Clumping factor A of Staphylococcus aureus inhibits phagocytosis by human polymorphonuclear leucocytes.

Abstract

Staphylococcus aureus is a major cause of nosocomial and community-acquired infection. It expresses several factors that promote avoidance of phagocytosis by polymorphonuclear leucocytes. Clumping factor A (ClfA) is a fibrinogen-binding surface protein of S. aureus that is an important virulence factor in several infection models. This study investigated whether ClfA is an antiphagocytic factor, and whether its antiphagocytic properties were based on its ability to bind fibrinogen. In S. aureus, ClfA was shown to be of equal importance to protein A, the antiphagocytic properties of which are well established. ClfA expressed in a surrogate Gram-positive host was also found to be antiphagocytic. A ClfA mutant that was unable to bind fibrinogen had a similar antiphagocytic effect to native ClfA in the absence of fibrinogen. ClfA inhibited phagocytosis in the absence of fibrinogen, and showed enhanced inhibition in the presence of fibrinogen.

Full text

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ClumpingfactorAofStaphylococcusaureus inhibitsphagocytosis
byhumanpolymorphonuclearleucocytes
Judy Higgins1, Anthony Loughman1, Kok P.M. van Kessel2, Jos A.G. van Strijp2& Timothy J. Foster1
1Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland; and2Eijkman Winkler Institute, UMC Utrecht, Utrecht,
The Netherlands
Correspondence: Timothy J. Foster, Moyne
Institute, Trinity College Dublin, Dublin 2,
Ireland. Tel.: 1353 1 6082014; fax: 1353 1
6799294; e-mail: tfoster@tcd.ie
Received 9 November 2005; revised 9 March
2006; accepted 10 March 2006.
First published online 4 April 2006.
doi:10.1111/j.1574-6968.2006.00229.x
Editor: Jan-Ingmar Flock
Keywords
Staphylococcus aureus; phagocytosis;
fibrinogen-binding protein.
Abstract
Staphylococcus aureus is a major cause of nosocomial and community-acquired
infection. It expresses several factors that promote avoidance of phagocytosis by
polymorphonuclear leucocytes. Clumping factor A (ClfA) is a fibrinogen-binding
surface protein of S. aureus that is an important virulence factor in several
infection models. This study investigated whether ClfA is an antiphagocytic factor,
and whether its antiphagocytic properties were based on its ability to bind
fibrinogen. In S. aureus, ClfA was shown to be of equal importance to protein A,
the antiphagocytic properties of which are well established. ClfA expressed in a
surrogate Gram-positive host was also found to be antiphagocytic. A ClfA mutant
that was unable to bind fibrinogen had a similar antiphagocytic effect to native
ClfA in the absence of fibrinogen. ClfA inhibited phagocytosis in the absence of
fibrinogen, and showed enhanced inhibition in the presence of fibrinogen.
Introduction
Staphylococcus aureus causes a wide variety of diseases,
including abscesses, septicaemia, septic arthritis and infective
endocarditis. Immunity to staphylococcal disease involves the
binding of complement and opsonizing antibodies to the
bacterial surface, which leads to activation of phagocytosis by
human polymorphonuclear leucocytes (PMNL) (Cunnion
et al., 2003; Rooijakkers et al., 2005). Once S. aureus pene-
trates the barrier of the skin PMNL are the critical first line of
defence (Verdrengh & Tarkowski, 1997).
Staphylococcus aureus expresses several factors that pro-
mote avoidance of phagocytosis by PMNL (Foster, 2005;
Rooijakkers et al., 2005). Protein A is a virulence factor in
experimental infection models of subcutaneous infection,
murine sepsis and septic arthritis and staphylococcal pneu-
monia (Patel et al., 1987; Gomez et al., 2004; Palmqvist et al.,
2002). It is believed to be antiphagocytic due to its ability to
bind the Fc region of immunoglobulin G (IgG) (Gemmell
et al., 1991). Capsular polysaccharide is a virulence factor in
a murine bacteraemia model (Thakker et al., 1998). The
antiphagocytic effect of capsule is due to its ability to
impede binding by opsonizing antibodies that recognize
antigens on the cell surface, as well as to inhibit complement
fixation (Thakker et al., 1998; Cunnion et al., 2003). This
contributes to the virulence of S. aureus by preventing
bacterial clearance from the bloodstream, liver and spleen
(Luong & Lee, 2002). Poly-N-acetylglucosamine also con-
tributes to virulence in animal models of bacteraemia and
renalabscessformation by inhibiting complement-mediated
phagocytic uptake by PMNL (Kropec et al., 2005).
