- Email: [email protected]
Distribution of virulence genes of Staphylococcus aureus isolated from stable nasal carriers
FEMS Microbiology Letters 233 (2004) 45–52 www.fems-microbiology.org
Distribution of virulence genes of Staphylococcus aureus isolated from stable nasal carriers Dimitar Nashev a, Katia Toshkova a, S. Isrina O. Salasia b, Abdulwahed A. Hassan c, €ck c Christoph L€ ammler d,*, Michael Zscho b
a National Center of Infectious and Parasitic Diseases, 26 Y. Sakazov Blvd, 1504 Sofia, Bulgaria Clinical Pathology Department, Faculty of Veterinary Medicine, Gadjah Mada University, Sekip Unit II, 55281 Yogyakarta, Indonesia c Staatliches Untersuchungsamt Hessen, Marburger Str. 54, 35396 Gießen, Germany d Institut f€ ur Pharmakologie und Toxikologie, Justus-Liebig-Universit€at Gießen, Frankfurter Straße 107, 35392 Gießen, Germany
Received 30 September 2003; received in revised form 22 December 2003; accepted 21 January 2004 First published online 6 February 2004
Abstract In the present study, we report data on virulence determinants of Staphylococcus aureus from stable nasal carriers, emphasizing on the genes encoding fibronectin (fnbA, fnbB) and collagen (cna) adhesive molecules. Of the 44 S. aureus isolates included, 32 isolates (16 pairs) were cultured from the anterior nares of 16 healthy carriers, eight isolates (four pairs) were collected from the nose of four patients with recurrent skin infections and four isolates were obtained from the infection site of these patients. The period between the two nasal swabs taken was 3–5 days. The persistency of carriage could be demonstrated by the indistinguishable genotypic chracteristics of the S. aureus isolates in each pair. This could be shown by determination of gene polymorphisms of coa gene and the X-region and IgG-binding region encoding segments of spa gene. In addition, the isolates within the pairs showed identical toxin patterns. This was determined by PCR amplification of the genes encoding staphylococcal enterotoxins (SEA to SEJ) and TSST-1. The genotypic properties also yielded an identity between persistent nasal carriage isolates and the corresponding skin infection isolates of the four patients. In addition, all S. aureus nasal and skin infection isolates were positive for gene fnbA, fnbB and cna could be found with a high frequency. Among the 44 isolates investigated, 16 isolates (36.7%) harbored gene fnbB and 21 isolates (47.7%) gene cna. The data in the present study showed a relatively wide distribution of the genes fnb and cna among the investigated isolates, indicating that the persistent carriage of strains harboring these virulence determinants may increase the risk for subsequent invasive infections in carriers. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Staphylococcus aureus; Nasal carriage; fnbA; fnbB; cna
1. Introduction The nasal carriage of Staphylococcus aureus represents a risk factor for subsequent invasive infection of patients and also for interpatient transmission. It was described that a nasal carrier state seems to be the origin for the * Corresponding author. Tel. +49-641-9938406; fax: +49-6419938409. E-mail address: [email protected] (C. L€ ammler).
development of staphylococcal infections in specific groups of patients, including patients on haemodialysis and continuous ambulatory peritoneal dialysis (CAPD), HIV/AIDS patients as well as patients with postsurgical wounds and patients with intravascular catheters [1,2]. In addition, the nasal carriage of S. aureus appears to play some role in the epidemiology and pathogenesis of staphylococcal skin infections [3]. The nasal carriage rate in adult populations have been reported to be between 30% and 50%. Among these individuals 20–35% carry the organism consistently,
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.01.032
46
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
designated as persistent or stable carriers, and 30–70% intermittently, designated as intermittent nasal carriers [1]. According to Doebbeling [4] the criteria for a stable carriage of S. aureus requires two positive and identical nasal swab cultures on two separate occasions, at least 24 h apart. However, the molecular basis for this persistent or intermittent nasal colonization is still insufficiently cleared. The microbial adherence to cells and extracellular matrix is considered as an essential first step in the process of colonization and infection [1,5]. A wellcharacterized family of staphylococcal surface adhesins, called MSCRAMMs (‘‘microbial surface components recognizing adhesive matrix molecules’’) are known to mediate adherence to host extracellular matrix components, such as fibrinogen, fibronectin and collagen [1,6]. Binding to fibronectin is mediated by two closely related proteins, FnBPA and FnBPB, encoded by the genes fnbA and fnbB [7,8]. The collagen binding protein CNA, as second important adhesive molecule is encoded by gene cna [9]. The S. aureus collagen adhesin occurs in at least four forms that differ in the number (one, two, three, or four) of B domains which contain 187 amino acids and are located between the domains that anchor CNA to the cell envelope and the ligand-binding A domain [10]. FnBPs mediate adhesion of S. aureus to human epithelial cells, including airway epithelium, endothelial cells and fibroblasts which may cause a subsequent internalization of the bacteria in these cells [11]. FnBPA and FnBPB are required for bacterial adhesion to fibronectin-coated surfaces. However, fnbA or fnbB defective single mutants showed no significant reduction in their adhesive properties, whereas the double mutants were completely unable to adhere [12]. In addition, S. aureus isolates associated with invasive diseases, including endocarditis, primary septic arthritis and/or osteomyelitis were more likely to have both genes fnbA and fnbB [13]. CNA of S. aureus was described as virulence factor in septic arthritis [14] and osteomyelitis [15]. CNA also seems to play an important role in the pathogenesis of S. aureus endocarditis and keratitis [6,16]. Recently, Peacock et al. [17] investigated the presence of 33 putative virulence determinants of S. aureus and found that the genes fnbA and cna were significantly more common in invasive isolates. In contrast to the relatively well-studied adherence mechanisms and surface components of S. aureus invasive isolates, the studies concerning the adhesive molecules in persistently or intermittently colonizing nasal mucosa strains are still limited in number [5,18]. In the present study, we report data on genotypic characteristics of paired isolates from persistent nasal carriers, especially emphasizing on the genes encoding adhesive molecules.
