Patrick Munro

René Clément

Jean-Philippe Lavigne

Céline Pulcini

Emmanuel Lemichez
email: lemichez@unice.fr

Luce Landraud
email: landraud.l@chu-nice.fr

S Aureus Infections

High prevalence of edin-C encoding RhoA-targeting toxin in clinical isolates of Staphylococcus aureus

Keywords: Staphylococcus aureus, EDIN, toxin, ADP-ribosyltransferase, virulence factors, Rho GTPases Staphylococcus aureus, EDIN, toxin, ADP-ribosyltransferase, virulence factors, Rho GTPases

Staphylococcus aureus, a major causative agent of human infection produces a large array of virulence factor including various toxins. Among them, the host RhoA GTPase ADPribosylating EDIN toxins are considered as potential virulence factors. Using the polymerase chain reaction, we analyzed the virulence profile of 256 S. aureus isolates from various clinical sites of infections. We developed specific primers to detect the three isoforms of edin encoding genes. We found a prevalence of 14% (36 bacteria) of edin encoding genes among these clinical isolates. Strikingly, we found that 90% of all edin-bearing S. aureus isolates carried the type-C allele. Both the spa types and the profile of virulence factors of these edinpositive isolates are highly variable. Notably, we show for the first time that edin-C positive isolates were more frequently recovered from deep-seated infections than other types of infections. Our present work thus strongly suggests that presence of edin-C is a risk factor of S. aureus dissemination in tissues and thus represents a predictive marker for a pejorative evolution of staphylococcal infections.

Staphylococcus aureus is a common bacterium, which is responsible for a unique variety of infections [START_REF] Lowy | Staphylococcus aureus infections[END_REF]. Development of pejorative forms of staphylococcal infections involves the combined action of numerous bacterial virulence factors, which corrupt host responses [2].

Bacterial virulence factors include specific adhesins, collectively referred as Microbial Surface Components Recognizing endothelial cell Adhesive Matrix Molecules (MSCRAMMs) and a large variety of toxins, such as the exfoliative toxins (ETs), hemolysin, leukocidin, enterotoxins and EDINs (epidermal cell differentiation inhibitors) [3][4][5][6].

EDINs belong to the family of Clostridium botulinum C3 exoenzyme [6,7]. They are members of a group of major bacterial virulence factors targeting host Rho GTPases [4,[6][7][8][9].

Rho proteins control essential cellular processes such as cell polarity, movement and phagocytosis, as well as cohesion of intercellular junctions [6,10,11]. Recent findings suggest that EDINs might favor bacterial dissemination in tissues, by a haematogenous route, through induction of large transcellular tunnels in endothelial cells named macroapertures [12][13][14]. Indeed, recent data show that S. aureus EDIN toxin promotes formation of infection foci in a mouse model of bacteremia [15]. To date, three isoforms of EDIN have been characterized. These comprise the first discovered EDIN isoform (EDIN-A), isolated from the E-1 strain of S. aureus [16], as well as EDIN-B [6,17] and EDIN-C [18]. The chromosomal gene encoding edin-B is located within a pathogenicity island frequently associated with the etD gene encoding the exfoliative toxin ET-D [17]. EDIN-C is encoded by the pETB plasmid, which also carries genes encoding ET-B and conferring cadmium resistance [18].

A first epidemiological survey, involving staphylococcal strains isolated from patients hospitalized for various infectious diseases demonstrated a higher prevalence of edinencoding genes in this group compared to nasal strains isolated from healthy students [19].

Another study shows that edin-B is present in 7% of bacteriemic S. aureus strains [17].

However, most other epidemiological data on edin are based on surveys focused on exfoliative toxins or PVL rather than EDIN toxin itself. For example, a genetic association between etD and edin-B was detected in the emerging ST80 clone Panton-Valentine Leukocidin (PVL)-positive and community-acquired (CA) methicillin-resistant S. aureus (MRSA) [20]. This clone is spreading throughout France and Tunisia and is most frequently associated with infections of the skin and soft tissues. Also, two-thirds of the strains belonging to the emerging ST123 epidemic European fusidic acid-resistant impetigo clone one of these strains suggested that it also bears edin-C [21].

In this study, we have developed a PCR-based method, to detect EDIN isoforms specifically. We demonstrate that 90% of all edin-bearing S. aureus isolates carry the type-C allele. We also show that these isolates are more significantly associated with deep-seated soft tissue infections than other types of infections (Fisher's exact test, p=0.03).

