Introduction

An Enterococcus faecium MLST scheme was established to provide a reference scheme for typing of E. faecium and to allow unambiguous comparison of data between different laboratories. The initial database contains the allelic profiles of 139 vancomycin-resistant and -susceptible isolates and will be updated with new profiles at intervals. Those performing MLST on this species are encouraged to submit their data to the curator so that the strain details can be added to the database. In this way the MLST database could be a powerful resource for global epidemiologic studies, recognition and tracking the worlwide interhospital spread of virulent, epidemic, and multiresistant clones.

The MLST scheme was developed in the laboratory of Rob Willems in The National Institute of Public Health and the Environment (RIVM), Netherlands in collaboration with David Tribe at The University of Melbourne, Australia

Homan WL, Tribe D, Poznanski S, Li M, Hogg G, Spalburg E, Van Embden JD, Willems RJ: Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol 2002 Jun;40(6):1963-71

Primers and PCR conditions for MLST of E. faecium

The primers that are used and the PCR conditions that we use in our laboratory are shown below. PCR conditions may need to be modified slightly in others laboratories. Since the same primers are used for the initial amplification, and for sequencing, it is important that PCR conditions are used which result in the amplification of only the desired fragment. In three cases we provide alternative primers (adk1n, adk2n, atpA1n,atpA2n, purK1n, purk2n, pstS1n) which may give better results.

The following primers are used for the amplification of the seven house-keeping gene fragments (1 is the forward and 2 the reversed primer):
adk1, 5’-TATGAACCTCATTTTAATGGG-3’
adk2, 5’-GTTGACTGCCAAACGATTTT-3’
adk1n, 5’-GAACCTCATTTTAATGGGG-3’
adk2n, 5’-TGATGTTGATAGCCAGACG-3’
atpA1, 5’-CGGTTCATACGGAATGGCACA-3’
atpA2, 5’-AAGTTCACGATAAGCCACGG-3’
atpA1n, 5’-TTCAAATGGCTCATACGG-3’
atpA2n, 5’-AGTTCACGATAAGCAACAGC-3’
ddl1, 5’-GAGACATTGAATATGCCTTATG-3’
ddl2, 5’-AAAAAGAAATCGCACCG-3’
gdh1, 5’-GGCGCACTAAAAGATATGGT-3’
gdh2, 5’-CCAAGATTGGGCAACTTCGTCCCA-3’
gyd-1, 5’-CAAACTGCTTAGCTCCAATGGC-3’
gyd2, 5’-CATTTCGTTGTCATACCAAGC-3’
purK1, 5’-GCAGATTGGCACATTGAAAGT-3’
purK2, 5’-TACATAAATCCCGCCTGTTTC/T-3’
purK1n, 5’-CAGATTGGCACATTGAAAG-3’
purK2n, 5’-TTCATTCACATATAGCCCG-3’
pstS1, 5’-TTGAGCCAAGTCGAAGCTGGAG-3’
pstS2, 5’-CGTGATCACGTTCTACTTCC-3’
pstS1n, 5’-TTGAGCCAAGTCGAAGC-3’.

PCR reactions are performed in 50 µl in PCR mixture. One reaction mixture contains 25 µL HotStar Taq Master Mix (Qiagen), 40 pmol of each primer, and milli-Q water to make a final volume of 50 µL. One µl of bacterial lysate is used as template for amplification. PCR conditions comprises an initial denaturation at 95^oC for 15 min, 35 cycles of 30 s at 94^oC, 30 s at 50^oC, and 30 s at 72^oC, followed by 5 min 72^oC. PCR products are purified with the Qiaquick PCR purification kit or the Qiaquick 96 PCR purification kit in accordance with manufacturer’s instructions. Approximately 5-20 ng PCR products, as estimated by gel electrophoresis, is used as template in the sequence reaction. Sequence reactions are performed in 20 µl sequence mixture. One reaction mixture contains 1 µL (5-20 ng) PCR product, 1 µL BigDye Terminator reaction kit, 7 µL reaction dilution buffer (200 mM Tris/HCl pH9,0 and 5 mM MgCl2), 1 µL (5 pmol) sequence primer, 4 µL Q-solution, and 6 µL milli-Q water. PCR products are sequenced with both PCR forward and reverse primers in separate sequence reactions. Cycle sequencing conditions comprises 25 cycles of 10 s at 96^oC, 5 s at 50^oC, and 4 min at 60^oC. Sequence reactions are purified by sephadex G50 in a 96 wells microtiter plate. Purified sequence reactions are injected directly out of water and loaded on a ABI PRISM 3700 DNA analyser. Run conditions are in accordance with manufacturer’s instructions.