Identification of Staphylococcus Species Micrococcus PDF

UK Standards for Microbiology Investigations
Identification of Staphylococcus species, Micrococcus

species and Rothia species
Issued by the Standards Unit, Microbiology Services, PHE

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 1 of 32
Crown copyright 2014
Identification of Staphylococcus species, Micrococcus species and Rothia species
Acknowledgments
UK Standards for Microbiology Investigations (SMIs) are developed under the
auspices of Public Health England (PHE) working in partnership with the National
Health Service (NHS), Public Health Wales and with the professional organisations
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medical content.
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Microbiology Services
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UK Standards for Microbiology Investigations are produced in association with:
Logos correct at time of publishing.

UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
Contents
ACKNOWLEDGMENTS .......................................................................................................... 2
AMENDMENT TABLE ............................................................................................................. 4
UK STANDARDS FOR MICROBIOLOGY INVESTIGATIONS: SCOPE AND PURPOSE ....... 6
SCOPE OF DOCUMENT ......................................................................................................... 9
INTRODUCTION ..................................................................................................................... 9
TECHNICAL INFORMATION/LIMITATIONS ......................................................................... 15
1 SAFETY CONSIDERATIONS .................................................................................... 17
2 TARGET ORGANISMS .............................................................................................. 17
3 IDENTIFICATION ....................................................................................................... 18
4 IDENTIFICATION OF STAPHYLOCOCCUS SPECIES, MICROCOCCUS SPECIES

AND ROTHIA SPECIES ............................................................................................. 23
5 REPORTING .............................................................................................................. 24
6 REFERRALS.............................................................................................................. 25
7 NOTIFICATION TO PHE OR EQUIVALENT IN THE DEVOLVED

ADMINISTRATIONS .................................................................................................. 25
REFERENCES ...................................................................................................................... 27

Amendment Table
Each SMI method has an individual record of amendments. The current amendments
are listed on this page. The amendment history is available from
standards@phe.gov.uk.
New or revised documents should be controlled within the laboratory in accordance
with the local quality management system.
Amendment No/Date. 5/12.11.14
Issue no. discarded. 2.3
Insert Issue no. 3
Section(s) involved Amendment
Whole document. Hyperlinks updated to gov.uk.

Page 2. Updated logos added.
Whole document. Document presented in a new format.
The scope has been updated to include webpage
Scope of document.
links for B 29 and ID 4 documents.
The taxonomy of Staphylococcus, Micrococcus
and Rothia species has been updated.
More information has been added to the
Characteristics section. The medically important
species are mentioned and their characteristics
Introduction. described.
Use of up-to-date references.
Section on Principles of Identification has been
updated to reflect rapid methods used for
identification.
Addition of information regarding agar media,
Technical
coagulase test and common issues with S. aureus
Information/Limitations.
has been described and referenced.
The section on the Target organisms has been
Target Organisms. updated and presented clearly. References have
been updated.
Minor amendments have been made to 3.1 and
3.2.
3.3 and 3.4 have been updated to reflect
Identification.
standards in practice.
Subsection 3.5 has been updated to include the
Rapid Molecular Methods.

Modification of flowchart for identification of

Identification Flowchart.
species has been made for easy guidance.
Subsection 5.1 - 5.4 has been updated to reflect
Reporting.
reporting practice.
The address of the reference laboratory has been
Referral.
updated.
References. Some references updated.

UK Standards for Microbiology Investigations#:

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Suggested Citation for this Document

Public Health England. (2014). Identification of Staphylococcus species, Micrococcus
species and Rothia species . UK Standards for Microbiology Investigations. ID 7 Issue
3. https://www.gov.uk/uk-standards-for-microbiology-investigations-smi-quality-and-
consistency-in-clinical-laboratories

Scope of Document
This SMI describes the identification of Staphylococcus species, Micrococcus species
and Rothia species. Details on MRSA screening can be found in B 29 - Investigation
of Specimens for Screening MRSA.
For the identification of catalase negative Gram positive cocci, see ID 4 - Identification
of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms.
This SMI should be used in conjunction with other SMIs.
Introduction
Taxonomy
Taxonomically, the genus Staphylococcus is in the bacterial family
Staphylococcaceae, which includes five lesser known genera, Gemella,
Jeotgalicoccus, Macrococcus, Nosocomiicoccus and Salinicoccus. There are currently
47 recognised species of staphylococci and 21 subspecies most of which are found
only in lower mammals1. 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 infection2.
The genus Micrococcus belongs to the bacterial family Micrococcaceae which
currently contains 16 species. These have been isolated from human skin, animal and
dairy products as well as environment (water, dust and soil)3. Some of these species
have been re-classified to other genera. Former members of the genus Micrococcus,
now assigned to other genera, include Arthrobacter agilis, Nesterenkonia halobia,
Kocuria kristinae, K. rosea, K. varians, Kytococcus sedentarius, and Dermacoccus
nishinomiyaensis. The Micrococcus species that are associated with infections are
Micrococcus luteus and Micrococcus lylae.
The genus Rothia belonged to the bacterial family Actinomycetaceae as described by
Georg and Brown in 1967 but more recent molecular studies placed the genus in the
family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, subclass
Actinobacteridae and class Actinobacteria. It is therefore in the same family as the
genera Micrococcus, Arthrobacter, Kocuria, Nesterenkonia, Renibacterium and
Stomatococcus, all of which show characteristic signature nucleotides in their 16S
rDNA sequences4. There are currently 6 species, Rothia dentocariosa and Rothia
mucilaginosa are the only two which have been known to cause infections in humans5.
Characteristics
Staphylococcus species are Gram positive, non-motile, non-sporing cocci of varying
size occurring singly, in pairs and in irregular clusters. Colonies are opaque and may
be white or cream and occasionally yellow or orange. The optimum growth
temperature is 30C-37C. They are facultative anaerobes and have a fermentative
metabolism. Staphylococcus species are usually catalase positive and are also
oxidase negative with the exception of the S. sciuri group (S. sciuri, S. lentus and
S. vitulinus), S. fleuretti and the Macrococcus group to which S. caseolyticus has been
assigned2,6,7. This is also a distinguishing factor from the genus streptococci, which
are catalase negative, and have a different cell wall composition to staphylococci.