Materials and methods

S. aureus isolates

A total of 256 isolates of S. aureus belonging to the collection of the Bacteriology department of the Hospital University of Nice were analyzed. These isolates were recovered from randomly consecutive episodes of S. aureus infections in patients hospitalized during 2005. These isolates were obtained from various types of clinical samples, comprising blood cultures (28 bacteria); skin infections including chronic ulcers, burns or wounds (83 bacteria); urine samples (41 bacteria); sputum samples (69 bacteria); and various deep-seated soft tissue infections such as subcutaneous or visceral abscesses, spontaneously or post operative soft tissue infections (35 bacteria). For the last group, bacteria were isolated from specimens obtained by guidance radiography needle biopsy or during endoscopic and surgical procedures. All isolates were characterized using routine methods according to each manufacturer's recommendations. All were positive for catalase, DNAse production and mannitol fermentation in Chapman medium, and confirmed to be S. aureus by specific 32rapidStaph (BioMérieux, Marcy-l'Etoile, France).

Antibiotic susceptibility determinations

Antimicrobial susceptibility testing was performed on all isolates obtained during the study using the disk diffusion method [22] on Mueller-Hinton medium (Difco Laboratories, Detroit, MI) according to the recommendations of the French Antibiogram Committee [http://www.sfm.asso.fr/nouv/general.php?pa=2]. Antibiotics tested were penicillin G, oxacillin, erythromycin, clindamycin and fusidic acid to focus on epidemiologic profiles.

DNA isolation and PCR-based detection of genes

For edin detection, total DNA was isolated from bacterial strains grown overnight at 37°C in BHI medium. Bacteria were lysed in 10 mM TrisHCl pH7.8, 100 mM NaCl, 1mM EDTA, 1% Triton X100. After incubation for 10 minutes at 100°C, DNA was collected and frozen. PCR amplification was used to detect the presence of edin-A, B and C using the primers described in Table 1. We have determined optimized thermal cycling conditions for edin-A (25 cycles of 30s at 94°C, 45s annealing at 58°C and 1 min elongation at 72°C), edin-B (25 cycles of 30s at 95°C, 1 min annealing at 50°C and 1 min elongation at 72°C) and edin-C (30 cycles of 30s at 94°C, 45s annealing at 54°C and 1 min elongation at 72°C). For the detection of other virulence genes, total DNA was isolated from bacterial strains grown three hours at 37°C in BHI medium. DNA was subsequently extracted with NucleoSpin Tissue (Macherey-Nagel GmbH, Düren, Germany) according to manufacturer's recommendations. Briefly, bacteria were pelleted by centrifugation at 8,000 ×g for 5 min, resuspended in 180µl of lysis buffer with 33µl of proteinase K (20mg/ml) (Invitrogen Life Technologies, Carlsbad, CA) and 3µl of recombinant lysostaphin (3U/µl) (Sigma-Aldrich, St Louis, MI), and incubated for 60 min at 37°C. DNA samples were eluted with 100 µl alkaline elution buffer (BE buffer, NucleoSpin Tissue, Macherey-Nagel). The presence of 30 genes, among the most prevalent virulenceassociated genes, was evaluated by PCR as described previously: staphylococcal enterotoxins (se) A, B, C, D, E, G, H, I, J, K, and Q, toxic shock syndrome toxin 1 (tst-1), exfoliative toxins A, B and D (etA, etB, etD), PVL (lukS-PV _ lukF-PV), LukDE leukocidin (lukE), nine MSCRAMM (bbp, cna, ebpS, clfA, clfB, fib, fnbA, fnbB, eno). The accessory gene regulator (agr) allele group was determined by multiplex PCR. spa sequencing spa typing was performed as described previously [23], using the spa typing website (http://www.spaserver.ridom.de/) that is developed by Ridom GmbH and curated by SeqNet.org (http://www.SeqNet.org/). Primers are indicated in Table 1.

Statistical analysis

The chi-square or Fisher's exact test for categorical variables was used to compare data as appropriate. A P value of less than 0.05 was considered significant.

Results

Detection of edin isoforms

S. aureus isolates analyzed in this study were collected at the university hospital of Nice from various infected patients. We designed primers with high sensitivity and specificity in order to detect, by PCR, edin-A, B and C alleles in these isolates. This was especially challenging for edin-C, which was poorly detected using a previously described pair of primers designed to amplify all edin isoforms. This is consistent with the fact that the sequence of edin-C has the most substantial sequence variations in regions recognized by these primer sequences (17% and 32% homology with the forward and reverse primers, respectively) (Fig. 1) [19]. As shown in figure 1, the three pairs of primers designed allowed us to amplify specifically a 455 bp DNA fragment for edin-A and B, and a 320 bp DNA fragment for edin-C.

