REVIEW ARTICLE
Gram-positive anaerobic cocci – commensals and opportunistic
pathogens
Elizabeth Carmel Murphy & Inga-Maria Frick
Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden
Correspondence: Elizabeth Murphy, BMC
B14, Tornavägen 10, Lund 221 84, Sweden.
Tel.: +46 46 2228168; fax: +46 46 157756;
e-mail: [email protected]
Received 27 February 2012; revised 30 July
2012; accepted 24 September 2012. Final
version published online 15 November 2012.
DOI: 10.1111/1574-6976.12005
Editor: Wilbert Bitter
MICROBIOLOGY REVIEWS
Keywords
Gram-positive anaerobic cocci; antibiotic
resistance; opportunistic pathogens;
taxonomic reclassification; skin infections;
Finegoldia magna.
Abstract
Among the Gram-positive anaerobic bacteria associated with clinical infections,
the Gram-positive anaerobic cocci (GPAC) are the most prominent and
account for approximately 25–30% of all isolated anaerobic bacteria from clinical specimens. Still, routine culture and identification of these slowly growing
anaerobes to the species level has been limited in the diagnostic laboratory,
mainly due to the requirement of prolonged incubation times and time-consuming phenotypic identification. In addition, GPAC are mostly isolated from
polymicrobial infections with known pathogens and therefore their relevance
has often been overlooked. However, through improvements in diagnostic and
in particular molecular techniques, the isolation and identification of individual
genera and species of GPAC associated with specific infections have been
enhanced. Furthermore, the taxonomy of GPAC has undergone considerable
changes over the years, mainly due to the development of molecular identification methods. Existing species have been renamed and novel species have been
added, resulting in changes of the nomenclature. As the abundance and significance of GPAC in clinical infections grow, knowledge of virulence factors and
antibiotic resistance patterns of different species becomes more important.
The present review describes recent advances of GPAC and what is known of
the biology and pathogenic effects of Anaerococcus, Finegoldia, Parvimonas,
Peptoniphilus and Peptostreptococcus, the most important GPAC genera isolated
from human infections.
Introduction
The normal microbiota that colonizes the skin and
mucosal surfaces of the human body consists of a plethora
of bacterial species of which anaerobic bacteria constitute
a large group. Usually, commensals do not breach these
protective barriers, but in case of a wound or when the
host becomes immuno-compromised, commensals and
pathogenic microorganisms can cause infection and disease. Among the Gram-positive anaerobic bacteria associated with clinical infections, the Gram-positive anaerobic
cocci (GPAC) are the most prominent. Of all isolated
anaerobic bacteria from clinical specimens, GPAC account
for approximately 25–30% (Murdoch, 1998; Boyanova
et al., 2004; Wildeboer-Veloo et al., 2007; Brazier et al.,
2008; Mikamo et al., 2011), see Fig. 1. In the literature,
GPAC in general have been described by various synonyms,
such as ‘anaerobic coccus’, ‘anaerobic streptococcus’,
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anaerobic Gram-positive coccus, and ‘Peptococcus and
Peptostreptococcus’. The term GPAC will be used in this
review. Constituting a major part of the normal microbiota, this heterogeneous group of bacteria colonise the skin
and mucosal surfaces of the mouth and upper respiratory
tract, the gastrointestinal tract and the female genitourinary tract (for references, see Murdoch, 1998). Clinically,
GPAC are often present in deep-seated anaerobic softtissue infections, infections of bones and joints and infections of the female genital tracts (Murdoch, 1998; Brazier
et al., 2008). Several previous studies have also reported
on the isolation of GPAC from wounds, both acute and
chronic wounds such as chronic ulcers (for references, see
Wall et al., 2002). For an overview of reported infections
associated with GPAC see Table 1.
Despite the fact that GPAC are frequently isolated from
infections involving anaerobic bacteria, the significance of
different isolates have not been well studied. The culture
FEMS Microbiol Rev 37 (2013) 520–553
521
Interactions of GPAC with the host
Fig. 1. Frequency of isolated anaerobic bacteria found in clinical
infection (1994–2004). Figure adapted from (Mikamo et al., 2011).
and identification of many GPAC strains in diagnostic laboratories remains difficult. The limitation of their isolation
is mainly due to their sensitivity to oxygen, which requires
appropriate methods of collection, transportation and
strictly anaerobic cultivation of specimens. Also, the slow
growth of these organisms combined with time-consuming
phenotypic identification methods have often resulted in
inconclusive identification of the GPAC species. In addition, GPAC are often isolated from polymicrobial infections with known pathogens and therefore, their relevance
has been largely overlooked. Furthermore, in clinical specimens, they have often been reported as anaerobic streptococci (Murdoch, 1998). However, the development and
application of molecular methods in clinical microbiology,
such as PCR (Lisby, 1998), multiplex PCR (Song et al.,
2003b), sequencing of the 16S rRNA gene (Li et al., 1994;
Conrads et al., 1997; Clarridge, 2004) and pyrosequencing
(Dowd et al., 2008a, b) have led to improved identification
of GPAC in clinical specimens, including chronic wounds
(Wolcott et al., 2009). It is thus evident that these bacteria
have been largely understudied and as the clinical significance of GPAC grows, it becomes essential to precisely
identify the isolated bacteria from clinical infections. Moreover, various genera and species of GPAC can express different virulence factors and they can also exhibit variations
in antimicrobial susceptibility emphasising the importance
of rapid and correct species identification in clinical samples. Thus, the introduction of newer diagnostic methods
may lead to improved treatment of GPAC infections. A
renewing interest in clinical microbiology to study anaerobes combined with the frequent isolation of GPAC from
FEMS Microbiol Rev 37 (2013) 520–553
clinical materials (Fig. 1 and Table 1) further emphasize
the need to study this important group of bacteria.
Data from molecular methods have led to extensive taxonomic changes during the last decades and also to the
occurrence of new genera and species. Currently, important genera of GPAC that may be isolated from humans
are Peptostreptococcus, Finegoldia (Murdoch & Shah,
1999), Parvimonas (Tindall & Euzeby, 2006), Anaerococcus
and Peptoniphilus (Ezaki et al., 2001). Other related GPAC
genera are Gallicola (Ezaki et al., 2001), Murdochiella
(Ulger-Toprak et al., 2010), Atopobium (Collins &
Wallbanks, 1992) and Anaerosphaera (Ueki et al., 2009).
In addition, genera such as Sarcina, Coprococcus and Blautia (previously Ruminococcus) (Ezaki et al., 1994; Liu et al.,
2008) are phylogenetically more distantly related GPAC.
This review describes what is known of the classification of
GPAC, clinical relevance of individual genera and species,
antibiotic resistance and a more in-depth description of
known virulence factors for some species that are more
commonly associated with clinical infections.
Classification
GPAC belongs to the Firmicutes phylum and are classified
in the order of Clostridiales having low DNA GC contents. Historically, the GPAC have undergone a considerable taxonomic revision. The genera Peptococcus and
Peptostreptococcus were originally classified on the basis of
morphological characteristics (Kluyver & van Niel, 1936);
peptococci were arranged in clusters and considered the
anaerobic equivalent of staphylococci, whereas peptostreptococci were arranged in long chains and thus
considered the anaerobic equivalent to streptococci. Peptococcus and Peptostreptococcus were for many years separated from each other by cellular arrangement, metabolic
end products, and utilisation of peptides and carbohydrates (Kluyver & van Niel, 1936; Rogosa, 1971, 1974;
Holdeman & Moore, 1974). Furthermore, they were
together with the genera Ruminococcus, Sarcina and
Coprococcus, assigned in the Peptococcaceae family of strict
anaerobic Gram-positive cocci or coccobacilli (Rogosa,
1971, 1974; Holdeman & Moore, 1974).
In 1983, Ezaki et al. reclassified the GPAC species on
the basis of DNA base composition, DNA–DNA hybridization data, the cellular fatty acid profiles and other
biochemical characteristics. As a result, four species of Peptococcus (Peptococcus asaccharolyticus, Peptococcus indolicus,
Peptococcus prevotii and Peptococcus magnus) were transferred to the genus Peptostreptococcus leaving Peptococcus
niger as the only remaining species in the genus Peptococcus. However, a later study using similar hybridization
techniques did not support this revision (Huss et al.,
1984), and in 1994, 16S rRNA sequence analysis
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Published by John Wiley & Sons Ltd. All rights reserved
522
E.C. Murphy & I.-M. Frick
Table 1. Overview of taxonomic changes in GPAC from 1997–2012
Genus
1997
(Murdoch, 1998)
2012
Peptococcus
P. niger
P. niger
Peptostreptococcus
P. anaerobius
P. anaerobius
P.
P.
P.
P.
asaccharolyticus
barnesae
harei
heliotrinreducens
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
hydrogenalis
indolicus
ivorii
lacrimalis
lactolyticus
magnus
micros
octavius
prevotii
tetradius
vaginalis
References
P. stomatis
New species proposed by Downes
and Wade (2006)
P. russellii
New species proposed by Whitehead
et al. (2011)
Clinical infections
Chronic wounds (Dowd et al.,
2008a)
Abscesses, infections of abdominal
cavity and female urogenitary
tract, pleural empyema chronic
wounds (for references see
Peptostreptococcus section)
Infections of oral cavity (Downes &
Wade, 2006; Rocas & Siqueira,
2008)
Reclassified to Slackia heliotrinreducens
in the family Coriobacteriaceae by
Wade et al. (1999)
Finegoldia
F. magna
P. magnus reclassified by Murdoch and
Shah (1999)
Micromonas
Parvimonas
P. micra
P. micros reclassified by Murdoch &
Shah, 1999; Renamed to Parvimonas
by Tindall and Euzeby (2006)
Anaerococcus
Reclassification to novel genus from
Peptostreptococcus by Ezaki et al. (2001)
A. hydrogenalis
A. lactolyticus
A. octavius
A. prevotii
A. tetradius
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Soft tissue and wound infections,
bone and joint infections,
vaginoses, chronic wounds, septic
arthritis, prosthetic valve
endocarditis, osteoarticular,
pleural empyema (for references
see Finegoldia section)
Oral infections, skin infections,
chronic wounds, joint infections,
abscesses, plueral empyema (for
references see Parvimonas section)
Vaginal discharges and ovarian
abscesses, skin and soft tissue
infections chronic wounds (for
references see Anaerococcus
section)
Urinary tract infections (Domann
et al., 2003), chronic ulcers (Dowd
et al., 2008a; Han et al., 2011)
Abscesses and vaginal infections
(Labutti et al., 2009), blood
infections (La Scola et al., 2011)
(Ezaki et al., 2001), pleural
empyema (Boyanova et al., 2004)
FEMS Microbiol Rev 37 (2013) 520–553
523
Interactions of GPAC with the host
Table 1. Continued
Genus
1997
(Murdoch, 1998)
2012
References
Clinical infections
A. vaginalis
A. murdochii
New species proposed by Song et al.
(2007b)
A. senegalensis
New species proposed by Lagier et al.
(2012)
Reclassification to novel genus from
Peptostreptococcus by Ezaki et al.
(2001)
Peptoniphilus
P. asaccharolyticus
P. harei
P. lacrimalis
P. indolicus
P. ivorii
P. gorbachii
P. olsenii
P. methioninivorax
P. tyrrelliae
P. coxii
P. duerdenii
P. koenoeneniae
Gallicola
G. barnesae
Murdochiella
M. asaccharolytica
Atopobium
A. parvulum
Anaerosphaera
FEMS Microbiol Rev 37 (2013) 520–553
New species proposed by Song et al.
(2007b)
New species proposed by Song et al.
(2007b)
New species proposed by Rooney et al.
(2011)
New species proposed by Citron et al.
(2012)
New species proposed by Citron et al.
(2012)
New species proposed by Ulger-Toprak
et al. (2012)
New species proposed by Ulger-Toprak
et al. (2012)
Reclassification to novel genus from
Peptostreptococcus by Ezaki et al.
(2001)
Genus proposed by Ulger-Toprak et al.
(2010)
A. parvulum
A. aminiphila
Chronic ulcers (Labutti et al.,
2009), blood infections
(La Scola et al., 2011)
Infected foot ulcers, soft tissue
infections, chronic wound
infection (Song et al., 2007b)
Chronic wounds, skin and soft
tissue, bone and genitourinary
tract, chronic rhinosinusitis (for
references see Peptoniphilus
section)
Osteoarticular samples (La Scola
et al., 2011), pleural empyema
(Boyanova et al., 2004)
Skin and soft tissue (Brazier et al.,
2008), pressure ulcers (Dowd
et al., 2008a), osteoarticular
samples (La Scola et al., 2011)
Vaginal infections (Murdoch,
1998), osteoarticular samples
(La Scola et al., 2011)
Pressure ulcers (Dowd et al.,
2008a)
Pressure ulcers (Dowd et al.,
2008a)
Low grade infections of lower
extremities (Song et al., 2007b)
Low grade infections of lower
extremities (Song et al., 2007b)
Leg infection, back cyst and
abscesses (Citron et al., 2012)
Leg infection, back cyst and
abscesses (Citron et al., 2012)
Wound infection (Ulger-Toprak
et al., 2012)
Wound infection (Ulger-Toprak
et al., 2012)
A case of abdominal wall abscess
and a case of sacral pilonidal cyst
(Ulger-Toprak et al., 2010)
Dental abscesses and abdominal
wounds (Olsen et al., 1991),
odontogenic infections (Downes
et al., 2001)
New genus proposed by Ueki et al.
(2009)
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524
E.C. Murphy & I.-M. Frick
Table 1. Continued
Genus
1997
(Murdoch, 1998)
Coprococcus
C. eutactus (T)
Sarcina
S. ventriculi
Ruminococcus
Blautia
S. maxima
R. productus
2012
References
Clinical infections
Intestinal GPAC rarely seen in
clinical infections (Maukonen
et al., 2012)
A case of emphysematous gastritis
(Laass et al., 2010)
B. producta
Proposed transfer of P. productus to
genus Ruminococcus by Ezaki et al.