Nitrate is often reduced to nitrite. Some species are susceptible to lysis by lysostaphin
but not lysozyme and are able to grow in 6.5% 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)8.
Coagulase positive staphylococci

Staphylococcus aureus
S. aureus are cocci that form irregular grape-like clusters. They are non-motile, non-
sporing and catalase positive. They grow rapidly and abundantly under aerobic
conditions. On blood agar, they appear as glistening, smooth, entire, raised,
translucent colonies that often have a golden pigment. The colonies are 2-3mm in
diameter after 24hr incubation and most strains show -haemolysis surrounding the
colonies.
There are currently 2 subspecies of S. aureus; these are S. aureus subspecies aureus
and S. aureus subspecies anaerobius.
S. aureus subspecies aureus is commonly isolated from human clinical specimens.
All strains are able to grow on thioglycolate medium within 24hr. Most strains produce
a wide zone of strong haemolysis within 24 to 36hr. They show positivity for D-
glucose, D-fructose, D-mannose, D-maltose, D-lactose, D-trehalose, D-mannitol,
sucrose, N-acetyl-glucosamine, D-celiobiose, and D-turanose; meanwhile, no acid
production was demonstrated by utilization of D-ribose, xylitol, xylose, D-melibiose,
raffinose, L-arabinose, and a-methyl-D-glucoside. They are also positive for catalase,
coagulase, and benzidine reactions and are capable of nitrate reduction and
acetylmethylcarbinol (acetoin) production. Results for DNase, clumping factor, urease,
arginine dihydrolase, pyrrolidonyl arylamidase, leucine arylamidase,
-N-acetylglucosaminidase, -chymotrypsin, -glucosidase, -glucosidase, alkaline
phosphatase, esterase C-4 and C-8, lipase (C-14), phosphatase acid, and
naphthol- AS-BI-phosphohydrolase are positive.
There is no production of oxidase, -galactosidase, - glucoronidase, -galactosidase,
valine arylamidase, cystine arylamidase, arginine arylamidase, trypsin, ornithine
decarboxylase, -mannosidase, and -fucosidase. All strains are resistant to
novobiocin9.
S. aureus subspecies anaerobius is rarely isolated from clinical specimens. They
are 0.8 to 1.0m in diameter and occur singly, in pairs, and predominantly in irregular
clusters. On the primary isolation medium, growth is obtained only in media that are
supplemented with blood, serum, or egg yolk and incubated microaerobically or
anaerobically. Colonies on blood agar after 2 days of incubation are very small (1 to
3mm in diameter), low convex, circular, entire, smooth, glistening, and opaque.
Pigment is not produced. Luxuriant growth is obtained on Dorset egg medium, with
colony diameters of 4 to 6mm. The strains produce unevenly disseminated growth on
brain heart infusion agar after 3 days of microaerophilic incubation. They grow as
dwarf colonies, among which a few colonies of normal size are observed10.
It grows poorly aerobically and growth may be CO2 dependent. It is slide coagulase
negative and thermonuclease negative and 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 the Reference Laboratory.

Staphylococcus aureus may be associated with severe infection and 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. It is also important to note that
coagulase negative strains of S. aureus have been reported11.
Other coagulase positive staphylococcal species such as S. hyicus, S. schleiferi
subspecies coagulans, S. pseudintermedius or S. intermedius may be coagulase
positive but have been found only occasionally to be associated with human infection
or carriage12,13. The production of coagulase and thermostable nuclease by these
staphylococci may lead to their misidentification as S. aureus. Staphylococcus delphini
is coagulase positive and thermostable nuclease positive (rarely isolated from
humans).
Carbon dioxide dependent strains of S. aureus can be recovered from clinical
material14. The significance of these strains in the laboratory is that they pose a
significant technical problem when performing antibiotic susceptibility testing as they
fail to grow in air. Therefore susceptibility testing should be performed in a CO2
enriched atmosphere. These strains although referred to as dwarf strains in the past
should not be confused with the slow-growing small colony variants (SCVs) of
S. aureus which have decreased metabolism and a defective electron transport
system and are auxotrophic for substrates such as haemin, menadione, thiamine or
thymidine15. Such strains are meticillin resistant and have an intrinsic resistance to
aminoglycoside antibiotics such as gentamicin and are most frequently identified in
patients with chronic or persistent infections16.
Multi resistance to antibiotics has most often been associated with meticillin resistant
strains16.
Staphylococcus aureus produces virulence factors such as protein A, capsular
polysaccharides and toxin. Some strains of S. aureus produce toxic shock
syndrome 1 toxin (TSST-1), Panton-Valentine Leucocidin or other toxins.
Coagulase negative staphylococci (CoNS)17