We next analyze the 256 clinical isolates of S. aureus. We found that 14 % (36) of these isolates were positive for one of the edin alleles. Among these 36 isolates, 90% were positive for edin-C and 5% were positive for either edin-A or B. To confirm the nature of the edin isoforms, we performed complete sequence analysis of five edin-C encoding genes from randomly selected isolates. We also sequenced edin-A and edin-B encoding genes. These results confirm the specificity of the new primers used and demonstrate that edin-C was more prevalent than other alleles of edin.

Detection of genes encoding virulence factors

The 36 isolates positive for edin genes were next analyzed for the distribution of major MSCRAMMs, various staphylococcal enterotoxins, exfoliative toxins, the toxic shock syndrome toxin 1 gene tst-1, as well as leucotoxin family encoding genes. Among the staphylococcal MSCRAMM genes, eno, clfA and clfB were detected in all edin-positive isolates (Table 2). bbp, fnbA and ebpS were the less frequently encountered MSCRAMMs among these isolates. However these adhesion factors had no preferential distribution among S. aureus isolated from various types of infections. Among the staphylococcal enterotoxins genes the most frequently encountered were seg, sei and sea (Table 2). In addition, 72% of edin-positive isolates (26 bacteria) contained a combination of these three genes. One edin-C bearing isolate, isolated from a urine sample, had only the sea enterotoxin gene. We detected the exfoliative toxin gene etD exclusively among the edin-B positive isolates. Two edin-A positive isolates carried the staphylococcal exfoliative gene etB. We found that 25 edin-C positive S. aureus (78%) were negative for etA, etB and etD exfoliative toxin encoding genes.

We observed that only 22% of the edin-C positive S. aureus (7 out of 32) had at least one of the two isoforms (etA or etB) of the exfoliative toxin gene. Five of these seven isolates carried both the etA and etB genes. No significant association was found between the presence of tst-1, exfoliative toxins or leucotoxin family encoded genes and the types of infection. Finally, a large number of edin-positive S. aureus belonged to the agr group 1 (50%, 18 bacteria), and to a lesser extent, to agr groups 2, 3 and 4 (17%, 6 bacteria each) (Table 2).

spa-typing

Determination of the spa type of 26 edin-positive isolates [23] allowed us to exclude the clonal origin of all edin-C positive S. aureus in our survey. Among them, only six isolates could be classified as ST45 (two isolates), ST30 (two isolates), ST59 or ST26. The other 20 isolates showed a high variability of their spa type (Table 3). For 14 isolates, we determined new repeat sequences including unidentified 24-bp repeats thus defining new spa types.

Among them, bacteria S7926 and S7262, isolated respectively in deep seated soft tissue and sputum sample from unrelated infected patients, presented the same spa type t6956 (Table 3).

Together these data excluded the clonal origin of all edin-C positive S. aureus in our survey.

Antibiotic susceptibility profiles

We next investigated whether edin-positive isolates were associated with specific antibiotic resistance profiles, such as community-acquired methicillin resistant S. aureus ST80 and fusidic acid-resistant impetigo clones [20,21]. In relation with these studies, we determined the minimum inhibitory concentrations (MICs) of edin-positive isolates for the following antibiotics: penicillin G, methicillin, erythromycin, clindamycin and fusidic acid. Results were presented in Table 2. Only one isolate, positive for edin-C, showed a methicillinresistance. Finally, edin-positive isolates did not show any specific resistance profile to classical antibiotics used to cure infections by S. aureus (Table 2).

Clinical origin of edin-positive S. aureus

any phenotypic profile or clonal origin, we further analyzed the distribution of edin-positive isolates with regard to the infectious sites. We noticed that isolates positive for edin-C were recovered at all sites of infection (Table 4). Strikingly, edin-positive S. aureus were more significantly associated with deep-seated infections of soft tissues than other types of infections (25.7%, Fischer exact test, p=0.03). No significant association was detected between edin-positive or edin-negative isolates and other types of infections (blood, urine, superficial soft tissue and sputum culture).

Discussion

Our study shows that edin-positive S. aureus isolates are found in all types of clinical infections included in this study, with a global prevalence of 14%. Moreover, we show that 90% of edin-positive isolates are positive for edin-C. This is consistent with a previous study performed with specific primers, which also reported a higher prevalence of edin-C in clinical isolates of S. aureus in Japan [24]. This points for the need of using specific primers to detect each edin isoform, especially edin-C, given its high prevalence. On the contrary, both studies point for a possible underestimation of the prevalence of edin-C in pathogenic S. aureus when consensus primers were used. Both the analysis of spa type and the distribution of various virulence factors among edin-positive S. aureus show their high genetic variability. Our results on the distribution of MSCRAMMs are consistent with previous findings [25].

