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UK Standards for Microbiology Investigations
Identification of Streptococcus species, Enterococcus
species and Morphologically Similar Organisms

Issued by the Standards Unit, Microbiology Services, PHE
Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
14 | Page: 1 of 36
© Crown copyright 2014

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms

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
whose logos are displayed below and listed on the website https://www
...
uk/ukstandards-for-microbiology-investigations-smi-quality-and-consistency-in-clinicallaboratories
...
gov
...

The contributions of many individuals in clinical, specialist and reference laboratories
who have provided information and comments during the development of this
document are acknowledged
...

For further information please contact us at:
Standards Unit
Microbiology Services
Public Health England
61 Colindale Avenue
London NW9 5EQ
E-mail: standards@phe
...
uk
Website: https://www
...
uk/uk-standards-for-microbiology-investigations-smi-qualityand-consistency-in-clinical-laboratories
UK Standards for Microbiology Investigations are produced in association with:

Logos correct at time of publishing
...
10
...
2
AMENDMENT TABLE
...
6
SCOPE OF DOCUMENT
...
9
TECHNICAL INFORMATION/LIMITATIONS
...
19

2

TARGET ORGANISMS
...
21

4

IDENTIFICATION OF STREPTOCOCCUS SPECIES, ENTEROCOCCUS SPECIES
AND MORPHOLOGICALLY SIMILAR ORGANISMS
...
28

6

REFERRALS
...
30

REFERENCES
...
10
...
The current amendments
are listed on this page
...
gov
...

New or revised documents should be controlled within the laboratory in accordance
with the local quality management system
...


10/28
...
14

Issue no
...


2
...


3

Section(s) involved

Amendment

Scope of document
...

The taxonomy of Streptococcus and Enterococcus
has been updated
...


More information has been added to the
Characteristics section
...
Other morphologically
similar organisms that are medically important are
also mentioned and their characteristics described
...


Technical
Information/Limitations
...


Safety considerations
...


Target Organisms
...


Identification
...
2, 3
...
4 to
reflect standards in practice
...

The table in 3
...

Subsection 3
...
10
...

Identification Flowchart
...


Reporting
...
1 has been updated to reflect
reporting practice
...


The addresses of the reference laboratories have
been updated
...


Document presented in a new format
...


Some references updated
...
10
...


•

SMIs provide clinicians with information about the available test repertoire and
the standard of laboratory services they should expect for the investigation of
infection in their patients, as well as providing information that aids the
electronic ordering of appropriate tests
...


Background to SMIs
SMIs comprise a collection of recommended algorithms and procedures covering all
stages of the investigative process in microbiology from the pre-analytical (clinical
syndrome) stage to the analytical (laboratory testing) and post analytical (result
interpretation and reporting) stages
...
Guidance notes cover the
clinical background, differential diagnosis, and appropriate investigation of particular
clinical conditions
...

Standardisation of the diagnostic process through the application of SMIs helps to
assure the equivalence of investigation strategies in different laboratories across the
UK and is essential for public health surveillance, research and development activities
...

The list of participating societies may be found at https://www
...
uk/uk-standards-formicrobiology-investigations-smi-quality-and-consistency-in-clinical-laboratories
...
Nominees of
professional societies are members of the Steering Committee and Working Groups
which develop SMIs
...
Nominees act as a conduit for two way reporting and dialogue
...

SMIs are developed, reviewed and updated through a wide consultation process
...


Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms

Quality Assurance
NICE has accredited the process used by the SMI Working Groups to produce SMIs
...
The
process for the development of SMIs is certified to ISO 9001:2008
...
SMIs are NICE accredited
and represent neither minimum standards of practice nor the highest level of complex
laboratory investigation possible
...
SMIs
help laboratories to meet accreditation requirements by promoting high quality
practices which are auditable
...

The performance of SMIs depends on competent staff and appropriate quality
reagents and equipment
...
Laboratories should
participate in external quality assessment schemes and undertake relevant internal
quality control procedures
...
By involving the public, health professionals, scientists and
voluntary organisations the resulting SMI will be robust and meet the needs of the
user
...


Information Governance and Equality
PHE is a Caldicott compliant organisation
...

The development of SMIs are subject to PHE Equality objectives
https://www
...
uk/government/organisations/public-health-england/about/equalityand-diversity
...


Legal Statement
Whilst every care has been taken in the preparation of SMIs, PHE and any supporting
organisation, shall, to the greatest extent possible under any applicable law, exclude
liability for all losses, costs, claims, damages or expenses arising out of or connected
with the use of an SMI or any information contained therein
...

The evidence base and microbial taxonomy for the SMI is as complete as possible at
the time of issue
...
These standards can only be superseded by revisions of the standard,
legislative action, or by NICE accredited guidance
...

Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms

Suggested Citation for this Document
Public Health England
...
Identification of Streptococcus species, Enterococcus
species and Morphologically Similar Organisms
...
ID 4 Issue 3
...
gov
...
10
...
Organisms morphologically similar to streptococci, which may be found in
clinical specimens, are also included
...
This SMI
adopts a simplified approach based on grouping organisms with similar phenotypic
attributes1
...

This SMI should be used in conjunction with other SMIs
...
There are also
some differences in opinion on the nomenclature of some of the streptococci between
identification systems in the UK and USA
...

The genus name Enterococcus, originally suggested in 1903 for bacteria previously
called Streptococcus faecalis and Streptococcus faecium, was revived in 1984 when
other bacteria were transferred to the genus1,3
...
Enterococcus faecalis and Enterococcus
faecium are the commonest enterococci isolated from human infections4
...
Streptococci are facultatively anaerobic and catalase negative1
...

Streptococci produce the enzyme leucine aminopeptidase (LAP), which has also been
called leucine arylamidase
...
Haemolysis produced by colonies on
blood agar and Lancefield serological grouping are important factors in presumptive
identification
...
10
...
This type of haemolysis can be
confused with β-haemolysis

Lancefield grouping:
Beta-hemolytic streptococci are further characterised via Lancefield serotyping, which
describes specific carbohydrates present on the bacterial cell wall5
...

Lancefield group A
Streptococcus pyogenes
Streptococcus pyogenes occurs in chains
...
5mm, domed, with an entire edge
...
Haemolysis is best observed by growing the
culture under anaerobic conditions because the haemolysins are more stable in the
absence of oxygen6
...
Pinpoint colony
forms of the S
...

Bacitracin susceptibility has been used presumptively for screening purposes but is
unreliable because it is not highly specific and methods vary between laboratories8-12
...

The pyrrolidonyl aminopeptidase (which has also been called the pyrrolidonyl
arylamidase or 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
...

Lancefield group B
Streptococcus agalactiae
Streptococcus agalactiae occurs in chains
...
Some strains may be non-haemolytic13
...

Islam’s medium, to detect orange pigment production, may be useful for primary
isolation and presumptive identification, but is not recommended in this SMI13
...
10
...
Large
colony forms of Lancefield groups C and G streptococci (≥0
...
Group C and G strains of S
...

Lancefield groups C and G streptococci will not grow on media containing bile
...
anginosus group can cross react with the Lancefield
groups C and G antibodies and may grow on media containing bile9
...
Colonies
on blood agar are small (≤0
...
Incubation conditions may be of some value for the presumptive
identification of the S
...

Organisms of this group may possess the Lancefield group A, C, F or G antigen or be
ungroupable17
...
intermedius possesses no group antigen
...
constellatus may
express group C, or F and S
...

Human isolates of streptococci which express the group F antigen are highly likely to
be members of the anginosus group
...

Resistance to sulphonamides and bacitracin may be used as screening tests for
organisms of the S
...

Identification of an isolate from a clinical specimen as being a member of this group is
potentially clinically significant, due to the propensity of this group to be associated
with invasive pyogenic infections
...
bovis group possess Lancefield
group D antigen
...

Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms
Microscopically the enterococci are Gram positive cocci, spherical or ovoid in shape
(0
...
5µm), usually occurring in pairs or short chains in broth culture
...
Most species will grow on nutrient agar at 45°C
...
6 and in 6
...
They can also survive at 60°C for
30min and are PYR positive which differentiates them from S
...
gallolyticus
...
Two species within the genus, Enterococcus
cassiflavus and Enterococcus gallinarum, are motile
...
Most species are catalase negative, but some
strains produce a pseudocatalase
...

E
...
However, vancomycin or
glycopeptide resistant enterococci (V/GRE) are becoming increasingly common and
this spread of resistance is thought to be due to transposons and plasmids moving
between bacterial species20
...
Currently, there are eight known vancomycin
resistant phenotypes: vanA, vanB, vanC, vanD, vanE, vanG, vanL, and vanM
...
The
strains with acquired resistance are the only ones aimed to control and reported in
VRE surveillance programmes
...
Strains that harbour
the vanA gene display high levels of resistance to vancomycin and teicoplanin,
whereas strains that harbour the vanB gene have variable levels of resistance to
vancomycin only21
...
bovis group, there are six species and they include: S
...
equinus,
S
...
bovis biotype I), S
...
bovis biotype
II/1), S
...
bovis biotype II/2) and S
...

Microscopically these species are Gram positive cocci, occurring in chains
...
Members of the S
...
It is
important to identify S
...
gallolyticus and S
...
The S
...

Streptococcus suis
S
...

They are commonly associated with the Lancefield groups R, S and T
...
suis I is
associated with group S and S
...
They do not grow in 6
...
Some strains are able to grow in the presence of 40% bile and all are able to
hydrolyse aesculin
...
10
...
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 35-37°C for 16-24hr
...

S
...

S
...

