contents)
Vibrio String Test
Vibrio species are positive, as are many other closely related organisms including
Shewanella & Plesiomonas species. Aermonas species are negative.
Serology
Commercial identification kit
These tests may require supplementation with NaCl. Refer to the manufacturer’s
instructions.
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Interpretation & Reporting of Results
•
Presumptive identification may be made
If appropriate growth characteristics, colonial appearance, Gram stain of the culture
and oxidase results are demonstrated.
•
Confirmation of identification may be made
Following 0129 sensitivity testing and/or Vibrio string testing, serology and
commercial identification kit results.
•
To medical microbiologist
Inform the medical microbiologist of all positive cultures from normally sterile sites, of
all presumptive and confirmed Vibrio species that are known to be pathogenic or
potentially pathogenic, and all isolates in outbreak situations.
Inform the medical microbiologist if the request card bears information which
suggests infection with V.cholerae or V.parahaemolyitcus, according to local
protocols e.g.
•
•
•
•
Severe water diarrhea
Suspected cholera
History of foreign travel,
Suspected food poisoning (especially cases involving consumption of
seafood).
The medical microbiologist should also be informed of presumptive or confirmed
Vibrio species in association with”
•
•
•
•
•
•
Wound infection or (necrotizing) myofascitis
Septicaemia history of foreign travel
History of foreign travel
Contact with (brackish) water, fishing/eating fish or seafood (suggestive of
infection with V.vulnificus, V.damsela or Aeromonas hydrophila sensu lato)
Alcoholism, substance abuse, immunodeficiency
Other serious medical conditions such as cancer, or persons receiving
treatment for cancer which induces neutropenia and/or mucositis.
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The Commonsense Bug Workbook
% Positive
V.
V.
V.
V.
V.
V
V.
V.
V.
V.
V.
V
cholerae
mimicus
metschnikovii
cincinnatiensis
hollisae
.damsela
fluvialis
furnissii
alginolyticus
parahaemolyticus
vulnificus
.carchariae
0% NaCl
100
100
0
0
0
0
0
0
0
0
0
0
1% NaCl
100
100
100
100
99
100
99
99
99
100
99
100
6% NaCl
53
49
78
100
83
95
96
100
100
99
65
100
8% NaCl
1
0
44
62
0
0
71
78
94
80
0
0
10% NaC
0
0
4
0
0
0
4
0
69
2
0
0
12% NaCl
0
0
0
0
0
0
0
0
17
1
0
0
Test
Growth in nutrient
broth with:
Reference:
Manual of Clinical Microbiology Sixth Ed. Page 470.
Shewanella algae
Shewanella putrefaciens
0% NaCl
-
+
1% NaCl
+
-
42ºC Growth
-
+
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Identification of Campylobacter and related species
Taxonomy
•
The family Campylobacteraceae (proposed in 1991) includes two colony related
genera, Campylobacter and Arcobacter. The genus Campylobacter contains 14
species.
TARGET ORGANISMS
Campylobacter species reported to have caused human gastrointestinal infection
•
•
•
•
Campylobacter jejuni
Campylobacter coli
Campylobacter lari
Campylobacter upsaliensis
Campylobacter species reported to have caused human extraintestinal infection
•
•
•
Campylobacter fetus
Campylobacter hyointestinalis
Campylobacter sputorum
Campylobacter species reported to have caused human dental infection
•
•
•
Campylobacter consisus
Campylobacter curvus
Campylobacter rectus
Characteristics
•
Members of the family Campylobacteraceae are curved, S-shaped or spiral Gram
negative rods: coccal forms may be seen under sub-optimal conditions.
•
On selective agar the colonies are grey/white or creamy grey and moist in
appearance. They are motile, Microaerophilic (optimum 5-10% oxygen) and
oxidase positive.
•
Campylobacter species do not ferment or oxidise carbohydrates. A well
recognized problem associated with identification of Campylobacter species is
the lack of effective discriminating tests.
•
The species most commonly associated with diarrheal disease in humans are
thermophilic i.e. they will grow at 42-43ºC and 37ºC but not at 25ºC.
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Principles of Identification
•
Preliminary identification of Campylobacter species from primary culture is by
colonial appearance, Gram stain, growth in oxygen and oxidase test.
