Development of a Multi-locus VNTR-based Method for Rapid and High-Throughput Genotyping of S. aureus

A reliable automated Variable Number Tandem Repeat (VNTR) method was developed for the rapid genotyping of Staphylococcus aureus. The assay permit automated real-time epidemiology allowing not only the genotyping of MRSA but also the characterization of MSSA. All steps were optimized to ensure rapid turnaround-time (4 hours, >100 strains/day) and moderate costs (<3-4 US$ /strain). The performance, discriminatory power and reproducibility of this method was estimated at least equivalent to the time-consuming PFGE (24 hours).
This assay is a valuable tool for the epidemiological surveillance of strain dissemination and for the study of relatedness between clinical isolates.

Development of MLVA
Development of a new automated genotyping assay allowing real-time epidemiology based on variable-number of tandem repeats - “multi locus variable number of tandem repeats analysis assay” (MLVA).
The main features of the assay are:

1. A multiplex PCR using 10 primer pairs targeting gene regions with variable number of tandem repeats.
2. The PCR amplification products are analysed using microcapillary electrophoresis.
3. Each strain is automatically assessed by cluster analysis using in-house developed software.
4. All steps were optimised to ensure rapid turnaround-time (4 hours, >100 strains/day) and moderate costs (<3-4 US$/strain).
5. MLVA was validated on a large collections of S. aureus clinical isolates and a panel of control strains previously characterised using standard methods. The performance of this method was estimated at least equivalent to PFGE (which requires 24 hours). Comparison of MLVA to several genotyping methods including comparative genome hybridisation on microarrays covering the whole S. aureus genome revealed that MLVA clearly discriminate clusters of hospital acquired MRSA from community acquired MRSA.

Comparison of MW2 and N315 electrophoregrams.

Implementation of the MLVA

1. In collaboration with Dr. S. Harbarth and Prof. D. Pittet, MLVA was performed on a collection of community-acquired MRSA (CA-MRSA) clinical isolates. The assay allowed to document that CA-MRSA recovered form patients at admission, displayed a large genetic diversity in the area of study.
2. In collaboration with Dr. H. Sax, MLVA was used to investigate outbreaks in the neonatology ward. MLVA revealed that 3 different strains were responsible for infections in 17 different patients, after transmission event.
3. In collaboration with Prof. S. Lacroix, MLVA allowed to show that patients suffering from chronic rhino-sinusitis carried the same strain of S. aureus for years.
4. In collaboration with Dr. S. Harbarth and Prof. D. Pittet, MLVA was used to characterise MRSA strains from community origin over a period of 13 years, strains identified as non-multiresistant to antibiotics. These strains showed a wide diversity of molecular profiles.

Analysis of genotyping patterns, multilocus sequence typing (ST) results, presence of Panton-Valentine leukocidin (PVL), staphylococcal cassette chromosome mec (SCCmec) type, and country of patient origin of 13 community-associated, methicillin-resistant Staphylococcus aureus isolates (CA-MRSA)

References

Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates.
Francois P, Huyghe A, Charbonnier Y, Bento M, Herzig S, Topolski I, Fleury B, Lew D, Vaudaux P, Harbarth S, van Leeuwen W, van Belkum A, Blanc DS, Pittet D, Schrenzel J.
J Clin Microbiol. 2005 Jul;43(7):3346-55.
[Abstract] - [PubMed]

Use of Oligoarrays for Characterization of Community-Onset Methicillin-Resistant Staphylococcus aureus.
Koessler T, Francois P, Charbonnier Y, Huyghe A, Bento M, Dharan S, Renzi G, Lew D, Harbarth S, Pittet D, Schrenzel J.
J Clin Microbiol. 2006 Mar;44(3):1040-8.
[Abstract] - [PubMed]

Community-associated methicillin-resistant Staphylococcus aureus, Switzerland.
Harbarth S, Francois P, Shrenzel J, Fankhauser-Rodriguez C, Hugonnet S, Koessler T, Huyghe A, Pittet D.
Emerg Infect Dis. 2005 Jun;11(6):962-5.
[Abstract] - [PubMed]