S
...
The 'Quellung reaction'
(capsular swelling) may be used microscopically to identify the specific types of
S
...
Commercial agglutination tests are also available for the rapid
detection of pneumococcal antigens, but these should be used with caution because
cross-reactions may occur with the S
...
mitis groups
...
These species are
Gram positive cocci occurring in chains, which are indistinguishable by Gram stain
from β-haemolytic streptococci
...
5-1
...

They possess no Lancefield antigens and are resistant to optochin
...

In the Streptococcus mitis subgroup, Streptococcus pseudopneumoniae has been
mistaken for S
...
pneumoniae:
•

There is no pneumococcal capsule (and is therefore not typable)

•

It is not soluble in bile

•

It is sensitive to optochin when incubated in ambient air, but appears resistant
or to have indeterminate susceptibility when incubated in 5% carbon dioxide

•

Commercial DNA probe hybridization tests are falsely positive23

Generally these streptococci would not require further identification, other than as an
α or non-haemolytic streptococci, when isolated from sites where they are considered
normal flora
...

Some species of streptococci, eg Streptococcus sanguinis and Streptococcus oralis
(formerly mitior), may account for up to 80% of all streptococcal endocarditis cases24
...
NVS require media supplemented with either pyridoxal or
cysteine for growth26,27
...
2-0
...
They can be either non-haemolytic or α-haemolytic
...
10
...
NVS should be suspected
when Gram positive cocci resembling streptococci are seen in positive blood cultures,
which subsequently fail to grow on subculture
...
Alternatively, media may
be supplemented with 10mg/L pyridoxal hydrochloride
...

Unusual Streptococcus species
Streptococcus acidominimus
Streptococcus acidominimus belongs to the Streptococcus viridans group and
microscopically, it occurs in short chains
...
They do not hydrolyse aesculin or arginine but ferments sucrose and
glucose
...

A few cases of deep-seated human infections by S
...
They are generally quite sensitive to β-lactam antibiotics
...

They are Aerococcus christensenii, Aerococcus sanguinicola, Aerococcus urinae,
Aerococcus urinaehominis and Aerococcus viridans
...

Sometimes a weak catalase or pseudocatalase reaction is produced
...
Some strains of Aerococcus viridans are bile aesculin positive
and PYR positive
...

Growth occurs both under aerobic and anaerobic conditions
...
viridans
...
sanguinicola as A
...

This makes the reports of infections caused by A
...

Most aerococci are sensitive to beta-lactams as well as to several other groups of
antibiotics
...

Facklamia species
There are six species of which four are from humans (Facklamia hominis, Facklamia
languida, Facklamia sourekii and Facklamia ignava)
...
Facklamia species resemble “viridans” streptococci on
culture
...
The Facklamia
species are facultatively anaerobic and grow best in an atmosphere of increased
Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms
carbon dioxide
...
They are catalase and oxidase negative but positive for pyrrolidonyl
arylamidase and leucine aminopeptidase
...
5% NaCl at 37°C but
fail to grow at 10 or 45°C
...
Acid is not
produced from glucose and other sugars and nitrate is not reduced38
...

Gemella species
There are currently five species isolated from human sources that are recognised:
Gemella haemolysans, Gemella morbillorum (formerly Streptococcus morbillorum),
Gemella bergeriae, Gemella sanguinis and Gemella asaccharolytica species nov39-42
...
Some strains easily
decolourise on Gram staining, occurring as Gram negative
...
They are either α-haemolytic or non-haemolytic on
blood agar and resemble colonies of viridans streptococci
...

In some commercial identification systems, “viridans” streptococci can be misidentified as Gemella species
...

Globicatella species
There are two species of Globicatella but the species that is implicated in human
infections is Globicatella sanguinis44-46
...
Microscopically, they are Gram
positive cocci occurring singly, in pairs or short chains
...
However, they do not produce leucine
aminopeptidase44
...

Globicatella species can be distinguished from aerococci by cellular morphology
...

Helcococcus species
There are currently three species of Helcococcus species isolated from humans
...

Helcococcus species are Gram positive cocci that are catalase negative and
facultatively anaerobic
...
They are slow
growing and appear like viridans streptococci on blood agar plate
...
Acid is produced but not
gas from glucose and other sugars
...
These
species are susceptible to vancomycin
...
10
...
kunzii produces tiny grey, non-haemolytic colonies; growth is stimulated by the
addition of serum or Tween 80 to the basal medium
...

H
...
This demonstrates relative vancomycin resistance like
Pediococcus species48
...
sueciensis produces pinpoint grey, non-haemolytic colonies after 48hr anaerobic
incubation
...

Lactococcus species
There are seven species of the Lactococcus currently recognised
...

They are facultatively anaerobic, α or non-haemolytic, Gram positive cocci which
occur singly, in pairs or chains
...

Leuconostoc species
The genus Leuconostoc consists of the following species (including re-classified and
synonymous species): Leuconostoc mesenteroides (type species), L
...
carnosum, L
...
cremoris, L
...
durionis,
L
...
ficulneum, L
...
garlicum, L
...
gelidum,
L
...
inhae, L
...
lactis, L
...
oeni, L
...
paramesenteroides, L
...
pseudomesenteroides
...