Coagulase negative staphylococci (CoNS) are normal commensals of the skin,
anterior nares, and ear canals of humans. They have long been considered as non-
pathogenic, and were rarely reported to cause severe infections. However, as a result
of the combination of increased use of intravascular devices and an increase in the
number of hospitalized immunocompromised patients, CoNS have emerged as a
major cause of nosocomial bloodstream infections.
They are opportunistic pathogens which lack many of the virulence factors associated
with S. aureus. There are more than 30 species of CoNS. The taxonomy of these
coagulase negative staphylococci (CoNS) fall into clusters based on 16s rRNA
sequences18.
S. epidermidis and S. saprophyticus are the species most often associated with
infection but Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus
haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus
sciuri, Staphylococcus schleiferi subspecies schleiferi, Staphylococcus simulans,
Staphylococcus saccharolyticus (previously known as Peptococcus saccharolyticus)
and Staphylococcus warneri have also been implicated19,20. Many of these species are
also thermostable nuclease negative. S. lugdunensis is coagulase negative but some
strains may be positive for the slide coagulase test or clumping factor 21.
Multi resistance to antibiotics also occurs in some strains of S. epidermidis which are
thermostable nuclease negative. S. saprophyticus, S. cohnii and S. sciuri groups are
generally novobiocin resistant as is S. hominis subsp. novobiosepticus22.
Staphylococcus pasteuri can be phenotypically distinguished from all of the other
novobiocin-susceptible staphylococci except S. warneri, from which it can only be
differentiated by genotyping23.
Staphylococcus epidermidis
S. epidermidis are approximately 0.5 to 1.5m in diameter and arranged in grape-like
clusters. They are facultative anaerobes that can grow by aerobic respiration or by
fermentation. Some strains may not ferment.
It forms greyish-white, raised, circular, smooth, glistening, and translucent to slightly
opaque, cohesive colonies approximately 12mm in diameter after overnight
incubation, and is non-haemolytic on blood agar. They grow well at NaCl
concentrations up to 7.5%, poorly at 10% and fail to grow at 15%. They are positive
for catalase, urease and exhibit a weak positive reaction for the nitrate reduction test.
They are negative for coagulase, oxidase and gelatin hydrolysis tests. They utilize
glucose, fructose, sucrose, and lactose to form acid products aerobically. In the
presence of lactose, they will also produce gas.
They are either susceptible or slightly resistant to lysostaphin and are resistant to
lysozyme.
S. epidermidis is sensitive to novobiocin, and this test distinguishes it from
Staphylococcus saprophyticus, which is coagulase negative, as well, but novobiocin
resistant24.
Staphylococcus saprophyticus
They are positive for catalase and urease tests while they are negative for motility,
coagulase, nitrate reduction and oxidase tests. They utilize fructose, maltose, sucrose
and trehalose to form acid products. They grow well on 10% NaCl agar, but only 11-
89% strains tolerate 15% NaCl. Colonies appear as raised to slightly convex, circular,
usually entire, 4.0 to 9.0mm in diameter, smooth, glistening, and usually opaque.
Colony pigment is variable; however, most strains are not pigmented or might have a
slight yellow tint which increases in intensity with age.
Two subspecies for S. saprophyticus exist: S. saprophyticus subsp. bovis and
S. saprophyticus subsp. saprophyticus, the latter is more commonly found in human
UTIs. S. saprophyticus subsp. saprophyticus is distinguished by its being nitrate
reductase and pyrrolidonyl arylamidase negative while S. saprophyticus subsp. bovis
is nitrate reductase and pyrolidonyl arymamidase positive25.
S. saprophyticus is resistant to the antibiotic novobiocin, a characteristic that is used in
laboratory identification to distinguish it from S. epidermidis, which is also coagulase
negative but novobiocin sensitive22.
Micrococcus species
Micrococcus species are strictly aerobic Gram positive cocci arranged in tetrads or
irregular clusters, not in chains and cells range from 0.5 to 3m in diameter. They are
seldom motile and are non-sporing. They are also catalase positive and often oxidase
positive, although weakly. Micrococci may be distinguished from staphylococci by a
modified oxidase test26,27. Their colonies are usually pigmented in shades of yellow or
red and grow on simple media. The optimum growth temperature is 25-37C. They
have a respiratory metabolism, often producing little or no acid from carbohydrates

and are usually halotolerant, growing in 5% NaCl. They contain cytochromes and are
resistant to lysostaphin8.
They are generally considered harmless saprophytes that inhabit or contaminate the
skin, mucosa, and also the oropharynx; however they can be opportunistic pathogens
in certain immunocompromised patients22.
There are currently 9 species of Micrococcus and 2 have been known to cause
infections in humans - Micrococcus lylae and Micrococcus luteus3.
Micrococcus lylae
They are mostly arranged in tetrads. They are positive for catalase and oxidase and
negative for urease. They grow in circular, entire, convex and usually not pigmented or
cream white colonies having diameters of approximately 4mm after 2-3 days on plate
at 37C. They assimilate D- Maltose, D- Trehalose, Maltitol, acetate, citrate,
d-Glucose, sucrose, d-fructose, d, fumarate, dl-3-hydroxybutyrate, dl-lactate, pyruvate,
l-aspartate, l-histidine, l-leucine, 3-hydroxybenzoate and 4-hydroxybenzoate and also
hydrolyse l-proline pNA, Tween 20 and Tween 8028.
M. lylae can be distinguished from the closely related species M. luteus by lysozyme
susceptibility, genetic compatibility, and the type of cell-wall peptidoglycan. There are
also some differences between these species in the parameters of pigmentation,
nitrogen requirements, nitrate reduction and acid from maltose and sucrose.
It has been isolated from human skin.
Micrococcus luteus
They are mostly arranged in tetrads. They are positive for catalase and oxidase. They
grow in circular, entire, convex and creamy yellow pigmented colonies having
diameters of approximately 4mm after 2-3 days at 37C. Several uncommon strains
produce raised colonies with translucent, depressed centres. Colony pigmentation
varies considerably but are usually different shades of yellow or cream-white29.
Growth or weak growth is observed at 45C, at pH 10 and in the presence of 10%
NaCl; no growth is observed in the presence of 15% NaCl. D-Glucose, sucrose and
D-mannose are assimilated while L-proline pNA, and Tween 20 are hydrolysed.
There are 3 biovars of M. luteus and they possess quite diverse chemotaxonomic
features with respect to their menaquinone systems, cell-wall compositions and
Fourier transform-infrared (FT-IR) spectroscopy (FT-IR) patterns, as well as
biochemical properties. The recognition of three different biovars within the species
M. luteus has the advantage that the three groups can be differentiated without
nomenclatural changes having to be introduced28.
It has been isolated from human skin.
Rothia species
Rothia species are Gram positive cocci with a variable microscopic morphology. Their
cells occur singly, in pairs, in clusters or in chains. They are weakly catalase positive
and weakly proteolytic. Rothia species are positive for nitrate and nitrite reduction,
liquefaction of gelatin and fermentation of sugars with the production of acid; while
negative for motility, urease and indole. Colonies on agar surface may appear
branched which rapidly fragment into bacillary or coccoid forms, resembling

