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Médecine et maladies infectieuses 38 (2008) 549–553

Original article

Genotypic screening of atypical Staphylococcus aureus strains isolatedfrom clinical samples for presence of selected adhesin genes

Screening génotypique des souches atypiques de Staphylococcus aureusisolées de divers prélèvements cliniques par détection des adhésines

sélectivesK. Wisniewska ∗, K. Garbacz, L. Piechowicz

Department of Medical Microbiology, Medical University of Gdansk, 38, Do Studzienki Street,80–227 Gdansk, Poland

Received 4 June 2007; accepted 3 June 2008Available online 30 July 2008

Abstract

Objective. – The aim of this study was to screen Staphylococcus aureus negative for production of coagulase or clumping factor and for presenceof selected adhesin genes.

Methods. – Sixty coagulase-negative and 20 clumping factor-negative S. aureus strains were studied. Detection of methicillin resistance wasperformed using the agar screen technique with 6 mg/L of oxacillin and was confirmed by amplification mec A gene. The presence of bone bindingprotein (bbp), collagen binding protein (cna), fibronectin A binding protein (fnbA), fibronectin B binding protein (fnbB) and clumping factor A(clfA) genes was detected by multiplex PCR.

Results. – 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 strainsfound. No correlation between presence of the particular genes and clinical samples was observed. The prevalence of fnbA, fnbB and cna wasstatistically higher in coagulase-negative than in clumping factor-negative strains (89, 89, 66 and 70, 70, 15%, respectively). Similarly, all of thesegenes 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. – Étude de la présence des gènes des adhésines sélectives chez des souches atypiques de Staphylococcus aureus à coagulase-négativeet le facteur agglutinant (clumping factor [CF])-négatif, résistants et sensibles à la méthicilline.

Méthodes. – Soixante souches de S. aureus à coagulase-négative et 20 avec CF-négatif ont été étudiées. La résistance à la méthicilline a étédéterminée par screening en millieu gélosé additionné de 6 mg/L d’oxacilline et ensuite a été confirmée par amplification du gène mecA. La présencedes gènes bone binding protein (bbp), collagen binding protein (cna), fibronectin A binding protein (fnbA), fibronectin B binding protein (fnbB) et

clumping factor A (clfA) a été détectée par réaction de polymérisation en chaîne.

Résultats. – Quatre-vingt-dix-huit pour cent des souches étudiées possédaient le gène clfA ; 85 % : fnbA et fnbB ; 54 % : cna et seulement5 % : bbp. Il n’y avait aucune corrélation entre la présence des gènes des adhésines et l’isolement de prélèvements cliniques particuliers. Laprévalence des gènes 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, fibronectin A binding protein; fnbB, fibonectin binding proteinB; clfA, clumpingfactorA; CNSA, coagulase-negative S. aureus; CFNSA, clumping factor-negative S. aureus.

∗ Corresponding author.E-mail address: kwis@amg.gda.pl (K. Wisniewska).

0399-077X/$ – see front matter © 2008 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.medmal.2008.06.003

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89, 89, 66 et 70, 70, 15 %, respectivement). De même, ces gènes ont été détectés plus fréquemment chez S. aureus résistants à la méthicillineSarm) que chez sensibles à la méthicilline (Sasm). La présence du gène cna n’a été observée que chez les S. aureus à coagulase-négative résistantsla méticilline.2008 Elsevier Masson SAS. All rights reserved.

eywords: Adhesion; Clumping factor; Coagulase; Staphylococcus aureus

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ots clés : Adhésion ; Coagulase ; Facteur agglutinant ; Staphylococcus aureu

. Introduction

Staphylococcus aureus is a common pathogen in a significantumber of community-acquired and nosocomial infections [1].he attachment of S. aureus to host cells and tissues is essen-

ial step in the colonization process and, thus, a risk factor fornvasive disease [2]. The adherence of S. aureus is mediatedy a family of proteins, in most cases covalently anchored tohe cell peptidoglycan, termed microbial surface componentsecognizing adhesive matrix molecules (MSCRAMM), whichpecifically binds to distinct extracellular components of hostissues or to serum-conditioned implanted biomaterials suchs catheters, artificial joints and vascular grafts [3]. There isvidence that some of the adhesive proteins of S. aureus aressociated with specific invasive infections [4–6].