Of these species, four of them (L
...
fructosum, L
...
pseudoficulneum) have been re-classified and transferred as belonging to the
genus Fructobacillus
...

These are non-pathogenic species and they prefer fructose but not glucose as growth
substrate
...

Leuconostoc mesenteroides has been divided into 4 subspecies; 2 of which were
reclassified (L
...
dextranicum have been renamed as Leuconostoc
mesenteroides subspecies cremoris and Leuconostoc mesenteroides subspecies
dextranicum respectively), Leuconostoc mesenteroides subspecies mesenteroides
and then a more recent addition, Leuconostoc mesenteroides subspecies
suionicum53
...
They
are catalase negative and colonies often are α-haemolytic on blood agar
...


Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms
Pediococcus species
Pediococcus species may resemble viridans streptococci on culture, but
microscopically they are similar to staphylococci
...
They are facultatively
anaerobic and catalase negative
...
They are leucine aminopeptidase–
positive, which distinguishes them from Leuconostoc species54
...


Principles of Identification
Isolates from primary culture are identified by colonial appearance, Gram stain,
catalase test, Lancefield grouping and optochin sensitivity
...

In some instances based on colonial morphology, clinical details and operator
experience, it may be possible to omit the early steps of identification (eg Gram stain
and catalase) and proceed to other tests
...

If Lancefield grouping does not provide sufficient identification for clinical
management, a full identification may be obtained using a commercial identification
system, in conjunction with the results of sensitivity testing
...

If confirmation of identification is required, isolates should be sent to a Reference
Laboratory where a referred (charged for) taxonomic identification service for
streptococci and other related Gram positive, catalase negative genera is available
...
There is also poor discrimination between
the S
...
mitis group as they are genetically inseparable, and so
Streptococcus mitis/oralis species can be erroneously identified as S
...

Another group that are difficult to differentiate are species belonging to the S
...
sanguinis groups which are often regarded as a single group, they give
discordant results due to the low quality of the identification system used
...

Despite this limitation, subtyping of Streptococcus pneumoniae strains by MALDI-TOF
MS can be reliably performed, even for immunologically non-typeable or nonencapsulated strains56
...
10
...


Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms

1

Safety Considerations57-73

Hazard Group 2 organisms
...

Appropriate personal protective equipment (PPE) and techniques designed to
minimise exposure of the laboratory workers should be worn and adhered to at all
time
...

Employers should ensure that personnel who are pregnant, immunocompromised or
immunosuppressed should be restricted from performing work with these highly
infectious microorganisms or from handling isolates requesting for identification of
these microorganisms and, in some situations, be restricted to a low-risk laboratory74
...

The above guidance should be supplemented with local COSHH and task specific risk
assessments
...


2

Target Organisms

Streptococcus species Reported to have Caused Human
Infection43,76
Streptococci possessing Lancefield group antigens A-G1
Group A
Streptococcus pyogenes (Streptococcus anginosus and Streptococcus constellatus
subspecies constellatus may cross react with the Lancefield group A antigen)
...

Group D3,15,19
Enterococcus species (see below)
Streptococcus bovis group
Taxonomy of the S
...
bovis biotype I), Streptococcus gallolyticus subsp pasteurianus
Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms
(S
...
bovis biotype II/1), Streptococcus
pasteurianus, Streptococcus lutetiensis
Group F
Streptococcus anginosus, Streptococcus constellatus subspecies constellatus
Group G
Group G streptococci (Streptococcus anginosus and Streptococcus constellatus
subspecies constellatus may cross react with the Lancefield group G antigen)
...
They are as follows:
Streptococcus anginosus group (also known as the S
...

*Taxonomically, this is shown to be within the mitis cluster but could be separated
from all other species
...
10
...
1

Microscopic Appearance

Gram stain (TP 39 - Staining Procedures)
Streptococcus, Enterococcus and Lactococcus species are Gram positive, round or
ovoid cells occurring in pairs, short or long chains or sometimes in clusters
...

Aerococcus, Pediococcus, Facklamia, and Helcococcus species are Gram positive
cocci in clusters or tetrads
...


3
...

CLED agar incubated aerobically at 35–37°C for 16-24hr
...


3
...
5mm, entire edged, may have a dry
appearance, colonies may be difficult to pick off the plate
...
5-1
...


Enterococci

α,β or non

Colonies are larger than those of streptococci, usually 1–
2mm, with a wet appearance
...


S
...
After anaerobic incubation colonies may be larger
and mucoid
...
anginosus”

α,β or non

Colonies are small (≤0
...

Some strains have a white “heaped” up colony

NVS

α or non

Colonies are small (≤0
...


Aerococcus species

α

Resemble “viridans” streptococci
...
10
...