Actinomyces or Nocardia species30. They exhibit good growth under aerobic or

microaerophilic conditions, but poor or no growth anaerobically.
Rothia species are susceptible to penicillin but because rare isolates may be resistant,
susceptibility testing should be performed.
There are currently 7 species of Rothia and 2 have been known to cause infections in
humans - Rothia dentocariosa and Rothia mucilaginosa5.
Rothia dentocariosa
R. dentocariosa cells occur singly, in pairs, in clusters or in chains. Colonial
pleomorphism can also be observed. Microscopically, the morphology varies from
coccoid to diphtheroid (with clavate ends) to filamentous. In broth cultures, cells may
be coccoid, which distinguishes them from Actinomyces species and appears in
filamentous forms on plates, but mixtures may appear in any culture4. They may show
rudimentary branching and loss of the Gram positive appearance in ageing cultures.
R. dentocariosa grows faster under aerobic than under anaerobic conditions, and
does not need CO2 or lipids for growth. It grows well on simple media (except
Sabouraud dextrose agar) and colonies may be creamy, dry, crumbly or mucoid, non-
haemolytic and may adhere to the agar surface. They are non-motile, catalase
positive and ferment carbohydrates with the end-products being lactic and acetic
acid31.
Catalase negative strains of R. dentocariosa have been reported and this will be more
difficult to recognise with traditional tests, since they may mimic the rare
Bifidobacterium strains that are able to grow aerobically, as well as Actinomyces and
Arcanobacterium species, Propionibacterium propionicum and catalase negative
Listeria strains4.
R. dentocariosa is distinct from Dermabacter species in that it is nitrate and
pyrazinamidase positive.
Rothia mucilaginosa (was previously known as Stomatococcus mucilaginosus,
Micrococcus mucilaginosus or Staphylococcus salivarius32,33.
This is found in clusters. Cells display variable catalase reactions ranging from
negative to weakly positive to strongly positive, oxidase negative, and exhibit
facultatively anaerobic metabolism. They are able to use glucose fermentatively.
Optimum growth temperature is 30-37C. Their white to greyish non-haemolytic
colonies may be mucoid, rubbery, or sticky in consistency and adherent to agar due to
the mucilagenous capsular material produced. The inability to grow in the presence of
5% NaCl distinguishes R. mucilaginosa from members of the genera Staphylococcus
and Micrococcus34.
It is isolated primarily from mouth and respiratory tract of humans, and is capable of
growth and producing diseases like endocarditis and meningitis in mammals.
Principles of Identification
Presumptive staphylococci need to be quickly differentiated into two groups:
Probable S. aureus - a potential pathogen when isolated from most sites
Other staphylococci - usually not significant in skin and superficial wound swab
sites, but a possible pathogen in some circumstances

Staphylococcus aureus has traditionally been identified by tube coagulase tests that
detect staphylocoagulase or "free coagulase". However, detection of surface proteins
such as clumping factor (slide coagulase test) and/or protein A (commercial latex
tests) may be used for rapid identification. Inclusion of latex particles sensitized with
antibodies against specific capsular antigens has enabled commercial manufacturers
to improve the sensitivity of latex tests to detect atypical strains of S. aureus and
MRSA that fail to express the major characteristics listed above35. Positive results or
suspected erroneous slide tests may be confirmed by a tube coagulase test.
Full molecular identification using for example, MALDI-TOF MS can be used to identify
CoNS isolates to species level.
Typing and differentiation between strains of S. aureus can be achieved using a range
of molecular techniques eg spa typing, Pulsed Field Gel Electrophoresis (PFGE),
Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA), Multi-locus
sequence typing (MLST), Microarrays, Next Generation Sequencing, etc36.
Technical Information/Limitations
Agar Media
The use of conventional media such as blood agar has the advantage that they may
also be useful for the simultaneous isolation of other pathogens such as streptococci.
The disadvantage of such media is that confirmatory tests are necessary to
differentiate S. aureus from other staphylococci35. Performing such tests on all
colonies resembling staphylococci can be time-consuming and labour intensive.
The use of chromogenic media, if sufficiently sensitive and specific, can potentially
reduce the number of confirmatory tests and achieve isolation and presumptive
identification in a single step. Another advantage is that they require fewer reagents
for confirmation of suspect colonies of S. aureus and hence may be cost effective37,38.
Chromogenic media for S. aureus may be supplemented with appropriate
antimicrobials (eg oxacillin or cefoxitin) for the detection of MRSA39.
Note: Chromogenic media are affected by direct light and plates should be stored in
the dark and not left in the light long before or after inoculation.
Coagulase test
S. aureus is differentiated from other staphylococci by the coagulase test. However it
is now known that not all S. aureus are coagulase positive and not all coagulase
positive staphylococci are S. aureus40.
S. lugdunensis is coagulase negative but some strains may be slide coagulase or
clumping factor positive.
For the tube coagulase test, citrate-utilizing organisms such as Enterococcus faecalis,
Pseudomonas species, Serratia marcescens, and strains of Streptococcus will clot
citrated plasma41.
S. hyicus, S. intermedius, S. pseudintermedius and S. schleiferi may be tube
coagulase positive.