2. Materials and methods 2.1. Bacterial isolates A total of 44 S. aureus isolates from 20 nasal carriers were included in the present study. Thirty two isolates (16 pairs) were collected from the anterior nares of 16 healthy carriers, eight isolates (four pairs) were cultured from the nose of four patients with recurrent skin infections. The corresponding isolates from the infection site of these four patients were also included in the study. The period between the two nasal swabs taken was 3–5 days. The swabs were immediately streaked on 5% sheep blood agar plates. After incubation for 24 h at 37 C, suspected colonies of S. aureus were isolated and further identified with the commercial identification system API Staph (Bio Merieux, Marcy-lÕEtoile, France) as recommended by the manufacturer. The strains were additionally investigated by tube-coagulase test (Bactident-coagulase, Merck, Darmstadt, Germany) and by clumping factor reaction with rabbit plasma on microscope slides. 2.2. PCR mediated identification and further characterization The determination of species specific parts of the genes encoding 23S rRNA and thermonuclease (nuc) was performed with oligonucleotide primers (MWG Biotech, Ebersberg, Germany) listed in Table 1. The PCR reaction mixture (20 ll) for both genes contained 0.7 ll primer 1 (10 pmol), 0.7 ll primer 2 (10 pmol), 0.4 ll dNTP (10 mM), 3.0 ll of 10· thermophilic buffer (Sigma–Aldrich Chemie, Steinheim, Germany), 1.2 ll MgCl2 (25 mM, Sigma), 0.1 ll Taq DNA polymerase (5 U/ll, Sigma) and 12.9 ll of distilled water. Finally, 2.0 ll of DNA preparation was added to each 0.2 ml reaction tube. The DNA of the isolates was prepared with the DNeasy Tissue Kit (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer with the modification that 5 ll of lysostaphin (1.8 U/ll, Sigma) was added to the cell lysis step. The DNA amplification was carried out with the cycling profiles shown in Table 1. The presence of PCR products was determined by electrophoresis of 8 ll of the reaction product in a 2% agarose gel, with Trisborate electrophoresis buffer (89 mM Tris-borate; 2 mM EDTA; pH 8.3) at 120 V and visualised under UV light using Gel Doc 1000 system (BioRad, M€ unchen, Germany). The product sizes were determined by using DNA molecular weight marker VI (0.154–2.18 kbp) or VIII (0.019–1.11 kbp) (both Roche Diagnostics, Mannheim, Germany). The determination of the genes encoding coagulase (coa), clumping factor (clfA), protein A (spa), the staphylococcal enterotoxins A (sea), B (seb), C (sec), D
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
47
Table 1 Oligonucleotide primers and PCR programs used for amplification of the genes encoding 23S rRNA and various other staphylococcal proteins including toxins and adhesive molecules Target gene
Sequence (50 –30 )
Size of amplified products (bp)
PCR program
Reference
23S rRNA
ACG GAG TTA CAA AGG ACG AC AGC TCA GCC TTA ACG AGT AC GCG ATT GAT GGT GAT ACG GTT ACG CAA GCC TTG ACG AAC TAA AGC ATA GAG ATG CTG GTA CAG G GCT TCC GAT TGT TCG ATG C GGC TTC AGT GCT TGT AGG TTT TCA GGG TCA ATA TAA GC CAA GCA CCA AAA GAG GAA CAC CAG GTT TAA CGA CAT CAC CTG CTG CAA ATG CTG CG GGC TTG TTG TTG TCT TCC TC AAA GTC CCG ATC AAT TTA TGG CTA GTA ATT AAC CGA AGG TTC TGT AGA TCG CAT CAA ACT GAC AAA CG GCA GGT ACT CTA TAA GTG CC GAC ATA AAA GCT AGG AAT TT AAA TCG GAT TAA CAT TAT CC CTA GTT TGG TAA TAT CTC CT TAA TGC TAT ATC TTA TAG GG TAG ATA AGG TTA AAA CAA GC TAA CTT ACC GTG GAC CCT TC AAT TAT GTG AAT GCT CAA CCC GAT C AAA CTT ATA TGG AAC AAA AGG TAC TAG TTC CAA TCA CAT CAT ATG CGA AAG CAG CAT CTA CCC AAA CAT TAG CAC C CTC AAG GTG ATA TTG GTG TAG G AAA AAA CTT ACA GGC AGT CCA TCT C CAT CAG AAC TGT TGT TCC GCT AG CTG AAT TTT ACC ATC AAA GGT AC ATG GCA GCA TCA GCT TGA TA TTT CCA ATA ACC ACC CGT TT GCG GAG ATC AAA GAC AA CCA TCT ATA GCT GTG TGG GGA GAA GGA ATT AAG GCG GCC GTC GCC TTG AGC GT ATA TGA ATT CGA GTA TAA GGA AGG GGT T TTT GGA TCC CTT TTT CAG TAT TAG TAA CCA AGT GGT TAC TAA TAC TG CAG GAT AGT TGG TTT A
1250
1a
[27]
279
2
[28]
Size polymorphisms
3
[29]
Size polymorphisms
4
[30]
Size polymorphisms
5
[20]
Size polymorphisms
3
[31]
216
6
[32]
478
6
[33]
257
6
[33]
317
6
[33]
170
6
[33]
642
6
[23]
375
6
[23]
576
6
[23]
142
7
[34]
350
6
[33]
1279
8
[35]
812
8
[8]
1609
8
[14]
1122
8
[14]
nuc coa clfA spa (X-region) spa (IgG-binding region) sea seb sec sed see seg seh sei sej tst fnbA fnbB cna (A domain) cna (B domain)
a 1, 37 · (94 C 40 s, 64 C 60 s, 72 C 75 s); 2, 37 · (94 C 60s, 55 C 30s, 72 C 30s); 3, 30 · (94 C 60 s, 58 C 60 s, 72 C 60 s); 4, 35 · (94 C 60 s; 57 C 60 s; 72 C 60 s); 5, 30 · (94 C 60 s, 60 C 60 s, 72 C 60 s); 6, 30 · (94 C 120 s, 55 C 120 s, 72 C 60 s); 7, 30 · (94 C 60 s, 62 C 60 s, 72 C 60 s); 8, 30 · (94 C 30 s, 50 C 30 s, 72 C 60 s).
(sed), E (see), G (seg), H (seh), I (sei) and J (sej), toxic shock syndrome toxin 1 (tst) and the adhesive molecules FnBPA, FnBPB and CNA domain A and B (fnbA, fnbB, cnaA and cnaB, respectively) were performed with the primers and thermocycler programs listed in Table 1. For control purposes the following S. aureus reference strains were used: 619/93 (sea), 62/92 (seb), 1229/93 (sec), 1644/93 (sed), FRI 918 (see), 161/93 (tst), Ly 990055 (seg, sei), Ly 990552 (seh). The strains were kindly provided by W. Witte, Robert-Koch-Insitut, Wernigerode, Germany and G. Lina, Centre Nationale des Toxemies a Staphylococques, Lyon, France. Positive controls for the remaining PCR reactions were obtained
from the strain collections of Institut f€ ur Pharmakologie und Toxikologie and Staatliches Untersuchungsamt Hessen.
3. Results According to cultural and biochemical properties, determined with the API Staph system, a positive clumping factor reaction and a positive tube coagulase test all 44 isolates used in the present study could be identified as S. aureus. The identification of the isolates could be confirmed by PCR amplification of species specific parts of the gene encoding 23S rRNA and by
48
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
Fig. 1. (a) Amplicons of the gene encoding staphylococcal coagulase. Isolates 17a, 17b and 17c (lanes 1, 2 and 3), isolates 5a and 5b (lanes 4 and 5), isolates 9a and 9b (lanes 6 and 7), isolate 18a (lane 8); M: DNA molecular weight marker VI (Roche). (b) Amplicons of the spa gene segment encoding X-region of protein A. Isolate 7a (lane 1), isolates 9a and 9b (lanes 2 and 3), isolates 15a and 15b (lanes 4 and 5); M: DNA molecular weight marker VIII (Roche). (c) Amplicons of the genes encoding fibronectin binding protein A (1300 bp, lanes 1 and 2) and B (900 bp, lane 3); fnbB negative isolate (lane 4); M: see (a). (d) Amplicons of the gene encoding collagen binding protein domain B (1200 bp, lanes 1 and 2) and domain A (1600 bp, lanes 3 and 4); M: see (a).
amplification of thermonuclease gene nuc. The amplicons of these genes showed an uniform size of approximately 1250 and 270 bp, respectively. In addition, the identification was confirmed by amplification of the gene coa encoding coagulase and gene spa encoding the Xand IgG-binding regions of protein A. Both genes or gene segments displayed typical gene polymorphisms (Figs. 1(a) and (b)) with indistinguishable sizes for the isolates of each pair. The exception were the isolates from patient 18, which showed a difference in the amplicon size of the spa gene segment encoding X-region of protein A. The amplification of the clumping factor gene clfA revealed a single amplicon with a size of approximately 1000 bp with the exception of two isolates (one pair), which displayed an amplicon size of 890 bp. Among the 44 S. aureus isolates examined, 12 isolates were positive for the genes encoding SEG and SEI, five isolates for the genes encoding SEB and SEI, four isolates for the genes encoding SEB, SEG, SEH and SEI, four isolates for the genes encoding SEA, SEG, SEI and TSST-1, two isolates for the genes encoding SEA, SED, SEG, SEI and SEJ, two isolates for the gene encoding SEB, and two isolates for the gene encoding SEH. The genes sea, seb, sec, sed, see, seg, seh, sei, sej and tst occurred pairwise. The four isolates from skin in-
fections and their corresponding nasal cultures showed indistinguishable size of coa gene and spa (X-region and IgG-binding region) gene amplicons and had the same toxin genotype (Table 2). The amplification of the genes fnbA, fnbB and cna (A domain) resulted in amplicons with sizes of approximately 1300, 900 and 1600 bp, respectively (Figs. 1(c) and (d)). The amplification of the B domain(s) of the gene cna revealed an uniform size of approximately 1200 bp, which corresponds to two copies of the 187-aminoacid repeat motif. All S. aureus isolates were found to be positive for gene fnbA. Among the 40 nasal isolates (20 pairs) investigated, 16 isolates (eight pairs, 40%) harbored gene fnbB, 18 isolates (nine pairs, 45%) harbored gene cna. Three of the four skin infection isolates were positive for cna and none of these strains was positive for fnbB. The genotypic characteristics of the S. aureus isolates are presented in Table 2.