Lactococcus species

α or non

Resemble enterococci

Leuconostoc species

α or non

Resemble “viridans” streptococci
...
4

Test Procedures

3
...
1 Biochemical tests
Catalase test (TP 8 - Catalase Test)
Streptococci and morphologically similar organisms are usually catalase negative
...

Bile Aesculin hydrolysis test (TP 2 - Aesculin Hydrolysis Test)
Enterococci, Lancefield Group D streptococci and lactococci hydrolyse aesculin in the
presence of 40% bile, other streptococci do not
...

Optochin sensitivity test (TP 25 - Optochin Test)
S
...

Occasional strains of S
...
mitis and S
...

Pyrrolidonyl arylamidase /PYR-aminopeptidase (PYR)9
Enterococci and S
...
bovis group and S
...

Bile solubility test (optional) (TP 5 - Bile Solubility Test)
S
...
pseudopneumoniae is partially soluble
and other α-haemolytic streptococci are insoluble
...
10
...
pneumoniae

-

S

-

-

d

Group D

+

R

-

+

-

Enterococci

+

R

v

+

+

S
...
anginosus group

v

R

-

v

-

“viridans”
streptococci

-

v

-

ND

ND

Facklamia species

-

R

-

-

+

Gemella species

-

R

w

-

+

Globicatella species

-

R

-

+

+

Helcococcus species

-

R

-

-

+

Leuconostoc species

-

R

d

D

Pediococcus species

cr

R

+

-

v-variable

R-resistant

w-weak reaction ND- no data

S-sensitive
+

-

PYR

cr-cross reacts d-6–84% strains positive

-

# - In the S
...
bovis biotype II/1) and S
...

These test results are consistent with taxonomy from three widely published systems

1,9,84

3
...
2 Streptococcal grouping (Commercial Identification Kits)
Lancefield showed that the majority of pathogenic streptococci possess specific
carbohydrate antigens, which permit the classification of streptococci into groups
...
The use of an enzymatic extraction
procedure considerably shortens the time required for antigen extraction and much
improves the antigen yield, partially for Group D streptococci
...
10
...
Latex test
particles are sensitised with group specific antibody and will agglutinate in the
presence of homologous antigen
...

The extract is then neutralized and the antigens are identified by agglutination
...
Laboratories
should follow manufacturer’s instructions and rapid tests and kits should be validated
and be shown to be fit for purpose prior to use
...
4
...
This has been shown to be a rapid and
powerful tool because of its reproducibility, speed and sensitivity of analysis
...
One limitation of MALDITOF is that it cannot readily distinguish between Streptococcus pneuomoniae from
other members of the Streptococcus mitis group
...
This has also
been used for identification of aerococci to the species level but however, the
accuracy of MALDI-TOF MS identification of bacterial species that are uncommon in
clinical samples, such as aerococci, needs to be further evaluated89
...
4
...
For Streptococcus species,
there are various PCRs for the different groups and their target genes and depending
on clinical details, the appropriate PCR will be performed
...

PCR has also been used for simultaneous detection of glycopeptide resistance
genotypes and identification to the species level of clinically relevant enterococci
(Enterococcus faecium, E
...
gallinarum, and E
...


3
...


Rapid Methods
A variety of 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, atpA Gene Sequence Analysis,
Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms
and Multilocus sequence typing (MLST)
...

However, some of these methods remain accessible to reference laboratories only
and are difficult to implement for routine bacterial identification in a clinical laboratory
...
pyogenes isolates94
...
avium,
E
...
cecorum, E
...
faecium species groups; but it fails to
discriminate closely related species, ie the members of E
...
faecium
species groups and Streptococcus species are not readily identified by the sequencing
of the16S rRNA gene4
...
However, PFGE was found to
be superior for interpretation of the interstrain relationships among enterococci but did
not result in species-specific discriminative DNA bands4
...

Multi-locus sequence typing (MLST)
Multi-locus sequence typing (MLST) is a highly discriminatory tool that is widely used
for phylogenetic typing of bacteria as well as to study the molecular epidemiology and
population genetic structure of microorganisms
...
MLST measures the
DNA sequence variations in a set of housekeeping genes directly and characterises
strains by their unique allelic profiles
...
Nucleotide differences between strains can be checked at a variable
number of genes depending on the degree of discrimination desired
...
Its advantages are that it is unambiguous and highly
portable and sequence data can be compared readily between laboratories and data
stored in a central database is easily accessible via the internet to produce a powerful
resource for global epidemiology96
...
10
...
This has also been used to identify the major clones associated with serious
invasive pneumococcal disease and for characterising Streptococcus pyogenes
(Lancefield group A streptococcus, GAS) isolates for epidemiological purposes by
using this method together with its dedicated web-based database and tools which
can be accessed on http://pubmlst
...

However, the drawbacks of MLST are the substantial cost and laboratory work
required to amplify, determine, and proofread the nucleotide sequence of the target
DNA fragments, making the method hardly suitable for routine laboratory testing
...
6

Storage and Referral

If required, subculture the pure isolate on a blood agar slope for referral to the
Reference Laboratory
...
10
...