Common issues with S. aureus

Yeasts can be misidentified as coagulase negative staphylococci on the basis of
colony morphology and a negative slide agglutination test. Speciation of staphylococci
should be considered for isolates from sterile sites and blood cultures to avoid missing
S. aureus, S. lugdunensis or yeasts.
Staphylococcus sciuri can give positive results with DNA and Staph aureus latex tests
and can have the mecA gene and therefore grow on chromogenic MRSA medium with
a blue green pigment. On blood agar, they appear as large yellow colonies resembling
S. aureus. It is easily distinguished from other staphylococcci as it is oxidase positive.
Other non S. aureus species such as S. intermedius could also be misidentified as
MRSA/MSSA.

1 Safety Considerations42-58
Refer to current guidance on the safe handling of all organisms documented in this
SMI.
Laboratory procedures that give rise to infectious aerosols must be conducted in a
microbiological safety cabinet50.
The above guidance should be supplemented with local COSHH and risk
assessments.
Compliance with postal and transport regulations is essential.
2 Target Organisms
Staphylococcus species reported to have caused human infections13,21,22,59-64
S. aureus group - Staphylococcus aureus subsp aureus, Staphylococcus aureus
subsp anaerobius
S. epidermidis group - Staphylococcus epidermidis, Staphylococcus capitis subsp
capitis, Staphylococcus capitis subsp urealyticus, Staphylococcus caprae,
Staphylococcus saccharolyticus
S. saprophyticus group - Staphylococcus saprophyticus subsp saprophyticus,
Staphylococcus cohnii subsp cohnii, Staphylococcus cohnii subsp urealyticus,
Staphylococcus xylosus
S. hyicus-intermedius group - Staphylococcus hyicus, Staphylococcus intermedius,
Staphylococcus pseudintermedius, Staphylococcus schleiferi subsp coagulans,
Staphylococcus schleiferi subsp schleiferi
S. simulans group - Staphylococcus simulans
S. haemolyticus group - Staphylococcus hominis subsp hominis, Staphylococcus
hominis subsp novobiosepticus, Staphylococcus haemolyticus
S. lugdunensis group - Staphylococcus lugdunensis
S. warneri group - Staphylococcus warneri, Staphylococcus pasteuri
S. auricularis group - Staphylococcus auricularis
S. carnosus group - Staphylococcus massiliensis, Staphylococcus pettenkoferi
S. sciuri group - Staphylococcus sciuri subsp sciuri, Staphylococcus sciuri subsp
rodentium,
Staphylococcus sciuri subsp carnaticus, Staphylococcus lentus, Staphylococcus
vitulinus
Other species reported to have caused human infections- Micrococcus luteus,
Micrococcus lylae, Micrococcus mortus (not officially recognised), Rothia
mucilaginosa, Rothia dentocariosa

3 Identification
3.1 Microscopic Appearance
Gram stain (TP 39 - Staining Procedures)
Gram positive cocci occurring singly, in pairs, tetrads or in irregular clusters.
3.2 Primary Isolation Media

Blood agar incubated in 5 - 10% CO2 at 35C - 37C for 16 - 24hr.
These organisms may be isolated from other media including CLED, Staph/Strep
selective and Mannitol Salt agar (MSA).
3.3 Colonial Appearance

Staphylococcus species usually grow as opaque, 1 5mm, smooth colonies; white,
cream or yellow to orange on blood agar. Haemolysis may be detected. They appear
as white or yellow-green, 1 - 2mm colonies on CLED agar.
Note: Small colony variant strains of S. aureus and strains resistant to vancomycin
(VRSA) may require 72hr incubation to become visible.
S. lugdunensis gives a prominent -haemolysis and a characteristic Eikenella-like
odour after 2 days of incubation on Columbia agar with 5% sheep blood, which,
combined with colony pleomorphism, helps in its initial recognition65.
Note: Avoid smelling or wafting the bacteria on the plates towards you because of
inhalation of spores and becoming contaminated. This is prohibited in the laboratory.
Micrococcus species produce yellow or red-pigmented colonies on blood agar.
Rothia species are round, convex, mucoidy and adhere to the agar. Colonial
morphology varies with species.
3.4 Test Procedures

3.4.1 Biochemical tests
Catalase test (see TP 8 - Catalase Test)
Staphylococcus, Micrococcus and Rothia species are catalase positive.
S. aureus subspecies anaerobius and S. saccharolyticus are catalase negative22.
Coagulase and other tests to detect S. aureus (see TP 10 - Coagulase Test)
Protein A, clumping factor (slide coagulase or latex), thermostable nuclease or tube
coagulase tests may be used. Positive results or suspected erroneous slide tests
(listed above) may be confirmed by a tube coagulase test.
S. aureus, some strains of S. hyicus, S. intermedius, and S. schleiferi subspecies
coagulans are coagulase positive and thermostable nuclease positive.
Other species of staphylococci are coagulase negative and thermostable nuclease
negative or weak positive. S. lugdunensis is coagulase negative but some strains may
be slide coagulase or clumping factor positive.