•
Species differentiation is difficult due to the lack of discriminating tests available
in most routine microbiology laboratories.
Primary isolation media
•
Blood agar or fastidious anaerobe agar incubated microaerobically or
anaerobically at 42ºC, for at least 40-48 hours.
•
Blood cultures may be incubated at 37ºC as there is unlikely to be competing
flora in these samples
•
Charcoal cefoperazone deoxycholate agar (CCDA) incubated microaerobically at
42ºC for 40-48 hours
•
Cultures may be incubated for a further 24 hours, if required
•
Some species may be inhibited by the antibiotics contained within the medium
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Differential characteristics of clinically relevant Campylobacter, Helicobacter, and Arcobacter species
Genus & species
C.coliº
C.concisus
C.curvus*
C.fetus subsp fetus
C.hyointestinalis
C.jejuni
C.jejuni subsp doylei
C.lari
C.rectus*
C.sputorum
C.upsaliensis
H.cinaedi
H.fennelliae
H.pylori^
#
A.butzleri
#
A.cryaerophilus
Growth at
25°C
42°C
+
+/+
+
+
+
+
-/+
+
+/-***
+/+
Slight +
+
+
-/+
+
-
Hippurate
Hydrolysis
Catalase
H2S in Triple
Sugar Iron Agar
Indoxyl Acetate
Hydrolysis
Nitrate to
Nitrite
+
+
-
+
+
+
+
+/- or weak +
+
-/+
-/weak +
+
+
+
-/weak +
+/-
+
+
+
+
+
-
+
+
+
+
+
+
-/+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+/+
+
Susceptible to 30µg disc
Cephalothin
Nalidixic acid
+**
ND
+
+
+
+**
+
+
ND
+
+
-/+
+
+
+/+
+
+
+
+/+/-
+, most strains positive; -, most strains negative; +/-, variable (more often positive); -/+, variable (more often negative); ND, test not done.
* Anaerobic, not microaerobic.
^Strong and rapid positive urease
#
Aerotolerant, not microaerobic; except for a few strains, A.cryaeophilus cannot grow on MacConkey agar, whereas A.butzleri grows on MacConkey agar.
**Isolates of quinolone resistant are becoming increasing more common.
***Hippurate negative strains exist.
ºShould be reported as Campylobacter species as hippurate negative C.jejuni strains exist.
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Identification of Helicobacter species
Target Organisms
Helicobacter species reported to have caused human infection
•
•
•
•
•
•
Helicobacter pylori
Helicobacter cinaedi
Helicobacter fennelliae
Helicobacter canis
Helicobacter pullorum
Helicobacter heilmannii (non-culturable)
Principles of Identification
•
Colonies from primary isolation plates are identified by colonial morphology, Gram
stain and biochemical test.
•
Isolates may be referred to the Reference laboratory for confirmation of identification
and typing.
Microscopy
•
Gram negative curved or comma-shaped rods. Spiral or helical shapes are less
evident. Older cultures may produce coccoid forms.
Primary Isolation Media
•
Chocolate agar incubated in 5% oxygen with 5-10% CO2 at 35-37ºC for up to 7 days.
Incubation for up to 10 days may be required post treatment. H.pylori is small (1mm)
and translucent grey, may be slightly haemolytic.
•
H.pylori selective agar incubated in 5% oxygen with 5-10% CO2 at 35-37ºC for up to 7
days. Incubation for up to 10 days may be required post treatment. H.pylori are
small (1-2mm) and convex on primary isolation.
Test Procedures
Organism
Oxidase
Urease
Nitrate
Growth at
42ºC
Catalase
H.pylori
H.cinaedi
H.fennelliae
H.canis
H.pullorum
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Identification of Haemophilus species and the HACEK
group of organisms
Introduction
This section describes the identification of Haemophilus species and other members of
theHACEK group (Haemophilus species, Actinobacillus actinomycetemcomitans,
Cardiobacterium hominis, Eikenella corrodens and Kingella species).