Consider clinical details)

Optochin

Lancefield Group
Rapid Aesculin hydrolysis

Sensitive

Resistant

A,B,C,D,F,G
Positive

S
...
pneumoniae may be
resistant to optochin: if there is a
clinical suspicion of pneumococcal
infection, confirm by performing
bile solubility
S
...


“viridans”
Streptococci:
Occasional strains of S
...
pseudopneumoniae optochin
resistant when incubated in 5%
CO2

Enterococcus sp
Lancefield group D
Lactococcus sp
Some strains of Aerococcus and
Leuconostoc sp
Pediococcus sp
Globicatella sp

Negative

Consider
Pediococcus sp
Lancefield Group B
S
...

Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
14 | Page: 27 of 36
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

(A,C,G, consider
S
...
1

Presumptive Identification

Presumptive identification can be made if appropriate growth characteristics, colonial
appearance, Gram stain of the culture; catalase and serological results are
demonstrated
...
2

Confirmation of Identification

Confirmation of identification and toxigenicity are undertaken only by the Respiratory
and Vaccine Preventable Bacteria Reference Unit (RVPBRU) PHE Colindale
...
3

Medical Microbiologist

Inform the medical microbiologist of all presumed and confirmed cultures of
Streptococcus and Enterococcus species and morphologically similar organisms
obtained from specimens from normally sterile sites
...

According to local protocols, consideration should also be given to informing the
medical microbiologist when the request bears relevant or additional information
suggestive of invasive or severe streptococcal infection eg:
•

Toxin mediated phenomena (Toxic Shock Syndrome or Scarlet Fever)

•

(Necrotising) fasciitis or myositis, puerperal sepsis

•

Endocarditis

•

Investigation of possible outbreaks or apparent cross-infection within a hospital
or other institution

•

Unusual antimicrobial resistance patterns, including vancomycin or other
glycopeptide resistant Enterococcus species and penicillin resistant
S
...


5
...


5
...


Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
14 | Page: 28 of 36
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms

5
...
Consideration should be
given to informing the relevant Infection Control staff of such isolates from patients
currently in the community (including nursing homes) in accordance with local
arrangements, notably if suspecting cross-transmission
...
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:
Streptococci
Streptococcus and Diphtheria Reference Section
WHO Global Collaborating Centre for Streptococcal and Diphtheria Infections
Respiratory and Vaccine Preventable Bacteria Reference Unit
Microbiology Services
Public Health England
61 Colindale Avenue
London
NW9 5EQ
https://www
...
uk/rvpbru-reference-and-diagnostic-services
Enterococci
Antimicrobial Resistance and Healthcare Associated Infections Reference Unit
(AMRHAI)
Microbiology Services
Public Health England
61 Colindale Avenue
London
NW9 5EQ
https://www
...
uk/amrhai-reference-unit-reference-and-diagnostic-services
Contact PHE’s main switchboard: Tel
...
gov
...
hps
...
nhs
...
aspx
Northern Ireland
http://www
...
hscni
...
htm

Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms

7 Notification to PHE100,101 or Equivalent in the
Devolved Administrations102-105
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
...
Urgent cases should be notified orally and
as soon as possible, recommended within 24 hours
...

For the purposes of the Notification Regulations, the recipient of laboratory
notifications is the local PHE Health Protection Team
...

Notification under the Health Protection (Notification) Regulations 2010 does not
replace voluntary reporting to PHE
...
This should continue
...

https://www
...
uk/government/organisations/public-health-england/about/ourgovernance#health-protection-regulations-2010
Other arrangements exist in Scotland102,103, Wales104 and Northern Ireland105
...
10
...


Hardie JM
...
In: Sneath PHA, editor
...
Vol 2
...
p
...


2
...
Streptococcal taxonomy based
on genome sequence analyses
...


3
...
Transfer of Streptococcus faecalis and Streptococcus faecium to
the Genus Enterococcus nom
...
as Enterococcus faecalis com
...
nov
...


4
...

Phylogeny and identification of Enterococci by atpA gene sequence analysis
...


5
...
What happened to the streptococci: overview of taxonomic and nomenclature
changes
...


6
...
Suitability of throat culture procedures for detection of group A streptococci and as
reference standards for evaluation of streptococcal antigen detection kits
...


7
...
The microbiologic aspects, including diagnosis, of beta-hemolytic
streptococcal and enterococcal infections
...


8
...
In: MacFaddin JF, editor
...
3rd ed
...
p
...


9
...
Presumptive identification of streptococci with a new
test system
...


10
...
Lack of reliability of primary grouping of beta-hemolytic streptococci by culture of
throat swabs with streptocult supplemented with bacitracin disks in general practice
...


11
...
Further studies on the reliability of the bacitracin inhibition
test for the presumptive identification of Lancefield group A streptococci
...


12
...
The bacitracin sensitivity test for identifying beta-haemolytic streptococci
of Lancefield group A
...