Coagulase and clumping factor are two species-specific pro-eins of S. aureus that were implicated in binding to fibrinogennd play important role in the ability of bacteria to cause infec-ions [2,3]. Clumping factor A (ClfA) is one of MSCRAMMhat promotes binding of fibrinogen to the bacterial cell surfacehile coagulase is an extracellular protein that binds pro-

hrombin to form staphylothrombin and this binding catalyseslasma clotting [3]. However, some clinical isolates, includingethicillin-resistant S. aureus (MRSA) strains, are not able to

roduce coagulase or clumping factor [7,8]. While the adhe-ion issue of S. aureus has been well examined in respect toarious pathological conditions [4–6], no studies of adherenceactors in atypical strains of this species has been performeds yet. Such investigations would permit better understandingf the ability of the strains, which are free from essential viru-ence factors for colonization host cells and tissues and might beseful in therapeutic aspect [9,10]. Thus, the aim of this studyas to examine coagulase-negative or clumping factor-negative. aureus strains for presence of genes encoding surface proteinsuch as clumping factor A (ClfA), bone binding protein (Bbp),ollagen binding protein (Cna) and fibrinogen A and B bindingroteins (FnbA and FnbB, respectively) the role of which as vir-lence factors has been well documented in typical strains ofhis species. In order to ascertain possible differences, the anal-sis of methicillin-resistant and methicillin-sensitive S. aureusMSSA) strains was performed.

. Materials and methods

.1. Bacterial isolates

The 80 S. aureus, including 60 coagulase-negative and 20lumping factor-negative strains used in this study, were clinical

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solates obtained from 21 polish medical centers, in majorityituated in Gdansk area. Clinical source of the strains was asollowing:

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 fluid (three strains).

The strains were isolated from varied patients over a 10 lastear period. All isolates were subcultured on bovine blood agarnd stored at −70 ◦C in trypticase soy broth (TSB) with 50%lycerol until investigations. The staphylococcal species wasdentified by clumping factor test, free coagulase test and API-taph ID 32 Kit (BioMèrieux, France). The clumping factoras detected in slide test with rabbit plasma (Biomed Warsaw,oland) [11]. Free coagulase test was performed in standard con-itions by incubating 0.8 ml of TSB culture with 0.2 ml rabbitlasma for 2, 4, 6 and 24 h at 37 ◦C [11]. S. aureus ATCC 12600train was used as positive control. The identification was con-rmed by PCR on the basis of presence of the species specificuclease gene nuc [12]. Detection of methicillin resistance waserformed by using the agar screen technique with 6 mg/L ofxacillin and was confirmed by amplification mec A gene [12].

.2. Detection of the adhesin genes

Detection of the following genes: bbp, cna, fnbA, fnbB, clfAas performed by multiplex PCR [13]. The staphylococcal DNAas extracted according to the procedure described previously

12]. Two primer sets were prepared for PCR: PCR1 to amplifybp and cna and PCR2 to amplify fnbA, fnbB and clfA. Theucleotide sequence of the primers and thermal cycling condi-ions were described by Tristan et al. [13].

.3. Statistical analysis

The data were analyzed by χ2 test. A P value of less than.05 was considered significant.

. Results

Almost all (98%) of the strains were positive for clfA gene.here were fnbA and fnbB in 85%, cna in 54% and bbp only

n 5% strains found. The fnbA, fnbB and cna were found in 89,

K. Wisniewska et al. / Médecine et maladies infectieuses 38 (2008) 549–553 551

Table 1Prevalence of the adhesin genes in coagulase-negative (CNSA) and clumping factor-negative (CFNSA) S. aureus strainsPrévalence des gènes 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 60 2 (3) 40 (66) 54 (89) 54 (89) 60 (100)C

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9 and 66% coagulase-negative strains and in 70, 70 and 15%lumping factor-negative strains, respectively (Table 1).