Clumping factor A (ClfA) is a fibrinogen-binding surface
proteinof S. aureus (McDevitt et al., 1997). Itcontains an N-
terminal secretory signal peptide, followed by a 520-residue
region A that contains the ligand-binding domain, region R
consisting primarily of SD dipeptide repeats and a C-
terminal domain that allows anchoring to the cell wall
peptidoglycan (McDevitt et al., 1995).
Clumping factor A is an important virulence factor in
several infection models, including rat experimental endocar-
ditis (Moreillon et al., 1995), murine sepsis and septic arthritis
(Josefsson et al., 2001) and rabbit infective endocarditis
(Vernachioetal.,2003).Bycomparingwild-typeS.aureuswith
ClfA-deficient mutants and by transfer of clfA to Streptococcus
gordonii (Stutzmann Meier et al., 2001) and to Lactococcus
lactis, ClfA was identified as an important factor in mediating
endovascularinfection (Queet al., 2001). ClfA-deficientstrains
are also attenuated in a murine model of sepsis and septic
arthritis (Josefsson et al., 2001; Vernachio et al., 2003). In this
model immunization with recombinant region A of ClfA was
protective, as was passive immunization with human IgG
containing a high titre against ClfA (Josefsson et al., 2001).
FEMS Microbiol Lett 258 (2006) 290–296
c ?2006 Federation of European Microbiological Societies
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It is possible that ClfA acts as a virulence factor in certain
infections by inhibiting phagocytosis, as well as promoting
adhesion to fibrin and fibrinogen. This study investigated
whether ClfA is antiphagocytic and if this effect is due to its
ability to bind fibrinogen.
Materials and methods
Bacterial strains and growth conditions
Bacterial strains are listed in Table 1. Staphylococcus aureus
strains were cultured on trypticase soy agar or broth (Oxoid,
Basingstoke, UK) at 371C with shaking at 200r.p.m. for liquid
cultures. Lactococcus lactis strains were cultured in M17
medium(Difco,Detroit,MI)containing0.5%glucosewithout
shaking at 301C. Antibiotics were incorporated where appro-
priate at the following concentrations: 10mgmL?1chloram-
phenicol,5mgmL?1erythromycinand10mgmL?1kanamycin.
Transduction
A null mutation in protein Awas transduced from S. aureus
8325-4 spa::KanR(Patel et al., 1987) by generalized phage
transduction using phage 85 (Foster, 1998) to strains New-
man (Duthie & Lorenz, 1952) and Newman clfA (McDevitt
et al., 1994) with selection for kanamycin resistance. The
fidelity of transductants was verified by Southern blotting
and by Western immunoblotting of solublized cell wall
proteins with anti-ClfA polyclonal antisera and with horse-
radish peroxidase-conjugated goat IgG.
PMNL isolation
Fresh whole blood was obtained from healthy volunteers,
heparinized and mixed with an equal volume of phosphate-
buffered saline (PBS). This was centrifuged through step
gradients of Histopaque (r=1.077, Sigma, St Louis, MO)
and Ficoll-paque (r=1.119, Amersham, Chalfont St Giles,
UK) and PMNL were aspirated from the buffy coat between
the Ficoll and Histopaque layers. Cells were washed in RPMI
1640 medium [containing 10mM 4-(2-hydroxyethyl)piper-
azine-1-ethanesulfonic acid (HEPES), 25mM glutamine
and 0.05% (v/v) human serum albumin] and resuspended
in water. After a 30s osmotic shock to lyse contaminating
red blood cells, 10? PBS was added and the cells were again
washed in RPMI. PMNLwere counted in a haemocytometer
(bright line, Neubauer) and adjusted to 5?106cells per mL
in RPMI. This procedure typically yielded 497% PMNL
with 495% viability, as determined by trypan blue exclu-
sion. All reagents used in PMNL isolation were certified
endotoxin-free.
Phagocytosis of bacteria by human PMNL
Whole bacterial cells grown to stationary phase were washed
twice in PBS and labelled with 30mgmL?1fluorescein
isothiocyanate (FITC) in PBS for 1h at 371C with shaking.