4. Discussion Many healthy people are persistently or intermittently colonized with S. aureus at their anterior nares and thus are at increased risk for certain staphylococcal
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
49
Table 2 Genotypic characteristics of the 44 S. aureus isolates from nares (n ¼ 40) and skin infections (n ¼ 4) Source
coa (bp)
spa (X-region)
spa (IgG-binding region)
clfA
Toxin gene pattern
fnbA
fnbB
cna
1
1a 1b
800a 800
240 240
700 700
1000 1000
seg, sei seg, sei
+ +
) )
+ +
2
2a 2b
600 600
320 320
900 900
1000 1000
) )
+ +
+ +
) )
3
3a 3b
600 600
350 350
900 900
1000 1000
) )
+ +
+ +
) )
4
4a 4b
500 500
320 320
900 900
1000 1000
) )
+ +
+ +
) )
5
5a 5b
690 690
320 320
900 900
1000 1000
) )
+ +
) )
) )
6
6a 6b
500 500
320 320
900 900
1000 1000
sea, seg, sei, tst sea, seg, sei, tst
+ +
) )
+ +
7
7a 7b
600 600
240 240
900 900
1000 1000
seg, sei seg, sei
+ +
) )
) )
8
8a 8b
600 600
240 240
900 900
1000 1000
seh seh
+ +
+ +
+ +
9
9a 9b
690 690
280 280
700 700
890 890
sea, sed, seg, sei, sej sea, sed, seg, sei, sej
+ +
+ +
) )
10
10a 10b
500 500
320 320
900 900
1000 1000
sea, seg, sei, tst sea, seg, sei, tst
+ +
) )
+ +
11
11a 11b
600 600
280 280
900 900
1000 1000
seb, seg, seh, sei seb, seg, seh, sei
+ +
+ +
) )
12
12a 12b
600 600
300 300
900 900
1000 1000
seb, seg, seh, sei seb, seg, seh, sei
+ +
+ +
) )
13
13a 13b
800 800
280 280
700 700
1000 1000
seb seb
+ +
) )
) )
14
14a 14b
800 800
320 320
700 700
1000 1000
) )
+ +
+ +
+ +
15
15a 15b
690 690
300 300
900 900
1000 1000
seb, sei seb, sei
+ +
) )
) )
16
16a 16b
500 500
240 240
900 900
1000 1000
seg, sei seg, sei
+ +
) )
+ +
17
17a 17b 17c
600 600 600
240 240 240
900 900 900
1000 1000 1000
) ) )
+ + +
) ) )
+ + +
18
18a 18b 18c
900 900 900
240 280 280
700 700
1000 1000 1000
seg, sei seg, sei seg, sei
+ + +
) ) )
+ + +
19
19a 19b 19c
690 690 690
300 300 300
900 900 900
1000 1000 1000
seb, sei seb, sei seb, sei
+ + +
) ) )
) ) )
20
20a 20b 20c
900 900 900
180 180 180
700 700 700
1000 1000 1000
seg, sei seg, sei seg, sei
+ + +
) ) )
+ + +
a
a and b, anterior nares; c, skin infection. Approximate size in bp.
infections. The exact reasons for these differences in colonization patterns are yet unknown. One reason might be a host predisposition. On the other hand, the
properties of the resident strain might be the leading factor in pathogenesis of nasal carriage. However, at present, relatively little is known about the basic surface
50
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
components of the staphylococcal cell wall which could be connected with the persistency of carriage. A study of van Belkum et al. [18] failed to prove a correlation between the polymorphisms of the protein A and coagulase genes and the persistency of colonization. In the present study, the persistency of carrier state could be demonstrated by the indistinguishable size of amplicons and toxin genotypes of the S. aureus isolates in each pair. In addition, comparable to previous studies [3], the relation between persistent nasal carriage isolates and corresponding skin infection isolates could be demonstrated for S. aureus isolates of four patients. The determination of the coa gene polymorphisms and the X-region and IgG-binding region encoding segments of spa gene revealed an indistinguishable size of amplicons in isolates of each pair. A difference in size of the Xregion encoding gene segment of one isolate of patient 18 could be explained by a deletion of segments in this region [19]. However, gene polymorphisms of the genes spa and coa had been commonly used for epidemiological typing of S. aureus [20]. The isolates in the present study were additionaly investigated for the genes encoding the classical and the newly described staphylococcal enterotoxins and TSST-1. As described previously, genotyping S. aureus by determination of toxin patterns could be an useful tool for comparing epidemiologically related strains [21]. According to the results of the present study, the isolates within the pairs were indistinguishable according to their toxin genotypes. The toxin genes seg and sei seemed to be the most prevalent toxin genes, occuring in combination in 20 of the 40 nasal isolates. These results are comparable to data shown by Jarraud et al. [22] in which 57% of the investigated strains from nasal carriers harbored the genes seg and sei. These authors previously reported that seg and sei are present in S. aureus in tandem orientation on a 3.2 kb DNA fragment [23]. The seg and sei genes were present in most strains (67%) associated with suppurative infections [22]. It was of interest that five cultures of the present study positive for sei were negative for seg. This could possibly be explained by a mutation of the seg primer binding site or a variation in the enterotoxin gene cluster. The gene seh, encoding SEH, also found among the strains of the present study, has been described to have sequence homology to the sea subfamily. In addition, SEH has the highest affinity ever measured for an enterotoxin for MHC class II molecules [24]. Among the genes encoding classical staphylococcal enterotoxins the most prevalent toxin gene was seb (25%), followed by sea (15%) and sed (5%). The gene encoding TSST-1 was only present in two cultures. These results are in contrast with the studies of Peacock et al. [17], who presented data that 44% of investigated carriage isolates harbored the gene tst, 30% were positive for sea, 13% for seb and 9% for sed. Both sed positive cultures in the present study were
simultaneously positive for sej. As shown in the studies of Zhang et al. [25], SED and SEJ are encoded by a plasmid and separated from each other by an intergenic region. Mongodin et al. [5] demonstrated the major role of FnBPs in S. aureus adherence to human airway epithelium, whereas staphylococcal protein A and clumping factor did not play any role in this process. These authors also reported that 97% of 32 clinical strains, isolated from the airway secretions of patients with cystic fibrosis and nosocomial pneumonia, possessed the two fnb genes. Thus, the presence of these genes seems to be an important factor for microbial pathogenesis. Booth et al. [26] investigated different S. aureus lineages associated with various sites of infection and nasal carriage and showed that 89.7% of the isolates, regardless of lineage identity, possesed gene fnbA, whereas fnbB was observed in 20.1% of the isolates. The latter was present only in the lineage associated with blood isolates. A positive signal for cna could be demonstrated for 44.9% of the isolates, regardless of the lineage. Peacock et al. [17] compared S. aureus isolates from healthy carriers and from patients with invasive disease for variation in the presence of 33 putative virulence determinant genes, also including fnbA and cna. The authors showed that 98% of invasive isolates harbored fnbA versus 87% in the carriage group. The gene cna appeared also significantly more common in disease isolates (52%) than in colonizing strains (32%). Comparable to these previous studies the data from the present study showed a relatively wide distribution of the genes fnb and cna among the isolates obtained from stable carriers. All 44 isolates harbored the gene fnbA and 36.7% of the isolates were positive for fnbB. The occurrence of gene cna also seems to be enhanced with 21 (47.7%) of the 44 isolates, indicating that both genes might play a role for the persistency of carriage. A further investigation of intermittently colonizing S. aureus might show the importance of this relation. The role of S. aureus nasal carriage for the pathogenesis of reccurent skin infections, as shown for four patients in the present study and in previous studies [3], indicated that an eradication of S. aureus nasal carriage is a strong demand for prevention of these infections. However, nasal colonization with strains carrying various virulence determinants, including fibronectin and collagen adhesins may also represent an increased risk factor for subsequent invasive infections in carriers.
Acknowledgements This work was supported by German Academic Exchange Service (DAAD) (D. Nashev) and by Alexander von Humboldt Foundation (S.I.O. Salasia).