13
...
Streptococcus and Enterococcus
...
Mackie & McCartney Practical Medical Microbiology
...
Edinburgh: Churchill
Livingstone; 1996
...
263-74
...


Kohler W
...
Int J
Med Microbiol 2007;297:133-50
...


Holt JG, Krieg N R, Sneath P H A, Staley J T, Williams S T, editors
...
9th ed
...
p
...
10
...


Jensen A, Hoshino T, Kilian M
...
whileyi subsp
...
and Streptococcus
constellatus subsp
...
nov
...


17
...
Color Atlas and
Textbook of Diagnostic Microbiology
...
Philadelphia: Lippincott Williams & Wilkins; 1997
...

98-102

18
...
Rapid differentiation of Streptococcus milleri from other beta-haemolytic
group A, C, and G streptococci by simple screening tests
...


19
...
Antibiotic susceptibility and biochemical properties of
Streptococcus faecalis strains reacting with both D and G antisera
...


20
...
Vancomycin-resistant enterococci
...


21
...
Current Rapid Screening Methods for Gastrointestinal Colonization of
Vancomycin-Resistant Enterococci
...


22
...
Association of
Streptococcus bovis with carcinoma of the colon
...


23
...
Characteristics of Streptococcus
pseudopneumoniae isolated from purulent sputum samples
...


24
...
Streptococci and aerococci associated with systemic infection in man
...


25
...
The genus Abiotrophia (Kawamura et al
...
nov
...
nov
...

nov
...
nov
...


26
...
Nutritionally variant streptococci
...


27
...
Antimicrobial
susceptibilities of invasive pediatric Abiotrophia and Granulicatella isolates
...


28
...
Guidelines for the antibiotic treatment of
endocarditis in adults: report of the Working Party of the British Society for Antimicrobial
Chemotherapy
...


29
...
Endocarditis caused by
nutritionally variant streptococci: a case report and literature review
...


30
...
Streptococcus acidominimus infections in
a human
...


31
...
Brain abscess due to Streptococcus acidominimus: first case
report
...


32
...
Aerococcus urinae in urinary tract infections
...


33
...
Aerococcus urinaehominis sp
...
, isolated from
human urine
...


Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
14 | Page: 32 of 36
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms
34
...
Aerococcus sanguicola sp
...
, isolated
from a human clinical source
...


35
...
Aerococci and aeococcal infections
...


36
...
Antimicrobial susceptibilities and clinical sources of Facklamia species
...


37
...
Facklamia languida sp
...
, isolated
from human clinical specimens
...


38
...
Phenotypic and phylogenetic
characterization of some Globicatella-like organisms from human sources: description of
Facklamia hominis gen
...
, sp
...
Int J Syst Bacteriol 1997;47:880-2
...


Collins MD, Hutson RA, Falsen E, Sjoden B, Facklam RR
...

nov
...
J Clin Microbiol 1998;36:3090-3
...


Ulger-Toprak N, Summanen PH, Liu C, Rowlinson MC, Finegold SM
...
nov
...
Int J Syst Evol Microbiol 2010;60:1023-6
...


Collins MD, Hutson RA, Falsen E, Sjoden B, Facklam RR
...
nov
...
J Clin Microbiol 1998;36:1290-3
...


Elsayed S, Zhang K
...
J Clin Microbiol 2004;42:4897-900
...


Facklam R, Elliott JA
...
Clin Microbiol Rev 1995;8:47995
...


Collins MD, Aguirre M, Facklam RR, Shallcross J, Williams AM
...
nov
...
nov
...
J Appl Bacteriol
1992;73:433-7
...


Jain N, Mathur P, Misra MC
...
J Infect Dev Ctries 2012;6:592-4
...


Seegmuller I, van der Linden M, Heeg C, Reinert RR
...
J Clin Microbiol 2007;45:666-7
...


Collins MD, Facklam RR, Rodrigues UM, Ruoff KL
...
nov
...
nov
...


48
...
Prosthetic joint
infection due to "Helcococcus pyogenes" [corrected]
...


49
...
Helcococcus sueciensis sp
...
, isolated from a
human wound
...


50
...
Odontogenic infection secondary
to Leuconostoc species
...


51
...
Nosocomial Outbreaks
Caused by Leuconostoc mesenteroides subsp
...
Emerg Infect Dis 2008;14:968-71
...


Endo A, Okada S
...
nov
...
nov
...
nov
...
10
...
nov
...
nov
...

53
...
List of Prokaryotic names with standing in nomenclature
...


Facklam R, Hollis D, Collins MD
...
J Clin Microbiol 1989;27:724-30
...


Ikryannikova LN, Filimonova AV, Malakhova MV, Savinova T, Filimonova O, Ilina EN, et al
...
Clin Microbiol Infect 2012
...


Murray PR
...
Clin Microbiol Infect 2010;16:1626-30
...