There were statistically differences between methicillin-esistant and MSSA strains observed. The fnbA, fnbB and cnaere in 90, 90 and 63% MRSA and, respectively, in 67, 67

nd 24% MSSA atypical strains detected. Among coagulase-egative strains, almost all MRSA and only 72, 72 and 15%SSA strains were positive for fnbA, fnbB and cna. Clumping

actor-negative MRSA strains were, in 82%, positive for fnbAnd fnbB but negative for cna (Table 2).

The most frequent adhesin genes as clfA and fnbA and Bere occurred in majority of the strains isolated from all clinical

amples (Table 3). Similarly, the strains positive for cna geneere obtained from all clinical source, but the frequency of cnaene varied from 25% for blood to 69% for nose, throat and ear.

. Discussion

The role of some of MSRAMMs as virulence determinants inhe pathogenesis of S. aureus disease is well documented [3–6].

oreover, the results obtained in experimental models suggesthat some of the adhesion proteins may be potential targets for therevention of staphylococcal infections [9,10]. For this aspect,enetic ability of S. aureus for presence of relevant adhesionediators must be well known, even with respect to atypical

trains. This study focused on getting familiar of the geneticbility to adhesion to host cells and tissues of S. aureus strains,

hich are negative in production of a crucial virulence deter-inants such clumping factor or coagulase. According to our

revious report, over 12.3 and 4.2% of S. aureus strains isolatedn Poland are coagulase- or clumping factor-negative, respec-

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able 2revalence of the adhesin genes in methicillin-resistant (MRSA) and methicillin-sensrévalence des gènes des adhesines dans les souches atypiques de S. aureus résistan

trains Number of strains Gene/Number of isol

bbp

RSACNSA 42 1 (2)CFNSA 17 0

Total 59 1 (2)

SSACNSA 18 1 (6)CFNSA 3 2 (67)

Total 21 3 (14)

3 (15) 14 (70) 14 (70) 18 (90)

43 (54) 68 (85) 68 (85) 78(98)

ively [7]. Moreover, most such strains are resistant to methicillin7,8].

As it has been described previously, ClfA promotes clumpingf bacterial cells in plasma and adherence of bacteria to bloodlots, to plasma conditioned biomaterials and to catheter dam-ged valves in a rat model of endocarditis [3]. Thus, it is probablysignificant factor in wound and foreign body infections anday be an excellent target for the generation of immune thera-

ies directed against S. aureus [10]. All clumping factor-negativetrains and almost all coagulase-negative strains examined in thistudy were positive for clfA gene. On the basis of this result, oneould assume that the negative reaction for clumping factor testight be caused by blocking up the expression of this gene.nother possible explanation of this effect is a too low concen-

ration of the expressed protein or reduced availability on theacterial surface.

Multiplicity of the adhesions necessary for the recognition ofarious receptors seems to be an important factor in the develop-ent of infection and may help to increase the pathogenicity ofgiven strain [2,3]. In comparison with the findings of others,

lumping factor- or coagulase-negative S. aureus strains seemot to be differed in genetic ability to poses crucial virulencedhesins from typical strains of this species [2,14,15]. In mostases of the present study, besides clfA, fnbA and B genes wereound together in the same isolate. It has been underlined byany authors, that the Fnb plays an important role in virulence

ction of S. aureus in human host [2,9,14]. Moreover, biological

ffect of Fnb has been confirmed in rat endocarditis infection [2].s it has been described, this protein is found to be very frequent

n clinical strains of S. aureus [2,14]. Our findings show thatenes responsible for production Fnb are widely distributed also

itive (MSSA) atypical S. aureus strains.ts (SARM) et sensibles à la méthicilline (SASM)

ates (%)

cna fnbA fnbB clfA

38 (90) 40 (95) 40 (95) 42 (100)0 14 (82) 14 (82) 15 (88)