(1994). Reclassified to a novel genus
Blautia by Liu et al. (2008)
confirmed that the genus Peptostreptococcus was phylogenetically disordered (Ezaki et al., 1994; Li et al., 1994),
thereby emphasising the need for a radical taxonomic
revision of the genus. In a comprehensive review of GPAC
by Murdoch in 1998, the taxonomic changes until 1997
were summerised and a possible revised classification was
discussed (Murdoch, 1998). The 1997 existing classification of the genera Peptococcus and Peptostreptococcus
(Murdoch, 1998) is shown in Table 1 together with an
overview of the taxonomic changes in GPAC up to 2012.
Since 1998, the genus Peptostreptococcus has been
divided into several novel genera. The type species of the
genus, Peptostreptococcus anaerobius, was found to be distantly related to other members of the genus, and thus a
division into six new groups was proposed (Murdoch &
Shah, 1999; Ezaki et al., 2001) (Table 1). Peptostreptococcus magnus and Peptostreptococcus micros were transferred
to two new genera, Finegoldia and Micromonas, respectively, where each strain is the only species in its respective genus (Murdoch & Shah, 1999). The genus name
Micromonas was found to be illegitimate, as Micromonas
are green algae, and has therefore, more recently been
replaced by Parvimonas (Tindall & Euzeby, 2006), with
Parvimonas micra being the only species present in this
genus. For the remaining peptostreptococci, three new
genera were proposed; Peptoniphilus, Anaerococcus and
Gallicola, which contains only one species, Gallicola
barnesae (Ezaki et al., 2001). Rajendram et al. (2001) proposed an alternative reclassification of the peptostreptococci, but in the current literature, the classification
according to Ezaki et al. (Ezaki et al., 2001) is used.
Other changes within the GPAC, as shown in Table 1,
include the reclassification of Ruminococcus productus to
Blautia producta (Liu et al., 2008) and the distantly
related Peptostreptococcus heliotrinreducans to the family
Coriobacteriaceae (Wade et al., 1999). Moreover, two
novel genera have been proposed; Anaerosphaera, with
the type species A. aminiphila most closely related to species of the genus Peptoniphilus (Ueki et al., 2009) and
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Intestinal GPAC rarely seen in
clinical infections (Maukonen et
al., 2012), a case of necrotising
fasciitis (Livaoglu et al., 2008)
Murdochiella with the type species M. asaccharolytica
most closely related to P. micra and Finegoldia magna
(Ulger-Toprak et al., 2010). For an overview of the
phylogenetic organisation within GPAC, see Fig. 2.
Fig. 2. Phylogenetic tree showing phylogenetic relationships within
GPAC. This tree was constructed by the neighbour-joining method,
by inputting 16S rRNA gene sequences into MacVector. Significant
bootstrap values, expressed as a percentage of 32 000 replications,
are indicated at branching points.
FEMS Microbiol Rev 37 (2013) 520–553
525
Interactions of GPAC with the host
Isolation and identification
Isolation of GPAC is usually performed on fastidious
anaerobe agar or blood agar anaerobically incubated
for 48 h or up to 7 days (Heginbothom et al., 1990;
Health-Protection-Agency, 2009). In most laboratories,
identification is phenotypically based on morphological
appearance, Gram’s stain reaction and sensitivity to metronidazole. However, resistance to metronidazole has
been reported for GPAC (Hecht, 2006) and such organisms may be overlooked by that approach. For classification to species or even genus level, further biochemical
identification tests, for instance inhibition by sodium
polyanethol sulphonate (SPS disc) (Graves et al., 1974),
pigment production, nitrate reduction, urease production, indole test and analysis of proteolytic enzyme profiles are required. Carbohydrate fermentation and
detection of volatile fatty acids by gas-liquid chromatography are other methods used for classification, but today
many laboratories do not have facilities to perform these
tests. In addition, identification and differentiation
between species by these conventional protocols are both
problematic and time-consuming. See Table 2 for an
overview of the biochemical characteristics of GPAC. In
the 1980s, a number of commercial biochemical assays,
such as RapID ANA (Innovative Diagnostic Systems,
Atlanta, GA), API 20A and AN-Ident (Analytlab Products, Plainview, NY), and Rapid ID 32A (bioMérieux,
Marcy l’Etoile, France), for detection of anaerobes, were
developed. These systems are based on the evaluation of
the action of a range of bacterial enzymes and other test
reactions.
With the introduction of new molecular approaches,
such as 16S rRNA gene sequencing and pyrosequencing,
analysis of bacterial composition in clinical samples without the need for culturing is allowed. 16S rRNA gene
sequencing represents an accurate method for both bacterial classification and identification, and through available
genotypic data, molecular techniques can be developed for
identification of GPAC. In a study by Song et al. (2003a),
sequence data for 13 type strains of GPAC from established species, including F. magna, P. micra, Peptoniphilus
harei and P. anaerobius, were determined. Based on these
data, a collection of clinical isolates previously identified
by phenotypic tests were reidentified by the use of 16S
rRNA gene sequencing. By this method, 84% of the clinical GPAC isolates were accurately identified to species level
(Song et al., 2003a). With assays such as 16S ribosomal
PCR (Riggio & Lennon, 2003), multiplex PCR (Song et al.,
2003b) and 16S rRNA gene-based fluorescent probes (Wildeboer-Veloo et al., 2007), rapid and reliable identification
of GPAC species is possible. Recently, a short biochemical
scheme was developed by Song et al. for simple identificaFEMS Microbiol Rev 37 (2013) 520–553
tion of GPAC in the clinical laboratory (Song et al.,
2007a). This scheme was based on the solid identification
of strains obtained from 16S rRNA gene sequencing (Song
et al., 2003a), and included both reference strains and
clinical isolates. Lin et al. (2010) developed an oligonucleotide array based on the 16S-23S rRNA intergenic
spacer region of clinically important anaerobes, including
Anaerococcus prevotii, Anaerococcus tetradius, F. magna,
Peptoniphilus asaccharolyticus, P. anaerobius and P. micra.
Reference strains and clinical isolates were identified by
the array and the sensitivity for identification of pure cultures was 99.7%, whereas the specificity was 97.1%.
Matrix-assisted laser desorption ionisation time-of-flight
mass spectrometry (MALDI-TOF MS) can also be utilised
for detection of proteins in bacteria isolated from clinical
specimens (Veloo et al., 2011c). By constructing a database, using commonly isolated strains of GPAC as reference, Veloo et al. could identify clinical isolates by
MALDI-TOF MS and results were then compared with
other identification methods. Of 107 unknown GPAC isolates, 96 could be identified with reliability. The other
strains (11/107) showed < 98% sequence similarities to
their closest reference strain, and therefore, it was concluded that these isolates probably represented a new species (Veloo et al., 2011c). With these methods, the
reliability of identification of slow-growing anaerobes will
most likely increase.
Clinical relevance
Apart from being major constituents of the normal anaerobic microbiota, GPAC are also considered as opportunistic pathogens. The introduction of foreign materials
(such as joint replacement, catheters, etc.), a growing
elderly population and number of immuno-compromised
individuals has contributed to a situation where the clinical significance of infections caused by opportunistic
pathogens has increased. Mostly, GPAC are isolated from
polymicrobial infections, but in many cases, the organisms are isolated in pure culture and these involve mainly
F. magna, although other species like P. micra, P. harei
and P. anaerobius also occur (Murdoch, 1998; WildeboerVeloo et al., 2007). The clinical importance of individual
GPAC species has not been extensively studied, probably
due to the mixed nature of infections and difficulties in
identifying many strains in the diagnostic laboratory. Special media and prolonged cultivation times are required
for isolation of anaerobes, thus culture results are delayed
and usually, treatment of the infection has already been
started. The polymicrobial nature of GPAC infections, in
addition to inadequate classification, has most likely contributed to the neglect of the clinical significance of individual species of GPAC.
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
ND
ND
A
A(B)
B
IC(IV)
ND
A
A
A
B
B
B
B
B
C
A/B
IV
B
C
A
A
B
B
+
+
+
+
v
/w
/w
/w
ALP
+
+
v
ADH
w
+
v
v
+
v
v
+
v
Indole
Production of*
+
+
+
v
Urease
+
+
aGAL
v
+
bGAL
+
v
+
+
+
+
aGLU
Saccharolytic enzymes*
+
+
bGUR
+
+
+
w
+
PheA
+/w
+/w
+
+
+
+
+
+
+
ProA
+
+
+
+
+
+
+
+
+
+
+
+
ArgA
Proteolytic enzymes*
/w
+/w
+
+
+
+
v
+
+
+
+
+
LeuA
w
+
+
+
+
w
PyrA
+
v
+
+
w
v
w
/w
w
w
+
TyrA
+
+
+
+
+
w
+
+/w
+
/w
+
w
+
HisA
+
+
+
+
+
+
+
/w
+
w
+
Glucose
ND
ND
+
ND
ND
ND
+
+
ND
Lactose
Carbohydrate
reactions†
v
+
+
+
+
v
+
+
w
Mannose
+
+
+
Raffinose
ND
ND
v
ND
ND
+
ND
+
ND
Ribose
fermentation
A, acetate; B, butyrate; IV, isovalerate; C, n-caproate; ALP, alkaline phosphatase; ADH, argininge dihydrolase; aGAL, a-galactosidase; bGAL, b-galactosidase; aGLU, a-glucosidase; bGUR, bglucuronidase; ArgA, arginine arylamidase (AMD); ProA, proline AMD; PheA, phenylalanine AMD; LeuA, leucine AMD; PyrA, pyroglutamyl AMD; TyrA, tyrosine AMD; HisA, histidine AMD;
, > 90% negative; w, weakly positive; +, > 90% positive; v, varied reactions; ND, not determined.
*Data on production of indole, urease, ALP, ADH, VFAs and saccharolytic and proteolytic enzymes taken mainly from Murdoch & Mitchelmore (1991), Murdoch (1998), Song et al. (2007a),
Citron et al. (2012), Downes and Wade (2006), Whitehead et al. (2011).
†
Data on carbohydrate fermentation reactions from Murdoch (1998), Holdeman et al. (1986), Murdoch et al. (1997), Citron et al. (2012), Downes and Wade (2006), Whitehead et al. (2011).
P. anaerobius (n = 63)
P. stomatis (n = 2)
P. russellii (n = 1)
Parvimonas micros (n = 31)
F. magna (n = 116)
A. prevotii (n = 1)
A. tetradius (n = 1)
A. lactolyticus (n = 1)
A. hydrogenalis (n = 14)
A. vaginalis (n = 29)
A. octavius (n = 6)
A. murdochii (n = 6)
Peptoniphilus ivorii (n = 4)
Peptoniphilus harei (n = 13)
Peptoniphilus niger (n = 1)
P. olsenii (n = 4)
P. gorbachii (n = 6)
Peptoniphilus indolicus (n = 6)
Peptoniphilus asaccharolyticus
(n = 52)
Peptoniphilus lacrimalis
(n = 1)
P. coxii (new) (n = 7)
P. tyrrelliae (n = 4)
B. producta (n = 1)
G. barnesae (n = 1)
Species (no. of strains
examined)
Terminal
major
VFA
Table 2. Biochemical characteristics of the majority of GPAC species
526
E.C. Murphy & I.-M. Frick
FEMS Microbiol Rev 37 (2013) 520–553
Interactions of GPAC with the host
A number of surveys have looked at the frequency of
anaerobes in clinical specimens and found that GPAC
constitute 24–31% of all anaerobic isolates (Holland et al.,
1977; Wren et al., 1977; Brook, 1988b; Murdoch et al.,
1994; Murdoch, 1998; Boyanova et al., 2004; WildeboerVeloo et al., 2007; Brazier et al., 2008; Mikamo et al.,
2011). They can be isolated from a wide variety of sites,
of which the dominating are abscesses and infections of
skin and soft tissue, mouth, bone and joint, upper respiratory and female genital tracts. In cases of pleural empyema, a life-threatening pleuropulmonary infection with
high mortality rate, P. micra has been the predominant
species, but also F. magna, P. anaerobius and Anaerococcus
vaginalis have been reported (Murdoch, 1998). When the
incidence of anaerobic bacteria in patients with pleural
empyema were investigated, GPAC were among the predominant anaerobic bacteria isolated from the infections
and found in 26.3% of all cases (Boyanova et al., 2004).
Among these, the incidences of P. micra, F. magna,
and P. anaerobius were similar, but P. asaccharolyticus,
A. tetradius and A. prevotii were detected as well (Boyanova et al., 2004). Other recent retrospective studies have
also reported on high incidences of GPAC isolated from
various clinical samples, such as infections of the abdominal cavity, skin, soft tissues, and bone (Mikamo et al.,
2011; Rodriguez-Cavallini et al., 2011). The frequency of
GPAC in relation to other anaerobic bacteria isolated
from clinical specimens is shown in Fig. 1.
A large part of human infectious diseases are comprised of chronic infections, including chronic wounds.
The colonisation of wounds often involves polymicrobial
biofilm communities and populating bacteria often
become resistant to many antibiotics. Numerous studies
have reported the recovery and isolation of GPAC from
both acute and chronic wounds (for references see Wall
et al., 2002). For instance, Bowler and Davies found that
more than 50% of anaerobes isolated from leg ulcers were
strains of GPAC belonging to the genus previously known
as Peptostreptococcus (Bowler & Davies, 1999a). More
recently, several reports have shed light about the bacterial profile associated with wound infections, and these
studies demonstrate that besides aerobic species, like
Staphylococcus spp., Streptococcus spp., Enterococcus spp.
and Pseudomonas aerugionosa, anaerobes, including
Peptoniphilus, Finegoldia and Anaerococcus are prominent
colonisers (Dowd et al., 2008a, b; Wolcott et al., 2009;
Gontcharova et al., 2010; Han et al., 2011). In these studies, molecular methods such as bacterial Tag-encoded
FLX amplicon pyrosequencing, 16S-based amplification
followed by pyrosequencing and shotgun Sanger sequencing were utilised.