Target Organisms
HACEK group reported to have caused human infection
•
•
•
•
•
•
Haemophilus influenzae
Haemophilus aphrophilus
Haemophilus ducreyi
Haemophilus parainfluenzae
Haemophilus paraphrophilus
Haemophilus segnis
Other species may be associated with human disease
•
•
•
•
Actinobacillus actinomycetemcomitans
Cardiobacterium hominis
Kingella kingae
Eikenella corrodens
Characteristics of Haemophilus species
•
Haemophilus are Gram negative spherical, oval or rod-shaped cells less than 1µm in
width, variable in length with marked pleomorphism and sometimes forming filaments.
Small, round, convex, colonies, which may be iridescent, develop in 24 hours on
chocolate blood agar. Iridescence is seen with capsulated strains.
•
All species require preformed growth factors present in blood, particularly X factor
(protoporphyrin IX or protoheme) and/or V factor [nicotinamide adenine dinucleotide
(NAD) or NAD phosphate (NADP)]. On blood agar H.influenzae exhibits satellitism
around colonies of haemolytic S.aureus (a source of V factor). Haemophilus
aphrophilus and Haemophilus paraphrophilus require CO2 for primary isolation.
Carbohydrates are catabolised with the production of acid. A few species produce
gas. The optimum growth temperature is 35-37ºC. They are facultatively anaerobic
and non-motile. Nitrates are reduced to nitrites.
•
The optimum temperature for growth of Haemophilus ducreyi is 33-34ºC but growth is
very slow and may be improved by the addition of Isovitalex to the culture medium.
Some strains require CO2 for primary isolation.
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Characteristics of the HACEK group of organisms
(For identification of Haemophilus species in the HACEK group see above_.
•
A systematic approach is used to differentiate the HACEK group of clinically
encountered morphologically similar, aerobic and facultatively anaerobic Gram
negative rods mainly associated with endocarditis and infections from normally sterile
sites. These organisms are oropharyngeal/respiratory tract commensals. The
identification is considered together with the clinical details and the isolates may be
identified further if clinically indicated.
•
Actinobacillus actinomycetemcomitans
A.actinomycetemcomitans is a Gram negative coccobacillus or a short rod 0.3-0.5 x
0.5-1.5 µm, which may exhibit irregular staining. A.actinomycetemcomitans is mostly
bacillary but cocci are interspersed. Occasional longer forms up to 6 µm may occur.
Cells are arranged singly, in pairs, or more rarely, in chains. Small amounts of
extracellular slime may be produced.
A.actinomycetemcomitans does not require X or V factors. After 24 hours incubation,
colonies on blood or chocolate agar may be less than 0.5 mm and enlarge to 1mm
after several days’ incubation. These colonies on blood or chocolate agar may be
firm, adherent, star-shaped, sometimes with rough surfaces and pitting and may be
difficult to remove from the agar surface. If extracellular slime is produced, cultures
may be sticky on primary isolation. Surface cultures have low viability and may die
within 5-7 days. It grows best under microaerophilic conditions with added CO2 and is
facultatively anaerobic. The optimal growth temperature is 37ºC. Cells are nonmotile and urease is not produced.
•
Cardiobacterium hominis
The genus Cardiobacterium contains only one species, Cardiobacterium hominis.
Cells are pleomorphic or straight rods 0.5-0.75 µm in diameter and 1-3 µm in length
with rounded ends, and long filaments may occur. Cells are arranged singly, in pairs,
in short chains and in rosette clusters. They are Gram negative, but parts of the cell
may stain Gram positive.
Growth on blood agar is poor. C.hominis does not require X or V factors. Very small
colonies are produced unless incubated in a humid aerobic or anaerobic atmosphere
with 5% CO2. After incubation for two days, colonies are 1 mm in diameter, smooth,
opaque and butyrous and some strains may pit the agar. C.hominis is facultatively
anaerobic, but CO2 may be required by some strains on primary isolation. The
optimum growth temperature is 30-37ºC. It is non-motile, oxidase positive and
catalase and urease negative.
•
Eikenella corrodens
The genus Eikenella contains only one species, Eikenella corrodens. Cells are
straight, unbranched, non-sporing, slender Gram negative rods 0.3-0.4 x 1.5-4 µm in
length.
