RÉSULTATS SUPPLÉMENTAIRES

♦ Au début de cette étude, une première banque de soustraction entre la souche utilisée comme ferment S. xylosus S04002 et la souche S. xylosus C2a a été constituée. Elle comprenait 576 clones. La taille et la présence d’inserts ont tout d’abord été détectées chez 192 clones et 33 ont été séquencés. L’analyse de ces séquences a révélée que plus de 70% des fragments soustraits étaient d’origine plasmidique. Or, le but de notre étude était l’identification des différences chromosomiques entre des souches de S. xylosus. Pour isoler et étudier les plasmides, il existe des méthodes plus adaptées comme la construction de banques plasmidiques et leur séquençage. Pour résoudre ce problème, nous avons donc

décidé de soustraire à chacune des souches cibles le génome de la souche S. xylosus C2a et les plasmides des souches cibles.

♦ Les plasmides des souches cibles ont été extraits et purifiés avec le kit QIAprep® Miniprep Handbook (Quiagen) selon les recommandations du fournisseur sauf qu’une incubation de 20 min à 37°C des cellules en présence de lysostaphine (0,5 mg/ml) a été réalisée avant l’étape de lyse. Le plasmide pSX267 de la souche S. xylosus DSM20267 d’une taille de 30 kb a servi de témoin. Les plasmides des souches d’E. coli Rsa (35 kb) et

E. coli V517 (55 kb, 7,4 kb, 5,7kb, 5,2 kb, 4 kb, 3,1 kb, 2,8 kb et 2,2 kb) ont été utilisés

comme marqueurs de taille, ainsi que le marqueur Lambda (Biorad) et Raoul (Qbiogene). La souche utilisée comme ferment possède un minimum de 7 plasmides de taille comprise entre 30 et 2,5 kb et la souche isolée de mammite un plasmide d’environ 30 kb. La souche isolée de dermite ne présente aucun plasmide apparent. Aux vues des différences du contenu plasmidique des souches de S. xylosus, nous avons voulu élargir cette étude à 15 autres souches d’origine diverse. Une grande variabilité du contenu plasmidique a été observée. La majorité des souches de S. xylosus hébergent entre 1 et 7 plasmides, de taille comprise entre 2 et 30kb. Cette première étude souligne l’intérêt de s’intéresser aux plasmides des souches de S. xylosus et à leur contenu pour mieux appréhender la diversité génétique au sein de l’espèce. Pour analyser le contenu plasmidique il sera alors nécessaire d’optimiser les méthodes d’extractions plasmidiques pour mieux évaluer la taille et le nombre de ces plasmides.

Genomic diversity in Staphylococcus xylosus species

Emilie Dordet-Frisoni, Géraud Dorchies, Cécilia De Araujo, Régine Talon, and Sabine Leroy*

INRA, Centre de Clermont-Ferrand Theix, Unité Microbiologie, Qualité et Sécurité des Aliments, 63122 Saint-Genès Champanelle, France.

RUNNING TITLE: Diversity in Staphylococcus xylosus

* Corresponding author. Tel.: +33-4-73-62-45-95; fax: +33-4-73-62-42-68. E-mail address: sleroy@clermont.inra.fr

ABSTRACT

Staphylococcus xylosus is a commensal of the skin of humans and animals and a ubiquitous

bacterium naturally present in food. It is one of the major starter cultures used for meat fermentation, but a few strains could potentially be hazardous and are related to animal opportunistic infections. To better understand the genetic diversity of S. xylosus intraspecies, suppressive and subtractive hybridization (SSH) was carried out with the S. xylosus C2a strain, a commensal of human skin, used as driver from three tester strains, S04002 used as a starter culture, S04009 isolated from cow mastitis or 00-1747 responsible for mouse dermatitis. SSH revealed 122 tester-specific fragments corresponding to 149 open reading frames (ORFs). A large proportion of these ORFs resembled genes involved in specific metabolisms. Analysis of the distribution of the tester-specific fragments in 20 S. xylosus strains of various origins showed that the S. xylosus species could be divided into two clusters with one only composed of potentially hazardous strains. The genetic content diversity of this species is colocalized in a region near the origin of replication of the chromosome. This region of speciation previously observed in Staphylococcus genus corresponded in S. xylosus species to a strain-specific region potentially implicated in ecological fitness.

INTRODUCTION

Staphylococcus xylosus is a commensal bacterium generally found inhabiting the skin and the

mucous membranes of mammals and birds (15;22). S. xylosus is ubiquitous, can be found in various niches (12;25;28) and persists in soils and on surfaces (32). This occurrence could be explained by the capacity of this species to form biofilms (29). S. xylosus is naturally present in raw meat and milk and is commonly used as starter culture for their fermentation (14;36). This species is virtually defined as a nonpathogenic Staphylococcus, but a few strains of

S. xylosus are related to animal and human opportunistic infections (27;33;37;39). Thus, most

strains are commensal and useful in food while others could potentially be hazardous, showing the versatility of this species.

Genomes, and consequently phenotypes, can be highly heterogeneous in bacterial strains, even if they belong to the same species. Genomic comparative studies between all

Staphylococcus sequenced genomes showed an average 78% identity (35). The 22% genetic

variability observed within the Staphylococcus genus is also observed within species. Indeed, genetic diversity between 36 S. aureus strains using whole-genome DNA microarrays, showed that 78% of genes were common to all strains and 22% of genes were strain-specific and might play a role in adaptation to specialized niches (7). In a previous study by genomic DNA restriction and pulsed field gel electrophoresis (PFGE) analysis of seven S. xylosus strains of various origins, we showed genomic diversity within this species, with a variation of chromosome size and ribosomal operon (rrn) number (5). S. xylosus isolated from opportunistic infections seemed to have chromosomes up to 11% larger than strains isolated from the food environment (5). Genomic differences were also found between S. xylosus strains isolated from meat products by PFGE and random amplified polymorphic DNA analyses (5), but much remains to be learned about the genetic content differences. Strain-specific genes of S. xylosus are of major interest in explaining the versatility of this species.

We expanded comparisons of S. xylosus strains, by using suppressive and subtractive hybridization (SSH). This method provides comprehensive surveys of genome differences among closely related strains by facilitating the identification of DNA segments present in one genome (tester) but absent in another (driver). It has been widely used for the analysis of bacterial pathogen genomes to discover new epidemiological markers, virulence factors, and host specificity determinants (38). SSH was also successfully used to study genome plasticity or intraspecies genomic diversity (23;30).

In this work, SSH was carried out between three S. xylosus tester strains: S. xylosus S04002, used as a starter culture, S. xylosus S04009 isolated from cow mastitis and S. xylosus 00-1747 responsible for mouse dermatitis (39), and S. xylosus C2a strain used as a driver, a commensal of human skin. The chromosomal organization of the S. xylosus C2a strain was known (5) and has constituted the starting point for studying the genetic diversity within this species. A total of 29.7, 25.4 and 22.8 kb specific DNA fragments were isolated from the 3 SSH libraries obtained from S. xylosus S04002, S04009 and 00-1747, respectively. Their distribution among 20 S. xylosus strains was analyzed and their location in the respective chromosomes of the S. xylosus tester strains was determined.

MATERIALS AND METHODS