When the bacterial diversity in chronic wound tissue
samples was examined using both standard culturing and
FEMS Microbiol Rev 37 (2013) 520–553
527
pyrosequencing, culturing revealed an average of three
common bacterial species in each wound. In contrast,
pyrosequencing revealed an average of 17 genera and
most of these were anaerobes, including Anaerococcus,
Finegoldia and Peptoniphilus (Han et al., 2011). Thus, it
can be concluded that the development of molecular
methods, such as bacterial Tag-encoded FLX amplicon
pyrosequencing, has lead to improved identification of
GPAC within various wound types. By symbiotically
existing together with aerobic colonisers, which use up
the oxygen, obligate anaerobes like GPAC may gain
advantages and also act synergistically in causing disease.
These bacteria and their metabolites may significantly
impair normal wound-healing processes, such as inflammation, remodelling of extracellular matrix and re-epithelialisation, and it is now evident that polymicrobial
biofilm communities constitute important barriers to the
healing of chronic wounds (Bjarnsholt et al., 2008). Considering that different virulence factors can be expressed
by various genera and species of GPAC (for instance
Finegoldia and Parvimonas, see below), a correct identification to the species level in clinical samples is important.
For clinicians, a better understanding of the bacterial
composition in a wound will naturally benefit the management of the particular wound.
A relative increase of GPAC in cases of anaerobic
bacteraemia has also been observed (Lassmann et al.,
2007), and by using 16S rRNA gene sequencing, it was
found that GPAC accounted for 7% of all anaerobic
bacteria isolated from bloodstream infections over a 5year period at Duke University Medical Center (Simmon
et al., 2008). Furthermore, in a recent study, anaerobes
isolated by routine culture of samples or biopsies
obtained from normally sterile sites, were identified
by the use of MALDI-TOF MS and 16S rRNA gene
sequencing (La Scola et al., 2011). By this method,
GPAC were identified in blood cultures and osteoarticular samples (La Scola et al., 2011). A case of bacteraemia
caused by Finegoldia was also recently reported (Rosenthal et al., 2012). Oral infections caused by GPAC genera, like Peptostreptococcus and Parvimonas have been
reported as well (described below). Also, Atopobium
spp., including Atopobium parvulum, occur in human
gingival crevices and may be isolated from dental
abscesses and abdominal wounds (Olsen et al., 1991).
Atopobium parvulum has not only been associated with
the saliva of healthy persons, but also with odontogenic
infections, such as dental implants (Downes et al.,
2001). In addition, members of A. parvulum are associated with patients suffering from halitosis (oral malodour) (Riggio et al., 2008).
An overview of infections associated with various GPAC
genera and species are shown in Table 1 and the clinical
ª 2012 Federation of European Microbiological Societies
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528
E.C. Murphy & I.-M. Frick
relevance of the genera Anaerococcus, Peptoniphilus,
Finegoldia, Peptostreptococcus and Parvimonas are
described in more detail below.
Antibiotic resistance
In general, GPAC have variable resistance to penicillins
(7–10%), clindamycin (7–20%), and metronidazole
(5–10%), whereas these bacteria are more susceptible to
b-lactam/b-lactamase inhibitors, cephalosporins, carbapenems, and chloramphenicol (Hecht, 2006). Also, resistance to tetracycline and erythromycin has been reported
(Brazier et al., 2003; Boyanova et al., 2004). Data describing differences in antimicrobial susceptibility between various species of GPAC are increasing (Bowker et al., 1996;
Brazier et al., 2003, 2008; Koeth et al., 2004; Roberts
et al., 2006; Könönen et al., 2007) and are described in
more detail for the major groups below.
Regarding the continuous rise in antibiotic resistance
amongst GPAC and anaerobes in general, more surveillance testing will be needed. Moreover, due to differences
in antibiotic susceptibility between GPAC species, it is
important to identify isolates in clinical specimens for
susceptibility testing to adapt the correct antibacterial
therapy.
Anaerococcus
Description and overview
The type species of the genus Anaerococcus is A. prevotii
(Ezaki et al., 2001) (see Fig. 3a). This strain was originally
designated as Micrococcus prevotii, then placed in the
genus Peptococcus (Foubert & Douglas, 1948), transferred
to the genus Peptostreptococcus in 1983 (Ezaki et al., 1983)
and finally to the genus Anaerococcus (Ezaki et al., 2001).
Anaerococcus prevotii and several other species have been
described, namely A. tetradius, Anaerococcus lactolyticus,
(a)
(b)
Fig. 3. Electron microscopy images of bacteria from the
Anaerococcus species. Scanning electron micrograph of (a)
Anaerococcus prevotii. Reproduced from (Labutti et al., 2009) (b)
Anaerococcus senegalensis. Reproduced from (Lagier et al., 2012).
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
Anaerococcus hydrogenalis, A. vaginalis, Anaerococcus
murdochii, Anaerococcus octavius and Anaerococcus
senegalensis (see Fig. 3b) (Ezaki et al., 2001; Song et al.,
2007a; Lagier et al., 2012). Cells occur in pairs, tetrads,
short chains or clumps and individual cells vary in size
from 0.6–0.9 lm in diameter, and on enriched blood agar,
colonies also vary in size (0.5–2 mm) (Labutti et al.,
2009). Peptones and amino acids are used as major energy
sources and butyrate is the major metabolite (Ezaki et al.,
2001). Most species are able to weakly ferment carbohydrates and are also indole-negative and coagulase-negative
(Ezaki et al., 2001).
The completed gene sequence of the type strain
A. prevotii PC1T, originally isolated from human plasma,
was recently published (Labutti et al., 2009). The genome
is 1 797 577 bp long (chromosome and one plasmid), has
an average G + C content of 35.6% and a total of 1913
open reading frames (ORFs). Of these, 1852 are proteincoding genes and 1399 of the genes have been assigned a
predicted function.
Clinical importance
Anaerococcus prevotii is frequently recovered from clinical specimens, such as vaginal discharges and ovarian,
peritoneal, sacral or lung abscesses (Labutti et al., 2009).
The species is also a common member of the normal
flora of skin, oral cavity and the gut (Ezaki et al.,
1983). In an rRNA gene-based study of the armpit
microbiota of healthy males, Anaerococcus spp. were
abundant (Egert et al., 2011). By using multiplex PCR,
16 out of 190 clinical isolates were identified as Anaerococcus spp., mainly A. vaginalis (Song et al., 2003b) and
in a European study on antimicrobial susceptibility
amongst 299 GPAC isolates, mainly from skin and
soft-tissue infections, 26 were identified as Anaerococcus
(Brazier et al., 2008). Using a culture-independent
molecular approach, A. lactolyticus was identified in urinary tract specimens in coinfection with both known
and unknown uropathogens (Domann et al., 2003). In a
recent survey of the bacterial diversity in biofilms of
various wound types, A. lactolyticus and A. vaginalis
were identified among the predominant species in
grouped samples of diabetic foot ulcers and pressure
ulcers using 16S rRNA gene-based molecular amplification followed by shotgun Sanger sequencing (Dowd
et al., 2008a). When the bacterial diversity in individual
chronic diabetic foot ulcers was investigated using a
pyrosequencing approach, Anaerococcus spp. were highly
prevalent and found in 22 of 40 samples (Dowd et al.,
2008b). Recently, A. vaginalis and A. prevotii were also
identified in blood cultures by mass spectrometry and
16S rRNA gene sequencing, (La Scola et al., 2011). In
FEMS Microbiol Rev 37 (2013) 520–553
529
Interactions of GPAC with the host
addition, five isolates of Anaerococcus were identified of
which at least two, based on sequence similarity with
known species, most likely belong to new species (La
Scola et al., 2011). Two of these isolates were obtained
from osteoarticular samples, one from cervical abscess
and the others from blood.
Antibiotic resistance
Anaerococcus prevotii is susceptible to penicillins (Murdoch, 1998) but resistant to SPS (Song et al., 2007a).
Brazier et al. (2003) also suggests that A. prevotii is resistant to tetracycline, erythromycin and clindamycin,
although the number of isolates in this study was very
low. Other studies have shown resistance of A. prevotii,
isolated from diabetic foot infections, to clindamycin,
levofloxacin and ceftazidine (Goldstein et al., 2006a;
Goldstein et al., 2006b). Clinical isolates of A. murdochii
(six strains) were reported to be resistant to colistin sulphate, two strains to kanamycin, one to clindamycin and
three showed intermediate resistance to penicillin (Song
et al., 2007b).
Peptoniphilus
Description and overview
The genus Peptoniphilus uses peptone as a major energy
source, butyrate is the major metabolic end-product and
carbohydrates are not fermented (Ezaki et al., 2001).
Cells vary in size depending upon species (from 0.5–
1.5 lm in diameter for P. harei), colonies are 1–2 mm,
circular, entire and opaque, the G+C content of DNA of
members of this genus is 30–34 mol% (Ezaki et al.,
1983). The type species is P. assacharolyticus, originally
classified in the genus Peptococcus, transferred to the
genus Peptostreptococcus in 1986 (Holdeman et al., 1986)
and finally reclassified to the genus Peptoniphilus (Ezaki
et al., 2001). However, the type strain of P. asaccharolyticus
(ATCC 14963) is not representative of the species and a
low DNA–DNA homology between clinical isolates
and the type strain was described. Thus, a number of
type strains were reidentified using 16S rRNA gene
sequencing and the closest relative for the strains was
P. harei (Veloo et al., 2011a). Peptoniphilus harei and
P. asaccharolyticus share the same biochemical features
and it was concluded that the isolates of P. asaccharolyticus
were misidentified. Veloo et al. (2011a) therefore suggested the incidence of P. asaccharolyticus in clinical
material to be highly overestimated. In a recent rRNAbased study of the armpit microbiota of healthy males,
Peptoniphilus spp. were also found to be abundant (Egert
et al., 2011).
FEMS Microbiol Rev 37 (2013) 520–553
Clinical relevance
With new molecular techniques like pyrosequencing, the
clinical importance of the genus Peptoniphilus has been
acknowledged. Several recent studies have found high frequency of Peptoniphilus spp. DNA within chronic wound
samples. For instance, Dowd et al. (2008a) found that
Peptoniphilus DNA comprises 38.4% of total sequences
within pressure ulcer samples, 7% in diabetic wounds,
but only 0.2% within venous leg ulcers. Dominating bacteria in pressure ulcers were Peptoniphilus ivorii, but also
high frequencies of P. harei and Peptoniphilus indolicus
were found (Dowd et al., 2008a). Other studies using pyrosequencing also report high prevalence of Peptoniphilus
spp. in diabetic ulcers (Dowd et al., 2008b; Gontcharova
et al., 2010). Recently, species of Peptonophilus, such as
P. harei, P. assacharolyticus and Peptoniphilus lacrimalis,
were also identified by mass spectrometry and 16S rRNA
gene sequencing in clinical material from osteoarticular
samples (La Scola et al., 2011). Other reported sites of
isolation of P. lacrimalis are from vaginal specimens and
discharge of the eye (Murdoch, 1998). Recently, two
novel species isolated from clinical specimens, including
leg infection, back cyst, and abscesses, were proposed,
Peptoniphilus coxii and Peptoniphilus tyrrelliae (Citron
et al., 2012). Also, two strains from human wound specimens were recently isolated and proposed to belong to
two novel species, Peptoniphilus duerdenii and Peptoniphilus koenoeneniae (Ulger-Toprak et al., 2012). Moreover, a
novel food-borne Peptoniphilus spp. was identified in a
study investigating microorganisms from retail ground
beef and was named Peptoniphilus methioninivorax (Rooney et al., 2011).
Interestingly, when the microbial flora was identified
with pyrosequencing in patients with chronic rhinosinusitis, anaerobic genera like Peptoniphilus predominated, in
contrast to conventional culturing methods, where mainly
Staphylococcus aureus and coagulase-negative Staphylococcus were detected (Stephenson et al., 2010). In a European
study on antimicrobial susceptibility among 299 GPAC
isolates, mainly from skin and soft-tissue infections, 70
isolates were identified as Peptoniphilus with P. harei being
the dominating species (Brazier et al., 2008). The clinical
importance of P. harei is further emphasised by Song et al.
(2003b) who identified 48 of 190 clinical isolates as
P. harei. A retrospective report on anaerobic isolates collected in Costa Rica between 1999 and 2008 revealed
approximately 60% Gram-positive bacteria and of these,
25% were cocci (Rodriguez-Cavallini et al., 2011). Species
were identified by the use of two commercial phenotypic
systems (RapID 32A and API 20A) and 12% were identified as Peptoniphilus spp. dominating in skin, soft tissue,
bone and genitourinary tract samples.
ª 2012 Federation of European Microbiological Societies
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530
Antibiotic resistance
A recent study reported that P. coxii strains were resistant
to doxycycline and 29% were resistant to moxifloxacin
and clindamycin, whereas all strains of P. tyrrelliae were
susceptible to doxycycline but resistant to moxifloxacin
and 25% to clindamycin (Citron et al., 2012). All strains
were susceptible to linezolid, metronidazole and penicillin
(Citron et al., 2012). In another study, the in vitro activity
of the broad-spectrum cephalosporin ceftobiprole was
compared with other antibiotics against 20 strains of
P. asaccharolyticus, isolated from diabetic foot infections
(Goldstein et al., 2006a). They were highly resistant to
levofloxacin and ceftazidine but sensitive to ceftobiprole.