Colonies may be very small on blood agar after overnight incubation or may not be
visible for several days. The colonies have moist, clear centres surrounded by flat,
and sometimes spreading, growth. Pitting of the medium may occur and yellow
colouration may be seen in older cultures due to cell density. There may be colonial
variation and spreading growth may vary between colonies of the same isolate.
E.corrodens is non-haemolytic but a slight greening may occur around the colonies.
Haemin is usually required for aerobic growth and rare strains remain X-dependent
after further subculture. The optimum growth temperature is 35-37ºC.
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E.corrodens is non-motile, but twitching motility may be produced on some media.
Strains are facultatively anaerobic, oxidase positive, catalase negative, urease
negative and capnophilic. It may be confused with Bacteroides ureolyticus, which
also exhibits pitting or corroding, but unlike E.corrodens is an obligate anaerobe and
urease positive.
•
Kingella species
The genus Kingella comprises two species, Kingella kingae and Kingella dentrificans.
Kingella indologenes has been transferred to a new genus and classified as
Suttonella indologenes.
Kingella species are straight rods, 1.0 µm in length with rounded or square ends.
They occur in pairs and sometimes short chains. Endospores are not formed. Cells
are Gram negative but tend to resist decolourisation.
Two types of colonies occur on blood agar, a spreading, corroding type and a
smooth, convex type. It does not require X or V factors. Growth is aerobic or
facultatively anaerobic. The optimum growth temperature is 33-37ºC. Kingella
species are non-motile, oxidase positive, catalase negative and urease negative.
Glucose and other carbohydrates are fermented with the production of acid but not
gas.
Kingella species may grow on Neisseria selective agar and therefore maybe
misidentified as pathogenic Neisseria species. They can be differentiated from
Moraxella and Neisseria species by a catalase test. Most Kingella species are
catalase negative: Moraxella and most Neisseria species (except Neisseria elongate)
are catalase positive.
Primary isolation media
•
Chocolate agar incubated in 5-10% CO2 at 35-37ºC for 16-48 hrs
Blood agar incubated in 5-10% CO2 at 35-37ºC for 16-48 hrs
H.ducreyi selective agar incubated in 5-10% CO2 at 33-34ºC for 5 days
Colonial appearance
•
Haemophilus species are small, round, convex colonies, which may be iridescent
and develop after 24 hours incubation on chocolate agar. Satellitism of
H.influenzae may be seen around colonies of S.aureus on blood agar.
•
Colonial morphology of other HACEK organisms varies with species and isolation
media (see introduction and below)
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SNP MICROBIOLOGY
HACEK group organisms
The Commonsense Bug Workbook
Characteristics of growth on blood agar after
aerobic incubation at 35-37ºC for 16-48 hrs
For descriptions of Haemophilus species see Haemophilus species
A.actinomycetemcomitans
Will not grow in air but grows in air + CO2. Minute
colonies at 24 hrs, 1 mm at 48 hrs. Firm, adherent,
star-shaped colonies with rough surface and which
may produce pitting of the agar. Some strains may be
sticky. Non-haemolytic.
C.hominis
Some strains will not grow without added CO2.
Colonies smooth, convex and opaque. 1-2 mm at 48
hrs. Slight alpha-haemolysis
E.corrodens
Colonies very small, moist, clear centres surrounded
by flat growth. Pitting may occur. Spreading is rare
and usually confined to a very small area around the
colony. Non-haemolytic. Colonies 0.5-1 mm after 48
hrs. Requires 5-10% CO2.
K.kingae
Two types of colony: a spreading, corroding type and
a smooth, convex type. Small zone of ß-haemolysis.
Cells are often capsulate, producing mucoid colonies.
Does not require 5-10% CO2.
K.denitrificans
Non-haemolytic. Two types of colony: a spreading,
corroding type and a smooth, convex type
Microscopic appearance
•
Gram stain:
Haemophilus species are small coccobacilli or longer rod-shaped Gram negative
cells, variable in length with marked pleomorphism and sometimes forming filaments.
Other HACEK organisms produce spherical, oval or rod-shaped Gram negative cells
which may be variable in length with marked pleomorphism or filament formation.
