Toxin Gene Content of the Lyon Methicillin-Resistant Staphylococcus aureus Clone Compared with That of Other Pandemic Clones
http://www.100md.com
《微生物临床杂志》
Centre National de Reference des Staphylocoques, INSERM E0230, IFR 62, Faculte de Medecine Laennec, 7 rue Guillaume Paradin, 69008 Lyon, France
ABSTRACT
The methicillin-resistant Staphylococcus aureus (MRSA) Lyon clone, detected throughout France, contains the enterotoxin A gene (sea), like other pandemic clones of clonal complex 8 (CC8). The egc locus was detected in MRSA pandemic clones of CC5, CC22, and CC45, occasionally with the toxic shock syndrome toxin 1 gene. The representative strain of the EMRSA-16 clone (CC30) harbored both sea and the egc locus.
TEXT
Methicillin-resistant Staphylococcus aureus (MRSA) strains are spreading through hospitals worldwide and are the leading hospital-acquired pathogen (1, 2). Population genetics studies based on multilocus sequence typing (MLST) have shown that major MRSA clones have emerged from five clonal complexes (CCs): CC5, CC8, CC22, CC30, and CC45 (16). Within each CC, prevalent clones (the Archaic clone, the Iberian clone, and the German clone, for example) have been genetically characterized by their sequence type, their spa type, and their SCCmec cassette (13). In France, the gentamicin-resistant Iberian clone was the major MRSA clone in the 1990s (11). Since 1992, French MRSA isolates have been increasingly susceptible to gentamicin, indicating a gradual decline of the Iberian clone (11).
MRSA clones have been characterized on the basis of several other characteristics. For instance, MRSA detected in Japan was shown to produce toxic shock syndrome toxin 1, a superantigenic toxin family member (7, 15). Moreover, Peacock et al. showed that the prevalence of the staphylococcal enterotoxin A gene (sea) was higher in invasive infections due to one of the MRSA clones (EMRSA-16) that predominate in the United Kingdom (14). Up to now, 18 superantigenic toxins have been described for S. aureus (7), but the distribution of the genes encoding superantigenic toxins is not known for all MRSA pandemic clones.
The aim of this study was to characterize the superantigenic gene content of the French and major pandemic MRSA clones. The study was conducted in three steps: (i) a local study characterizing MRSA isolates collected by blood culture in 2003 in intensive care units of Edouard Herriot Hospital in Lyon, France, permitting the recognition of the major MRSA clone; (ii) a nationwide study of the spread of the major clone identified in Lyon (designated the Lyon clone); (iii) a worldwide study comparing the characteristics and toxin gene content of the Lyon clone and the major pandemic MRSA clones.
All S. aureus isolates included in this study were identified by means of standard microbiological methods, and drug resistance was determined by using the Phoenix Automated Microbiology System (BD Biosciences). MRSA isolates were first genetically characterized by means of MLST as described by Enright et al. (4, 5). spa types were determined with the assistance of Ridom Staph Type software (Ridom GmbH, Würzburg, Germany [http://www.ridom.de/staphtype/]) (6). Pulsed-field gel electrophoresis patterns were prepared as previously described (17) and were digitized and analyzed with the Taxotron typing system (Institut Pasteur, France). The accessory gene regulator (agr) allele group (1 to 4), the presence of the mecA gene, and the toxin gene content were determined by multiplex PCR as previously described (8-10). The type of the staphylococcal chromosomal cassette (SCCmec) was determined as described by Oliveira and de Lencastre (12).
In 2003, 17 MRSA blood isolates were collected at Edouard Herriot Hospital in Lyon, France. They belonged to a major lineage, designated sequence type 8 (ST8) (14 isolates), and a minor lineage, designated ST5 (3 isolates). Clone ST8, named the Lyon clone, included 13 of the 14 ST8 isolates. This clone shared ST8 within the CC8, spa type t008 or a minor variation of spa type t008 (t068 or t569, as shown in Table 1), agr allele type 1, the sea gene, the leucocidin lukE-lukD genes, the hemolysin hlgv gene, and the SCCmec type IVA cassette. The staphylococcal enterotoxin B and D genes (seb and sed) were not detected in all of the isolates. Almost all of the Lyon clone isolates were resistant to penicillin, oxacillin, kanamycin, tobramycin, erythromycin, and ofloxacin. One ST8 isolate (HT20040113) was not considered to belong to the Lyon clone, owing to the multiple variations of the spa type and to the absence of the sea gene (Table 1). Two of the three ST5 MRSA isolates contained the tst gene and corresponded to an emerging MRSA clone related to the New York/Japan (NY/J) clone (3).