Peptoniphilus harei has been reported as resistant to tetracycline, in contrast to P. lacrimalis and P. ivorii (Brazier
et al., 2003). Clindamycin resistance by strains of
P. asaccharolyticus has also been reported (Citron et al.,
2005; Goldstein et al., 2006b). Song et al. (2007a)
reported that Peptoniphilus gorbacchi showed resistance to
clindamycin (2 of 6 strains) and one strain showed intermediate resistance to penicillin.
Finegoldia
Description and overview
The genus Finegoldia is named after the American microbiologist S. M. Finegold and the type species is F. magna
(Murdoch & Shah, 1999). The original classification of
F. magna remains unclear, but it might have been
described first as Diplococcus magnus in 1933 (Prevot,
1933), and in 1974, given the name Peptococcus magnus
(Rogosa, 1974). The taxonomic revision by Ezaki and
coworkers transferred the species to the genus Peptostreptococcus (Ezaki et al., 1983). In 1999, Peptostreptococcus
E.C. Murphy & I.-M. Frick
magnus was reclassified in the current genus Finegoldia as
F. magna (Murdoch & Shah, 1999).
Finegoldia magna cells vary from 0.8 to 1.6 lm in
diameter and occur predominantly in masses but occasionally in pairs or short chains (see Fig. 4). The growth
rate in vitro is relatively slow. In liquid Todd-Hewitt
medium (supplemented with 0.5% Tween-80), the bacteria reach stationary phase after incubation for 70–90 h
(Karlsson et al., 2007). Following growth on enriched
blood agar for 2–5 days, colonies range 1–2 mm in diameter. The colour of the colonies is most frequently translucent, but can vary from white to grey and even yellow
(Murdoch & Mitchelmore, 1991; Murdoch, 1998).
Finegoldia magna is an anaerobic bacterium requiring an
oxygen-free environment for growth. However, F. magna
isolates on enriched blood agar plates that were exposed
to air still had some viable cells after 48 h, indicating that
resting cells may be relatively aerotolerant (Murdoch,
1998). Acetic acid is the major fermentation product and
most strains produce weak acid from fructose and only a
few strains from glucose (Ezaki et al., 1983; Murdoch,
1998). Instead peptones and amino acids can be used as
major energy sources. All strains produce ammonia from
glycine and most strains produce ammonia from threonine and serine (Ezaki et al., 1983). Aminopeptidase activities have been reported (Ng et al., 1998) and also catalase
activity (Murdoch & Mitchelmore, 1991; Krepel et al.,
1992). Coagulase, indole and urease are not formed, and
no strain reduces nitrate (Ezaki et al., 1983; Murdoch,
1998). For a summary of the biochemical features and
major characteristics of F. magna, see Table 2.
Genome description
The first complete genome sequence of F. magna strain
ATCC 29328, originally isolated from an abdominal
Fig. 4. Electron microscopy images of
Finegoldia magna ALB8. Left: Scanning
electron micrographs of F. magna strains ALB8
(top row) and 505 (non-FAF expressing strain)
(bottom row) Bar represents 10 lm. Right:
Transmission electron micrographs of same.
Bar represents 5 lm. Reproduced with
premission from (Frick et al., 2008).
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Rev 37 (2013) 520–553
Interactions of GPAC with the host
wound, was published in 2008 (Goto et al., 2008). The
genome consists of a circular chromosome (1.8 Mb) and
a plasmid pPEP1 (0.2 Mb) with a GC content of 32.3%
for the chromosome and 29.7% for the plasmid. A total
of 1813 ORFs were found, 1631 in the chromosome and
182 in the plasmid. Genomic analysis revealed that only
F. magna has a complete glycolysis pathway for fructose
in accordance with previous reports that most strains
produce weak acid from fructose and only few strains
from glucose (Ezaki et al., 1983; Murdoch, 1998). The
genome has many aminopeptidases and amino acid/oligopeptide transporters that may facilitate the uptake of
amino acids from the environment, thereby, amino acids
can be used as major energy sources. As compared to
other GPAC species, F. magna was reported to possess
more aminopeptidase activities (Ng et al., 1998), implicating a higher pathogenicity for this species. Other types
of transporters found include electron, ion, multidrugefflux and ATP-binding cassette transporters. Genes for
superoxide reductase, NADH oxidase and a putative
NADH dehydrogenase are also detected and these genes
may help F. magna to survive in aerobic conditions.
In the F. magna genome, four genes encoding the functional albumin-binding domain (GA module) are identified. The GA module is found in the Peptostreptococcal
Albumin Binding (PAB) protein, originally isolated from
F. magna in 1994 (de Château & Björck, 1994). The isolation and characterisation of protein PAB is described in
more detail in the following section. Also, collagen adhesion homologues, amidase homologues, a serine proteinase precursor and a putative biofilm-associated surface
protein were identified. Moreover, in silico analysis of the
genome identified eleven genes encoding sortases; four on
the chromosome and seven on the plasmid. Sortases are
extracellular transpeptidases, that catalyse the covalent
anchoring of proteins with LPXTG-like motifs to the
bacterial cell wall by cleaving the threonine and glycine
residues (Navarre & Schneewind, 1999; Novick, 2000;
Mazmanian et al., 2001). The presence of seven sortase
genes on the plasmid seems to be a unique feature of
F. magna, as searching of sortases in 29 plasmids of 14
Gram-positive bacterial species revealed only one sortase
gene present on a Clostridium perfringens plasmid (Goto
et al., 2008). Thus, the plasmid-encoded sortases in
F. magna might be of importance in terms of pathogenicity through enrichment of the variety of surface proteins
leading to enhancement of the bacterial interaction with
host tissues (Goto et al., 2008).
Clinical importance
Amongst the GPAC, F. magna is probably the most
pathogenic organism and is the species most frequently
FEMS Microbiol Rev 37 (2013) 520–553
531
isolated in pure culture from various clinical infection
sites (Bourgault et al., 1980; Murdoch, 1998). Typical
infections connected with F. magna are soft-tissue
abscesses, wound infections, bone and prosthetic joint
infections (Fitzgerald et al., 1982; Davies et al., 1988;
Murdoch, 1998; Brook & Frazier, 2000; Brazier et al.,
2008; Brook, 2008; Holst et al., 2008; Levy et al., 2009;
Martin et al., 2009). The bacterium has also been
described in septic arthritis (Fitzgerald et al., 1982; Hunter & Chow, 1988), nonpuerperal breast abscesses (Edmiston et al., 1990; Krepel et al., 1992; Castello et al., 2007)
and in vaginoses (Kastern et al., 1990; Ricci et al., 2001;
Aggarwal et al., 2003). In addition, rare cases of infectious
endocarditis on prosthetic valves (Cofsky & Seligman,
1985; Pouëdras et al., 1992; van der Vorm et al., 2000;
Bassetti et al., 2003; Fournier et al., 2008) and postoperative mediastinitis (Kernéis et al., 2009) caused by
F. magna have been described. In a clinical study, the
incidence of anaerobic bacteria in patients with pleural
empyema were analysed (Boyanova et al., 2004). GPAC
were found in 35.4% of the patients that were positive
for anaerobic bacteria (147 of 198 patients), and among
those F. magna accounted for 7.5%. Also a case of necrotising pneumonia caused by F. magna was recently
described (Sedano Gómez et al., 2011).
Most likely, the incidence of F. magna is highly underestimated due to problems of obtaining good quality
anaerobic clinical specimens. For instance, detection of
F. magna in blood cultures was found to be dependent
on the blood culture system used (Bassetti et al., 2003).
In this case of prosthetic valve endocarditis caused by
F. magna, several blood cultures incubated in BacT/
ALERT (BioMérieux) and BACTEC 9240 (Becton Dickinson) systems were negative despite growth of F. magna
from biopsies of the aortic wall of the patient (Bassetti
et al., 2003). Additional tests demonstrated that the isolated strain did grow in other blood culture systems like
SEPTI-CHEK BHI-S (Becton-Dickinson) and ISOLATOR
(Du Pont Co.) or in thioglycolate medium and on blood
agar (Bassetti et al., 2003). This is consistent with other
reports on prosthetic valve endocarditis caused by
F. magna, where the bacterium could only be detected in
cultures from the infected valve (Pouëdras et al., 1992;
van der Vorm et al., 2000). Thus, the relevance of
F. magna as the infectious agent in patients with apparent
culture-negative endocarditis has to be considered. Interestingly, by the use of molecular amplification in combination with traditional culturing of samples from
prosthetic joint infections, correct diagnoses was made by
PCR in cases where culturing was negative (Holst et al.,
2008; Levy et al., 2009). A more accurate detection and
identification of the bacteria might also lead to a changed
view of the frequency of F. magna in relation to certain
ª 2012 Federation of European Microbiological Societies
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532
clinical conditions. The prospect of giving the correct
antibiotic therapy will also be improved.
Finegoldia magna has been shown to be one of the
most common anaerobes isolated from skin specimens
(Higaki & Morohashi, 2003), and several surveys have
reported F. magna as highly prevalent in chronic wounds,
including diabetic ulcers and pressure ulcers (Hansson
et al., 1995; Stephens et al., 2003; Dowd et al., 2008a, b;
Gontcharova et al., 2010). In a study by Hansson et al.
(1995), F. magna was present in 29% of patients suffering
from venous leg ulcers (58 patients) without any clinical
sign of infection. In another study, the bacterial flora
was characterised in venous leg ulcers of 178 patients
during 12 weeks and 153 individual bacterial species
were identified. Finegoldia magna was among the most
frequently isolated species and was found in 21.4% of
the isolates (Moore et al., 2010). Furthermore, in samples from 40 diabetic foot ulcers, F. magna was present
in 23 of the samples (Dowd et al., 2008b). Recently, anaerobes isolated by routine culture from deep samples, were
identified by the use of MALDI-TOF MS and 16S rRNA
gene sequencing (La Scola et al., 2011). From a total
of 544 isolates, from various sampling sites, 332 isolates
(61%) were identified by MALDI-TOF MS whereas
the remaining 212 isolates (39%) could be identified
by 16S rRNA gene sequencing. Finegoldia magna was
amongst the most common anaerobes and mainly isolated
from osteoarticular samples (La Scola et al., 2011).
Finegoldia magna has recently been implicated in a case
of toxic shock syndrome (Rosenthal et al., 2012). The case
involved a fatal monomicrobial F. magna bacteraemia and
it was believed to be caused by the superantigen activity of
protein L, binding to the variable domain of the j light
chains of IgG. This report suggests that the overall significance of F. magna as a pathogen is underestimated and
that more sensitive detection methods will see it being
identified more frequently in clinical infections in the
future (Rosenthal et al., 2012).
Antibiotic resistance
In general, F. magna is susceptible to the antibiotics that
are used for treatment of anaerobic infections, but lower
antibiotic resistance rates (10–20%) to clindamycin, metronidazole, penicillin and higher resistance rates (> 20%)
to erythromycin and tetracycline have been reported
(Aldridge et al., 2001; Brazier et al., 2003, 2008; Martin
et al., 2009; Hawser, 2010). Various rates of resistance to
some fluoroquinolones (levofloxacin and moxifloxacin)
and cephalosporins (cefotaxime, cefepime and ceftazidime) have also been described (Goldstein et al., 2006a;
Veloo et al., 2011b). In the study by Veloo et al. (2011b),
F. magna showed the highest MIC50 (Minimum Inhibiª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
E.C. Murphy & I.-M. Frick
tory Concentration required to inhibit the growth of 50%
of the organisms) and MIC90 values for penicillin G,
amoxicillin–clavulanic acid, clindamycin, and tigecycline
and it also had the highest MIC90 values for levofloxacin
and moxifloxacin as compared to other GPAC, like
P. micra and P. harei, also tested. Furthermore, F. magna
isolates displaying various rates of resistance to erythromycin, azithromycin, ampicillin and levofloxacin were
found susceptible to telithromycin, a ketolide structurally
related to clarithromycin (Mikamo et al., 2003). Telithromycin was shown to exhibit a rapid and prolonged inhibitory activity against F. magna and other common
anaerobic and aerobic pathogens, suggesting a clinical use
in mixed respiratory infections (Stein et al., 2006).
Metronidazole, a 5-nitroimidazole, has been one of the
preferred antimicrobials for serious anaerobic infections;
however, resistance has been noted for some anaerobic
species, for instance Bacteroides fragilis, Clostridium spp.
and Peptostreptococcus spp. (Pankuch et al., 1993). Resistance mechanisms have not been conclusively identified,
but in Bacteroides the presence of nim genes encoding
nitroimidazole reductases has been implicated as a possible cause of resistance (Trinh & Reysset, 1996). Out of
nine tested F. magna strains, two were found highly resistant to metronidazole (MIC > 128 mg L 1), and also
possessed a nimB gene (Theron et al., 2004). The nimB
gene was however, also found in three of the seven susceptible F. magna strains, implying the possibility of a
silent nimB gene (Theron et al., 2004).
Virulence factors
The most well-described virulence factors of F. magna are
protein L (Björck, 1988), PAB (de Château & Björck,
1994), SufA (Karlsson et al., 2007) and FAF (Frick et al.,
2008) (Fig. 5). These four proteins are described in more
detail in the following section. Other virulent properties
have been described in F. magna. However, the proteins
responsible for these actions have not yet been identified.
Finegoldia magna strains with enhanced collagenase, gelatinase and hippurate hydrolase activity have been isolated
from nonpuerperal abscesses and diabetic foot infections
(Krepel et al., 1991, 1992). Furthermore, supernatant
from strains isolated from chronic leg ulcers inhibited
keratinocyte wound repopulation and endothelial tubule
formation in vitro (Stephens et al., 2003).
Finegoldia magna has recently been described to have
the ability to form biofilm (Donelli et al., 2012). It was
found to be strongly adherent and could develop as a
dual-species biofilm with both B. fragilis and Clostridium
difficile. The ability of F. magna to form a biofilm could
protect it from host immune defences, as well as targeted
antibiotic therapies (Donelli et al., 2012).
