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MĂ©decine et maladies infectieuses 38 (2008) 549â553
Original article
Genotypic screening of atypical Staphylococcus aureus strains isolated
from clinical samples for presence of selected adhesin genes
Screening génotypique des souches atypiques de Staphylococcus aureus
isolées de divers prélÚvements cliniques par détection des adhésines
sélectives
K. WiÂŽniewska â , K. Garbacz, L. Piechowicz
s
Department of Medical Microbiology, Medical University of GdaÂŽ sk, 38, Do Studzienki Street,
n
80â227 GdaÂŽ sk, Poland
n
Received 4 June 2007; accepted 3 June 2008
Available online 30 July 2008
Abstract
Objective. â The aim of this study was to screen Staphylococcus aureus negative for production of coagulase or clumping factor and for presence
of selected adhesin genes.
Methods. â Sixty coagulase-negative and 20 clumping factor-negative S. aureus strains were studied. Detection of methicillin resistance was
performed using the agar screen technique with 6 mg/L of oxacillin and was conïŹrmed by ampliïŹcation mec A gene. The presence of bone binding
protein (bbp), collagen binding protein (cna), ïŹbronectin A binding protein (fnbA), ïŹbronectin B binding protein (fnbB) and clumping factor A
(clfA) genes was detected by multiplex PCR.
Results. â Almost all (98%) of the strains were positive for clfA gene. There were fnbA and fnbB in 85%, cna in 54% and bbp in 5% of strains
found. No correlation between presence of the particular genes and clinical samples was observed. The prevalence of fnbA, fnbB and cna was
statistically higher in coagulase-negative than in clumping factor-negative strains (89, 89, 66 and 70, 70, 15%, respectively). Similarly, all of these
genes were more often observed in MRSA than in MSSA atypical strains. The cna was detected only in coagulase-negative MRSA.
© 2008 Elsevier Masson SAS. All rights reserved.
Résumé
Objectif. â Ătude de la prĂ©sence des gĂšnes des adhĂ©sines sĂ©lectives chez des souches atypiques de Staphylococcus aureus Ă coagulase-nĂ©gative
et le facteur agglutinant (clumping factor [CF])-négatif, résistants et sensibles à la méthicilline.
MĂ©thodes. â Soixante souches de S. aureus Ă coagulase-nĂ©gative et 20 avec CF-nĂ©gatif ont Ă©tĂ© Ă©tudiĂ©es. La rĂ©sistance Ă la mĂ©thicilline a Ă©tĂ©
dĂ©terminĂ©e par screening en millieu gĂ©losĂ© additionnĂ© de 6 mg/L dâoxacilline et ensuite a Ă©tĂ© conïŹrmĂ©e par ampliïŹcation du gĂšne mecA. La prĂ©sence
des gĂšnes bone binding protein (bbp), collagen binding protein (cna), ïŹbronectin A binding protein (fnbA), ïŹbronectin B binding protein (fnbB) et
clumping factor A (clfA) a été détectée par réaction de polymérisation en chaßne.
RĂ©sultats. â Quatre-vingt-dix-huit pour cent des souches Ă©tudiĂ©es possĂ©daient le gĂšne clfA ; 85 % : fnbA et fnbB ; 54 % : cna et seulement
5 % : bbp. Il nây avait aucune corrĂ©lation entre la prĂ©sence des gĂšnes des adhĂ©sines et lâisolement de prĂ©lĂšvements cliniques particuliers. La
prévalence des gÚnes fnbA, fnbB et cna chez les souches à coagulase-négative était statistiquement plus élevée que chez les souches avec CF-négatif
Abbreviations: bbp, bone binding protein; can, collagen binding protein; fnbA, ïŹbronectin A binding protein; fnbB, ïŹbonectin binding proteinB; clfA, clumping
factorA; CNSA, coagulase-negative S. aureus; CFNSA, clumping factor-negative S. aureus.
â Corresponding author.
E-mail address: kwis@amg.gda.pl (K. WiÂŽniewska).
s
0399-077X/$ â see front matter © 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.medmal.2008.06.003
- 2. 550
K. WiÂŽniewska et al. / MĂ©decine et maladies infectieuses 38 (2008) 549â553
s
(89, 89, 66 et 70, 70, 15 %, respectivement). De mĂȘme, ces gĂšnes ont Ă©tĂ© dĂ©tectĂ©s plus frĂ©quemment chez S. aureus rĂ©sistants Ă la mĂ©thicilline
(Sarm) que chez sensibles Ă la mĂ©thicilline (Sasm). La prĂ©sence du gĂšne cna nâa Ă©tĂ© observĂ©e que chez les S. aureus Ă coagulase-nĂ©gative rĂ©sistants
à la méticilline.