DNAse test (see TP 12 Deoxyribonuclease Test)

Commercially available DNA containing agars are used to detect thermolabile
nuclease activity. Addition of a weak acid (1N HCl) solution to an 18 24hr culture
plate will demonstrate clearing around colonies of DNAse positive species and if
toluidine blue O solution is added, a bright rose-pink zone around colonies of DNAse
positive species can be seen.
Note: It is important that both positive and negative screening tests for S. aureus are
verified using a second confirmatory test to detect false positive and false negative
primary screens eg Protein A latex and DNAse.
3.4.2 Commercial identification Systems
Several commercial identification systems are available for the speciation of
Staphylococci. Results should be interpreted in conjunction with the key test results
indicated above.
3.4.3 Matrix-Assisted Laser Desorption/Ionisation - Time of Flight
(MALDI-TOF)
Matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-
TOF MS), which can be used to analyse the protein composition of a bacterial cell,
has emerged as a new technology for species identification. This has been shown to
be a rapid and powerful tool because of its reproducibility, speed and sensitivity of
analysis. The advantage of MALDI-TOF as compared with other identification methods
is that the results of the analysis are available within a few hours rather than several
days. The speed and the simplicity of sample preparation and result acquisition
associated with minimal consumable costs make this method well suited for routine
and high-throughput use66.
MALDI-TOF MS method has been found to be useful as an additional test for the
description of new staphylococcal species and in the profiling of staphylococcal strains
and it has also revealed different clonal lineages of S. epidermidis that were of either
human or environmental origin67. However, further studies are required to test this
technology with a large collection of staphylococci of diverse origins.
3.4.4 Nucleic Acid Amplification Tests (NAATs)
PCR is usually considered to be a good method for bacterial detection as it is simple,
sensitive and specific. However, it does have limitations. Although the 16S rRNA gene
is generally targeted for the design of species-specific PCR primers for identification,
designing primers is difficult when the sequences of the homologous genes have high
similarity. In the case of staphylococci, therefore, ribotyping, internal transcribed
spacer PCR and various other methods have been used. There are different PCRs for
the different groups (coagulase positive and the coagulase negative staphylococcal
species) and their target genes and depending on clinical details, the appropriate PCR
will be performed68,69. However, the development of a species-specific quantitative
PCR methodology has proved difficult.
Multiplex PCR assay has also been used for detection of genes encoding surface
protein adhesins, toxins or antibiotic resistance in Staphylococci and more recently, for
species identification of coagulase positive Staphylococci by targeting the
thermonuclease (nuc) gene locus70-72. This has also been used to discriminate
simultaneously between mecA and mecALGA251 alongside the detection of Panton
Valentine leucocidin (PVL) and nuc genes of Meticillin resistant S. aureus and it
provides a valuable tool for the rapid and accurate characterization of staphylococci
which is essential in modern hospital practice73. This approach would also be valuable
for surveys.
3.5 Further Identification

Toxin studies
Occasionally S. aureus is recovered from cases of suspected toxin-mediated disease
eg staphylococcal scalded skin syndrome, toxic shock syndrome, PVL toxin,
necrotising pneumonia, bullous impetigo and food poisoning. The Staphylococcus
Reference Laboratory welcomes such isolates being referred for toxin gene profiling
and typing studies.
Rapid Methods
A variety of current rapid typing methods have been developed for isolates from
clinical samples; these include molecular techniques such as Pulsed- Field Gel
Electrophoresis (PFGE), 16S rRNA gene sequencing, PCR- restriction fragment
length Polymorphism (PCR-RFLP), spa typing, Multiple-Locus Variable Number
Tandem Repeat Analysis (MLVA) and Multi-locus sequence typing (MLST). All of
these approaches enable subtyping of strains, but do so with different accuracy,
discriminatory power, and reproducibility.
For further molecular investigation, Microarray analysis, single nucleotide
polymorphism (SNPs) these are looking to replace PFGE and MLVA) and even
whole-genome sequencing (WGS) may be options, as new technology (eg Ion Torrent
Sequencing) allows WGS results within a few days36.
However, some of these methods remain accessible to reference laboratories only
and are difficult to implement for routine bacterial identification in a clinical laboratory.
16S rRNA gene sequencing
16S rRNA gene sequences has been useful in phylogenetic studies at the genus
level, its use has been questioned in studies at the Staphylococcus species level18.
This stems from the fact that closely related species may have identical 16S rRNA
sequences or, alternatively, that divergent 16S rRNA sequences may exist within a
single organism74.
S. caprae and S. capitis cannot be distinguished by their 16S rRNA gene sequences.
Similarly, some Staphylococcus taxa have the same 16S rRNA gene sequences in
variable regions V1, V3, V7, and V9, with identical sequences occurring in, eg
S. vitulinus, S. saccharolyticus, S. capitis subsp urealyticus, S. caprae, the two
subspecies of S. aureus, and the two subspecies of S. cohnii75.
PCR- restriction fragment length Polymorphism (PCR-RFLP)
Due to the limited number of stable features that can be used for species
discrimination, many taxa remain difficult to distinguish from one another and are
misidentified by phenotypic tests.
However, restriction fragment length polymorphism (RFLP) analysis of the dnaJ gene
of PCR products has been reported for use for the identification of staphylococci. This
has proved to be an adequate tool for the correct identification of almost all prevalent
species and subspecies of Staphylococcus, irrespective of their phenotypic
characterization. This method requires only PCR and one or two enzymes and thus is
technically less demanding than the majority of other molecular approaches. It is