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
References [1] Kluytmans, J., van Belkum, A. and Verbrugh, H. (1997) Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin. Microbiol. Rev. 10, 505– 520. [2] Wenzel, R. and Pearl, T. (1995) The significance of nasal carriage of Staphylococcus aureus and the incidence of postoperative wound infection. J. Hosp. Infect. 31, 13–24. [3] Toshkova, K., Annem€ uller, C. and L€ammler, C. (2001) The significance of nasal carriage of Staphylococcus aureus as risk factor for human skin infections. FEMS Microbiol. Lett. 202, 17–24. [4] Doebbeling, B.N. (1994) Nasal and hand carriage of Staphylococcus aureus in healthcare workers. J. Chemotherapy VI (Suppl. 2), 11–17. [5] Mongodin, E., Bajolet, O., Cutrona, J., Bonnet, N., Dupuit, F., Puchelle, E. and de Bentzmann, S. (2002) Fibronectin-binding proteins of Staphylococcus aureus are involved in adherence to human airway epithelium. Infect. Immun. 70, 620–630. [6] Navarre, W. and Schneewind, O. (1999) Surface proteins of Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63, 174–229. [7] Flock, J., Fromann, G., Jonsson, K., Guss, B., Sign€as, C., Nilsson, € k, M., Wadstr€ B., Raucci, G., H€ oo om, T. and Lindberg, M. (1987) Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus. EMBO J. 6, 2351–2357. [8] Jonsson, K., Sign€ as, H., M€ uller, H. and Lindberg, M. (1991) Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene. Eur. J. Biochem. 202, 1041–1048. [9] Patti, J.M., Jonsson, H., Guss, B., Switalski, L.M., Wiberg, K., € k, M. (1992) Molecular characterization Lindberg, M. and H€ oo and expression of a gene encoding a Staphylococcus aureus collagen adhesin. J. Biol. Chem. 267, 4766–4772. [10] Gillaspy, A., Patti, J., Pratt, F., Iandolo, J. and Smeltzer, M. (1997) The Staphylococcus aureus collagen adhesin-encoding gene (cna) is within a discrete genetic element. Gene 196, 239–248. [11] Dziewanowska, K., Patti, J., Deobald, C., Bayles, K., Trumble, W. and Bonach, G. (1999) Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells. Infect. Immun. 67, 4673–4678. [12] Greene, C., McDevitt, D., Francois, P., Vaudaux, P., Lew, D. and Foster, T. (1995) Adhesion properties of mutants of Staphyloccus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes. Mol. Microbiol. 17, 1143–1152. [13] Peacock, S., Day, N., Thomas, M., Berendt, A. and Foster, T. (2000) Clinical Isolates of Staphylococcus aureus exhibit diversity in fnb genes and adhesion to human fibronectin. J. Infect. 41, 23–31. [14] Switalski, L., Patti, J., Butcher, W., Gristina, A., Speziale, P. and € k, M. (1993) A collagen receptor on Staphyloccus aureus H€ oo strains isolated from patients with septic arthritis mediates adhesion to cartilage. Mol. Microbiol. 7, 99–107. [15] Elasri, M., Thomas, J., Skinner, R., Blevins, J., Beenken, K., Nelson, C. and Smeltzer, M. (2002) Staphyloccus aureus collagen adhesin contributes to the pathogenesis of osteomyelitis. Bone 30, 275–280. €k, M., Jones, D. [16] Rhem, M., Lech, E., Patti, J., McDevitt, D., H€ oo and Wilhelmus, K. (2000) The collagen-binding adhesin is a virulence factor in Staphylococcus aureus keratitis. Infect. Immun. 68, 3776–3779. [17] Peacock, S., Moore, C., Justice, A., Kantazanou, M., Story, L., Mackie, K., OÕNeil, G. and Day, N. (2002) Virulent combinations of adhesin and toxin genes in natural populations of Staphyloccus aureus. Infect. Immun. 70, 4987–4996. [18] van Belkum, A., Riewarts Eriksen, N.H., Sijmons, M., van Leeuwen, W., van den Bergh, M., Kluytmans, J., Espersen,
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
51
F. and Verbrugh, H. (1997) Coagulase and protein A polymorphisms do not contribute to persistence of nasal colonization by Staphylococcus aureus. J. Med. Microbiol. 46, 222–232. Toshkova, K., Savov, E., Soedarmanto, I., L€ammler, C., Chankova, D., van Belkum, A., Verbrugh, H.A. and van Leeuwen, W. (1997) Typing of Staphylococcus aureus isolated from nasal carriers. Zbl. Bakt. 286, 547–559. Frenay, H.M.E., Bunschoten, A.E., Schouls, L.M., van Leeuwen, W.J., Vandenbrouke-Grauls, C.M.J.E., Verhoef, J. and Mooi, F.R. (1996) Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism. Eur. J. Clin. Microbiol. Infect. Dis. 15, 60–64. Nishi, J., Yoshinaga, M., Miyanohara, H., Kawahara, M., Kawabata, M., Motoya, T., Owaki, T., Oiso, S., Kawakami, M., Kamewari, S., Koyama, Y., Wakimoto, N., Tokuda, K., Manago, K. and Maruyama, I. (2002) An epidemiologic survey of methicillin-resistant Staphylococcus aureus by combined use of mec-HVR genotyping and toxin genotyping in a university hospital in Japan. Infect. Control. Hosp. Epidemiol. 23, 506– 510. Jarraud, S., Peyrat, M.A., Lim, A., Tristan, A., Bes, M., Mougel, C., Etienne, J., Vandenesch, F., Bonneville, M. and Lina, G. (2001) Egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J. Immunol. 166, 669–677. Jarraud, S., Cozon, G., Vandenesch, F., Bes, M., Etienne, J. and Lina, G. (1999) Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J. Clin. Microbiol. 37, 2446–2449. Nilson, H., Bj€ ork, P., Dohlsten, M. and Antonsson, P. (1999) Staphylococcal enterotoxin H displays unique MHC class IIbinding properties. J. Immunol. 163, 6686–6693. Zhang, S., Iandolo, J.J. and Stewart, G.C. (1998) The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej). FEMS Microbiol. Lett. 168, 227–233. Booth, M., Pence, L., Mahasreshti, P., Callegan, M. and Gilmore, M. (2001) Clonal associations among Staphylococcus aureus isolates from various sites of infection. Infect. Immun. 69, 345– 352. Straub, J.A., Hertel, C. and Hammes, W.P. (1999) A 23S rRNAtargeted polymerase chain reaction-based system for detection of Staphylococcus aureus in meat starter cultures and dairy products. J. Food Prot. 62, 1150–1156. Brakstad, O., Aasbakk, K. and Maeland, J. (1992) Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene J. Clin. Microbiol. 30, 1654–1660. Hookey, J.V., Richardson, J.F. and Cookson, B.D. (1998) Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. J. Clin. Microbiol. 36, 1083–1089. Stephan, R., Annem€ uller, C., Hassan, A.A. and L€ammler, C. (2000) Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north-east Switzerland. Vet. Microbiol. 2001, 1–10. Seki, K., Sakurada, J., Seong, H., Murai, M., Tachi, H., Ishii, H. and Masuda, S. (1998) Occurrence of coagulase serotype among Staphylococcus aureus strains isolated from healthy individualsspecial reference to correlation with size of protein-A gene. Microbiol. Immunol. 42, 407–409. Tsen, H.Y. and Chen, T.R. (1992) Use of the polymerase chain reaction for specific detection of type A, D, and E enterotoxigenic Staphylococcus aureus in foods. Appl. Microbiol. Biotechnol. 37, 685–690. Johnson, W.M., Tyler, S.D., Ewan, E.P., Ashton, F.E., Pollard, D.R. and Rozee, K.R. (1991) Detection of genes for enterotoxins,
52
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction. J. Clin. Microbiol. 29, 426–430. [34] Monday, S. and Bonach, G. (1999) Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates. J. Clin. Microbiol. 37, 3411–3414.
[35] Sign€as, C., Raucci, G., Jonsson, K., Lindgren, P.E., Ananthar€ k, M. and Lindberg, M. (1989) Nucleotide amaiah, G.M., H€ oo sequence of the gene for a fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides. Proc. Natl. Acad. Sci. USA 86, 699–703.