Since our multiplex PCR method based on the detection of more than 15 virulence factor genes and the agr gene regulator reveals the genetic background of a given strain (9), we considered that hospital-acquired MRSA strains sharing the agr-1, sea, lukE-lukD, and hlgv genes potentially belonged to the Lyon clone. To evaluate the spread of the Lyon clone throughout France, we analyzed the 2001 to 2005 database of the French National Reference Center for staphylococci, excluding Panton-Valentine-leucocidin-positive MRSA isolates, which belong to community-acquired MRSA clones. We selected 320 Panton-Valentine-leucocidin-negative MRSA isolates, of which 165 (51.6%), from 20 French towns, matched the characteristics of the Lyon clone. Thirty-two of these isolates were fully characterized by MLST and spa typing. They were ST8 and had an SCCmec cassette of type IVA (except for two strains, which had an SCCmec cassette of type IV) and spa type t008, t024, t068, t121, t569 or t681 (all considered minor variants of the major spa type t008, differing by a single repeat [t024, t068, t121, and t681] or two repeats [t569]), confirming that they belonged to the Lyon clone. This suggests that the Lyon clone is present throughout France.
We then compared the characteristics of the Lyon clone with those of other pandemic MRSA clones (Fig. 1). The Lyon clone and New York clone V belonged to CC8, were ST8, and had an agr type 1 allele, a similar toxin gene profile, and related spa types. Other MRSA clones of CC8 had an agr-1 allele. The sea gene was also detected in representatives of the Iberian and Hungarian clones. All representatives of MRSA clones belonging to CC5, CC22, and CC45 shared the enterotoxin gene cluster locus (egc) encoding five superantigenic enterotoxin genes (seg, sei, sem, sen, and seo) and occasionally the tst gene. The representative strain of the EMRSA-16 clone, which represents CC30, shared both the sea gene and the egc locus. The Brazilian clone was the only clone that lacked all of the tested superantigenic toxin genes. Note that all strains of the same CC had the same agr allele [1, 2, or 3].
FIG. 1. Dendrogram constructed from the schematic representation of the pulsed-field gel electrophoresis types of pandemic MRSA clones and other MRSA isolates included in this study together with their genetic characteristics, such as ST, spa type, SCCmec type, agr allele group, and toxin gene content (sea-sed, staphylococcal enterotoxin A to D genes; sel, staphylococcal enterotoxin L gene; egc, enterotoxin gene cluster; tst, toxic shock syndrome toxin 1 gene). CCs were assigned according to the MLST database at http://www.mlst.net. Blue isolates correspond to the Lyon clone and red isolates to an emerging clone (3). Drug resistance is given in parentheses (KAN, kanamycin; TOB, tobramycin; GEN, gentamicin; ERY, erythromycin; CLI, clindamycin; OFX, ofloxacin; SXT, trimethoprim-sulfamethoxazole; FUS, fusidic acid; FOF, fosfomycin). NT, nontypeable. The fragment size (kb) of the reference strain NCTC8325 is indicated on the bottom line.
In conclusion, the Lyon clone is detected all over France and is related to other pandemic MRSA clones that share the sea gene. With the exception of the Brazilian clone, all pandemic MRSA clones contain at least one gene encoding a superantigenic toxin. Superantigenic toxins could partly account for the virulence of hospital-acquired MRSA infections.
ACKNOWLEDGMENTS
We thank H. de Lencastre, A. Tomasz, and J. Perry for kindly providing pandemic MRSA reference strains; Christine Gardon, Christine Courtier, and Celine Spinelli (Centre National de Reference des Staphylocoques) for their technical support; and David Young for editorial assistance.
FOOTNOTES
REFERENCES
Astiz, M. E., and E. C. Rackow. 1998. Septic shock. Lancet 351:1501-1505.
Brun-Buisson, C., P. Meshaka, P. Pinton, and B. Vallet. 2004. EPISEPSIS: a reappraisal of the epidemiology and outcome of severe sepsis in French intensive care units. Intensive Care Med. 30:580-588.
Durand, G., M. Bes, H. Meugnier, M. C. Enright, F. Forey, N. Liassine, A. Wenger, K. Kikuchi, G. Lina, F. Vandenesch, and J. Etienne. 2006. Detection of new methicillin-resistant Staphylococcus aureus clones containing the toxic shock syndrome toxic shock syndrome 1 gene responsible for hospital- and community-acquired infections in France. J. Clin. Microbiol. 44:847-853.
Enright, M. C., N. P. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008-1015.
