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MLVA

Staphylococcus aureus

Staphylococcus aureus is an important bacterial pathogen that is associated with serious community-acquired and nosocomial diseases. Although S. aureus can cause a variety of serious clinical syndromes such as bacteremia, pneumonia and endocarditis, it seems to be omnipresent and is often carried without any clinical symptoms. Carriers may spread the pathogen infecting individuals who may develop disease. The introduction of methicillin has led to the rapid emergence of methicillin-resistant S. aureus (MRSA) and is posing a major clinical problem within hospitals worldwide

Multiple-Locus Variable number of tandem repeat Analysis (MLVA) of Staphylococcus aureus
In order to understand the population biology of S. aureus and to study the impact of measures to control MRSA infections unambiguous characterization of S. aureus isolates is required. Many typing techniques have been employed for the analysis of S. aureus of which multi-locus sequence typing (MLST) is considered to be the current gold standard for molecular typing of S. aureus. However, MLST is expensive, labor intensive and therefore not the method of choice for high throughput typing for most laboratories. Spa-sequence typing is a portable technique and is relatively easy to perform. A disadvantage of this technique is that it is based on a single locus in the genome only and clustering and tree-building of S. aureus isolates based on spa-data is complex. For these reasons we developed a typing technique based on the composition of genomic loci containing tandem repeats. Several MLVA schemes for S. aureus have been designed and used to type this pathogen. However, these MLVA schemes rely on analysis in agarose gels making them less accurate and often not portable. The MLVA used here is based on accurate band sizing using an automated DNA sequencer.

MLVA for S. aureus has been developed in the Laboratory for Infectious Diseases and Perinatal Screening (LIS) of the Centre for Infectious Disease Control Netherlands (CIb) at the National Institute for Public Health and the Environment (RIVM).
Schouls, L. M., E. C. Spalburg, M. van Luit, X. W. Huijsdens, G. N. Pluister, M. G. van Santen-Verheuvel, H. G. van der Heide, H. Grundmann, M. E. Heck, and A. J. de Neeling. 2009. Multiple-locus variable number tandem repeat analysis of Staphylococcus aureus: comparison with pulsed-field gel electrophoresis and spa-typing. PLoS One 4:e5082.
In this paper there are some mistakes in the MLVA profiles displayed in figure 7. The table with the correct assignment of the MLVA profiles of the S. aureus strains for which an annotated whole genome sequences have been published can be downloaded from the ‘Protocols and Tables’ page. Also a .pdf document that displays the sequence of the VNTR loci in S. aureus strain N315 has now been added as a downloadable file on the ‘Protocols and Tables’ page.

Profiles and types
In the MLVA of S. aureus 8 VNTR loci are used. On the 26th of May 2011 the S. aureus MLVA type list contained 2793 distinct allelic profiles and their corresponding MLVA type designation. These profiles were obtained from nearly 23,000 S. aureus isolates. Approximately 75% of these isolates were MRSA, the remaining 25% were methicillin-sensitive (MSSA) strains. They also include MLVA types obtained from 2500 MRSA and MSSA isolates collected in a 23 different European countries. The majority of the isolates were from collected from patients that carried MRSA rather than from patients with invasive disease. The current number of types and complexes is displayed at the top of this page on the right hand side under ‘Statistics’. The table with the currently assigned MLVA types is downloadable from the ‘Protocols and Tables’ page.

Detection of the mecA gene and lukF gene (Panton-Valentine leukocidin, PVL) in the MLVA
Recently the method was expanded to facilitate the detection of the mecA and lukF genes simultaneous with the MLVA. For the detection of the mecA and lukF genes mecA-specific primers were included in the first multiplex mixture and similarly lukF-specific primers were included in the second multiplex mixture. The use of these primer sets results in 150 base pair PCR products which are smaller than the smallest PCR product generated by the MLVA primers. Both the downloadable protocol and the GeneMarker panel files on the ‘Protocols and Tables’ page have been updated and now include the mecA and lukF designation.