Distribution of virulence genes of Staphylococcus aureus isolated from stable nasal carriers Dimitar Nashev a, Katia Toshkova a, S. Isrina O. Salasia b, Abdulwahed A. Hassan c, €ck c Christoph L€ ammler d,*, Michael Zscho b
a National Center of Infectious and Parasitic Diseases, 26 Y. Sakazov Blvd, 1504 Sofia, Bulgaria Clinical Pathology Department, Faculty of Veterinary Medicine, Gadjah Mada University, Sekip Unit II, 55281 Yogyakarta, Indonesia c Staatliches Untersuchungsamt Hessen, Marburger Str. 54, 35396 Gießen, Germany d Institut f€ ur Pharmakologie und Toxikologie, Justus-Liebig-Universit€at Gießen, Frankfurter Straße 107, 35392 Gießen, Germany
Received 30 September 2003; received in revised form 22 December 2003; accepted 21 January 2004 First published online 6 February 2004
Abstract In the present study, we report data on virulence determinants of Staphylococcus aureus from stable nasal carriers, emphasizing on the genes encoding fibronectin (fnbA, fnbB) and collagen (cna) adhesive molecules. Of the 44 S. aureus isolates included, 32 isolates (16 pairs) were cultured from the anterior nares of 16 healthy carriers, eight isolates (four pairs) were collected from the nose of four patients with recurrent skin infections and four isolates were obtained from the infection site of these patients. The period between the two nasal swabs taken was 3–5 days. The persistency of carriage could be demonstrated by the indistinguishable genotypic chracteristics of the S. aureus isolates in each pair. This could be shown by determination of gene polymorphisms of coa gene and the X-region and IgG-binding region encoding segments of spa gene. In addition, the isolates within the pairs showed identical toxin patterns. This was determined by PCR amplification of the genes encoding staphylococcal enterotoxins (SEA to SEJ) and TSST-1. The genotypic properties also yielded an identity between persistent nasal carriage isolates and the corresponding skin infection isolates of the four patients. In addition, all S. aureus nasal and skin infection isolates were positive for gene fnbA, fnbB and cna could be found with a high frequency. Among the 44 isolates investigated, 16 isolates (36.7%) harbored gene fnbB and 21 isolates (47.7%) gene cna. The data in the present study showed a relatively wide distribution of the genes fnb and cna among the investigated isolates, indicating that the persistent carriage of strains harboring these virulence determinants may increase the risk for subsequent invasive infections in carriers. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Staphylococcus aureus; Nasal carriage; fnbA; fnbB; cna
1. Introduction The nasal carriage of Staphylococcus aureus represents a risk factor for subsequent invasive infection of patients and also for interpatient transmission. It was described that a nasal carrier state seems to be the origin for the * Corresponding author. Tel. +49-641-9938406; fax: +49-6419938409. E-mail address: [email protected] (C. L€ ammler).
development of staphylococcal infections in specific groups of patients, including patients on haemodialysis and continuous ambulatory peritoneal dialysis (CAPD), HIV/AIDS patients as well as patients with postsurgical wounds and patients with intravascular catheters [1,2]. In addition, the nasal carriage of S. aureus appears to play some role in the epidemiology and pathogenesis of staphylococcal skin infections [3]. The nasal carriage rate in adult populations have been reported to be between 30% and 50%. Among these individuals 20–35% carry the organism consistently,
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.01.032
46
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
designated as persistent or stable carriers, and 30–70% intermittently, designated as intermittent nasal carriers [1]. According to Doebbeling [4] the criteria for a stable carriage of S. aureus requires two positive and identical nasal swab cultures on two separate occasions, at least 24 h apart. However, the molecular basis for this persistent or intermittent nasal colonization is still insufficiently cleared. The microbial adherence to cells and extracellular matrix is considered as an essential first step in the process of colonization and infection [1,5]. A wellcharacterized family of staphylococcal surface adhesins, called MSCRAMMs (‘‘microbial surface components recognizing adhesive matrix molecules’’) are known to mediate adherence to host extracellular matrix components, such as fibrinogen, fibronectin and collagen [1,6]. Binding to fibronectin is mediated by two closely related proteins, FnBPA and FnBPB, encoded by the genes fnbA and fnbB [7,8]. The collagen binding protein CNA, as second important adhesive molecule is encoded by gene cna [9]. The S. aureus collagen adhesin occurs in at least four forms that differ in the number (one, two, three, or four) of B domains which contain 187 amino acids and are located between the domains that anchor CNA to the cell envelope and the ligand-binding A domain [10]. FnBPs mediate adhesion of S. aureus to human epithelial cells, including airway epithelium, endothelial cells and fibroblasts which may cause a subsequent internalization of the bacteria in these cells [11]. FnBPA and FnBPB are required for bacterial adhesion to fibronectin-coated surfaces. However, fnbA or fnbB defective single mutants showed no significant reduction in their adhesive properties, whereas the double mutants were completely unable to adhere [12]. In addition, S. aureus isolates associated with invasive diseases, including endocarditis, primary septic arthritis and/or osteomyelitis were more likely to have both genes fnbA and fnbB [13]. CNA of S. aureus was described as virulence factor in septic arthritis [14] and osteomyelitis [15]. CNA also seems to play an important role in the pathogenesis of S. aureus endocarditis and keratitis [6,16]. Recently, Peacock et al. [17] investigated the presence of 33 putative virulence determinants of S. aureus and found that the genes fnbA and cna were significantly more common in invasive isolates. In contrast to the relatively well-studied adherence mechanisms and surface components of S. aureus invasive isolates, the studies concerning the adhesive molecules in persistently or intermittently colonizing nasal mucosa strains are still limited in number [5,18]. In the present study, we report data on genotypic characteristics of paired isolates from persistent nasal carriers, especially emphasizing on the genes encoding adhesive molecules.
2. Materials and methods 2.1. Bacterial isolates A total of 44 S. aureus isolates from 20 nasal carriers were included in the present study. Thirty two isolates (16 pairs) were collected from the anterior nares of 16 healthy carriers, eight isolates (four pairs) were cultured from the nose of four patients with recurrent skin infections. The corresponding isolates from the infection site of these four patients were also included in the study. The period between the two nasal swabs taken was 3–5 days. The swabs were immediately streaked on 5% sheep blood agar plates. After incubation for 24 h at 37 C, suspected colonies of S. aureus were isolated and further identified with the commercial identification system API Staph (Bio Merieux, Marcy-lÕEtoile, France) as recommended by the manufacturer. The strains were additionally investigated by tube-coagulase test (Bactident-coagulase, Merck, Darmstadt, Germany) and by clumping factor reaction with rabbit plasma on microscope slides. 