Classically, edin and exfoliative toxin encoding genes are associated in specific lineages responsible for skin infections [17,21,24]. Interestingly, here we show that edin-C is not strictly associated with genes encoding exfoliative toxin of serotypes A/B/D (7 bacteria edin-C-positive and et-positive, versus 25 bacteria edin-C-positive and et-negative). The high prevalence of edin-C-positive and et-negative isolates observed in our study might be explained by the plasticity of the pETB plasmid, which has been previously reported in two different variants [18,21]. Also, a recent study shows genetic variations in pathogenicity islands encoding EDIN-B [17].

Given that spa analysis constitutes a good tool for epidemiological typing of S. aureus [26], the use of this method allowed us to exclude the clonal origin of edin-C positive isolates.

The fact that edin-C is plasmid born might explain its presence in isolates of various genetic backgrounds.

S. aureus positive for edin are more frequently associated with deep-seated infections of soft tissues. We have previously established that infection of endothelial cells, and other cell types, by EDIN-producing S. aureus triggers the formation of transcellular tunnels named macroapertures [12,13]. Opening of transcellular tunnels in the endothelium suggested that EDIN might favour bacterial extravasation in tissues during bacteremia. In a mouse model of intravascular injection of S. aureus, we have observed that EDIN plays no detectable role in the persistence of bacteria in the blood stream [15]. This data is consistent with the absence of a higher prevalence of edin positive S. aureus recovered from patients associated bacteriemia in this study. In contrast, in this model of mouse infection EDIN toxin promotes formation of infection foci [15]. This suggested that EDIN might enhance the invasive capacity of S. aureus. The hypothesis of a role of EDIN in S. aureus infection is also supported by our present findings showing that S. aureus associated with deep-seated infections of soft tissues have a higher prevalence of edin. However, whether or not edin-positive S. aureus is preferentially associated with a specific type of deep seated infection remains to be further determined".

Figure legend

Fig. 1 Characterization of the three edin alleles. A) PCR amplification of edin-A, edin-B and edin-C from S. aureus strains S25edin-A(+) [15], S7256edin-B(+) and S7475edin-C(+) (this study), respectively, using specific oligonucleotides edinA, edinB, edinC (Table 1) and the previously described edin oligonucleotides referred as edinX [19]. B) Sequence alignments of edin-A, edin-B and edin-C showing sequence homologies and localization of each oligonucleotide (underlined: edinX; highlighted: edinA, edinB and edinC). 19

Strains EDIN type

Spa-type or repeat sequences MLST

Blood S25 A t6403 - S7232 C NT $ - Deep-seated soft tissue S7272 C t6953 - S7404 C t6649 - S7408 C t6484 - S7466 C t012 ST-30 S7595 C NT - S7600 C t6677 - S7926 C t6956 - S8028* C NT - S8087 C t6481 - Sputum sample S7225 C t2726 - S7259 C t2088 - S7262 C t6956 - S7413 C t6483 - S7535 C t2647 - S7634 C t6954 - S7649 B NT - S7920 C NT - S7965 C NT - S8100 C t015 ST-45 Superficial soft tissue S7181 C t6650 - S7183 C NT - S7436 C t6957 - S7475 C t6480 - S7526 C t137 - S7539 C NT - S7569 A t031 ST-45 S7599 C t6482 - S7932 C NT - S7938 C t620 - S7977 C NT - Urine sample S7256 B t078 ST-26 S7322 C t012 ST-30 S7906 C t645 - S7946 C t216 ST-59 * MRSA bacteria $ Not typable

Introduction

Staphylococcus aureus is a common bacterium, which is responsible for a unique variety of infections [START_REF] Lowy | Staphylococcus aureus infections[END_REF]. Development of pejorative forms of staphylococcal infections involves the combined action of numerous bacterial virulence factors, which corrupt host responses [2].

Bacterial virulence factors include specific adhesins, collectively referred as Microbial Surface Components Recognizing endothelial cell Adhesive Matrix Molecules (MSCRAMMs) and a large variety of toxins, such as the exfoliative toxins (ETs), hemolysin, leukocidin, enterotoxins and EDINs (epidermal cell differentiation inhibitors) [3][4][5][6].

EDINs belong to the family of Clostridium botulinum C3 exoenzyme [6,7]. They are members of a group of major bacterial virulence factors targeting host Rho GTPases [4,[6][7][8][9].