Enright, M. C., and B. G. Spratt. 1999. Multilocus sequence typing. Trends Microbiol. 7:482-487.
Harmsen, D., H. Claus, W. Witte, J. Rothganger, D. Turnwald, and U. Vogel. 2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol. 41:5442-5448.
Holtfreter, S., and B. M. Broker. 2005. Staphylococcal superantigens: do they play a role in sepsis Arch. Immunol. Ther. Exp. (Warsaw) 53:13-27.
Jarraud, S., G. Cozon, F. Vandenesch, M. Bes, J. Etienne, and G. Lina. 1999. Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J. Clin. Microbiol. 37:2446-2449.
Jarraud, S., C. Mougel, J. Thioulouse, G. Lina, H. Meugnier, F. Forey, X. Nesme, J. Etienne, and F. Vandenesch. 2002. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect. Immun. 70:631-641.
Jarraud, S., M. A. Peyrat, A. Lim, A. Tristan, M. Bes, C. Mougel, J. Etienne, F. Vandenesch, M. Bonneville, and G. Lina. 2001. egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J. Immunol. 166:669-677.
Lelièvre, H., G. Lina, M. K. Jones, C. Olive, F. Forey, M. Roussel- Delvallez, M. H. Nicolas-Chanoine, C. M. Bebear, V. Jarlier, A. Andremont, F. Vandenesch, and J. Etienne. 1999. Emergence and spread in French hospital of methicillin-resistant Staphylococcus aureus with increasing susceptibility to gentamycin and other antibiotics. J. Clin. Microbiol. 37:3452-3457.
Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2155-2161.
Oliveira, D. C., A. Tomasz, and H. de Lencastre. 2002. Secrets of success of a human pathogen: molecular evolution of pandemic clones of methicillin-resistant Staphylococcus aureus. Lancet Infect. Dis. 2:180-189.
Peacock, S. J., C. E. Moore, A. Justice, M. Kantzanou, L. Story, K. Mackie, G. O'Neill, and N. P. Day. 2002. Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus. Infect. Immun. 70:4987-4996.
Piao, C., T. Karasawa, K. Totsuka, T. Uchiyama, and K. Kikuchi. 2005. Prospective surveillance of community-onset and healthcare-associated methicillin-resistant Staphylococcus aureus isolated from a university-affiliated hospital in Japan. Microbiol. Immunol. 49:959-970.
Robinson, D. A., and M. C. Enright. 2003. Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 47:3926-3934.
Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.(Tristan Ferry, Michèle Be)
ABSTRACT
The methicillin-resistant Staphylococcus aureus (MRSA) Lyon clone, detected throughout France, contains the enterotoxin A gene (sea), like other pandemic clones of clonal complex 8 (CC8). The egc locus was detected in MRSA pandemic clones of CC5, CC22, and CC45, occasionally with the toxic shock syndrome toxin 1 gene. The representative strain of the EMRSA-16 clone (CC30) harbored both sea and the egc locus.
TEXT
Methicillin-resistant Staphylococcus aureus (MRSA) strains are spreading through hospitals worldwide and are the leading hospital-acquired pathogen (1, 2). Population genetics studies based on multilocus sequence typing (MLST) have shown that major MRSA clones have emerged from five clonal complexes (CCs): CC5, CC8, CC22, CC30, and CC45 (16). Within each CC, prevalent clones (the Archaic clone, the Iberian clone, and the German clone, for example) have been genetically characterized by their sequence type, their spa type, and their SCCmec cassette (13). In France, the gentamicin-resistant Iberian clone was the major MRSA clone in the 1990s (11). Since 1992, French MRSA isolates have been increasingly susceptible to gentamicin, indicating a gradual decline of the Iberian clone (11).
MRSA clones have been characterized on the basis of several other characteristics. For instance, MRSA detected in Japan was shown to produce toxic shock syndrome toxin 1, a superantigenic toxin family member (7, 15). Moreover, Peacock et al. showed that the prevalence of the staphylococcal enterotoxin A gene (sea) was higher in invasive infections due to one of the MRSA clones (EMRSA-16) that predominate in the United Kingdom (14). Up to now, 18 superantigenic toxins have been described for S. aureus (7), but the distribution of the genes encoding superantigenic toxins is not known for all MRSA pandemic clones.
The aim of this study was to characterize the superantigenic gene content of the French and major pandemic MRSA clones. The study was conducted in three steps: (i) a local study characterizing MRSA isolates collected by blood culture in 2003 in intensive care units of Edouard Herriot Hospital in Lyon, France, permitting the recognition of the major MRSA clone; (ii) a nationwide study of the spread of the major clone identified in Lyon (designated the Lyon clone); (iii) a worldwide study comparing the characteristics and toxin gene content of the Lyon clone and the major pandemic MRSA clones.