2.2. PCR mediated identification and further characterization The determination of species specific parts of the genes encoding 23S rRNA and thermonuclease (nuc) was performed with oligonucleotide primers (MWG Biotech, Ebersberg, Germany) listed in Table 1. The PCR reaction mixture (20 ll) for both genes contained 0.7 ll primer 1 (10 pmol), 0.7 ll primer 2 (10 pmol), 0.4 ll dNTP (10 mM), 3.0 ll of 10· thermophilic buffer (Sigma–Aldrich Chemie, Steinheim, Germany), 1.2 ll MgCl2 (25 mM, Sigma), 0.1 ll Taq DNA polymerase (5 U/ll, Sigma) and 12.9 ll of distilled water. Finally, 2.0 ll of DNA preparation was added to each 0.2 ml reaction tube. The DNA of the isolates was prepared with the DNeasy Tissue Kit (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer with the modification that 5 ll of lysostaphin (1.8 U/ll, Sigma) was added to the cell lysis step. The DNA amplification was carried out with the cycling profiles shown in Table 1. The presence of PCR products was determined by electrophoresis of 8 ll of the reaction product in a 2% agarose gel, with Trisborate electrophoresis buffer (89 mM Tris-borate; 2 mM EDTA; pH 8.3) at 120 V and visualised under UV light using Gel Doc 1000 system (BioRad, M€ unchen, Germany). The product sizes were determined by using DNA molecular weight marker VI (0.154–2.18 kbp) or VIII (0.019–1.11 kbp) (both Roche Diagnostics, Mannheim, Germany). The determination of the genes encoding coagulase (coa), clumping factor (clfA), protein A (spa), the staphylococcal enterotoxins A (sea), B (seb), C (sec), D
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
47
Table 1 Oligonucleotide primers and PCR programs used for amplification of the genes encoding 23S rRNA and various other staphylococcal proteins including toxins and adhesive molecules Target gene
Sequence (50 –30 )
Size of amplified products (bp)
PCR program
Reference
23S rRNA
ACG GAG TTA CAA AGG ACG AC AGC TCA GCC TTA ACG AGT AC GCG ATT GAT GGT GAT ACG GTT ACG CAA GCC TTG ACG AAC TAA AGC ATA GAG ATG CTG GTA CAG G GCT TCC GAT TGT TCG ATG C GGC TTC AGT GCT TGT AGG TTT TCA GGG TCA ATA TAA GC CAA GCA CCA AAA GAG GAA CAC CAG GTT TAA CGA CAT CAC CTG CTG CAA ATG CTG CG GGC TTG TTG TTG TCT TCC TC AAA GTC CCG ATC AAT TTA TGG CTA GTA ATT AAC CGA AGG TTC TGT AGA TCG CAT CAA ACT GAC AAA CG GCA GGT ACT CTA TAA GTG CC GAC ATA AAA GCT AGG AAT TT AAA TCG GAT TAA CAT TAT CC CTA GTT TGG TAA TAT CTC CT TAA TGC TAT ATC TTA TAG GG TAG ATA AGG TTA AAA CAA GC TAA CTT ACC GTG GAC CCT TC AAT TAT GTG AAT GCT CAA CCC GAT C AAA CTT ATA TGG AAC AAA AGG TAC TAG TTC CAA TCA CAT CAT ATG CGA AAG CAG CAT CTA CCC AAA CAT TAG CAC C CTC AAG GTG ATA TTG GTG TAG G AAA AAA CTT ACA GGC AGT CCA TCT C CAT CAG AAC TGT TGT TCC GCT AG CTG AAT TTT ACC ATC AAA GGT AC ATG GCA GCA TCA GCT TGA TA TTT CCA ATA ACC ACC CGT TT GCG GAG ATC AAA GAC AA CCA TCT ATA GCT GTG TGG GGA GAA GGA ATT AAG GCG GCC GTC GCC TTG AGC GT ATA TGA ATT CGA GTA TAA GGA AGG GGT T TTT GGA TCC CTT TTT CAG TAT TAG TAA CCA AGT GGT TAC TAA TAC TG CAG GAT AGT TGG TTT A
1250
1a
[27]
279
2
[28]
Size polymorphisms
3
[29]
Size polymorphisms
4
[30]
Size polymorphisms
5
[20]
Size polymorphisms
3
[31]
216
6
[32]
478
6
[33]
257
6
[33]
317
6
[33]
170
6
[33]
642
6
[23]
375
6
[23]
576
6
[23]
142
7
[34]
350
6
[33]
1279
8
[35]
812
8
[8]
1609
8
[14]
1122
8
[14]
nuc coa clfA spa (X-region) spa (IgG-binding region) sea seb sec sed see seg seh sei sej tst fnbA fnbB cna (A domain) cna (B domain)
a 1, 37 · (94 C 40 s, 64 C 60 s, 72 C 75 s); 2, 37 · (94 C 60s, 55 C 30s, 72 C 30s); 3, 30 · (94 C 60 s, 58 C 60 s, 72 C 60 s); 4, 35 · (94 C 60 s; 57 C 60 s; 72 C 60 s); 5, 30 · (94 C 60 s, 60 C 60 s, 72 C 60 s); 6, 30 · (94 C 120 s, 55 C 120 s, 72 C 60 s); 7, 30 · (94 C 60 s, 62 C 60 s, 72 C 60 s); 8, 30 · (94 C 30 s, 50 C 30 s, 72 C 60 s).
(sed), E (see), G (seg), H (seh), I (sei) and J (sej), toxic shock syndrome toxin 1 (tst) and the adhesive molecules FnBPA, FnBPB and CNA domain A and B (fnbA, fnbB, cnaA and cnaB, respectively) were performed with the primers and thermocycler programs listed in Table 1. For control purposes the following S. aureus reference strains were used: 619/93 (sea), 62/92 (seb), 1229/93 (sec), 1644/93 (sed), FRI 918 (see), 161/93 (tst), Ly 990055 (seg, sei), Ly 990552 (seh). The strains were kindly provided by W. Witte, Robert-Koch-Insitut, Wernigerode, Germany and G. Lina, Centre Nationale des Toxemies a Staphylococques, Lyon, France. Positive controls for the remaining PCR reactions were obtained
from the strain collections of Institut f€ ur Pharmakologie und Toxikologie and Staatliches Untersuchungsamt Hessen.
3. Results According to cultural and biochemical properties, determined with the API Staph system, a positive clumping factor reaction and a positive tube coagulase test all 44 isolates used in the present study could be identified as S. aureus. The identification of the isolates could be confirmed by PCR amplification of species specific parts of the gene encoding 23S rRNA and by
48
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
Fig. 1. (a) Amplicons of the gene encoding staphylococcal coagulase. Isolates 17a, 17b and 17c (lanes 1, 2 and 3), isolates 5a and 5b (lanes 4 and 5), isolates 9a and 9b (lanes 6 and 7), isolate 18a (lane 8); M: DNA molecular weight marker VI (Roche). (b) Amplicons of the spa gene segment encoding X-region of protein A. Isolate 7a (lane 1), isolates 9a and 9b (lanes 2 and 3), isolates 15a and 15b (lanes 4 and 5); M: DNA molecular weight marker VIII (Roche). (c) Amplicons of the genes encoding fibronectin binding protein A (1300 bp, lanes 1 and 2) and B (900 bp, lane 3); fnbB negative isolate (lane 4); M: see (a). (d) Amplicons of the gene encoding collagen binding protein domain B (1200 bp, lanes 1 and 2) and domain A (1600 bp, lanes 3 and 4); M: see (a).
amplification of thermonuclease gene nuc. The amplicons of these genes showed an uniform size of approximately 1250 and 270 bp, respectively. In addition, the identification was confirmed by amplification of the gene coa encoding coagulase and gene spa encoding the Xand IgG-binding regions of protein A. Both genes or gene segments displayed typical gene polymorphisms (Figs. 1(a) and (b)) with indistinguishable sizes for the isolates of each pair. The exception were the isolates from patient 18, which showed a difference in the amplicon size of the spa gene segment encoding X-region of protein A. The amplification of the clumping factor gene clfA revealed a single amplicon with a size of approximately 1000 bp with the exception of two isolates (one pair), which displayed an amplicon size of 890 bp. Among the 44 S. aureus isolates examined, 12 isolates were positive for the genes encoding SEG and SEI, five isolates for the genes encoding SEB and SEI, four isolates for the genes encoding SEB, SEG, SEH and SEI, four isolates for the genes encoding SEA, SEG, SEI and TSST-1, two isolates for the genes encoding SEA, SED, SEG, SEI and SEJ, two isolates for the gene encoding SEB, and two isolates for the gene encoding SEH. The genes sea, seb, sec, sed, see, seg, seh, sei, sej and tst occurred pairwise. The four isolates from skin in-
fections and their corresponding nasal cultures showed indistinguishable size of coa gene and spa (X-region and IgG-binding region) gene amplicons and had the same toxin genotype (Table 2). The amplification of the genes fnbA, fnbB and cna (A domain) resulted in amplicons with sizes of approximately 1300, 900 and 1600 bp, respectively (Figs. 1(c) and (d)). The amplification of the B domain(s) of the gene cna revealed an uniform size of approximately 1200 bp, which corresponds to two copies of the 187-aminoacid repeat motif. All S. aureus isolates were found to be positive for gene fnbA. Among the 40 nasal isolates (20 pairs) investigated, 16 isolates (eight pairs, 40%) harbored gene fnbB, 18 isolates (nine pairs, 45%) harbored gene cna. Three of the four skin infection isolates were positive for cna and none of these strains was positive for fnbB. The genotypic characteristics of the S. aureus isolates are presented in Table 2.