Test procedures
•
Growth requirement for X and V factors or porphyrin synthesis test
•
Serotyping capsular H.influenzae strains with commercial type-specific antisera
•
Commercial identification kit
Presumptive identification may be made
•
If appropriate growth characteristics, colonial appearance, Gram stain of the culture,
oxidase and tributyrin test results are demonstrated
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Confirmation of identification may be made
•
Following commercial identification kit or other biochemical test results.
Summary of X and V test results
X factor
V factor
X + V factor
Porphyrin
No growth
No growth
Growth
Negative
H.haemolyticus
No growth
No growth
Growth
Negative
H.parainfluenzae
No growth
Growth
Growth
Positive
Growth
Growth
Growth
Positive
(weak)
H.paraphrophilus
No growth
Growth
Growth
Negative
H.segnis
No growth
Growth
Growth
Negative
a
H.influenzae
b
H.aphrophilus
a
H.influenzae biovar aegyptius is indistinguishable from H.influenzae biotype III in normal
laboratory tests
b
ß-haemolytic on horse blood agar
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SNP MICROBIOLOGY
The Commonsense Bug Workbook
Identification of Pasteurella Species
Introduction
•
This section describes the procedure for the identification of Pasteurella species and
distinguishes these from morphologically similar species.
Taxonomy
•
Currently some 20 species are included in the genus Pasteurella. Not all of these are
true members. DNA-DNA hybridization indicates that some of the species are more
closely related to the genus Actinobacillus.
•
Pasteurella multocida is the type species of the genus.
Characteristics of Pasteurella species
•
Pasteurella species are spherical, ovoid or rod-shaped cells 0.3-1.0 µm in diameter
and 1.0-2.0 µm in length. Cells are Gram negative, and occur singly, or in pairs or
short chains. Bipolar staining may be seen. Capsules may be present. Pasteurella
species are non-motile, and are facultatively anaerobic.
•
Pasteurella species have both an oxidative and fermentative metabolisms. The
optimum growth temperature is 37ºC. Glucose and other carbohydrates are
catabolised with the production of acid but no gas. Most species are catalase
positive and oxidase positive; nitrates are reduced to nitrites by almost all species.
•
Colonies of Pasteurella species are usually grey and viscous, with a strong mucinous
odour. Rough, irregular colonies may also occur. Freshly isolated strains of
Pasteurella haemolytica produce clear zones of ß-haemolysis on blood agar – this
organism is a cause of mastitis and septicaemia in some peridomestic animals, but is
very rarely the cause of human infection.
•
Pasteurella and Actinobacillus species are so similar, that no single phenotypic
feature reliable distinguishes between the two genera. In clinical practice, however,
an organism with characteristics corresponding to the genus Pasteurella is highly
likely to be so, if recovered from clinical specimens in association with a bite from a
cat or dog.
•
The genus Actinobacillus now includes Actinobacillus ureae – formerly Pasteurella
ureae. A.ureae is thought to be a commensal or opportunist pathogen of human
beings, and is principally reported in connection with disease of the respiratory tract
(e.g. cases of pneumonia, lung abscess).
Occasionally, invasive infections
(septicaemia, meningitis) are also reported.
•
As the name suggest, A.ureae is urease positive. Most species of Pasteurella are
urease negative (including P.multocida). Thus, a Pasteurella – like organism, urease
positive, recovered in association with human respiratory tract disease, is likely to be
A.ureae.
•
Phenotypically, Pasteurella species may resemble Haemophilus species – but
Pasteurella species will not regularly exhibit satellitism around colonies of
Staphylococcus species, nor are they regularly auxotropic for X or V factors; growth
is not especially enhanced by use of chocolatised blood agar.
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Identification
•
Colonies on blood agar are identified by colonial morphology, Gram stain, and
oxidase test and catalase production. Additional tests are needed for confirmation
and/or isolates should be referred to the Reference laboratory.
Target Organisms
Pasteurella species reported to have caused human infection
•
•
•
•
•
•
•
•
•
•
•
P.aerogenes
P.bettyae
P.canis
P.dagmatis
P.gallinarum
P.haemolytica (Biotype A)
P.multocida subspecies gallicida
P.multocida subspecies multocida
P.multocida subspecies septica
P.pneumotropica
P.stomatis
Microscopic Appearance
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Gram stain
Spherical, ovoid or rod-shaped Gram negative cells which occur singly or in pairs or short
chains. Bipolar staining is common. Capsules may be present.