38 (63) 54 (90) 54 (90) 57 (97)

3 (17) 13 (72) 13 (72) 18 (100)3 (100) 0 0 3 (100)

5 (24) 14 (67) 14 (67) 21(100)

552 K. Wisniewska et al. / Médecine et maladies infectieuses 38 (2008) 549–553

Table 3Distribution of adhesin genes among atypical S. aureus strains isolated from various clinical samplesDistribution de gènes des adhésines dans les souches atypiques de S. aureus isolées de différents prélèvements cliniques

Clinical source Number of isolates (100%) Genes/Number of strains (%)

bbp cna fnbA fnbB clfA

Pus and purulent lesions 31 0 14 (45) 29 (94) 29 (94) 31 (100)Burn wound 22 1 (5) 15 (68) 18 (82) 18 (82) 22 (100)Nose, throat, ear 13 3 (23) 9 (69) 9 (69) 9 (69) 13 (100)Tracheostomy tube, bronchial fluid 7 0 2 (29) 7 (100) 7 (100) 7 (100)Blood 4 0 1 (25) 3 (75) 3 (75) 2 (50)Catheters 3 0 2 (67) 2 (67) 2 (67) 3 (100)

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n atypical strains of this species. Additionally research is neededo answer why coagulase-negative strains are statistically morerequent positive for fnb genes than clumping factor-negativetrains.

In contrast, bbp were found only in 5% of examined strains.here is clear evidence that the Bbp is a crucial factor in bonend joint infections caused by S. aureus [4,16]. Thus, the gene ofhis protein would be expected to be present mostly in isolatesausing such infections. Some of the strains of our collectionere isolated from infections observed in patients from ortho-edic unit where bone and joint infections are most common.nexpectedly, no one strain positive for the bbp was derived

rom orthopedic unit. Taking the results obtained by Tung etl. into account, specificity of Bbp for a factor other than boneialoprotein cannot be excluded [16].

The other MSCRAMM that is possibly important in staphy-ococcal infection is Cna. The Cna protein mediates bacterialdherence to collagen substrates and collagenous tissues and its necessary for S. aureus cells to adhere to cartilage in vitro [2].s it has been described, the presence of cna is not generally

xpressed by the majority of strains and may differ from 38 to6% positive results [2,17,18]. There is evidence that strains iso-ated from healthy nasal carriers harbouring cna gene were foundo be about 48% [19]. As shown by the results of our findings,nly 54% of atypical S. aureus possesses genetic ability to pro-uce Cna. Moreover, similarly to fnb genes, cna gene is found toe more common in coagulase-negative than in clumping factor-egative strains and exists without any correlation with clinicalamples.

The vast majority of the examined strains were resistant toethicillin. This is in agreement with other reports giving evi-

ence that the negative result of clumping factor or coagulaseetection is more common in MRSA than in MSSA strains7,8]. This phenomenon can be explained by the insertion intoacterial genome sequence of methicillin resistance that maynclude DNA elements altering some bacterial properties [7].he results of our study show that atypical strains resistant

o methicillin much more often possess genetic ability to pro-

uce cna and both fnb A and B genes than strains sensitiveo this antibiotic. The indications of Rice et al. would sup-ort this observation, with respect to fnb genes and typical. aureus strains [20]. On the basis of the results of our examina-

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4 (5) 43 (54) 68 (85) 68 (85) 78 (98)

ion, one may assume that different virulence determinants arenvolved to initiate colonization in MRSA coagulase-negativend clumping factor-negative strains as cna gene was presentnly in coagulase-negative strains. Further research is necessaryo explain the presence of the bbp only in MSSA strains.

To summarize, we have shown that atypical, as coagulase-egative and clumping factor-negative, S. aureus strains iso-ated from clinical samples in Poland possess the genes of therucial staphylococcal adhesive proteins. The observed featurencourages the development of new strategies for preventionf bacterial colonization caused by such strains in hospitalizedatients, especially in relation to MRSA.

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