easier to use, less expensive and less equipment dependent than sequencing.
This method is also able to discriminate subspecies of the species S. capitis,
S. carnosus, S. cohnii, and S. hominis74.
Pulsed Field Gel Electrophoresis (PFGE)
PFGE detects genetic variation between strains using rare-cutting restriction
endonucleases, followed by separation of the resulting large genomic fragments on an
agarose gel. PFGE is known to be highly discriminatory and a frequently used
technique for outbreak investigations. However, the stability of PFGE may be
insufficient for reliable application in long-term epidemiological studies. Due to its time-
consuming nature (30hr or longer to perform) and its requirement for special
equipment and the interpretation of its results often being subjective, PFGE is not
used widely outside reference laboratories. These problems make the exchange of
strain typing information difficult and complicate the creation of an S. aureus and
MRSA typing database76.
Presently, pulsed-field gel electrophoresis (PFGE) remains the most discriminatory
technique for S. aureus typing, but it allows the constitution of shared databases only
at the national level and is not appropriate for population studies77.
More recently, PFGE has been used for epidemiological typing of Meticillin resistant
Staphylococcus aureus (MRSA)78.
Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA)
Multiple-Locus Variable number tandem repeat Analysis (MLVA) is a method used to
perform molecular typing of particular microorganisms. It utilizes the naturally
occurring variation in the number of tandem repeated DNA sequences found in many
different loci in the genome of a variety of organisms. The molecular typing profiles are
used to study transmission routes, to assess sources of infection and also to assess
the impact of human intervention such as vaccination and use of antibiotics on the
composition of bacterial populations.
This has been used successfully for the genotyping of S. aureus77.
spa sequence typing
spa sequencing appears to be a highly effective rapid typing tool for S. aureus that,
despite some expense of specificity, has significant advantages in terms of speed,
ease of use, ease of interpretation, and standardization among laboratories. It
provides a suitable discrimination for outbreak investigation. Another technique that
can be used in outbreaks is SCCmec typing depending on the local epidemiology
although better SCCmec methods need to be developed36. An additional advantage of
spa typing is that adequate typing information is obtained from a single locus, as
opposed to MLST, which requires the combination of allelic information from many
genes.
It has been documented that spa repeat sequences by themselves define excellent
resolving power among strains of S. aureus79,80. spa typing correctly assigns
staphylococcal strains to the appropriate phylogenetic groups and performs better
than multi locus enzyme electrophoresis (MLEE) and PFGE, and it facilitates the
detection of both macro- and micro-variation81.

However, spa does have some disadvantages. It is insufficiently discriminatory in

regions where a particular clone/small number of clones are endemic; it is not
recommended for smaller local hospital laboratories and is currently not yet used
universally36.
Multi-locus sequence typing (MLST)
MLST measures the DNA sequence variations in a set of housekeeping genes directly
and characterizes strains by their unique allelic profiles. The principle of MLST is
simple: the technique involves PCR amplification followed by DNA sequencing.
Nucleotide differences between strains can be checked at a variable number of genes
depending on the degree of discrimination desired.
This has been used to provide a reliable method of characterising MRSA clones as
well as investigating the epidemiology and phylogeny of S. lugdunensis82. It has also
been used for analysing the evolution of S. epidermidis as well.
Microarrays
DNA microarray technology can provide detailed, clinically relevant information on the
isolate by detecting the presence or absence of a large number of virulence-
associated genes simultaneously in a single assay; however, their clinical value has
been limited by a complicated methodology that is unsuitable for routine use in
diagnostic microbiology laboratories.
This has been used to successfully differentiate between isolates representative of a
spectrum of S. aureus types, including meticillin susceptible, meticillin resistant,
community-acquired, and vancomycin resistant S. aureus, and to simultaneously
detect clinically relevant virulence determinants83.
Whole Genome Sequencing (WGS)
This is also known as full genome sequencing, complete genome sequencing, or
entire genome sequencing. It is a laboratory process that determines the complete
DNA sequence of an organism's genome at a single time. There are several high-
throughput techniques that are available and used to sequence an entire genome
such as pyrosequencing, nanopore technology, IIIumina sequencing, Ion Torrent
sequencing, etc. This sequencing method holds great promise for rapid, accurate, and
comprehensive identification of bacterial transmission pathways in hospital and
community settings, with concomitant reductions in infections, morbidity, and costs84.
This has been useful in the detection of meticillin resistant S. aureus in an outbreak85.
It has also been used to highlight extensive differences in genome content between
the closely related Staphylococcus intermedius group (S. intermedius,
S. pseudintermedius and S. delphini) inhabiting distinct host niches as well as
providing new avenues for research into pathogenesis and bacterial host-adaptation13.
3.6 Storage and Referral

If required, save pure isolate on a nutrient agar slope for referral to the Reference
Laboratory.
Any strain of S. aureus suspected of demonstrating unusual resistance eg vancomyin,
linezolid must be referred to the Staphylococcal Reference Service for further
examination.

4 Identification of Staphylococcus Species,

Micrococcus species and Rothia species
Clinical specimens
Primary isolation plate
Opaque, white, cream, yellow or orange colonies on blood agar
Gram stain
Gram positive cocci in clusters Suspected
If there is a different Gram stain S. aureus
appearance refer to the appropriate UKSMI
Catalase
Negative Positive
Catalase negative
S. aureus subsp anaerobius S.capitis Catalase positive
S. saccharolyticus All Staphylococcus,
(grows anaerobically) Micrococcus and Rothia
Rothia dentocariosa* species
Consider other organisms
Novobiocin
sensitivity test
Resistant
S. saprophyticus,
S. sciuri and
Sensitive
S. cohnii groups
All Staphylococcus species
S. hominis subsp.
novobiosepticus
S. cohnii group
Coagulase (slide or tube)

DNAse test, Commercial
identification system
Negative Positive
S. aureus
Coagulase-negative S. hyicus*
Staphylococcus species S. intermedius*
S. schleiferi subsp coagulans*
Confirm with tube

coagulase if required
Further identification if clinically indicated

Refer to the Reference Laboratory
If required, save the pure isolate
onto a nutrient agar slope
* Strains may be positive to certain tests

5 Reporting
5.1 Presumptive Identification
If appropriate growth characteristics, colonial appearance, Gram stain of the culture,
catalase and slide coagulase or latex agglutination results are demonstrated.
5.2 Confirmation of Identification