Primary Isolation
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Blood agar 16-48 hrs incubation in 5-10% CO2 at 35-37ºC
Colonial Appearance
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Colonies are grey and viscous but rough irregular colonies occur frequently. Freshly
isolated strains of P.haemolytica produce clear zones of ß-haemolysis on blood agar.
Identification
Oxidase test – Positive (almost always)
Catalase test – Positive
Sensitivity to penicillin – Sensitive
Commercial identification kit
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Flowchart: Identification of Pasteurella species – summary
Clinical specimens
Primary isolation plate (blood agar)
Pasteurella species are grey, viscous, rough, irregular, non-haemolytic colonies on blood agar
P.haemolytica are beta-haemolytic on blood agar
Gram stain
Gram negative rods or cocco-bacilli
Oxidase*
Positive
Possible Pasteurella species
Negative
Not Pasteurella species**
Catalase*
Positive
Possible Pasteurella species
Negative
Not Pasteurella species***
Sensitivity to Penicillin
Sensitive
Possible Pasteurella species
Resistant
Not Pasteurella species
Commercial identification kit
*All oxidase and catalase reactions may be weak
**P.bettyae is oxidase negative
***P.haemolytica Biotype T is catalase negative
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Results & Reporting
Presumptive identification may be made:
If appropriate growth characteristics, colonial appearance, Gram stain of the culture, oxidase
and catalase test results are demonstrated
Confirmation of identification may be made:
Following use of a commercial kit and/or referral to a Reference laboratory
To medical microbiologist
The medical microbiologist should be informed of presumptive or confirmed Pasteurella
species when the request bears relevant information e.g.
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Animal bite
Wound infection or septic arthritis
Septicaemia
Meningeoencephalitis
Pneumonia, empyema thoracis or lung abscess
History of farming or veterinary work
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Identification of Moraxella species and
Morphologically similar species
Introduction
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This section describes the identification of Moraxella species and those species
which are morphologically similar.
Target Organisms
Moraxella species and morphologically similar organisms reported to have caused
human infection
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M.atlantae
M.catarrhalis
M.lacunata
M.nonliquefaciens
M.osloensis
K.denitrificans
K.kingae
O.urethralis
P.immobilis
P.phenylpyruvicus
Taxonomy
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The genera Moraxella (including the former Branhamella), Acinetobacter and
Psychrobacter currently belong to the family Moraxellaceae; the classification is still
under review. The genus Oligella includes the previous Moraxella urethralis and
CDC group M-4 now both classified as Oligella ureolytica. Kingella kingae (formally
referred to as Moraxella kingii or Moraxella new species I) has been placed in the
genus Kingella. Kingella denitrificans, previously designated CDC group TM-1 has
also been placed in this genus. Psychrobacter phenylpyruvicus (formally Moraxella
phenylpyruvica) is phenotypically similar to Moraxella lincolnii and M.osloensis.
P.phenylpyruvicus is urease positive. Brucella species can be misidentified as
P.phenylpyruvicus in some commercial identification kits.
Microscopic Appearance
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Gram stain
Gram negative with a tendency to resist decolourisation
Moraxella subgenus Moraxella
Rods often coccobacilli. Usually occur in pairs or short chains with one plane of
division
Moraxella subgenus Branhamella
Cocci occur singly or in pairs with adjacent sides flattened, sometimes forming tetrads
Kingella species
Plump rods or coccobacilli occurring in pairs or chains
Oligella species
Small rods or coccobacilli, often occurring in pairs. Cells lack the typical plumpness
of Moraxella species.
Psychrobacter phenylpyruvicus
Rods, often coccobacilli. Usually occur in planes with one plane of division.
Microscopy can differentiate Brucella species (very small coccobacilli) from
P.phenylpyruvicus
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Characteristics of Moraxella species
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Genus Moraxella
Moraxella species are Gram negative and cells may be capsulated. They are nonmotile and aerobic, but some strains may grow weakly under anaerobic conditions.
