A novel high-resolution single locus sequence typing scheme for mixed populations of Propionibacterium acnes in vivo

Abstract

The Gram-positive anaerobic bacterium Propionibacterium acnes is a prevalent member of the normal skin microbiota of human adults. In addition to its suspected role in acne vulgaris it is involved in a variety of opportunistic infections. Multi-locus sequence-typing (MLST) schemes identified distinct phylotypes associated with health and disease. Being based on 8 to 9 house-keeping genes these MLST schemes have a high discriminatory power, but their application is time- and cost-intensive. Here we describe a single-locus sequence typing (SLST) scheme for P. acnes. The target locus was identified with a genome mining approach that took advantage of the availability of representative genome sequences of all known phylotypes of P. acnes. We applied this SLST on a collection of 188 P. acnes strains and demonstrated a resolution comparable to that of existing MLST schemes. Phylogenetic analysis applied to the SLST locus resulted in clustering patterns identical to a reference tree based on core genome sequences. We further demonstrate that SLST can be applied to detect multiple phylotypes in complex microbial communities by a metagenomic pyrosequencing approach. The described SLST strategy may be applied to any bacterial species with a basically clonal population structure to achieve easy typing and mapping of multiple phylotypes in complex microbiotas. The P. acnes SLST database can be found at http://medbac.dk/slst/pacnes.

Figure 1. The SLST scheme discriminates the phylogenetic clusters of P. acnes.

Minimum Evolution phylogenetic tree based on 86 complete and draft P. acnes genomes, generated in MEGA v. 5.22 and based on a 1,964,522-bp concatemer of core sequences. Only bootstrap values of 100% are shown. The leftmost column depicts the identified SLST type. Type designations according to MLST9 and MLST8 schemes and ribotyping are also given, in addition to the traditional type assignments based on recA and tly sequence analysis. * indicates unknown types.

Figure 2. Strategy for the identification of SLST candidates in P. acnes.

The first column shows the applied filter. The second column indicates the location of the remaining candidate fragments across the genome. The third column gives the number of remaining candidates.

Figure 3. Distribution of P. acnes clones on skin and mucosal sites of a healthy subject revealed by application of the SLST scheme to a pyrosequencing-based analysis of complex microbiotas.

Identified SLST types are color-coded. Major types IA₁, IB, II and III are depicted in blue, red, yellow, and green and nuances thereof, respectively. The rightmost column indicates the SLST type distribution of 74 P. acnes isolates from a parallel sample of the skin of the cheek of the same subject. SLSTs represented by less than 0.03% of reads and reads that remained unassigned are not shown. See table S2 for distribution of all reads.

Figure 4. Minimum evolution tree generated from SLST target sequences of all 41 identified SLST types.

Bootstrap values based on 500 replications and higher than 75% are shown.