Genomic analyses of Bacteroides fragilis: subdivisions I and II represent distinct species.

Introduction. Bacteroides fragilis is a Gram-negative anaerobe that is a member of the human gastrointestinal microbiota and is frequently found as an extra-intestinal opportunistic pathogen. B. fragilis comprises two distinct groups - divisions I and II - characterized by the presence/absence of genes [cepA and ccrA (cfiA), respectively] that confer resistance to ß-lactam antibiotics by either serine or metallo-ß-lactamase production. No large-scale analyses of publicly available B. fragilis sequence data have been undertaken, and the resistome of the species remains poorly defined.Hypothesis/Gap Statement. Reclassification of divisions I and II B. fragilis as two distinct species has been proposed but additional evidence is required.Aims. To investigate the genomic diversity of GenBank B. fragilis genomes and establish the prevalence of division I and II strains among publicly available B. fragilis genomes, and to generate further evidence to demonstrate that B. fragilis division I and II strains represent distinct genomospecies.Methodology. High-quality (n=377) genomes listed as B. fragilis in GenBank were included in pangenome and functional analyses. Genome data were also subject to resistome profiling using The Comprehensive Antibiotic Resistance Database.Results. Average nucleotide identity and phylogenetic analyses showed B. fragilis divisions I and II represent distinct species: B. fragilis sensu stricto (n=275 genomes) and B. fragilis A (n=102 genomes; Genome Taxonomy Database designation), respectively. Exploration of the pangenome of B. fragilis sensu stricto and B. fragilis A revealed separation of the two species at the core and accessory gene levels.Conclusion. The findings indicate that B. fragilis A, previously referred to as division II B. fragilis, is an individual species and distinct from B. fragilis sensu stricto. The B. fragilis pangenome analysis supported previous genomic, phylogenetic and resistome screening analyses collectively reinforcing that divisions I and II are two separate species. In addition, it was confirmed that differences in the accessory genes of B. fragilis divisions I and II are primarily associated with carbohydrate metabolism and suggest that differences other than antimicrobial resistance could also be used to distinguish between these two species.

The potential role of molecular mimicry by the anaerobic microbiota in the aetiology of autoimmune disease.

Autoimmune diseases are thought to develop as a consequence of various environmental and genetic factors, each of which contributes to dysfunctional immune responses and/or a breakdown in immunological tolerance towards native structures. Molecular mimicry by microbial components is among the environmental factors thought to promote a breakdown in immune tolerance, particularly through the presence of cross-reactive epitopes shared with the human host. While resident members of the microbiota are essential promoters of human health through immunomodulation, defence against pathogenic colonisation and conversion of dietary fibre into nutritional resources for host tissues, there may be an underappreciated role of these microbes in the aetiology and/or progression of autoimmune disease. An increasing number of molecular mimics are being identified amongst the anaerobic microbiota which structurally resemble endogenous components and, in some cases, for example the human ubiquitin mimic of Bacteroides fragilis and DNA methyltransferase of Roseburia intestinalis, have been associated with promoting antibody profiles characteristic of autoimmune diseases. The persistent exposure of molecular mimics from the microbiota to the human immune system is likely to be involved in autoantibody production that contributes to the pathologies associated with immune-mediated inflammatory disorders. Here-in, examples of molecular mimics that have been identified among resident members of the human microbiota and their ability to induce autoimmune disease through cross-reactive autoantibody production are discussed. Improved awareness of the molecular mimics that exist among human colonisers will help elucidate the mechanisms involved in the breakdown of immune tolerance that ultimately lead to chronic inflammation and downstream disease.

Point-Counterpoint: What's in a Name? Clinical Microbiology Laboratories Should Use Nomenclature Based on Current Taxonomy.

INTRODUCTION The mnemonic SPICE (Serratia, Pseudomonas, indole-positive Proteus, Citrobacter, and Enterobacter) has served as a reminder to consider when a Gram-negative organism may carry a chromosomal copy of blaampC, with the associated risk of developing resistance to first-, second-, and third-generation cephalosporins. However, in 2017, there was a well-founded proposal to rename Enterobacter aerogenes to Klebsiella aerogenes, based on whole-genome sequencing (WGS), and the SPICE mnemonic lost its relevance. With the increased use of WGS for taxonomy, it seems like bacteria and fungi are undergoing constant name changes. These changes create unique challenges for clinical microbiology laboratories, who would like to issue reports that are readily understood and that help clinicians determine empirical antibiotic therapy, interpret antimicrobial resistance, and understand clinical significance. In this Point-Counterpoint, Drs. Karen Carroll and Erik Munson discuss the pros of updating bacterial taxonomy and why clinical labs must continue to update reporting, while Drs. Susan Butler-Wu and Sheila Patrick argue for caution in adopting new names for microorganisms.

A tale of two habitats: Bacteroides fragilis, a lethal pathogen and resident in the human gastrointestinal microbiome.

Bacteroides fragilis is an obligately anaerobic Gram-negative bacterium and a major colonizer of the human large colon where Bacteroides is a predominant genus. During the growth of an individual clonal population, an astonishing number of reversible DNA inversion events occur, driving within-strain diversity. Additionally, the B. fragilis pan-genome contains a large pool of diverse polysaccharide biosynthesis loci, DNA restriction/modification systems and polysaccharide utilization loci, which generates remarkable between-strain diversity. Diversity clearly contributes to the success of B. fragilis within its normal habitat of the gastrointestinal (GI) tract and during infection in the extra-intestinal host environment. Within the GI tract, B. fragilis is usually symbiotic, for example providing localized nutrients for the gut epithelium, but B. fragilis within the GI tract may not always be benign. Metalloprotease toxin production is strongly associated with colorectal cancer. B. fragilis is unique amongst bacteria; some strains export a protein >99 % structurally similar to human ubiquitin and antigenically cross-reactive, which suggests a link to autoimmune diseases. B. fragilis is not a primary invasive enteric pathogen; however, if colonic contents contaminate the extra-intestinal host environment, it successfully adapts to this new habitat and causes infection; classically peritoneal infection arising from rupture of an inflamed appendix or GI surgery, which if untreated, can progress to bacteraemia and death. In this review selected aspects of B. fragilis adaptation to the different habitats of the GI tract and the extra-intestinal host environment are considered, along with the considerable challenges faced when studying this highly variable bacterium.