© 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Adhesion; Clumping factor; Coagulase; Staphylococcus aureus
Mots clés : Adhésion ; Coagulase ; Facteur agglutinant ; Staphylococcus aureus
1. Introduction
Staphylococcus aureus is a common pathogen in a signiïŹcant
number of community-acquired and nosocomial infections [1].
The attachment of S. aureus to host cells and tissues is essential step in the colonization process and, thus, a risk factor for
invasive disease [2]. The adherence of S. aureus is mediated
by a family of proteins, in most cases covalently anchored to
the cell peptidoglycan, termed microbial surface components
recognizing adhesive matrix molecules (MSCRAMM), which
speciïŹcally binds to distinct extracellular components of host
tissues or to serum-conditioned implanted biomaterials such
as catheters, artiïŹcial joints and vascular grafts [3]. There is
evidence that some of the adhesive proteins of S. aureus are
associated with speciïŹc invasive infections [4â6].
Coagulase and clumping factor are two species-speciïŹc proteins of S. aureus that were implicated in binding to ïŹbrinogen
and play important role in the ability of bacteria to cause infections [2,3]. Clumping factor A (ClfA) is one of MSCRAMM
that promotes binding of ïŹbrinogen to the bacterial cell surface
while coagulase is an extracellular protein that binds prothrombin to form staphylothrombin and this binding catalyses
plasma clotting [3]. However, some clinical isolates, including
methicillin-resistant S. aureus (MRSA) strains, are not able to
produce coagulase or clumping factor [7,8]. While the adhesion issue of S. aureus has been well examined in respect to
various pathological conditions [4â6], no studies of adherence
factors in atypical strains of this species has been performed
as yet. Such investigations would permit better understanding
of the ability of the strains, which are free from essential virulence factors for colonization host cells and tissues and might be
useful in therapeutic aspect [9,10]. Thus, the aim of this study
was to examine coagulase-negative or clumping factor-negative
S. aureus strains for presence of genes encoding surface proteins
such as clumping factor A (ClfA), bone binding protein (Bbp),
collagen binding protein (Cna) and ïŹbrinogen A and B binding
proteins (FnbA and FnbB, respectively) the role of which as virulence factors has been well documented in typical strains of
this species. In order to ascertain possible differences, the analysis of methicillin-resistant and methicillin-sensitive S. aureus
(MSSA) strains was performed.
isolates obtained from 21 polish medical centers, in majority
situated in Gdansk area. Clinical source of the strains was as
following:
âą
âą
âą
âą
âą
âą
âą
pus and purulent lesions (31 strains);
burn wound (23 strains);
nose, throat, ear (13 strains);
tracheostomy tube (four strains);
blood (four strains);
catheters (three strains);
bronchial ïŹuid (three strains).
The strains were isolated from varied patients over a 10 last
year period. All isolates were subcultured on bovine blood agar
and stored at â70 ⊠C in trypticase soy broth (TSB) with 50%
glycerol until investigations. The staphylococcal species was
identiïŹed by clumping factor test, free coagulase test and APIStaph ID 32 Kit (BioMĂšrieux, France). The clumping factor
was detected in slide test with rabbit plasma (Biomed Warsaw,
Poland) [11]. Free coagulase test was performed in standard conditions by incubating 0.8 ml of TSB culture with 0.2 ml rabbit
plasma for 2, 4, 6 and 24 h at 37 ⊠C [11]. S. aureus ATCC 12600
strain was used as positive control. The identiïŹcation was conïŹrmed by PCR on the basis of presence of the species speciïŹc
nuclease gene nuc [12]. Detection of methicillin resistance was
performed by using the agar screen technique with 6 mg/L of
oxacillin and was conïŹrmed by ampliïŹcation mec A gene [12].
2.2. Detection of the adhesin genes
Detection of the following genes: bbp, cna, fnbA, fnbB, clfA
was performed by multiplex PCR [13]. The staphylococcal DNA
was extracted according to the procedure described previously
[12]. Two primer sets were prepared for PCR: PCR1 to amplify
bbp and cna and PCR2 to amplify fnbA, fnbB and clfA. The
nucleotide sequence of the primers and thermal cycling conditions were described by Tristan et al. [13].
2.3. Statistical analysis
The data were analyzed by Ï2 test. A P value of less than
0.05 was considered signiïŹcant.