Rho proteins control essential cellular processes such as cell polarity, movement and phagocytosis, as well as cohesion of intercellular junctions [6,10,11]. Recent findings suggest that EDINs might favor bacterial dissemination in tissues, by a haematogenous route, through induction of large transcellular tunnels in endothelial cells named macroapertures [12][13][14]. Indeed, recent data show that S. aureus EDIN toxin promotes formation of infection foci in a mouse model of bacteremia [15]. To date, three isoforms of EDIN have been characterized. These comprise the first discovered EDIN isoform (EDIN-A), isolated from the E-1 strain of S. aureus [16], as well as EDIN-B [6,17] and EDIN-C [18]. The chromosomal gene encoding edin-B is located within a pathogenicity island frequently associated with the etD gene encoding the exfoliative toxin ET-D [17]. EDIN-C is encoded by the pETB plasmid, which also carries genes encoding ET-B and conferring cadmium resistance [18].

A first epidemiological survey, involving staphylococcal strains isolated from patients hospitalized for various infectious diseases demonstrated a higher prevalence of edinencoding genes in this group compared to nasal strains isolated from healthy students [19].

Another study shows that edin-B is present in 7% of bacteriemic S. aureus strains [17].

However, most other epidemiological data on edin are based on surveys focused on exfoliative toxins or PVL rather than EDIN toxin itself. For example, a genetic association between etD and edin-B was detected in the emerging ST80 clone Panton-Valentine Leukocidin (PVL)-positive and community-acquired (CA) methicillin-resistant S. aureus (MRSA) [20]. This clone is spreading throughout France and Tunisia and is most frequently associated with infections of the skin and soft tissues. Also, two-thirds of the strains belonging to the emerging ST123 epidemic European fusidic acid-resistant impetigo clone one of these strains suggested that it also bears edin-C [21].

In this study, we have developed a PCR-based method, to detect EDIN isoforms specifically. We demonstrate that 90% of all edin-bearing S. aureus strains carry the type-C allele. We also show that these strains are more significantly associated with deep-seated soft tissue infections than other types of infections (Fisher's exact test, p=0.03).

Materials and methods

S. aureus isolates

A total of 256 strains of S. aureus were retrospectively collected from patients hospitalized at the university hospital of Nice during 2005. These strains were obtained from various types of clinical samples, comprising blood cultures (28 strains); skin infections including chronic ulcers, burns or wounds (83 strains); urine samples (41 strains); sputum samples (69 strains);

and various deep-seated soft tissue infections such as subcutaneous or visceral abscesses, spontaneously or post operative soft tissue infections (35 strains). All isolates were characterized using routine methods according to each manufacturer's recommendations. All were positive for catalase, DNAse production and mannitol fermentation in Chapman medium, and confirmed to be S. aureus by specific 32rapidStaph (BioMérieux, Marcyl'Etoile, France).

Antibiotic susceptibility determinations

Antimicrobial susceptibility testing was performed on all isolates obtained during the study using the disk diffusion method [22] on Mueller-Hinton medium (Difco Laboratories, Detroit,

MI) according to the recommendations of the French Antibiogram Committee

[http://www.sfm.asso.fr/nouv/general.php?pa=2]. Antibiotics tested were penicillin G, oxacillin, erythromycin, clindamycin and fusidic acid to focus on epidemiologic profiles.

DNA isolation and PCR-based detection of genes

For edin detection, total DNA was isolated from bacterial strains grown overnight at 37°C in BHI medium. Bacteria were lysed in 10 mM TrisHCl pH7.8, 100 mM NaCl, 1mM EDTA, 1%

Triton X100. After incubation for 10 minutes at 100°C, DNA was collected and frozen. PCR amplification was used to detect the presence of edin-A, B and C using the primers described in Table 1. We have determined optimized thermal cycling conditions for edin-A (25 cycles of 30s at 94°C, 45s annealing at 58°C and 1 min elongation at 72°C), edin-B (25 cycles of 30s at 95°C, 1 min annealing at 50°C and 1 min elongation at 72°C) and edin-C (30 cycles of 30s at 94°C, 45s annealing at 54°C and 1 min elongation at 72°C). For the detection of other virulence genes, total DNA was isolated from bacterial strains grown three hours at 37°C in BHI medium. DNA was subsequently extracted with NucleoSpin Tissue (Macherey-Nagel GmbH, Düren, Germany) according to manufacturer's recommendations. Briefly, bacteria were pelleted by centrifugation at 8,000 ×g for 5 min, resuspended in 180µl of lysis buffer with 33µl of proteinase K (20mg/ml) (Invitrogen Life Technologies, Carlsbad, CA) and 3µl of recombinant lysostaphin (3U/µl) (Sigma-Aldrich, St Louis, MI), and incubated for 60 min at 37°C. DNA samples were eluted with 100 µl alkaline elution buffer (BE buffer, NucleoSpin Tissue, Macherey-Nagel). The presence of 30 genes, among the most prevalent virulenceassociated genes, was evaluated by PCR as described previously: staphylococcal enterotoxins (se) A, B, C, D, E, G, H, I, J, K, and Q, toxic shock syndrome toxin 1 (tst-1), exfoliative toxins A, B and D (etA, etB, etD), PVL (lukS-PV _ lukF-PV), LukDE leukocidin (lukE), nine MSCRAMM (bbp, cna, ebpS, clfA, clfB, fib, fnbA, fnbB, eno). The accessory gene regulator (agr) allele group was determined by multiplex PCR.

spa sequencing spa typing was performed as described previously [23], using the spa typing website (http://www.spaserver.ridom.de/) that is developed by Ridom GmbH and curated by SeqNet.org (http://www.SeqNet.org/). Primers are indicated in Table 1.