Cells were washed three times in PBS and enumerated in a
Neubauer haemocytometer (Brand GmbH, Wertheim, Ger-
many), adjusted to 1?109CFUmL?1in RPMI and stored
frozen at ?201C. FITC-labelling of bacteria did not affect
their ability to bind fibrinogen (data not shown). Bacteria
were thawed on ice and diluted to 5?107CFUmL?1in
RPMI. Pooled human serum was diluted in RPMI and
fibrinogen (Calbiochem, Schwalbach, Germany) was added
to serum where necessary. The final concentration of
fibrinogen, where present, was the same percentage as the
serum concentration, calculated as 3mgmL?1=100%. Bac-
teria (50mL) wereopsonized in 10mL diluted serum (with or
Table 1. Strains and plasmids used in this study
PropertiesReferences
Strain
Staphylococcus aureus
Newman
Newman clfA
Newman spa
Newman spa clfA
Lactococcus lactis
MG1363
NZ9800
Plasmid
pKS80
pKS80 clfA
pKS80 spa
pNZ8037
pNZ8037 clfA
pNZ8037 clfAPY
NCTC 8178
clfA::ErmR?
spa::KanR
spa::KanRclfA::ErmR
Duthie & Lorenz (1952)
McDevitt et al. (1994)
This study
This study
Plasmid-free derivative of NCD 0712
MG1363 bearing nisin resistance plasmid [CamR]
Gasson (1983)
Kuipers et al. (1993)
L. lactis expression vector [ErmR]
pKS80 containing clfA coding sequence [ErmR]
pKS80 containing spa coding sequence [ErmR]
Nisin-inducible L. lactis expression vector [CamR]
pNZ8037 containing clfA coding sequence [CamR]
pNZ8037 containing clfA coding sequence, incorporating mutations [CamR]
Wells et al. (1993)
O’Brien et al. (2002)
O’Brien et al. (2002)
de Ruyter et al. (1996)
Loughman et al. (2005)
Loughman et al. (2005)
?ErmR, KanRand CamR; resistance to erythromycin, kanamycin and chloramphenicol, respectively.
FEMS Microbiol Lett 258 (2006) 290–296
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Clumping factor A of Staphylococcus aureus

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without fibrinogen) for 10min at 371C, followed by addi-
tion of 50mL prewarmed PMNL and incubation at 371C
with vigorous shaking. The final bacteria:PMNL ratio was
10:1. Reactions were stopped after 5–15 min by addition of
100mL ice-cold 1% (w/v) paraformaldehyde in PBS. The
percentage of PMNL bearing FITC-labelled bacteria (%
phagocytosis) was determined by flow cytometric analysis
of 5000 cells with manual gating using a FACScan flow
cytometer (Becton Dickinson, Oxford, UK). The percentage
of internalized bacteria was determined by trypan blue
quenching of extracellular fluorescence (Benne et al., 1997;
Su et al., 2001). Addition of 20mgmL?1(final concentra-
tion) trypan blue (Merck, Schwalbach, Germany) demon-
strated that 485% bacteria associated with PMNL were
internalized (data not shown). The absence of bacterial
clumping during the assay was verified microscopically.
Samples were prepared in triplicate and all experiments
were repeated a minimum of three times using the blood of
different donors. Statistical analyses were performed using
the Student’s t-test for paired data in Kaleidagraph (Synergy
software). P values o0.01 were considered significant.
Whole cell dot immunoblotting
Quantitation of the relative amounts of protein expressed by
L. lactis strains was carried out by whole cell dot immuno-
blotting, as described previously (Loughman et al., 2005).
Results
Inorder toinvestigatethe antiphagocytic effects of protein A
and ClfA, strains Newman, Newman clfA, Newman spa and
Newman spa clfA were tested for their uptake by fresh
human PMNL in the presence of human serum opsonins.
Increasing concentrations of serum resulted in increased
phagocytic uptake of all strains, indicating that the process
was dependent on serum opsonins. At 1% normal human
serum (NHS) strain Newmanwas phagocytosed by approxi-
mately 46% of PMNL (Fig. 1). Newman spa and Newman
clfA were taken up by approximately 45% of PMNL at this
serum concentration, whereas the Newman spa clfA double
mutant was taken up by 72% of PMNL (Po0.001). This
demonstrated that both protein A and ClfA can contribute
to the inhibition of phagocytosis of Staphylococcus aureus
Newman, and that the absence of one factor was compen-
sated for by the presence of the other. The absence of both
factors led to a significant increase in phagocytic uptake.
