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  • 1. Disponible en ligne sur Médecine et maladies infectieuses 38 (2008) 549–553 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 prélèvements cliniques par détection des adhésines sélectives K. Wi´niewska ∗ , K. Garbacz, L. Piechowicz s Department of Medical Microbiology, Medical University of Gda´ sk, 38, Do Studzienki Street, n 80–227 Gda´ sk, Poland n Received 4 June 2007; accepted 3 June 2008 Available 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 presence of selected adhesin genes. Methods. – 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 confirmed by amplification mec A gene. The presence of bone binding protein (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 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. – Étude de la présence des gènes des adhésines sélectives chez des souches atypiques de Staphylococcus aureus à coagulase-négative et 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 été déterminée par screening en millieu gélosé additionné de 6 mg/L d’oxacilline et ensuite a été confirmée par amplification du gène mecA. La présence des 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 seulement 5 % : bbp. Il n’y avait aucune corrélation entre la présence des gènes des adhésines et l’isolement de prélèvements cliniques particuliers. La prévalence 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, clumping factorA; CNSA, coagulase-negative S. aureus; CFNSA, clumping factor-negative S. aureus. ∗ Corresponding author. E-mail address: (K. Wi´niewska). s 0399-077X/$ – see front matter © 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.medmal.2008.06.003
  • 2. 550 K. Wi´niewska et al. / Médecine et maladies infectieuses 38 (2008) 549–553 s (89, 89, 66 et 70, 70, 15 %, respectivement). De même, ces gènes ont été détectés plus fréquemment chez S. aureus résistants à la méthicilline (Sarm) que chez sensibles à la méthicilline (Sasm). La présence du gène cna n’a été observée que chez les S. aureus à coagulase-négative résistants 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 significant 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 specifically binds to distinct extracellular components of host tissues or to serum-conditioned implanted biomaterials such as catheters, artificial joints and vascular grafts [3]. There is evidence that some of the adhesive proteins of S. aureus are associated with specific invasive infections [4–6]. Coagulase and clumping factor are two species-specific proteins of S. aureus that were implicated in binding to fibrinogen 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 fibrinogen 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 [4–6], 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 fibrinogen 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: • • • • • • • 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 last year period. All isolates were subcultured on bovine blood agar and stored at −70 ◦ C in trypticase soy broth (TSB) with 50% glycerol until investigations. The staphylococcal species was identified by clumping factor test, free coagulase test and APIStaph ID 32 Kit (BioMèrieux, 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 identification was confirmed by PCR on the basis of presence of the species specific nuclease gene nuc [12]. Detection of methicillin resistance was performed by using the agar screen technique with 6 mg/L of oxacillin and was confirmed by amplification 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 χ2 test. A P value of less than 0.05 was considered significant. 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. Wi´niewska et al. / Médecine et maladies infectieuses 38 (2008) 549–553 s 551 Table 1 Prevalence of the adhesin genes in coagulase-negative (CNSA) and clumping factor-negative (CFNSA) S. aureus strains Prévalence 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 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 [3–6]. 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 significant 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 findings 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 confirmed 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 findings 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 gènes 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)