FEMS Microbiol Rev 37 (2013) 520–553
Interactions of GPAC with the host
533
Fig. 5. Overview of Finegoldia magna surface
proteins and SufA and their effects on the
host during infection.
Protein L
Protein L – an F. magna surface protein with
affinity for Ig L chains
Protein L (76–106 kDa depending on the strain from
which it is isolated) was initially isolated from F. magna
strain 312 and could not be identified in other investigated strains (Björck, 1988). In binding experiments,
125
I-radiolabelled protein L was able to bind IgG, F(ab’)2
fragments and Fab fragments of IgG, j and k L chains, IgM
and IgA. In binding assays with human plasma, it binds
exclusively and highly specifically with human Ig L chains
(Björck, 1988). Protein L binds to IgM, IgA and IgG with
an equilibrium constant of approximately 1010 M 1 for
each, indicating a similar binding to all three immunoglobulin classes (Åkerstrom & Björck, 1989).
Protein L interacts with the light chain of Ig through
the j variable domain (Nilson et al., 1992). The ability
of protein L to bind Vj domains makes it of potential
value in the isolation of antigen-binding VL domains and
Fv fragments prepared from monoclonal antibodies
(Nilson et al., 1992).
Structure of protein L and interaction with
domains of Ig
Protein L was the first gene in F. magna to be sequenced.
Its basic structure bears both similarities and differences
with cell-wall proteins of Gram-positive bacteria. Its signal sequence is considerably shorter, consisting of 18
amino acid residues compared to at least 33 in most
other Gram-positive cell-wall proteins (see schematic representation Fig. 6a). Following the signal sequence, there
is region A which consists of 79 residues. The j-binding
FEMS Microbiol Rev 37 (2013) 520–553
property was mapped to the B repeats, which consist of
72–76 amino acid residues each (Kastern et al., 1992).
This is analogous to protein A and protein G whose IgG
Fc-binding activity is also located in repeat units (Sjödahl, 1977; Fahnestock et al., 1986; Guss et al., 1986).
The j-binding B repeats of protein L shared no homology with Ig-binding domains of any other bacterial protein, which is expected, due to its unique specificity for
Ig light chains. Protein L also contains two twin-C
repeats (52 amino acid residues each) with unknown
function and shares no homology with protein or gene
sequences in major databanks. Following on from the C
repeats comes the cell wall-spanning region (W), an
LPXTG-type motif and a hydrophobic membrane-spanning region (Fischetti et al., 1990; Kastern et al., 1992)
(Fig. 6a).
The three dimensional structure of the 76 amino acid
residue domain B1 of protein L was resolved using NMR
spectroscopy and distance geometry-restrained stimulated
annealing. It comprises a 15-amino acid residue disordered NH2-terminus followed by an a-helix packed
against a four-stranded b-sheet. It shares very limited
sequence homology with the protein G domains interacting with the heavy chains of IgG (Wikström et al., 1994).
However, despite their different binding properties, the
B1 domain and the IgG-binding protein G domain
have similar folds (see Fig. 6c) (Wikström et al., 1993).
The B1 domain of protein L is thought to interact
with the human Ig j chain through most of the residues
in b-sheet 2, the COOH-terminal residues of the
a-helix and the loop connecting the a-helix with the
third b-strand (Wikström et al., 1995).
The crystal structure of the complex between a human
Ig Fab fragment and a single domain of protein L was
refined to 2.7 Å (Graille et al., 2001). The protein
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
534
E.C. Murphy & I.-M. Frick
(a)
(b)
(c)
L domain used in the study, C* (61 residues), has the
same three-dimensional structure as domain B1 as determined by NMR (Wikström et al., 1994) and the two
structures superimpose with an rmsd of 1.31 Å over 59
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
Fig. 6. (a) Overview of domain layout in
Finegoldia magna proteins. S, signal peptide;
W, wall spanning region; M, membrane
spanning region; S8, peptidase S8 domain; PA,
protease associated domain; DUF, domain of
unknown function; Ala, alanine rich region. (b)
Structure showing interaction of protein L C*
domain (same three dimensional structure as
domain B1) with two human IgM Fab 2A2
domains. The protein L domain (yellow) is
sandwiched between and interacting with the
light chains of both Fab domains (light blue).
Structure was built using the 1HEZ pdb file
(Graille et al., 2001). This image was made
with VMD. VMD is developed with NIH
support by the Theoretical and Computational
Biophysics group at the Beckman Institute,
University of Illinois at Urbana-Champaign. (c)
Comparison of protein L (PpL) with protein G
(SpG) fold. The b sheets are orientated to
show the structural similarities in both
domains. The b2 sheet of both domains
interacts with an Fab VL (protein L) or an Fab
CH1 (protein G) b sheet, through a b zipper
interaction. Coloured lines represent hydrogen
bonds between the main-chain atoms. Figure
reproduced with permission from (Graille
et al., 2001)
residues (Graille et al., 2001). The crystal structure
revealed that C* has two separate regions that can interact with j-light chains (see Fig. 6b). Therefore, it can
bind two Fab fragments simultaneously, involving similar
FEMS Microbiol Rev 37 (2013) 520–553
Interactions of GPAC with the host
sites on the VL domains of the j-chain, but with markedly different affinities. The possibility of a single protein
L domain interacting with two VL regions suggests that
protein L could bridge two Ig molecules anchored at the
membrane of B cells (Graille et al., 2001).
Protein L as a tool for antibody detection
Two proteins widely used for binding and detection of
IgG antibodies are protein A from staphylococcal strains
(Forsgren & Sjöquist, 1966) and protein G from group C
and G streptococci (Björck & Kronvall, 1984; Reis et al.,
1984). These proteins that have been well-characterised
have affinity for the Fc part of mammalian IgG. They display similar physiochemical properties and are fibrous,
elongated, monomeric proteins with several binding sites
for IgG (Åkerstrom & Björck, 1986). Protein G binds to
all human IgG subclasses and has a higher affinity for
human IgG than protein A (Langone, 1982; Åkerstrom &
Björck, 1986). However, the ability of protein L to bind
to the light chain of Ig means that it can bind all classes
of Ig with a j-light chain. Therefore, protein L represents
a new tool for the binding and detection of antibodies
due to its broader Ig-binding spectrum (Åkerstrom &
Björck, 1989). Furthermore, as protein L does not bind
bovine immunoglobulins (Château et al., 1993), the protein provides a convenient way to purify j-light chaincontaining monoclonal antibodies produced from culture
supernatant or ascites where foetal calf serum is used.
Biological implications for protein L – Ig light
chain interactions
By expressing protein L on the surface of some strains,
F. magna is able to bind different classes of immunoglobulin on its cell surface with high affinity during infection.
As a result, the bacterium could influence cellular and
molecular events (Kastern et al., 1990). Although most of
protein L is bound at the bacterial surface, some of it is
also released into the medium during growth (Björck,
1988; Kastern et al., 1990). In a study, Kastern et al.
found a significant correlation between expression of protein L and F. magna isolated from bacterial vaginosis
(Kastern et al., 1990). Protein L and Protein L-expressing
bacteria were found to induce an in vitro mediator release
from human basophils and mast cells from lung and skin
tissues (Patella et al., 1990), most likely through an interaction with IgE on the surface of the cells. Mediator
release was dose- dependent with histamine secretion
gradually increasing with increase in bacterial concentration (Patella et al., 1990).
Protein L had a further effect on human basophils
through inducing the de novo synthesis of leukotriene C4
FEMS Microbiol Rev 37 (2013) 520–553
535
(LTC4) (Patella et al., 1990). LTC4 is a proinflammatory
chemical mediator that possesses many biological and
vasoactive properties in humans (Samuelsson et al.,
1987; Marone et al., 1988). In human skin mast cells
(HSMC), protein L induced a greater histamine release
than anti-IgE, whereas protein A and protein G failed to
have any effect. In addition, protein L also induced the
de novo synthesis of the chemical mediator, prostaglandin D2 (PGD2), from HSMC. Both LTC4 and PGD2
have significant biological importance in inflammation,
suggesting that protein L-expressing F. magna could have
increased virulence during infection. In the same study
by Patella et al. (1990), intradermal injection of protein
L induced a wheal-and-flare reaction in nonallergic subjects. These results abide by previous findings that
expression of protein L is correlated with virulence
(Kastern et al., 1990).
To investigate the virulent effects of protein L in infection, the human oral commensal, Streptococcus gordonii
GP1291, was used to construct a recombinant bacterium
expressing four of the Ig-binding domains of protein L
(B1–B4) (Ricci et al., 2001). As a control, a GP1292 strain
was used which expressed another M6-based fusion protein. After 6 weeks, 33.3% of the mice inoculated with
GP1291 were still colonised, as compared to 5.5% of
GP1292 inoculated mice. Furthermore, in the murine
vagina, S. gordonii GP1291 persisted for 14 weeks, whereas
GP1292 persisted for 8 weeks. These results suggest that
Ig-binding domains of protein L on the surface of
S. gordonii enhance the duration of vaginal colonisation
(Ricci et al., 2001). Ricci et al. hypothesise that the effect
seen could be due to protein L facilitating the adherence
of F. magna to the vaginal mucosa through interaction
with surface associated IgA during oestrus, as S. gordonii
colonisation occurs during this time. However, during
proestrus, colonisation is at its lowest and IgG and IgA
reach their highest concentrations in vaginal fluid. During
this time, protein L could mainly interfere with the
defence functions of soluble Ig and thus, prevent bacterial
clearance (Ricci et al., 2001).
Another biological function for protein L is that it is a
potent inducer of the synthesis and release of IL-4 and
IL-13 from human FceRI+ cells (human basophils and
mast cells expressing the FceRI high affinity receptor for
IgE) (Genovese et al., 2003). This activity classifies protein L as a bacterial superantigen due to its ability to
induce the release of two cytokines critical for Th2 polarisation from human FceRI+ cells. This action is mediated
by binding to the j-chains of IgE present on human
basophils (Genovese et al., 2003). A recent study by
Nunomura et al. (2012) suggests that cell-surface FceRI
expression is an important participant in protein L-mediated full activation of mast cells. Engagement of FceRI
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
536
with the j-chains of IgE and protein L induces tyrosine
phosphorylation of ITAM in the FcRb- and c-signalling
subunits leading to intracellular signalling and the release
of proinflammatory mediators such as TNF-a and LTC4.
Both TNF-a and LTC4 play important roles in adaptive
immunity (Nunomura et al., 2012). The ability of protein
L to induce de novo synthesis of immunoregulatory cytokines has an important relevance as to the virulence of
protein L expressing F. magna strains. Furthermore, protein L appears to be the first bacterial protein capable of
inducing de novo synthesis and release of IL-4 and IL-13
from basophils (Genovese et al., 2003). During infection,
this could be both beneficial and harmful to the bacterium. Inflammation leads to an increase in vascular permeability causing an influx of nutrient-rich plasma.
However, inflammation also induces the production of
many antimicrobial peptides and chemokines. Nevertheless, if the bacterium is capable of counteracting these
mechanisms more effectively than other bacterial species
in the same locality, this will create a selective advantage
(Åkerstrom & Björck, 1989).
To investigate in vivo trafficking of protein L, Smith
et al. (2004a) used whole body autoradiography in a
murine model system. Analysis showed that protein L
primarily targets secondary lymphoid organs, the spleen
and lymph nodes. The main target of protein L was the
white pulp of the spleen, which is composed of highly
organised lymphoid tissue containing T and B lymphocytes (Picker & Siegelman, 1999). Therefore, protein L
preferentially targets cells expressing surface Ig or cells
that can interact with Ig, which agrees with previous
studies. The major cell population targeted by protein L
was B lymphocyte B220+ cells, whose interaction was
rapid and transient (Smith et al., 2004a). This targeting is
most likely mediated through direct interaction of protein
L to surface Ig on B cells. In addition, this interaction in
vivo resulted in B-cell activation as splenic B cells showed
upregulation of MHC-II and CD86 after administration
of protein L (Smith et al., 2004a). The ability of protein
L to target a specific cell population in vivo could lead to
the development of new therapeutic disease treatments.
Further investigations in the effects of protein L on
B-cell subpopulations discovered that in a mature pool of
B cells, addition of protein L caused a reduction of splenic marginal zone B cells and peritoneal B-1 cells (Viau
et al., 2004). These two subsets of B cells are important
in innate B-cell immunity and induce the rapid clearance
of pathogens. Therefore, F. magna may use protein L as a
tool to subvert the first line of the host’s immune defence
(Viau et al., 2004).
Apart from the B1–B5 repeats which have been shown
to bind to human Ig L chains, a function has also been
determined for the A domain. The A domain is the outª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
E.C. Murphy & I.-M. Frick
ermost domain of protein L and a short segment (< 10
aa) most proximal to the B1 domain was found to have
high affinity for the neutrophil cytosolic proteins S100A8
and S100A9 (Åkerstrom & Björck, 2009). S100A8 and
S100A9 make up almost 40% of the total protein cytosolic content of neutrophils and monocytes (Odink et al.,
1987). At certain Ca levels, the two proteins form the
tetramer, (S100A8/A9)2, known as calprotectin, which has
the ability to inhibit the growth of various bacterial and
fungal species (Steinbakk et al., 1990). S100A8/A9 was
found to kill F. magna 505 (a strain not expressing protein L), but not F. magna strain 312, which further suggests that resistance to killing is due to surface protein L
(Åkerstrom & Björck, 2009). The resistance is thought to
be due to protein L forming a barrier by binding the
neutrophil proteins at a distance from the cell surface
(Åkerstrom & Björck, 2009). S100A8/A9 action on
F. magna 505 was seen at pH 5.5 and not pH 7.5 which
is relevant to where F. magna are most commonly
isolated – the skin, mucosal surfaces in the vagina and in
mouth and tissue abscesses (Rentzsch & Wilke, 1970;
Bryant et al., 1980; Kastern et al., 1990; Grinstein et al.,
1991; Ohman & Vahlquist, 1994; Robinson et al., 2002).