Most species except Moraxella osloensis are nutritionally fastidious. The optimum
growth temperature is 33-35ºC. Moraxella species are usually catalase positive,
oxidase positive and do not produce acid from carbohydrates. Colonies of Moraxella
lacunata and Moraxella nonliquefaciens are small on blood agar. Some strains of
M.lacunata are haemolytic. Moraxella catarrhalis is the most frequently isolated
species of Moraxella and can be differentiated form Neisseria species by the tributyrin
test: M.catarrhalis is positive and Neisseria species are negative. However, as the
tributyrin test is positive for Moraxella species other than M.catarrhalis, it cannot be
used to differentiate among the Moraxellae. Ninety percent of M.catarrhalis are ßlactamase positive.
The genus is divided into Moraxella subgenus Moraxella that includes all the rod
shaped species, and Moraxella subgenus Branhamella which contains the cocci.
•
Moraxella subgenus Moraxella
Rods are often very short and plump, approaching a coccus shape 1.0-1.5 x 1.5-2.5
µm. Cells usually occur in pairs or short chains with one plane of division.
Pleomorphism is enhanced by lack of oxygen and by incubation at temperatures
above the optimum. The medically important species are Moraxella atlantae,
M.lacunata, M.nonliquefaciens and M.osloensis.
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Moraxella subgenus Branhamella
Cocci 0.6-1.0 µm in diameter occur singly or in pairs with adjacent sides flattened and
sometimes from tetrads. There is one medically important species M.catarrhalis.
Identification
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Colonies isolated on chocolate or blood agar plates are identified by colonial
morphology, Gram stain and oxidase reaction. Further biochemical identification may
be performed. If required, isolates may be referred to the Reference laboratory for
confirmation and further identification.
Primary isolation media
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Blood or chocolate agar 16-48 hrs incubation in 5-10% CO2 at 35-37ºC
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Identification of Moraxella species and morphologically similar species
Clinical specimens
Primary isolation plate
Moraxella species are white or buff, convex colonies on blood agar
Kingella species are smooth entire convex or spreading colonies 0.5-1 mm in
diameter. K.kingae is haemolytic
Oligella species are small, white opaque and non-haemolytic
Psychrobacter species are non-pigmented smooth opaque colonies
Gram stain
Gram negative rods or coccobacilli
Oxidase
Positive
Possible Moraxella species
or P.phenylpyruvicus
Negative
Possible Acinetobacter species
Tributyrin
Negative
Neisseria species
Positive
Moraxella species
If clinically indicated
If clinically indicated
Commercial identification kit*
or other identification
*Note: Commercial kits may misidentify Brucella species as P.phenylpyruvicus
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Colonial appearance
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Moraxella subgenus Moraxella
Smooth, flat, uniform, buff colonies 1-2mm in diameter
Colonies of M.lacunata, M.atlantae and M.nonliquefaciens are small on blood agar.
M.lacunata and M.atlantae also pit the agar. Some strains of M.lacunata are
haemolytic.
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Moraxella subgenus Branhamella
Smooth, round, uniform, grey/brown colonies 1 mm in diameter
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Kingella species
Two types of colonies occur on blood agar, a smooth entire convex type or a
spreading colony. Colonies are small 0.5-1 mm in diameter after 48 hrs. Kingella
kingae produce distinct zones of beta-haemolysis.
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Oligella species
Colonies are small after 24 hrs incubation. They are white, opaque, entire and nonhaemolytic.
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Psychrobacter phenylpyruvicus
Requires incubation at 20-25ºC. Colonies of P.phenylpyruvicus are small on blood
agar. Growth is enhanced by bile salts to form non-pigmented, smooth, opaque
colonies.
Identification
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Oxidase test – positive
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Tributyrin test - positive
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Commercial identification kit – Additional biochemical / other tests
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References
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Bergey’s Manual of Systematic Bacteriology, Volume 2.
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Identification of Unusual Pathogenic Gram-Negative Aerobic and Facultatively Anaerobic
Bacteria, Second Edition.
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Manual of Clinical Microbiology, 7 Edition.
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Manual of Clinical Microbiology, 8 Edition.
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Cowan and Steel’s Manual for the Identification of Medical Bacteria, 3 Edition.
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British National Standard Methods – Bacteriology 2006.
th
th
rd
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