4. Discussion Many healthy people are persistently or intermittently colonized with S. aureus at their anterior nares and thus are at increased risk for certain staphylococcal
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
49
Table 2 Genotypic characteristics of the 44 S. aureus isolates from nares (n ¼ 40) and skin infections (n ¼ 4) Source
coa (bp)
spa (X-region)
spa (IgG-binding region)
clfA
Toxin gene pattern
fnbA
fnbB
cna
1
1a 1b
800a 800
240 240
700 700
1000 1000
seg, sei seg, sei
+ +
) )
+ +
2
2a 2b
600 600
320 320
900 900
1000 1000
) )
+ +
+ +
) )
3
3a 3b
600 600
350 350
900 900
1000 1000
) )
+ +
+ +
) )
4
4a 4b
500 500
320 320
900 900
1000 1000
) )
+ +
+ +
) )
5
5a 5b
690 690
320 320
900 900
1000 1000
) )
+ +
) )
) )
6
6a 6b
500 500
320 320
900 900
1000 1000
sea, seg, sei, tst sea, seg, sei, tst
+ +
) )
+ +
7
7a 7b
600 600
240 240
900 900
1000 1000
seg, sei seg, sei
+ +
) )
) )
8
8a 8b
600 600
240 240
900 900
1000 1000
seh seh
+ +
+ +
+ +
9
9a 9b
690 690
280 280
700 700
890 890
sea, sed, seg, sei, sej sea, sed, seg, sei, sej
+ +
+ +
) )
10
10a 10b
500 500
320 320
900 900
1000 1000
sea, seg, sei, tst sea, seg, sei, tst
+ +
) )
+ +
11
11a 11b
600 600
280 280
900 900
1000 1000
seb, seg, seh, sei seb, seg, seh, sei
+ +
+ +
) )
12
12a 12b
600 600
300 300
900 900
1000 1000
seb, seg, seh, sei seb, seg, seh, sei
+ +
+ +
) )
13
13a 13b
800 800
280 280
700 700
1000 1000
seb seb
+ +
) )
) )
14
14a 14b
800 800
320 320
700 700
1000 1000
) )
+ +
+ +
+ +
15
15a 15b
690 690
300 300
900 900
1000 1000
seb, sei seb, sei
+ +
) )
) )
16
16a 16b
500 500
240 240
900 900
1000 1000
seg, sei seg, sei
+ +
) )
+ +
17
17a 17b 17c
600 600 600
240 240 240
900 900 900
1000 1000 1000
) ) )
+ + +
) ) )
+ + +
18
18a 18b 18c
900 900 900
240 280 280
700 700
1000 1000 1000
seg, sei seg, sei seg, sei
+ + +
) ) )
+ + +
19
19a 19b 19c
690 690 690
300 300 300
900 900 900
1000 1000 1000
seb, sei seb, sei seb, sei
+ + +
) ) )
) ) )
20
20a 20b 20c
900 900 900
180 180 180
700 700 700
1000 1000 1000
seg, sei seg, sei seg, sei
+ + +
) ) )
+ + +
a
a and b, anterior nares; c, skin infection. Approximate size in bp.
infections. The exact reasons for these differences in colonization patterns are yet unknown. One reason might be a host predisposition. On the other hand, the
properties of the resident strain might be the leading factor in pathogenesis of nasal carriage. However, at present, relatively little is known about the basic surface
50
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
components of the staphylococcal cell wall which could be connected with the persistency of carriage. A study of van Belkum et al. [18] failed to prove a correlation between the polymorphisms of the protein A and coagulase genes and the persistency of colonization. In the present study, the persistency of carrier state could be demonstrated by the indistinguishable size of amplicons and toxin genotypes of the S. aureus isolates in each pair. In addition, comparable to previous studies [3], the relation between persistent nasal carriage isolates and corresponding skin infection isolates could be demonstrated for S. aureus isolates of four patients. The determination of the coa gene polymorphisms and the X-region and IgG-binding region encoding segments of spa gene revealed an indistinguishable size of amplicons in isolates of each pair. A difference in size of the Xregion encoding gene segment of one isolate of patient 18 could be explained by a deletion of segments in this region [19]. However, gene polymorphisms of the genes spa and coa had been commonly used for epidemiological typing of S. aureus [20]. The isolates in the present study were additionaly investigated for the genes encoding the classical and the newly described staphylococcal enterotoxins and TSST-1. As described previously, genotyping S. aureus by determination of toxin patterns could be an useful tool for comparing epidemiologically related strains [21]. According to the results of the present study, the isolates within the pairs were indistinguishable according to their toxin genotypes. The toxin genes seg and sei seemed to be the most prevalent toxin genes, occuring in combination in 20 of the 40 nasal isolates. These results are comparable to data shown by Jarraud et al. [22] in which 57% of the investigated strains from nasal carriers harbored the genes seg and sei. These authors previously reported that seg and sei are present in S. aureus in tandem orientation on a 3.2 kb DNA fragment [23]. The seg and sei genes were present in most strains (67%) associated with suppurative infections [22]. It was of interest that five cultures of the present study positive for sei were negative for seg. This could possibly be explained by a mutation of the seg primer binding site or a variation in the enterotoxin gene cluster. The gene seh, encoding SEH, also found among the strains of the present study, has been described to have sequence homology to the sea subfamily. In addition, SEH has the highest affinity ever measured for an enterotoxin for MHC class II molecules [24]. Among the genes encoding classical staphylococcal enterotoxins the most prevalent toxin gene was seb (25%), followed by sea (15%) and sed (5%). The gene encoding TSST-1 was only present in two cultures. These results are in contrast with the studies of Peacock et al. [17], who presented data that 44% of investigated carriage isolates harbored the gene tst, 30% were positive for sea, 13% for seb and 9% for sed. Both sed positive cultures in the present study were
simultaneously positive for sej. As shown in the studies of Zhang et al. [25], SED and SEJ are encoded by a plasmid and separated from each other by an intergenic region. Mongodin et al. [5] demonstrated the major role of FnBPs in S. aureus adherence to human airway epithelium, whereas staphylococcal protein A and clumping factor did not play any role in this process. These authors also reported that 97% of 32 clinical strains, isolated from the airway secretions of patients with cystic fibrosis and nosocomial pneumonia, possessed the two fnb genes. Thus, the presence of these genes seems to be an important factor for microbial pathogenesis. Booth et al. [26] investigated different S. aureus lineages associated with various sites of infection and nasal carriage and showed that 89.7% of the isolates, regardless of lineage identity, possesed gene fnbA, whereas fnbB was observed in 20.1% of the isolates. The latter was present only in the lineage associated with blood isolates. A positive signal for cna could be demonstrated for 44.9% of the isolates, regardless of the lineage. Peacock et al. [17] compared S. aureus isolates from healthy carriers and from patients with invasive disease for variation in the presence of 33 putative virulence determinant genes, also including fnbA and cna. The authors showed that 98% of invasive isolates harbored fnbA versus 87% in the carriage group. The gene cna appeared also significantly more common in disease isolates (52%) than in colonizing strains (32%). Comparable to these previous studies the data from the present study showed a relatively wide distribution of the genes fnb and cna among the isolates obtained from stable carriers. All 44 isolates harbored the gene fnbA and 36.7% of the isolates were positive for fnbB. The occurrence of gene cna also seems to be enhanced with 21 (47.7%) of the 44 isolates, indicating that both genes might play a role for the persistency of carriage. A further investigation of intermittently colonizing S. aureus might show the importance of this relation. The role of S. aureus nasal carriage for the pathogenesis of reccurent skin infections, as shown for four patients in the present study and in previous studies [3], indicated that an eradication of S. aureus nasal carriage is a strong demand for prevention of these infections. However, nasal colonization with strains carrying various virulence determinants, including fibronectin and collagen adhesins may also represent an increased risk factor for subsequent invasive infections in carriers.
Acknowledgements This work was supported by German Academic Exchange Service (DAAD) (D. Nashev) and by Alexander von Humboldt Foundation (S.I.O. Salasia).