2. Materials and methods
3. Results
2.1. Bacterial isolates
The 80 S. aureus, including 60 coagulase-negative and 20
clumping factor-negative strains used in this study, were clinical
Almost all (98%) of the strains were positive for clfA gene.
There were fnbA and fnbB in 85%, cna in 54% and bbp only
in 5% strains found. The fnbA, fnbB and cna were found in 89,
- 3. K. WiÂŽniewska et al. / MĂ©decine et maladies infectieuses 38 (2008) 549â553
s
551
Table 1
Prevalence of the adhesin genes in coagulase-negative (CNSA) and clumping factor-negative (CFNSA) S. aureus strains
Prévalence des gÚnes des adhesines dans les souches de S. aureus coagulase-négatifs (CNSA) et CF-négatifs (CFNSA)
Strains
Number of strains (100%)
Gene/Number of strains (%)
bbp
cna
fnbA
fnbB
clfA
CNSA
CFNSA
60
20
2 (3)
2 (10)
40 (66)
3 (15)
54 (89)
14 (70)
54 (89)
14 (70)
60 (100)
18 (90)
Total
80
4 (5)
43 (54)
68 (85)
68 (85)
78(98)
89 and 66% coagulase-negative strains and in 70, 70 and 15%
clumping factor-negative strains, respectively (Table 1).
There were statistically differences between methicillinresistant and MSSA strains observed. The fnbA, fnbB and cna
were in 90, 90 and 63% MRSA and, respectively, in 67, 67
and 24% MSSA atypical strains detected. Among coagulasenegative strains, almost all MRSA and only 72, 72 and 15%
MSSA strains were positive for fnbA, fnbB and cna. Clumping
factor-negative MRSA strains were, in 82%, positive for fnbA
and fnbB but negative for cna (Table 2).
The most frequent adhesin genes as clfA and fnbA and B
were occurred in majority of the strains isolated from all clinical
samples (Table 3). Similarly, the strains positive for cna gene
were obtained from all clinical source, but the frequency of cna
gene varied from 25% for blood to 69% for nose, throat and ear.
4. Discussion
The role of some of MSRAMMs as virulence determinants in
the pathogenesis of S. aureus disease is well documented [3â6].
Moreover, the results obtained in experimental models suggest
that some of the adhesion proteins may be potential targets for the
prevention of staphylococcal infections [9,10]. For this aspect,
genetic ability of S. aureus for presence of relevant adhesion
mediators must be well known, even with respect to atypical
strains. This study focused on getting familiar of the genetic
ability to adhesion to host cells and tissues of S. aureus strains,
which are negative in production of a crucial virulence determinants such clumping factor or coagulase. According to our
previous report, over 12.3 and 4.2% of S. aureus strains isolated
in Poland are coagulase- or clumping factor-negative, respec-
tively [7]. Moreover, most such strains are resistant to methicillin
[7,8].
As it has been described previously, ClfA promotes clumping
of bacterial cells in plasma and adherence of bacteria to blood
clots, to plasma conditioned biomaterials and to catheter damaged valves in a rat model of endocarditis [3]. Thus, it is probably
a signiïŹcant factor in wound and foreign body infections and
may be an excellent target for the generation of immune therapies directed against S. aureus [10]. All clumping factor-negative
strains and almost all coagulase-negative strains examined in this
study were positive for clfA gene. On the basis of this result, one
could assume that the negative reaction for clumping factor test
might be caused by blocking up the expression of this gene.
Another possible explanation of this effect is a too low concentration of the expressed protein or reduced availability on the
bacterial surface.
Multiplicity of the adhesions necessary for the recognition of
various receptors seems to be an important factor in the development of infection and may help to increase the pathogenicity of
a given strain [2,3]. In comparison with the ïŹndings of others,
clumping factor- or coagulase-negative S. aureus strains seem
not to be differed in genetic ability to poses crucial virulence
adhesins from typical strains of this species [2,14,15]. In most
cases of the present study, besides clfA, fnbA and B genes were
found together in the same isolate. It has been underlined by
many authors, that the Fnb plays an important role in virulence
action of S. aureus in human host [2,9,14]. Moreover, biological
effect of Fnb has been conïŹrmed in rat endocarditis infection [2].
As it has been described, this protein is found to be very frequent
in clinical strains of S. aureus [2,14]. Our ïŹndings show that
genes responsible for production Fnb are widely distributed also
Table 2
Prevalence of the adhesin genes in methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) atypical S. aureus strains.