How to ensure accurate sizing of the PCR products
Standardizing typing techniques that are not sequence based has proven to be difficult. Although the separation of the PCR products obtained in MLVA is performed on a DNA sequencer standardization may pose a problem for MLVA as well. The results of sizing of PCR products performed in one lab may differ from the results obtained in other labs. Such differences may have been caused by the use of different sequencers, different buffers, different capillaries etc. In order to eliminate such problems a calibration set may used which we may provide if you want to implement the MLVA in your lab. This calibration set is composed of a number of allelic ladders for each of the 8 VNTR loci used. These allelic ladders contain each allele that we currently have identified. If obtain the allelic ladders from us you can perform the MLVA PCRs and separate the PCR products on you sequencer. This will reveal the positions to which the alleles will migrate on you sequencer. Once you have established this, you can use this to define the bin positions for your own sequencing system. Although we do not recommend this, this would in principle allow for the use of other fluorescent labels on the primers. A detailed protocol how to use this calibration set can be found on the Protocols and Tables’ page. If you require such a calibration set please contact us.

Protocol and tables for data analysis.

A number of files that may be useful if you want to perform MLVA can be downloaded here. These files may be updated if alterations in the protocol or allele tables have been made.

  • Protocol MLVA Saureus MLVA.pdf A detailed protocol for the MLVA scheme containing details on the reagents, primers
  • Use of the S. aureus MLVA calibration set.pdf A detailed protocol on how to use the MLVA calibration set
  • VNTR loci in N315.pdf DNA sequence of the 8 VNTR loci in S. aureus strain N315
  • MLVA profiles sequenced strains.xls MLVA profiles of the S. aureus strains for which the whole genome has been sequenced; corrected version of Table 7 of the MLVA paper
  • Saureus MLVA type table A file containing all currently known MLVA types, complexes and profiles in CSV format.
  • Saureus_MLVA_allele_size_table.xls A file containing the theoretical size of all currently known alleles.
  • SGPanel_MLVA_mrsa_mix1_MecA.xml Panel file for use with the GeneMarker software. This file contains all allele sizes and their bin sizes and is used to assign the number of repeats in the VNTRs amplified in multiplex 1.
  • SGPanel_MLVA_mrsa_mix2_PVL.xml Panel file for use with the GeneMarker software. This file contains all allele sizes and their bin sizes and is used to assign the number of repeats in the VNTRs amplified in multiplex 2.
  • SGSize_GS1200LIZ.xml Size standard file for use with the GeneMarker software.
  • Saureus_MST.pdf Minimum spanning tree based on MLVA profiles.
  • Older downloads
  • Protocol_MLVA_Saureus old.pdf A detailed protocol for the MLVA scheme containing details on the reagents, primers. THIS PROTOCOL DOES NOT INCLUDE MECA AND PVL PCR!
  • SGPanel_MLVA_mrsa_mix1.xml Panel file for use with the GeneMarker software. This file contains all allele sizes and their bin sizes and is used to assign the number of repeats in the VNTRs amplified in multiplex 1. THIS PANEL FILE DOES NOT CONTAIN THE ASSIGNMENT FOR THE MECA PRODUCT!
  • SGPanel_MLVA_mrsa_mix2.xml Panel file for use with the GeneMarker software. This file contains all allele sizes and their bin sizes and is used to assign the number of repeats in the VNTRs amplified in multiplex 2. THIS PANEL FILE DOES NOT CONTAIN THE ASSIGNMENT FOR THE LUKF PRODUCT!

Please enter your MLVA profile in the following text boxes and click submit.

If you discover a new allele or MLVA type please which you would like to be added to the list, please contact Leo Schouls. You probably will need to send either the .fsa trace files or DNA/lysate for confirmation and official assignment of a new allele or MLVA type.

V09_01
V61_01
V61_02
V67_01
V21_01
V24_01
V63_01
V81_01

Please enter MLVA profiles with an isolate identifier in TAB delimited format.
(Click here for example data)

If you discover a new allele or MLVA type please which you would like to be added to the list please contact Leo Schouls. You probably will need to send either the .fsa trace files or DNA/lysate for confirmation and official assignment of a new allele or MLVA type.

 
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Statistics

24/09/2017

Total - 5651 profiles

Total - 62 complexes

Contact:
Leo Schouls