European Parliament
...
The requirements for specimen containers are
given in the EU in vitro Diagnostic Medical Devices Directive (98/79/EC Annex 1 B 2
...
The manufacturing processes must be
appropriate for these purposes"
...


Official Journal of the European Communities
...
7-12-1998
...
1-37
...


Health and Safety Executive
...
9/99
...


Department for transport
...
2011
...


World Health Organization
...
2012
...


Home Office
...
2001 (as amended)
...


Advisory Committee on Dangerous Pathogens
...
Health
and Safety Executive
...
p
...


Advisory Committee on Dangerous Pathogens
...
Her
Majesty's Stationery Office
...


65
...
Biological agents: Managing the risks in
laboratories and healthcare premises
...
2005
...


Advisory Committee on Dangerous Pathogens
...
Appendix 1
...
Health and Safety Executive
...


67
...
Guidelines for Safe Work Practices in Human and
Animal Medical Diagnostic Laboratories
...


68
...
Control of Substances Hazardous to Health Regulations
...
5th ed
...


69
...
Five Steps to Risk Assessment: A Step by Step Guide to a Safer
and Healthier Workplace
...
2002
...
10
...


Health and Safety Executive
...
HSE Books
...


71
...
Safe Working and the Prevention of Infection in Clinical
Laboratories and Similar Facilities
...
2003
...


British Standards Institution (BSI)
...
2000
...


British Standards Institution (BSI)
...
Information to
be supplied by the purchaser and to the vendor and to the installer, and siting and use of
cabinets
...
24-3-2005
...
1-14

74
...
Laboratory-associated infections and biosafety
...


75
...
Infections in British clinical laboratories, 1984-5
...


76
...
Classification and overview of the genera Streptococcus and
Enterococcus
...


77
...
Reappraisal of the taxonomy of the
Streptococcus bovis/Streptococcus equinus complex and related species: description of
Streptococcus gallolyticus subsp
...
nov
...
gallolyticus subsp
...
nov
...
gallolyticus subsp
...
nov
...


78
...
Infections caused by viridans streptococci in patients with neutropenia
...


79
...
Streptococcus massiliensis sp
...
, isolated from a patient
blood culture
...


80
...
Streptococcus peroris sp
...
and Streptococcus infantis sp
...
, new members of the Streptococcus mitis group, isolated
from human clinical specimens
...


81
...
Streptococcus sinensis sp
...
, a novel species isolated from a patient with infective endocarditis
...


82
...
Human Infections Due to Streptococcus acidominimus
...


83
...
Granulicatella infection: diagnosis and
management
...


84
...
Characters of Gram Positive Bacteria
...
3rd ed
...
p
...


85
...
A serological differentiation of human and other groups of hemolytic streptococci
...


86
...
The formamide method for the extraction of polysaccharides from haemolytic
streptococci
...


87
...
Preparation of streptococcal extracts for Lancefield grouping
...


Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
14 | Page: 35 of 36
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England

Identification of Streptococcus species, Enterococcus species and Morphologically Similar
Organisms
88
...
Evaluation of species-specific PCR, Bruker MS,
VITEK MS and the VITEK 2 system for the identification of clinical Enterococcus isolates
...


89
...
Matrix-assisted laser desorption ionizationtime of flight mass spectrometry is a sensitive and specific method for identification of aerococci
...


90
...
Development of a
multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey
for virulence determinants among European hospital isolates of Enterococcus faecium
...


91
...
A multiplex PCR assay for
the direct identification of the capsular type (Ia to IX) of Streptococcus agalactiae
...


92
...
Sequential multiplex PCR approach for determining capsular serotypes
of Streptococcus pneumoniae isolates
...


93
...
Detection of glycopeptide resistance genotypes and
identification to the species level of clinically relevant enterococci by PCR
...


94
...
Sequencing emm-specific PCR products for routine and
accurate typing of group A streptococci
...


95
...
Pulsed-field gel electrophoresis as an epidemiological tool for enterococci
and streptococci
...


96
...
Recombination and the population structures of bacterial pathogens
...


97
...
Multilocus sequence
typing system for group B streptococcus
...


98
...
A multilocus sequence typing scheme for Streptococcus pneumoniae:
identification of clones associated with serious invasive disease
...


99
...
Multilocus sequence typing of
Streptococcus pyogenes and the relationships between emm type and clone
...


100
...
Laboratory Reporting to Public Health England: A Guide for Diagnostic
Laboratories
...
p
...

101
...
Health Protection Legislation (England) Guidance
...
p
...

102
...
Public Health (Scotland) Act
...

103
...
Public Health etc
...
Implementation of Part 2: Notifiable
Diseases, Organisms and Health Risk States
...

104
...
Health Protection Legislation (Wales) Guidance
...

105
...
Public Health Act (Northern Ireland) 1967 Chapter 36
...


Bacteriology – Identification | ID 4 | Issue no: 3 | Issue date: 28
...
14 | Page: 36 of 36
UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England



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