The addition of a physiologically relevant concentration
of fibrinogen had no significant impact on the level of
uptake of the ClfA-deficient strains Newman clfA and New-
man spa clfA (Fig. 1). However, the levels of uptake of both
the wild-type strain and of Newman spa in 1% NHS were
significantlyreducedin the
(Po0.005 and Po0.01, respectively). This indicated that
presence of fibrinogen
strains bearing ClfA on their cell surface had enhanced anti-
phagocytic properties in the presence of soluble fibrinogen.
The presence of fibrinogen had no effect on the phagocytic
uptake of cells lacking ClfA.
Bacterial cells used in this assay were grown to stationary
phase to minimize any possible contribution of the fibrino-
gen-binding protein clumping factor B, which is absent in
stationary phase due to proteolysis and cessation of clfB
transcription (McAleese et al., 2001). The fibronectin-bind-
ing proteins are also predominantly expressed during the
exponential phase of growth and can also bind fibrinogen.
However, the fnbA and fnbB genes of strain Newman each
have a frameshift mutation that results in secretion of
truncated forms of the proteins (Grundmeier et al., 2004).
Thus, ClfA was the only known fibrinogen-binding surface
protein expressed by the cells in this assay.
In order to investigate further the antiphagocytic proper-
ties of ClfA, the protein was expressed on the surface of the
Gram-positive surrogate host Lactococcus lactis (O’Brien
et al., 2002). Increasing concentrations of serum led to
increased uptake of L. lactis cells (Fig. 2), indicating that
efficient opsonization of the bacteria was required to facil-
itate phagocytosis. Lactococcus lactis cells bearing an empty
plasmid vector (pKS80) were taken up by 50% of PMNL at
0
10
20
30
40
50
60
70
80
0.11
% Phagocytosis
% NHS
Fig. 1. Phagocytosis of Newman strains by human polymorphonuclear
leucocytes (PMNL). Freshly isolated human PMNL were incubated with
Staphylococcus aureus Newman strains grown to stationary phase and
opsonized with varying concentrations of normal human serum in the
absence (solid lines) or presence (dashed lines) of physiologically relevant
concentrations of fibrinogen. Wild-type Newman (squares) expresses
both clumping factor A (ClfA) and protein A, Newman spa (diamonds) is
protein A-deficient, Newman clfA (circles) is ClfA-deficient and Newman
spa clfA (triangles) is deficient in both protein A and ClfA. The
percentage of PMNL containing bacteria is expressed as % phagocytosis.
All samples were prepared in triplicate. Data are representative of at least
three independent experiments using three different donors.
FEMS Microbiol Lett 258 (2006) 290–296
c ?2006 Federation of European Microbiological Societies
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292
J. Higgins et al.

Page 4

1% NHS. The addition of fibrinogen had no significant
impact on the level of uptake of these bacteria. L. lactis
expressing ClfA (pKS80 clfA) were taken up by significantly
fewer PMNL (25% at 1% NHS, P=0.002 compared with
L. lactis pKS80). The addition of fibrinogen further reduced
this level to 2% (P=0.001). Lactococcus lactis expressing
protein A were taken up by only 9% of PMNL at 1% NHS
(P=0.003 compared with L. lactis pKS80), however the
addition of fibrinogen did not affect the level of uptake of
this strain. Protein A showed very potent inhibition of
phagocytosis, but showed no fibrinogen-dependent reduc-
tion of uptake. ClfA was capable of inhibition of phagocy-
tosis in the absence of fibrinogen, and showed increased
inhibition in the presence of fibrinogen.