All S. aureus isolates included in this study were identified by means of standard microbiological methods, and drug resistance was determined by using the Phoenix Automated Microbiology System (BD Biosciences). MRSA isolates were first genetically characterized by means of MLST as described by Enright et al. (4, 5). spa types were determined with the assistance of Ridom Staph Type software (Ridom GmbH, Würzburg, Germany [http://www.ridom.de/staphtype/]) (6). Pulsed-field gel electrophoresis patterns were prepared as previously described (17) and were digitized and analyzed with the Taxotron typing system (Institut Pasteur, France). The accessory gene regulator (agr) allele group (1 to 4), the presence of the mecA gene, and the toxin gene content were determined by multiplex PCR as previously described (8-10). The type of the staphylococcal chromosomal cassette (SCCmec) was determined as described by Oliveira and de Lencastre (12).
In 2003, 17 MRSA blood isolates were collected at Edouard Herriot Hospital in Lyon, France. They belonged to a major lineage, designated sequence type 8 (ST8) (14 isolates), and a minor lineage, designated ST5 (3 isolates). Clone ST8, named the Lyon clone, included 13 of the 14 ST8 isolates. This clone shared ST8 within the CC8, spa type t008 or a minor variation of spa type t008 (t068 or t569, as shown in Table 1), agr allele type 1, the sea gene, the leucocidin lukE-lukD genes, the hemolysin hlgv gene, and the SCCmec type IVA cassette. The staphylococcal enterotoxin B and D genes (seb and sed) were not detected in all of the isolates. Almost all of the Lyon clone isolates were resistant to penicillin, oxacillin, kanamycin, tobramycin, erythromycin, and ofloxacin. One ST8 isolate (HT20040113) was not considered to belong to the Lyon clone, owing to the multiple variations of the spa type and to the absence of the sea gene (Table 1). Two of the three ST5 MRSA isolates contained the tst gene and corresponded to an emerging MRSA clone related to the New York/Japan (NY/J) clone (3).
Since our multiplex PCR method based on the detection of more than 15 virulence factor genes and the agr gene regulator reveals the genetic background of a given strain (9), we considered that hospital-acquired MRSA strains sharing the agr-1, sea, lukE-lukD, and hlgv genes potentially belonged to the Lyon clone. To evaluate the spread of the Lyon clone throughout France, we analyzed the 2001 to 2005 database of the French National Reference Center for staphylococci, excluding Panton-Valentine-leucocidin-positive MRSA isolates, which belong to community-acquired MRSA clones. We selected 320 Panton-Valentine-leucocidin-negative MRSA isolates, of which 165 (51.6%), from 20 French towns, matched the characteristics of the Lyon clone. Thirty-two of these isolates were fully characterized by MLST and spa typing. They were ST8 and had an SCCmec cassette of type IVA (except for two strains, which had an SCCmec cassette of type IV) and spa type t008, t024, t068, t121, t569 or t681 (all considered minor variants of the major spa type t008, differing by a single repeat [t024, t068, t121, and t681] or two repeats [t569]), confirming that they belonged to the Lyon clone. This suggests that the Lyon clone is present throughout France.
We then compared the characteristics of the Lyon clone with those of other pandemic MRSA clones (Fig. 1). The Lyon clone and New York clone V belonged to CC8, were ST8, and had an agr type 1 allele, a similar toxin gene profile, and related spa types. Other MRSA clones of CC8 had an agr-1 allele. The sea gene was also detected in representatives of the Iberian and Hungarian clones. All representatives of MRSA clones belonging to CC5, CC22, and CC45 shared the enterotoxin gene cluster locus (egc) encoding five superantigenic enterotoxin genes (seg, sei, sem, sen, and seo) and occasionally the tst gene. The representative strain of the EMRSA-16 clone, which represents CC30, shared both the sea gene and the egc locus. The Brazilian clone was the only clone that lacked all of the tested superantigenic toxin genes. Note that all strains of the same CC had the same agr allele [1, 2, or 3].
