Argemi, X.

Dahyot, S

Dubos, C.

François, P.

Laumay, F

Lebeurre, J

Lemée, L.

Pestel-Caron, M.

Prevost, G.

English

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HAL Id: hal-02264266
https://hal-normandie-univ.archives-ouvertes.fr/hal-02264266
Submitted on 6 Aug 2019
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fbl-typing of Staphylococcus lugdunensis: a frontline
tool for epidemiological studies
S Dahyot, J Lebeurre, F Laumay, X. Argemi, C. Dubos, L. Lemée, G.
Prevost, P. François, M. Pestel-Caron
To cite this version:
S Dahyot, J Lebeurre, F Laumay, X. Argemi, C. Dubos, et al.. fbl-typing of Staphylococcus lug-
dunensis: a frontline tool for epidemiological studies. ECCMID, Apr 2019, Amsterdam, Netherlands.
￿hal-02264266￿
fbl-typing of Staphylococcus lugdunensis: a frontline tool for epidemiological studies
S. Dahyot1, J. Lebeurre2, F. Laumay3, X. Argemi4, 5, C. Dubos1, L. Lemée1, G. Prévost4, P. François3, M. Pestel-Caron1
1Normandie Univ, UNIROUEN, GRAM EA2656, Rouen University Hospital, F-76000 Rouen, France; 2Normandie Univ, UNIROUEN, GRAM EA2656, F-76000 Rouen, France; 3University of Geneva Hospitals, Genomic Research Laboratory, Service of Infectious Diseases, Geneva, 
Switzerland; 4Université de Strasbourg, CHRU de Strasbourg, VBP EA7290, Fédération de Médecine Translationnelle de Strasbourg, Institut de Bactériologie, F-67000 Strasbourg, France; 5Hôpitaux Universitaires, Maladies Infectieuses et Tropicales, F-67000 Strasbourg, France.
P2690 session «Molecular & genomic surveillance 
of Gram-positive cocci »
Introduction
 fbl R-domain is a reliable target as a frontline tool for the single locus 
genotyping of clinical isolates of S. lugdunensis.
 fbl-typing is an easy to use, cost-effective, rapid and portable method, 
suitable for local and international epidemiological studies.
 Impact of fbl polymorphisms on the structure of the protein and in 
virulence remains to be determined.
Conclusion
 fbl-typing results
 Concordance between typing methods
 Development of « fbl-typing Server »
1Chassain B. et al. Multilocus sequence typing analysis of S. lugdunensis implies a clonal population structure. 2012. J. Clin. Microbiol.
2 Dahyot S. et al. Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and Tandem Repeat Sequence Typing (TRST), helpful tools for subtyping S. lugdunensis. 2018. Sci. Rep.
3Argemi X. et al. VISLISI trial, a prospective clinical study allowing identification of a new metalloprotease and putative virulence factor from S. lugdunensis. 2017. Clin Microbiol Infect.
• Sequencing and assembling of fbl repeats obtained for the 240 isolates:
- length of the fbl R-domain : 9 to 52 repeats 
- number of sequences of 18-bp repeat : 54 individual sequences 
 number of unique combination : 92 fbl-types (fbl9a to fbl52a)
• Most common fbl-types : fbl47b (n = 43), fbl45f (n = 24) and fbl41a (n = 13)
• fbl-typing clustering (Figure 2):
- 10 clusters identified (1 to 10)
- main clusters: cluster 1  (57 isolates, 20 fbl-types) and cluster 2 (52 isolates, 6 fbl-types)
• Comparison of fbl-typing clustering with MLST and TRST data for 128 isolates 
• Discriminatory power of fbl-typing (DIfbl = 0.964) (Table 1) :
- higher than MLST (DIMLST = 0.899)
- equivalent to TRST (DITRST = 0.943)
• Clustering results of fbl-typing :
- congruent with MLST
- fbl-types predict MLST clonal complexes (CCs) with 100% of probability (Figure 2)
• Development of a Web tool publicly available: http://fbl-typing.univ-rouen.fr
• Allows identification of fbl-types from sequencing data (FASTA format)
Strains & Methods
Results
Figure 2. Minimum spanning tree analysis of the 240 S. lugdunensis isolates based on fbl-types.
Cluster analysis was performed using the polymorphic VNTR typing plugin of BioNumerics. fbl-types separated by a minimum
spanning tree distance of ≤ 2 (i.e., if they were ≥ 97% similar) were considered closely related and assigned to the same cluster. Each
circle represents a fbl-type and its size is proportional to the number of isolates. The length of the branches expressed the minimum
spanning tree distance between two fbl-types. Gray zones around circles delineate fbl clusters. The colors used are based on clonal
complexes (CCs) defined by MLST, undetermined = unknown CC (isolates non characterized by MLST).
• Staphylococcus lugdunensis is increasingly recognized as a virulent pathogen, responsible for severe 
infections with an outcome resembling that of Staphylococcus aureus rather than that caused by 
coagulase-negative staphylococci
• Molecular typing methods based on Sanger sequencing have been developed to characterize 
S. lugdunensis genetic diversity:
- MultiLocus Sequence Typing (MLST)1 : 7 housekeeping genes
- Tandem Repeat Sequence Typing (TRST)2 : 7 Variable Number of Tandem Repeats (VNTRs) 
• However, MLST and TRST daily use is time consuming and expensive
We developed a single locus typing scheme for S. lugdnensis, based on the DNA sequence 
analysis of the R-domain within the fbl gene encoding the fibrinogen-binding protein Fbl
• Analysis of 240 S. lugdunensis isolates recovered from 230 patients (various clinical and geographical 
origins):
- 128 isolates previously characterized by MLST and TRST, collected from five French regions and 
Sweden 2,3
- 106 isolates collected at University Hospital of Rouen in 2016
• fbl-typing:
- amplification and sequencing of the fbl R-domain (18-bp repeats region) (Figure 1)
- determination of the numbers as well as the sequences of each fbl repeats (BioNumerics 7.6) 
- each unique combination of repeats = a fbl-type 
• Deduction of genetic relationships by minimum spanning tree analysis
• Evaluation of the discriminatory power by calculation of Simpson's Index of diversity (DI)  and 
associated Confidence Intervals (CI)
Figure 1. Schematic representation of the fbl gene of S. lugdunensis.
Boxes indicate segments of the gene: S, signal peptide, N1-, N2-, and N3-
domains, R, repeat domain, WMC: W, wall spanning; M, membrane spanning;
C, cytoplasmic positively charge tail. The locations of the forward and reverse
primers used to amplify and sequence the R-domain are shown at the top.
Table 1. Discriminatory power of the three typing methods for unrelated isolates.
Only one genotype by patient was included to avoid any bias.
aDI: Simpson's Diversity index. bCI: Confidence Interval. cND: Not Determined.


fbl-typing of Staphylococcus lugdunensis: a frontline tool for epidemiological studies

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fbl-typing of Staphylococcus lugdunensis: a frontline tool for epidemiological studies

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