In order to investigate whether the effect of fibrinogen
was due to direct binding to ClfA, a derivative of ClfA with
substitutions in residues essential for fibrinogen binding
(ClfAPY) was used (Loughman et al., 2005). Lactococcus
lactis NZ9800 allows inducible expression of genes cloned
into the vector pNZ8037. The relative levels of expression of
ClfA and ClfAPY were monitored by whole cell dot im-
munoblotting and shown to be equal before cell labelling
(data not shown). Lactococcus lactis NZ9800 bearing the
empty vector (pNZ8037) was taken up by approximately
48% of PMNL at 1% NHS both in the presence and absence
of fibrinogen (Fig. 3). Lactococcus lactis NZ9800 expressing
ClfA (pNZ8037 clfA) was taken up by approximately 32% of
PMNL at the same serum concentration in the absence of
fibrinogen, and the level of uptake was further reduced to
approximately 4% in the presence of fibrinogen (Po0.001
compared with L. lactis pNZ8037). Lactococcus lactis expres-
sing ClfAPY (pNZ8037 clfAPY), which does not bind fibrino-
gen (Loughman et al., 2005) was taken up by approximately
27% of PMNL at 1% NHS. However, the addition of soluble
fibrinogen did not influence the level of uptake of this strain
(Fig. 3). This indicated that direct binding of fibrinogen to
ClfAonthesurface ofthebacteriacausedincreasedinhibition
of phagocytosis by human PMNL.
Discussion
The importance of ClfA in the virulence of Staphylococcus
aureus in a variety of animal models suggests that the
protein might have an antiphagocytic effect. This study
investigated a possible role for ClfA in the resistance of S.
aureus to phagocytosis by human PMNL. The experiments
described here show clearly that the well known antiphago-
cytic action of protein A (Rooijakkers et al., 2005) can be
clearly demonstrated in our in vitro phagocytosis assay. Null
mutation of the gene responsible in virulent staphylococci
0
10
20
30
40
50
60
0.11
% Phagocytosis
% NHS
Fig. 2. Phagocytosis of Lactococcus lactis strains expressing clumping
factor A (ClfA) or protein A. Freshly isolated human polymorphonuclear
leucocytes (PMNL) were incubated with L. lactis MG1363 strains expres-
sing no protein (pKS80, squares), ClfA (pKS80 clfA, diamonds) or protein
A (pKS80 spa, circles) grown to stationary phase and opsonized with
varying concentrations of normal human serum in the absence (solid
lines) or presence (dashed lines) of physiologically relevant concentra-
tions of fibrinogen. The percentage of PMNL containing bacteria is
expressed as % phagocytosis. All samples were prepared in triplicate.
Data are representative of at least three independent experiments using
three different donors.
0
10
20
30
40
50
60
0.11
% Phagocytosis
% NHS
Fig. 3. Phagocytosis of Lactococcus lactis strains expressing clumping
factor A (ClfA) or ClfAPY. Freshly isolated human PMNL were incubated
with L. lactis NZ9800 strains expressing no protein (pNZ8037, squares),
ClfA (pNZ8037 clfA, diamonds) or ClfAPY (pNZ8037 clfAPY, circles)
grown to stationary phase and opsonized with varying concentrations
of normal human serum in the absence (solid lines) or presence (dashed
lines) of physiologically relevant concentrations of fibrinogen. The per-
centage of polymorphonuclear leucocytes (PMNL) containing bacteria is
expressed as % phagocytosis. All samples were prepared in triplicate.
Data are representative of at least three independent experiments using
three different donors.
FEMS Microbiol Lett 258 (2006) 290–296
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Clumping factor A of Staphylococcus aureus

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(Newman spa) resulted in increased sensitivity to phagocy-
tosis. Moreover, introduction of the gene into the nonviru-
lent Lactococcus lactis (pKS80 spa) showed clear inhibition
of phagocytosis. In the same system, it was also shown that
ClfA is an important antiphagocytic factor for S. aureus, with
both fibrinogen-dependent and fibrinogen-independent
mechanisms. In S. aureus Newman, ClfA was shown to be
of equal importance to protein A. Using ClfA expressed in a
surrogate Gram-positive host we could also show that it
possesses strong antiphagocytic properties. A ClfA mutant
that was unable to bind fibrinogen had a similar antiphago-
cytic effect as native ClfA in the absence of fibrinogen, fur-
ther establishing the existence of a fibrinogen-independent
mechanism.
When S. aureus is grown in plasma it becomes coated
with soluble plasma components that interfere with bacter-
ial adherence to immobilized ligands (Massey et al., 2002).