Prévalence des gÚnes des adhesines dans les souches atypiques de S. aureus résistants (SARM) et sensibles à la méthicilline (SASM)
Strains
Number of strains
Gene/Number of isolates (%)
bbp
cna
fnbA
fnbB
clfA
MRSA
CNSA
CFNSA
42
17
1 (2)
0
38 (90)
0
40 (95)
14 (82)
40 (95)
14 (82)
42 (100)
15 (88)
Total
59
1 (2)
38 (63)
54 (90)
54 (90)
57 (97)
MSSA
CNSA
CFNSA
18
3
1 (6)
2 (67)
3 (17)
3 (100)
13 (72)
0
13 (72)
0
18 (100)
3 (100)
Total
21
3 (14)
5 (24)
14 (67)
14 (67)
21(100)
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s
Table 3
Distribution of adhesin genes among atypical S. aureus strains isolated from various clinical samples
Distribution de gÚnes des adhésines dans les souches atypiques de S. aureus isolées de différents prélÚvements cliniques
Clinical source
Number of isolates (100%)
Genes/Number of strains (%)
bbp
cna
fnbA
fnbB
clfA
Pus and purulent lesions
Burn wound
Nose, throat, ear
Tracheostomy tube, bronchial ïŹuid
Blood
Catheters
31
22
13
7
4
3
0
1 (5)
3 (23)
0
0
0
14 (45)
15 (68)
9 (69)
2 (29)
1 (25)
2 (67)
29 (94)
18 (82)
9 (69)
7 (100)
3 (75)
2 (67)
29 (94)
18 (82)
9 (69)
7 (100)
3 (75)
2 (67)
31 (100)
22 (100)
13 (100)
7 (100)
2 (50)
3 (100)
Total
80
4 (5)
43 (54)
68 (85)
68 (85)
78 (98)
in atypical strains of this species. Additionally research is needed
to answer why coagulase-negative strains are statistically more
frequent positive for fnb genes than clumping factor-negative
strains.
In contrast, bbp were found only in 5% of examined strains.
There is clear evidence that the Bbp is a crucial factor in bone
and joint infections caused by S. aureus [4,16]. Thus, the gene of
this protein would be expected to be present mostly in isolates
causing such infections. Some of the strains of our collection
were isolated from infections observed in patients from orthopedic unit where bone and joint infections are most common.
Unexpectedly, no one strain positive for the bbp was derived
from orthopedic unit. Taking the results obtained by Tung et
al. into account, speciïŹcity of Bbp for a factor other than bone
sialoprotein cannot be excluded [16].
The other MSCRAMM that is possibly important in staphylococcal infection is Cna. The Cna protein mediates bacterial
adherence to collagen substrates and collagenous tissues and it
is necessary for S. aureus cells to adhere to cartilage in vitro [2].
As it has been described, the presence of cna is not generally
expressed by the majority of strains and may differ from 38 to
56% positive results [2,17,18]. There is evidence that strains isolated from healthy nasal carriers harbouring cna gene were found
to be about 48% [19]. As shown by the results of our ïŹndings,
only 54% of atypical S. aureus possesses genetic ability to produce Cna. Moreover, similarly to fnb genes, cna gene is found to
be more common in coagulase-negative than in clumping factornegative strains and exists without any correlation with clinical
samples.
The vast majority of the examined strains were resistant to
methicillin. This is in agreement with other reports giving evidence that the negative result of clumping factor or coagulase
detection is more common in MRSA than in MSSA strains
[7,8]. This phenomenon can be explained by the insertion into
bacterial genome sequence of methicillin resistance that may
include DNA elements altering some bacterial properties [7].
The results of our study show that atypical strains resistant
to methicillin much more often possess genetic ability to produce cna and both fnb A and B genes than strains sensitive
to this antibiotic. The indications of Rice et al. would support this observation, with respect to fnb genes and typical
S. aureus strains [20]. On the basis of the results of our examina-
tion, one may assume that different virulence determinants are
involved to initiate colonization in MRSA coagulase-negative
and clumping factor-negative strains as cna gene was present
only in coagulase-negative strains. Further research is necessary
to explain the presence of the bbp only in MSSA strains.
To summarize, we have shown that atypical, as coagulasenegative and clumping factor-negative, S. aureus strains isolated from clinical samples in Poland possess the genes of the
crucial staphylococcal adhesive proteins. The observed feature
encourages the development of new strategies for prevention
of bacterial colonization caused by such strains in hospitalized
patients, especially in relation to MRSA.
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