  • 4. 552 K. Wi´niewska et al. / Médecine et maladies infectieuses 38 (2008) 549–553 s Table 3 Distribution of adhesin genes among atypical S. aureus strains isolated from various clinical samples Distribution 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 Burn wound Nose, throat, ear Tracheostomy tube, bronchial fluid 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, specificity 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 findings, 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. References [1] Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:520–32. [2] Foster TJ, Hook M. Surface protein adhesions of Staphylococcus aureus. Trends Microbiol 1998;12:484–8. [3] Patti JM, Allen BL, McGavin MJ, Hook M. MSCRAMM-mediated adherence of microorganisms to host tissues. Ann Rev Microbiol 1994;48:585–617. [4] Hudson MC, Ramp WK, Frankenburg KP. Staphylococcus aureus adhesion to bone matrix and bone-associated biomaterials. FEMS 1999;173:279–84. [5] Johansson A, Flock JI, Svensson O. Collagen and fibronectin binding in experimental staphylococcal osteomyelitis. Clin Orthop 2001;382:241–6. [6] Patti JM, Bremmell T, Krajewska–Pietrasik D, Abdelnour A, Tarkowski A, Ryden C, et al. The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis. Infect Immun 1994;62:152–61. [7] Garbacz K, Piechowicz L, Wi´niewska K, Gali´ ski J. Charakterystyka s n defektywnych szczepów MRSA nie wytwarzaj˛ cych koagulazy lub czyna nika CF. Med Dosw Mikrobiol 2002;54:1–8. [8] Neville LO, Billington OJ, Kibbler CC, Gillespie SH. Methicillin resistant Staphylococcus aureus without clumping factor, protein A and Dnase. Lancet 1991;338:518. [9] Flock JI. Extracellular-matrix-binding proteins as targets for the prevention of Staphylococcus aureus infections. Mol Med Today 1999;5:532–7. [10] Hall AE, Domanski PJ, Patel PR, Vernachio JH, Syribeys PJ, Gorovits EL, et al. Characterization of protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A. Infect Immun 2003;71:6864–70. [11] Kloos WE, Lambe DW. Staphylococcus. Manual of clinical microbiology, 5th ed. American Society for Microbiology. Washington 1991 D.C. 222–237. [12] Barski P, Piechowicz L, Gali´ ski J, Kur J. Rapid assay for detection of n methicillin-resistant Staphylococcus aureus using multiplex PCR. Mol Cell Probes 1996;10:471–5.
  • 5. K. Wi´niewska et al. / Médecine et maladies infectieuses 38 (2008) 549–553 s [13] Tristan A, Ying L, Bes M, Etienne J, Vandenesch F, Lina G. Use of multiplex PCR to identify Staphylococcus aureus adhesions involved in human hematogenous infections. J Clin Microbiol 2003;41:4465–7. [14] Aricola CR, Campoccia D, Gamberini S, Baldassarri L, Montanaro L. Prevalence of cna, fnbA and fnbB adhesions genes among Staphylococcus aureus isolates from orthopedic infections associated to different types of implant. FEMS Microbiol Lett 2005;246:81–6. [15] Vancraeynest D, Hermans K, Haesebrouck F. Genotypic and phenotypic screening of high and low virulence Staphylococcus aureus isolates from rabbits for biofilm formation and MSCRAMMs. Vet Microbiol 2004;103:241–7. [16] Tung HS, Guss B, Hellman U, Persson L, Rubin K, Ryden C. A bone sialopotein-binding protein from Staphylococcus aureus: a member of the staphylococcal Sdr family. Biochem J 2000;345:611–9. 553 [17] Holderbaum D, Spech T, Ehrhart LA, Keys T, Hall GS. Collagen binding in clinical isolates of Staphylococcus aureus. J Clin Microbiol 1987;25:2258–61. [18] Thomas MG, Peacock S, Daenke S, Berent AR. Adhesion of Staphylococcus aureus to collagen is not a major virulence determinant for septic arthritis, osteomyelitis or endocarditis. J Infect Dis 1999;179:291–3. [19] Nashev D, Toshkova K, Salasia SI, Hassan AA, Lammler C, Zschock M. Distribution of virulence genes of Staphylococcus aureus isolated from stable nasal carriers. FEMS Microbiol Lett 2004;223:45–52. [20] Rice K, Huesca M, Vaz D, Mc Gavin MJ. Variance in fibronectin binding and fnb locus polymorphisms in Staphylococcus aureus: identification of antigenic variation in a fibronectin binding protein adhesin of the epidemic CMRSA-1 strain of methicillin-resistant S. aureus. Infect Immun 2001;69:3791–9.