Statistical analysis

The chi-square or Fisher's exact test for categorical variables was used to compare data as appropriate. A P value of less than 0.05 was considered significant.

Results

Detection of edin isoforms

S. aureus isolates analyzed in this study were collected at the university hospital of Nice from various infected patients. We designed primers with high sensitivity and specificity in order to detect, by PCR, edin-A, B and C alleles in these strains. This was especially challenging for edin-C, which was poorly detected using a previously described pair of primers designed to amplify all edin isoforms. This is consistent with the fact that the sequence of edin-C has the most substantial sequence variations in regions recognized by these primer sequences (17% and 32% homology with the forward and reverse primers, respectively) (Fig. 1) [19]. As shown in figure 1, the three pairs of primers designed allowed us to amplify specifically a 455 bp DNA fragment for edin-A and B, and a 320 bp DNA fragment for edin-C.

We next analyze the 256 clinical strains of S. aureus. We found that 14 % (36) of these strains were positive for one of the edin alleles. Among these 36 strains, 90% were positive for edin-C and 5% were positive for either edin-A or B. To confirm the nature of the edin isoforms, we performed complete sequence analysis of five edin-C encoding genes from randomly selected strains. We also sequenced edin-A and edin-B encoding genes. These results confirm the specificity of the new primers used and unravel that edin-C was more prevalent than other alleles of edin.

Detection of genes encoding virulence factors

The 36 strains positive for edin genes were next analyzed for the distribution of major MSCRAMMs, various staphylococcal enterotoxins, exfoliative toxins, the toxic shock syndrome toxin 1 gene tst-1, as well as leucotoxin family encoding genes. Among the staphylococcal MSCRAMM genes, eno, clfA and clfB were detected in all edin-positive strains (Table 2). bbp, fnbA and ebpS were the less frequently encountered MSCRAMMs among these strains. However these adhesion factors had no preferential distribution among S. aureus isolated from various types of infections. Among the staphylococcal enterotoxins genes the most frequently encountered were seg, sei and sea (Table 2). In addition, 72% of edin-positive strains (26 strains) presented a combination of these three genes. One edin-C bearing strain, isolated from a urine sample, had only the sea enterotoxin gene. We detected the exfoliative toxin gene etD exclusively among the edin-B positive strains. Two edin-A and etB. Finally, edin-positive strains did not show any specific resistance profile to classical antibiotics used to cure infections by S. aureus (Table 2).

Clinical origin of edin-positive S. aureus

Having shown no link between a high prevalence of edin-C within our S. aureus strains and any phenotypic profile or clonal origin, we further analyzed the distribution of edin-positive strains with regard to the infectious sites. We noticed that strains positive for edin-C were recovered at all sites of infection (Table 4). Strikingly, edin-positive S. aureus were more significantly associated with deep-seated infections of soft tissues than other types of infections (25.7%, Fischer exact test, p=0.03). No significant association was detected between edin-positive or edin-negative strains and other types of infections (blood, urine, superficial soft tissue and sputum culture).

Discussion

Our study shows that edin-positive S. aureus strains are found in all types of clinical infections included in this study, with a global prevalence of 14%. Moreover, we show that 90% of edin-positive strains are positive for edin-C. This is consistent with a previous study performed with specific primers, which also reported a higher prevalence of edin-C in clinical strains of S. aureus in Japan [24]. Both studies point for a possible underestimation of the prevalence of edin-C in pathogenic S. aureus. This points for the need of using specific primers to detect each edin isoform, especially edin-C, given its high prevalence.

Both the analysis of spa type and the distribution of various virulence factors among edin-positive S. aureus show their high genetic variability. Our results on the distribution of MSCRAMMs are consistent with previous findings [25]. Classically, edin and exfoliative toxin encoding genes are associated in specific lineages responsible for skin infections [17,21,24]. Interestingly, here we show that edin-C is not strictly associated with genes encoding exfoliative toxin of serotypes A/B/D (7 strains edin-C-positive and et-positive, versus 25 strains edin-C-positive and et-negative). The high prevalence of edin-C-positive and etnegative strains observed in our study might be explained by the plasticity of the pETB plasmid, which has been previously reported in two different variants [18,21]. Also, a recent study shows genetic variations in pathogenicity islands encoding EDIN-B [17].