In addition, an acidic pH is found in tissues and wound
fluids during inflammation and infection (Johne et al.,
1997).
PAB – peptostreptococcal albumin binding
protein
Protein PAB: mosaic organisation and a product
of intergenic interspecies recombination of a
functional domain
Protein PAB is a human serum albumin (HSA)-binding
protein isolated from a HSA-binding strain of F. magna
(de Château & Björck, 1994) (see schematic representation Fig. 6a). It could be purified from F. magna ALB8
from mutanolysin-released cell-surface proteins and from
the growth medium. Protein PAB is composed of 387
amino acids and harbours an amino acid stretch comprised of 45 amino acids sharing 60% homology with the
repeated albumin-binding sequence of protein G of group
C and G streptococci. This section is known as the GA
module in protein PAB and it corresponds to the HSAbinding domain of the protein (protein G-related albumin-binding module) (de Château & Björck, 1994). PAB
contains no repeated domains, in contrast to proteins L
and G and most other surface proteins of Gram-positive
bacteria. When the amino acid sequence of the GA
module is lined up with two HSA-binding domains from
protein DG12 from a bovine group G streptococcal
strain, three HSA-binding protein G domains of the G148
FEMS Microbiol Rev 37 (2013) 520–553
537
Interactions of GPAC with the host
strain and protein PAB from F. magna ALB1, there is a
high degree of homology and 14 conserved residues.
Hence, this module appears to have been transferred
across species borders from one prokaryotic gene to
another, possibly originating in group C or G streptococcus and transferred to F. magna through the pCF10 conjugated plasmid from Enterococcus faecalis (de Château &
Björck, 1994). See Fig. 7a for a model of GA module
shuffling between different bacteria.
Structure of the GA module
The GA module consists of 45 amino acid residues and
was the first example of module shuffling in prokaryotes.
Prokaryotes lack introns, making exon or module shuffling less probable. However, the gene sequences of bacterial HSA-binding domains were analysed with regard to
their global fold and this led to the identification of
‘recer’ sequences (de Château & Björck, 1996). Recer
sequences act as structureless spacer sequences of 15 nucleotides in length flanking the different modules, that
promote flexibility and interdomain in-frame recombination in the corresponding protein (de Château & Björck,
1996). The presence of recer sequences in HSA-binding
domains of bacterial proteins could help explain how the
GA module was incorporated into protein PAB. These
recer sequences are quite rare and after their identification, they have not been found on a wide scale in other
genomes.
Protein PAB contains two GA modules – GA and uGA
– which share 38% identity and provide two binding sites
(a)
for HSA (de Château & Björck, 1996). The uGA domain
has a much weaker binding affinity for HSA, if the same
binding site for HSA on different GA modules is
assumed (de Château et al., 1996). uGA is present on
protein PAB’s predecessor – protein urPAB, which
contains uGA, but not the GA module (de Château &
Björck, 1996).
Initial studies on the structure of the GA module in
F. magna ALB8 using NMR spectroscopy showed that it
consisted of a three-helix-bundle with a left-handed folding topology (Johansson et al., 1995). The presence of
similar folds in different proteins with different proteinbinding capabilities suggests that this is an energetically
favourable fold that is conserved during evolution
(Johansson et al., 1995). Further NMR studies on the
structure of the same GA module using NMR spectroscopy agreed with the structural prediction produced in
1995 (Johansson et al., 1997). The region responsible for
binding to HSA was predicted to be the NH2-terminal
well-conserved region of the GA module (Johansson
et al., 1997).
The crystal structure of the GA module in complex
with HSA was resolved at 2.7 Å (pdb code 1tf0) (Lejon
et al., 2004). The GA module binds to HSA at a site in
domain II of the albumin molecule (see Fig. 7b). In the
GA module, the region involved in binding was discovered
to be the residues from the second helix and the two
loops surrounding it (Lejon et al., 2004). The interaction
between Phe-27 in the GA module and Met-329 in HSA
was found to be crucial for the hydrophobic interaction.
Binding of HSA by protein PAB in F. magna appears to
(b)
Fig. 7. (a) Possible modes of shuffling of the GA module to form the pab gene in Finegoldia magna. Putative methods of uptake of the GA
module include: (1) direct uptake of GA module fragment from cell debris and subsequent homologous/nonhomologous recombination with
F. magna chromosome. (2) Release of pCF10 from E. faecalis into cell debris from group G streptococcus, recombination with the GA module
and transformation into F. magna. (3) Conjugational plasmid pCF10 is transferred by conjugation from E. faecalis to group C or G streptococcus,
homologous recombination with the protein G gene (spg) on the chromosome and finally, conjugation and homologous recombination with
F. magna. Figure adapted from (de Château & Björck, 1994) (b) Structure of the HSA-GA complex, showing the GA module binding to a novel
site on albumin. Structure was built using the 1TF0 pdb file (Lejon et al., 2004). This image was made with VMD. VMD is developed with NIH
support by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign.
FEMS Microbiol Rev 37 (2013) 520–553
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
538
enhance it’s pathogenicity as protein PAB-expressing
strains were mostly isolated from patients with localised
suppurative infections. Binding of HSA could provide the
bacteria with fatty acids and other nutrients carried by
HSA, causing faster growth rates at an infection site
(Lejon et al., 2004). The biological implications of HSA
binding will be discussed in more detail in the next
section.
Biological functions and clinical implications of
protein PAB
Protein PAB and PAB-expressing F. magna were found to
bind HSA with high specificity (de Château et al., 1996).
In a study of 30 isolates from localised suppurative infections (abscesses, soft-tissue and wound infections), 16
were found to bind significant amounts of HSA. Vaginosis and commensal isolates showed no affinity for HSA
(de Château et al., 1996). These results suggest a link
between the albumin binding phenotype and suppurative
infections. Therefore, protein PAB plays an important
role in enhancing bacterial virulence during infection.
Furthermore, strains found to express protein PAB did
not express Ig light-chain binding protein L (de Château
et al., 1996).
Studies carried out by de Château et al. (1996) showed
that by the addition of HSA to the growth medium, the
growth rates and maximum cell densities of HSA-binding
strains of F. magna were significantly increased. This was
not the case for nonbinding strains of F. magna. The reason for this growth increase is thought to be the access of
the bacteria to diverse ligands carried by HSA. HSA is a
major transporter of long and short-chain fatty acids,
tryptophan, thyroxine, calcium ions, etc. By binding HSA
to the bacterial surface through protein PAB, these
ligands are made available to the bacteria. Moore et al.
(1977) have shown that F. magna grows better in the
presence of Tween 80, a detergent containing an oleic
acid tail. Free fatty acids (FFAs) are present at a concentration of 0.5 mM in human plasma and 99% of it is
tightly bound to the FFA-binding sites of HSA. This is
due to FFAs having a rapid turnover, t1/2, of 2 min in
human plasma. HSA is present extravascularly in most
tissues and in inflammatory secretions at mucosal surfaces. Therefore, HSA-binding F. magna will have ample
access to HSA and its ligands. This will give the bacteria
selective advantages and faster growth rates when
establishing infection.
In a recent study by Egesten et al. (2011), binding of
HSA by the GA module was found to have a function in
addition to nutrient access and fast growth. Group G
streptococci, carrying the HSA-binding protein G, bound
HSA from both saliva and plasma, and the bound HSA
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
E.C. Murphy & I.-M. Frick
inactivated the antibacterial peptide, MIG/CXCL9. This
protects the bacterium from harmful antibacterial peptides that are released during bacterial invasion, through
a protein G-dependent HSA coating (Egesten et al.,
2011). This could also be a survival strategy used by
F. magna to survive on an activated epithelial surface, via
a protein PAB-dependent HSA coating.
The GA module of protein PAB was found to bind
HSA with much higher affinity than its predecessor,
G148-GA3, reflecting the power of bacterial evolution
(Johansson et al., 2002). Worryingly, PAB-expressing
F. magna strains were found to be tetracycline- resistant,
suggesting that antibiotics are providing the selective
pressure behind module shuffling (Johansson et al.,
2002). If this trend continues, F. magna could turn from
being a member of the normal bacterial flora into a
potential pathogen. Finegoldia magna’s current role as a
commensal and opportunistic pathogen means it utilises
every opportunity to establish infection. Continued selective pressure through the use of antibiotics could mean
that F. magna acquires more virulent traits, making
infections more serious and difficult to treat.
SufA – subtilase of F. magna
SufA is a subtilisin-like proteinase of F. magna that was
found to have a wide range of functions, which could
result in enhanced pathogenicity of F. magna during
infection. SufA was initially identified as a papain-released
bacterial surface protein with a putative molecular mass
of 127 kDa (Karlsson et al., 2007). The putative locations
of the catalytic triad are Asp181, His247 and Ser578. The
consensus pattern and order of this catalytic triad is consistent with peptidases of the S8 family – subtilisin family
(Siezen et al., 1991; Siezen & Leunissen, 1997). See Fig. 6a
for an illustrated domain layout of SufA.
Investigations were carried out to determine the capability of SufA to degrade antibacterial peptides, as this
appears to be an important characteristic of proteinases
of major human pathogens (Schmidtchen et al., 2002;
Sieprawska-Lupa et al., 2004). SufA was shown to completely degrade the antibacterial peptide, LL-37, during an
incubation of 1–3 h (Karlsson et al., 2007). LL-37 is a
human cathelicidin, which kills target microorganisms by
disrupting membrane integrity (Turner et al., 1998). SufA
was also capable of degrading the chemokine MIG/
CXCL9 (Monokine Induced by Gamma-interferon) (Cole
et al., 2001; Egesten et al., 2007) into small fragments.
SufA cleavage of LL-37 and MIG/CXCL9 resulted in
enhanced survival of F. magna during antibacterial assays
with these peptides (Karlsson et al., 2007).
SufA was found to be unable to cleave antibacterial
peptides of the defensin family, possibly due the presence
FEMS Microbiol Rev 37 (2013) 520–553
539
Interactions of GPAC with the host
of cysteines in the defensin family, which protect the peptides from SufA proteolysis (Karlsson et al., 2007). LL-37,
on the other hand, is a linear a-helical peptide without
any cysteines (Bals & Wilson, 2003) and can be completely degraded by SufA. In addition, the COOH-terminal end of MIG/CXCL9 where SufA is predicted to cleave
has been reported to have an a-helical structure (Egesten
et al., 2007). The ability of SufA to cleave and inactivate
certain antimicrobial peptides could lead to enhanced
survival and proliferation during infection. This theory
was further investigated by a study carried out by Frick
et al. (2011) using the antibacterial proteins Midkine
(MK) and BRAK/CXCL14, which are both expressed constitutively in the epidermal layer of skin. Here, SufA-generated fragments of MK and BRAK/CXCL14, after a 1 h
digestion, were found to still efficiently kill the virulent
human pathogen, Streptococcus pyogenes. Finegoldia
magna was also killed, but to a much lesser extent (Frick
et al., 2011). This could provide selective advantages to
F. magna during the early stages of infection.
Further studies on SufA interaction with MIG/CXCL9
showed that SufA was able to modulate the chemokine’s activities to promote bacterial survival during epithelial inflammation. MIG/CXCL9 is an ELR-negative
CXC- chemokine and is produced by human keratinocytes in response to inflammatory stimuli, e.g. the cytokine IFN-c (Liao et al., 1995). In IFN-c-stimulated
keratinocytes, F. magna failed to increase production of
MIG/CXCL9, in sharp contrast to S. pyogenes (Karlsson
et al., 2009a). This may be due to PAMPs on the surface of
F. magna being less exposed, resulting in a decreased
inflammatory response and allowing F. magna to survive
as a commensal.
MIG/CXCL9 exerts its bactericidal activity through
membrane perturbation and translocation through the
bacterial membrane into the cytoplasm resulting in possible inhibition of essential enzymatic activities (Karlsson
et al., 2009b). SufA-degraded fragments of MIG/CXCL9
still retained the ability to enter the cytoplasm of S. pyogenes, but not that of F. magna. This may be explained, in
part, by the fact that SufA-processed MIG/CXCL9 loses
its ability to form dimers, which is essential for the activity of many chemokines (Proudfoot et al., 2003; Campanella et al., 2006). In addition, differences in cell-wall
architecture and membrane composition between the two
bacterial species, may explain their stark difference in susceptibility to the processed chemokine (Karlsson et al.,
2009b). As shall be discussed in the following section,
FAF, the adhesion protein of F. magna, when released
from the bacterial surface by SufA, is capable of binding
and neutralising the effect of both MIG/CXCL9 and
LL-37 in the growth medium (Frick et al., 2008). Therefore, SufA and FAF appear to play a dual role in protectFEMS Microbiol Rev 37 (2013) 520–553
ing F. magna from antibacterial peptides present in the
environment.
The role that SufA plays in the interaction with the host
coagulation system was also explored. Many pathogenic
bacteria manipulate the host coagulation system through
proteolytic degradation or binding of its components
(Sun, 2006). SufA was found to specifically and rapidly
cleave fibrinogen, by removing the COOH-terminal part
of the fibrinogen Aa chains (aC) (Karlsson et al., 2009b).
Further processing by SufA results in an attack on the
NH2-terminal part of the Bb chains and further processing
of the Aa chains. The aC chains are important for lateral
fibril associations and clot formation and stabilisation.
SufA-treated plasma showed an increased thrombin clotting time (TCT assay) and this could be explained by the
removal of the aC chains (Karlsson et al., 2009b). The
cleavage of fibrinogen by SufA could not only affect fibrin
polymerisation and clotting but also some of the other
important functions of fibrinogen, such as wound healing.
