Multiplex Touchdown PCR for Rapid Typing of the Opportunistic Pathogen Propionibacterium acnes

The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA(1), IA(2), 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 IA(1), IA(2), and IC), sodA (types IA(2) 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 IA(1) (n = 145), IA(2) (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

Complete Genome Sequence of Propionibacterium avidum Strain 44067, Isolated from a Human Skin Abscess

Propionibacterium avidum is an anaerobic Gram-positive bacterium that forms part of the normal human cutaneous microbiota, colonizing moist areas such as the vestibule of the nose, axilla, and perineum. Here we present the complete genome sequence of P. avidum strain 44067, which was isolated from a carbuncle of the trunk

The Nrd1-like protein Seb1 coordinates cotranscriptional 3′ end processing and polyadenylation site selection

Termination of RNA polymerase II (RNAPII) transcription is associated with RNA 3 end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in Saccharomyces cerevisiae does not rely on RNA cleavage for termination but instead terminates via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the Schizosaccharomyces pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3 end processing factors, is enriched at the 3 end of genes, and binds RNA motifs downstream from cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3 untranslated region (UTR) length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3 end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 cotranscriptionally controls alternative polyadenylation

MALDI-TOF MS fingerprinting facilitates rapid discrimination of phylotypes I, II and III of Propionibacterium acnes.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used today for species determination of bacteria and fungi in routine microbiological laboratories, and can also be used for subtyping of bacteria, such as Bacteroides fragilis. Propionibacterium acnes is frequently referred to as an anaerobic skin commensal of relatively low pathogenicity. In addition to its accepted pathogenic role in acne, P. acnes is now emerging as an important opportunistic pathogen in many other clinical situations, including late-stage prosthetic joint infections, osteomyelitis, endocarditis, endophthalmitis, post-neurosurgical infections and possibly prostate cancer. At the population genetic level, P. acnes can be differentiated into a number of distinct phylogroups, known as types IA1, IA2, IB, IC, II and III, which may be associated with different types of infections and clinical conditions. The aim of the present study was to evaluate MS-based typing for resolution of these genetic groups after routine identification by MALDI-TOF MS (Bruker MALDI Biotyper). The software package ClinProTools 2.2 was used to analyze the protein based mass spectra of reference strains belonging to types IA, IB, IC, II and III. Phylogroup-specific peaks and peak shifts were then identified visually. In addition, peak variations between the different types of P. acnes were investigated by using FlexAnalysis 3.3 software (Bruker). A differentiating library was created, which was used to type further 48 clinical isolates of P. acnes. Typing data obtained by MALDI-TOF MS were then compared with the results from Multilocus Sequence Typing (MLST). Most of the clinical isolates (n = 19) belonged to the type IA grouping according to MALDI-TOF MS. By MLST, all isolates were identified as type IA1. Twenty-one clinical isolates belonged to the type IB cluster based on both MALDI-TOF MS and MLST typing. Eight clinical isolates were identified as type II strains by both typing methods and all the type III reference strains could be distinguished by the presence of a unique type III-specific peak (7238 Da) by the MALDI-TOF MS. Our study demonstrates that MALDI-TOF MS is a reliable and powerful tool for rapid identification and typing of P. acnes strains from the main genetic divisions of the species