FIG. 1. Dendrogram constructed from the schematic representation of the pulsed-field gel electrophoresis types of pandemic MRSA clones and other MRSA isolates included in this study together with their genetic characteristics, such as ST, spa type, SCCmec type, agr allele group, and toxin gene content (sea-sed, staphylococcal enterotoxin A to D genes; sel, staphylococcal enterotoxin L gene; egc, enterotoxin gene cluster; tst, toxic shock syndrome toxin 1 gene). CCs were assigned according to the MLST database at http://www.mlst.net. Blue isolates correspond to the Lyon clone and red isolates to an emerging clone (3). Drug resistance is given in parentheses (KAN, kanamycin; TOB, tobramycin; GEN, gentamicin; ERY, erythromycin; CLI, clindamycin; OFX, ofloxacin; SXT, trimethoprim-sulfamethoxazole; FUS, fusidic acid; FOF, fosfomycin). NT, nontypeable. The fragment size (kb) of the reference strain NCTC8325 is indicated on the bottom line.
In conclusion, the Lyon clone is detected all over France and is related to other pandemic MRSA clones that share the sea gene. With the exception of the Brazilian clone, all pandemic MRSA clones contain at least one gene encoding a superantigenic toxin. Superantigenic toxins could partly account for the virulence of hospital-acquired MRSA infections.
ACKNOWLEDGMENTS
We thank H. de Lencastre, A. Tomasz, and J. Perry for kindly providing pandemic MRSA reference strains; Christine Gardon, Christine Courtier, and Celine Spinelli (Centre National de Reference des Staphylocoques) for their technical support; and David Young for editorial assistance.
FOOTNOTES
REFERENCES
Astiz, M. E., and E. C. Rackow. 1998. Septic shock. Lancet 351:1501-1505.
Brun-Buisson, C., P. Meshaka, P. Pinton, and B. Vallet. 2004. EPISEPSIS: a reappraisal of the epidemiology and outcome of severe sepsis in French intensive care units. Intensive Care Med. 30:580-588.
Durand, G., M. Bes, H. Meugnier, M. C. Enright, F. Forey, N. Liassine, A. Wenger, K. Kikuchi, G. Lina, F. Vandenesch, and J. Etienne. 2006. Detection of new methicillin-resistant Staphylococcus aureus clones containing the toxic shock syndrome toxic shock syndrome 1 gene responsible for hospital- and community-acquired infections in France. J. Clin. Microbiol. 44:847-853.
Enright, M. C., N. P. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008-1015.
Enright, M. C., and B. G. Spratt. 1999. Multilocus sequence typing. Trends Microbiol. 7:482-487.
Harmsen, D., H. Claus, W. Witte, J. Rothganger, D. Turnwald, and U. Vogel. 2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol. 41:5442-5448.
Holtfreter, S., and B. M. Broker. 2005. Staphylococcal superantigens: do they play a role in sepsis Arch. Immunol. Ther. Exp. (Warsaw) 53:13-27.
Jarraud, S., G. Cozon, F. Vandenesch, M. Bes, J. Etienne, and G. Lina. 1999. Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J. Clin. Microbiol. 37:2446-2449.
Jarraud, S., C. Mougel, J. Thioulouse, G. Lina, H. Meugnier, F. Forey, X. Nesme, J. Etienne, and F. Vandenesch. 2002. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect. Immun. 70:631-641.
Jarraud, S., M. A. Peyrat, A. Lim, A. Tristan, M. Bes, C. Mougel, J. Etienne, F. Vandenesch, M. Bonneville, and G. Lina. 2001. egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J. Immunol. 166:669-677.
Lelièvre, H., G. Lina, M. K. Jones, C. Olive, F. Forey, M. Roussel- Delvallez, M. H. Nicolas-Chanoine, C. M. Bebear, V. Jarlier, A. Andremont, F. Vandenesch, and J. Etienne. 1999. Emergence and spread in French hospital of methicillin-resistant Staphylococcus aureus with increasing susceptibility to gentamycin and other antibiotics. J. Clin. Microbiol. 37:3452-3457.
Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2155-2161.
Oliveira, D. C., A. Tomasz, and H. de Lencastre. 2002. Secrets of success of a human pathogen: molecular evolution of pandemic clones of methicillin-resistant Staphylococcus aureus. Lancet Infect. Dis. 2:180-189.
Peacock, S. J., C. E. Moore, A. Justice, M. Kantzanou, L. Story, K. Mackie, G. O'Neill, and N. P. Day. 2002. Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus. Infect. Immun. 70:4987-4996.
Piao, C., T. Karasawa, K. Totsuka, T. Uchiyama, and K. Kikuchi. 2005. Prospective surveillance of community-onset and healthcare-associated methicillin-resistant Staphylococcus aureus isolated from a university-affiliated hospital in Japan. Microbiol. Immunol. 49:959-970.
Robinson, D. A., and M. C. Enright. 2003. Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 47:3926-3934.
Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.(Tristan Ferry, Michèle Be)