Given that spa analysis constitutes a good tool for epidemiological typing of S. aureus [26], the use of this method allowed us to exclude the clonal origin of edin-C positive strains.

The fact that edin-C is plasmid born might explain its presence in strains of various genetic backgrounds.

S. aureus positive for edin are more frequently associated with deep-seated infections of soft tissues. Infection of endothelial cells, and other cell types, by EDIN-producing S. aureus triggers the formation of transcellular tunnels named macroapertures [12,13]. Moreover, we recently reported that S. aureus EDIN toxin promotes formation of infection foci in a mouse model of bacteremia [15]. Together with the present study, this suggests that EDIN might enhance the invasive capacity of S. aureus. However, whether or not edin-positive S. aureus is preferentially associated with a specific type of deep seated infection remains to be further determined.

Figure legend

Fig. 1 Characterization of the three edin alleles. A) PCR amplification of edin-A, edin-B and edin-C from S. aureus strains S25edin-A(+) [15], S7256edin-B(+) and S7475edin-C(+) (this study), respectively, using specific oligonucleotides edinA, edinB, edinC (Table 1) and the previously described edin oligonucleotides referred as edinX [19]. B) Sequence alignments of edin-A, edin-B and edin-C showing sequence homologies and localization of each oligonucleotide (underlined: edinX; highlighted: edinA, edinB and edinC).

Table 1 :agr2

 1 Oligonucleotides primers used in this study Sense 5'-ATGCACATGG TGCACATGC-3' Antisense 5'-TATTACTAATTGAAAAGTGC CATAGC-3'[30]

Table 1 :

 1 Oligonucleotides primers used in this study

  Figure1

Table 2 :

 2 Virulence profile and antibiotic susceptibility of clinical edin-positive S. aureus isolates.

		blood (n=2)	superficial soft tissue (n=11)	urine sample (n=4)	sputum sample (n=10)	deep-seated soft tissue (n=9)	total (n=36)
		n	%	n	%	n	%	n	%	n	%	n	%
	Virulence profile
	MSCRAMMs
	bbp	0	0	3	27	3	75	3	30		67	15	42
	cna	1	50	9	82	4	100	10	100		89	32	89
	ebpS	0	0	5	45	1	25	6	60		67	18	50
	fnbA	1	50	7	64	1	25	5	50		56	19	53
	fnbB	1	50	11	100	4	100	7	70		89	31	86
	fib	2	100	10	91	4	100	7	70		100	32	89
	clfA	2	100	11	100	4	100	10	100		100	36	100
	clfB	2	100	11	100	4	100	10	100		100	36	100
	eno	2	100	11	100	4	100	10	100		100	36	100
	SEs
	sea	1	50	10	91	3	75	9	90		100	32	89
	seb	1	50	7	64	2	50	8	80		67	24	67
	sec	1	50	7	64	2	50	10	100		67	26	72
	sed	0	0	8	73	3	75	6	60		67	23	64
	see	0	0	0	0	0	0	0	0		0	0	0
	sek	1	50	5	45	1	25	7	70		22	16	44
	seq	1	50	3	27	1	25	5	50		56	15	42
	seg	2	100	10	91	2	50	10	100		100	33	92
	seh	0	0	0	0	0	0	0	0		11	1	3
	sei	2	100	11	100	2	50	9	90		89	32	89
	sej	0	0	1	9	0	0	2	20		11	4	11
	tst-1	2	100	6	55	1	25	7	70		56	21	58
	etA	0	0	2	18	0	0	2	20		22	6	17
	etB	1 &	50	4 $	36	0	0	2	20		11	8	22
	etD	0	0	0	0	1*	25	1*	10		0	2	6
	lukPV	0	0	1	9	1	25	0	0		33	5	14
	lukE	2	100	8	73	3	75	5	50		44	24	67
	agr group
	agr1	1	50	3	27	3	75	6	60		56	18	50
	agr2	1	50	3	27	1	25	1	10		0	6	17
	agr3	0	0	3	27	0	0	0	0		33	6	17
	agr4	0	0	2	18	0	0	3	30		11	6	17
	antibiotic resistance profile
	penicillin G	1	50	5	45	2	50	8	80		67	22	61
	Methicillin	0	0	0	0	0	0	0	0		11	1	3
	Erythromycin	0	0	1	9	0	0	1	10		11	3	8
	Clindamycin	0	0	0	0	0	0	0	0		11	1	3
	Fusidic acid	0	0	0	0	1¤	25	0	0	1¤	11	2	6
	* Edin B positive strains

$

Edin A positive for one strain & EdinA positive strain ¤ Increase in fusidic acid MIC, classified as intermediary sensibility

Table 3 :

 3 spa-type of 36 edin-positive Staphylococcus aureus isolates.

