Multiplex touchdown PCR for rapid typing of the opportunistic pathogen Propionibacterium acnes

Abstract

The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II, and III, which vary in their production of putative virulence factors, their inflammatory potential, and their biochemical, aggregative, and morphological characteristics. Although multilocus sequence typing (MLST) currently represents the gold standard for unambiguous phylogroup classification and individual strain identification, it is a labor-intensive and time-consuming technique. As a consequence, we developed a multiplex touchdown PCR assay that in a single reaction can confirm the species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA gene (all isolates), ATPase (types IA1, IA2, and IC), sodA (types IA2 and IB), atpD (type II), and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterized by MLST and representing types IA1 (n=145), IA2 (n=20), IB (n=65), IC (n=7), II (n=45), and III (n=30), the multiplex displayed 100% sensitivity and 100% specificity for detecting isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. This multiplex assay will provide researchers with a rapid, high-throughput, and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, and it will serve as a prescreening tool to maximize the number of genetically diverse isolates selected for downstream higher-resolution sequence-based analyses.

FIG 1

Multiplex PCR analysis of P. acnes strains (except lane 19) representing different phylogroups and STs. Lane 1, strain hdn-1 (ST1, type IA₁); lane 2, strain PRP-60 (ST20, type IA₁); lane 3, strain 76793 (ST101, type IA₁); lane 4, strain Pacn33 (ST2, type IA₂); lane 5, strain P.acn17 (ST2, type IA₂); lane 6, strain P. acn31 (ST2, type IA₂); lane 7, strain 6609 (ST5, type IB); lane 8, strain VA3/4 (ST78, type IB); lane 9, strain 74874 (ST43, type IB); lane 10, strain PRP-38 (ST70, type IC); lane 11, strain PV66 (ST85, type IC); lane 12, strain 5/1/3 (ST107, type IC); lane 13, strain ATCC 11828 (ST27, type II); lane 14, strain VA2/9N (ST28, type II); lane 15, strain 6187 (ST30, type II); lane 16, strain 12S (ST32, type III); lane 17, strain Asn12 (ST33, type III); lane 18, strain Asn10 (ST81, type III); lane 19, Propionibacterium avidum strain 44067. Ma, molecular size markers. ST is based on the eight-gene MLST scheme of McDowell et al. (21) and the database at http://pubmlst.org/pacnes/. Gene amplicons (left to right): a, 16S rRNA; b, ATPase; c, sodA; d, toxin; e, atpD; f, recA.