During wound healing, the fibrin network provides a temporary matrix into which cells can proliferate (Drew et al.,
2001; Laurens et al., 2006). SufA inhibition of this network could significantly delay wound repair.
SufA was shown to prevent the formation of a fibrin
network around F. magna which had adhered to human
keratinocytes (Karlsson et al., 2009b). On the other hand,
an intact fibrin network was formed around F. magna
bacteria, from which the SufA gene had been knocked
out. These results suggest that cleavage of fibrinogen by
SufA prevents the formation of a fibrin network around
F. magna that have adhered to human keratinocytes. This
could prevent the bacteria from becoming trapped and
facilitate the establishment of infection, promoting virulence (Karlsson et al., 2009a). In addition, it could add
selective advantages to the bacteria as a member of the
normal flora.
FAF – F. magna adhesion factor
FAF is an alpha helical coiled coil protein that mediates
bacterial aggregation by interacting with FAF molecules
on neighbouring F. magna bacteria (see Fig. 6a for a
domain layout). The structure and location of FAF and
the virulent M proteins of S. pyogenes are highly similar,
suggesting that both of these proteins share comparable
functions. In addition, both proteins are released from
the bacterial surface. FAF is released in the form of a
53 kDa fragment by the subtilisin-like protease, SufA,
which was described in the previous section (Frick et al.,
2008). FAF is present at the surface of most F. magna
strains; however, the gene is not identical. There was
found to be strain-to-strain sequence variation in FAF,
mainly in the NH2-terminal part.
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
540
Studies on the biological function of FAF are still at a
preliminary stage. However, initial studies have identified
ligands for FAF. FAF was found to bind to BM-40, a
noncollagenous glycoprotein, which is a linking molecule
in the basement membrane of skin. The binding site
region for BM-40 was localised to the COOH-terminal
end of the molecule, which is conserved amongst FAF
homologues (Frick et al., 2008). This sequence conservation suggests an evolutionary pressure to maintain this
region of the FAF molecule, signifying the importance for
a FAF-BM40 interaction in infection. The biological
significance of this interaction was further illustrated by
the coincubation of F. magna bacteria with human skin
biopsies, which showed bacteria present at the basement
membrane through the colocalisation of gold-labelled
FAF and BM-40 (Frick et al., 2008). Interestingly, most of
the FAF-expressing strains in this study (20 of 28) were
also found to express PAB, the albumin-binding surface
protein. As previously described, binding of albumin
promotes growth of F. magna and BM-40 has been
reported to increase albumin transport across the epithelium (Goldblum et al., 1994). Therefore, BM-40 bound
by FAF could influence transport and as a result, increase
bacterial multiplication. BM-40 has also been found in
soluble form in wound fluid and has been shown to
stimulate wound healing and cell proliferation (Brekken
& Sage, 2000). Therefore, through its interaction with
BM-40, both soluble FAF and FAF-expressing F. magna
could impair wound healing in chronic wounds, which
would explain why these bacteria are so effective at
causing infection in this niche.
LL-37 was identified as another ligand for FAF and both
the NH2- and COOH-terminal regions of FAF were
required for full binding. LL-37 was shown to be able to
effectively kill a non-FAF expressing strain, but, addition
of exogenous FAF significantly lowered killing (Frick
et al., 2008). FAF-expressing F. manga were found to be
much more resistant to killing from LL-37 than non-FAF
expressing bacteria. FAF was also found to have a neutralising capacity on other bactericidal proteins – MK, BRAK/
CXCL14 and hBD-3 – in a dose-dependent manner (Frick
et al., 2011). Interestingly, hBD-3, which is resistant to
SufA cleavage, was the peptide most efficiently neutralised
by FAF. The ability of F. magna to effectively block the
activity of antimicrobial peptides could help it to survive
as a commensal at the skin epidermis (Frick et al., 2011).
Large amounts of FAF were found to be released from
F. magna and this exogenous FAF could act as a protective barrier around the bacteria during infection (Frick
et al., 2008). FAF appears to significantly affect the interaction between the bacteria and the host and helps in
understanding how F. magna is able to colonise and
survive in the human host.
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
E.C. Murphy & I.-M. Frick
Peptostreptococcus
Description and overview
The genus Peptostreptococcus used to be genetically and
phenotypically heterogeneous, but has undergone extensive taxonomic change in the last decade. A number of
new genera have been proposed to account for the many
differences in fundamental characteristics between the
species, including Finegoldia, Parvimonas, Gallicola,
Peptoniphilus and Anaerococcus (Murdoch & Shah, 1999;
Ezaki et al., 2001; Tindall & Euzeby, 2006). The
genus now consists of just three species – the type species
P. anaerobius and the recently identified Peptostreptococcus
stomatis and Peptostreptococcus russellii (Downes & Wade,
2006; Whitehead et al., 2011). Peptostreptococcus anaerobius have a cell morphology, which is usually coccobacillary
with a diameter of 0.5–0.7 lm and occurs in short chains
(Holdeman et al., 1986; Murdoch & Mitchelmore, 1991).
Colonies are grey with a slightly raised off-white centre
and grow on enriched blood agar more rapidly than other
GPAC species, giving off a distinctive, sickly sweet odour
(Murdoch & Mitchelmore, 1991). It is found in the
gastrointestinal and vaginal flora (Neut et al., 1985;
Holdeman et al., 1986; Hillier et al., 1993).
Peptostreptococcus stomatis cells (0.8 9 0.8–0.9 lm) are
arranged in pairs or short chains. Their colonies have a
diameter of 0.8–1.8 mm and are circular, high convex to
pyramidal in the centre, opaque, shiny and cream to offwhite with a narrow, grey peripheral outer ring in colour
(Downes & Wade, 2006). Peptostreptococcus stomatis grow
moderately in broth media and growth can be enhanced
through the addition of fermentable carbohydrates. Cells
ferment fructose, glucose and maltose weakly and are
mildly saccharolytic. End products of metabolism include
major amounts of acetic and isocaproic acids, minor
amounts of isobutyric and isovaleric acids and trace to
minor amounts of butyric acid. It has a G + C content of
36 mol% (Downes & Wade, 2006). It is a member of the
oral commensal flora and probably accounts for isolates
that were previously thought to be P. anaerobius, as it is
now hypothesised that P. anaerobius is not, in fact, an
oral commensal (Downes & Wade, 2006).
Peptostreptococcus russellii was isolated from a swine
manure storage pit and cells (0.8–1 lm) occur in pairs
and chains of 3–10 cells. Colonies are 2–3 mm in diameter, convex, opaque, smooth and whitish in colour
(Whitehead et al., 2011). Cells are weakly saccharolytic
and the major end-product of glucose metabolism is acetate. Copious amounts of ammonia are produced
(> 40 mM) from various nitrogen sources. The type
strain is RT-10BT and has a G + C content of 35.6 mol%
(Whitehead et al., 2011).
FEMS Microbiol Rev 37 (2013) 520–553
541
Interactions of GPAC with the host
Clinical importance
Peptostreptococcus anaerobius is one of the most common
GPAC associated with infections of the abdominal cavity
and female urogenitary tract (Brook, 1988b, 1989a;
Murdoch et al., 1994). Infections of the female urogenitary tract include bacterial vaginosis, which is a polymicrobial syndrome, where Lactobacillus populations are
replaced by a mixture of bacteria including P. anaerobius, Gram-negative anaerobic rods such as Prevotella spp.,
the facultative Gardnerella vaginalis and the genital
mycoplasmas Mycoplasma hominis and Ureaplasma urealyticum (Hill, 1993). Pybus et al. discovered that P. anaerobius was unable to grow in vaginal defined medium
that had not been conditioned by peptone-supplementation through prior incubation with Prevotella bivia.
Prevotella bivia culture supernatants have a net accumulation of amino acids which supported the growth of
P. anaerobius. This commensal symbiosis is thought to
exist in bacterial vaginoses whereby Prevotella bivia supports the growth of P. anaerobius (Pybus & Onderdonk,
1998).
It has also been isolated from abscesses from a wide
range of human clinical specimens including the brain,
ear, jaw, pleural cavity, blood, spinal and joint fluid and
pelvic, urogenital and abdominal regions (Holdeman
et al., 1986). It is mostly associated with mixed infection
sites but there have been some reports of isolation from
pure culture (Brook & Frazier, 1993; Montejo et al.,
1995).
It was also isolated in pure culture from a case of orbital cellulitis, suggesting it has the potential to cause severe
orbital disease (Malik et al., 2004). Peptostreptococcus anaerobius has recently been associated with some cases of
infective endocarditis, however, the occurrence is rare and
there is a more favourable prognosis than with patients
with infective endocarditis from other anaerobic bacteria
(Minces et al., 2010; Wu et al., 2011). It has been isolated
from some cases of skin infection, in association with
aerobic bacteria, such as S. aureus (Higaki et al., 2000).
It has also been reported to be isolated from a range of
acute and chronic wounds (Sanderson et al., 1979; Bowler
& Davies, 1999b) including dermal ulcers (Alper et al.,
1983), leg ulcers (Bowler & Davies, 1999a), post-thoractomy
sternal wound (Brook, 1989b), chronic venous leg ulcers
(Gilchrist & Reed, 1989; Hansson et al., 1995; Brook &
Frazier, 1998), nonpuerperal breast infection (Edmiston
et al., 1990), diabetic foot ulcers (Johnson et al., 1995),
burn wound infections (Mousa, 1997), surgical wounds
(Steffen & Hentges, 1981) and diabetic foot infections
(Wheat et al., 1986).
Peptostreptococcus stomatis is associated with infections
of the human oral cavity, such as dentoalveolar abscesses
FEMS Microbiol Rev 37 (2013) 520–553
and endodontic infections (Downes & Wade, 2006).
However, it is only a recently described species, so there
are limited clinical data available. As mentioned in the
previous section, oral infections reported to be caused by
P. anaerobius were probably incorrectly identified and
were in fact caused by P. stomatis. It was detected in
one-quarter of the cases from a large number of necrotic
root canal samples and was one of the most dominant
taxa in some of the communities (Rocas & Siqueira,
2008).
Antibiotic resistance
Antibiotic susceptibility data of Peptostreptococcus are not
widely available. However, a recent study looked at the
antibiotic susceptibilities of 61 isolates of P. anaerobius
and P. stomatis against amoxicillin, amoxicillin-clavulanic
acid, cefoxitin, ertapenem, azithromycin, clindamycin,
metronidazole and moxifloxacin (Könönen et al., 2007).
The isolates originated from various clinical specimens
from anatomical sites such as ulcer and skin specimens,
pus specimens of the genitourinary tract and oropharyngeal and gastrointestinal specimens. Peptostreptococcus
stomatis was found to be susceptible to all antibiotics with
a slightly increased resistance to clindamycin. Thirteen
per cent of P. anaerobius isolates displayed intermediateto-resistant MICs to one or more drugs including amoxicillin, amoxicillin-clavulanic acid, cefoxitin and moxifloxacin (Könönen et al., 2007). This study also found most
P. anaerobius isolates to be from infection sites of the
lower extremities and genitourinary tract, whereas, most
P. stomatis isolates were from oral, pharyngeal and gastrointestinal specimens. These findings are in line with
the studies of Downes and Wade and Riggio and Lennon,
suggesting that P. anaerobius is not involved in colonisation/infection of the oral and pharyngeal areas (Riggio &
Lennon, 2003; Downes & Wade, 2006). Metronidazole
proved to be one of the most effective antibiotics against
both Peptostreptococcus spp.. However, the nitroimidazole
resistance gene – nimB – has been found in 31% of
P. anaerobius strains in another study (Theron et al.,
2004). Therefore, metronidazole resistance in Peptostreptococcus will have to be monitored in the future.
Peptostreptococcus anaerobius has also been shown to
be extremely susceptible to a new antibiotic, oritavancin,
which is being developed for infections caused by
vancomycin-susceptible and -resistant organisms (Citron
et al., 2005). It is a glycopeptide antibiotic that inhibits
bacterial cell-wall formation through blocking the transglycosylation step in peptidoglycan hydrolysis. It was
found to be twofold more active than vancomycin
against eleven strains of P. anaerobius (Citron et al.,
2005).
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
542
Parvimonas
Description and overview
The Parvimonas genus contains just one species – P. micra
– which has undergone much reclassification in recent
times. Originally classified as Peptostreptococcus micros, it
was transferred to the Micromonas genus in 1999 by Murdoch and Shah and known as Micromonas micros (Murdoch & Shah, 1999). The Micromonas genus was replaced
by Parvimonas by Tindall and Euzeby in 2006, with P. micra as it’s sole species (Tindall & Euzeby, 2006).
Parvimonas micra cells (0.3 –0.7 lm) are usually
arranged in pairs and chains (Holdeman et al., 1986).
Their colonies have a diameter of 1 mm and are usually
white in colour, domed, glistening and typically surrounded by a yellow-brown halo of discoloured agar up
to 2 mm wide on enriched blood agar plates (Murdoch
et al., 1988; Murdoch & Mitchelmore, 1991; van Dalen
et al., 1993). There is both a rough and a smooth
morphotype of P. micra, which differ with regard to
the presence of fibrillar structures on the cell wall,
hydrophobic activity, the ability to lyse erythrocytes and
the composition of cell-wall proteins (van Dalen et al.,
1993). The smooth morphotype appear as small, domed,
bright white, nonhaemolytic colonies and the rough morphotype appear as white, dry, haemolytic colonies with
wrinkled edges and long, thin fibrillar structures outside
the cell envelope. Repeated subculturing in broth results
in the rough morphotype changing into the smooth colony morphology with no fibrillar structures (van Dalen
et al., 1993).