Table 4 :

 4 Presence of edin genes in 256 Staphylococcus aureus isolates associated with various clinical syndromes.

	Source of isolates (N)	Number of edin isolates (%)	edinA	edin-isoforms edinB	edinC
	Blood (28)	2 (7.1)	1	0	1
	Urine (41)	4 (9.8)	0	1	3
	Superficial soft tissue (83)	11 (13.3)	1	0	10
	Deep-seated soft tissue (35)	9 (25.7)*	0	0	9
	Sputum (69)	10 (14.5)	0	1	9
	Total (256)	36 (14)	2 (5)	2 (5)	32 (90)

Table 4 :

 4 Presence of edin genes in 256 Staphylococcus aureus strains associated with various clinical syndromes.

	Source of isolates (N)	Number of edin isolates (%)	edinA	edin-isoforms edinB	edinC
	Blood (28)	2 (7.1)	1	0	1
	Urine (41)	4 (9.8)	0	1	3
	Superficial soft tissue (83)	11 (13.3)	1	0	10
	Deep-seated soft tissue (35)	9 (25.7)*	0	0	9
	Sputum (69)	10 (14.5)	0	1	9
	Total (256)	36 (14)	2 (5)	2 (5)	32 (90)
	* p<0,05 (Fisher's exact test)

Acknowledgments

We are grateful to Fernand Girard-Pipau and Claire Poyart for providing various strains of S. aureus and Pr Jean-Louis Mege for critical reading of the manuscript.

Our laboratory is supported by an institutional funding from the INSERM, a grant from the Agence Nationale de la Recherche (ANR RPV07055ASA) and the Association pour la Recherche sur le Cancer (ARC 4906).

Acknowledgments

We are grateful to Fernand Girard-Pipau and Claire Poyart for providing various strains of S. aureus and Pr Jean-Louis Mege for critical reading of the manuscript.

Our laboratory is supported by an institutional funding from the INSERM, a grant from the Agence Nationale de la Recherche (ANR RPV07055ASA) and the Association pour la Recherche sur le Cancer (ARC 4906).

positive strains carried the staphylococcal exfoliative gene etB. We found that 25 edin-C positive S. aureus (78%) were negative for etA, etB and etD exfoliative toxin encoding genes.

We observed that only 22% of the edin-C positive S. aureus (7 out of 32) had at least one of the two isoforms (etA or etB) of the exfoliative toxin gene. Five of these seven strains carried both the etA and etB genes. Finally, a large number of edin-positive S. aureus belonged to the agr group 1 (50%, 18 strains), and to a lesser extent, to agr groups 2, 3 and 4 (17%, 6 strains each) (Table 2).

spa-typing

Determination of the spa type of 26 edin-positive strains [23] allowed us to exclude the clonal origin of all edin-C positive S. aureus in our survey. Among them, only six strains could be classified as ST45 (two isolates), ST30 (two isolates), ST59 or ST26. The other 20 strains showed a high variability of their spa type (Table 3). For 14 strains, we determined new repeat successions including unidentified 24-bp repeats thus defining new spa types. Among them, strains S7926 and S7262, isolated respectively in deep seated soft tissue and sputum sample from unrelated infected patients, presented the same spa type t6956 (Table 3).

Together these data excluded the clonal origin of all edin-C positive S. aureus in our survey.

Antibiotic susceptibility profiles

We next investigated whether edin-positive strains were associated with specific antibiotic resistance profiles, such as community-acquired methicillin resistant S. aureus ST80 and fusidic acid-resistant impetigo clones [20,21]. In relation with these studies, we determined the minimum inhibitory concentrations (MICs) of edin-positive strains for the following antibiotics: penicillin G, methicillin, erythromycin, clindamycin and fusidic acid. Fourteen edin-positive strains were susceptible to all tested antimicrobial molecules (41.6 %), except fusidic acid. Indeed, one of 14 isolates tested presented only an increase in fusidic acid MIC, classified as intermediary sensibility. Twenty two isolates were resistant to penicillin G. Among them, 19 were susceptible to all other antimicrobial molecules tested (83%) and two strains also presented erythromycin resistance (8.7%). Only one strain, positive for edin-C, showed a methicillin-resistance associated to additional resistance, i.e. erythromycin and clindamycin as well as an increase of MIC to fusidic acid. This strain was negative for etA