Following confirmatory test results.
5.3 Medical Microbiologist

Inform the medical microbiologist of presumptive and confirmed Staphylococcus
aureus when the request bears relevant information, eg:
Toxin-mediated phenomena (eg Toxic Shock Syndrome, scalded skin
syndrome, epidermal necrolysis, bullous impetigo, necrotising pneumonia, food
poisoning)
Suspected outbreaks or instances of cross-infection
The medical microbiologist should also be informed of presumptive and confirmed
isolates of Staphylococcus species under the following circumstances:
Osteomyelitis and septic arthritis
Infections involving indwelling medical devices, eg prosthetic valves,
pacemakers, CSF shunts, peritoneal or vascular catheters
Endocarditis, haematogenous dissemination of infection, septicaemia
Isolates from normally sterile sites
Serious soft-tissue infections (cellulitis, erysipelas, necrotising myofasciitis,
puerperal sepsis, surgical wound infection, pneumonia, peritonitis, meningitis,
formation of abscesses or empyemas)
Follow local protocols for reporting to clinician.
5.4 CCDC
Refer to local Memorandum of Understanding.
5.5 Public Health England86

Refer to current guidelines on CIDSC and COSURV reporting.
5.6 Infection Prevention and Control Team

Inform the infection prevention and control team of isolates of meticillin resistant
Staphylococcus aureus and any S. aureus bacteremia (MSSA) in accordance with
local protocols.

6 Referrals
6.1 Reference Laboratory
Contact appropriate devolved national reference laboratory for information on the tests
available, turnaround times, transport procedure and any other requirements for
sample submission:
Staphylococcus Reference Service
Antimicrobial Resistance and Healthcare Associated Infections Reference Unit
Microbiology Services
Public Health England
61 Colindale Avenue
London
NW9 5EQ
Contact PHEs main switchboard: Tel. +44 (0) 20 8200 4400
England and Wales
https://www.gov.uk/specialist-and-reference-microbiology-laboratory-tests-and-
services
Scotland
http://www.hps.scot.nhs.uk/reflab/index.aspx
Northern Ireland
http://www.belfasttrust.hscni.net/Laboratory-MortuaryServices.htm
7 Notification to PHE86,87 or Equivalent in the

Devolved Administrations88-91
The Health Protection (Notification) regulations 2010 require diagnostic laboratories to
notify Public Health England (PHE) when they identify the causative agents that are
listed in Schedule 2 of the Regulations. Notifications must be provided in writing, on
paper or electronically, within seven days. Urgent cases should be notified orally and
as soon as possible, recommended within 24 hours. These should be followed up by
written notification within seven days.
For the purposes of the Notification Regulations, the recipient of laboratory
notifications is the local PHE Health Protection Team. If a case has already been
notified by a registered medical practitioner, the diagnostic laboratory is still required
to notify the case if they identify any evidence of an infection caused by a notifiable
causative agent.
Notification under the Health Protection (Notification) Regulations 2010 does not
replace voluntary reporting to PHE. The vast majority of NHS laboratories voluntarily
report a wide range of laboratory diagnoses of causative agents to PHE and many
PHE Health protection Teams have agreements with local laboratories for urgent
reporting of some infections. This should continue.
Note: The Health Protection Legislation Guidance (2010) includes reporting of Human
Immunodeficiency Virus (HIV) & Sexually Transmitted Infections (STIs), Healthcare
Associated Infections (HCAIs) and CreutzfeldtJakob disease (CJD) under

Notification Duties of Registered Medical Practitioners: it is not noted under

Notification Duties of Diagnostic Laboratories.
https://www.gov.uk/government/organisations/public-health-england/about/our-
governance#health-protection-regulations-2010
Other arrangements exist in Scotland88,89, Wales90 and Northern Ireland91.

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UK Standards for Microbiology Investigations

Identification of Staphylococcus species, Micrococcus

Issued by the Standards Unit, Microbiology Services, PHE

Logos correct at time of publishing.

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 2 of 32

AMENDMENT TABLE ............................................................................................................. 4

UK STANDARDS FOR MICROBIOLOGY INVESTIGATIONS: SCOPE AND PURPOSE ....... 6

SCOPE OF DOCUMENT ......................................................................................................... 9

TECHNICAL INFORMATION/LIMITATIONS ......................................................................... 15

1 SAFETY CONSIDERATIONS .................................................................................... 17

2 TARGET ORGANISMS .............................................................................................. 17

4 IDENTIFICATION OF STAPHYLOCOCCUS SPECIES, MICROCOCCUS SPECIES

7 NOTIFICATION TO PHE OR EQUIVALENT IN THE DEVOLVED

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 3 of 32

Amendment No/Date. 5/12.11.14

Issue no. discarded. 2.3

Insert Issue no. 3

Section(s) involved Amendment

Whole document. Hyperlinks updated to gov.uk.

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 4 of 32

Modification of flowchart for identification of

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 5 of 32

UK Standards for Microbiology Investigations#:

Equal Partnership Working

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 6 of 32

Patient and Public Involvement

Information Governance and Equality

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 7 of 32

Suggested Citation for this Document

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 8 of 32

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 9 of 32

Coagulase positive staphylococci

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Staphylococcus aureus may be associated with severe infection and it is important to

Coagulase negative staphylococci (CoNS)17

have a respiratory metabolism, often producing little or no acid from carbohydrates

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Actinomyces or Nocardia species30. They exhibit good growth under aerobic or

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 14 of 32

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 15 of 32

Common issues with S. aureus

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3.2 Primary Isolation Media

3.3 Colonial Appearance

3.4 Test Procedures

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DNAse test (see TP 12 Deoxyribonuclease Test)

3.5 Further Identification

technically less demanding than the majority of other molecular approaches. It is

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 21 of 32

However, spa does have some disadvantages. It is insufficiently discriminatory in

3.6 Storage and Referral

Bacteriology Identification | ID 7 | Issue no: 3 | Issue date: 12.11.14 | Page: 22 of 32

4 Identification of Staphylococcus Species,

Coagulase (slide or tube)

Confirm with tube

Further identification if clinically indicated

* Strains may be positive to certain tests