Clinical importance
Parvimonas micra is part of the normal commensal flora
of the gastrointestinal tract (Finegold et al., 1974; Bartlett, 1990) and the gingival crevice (Holdeman et al.,
1986; Rams et al., 1992). With regard to its role in clinical infections, it is mainly recognised as an oral pathogen and is particularly isolated from polymicrobial
infections such as periodontitis. Periodontitis is caused
by a group of bacteria and results in destruction of the
periodontal tissues (Socransky et al., 1999). Recent
advances in bacterial identification, such as microarray
and 16S rRNA gene sequencing have confirmed the association of P. micra with periodontal infections. Nonnenmacher et al. targeted the 16S rRNA gene sequences
of a range of periodontal pathogens using real-time PCR
from 50 subgingival plaque samples from periodontitis
patients and 33 from periodontally healthy subjects. They
found higher counts of P. micra in samples from periodontitis patients (Nonnenmacher et al., 2004). A microª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
E.C. Murphy & I.-M. Frick
array study carried out by Vianna et al. (2005) looked at
the microbial composition of necrotic root canals from
20 patients and compared it with methods of culture
identification. From the culture-based identification,
P. micra was present in 10% of samples. However, using
microarray, P. micra was found in 50% of samples and
was the most prevalent organism detected (Vianna et al.,
2005). This study shows that the prevalence of P. micra
in oral infections could have previously been underestimated due to the use of culture-based techniques for
identification. A nucleic acid-based technique will provide a more comprehensive picture of bacteria associated
with necrotic teeth.
Parvimonas micra has been identified as a prominent
oral pathogen in endoperiodontal lesions, apical abscesses
and periodontitis in many recent studies (Lee et al., 2003;
Fritschi et al., 2008; Siqueira et al., 2009; Rocas et al.,
2011; Didilescu et al., 2012). Rocas and Siqueira (2008)
identified P. micra in 28% of samples from infected root
canals of 43 teeth with chronic apical periodontitis.
Another study found a positive association between
P. micra and severe gingival overgrowth in patients taking
cyclosporin A following organ transplantation, where
P. micra was found in 66% of subgingival samples
(Romito et al., 2004). Parvimonas micra was found to be
present in 5.9% of isolates in a study looking at the profiling of the microbiota in infected root canals (Sato
et al., 2012). Sato et al. used restriction fragment length
polymorphism analysis of PCR-amplified 16S ribosomal
RNA genes and sequencing, for identification of live
bacterial cells.
It has also been implicated in infection in other parts
of the body. It has been reported to have been isolated
from a range of skin infections including chronic wounds
(Bowler & Davies, 1999b), leg ulcers (Bowler & Davies,
1999a), nonpuerperal breast infection (Edmiston et al.,
1990), burn wound infections (Mousa, 1997), acute (surgical) wounds (Sanderson et al., 1979) and diabetic foot
infections (Wheat et al., 1986). Riesbeck and Sanzen
(1999) identified P. micra as the causative microorganism
in a case of destructive knee joint infection with rapid
progress of cartilage destruction. Bartz et al. (2005) found
P. micra to be the causative microorganism in a prosthetic joint infection of the hip, which was linked to a
tooth extraction. A study by Lafaurie et al. (2007) looked
at the frequency of periodontic and subgingival anaerobic
and facultative bacteria in the bloodstream following
scaling and root planing as a result of preventative dental
procedures and periodontal therapy. They found that
P. micra was one of the most frequently identified periodontopathogens in peripheric blood. It was still present in
the bloodstream, in some cases, 30 min after the dental
procedure. As the capability of neutralising a bacterial
FEMS Microbiol Rev 37 (2013) 520–553
Interactions of GPAC with the host
threat in the bloodstream varies between patients, this
may represent a risk factor for developing remote infections (Lafaurie et al., 2007). Studies by Murdoch et al.
(1988, 1994) isolated P. micra from abscesses from
numerous infection sites, including soft-tissue abscesses,
plueral empyemas, anorectal abscess and from an intrauterine contraceptive device and patients with chronic
sinusitis. They found that it usually occurred in a particular flora at the infection site which consisted of microaerophilic streptococci, Fusobacterium spp. and Bacteroides
spp. (Murdoch et al., 1988, 1994).
Antibiotic resistance
Overall, P. micra is highly susceptible to antibiotics (Aldridge et al., 1983, 2001; Mitchelmore et al., 1995; Bowker
et al., 1996). A recent study looked at the antimicrobial
susceptibility of GPAC of clinical strains isolated in 10
European countries, found P. micra to be present in
17.7% of isolates (53 strains) (Brazier et al., 2008). All
P. micra strains were found to be susceptible to imipenem, metroniazole, vancomycin, linezolid and metronidazole. Two strains were found to be resistant to either
penicillin or clindamycin. The penicillin-resistant strain
did not produce b-lactamase, so penicillin resistance
could be due to modifications in the penicillin-binding
proteins (Reig & Baquero, 1994). Clindamycin resistance
is caused by an RNA methylase that modifies the site of
action of the antibiotic (Garcia-Rodriguez et al., 1995).
A study by Veloo et al. (2011) looked at the
antimicrobial susceptibilities of 115 isolates of clinically relevant GPAC over a 3-year period in the Netherlands. One
strain of P. micra was identified that was resistant to
metronidazole, but no significant resistances to any other
antibiotics were found (Veloo et al., 2011). Rams et al.
looked at spiramycin, amoxicillin and metronidazole resistance in human periodontitis microbiota from 37 patients
with untreated severe periodontitis (Rams et al., 2011). A
10.8% of P. micra isolates were found to be resistant to spiramycin, 2.7% to amoxicillin and none to metronidazole.
Whereas antibiotic resistance in P. micra is not yet a significant issue when treating infection, monitoring through
continuous antimicrobial susceptibility testing still seems
highly justified.
Virulence factors
Parvimonas micra is one of the best studied of the GPAC
in terms of characterisation of its virulence factors. The
demonstration of synergy with facultative and anaerobic
bacteria during the growth of abscesses, capsule formation
and the ability to form hydrogen sulphide from glutathione have been described as important virulence factors
FEMS Microbiol Rev 37 (2013) 520–553
543
(Brook & Walker, 1985; Brook, 1987, 1988a; Carlsson
et al., 1993). Furthermore, it has been described to adhere
to gingival epithelial cells (Dzink et al., 1989) and to
express immunoglobulin Fc-binding proteins (Grenier &
Michaud, 1994). As described earlier, P. micra cells exist
in either a rough or a smooth morphotype, with the
rough morphotype having fibrillar material on their surface (van Dalen et al., 1993). A study by van Dalen et al.
(1998) looking at the pathogenicity of the two different
morphotypes, found that abscesses induced by the rough
morphotype were slightly, but significantly larger. The
same study also looked at virulence of both morphotypes
in a mixed infection with two Prevotella spp. and found
that the most effective combination in terms of transmissibility of infection, bacterial counts from pus and larger
abscesses produced, was the rough morphotype of
P. micra with Prevotella intermedia. They also discovered
that the rough morphotype had an increased interaction
with polymorphonuclear leukocytes in the absence of
serum and that this could be due to the increased
amount of fibrillar material on the surface of the bacteria
(van Dalen et al., 1998).
Kremer et al. (1999a) looked at the function of the
fibrillar surface appendages in adherence to epithelial
cells. They discovered that adherence of the rough morphotype was significantly lower than the smooth and the
smooth variant of the rough type. Thus, the fibril-like
surface structures appear to have an obstructive effect on
adherence. This difference was abolished by protease
removal of the fibrillar appendages on the rough morphotype. The adherence was pH-independent and is
probably mediated by periodate-sensitive extracellular
polysaccharides (Kremer et al., 1999a). Kremer et al.
cloned a component of the fibril-like structure and designated it fibA (Kremer et al., 1999b). It encodes a 45 kDa
protein that is recognised by fibril-specific antibodies and
has a 38-residue leader peptide, which directs exportation
of the protein. Its COOH-terminus comprises of a large
number of aromatic amino acids, which may be involved
in anchoring the protein to cell-surface carbohydrate
structures (Kremer et al., 1999b).
Data on proteolytic activity in the virulence of P. micra
have started to emerge. Ng et al. (1998) found that 15 of
19 strains of P. micra tested possessed cell-associated gelatinase activity. Mikamo et al. (1999) reported that nine
out of 18 strains of P. micra that were isolated from
amniotic fluid with preterm premature rupture of membranes demonstrated elastase activity. A study by Grenier
and Bouclin (2006) investigated the contribution of
proteases and plasmin-acquired activity to P. micra virulence. They discovered the production of a chymotrypsinlike enzyme from P. micra that is both cell-bound and
secreted. Furthermore, they verified the gelatinase activity
ª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
544
previously reported by Ng et al. and found that it was
restricted to the rough morphotype only, which expresses
three gelatinase bands. An in vitro model of bacterial virulence using a reconstituted basement membrane showed
that the chymotrypsin-like protease and gelatinases
directly contributed to bacterial penetration of the rough
morphotype. Both the rough and smooth morphotype
were found to bind human plasminogen to the cell surface and once bound, plasminogen activators of bacterial
(streptokinase) and human (urokinase) origin activated
plasminogen to plasmin. Activated plasmin on the bacterial surface may interact with extracellular matrix and
P. micra cells coated with plasmin were found to have a
significantly greater tissue penetration activity through an
in vitro basement membrane model (Grenier & Bouclin,
2006). A more recent study by Ota-Tsuzuki and Alves
Mayer (2010) investigated collagenase production and
haemolytic activity in P. micra oral isolates. All of the
studied isolates demonstrated collagenolytic activity to
some extent. Only two of the 38 isolates tested demonstrated elastase activity, which is in stark contrast to the
study on elastase activity in P. micra isolates from amniotic fluids in uterine infections (Mikamo et al., 1999).
This finding suggests a difference in the proteolytic profile
of strains isolated from different sites of infection. Furthermore, oral P. micra isolates demonstrated a high haemolytic activity against chicken blood erythrocytes and to
a less extent, rabbit, but none towards sheep blood
(Ota-Tsuzuki & Alves Mayer, 2010).
Some studies have also been carried out looking at the
ability of P. micra to stimulate an inflammatory response
in cells. Nonnenmacher et al. (2003) found that a DNA
preparation containing unmethylated CpG motifs from
P. micra stimulated macrophages and gingival fibroblasts
to produce TNF-a and IL-6. A study by Yoshioka et al.
(2005) reported that P. micra cells bound Actinobacillus
actinomycetemcomitans lipopolysaccharide. Binding of
Gram-negative lipopolysaccharide significantly increased
its capacity to induce TNF-a production by human
macrophages.
Further studies into P. micra virulence were carried out
by Tanabe et al. (2007), who investigated the response of
human macrophages to a cell-wall preparation of P. micra. They reported that the cell-wall preparation stimulated the production of TNF-a and IL-1b, both of which
are important determinants of the progression of periodontitis (Graves & Cochran, 2003). In addition, the cellwall preparation induced significant amounts of IL-6, IL8 and RANTES secretion by macrophages (Tanabe et al.,
2007). In vivo effects of IL-6 secretion during periodontitis may include promotion of bone resorption (Ishimi
et al., 1990). IL-8 and RANTES are both potent chemokines which favour the accumulation of chemokines durª 2012 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
E.C. Murphy & I.-M. Frick
ing inflammation (Luster, 1998). This could help
contribute to periodontal tissue destruction during infection. Furthermore, RANTES has been reported to be
important in the initiation and progression of periodontitis (Gamonal et al., 2000; Johnson et al., 2004). The
induction of proinflammatory cytokines and chemokines
by the P. micra cell wall activate host-mediated destructive processes that are seen during periodontitis (Tanabe
et al., 2007). Significantly, P. micra cell-wall extracts were
also found to induce increased expression of active
MMP-9. MMP-9 has been linked to periodontal tissue
destruction and is highly expressed in inflamed gingival
tissues (Teng et al., 1992; Smith et al., 2004b). The same
study also found that various intracellular signalling pathways are induced by the P. micra cell wall, including
PKA, ERK2, JNK and p38, leading to an increased production of the proinflammatory cytokines seen above and
possibly contributing to periodontal tissue destruction
(Tanabe et al., 2007).
Conclusions and future work
GPAC are part of the commensal microbiota of humans in
multiple sites of the body and account for approximately
one-third of isolated anaerobic bacteria from clinical
specimens (Murdoch et al., 1994). GPAC have undergone
extensive taxonomic reclassification in the past 15 years
with the Peptostreptococcus genus being subdivided into six
new groups, along with the identification of newly
described species. Although mainly isolated from polymicrobial infections, some species have been isolated in pure
culture. The most common GPAC isolated from clinical
infections are F. magna, P. anaerobius, P. micra and
P. asaccharolyticus (Wren, 1996). Previously, the lack of
efficient and specific identification techniques for the
detection of GPAC in infection meant that some species
were misidentified or even overlooked. The advent of the
multiplex PCR assay, 16S rRNA gene-based probes and
MALDI-TOF mass spectrometry through the availability
of genotypic data mean that, in the future, GPAC will be
more correctly and comprehensively identified from clinical samples. This will ensure correct treatment of infection
and provide more extensive data on the clinical importance of some species. Recent data on the antibiotic resistance of GPAC have shown that variable resistance exists
towards penicillins, clindamycin and metronidazole.
Reports on differences in antimicrobial susceptibility
between various species of GPAC is increasing and should
continued to be studied, to monitor a potential increasing
resistance of GPAC to antibiotics. Whereas there are some
data on the virulence factors and pathogenic mechanisms
of F. magna and P. micra, these studies should be
extended to the other clinically relevant GPAC species.
FEMS Microbiol Rev 37 (2013) 520–553
Interactions of GPAC with the host
Acknowledgements
This work was supported by the Swedish Research Council
(project 7480), the Foundations of Crafoord, Bergvall and
Österlund, the Royal Physiographic Society, and Hansa
Medical AB.
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