In this study, inactivation of both clfA and spa in a micro-
encapsulated strain of S. aureus caused a significant increase
in the level of phagocytosis. Although Newman can express
a type 5 capsular polysaccharide, the growth conditions used
in this study would not have promoted its expression at high
levels. The presence of either ClfAor protein Aalone seemed
to be sufficient to inhibit phagocytosis. It appears that being
coated with either immunoglobulin or fibrinogen, or a
mixture of both, is sufficient to impede phagocytosis. This
indicates that protein A and ClfA are two major antiphago-
cytic factors for S. aureus.
Anti-ClfA antibodies are present both in normal and
convalescent sera (Colque-Navarro et al., 2000; Dryla et al.,
2005). This implies that normal exposure to S. aureus as a
commensal of the skin causes the formation of anti-ClfA
antibodies, but that the antibodies in normal sera are not
protective against invasive S. aureus disease. These antibo-
dies have opsonizing activity (Josefsson et al., 2001; Verna-
chio et al., 2003; Patti, 2004). Immunization of mice with
recombinant ClfA induced anti-ClfA antibody production,
whereas infection of naı ¨ve mice with S. aureus Newman did
not (Josefsson et al., 2001). Perhaps the abilityof ClfAon the
surface of S. aureus cells to bind fibrinogen in the blood-
stream prevents the development of high levels of specific
antibodies directed against it. In addition, the recent ob-
servation that low levels of anti-ClfA antibodies stimulate
platelet aggregation may go some way to explaining why the
low levels of antibodies in normal serum are not protective
against staphylococcal infection (Loughman et al., 2005).
Itis likely that the pooled human serum used in this study
contained low levels of antibodies against ClfA (Dryla et al.,
2005; Loughman et al., 2005). They might interact with
ClfA in one of two ways. They may compete with fibrinogen
to bind ClfA and promote phagocytosis, either alone via
Fc receptors on the PMNL surface or by stimulating
complement activation. Alternatively they may promote
phagocytosis by binding to ClfA along with bound
fibrinogen.
However, ClfA is antiphagocytic despite the presence of
low levels of anti-ClfA antibodies in normal human sera.
Perhaps opsonins bound to bacteria are prevented from
interacting productively with Fc receptors and complement
receptors on the PMNL surface, possibly through steric
hindrance by bound fibrinogen, immunoglobulins bound
to protein A, or other serum or bacterial factors. These may
form a protective layer of host proteins around the bacter-
ium, preventing access by antibodies to antigens on the cell
wall surface such as peptidoglycan, teichoic acids and other
proteins, or preventing interactions between antibodies
bound to these antigens and Fc receptors on the PMNL
surface. Such a protective layer might also interfere with
complement deposition.
Attempts to measure the survival of bacteria following
uptake gave paradoxical results. After 30min c. 50% of
wild-type bacteria were killed, however the ClfA and
protein A mutants were killed less than wild type during
this time (unpublished data), despite being taken up
more efficiently (Fig. 1). This may have been caused by
alteration of the fidelity of the phagosome due to changes in
major surface proteins on the bacterial surface, as was
observed by Gresham et al. (2000). The fluorescence-based
assay used in this study allows analysis of bacterial uptake
independently from the complex interactions involved in
bacterial killing.
The presence of two mechanisms of resistance to phago-
cytic uptake, one fibrinogen-dependent and the other fibri-
nogen-independent may reflect the presence of different
niches for the bacteria within the host during disease
progression, in which fibrinogen is available or unavailable.
For example, it appears likely that the ability of ClfA to
inhibit phagocytosis independently of fibrinogen may be
important in the murine sepsis and septic arthritis model, as
depletion of free fibrinogen in the bloodstream led to
aggravation of septic arthritis caused by ClfA-producing S.
aureus strains (Palmqvist et al., 2004). This fibrinogen-
independent inhibition may also be important in vivo in
environments with an elevated Ca21concentration, as Ca21
inhibits fibrinogen binding by ClfA with an IC50of 2.5mM
(O’Connell et al., 1998).
The ability of ClfA to inhibit phagocytosis by human
PMNL mayexplain its importance in S. aureus virulence in a
variety of animal models of infection. It is likely also to be
the reason that high-titre, but not low-titre anti-ClfA anti-
bodies are protective.
Acknowledgements
This work was supported by the Health Research Board of
Ireland and Science Foundation Ireland.
FEMS Microbiol Lett 258 (2006) 290–296
c ?2006 Federation of European Microbiological Societies
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294
J. Higgins et al.

Page 6

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