MLST of Propionibacterium acnes
Genes
The Propionibacterium acnes MLST scheme uses partial sequences from seven core housekeeping genes (3135 bp). The scheme will resolve query isolates into sequence types from the genetic divisions IA1, IA2, IB, II & III as illustrated.
The seven genes used are:
Shikimate 5-dehydrogenase (aroE)
ATP synthase beta chain (atpD)
Guanylate kinase (gmk)
GMP synthase (guaA)
GTP-binding protein (lepA)
recA protein (recA)
Superoxide dismutase (sodA)
Amplification primers and product length
| Housekeeping Gene | Amplification Forward primers (5'-3') | Amplification Reverse primers (5'-3') | Sequence length |
|---|---|---|---|
| aroE | GTGATTGGCCATCCAGTG | CGCTGTGGACCTCAAAAC | 762 |
| recA | GCTCTATCATGCGCCTTG | GCAACATCCGGGTTACCT | 877 |
| atpD | AATTACCCCCGAGACGAA | CGTGTTCTGGGACAGGAA | 1261 |
| guaA | TCGCCTTCATGGAACAAC | CCATAAGTACGCCCGTCA | 1384 |
| lepA | TCGCGCCCAGTACTTAGA | CGGATTTCCACTCGATCA | 1218 |
| sodA | TGGAACTGCACCATGACA | GCTAACGACGTTCCACCA | 488 |
| gmk | TAGCCATCCGGAGATCGT | GCGCAACTGCGTGATCTA | 444 |
Sequencing primers and product length
| Housekeeping Gene | Sequencing Forward primers (5'-3') | Sequencing Reverse primers (5'-3') | Sequence length |
|---|---|---|---|
| aroE | GGGCTATCAGTGACGATG | GATCTTCAGCACGCCTTA | 424 |
| atpD | TAAGGGTCACGTCTGGAA | ACATCGCGGAAGTACTCA | 453 |
| gmk | AGATCGTCGTTTCCAGGT | ACAACGGCGTCAAATTC | 400 |
| guaA | GCGTTTGAAGACGTTGAG | GCTGGTCAGCATTGAGAC | 493 |
| lepA | GTCAAGGATGTCCGTCAA | GCAGGACTGAGAATGGTG | 452 |
| recA | GGGGTCGATACAGATTCC | TGTCAAACTCTGCCTGCT | 463 |
| sodA | ACAAGCACCACAACACCT | TAACGTAGTCGGCCTTGA | 450 |
PCR amplification & sequencing:
Reaction conditions for all the primers are as follows: initial denaturation at 94 °C for 1 min; 30 cycles of denaturation at 94 °C for 1 min, primer annealing at 58 °C for 1 min and extension at 72 °C for 2 min; followed by a final extension step at 72 °C for 10 min. Each 50 µl amplification reaction mixture comprises ~10 ng DNA, 20 pmol forward and reverse primer and x1 PCR buffer (Qiagen) containing 1.5 mM MgCl2, 0.8 mM dNTPs (Qiagen),1xQ solution (Qiagen) and 1.25 U Taq (Qiagen).
The amplification products are purified using MinElute UF plates (Qiagen) following the manufacturer's protocol before being used in the sequencing reaction. Sequencing is carried out on each DNA strand with BigDye Terminator Ready Reaction Mix v3.1 (PE Biosystems, Foster City, US) under standard sequencing conditions according to the manufacturer's protocol. Unincorporated dye terminators are removed by precipitation with 95% alcohol. The reaction products are then separated and detected on an ABI PRISM genetic analyser 3100 (PE Biosystems).
Expanded MLST scheme for Subtyping of Clones & Clonal Complexes:
Further subtyping of specific clones can be achieved by the addition of the complete gene sequences from the putative virulence determinants tly (haemolysin/ cytotoxin; 777bp) and camp2 (Camp factor; 804bp) to the MLST scheme.
tly primer sequences:
PAT-1: CAGGACGTGATGGCAATGCGA PAT-2: TCGTTCACAAGACCACAGTAGC
camp-2 primer sequences:
C2-F: GTCGTAGCCATACACCACACG C2-R: GCACCGAGTGTTGATGTCAATTAGC
Amplification conditions are as described in:
- McDowell, Valanne S, Ramage G, Tunney MM, Glenn JV, et al. (2005) Propionibacterium acnes types I and II represent phylogenetically distinct groups. J Clin Microbiol 43: 326-334.
- Valanne S, McDowell A, Ramage G, Tunney MM, Einarsson GG, et al. (2005) CAMP factor homologues in Propionibacterium acnes: a new protein family differentially expressed by types I and II. Microbiology 151: 1369-1379.
The P. acnes MLST database also includes all allele sequences for both virulence genes, and alleles associated with each isolate are available via the isolate database. To date (2012), a total of 89 expanded sequence types (eST) have been determined from a total of 280 isolates using the nine locus scheme.