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
References [1] Kluytmans, J., van Belkum, A. and Verbrugh, H. (1997) Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin. Microbiol. Rev. 10, 505– 520. [2] Wenzel, R. and Pearl, T. (1995) The significance of nasal carriage of Staphylococcus aureus and the incidence of postoperative wound infection. J. Hosp. Infect. 31, 13–24. [3] Toshkova, K., Annem€ uller, C. and L€ammler, C. (2001) The significance of nasal carriage of Staphylococcus aureus as risk factor for human skin infections. FEMS Microbiol. Lett. 202, 17–24. [4] Doebbeling, B.N. (1994) Nasal and hand carriage of Staphylococcus aureus in healthcare workers. J. Chemotherapy VI (Suppl. 2), 11–17. [5] Mongodin, E., Bajolet, O., Cutrona, J., Bonnet, N., Dupuit, F., Puchelle, E. and de Bentzmann, S. (2002) Fibronectin-binding proteins of Staphylococcus aureus are involved in adherence to human airway epithelium. Infect. Immun. 70, 620–630. [6] Navarre, W. and Schneewind, O. (1999) Surface proteins of Gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63, 174–229. [7] Flock, J., Fromann, G., Jonsson, K., Guss, B., Sign€as, C., Nilsson, € k, M., Wadstr€ B., Raucci, G., H€ oo om, T. and Lindberg, M. (1987) Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus. EMBO J. 6, 2351–2357. [8] Jonsson, K., Sign€ as, H., M€ uller, H. and Lindberg, M. (1991) Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene. Eur. J. Biochem. 202, 1041–1048. [9] Patti, J.M., Jonsson, H., Guss, B., Switalski, L.M., Wiberg, K., € k, M. (1992) Molecular characterization Lindberg, M. and H€ oo and expression of a gene encoding a Staphylococcus aureus collagen adhesin. J. Biol. Chem. 267, 4766–4772. [10] Gillaspy, A., Patti, J., Pratt, F., Iandolo, J. and Smeltzer, M. (1997) The Staphylococcus aureus collagen adhesin-encoding gene (cna) is within a discrete genetic element. Gene 196, 239–248. [11] Dziewanowska, K., Patti, J., Deobald, C., Bayles, K., Trumble, W. and Bonach, G. (1999) Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells. Infect. Immun. 67, 4673–4678. [12] Greene, C., McDevitt, D., Francois, P., Vaudaux, P., Lew, D. and Foster, T. (1995) Adhesion properties of mutants of Staphyloccus aureus defective in fibronectin-binding proteins and studies on the expression of fnb genes. Mol. Microbiol. 17, 1143–1152. [13] Peacock, S., Day, N., Thomas, M., Berendt, A. and Foster, T. (2000) Clinical Isolates of Staphylococcus aureus exhibit diversity in fnb genes and adhesion to human fibronectin. J. Infect. 41, 23–31. [14] Switalski, L., Patti, J., Butcher, W., Gristina, A., Speziale, P. and € k, M. (1993) A collagen receptor on Staphyloccus aureus H€ oo strains isolated from patients with septic arthritis mediates adhesion to cartilage. Mol. Microbiol. 7, 99–107. [15] Elasri, M., Thomas, J., Skinner, R., Blevins, J., Beenken, K., Nelson, C. and Smeltzer, M. (2002) Staphyloccus aureus collagen adhesin contributes to the pathogenesis of osteomyelitis. Bone 30, 275–280. €k, M., Jones, D. [16] Rhem, M., Lech, E., Patti, J., McDevitt, D., H€ oo and Wilhelmus, K. (2000) The collagen-binding adhesin is a virulence factor in Staphylococcus aureus keratitis. Infect. Immun. 68, 3776–3779. [17] Peacock, S., Moore, C., Justice, A., Kantazanou, M., Story, L., Mackie, K., OÕNeil, G. and Day, N. (2002) Virulent combinations of adhesin and toxin genes in natural populations of Staphyloccus aureus. Infect. Immun. 70, 4987–4996. [18] van Belkum, A., Riewarts Eriksen, N.H., Sijmons, M., van Leeuwen, W., van den Bergh, M., Kluytmans, J., Espersen,
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
51
F. and Verbrugh, H. (1997) Coagulase and protein A polymorphisms do not contribute to persistence of nasal colonization by Staphylococcus aureus. J. Med. Microbiol. 46, 222–232. Toshkova, K., Savov, E., Soedarmanto, I., L€ammler, C., Chankova, D., van Belkum, A., Verbrugh, H.A. and van Leeuwen, W. (1997) Typing of Staphylococcus aureus isolated from nasal carriers. Zbl. Bakt. 286, 547–559. Frenay, H.M.E., Bunschoten, A.E., Schouls, L.M., van Leeuwen, W.J., Vandenbrouke-Grauls, C.M.J.E., Verhoef, J. and Mooi, F.R. (1996) Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism. Eur. J. Clin. Microbiol. Infect. Dis. 15, 60–64. Nishi, J., Yoshinaga, M., Miyanohara, H., Kawahara, M., Kawabata, M., Motoya, T., Owaki, T., Oiso, S., Kawakami, M., Kamewari, S., Koyama, Y., Wakimoto, N., Tokuda, K., Manago, K. and Maruyama, I. (2002) An epidemiologic survey of methicillin-resistant Staphylococcus aureus by combined use of mec-HVR genotyping and toxin genotyping in a university hospital in Japan. Infect. Control. Hosp. Epidemiol. 23, 506– 510. Jarraud, S., Peyrat, M.A., Lim, A., Tristan, A., Bes, M., Mougel, C., Etienne, J., Vandenesch, F., Bonneville, M. and Lina, G. (2001) Egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J. Immunol. 166, 669–677. Jarraud, S., Cozon, G., Vandenesch, F., Bes, M., Etienne, J. and Lina, G. (1999) Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J. Clin. Microbiol. 37, 2446–2449. Nilson, H., Bj€ ork, P., Dohlsten, M. and Antonsson, P. (1999) Staphylococcal enterotoxin H displays unique MHC class IIbinding properties. J. Immunol. 163, 6686–6693. Zhang, S., Iandolo, J.J. and Stewart, G.C. (1998) The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej). FEMS Microbiol. Lett. 168, 227–233. Booth, M., Pence, L., Mahasreshti, P., Callegan, M. and Gilmore, M. (2001) Clonal associations among Staphylococcus aureus isolates from various sites of infection. Infect. Immun. 69, 345– 352. Straub, J.A., Hertel, C. and Hammes, W.P. (1999) A 23S rRNAtargeted polymerase chain reaction-based system for detection of Staphylococcus aureus in meat starter cultures and dairy products. J. Food Prot. 62, 1150–1156. Brakstad, O., Aasbakk, K. and Maeland, J. (1992) Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene J. Clin. Microbiol. 30, 1654–1660. Hookey, J.V., Richardson, J.F. and Cookson, B.D. (1998) Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. J. Clin. Microbiol. 36, 1083–1089. Stephan, R., Annem€ uller, C., Hassan, A.A. and L€ammler, C. (2000) Characterization of enterotoxigenic Staphylococcus aureus strains isolated from bovine mastitis in north-east Switzerland. Vet. Microbiol. 2001, 1–10. Seki, K., Sakurada, J., Seong, H., Murai, M., Tachi, H., Ishii, H. and Masuda, S. (1998) Occurrence of coagulase serotype among Staphylococcus aureus strains isolated from healthy individualsspecial reference to correlation with size of protein-A gene. Microbiol. Immunol. 42, 407–409. Tsen, H.Y. and Chen, T.R. (1992) Use of the polymerase chain reaction for specific detection of type A, D, and E enterotoxigenic Staphylococcus aureus in foods. Appl. Microbiol. Biotechnol. 37, 685–690. Johnson, W.M., Tyler, S.D., Ewan, E.P., Ashton, F.E., Pollard, D.R. and Rozee, K.R. (1991) Detection of genes for enterotoxins,
52
D. Nashev et al. / FEMS Microbiology Letters 233 (2004) 45–52
exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction. J. Clin. Microbiol. 29, 426–430. [34] Monday, S. and Bonach, G. (1999) Use of multiplex PCR to detect classical and newly described pyrogenic toxin genes in staphylococcal isolates. J. Clin. Microbiol. 37, 3411–3414.
[35] Sign€as, C., Raucci, G., Jonsson, K., Lindgren, P.E., Ananthar€ k, M. and Lindberg, M. (1989) Nucleotide amaiah, G.M., H€ oo sequence of the gene for a fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides. Proc. Natl. Acad. Sci. USA 86, 699–703.