Peptoids with Antibiofilm Activity against the Gram Negative Obligate Anaerobe, Fusobacterium nucleatum.

Peptoids (oligo N-substituted glycines) are peptide analogues, which can be designed to mimic host antimicrobial peptides, with the advantage that they are resistant to proteolytic degradation. Few studies on the antimicrobial efficacy of peptoids have focused on Gram negative anaerobic microbes associated with clinical infections, which are commonly recalcitrant to antibiotic treatment. We therefore studied the cytotoxicity and antibiofilm activity of a family of peptoids against the Gram negative obligate anaerobe Fusobacterium nucleatum, which is associated with infections in the oral cavity. Two peptoids, peptoid 4 (NaeNpheNphe)4 and peptoid 9 (NahNspeNspe)3 were shown to be efficacious against F. nucleatum biofilms at a concentration of 1 µM. At this concentration, peptoids 4 and 9 were not cytotoxic to human erythrocytes or primary human gingival fibroblast cells. Peptoids 4 and 9 therefore have merit as future therapeutics for the treatment of oral infections.

Longitudinal time-lapse in vivo micro-CT reveals differential patterns of peri-implant bone changes after subclinical bacterial infection in a rat model.

Subclinical infection associated with orthopedic devices can be challenging to diagnose. The goal of this study was to evaluate longitudinal, microcomputed tomography (microCT) imaging in a rat model of subclinical orthopedic device-related infection caused by Staphylococcus epidermidis and four different Cutibacterium (previously Propionibacterium) acnes strains, and compare outcomes with non-inoculated and historical S. aureus-inoculated controls. Sterile screws or screws colonized with bacteria were placed in the tibia of 38 adult Wistar rats [n = 6 sterile screws; n = 6 S. epidermidis-colonized screws; n = 26 C. acnes-colonized screws (covering all three main subspecies)]. Regular microCT scans were taken over 28 days and processed for quantitative time-lapse imaging with dynamic histomorphometry. At euthanasia, tissues were processed for semiquantitative histopathology or quantitative bacteriology. All rats receiving sterile screws were culture-negative at euthanasia and displayed progressive bony encapsulation of the screw. All rats inoculated with S. epidermidis-colonized screws were culture-positive and displayed minor changes in peri-implant bone, characteristic of subclinical infection. Five of the 17 rats in the C. acnes inoculated group were culture positive at euthanasia and displayed bone changes at the interface of the screw and bone, but not deeper in the peri-implant bone. Dynamic histomorphometry revealed significant differences in osseointegration, bone remodeling and periosteal reactions between groups that were not measurable by visual observation of still microCT images. Our study illustrates the added value of merging 3D microCT data from subsequent timepoints and producing inherently richer 4D data for the detection and characterization of subclinical orthopedic infections, whilst also reducing animal use.

Draft Genome Sequence of Propionibacterium acnes subsp. elongatum Strain Asn12.

Propionibacterium acnes, a non-spore-forming anaerobic Gram-positive bacterium, has been linked to a wide range of opportunistic human infections and conditions, most notably acne vulgaris. Here, we present the draft genome sequence of P. acnes subsp. elongatum strain Asn12, isolated from spinal disc tissue (in the United Kingdom).

Surface-Enhanced Raman Spectroscopy for the Detection of a Metabolic Product in the Headspace Above Live Bacterial Cultures.

In situ surface-enhanced Raman spectra of the headspace above cultures of six bacterial species showed strong characteristic bands from chemisorbed methyl sulfide. This marker compound is created by dissociation of dimethyl disulfide (DMDS), a fermentative metabolite of bacteria, on the surface of the enhancing Au or Ag nanoparticle films. Kinetic binding plots of media spiked with DMDS and of live cultures showed that the Au-based substrates were more suitable for the rapid detection of bacteria than Ag-based substrates. For E. coli DH5α, the sensitivity limit for headspace SERS detection was 1.5×107  CFU mL-1 , which corresponded to detection 15 min after inoculation of the growth medium. Since the metabolites are only produced by viable bacteria, antibiotic (gentamicin) treatment stopped the normal signal growth of the marker peak. This work is a promising step towards rapid bedside detection of bacterial infections and rapid screening of antibiotics against bacteria.

Novel large-scale chromosomal transfer in Bacteroides fragilis contributes to its pan-genome and rapid environmental adaptation.

Bacteroides fragilis, an important component of the human gastrointestinal microbiota, can cause lethal extra-intestinal infection upon escape from the gastrointestinal tract. We demonstrated transfer and recombination of large chromosomal segments from B. fragilis HMW615, a multidrug resistant clinical isolate, to B. fragilis 638R. In one example, the transfer of a segment of ~435 Kb/356 genes replaced ~413 Kb/326 genes of the B. fragilis 638R chromosome. In addition to transfer of antibiotic resistance genes, these transfers (1) replaced complete divergent polysaccharide biosynthesis loci; (2) replaced DNA inversion-controlled intergenic shufflons (that control expression of genes encoding starch utilization system outer membrane proteins) with more complex, divergent shufflons; and (3) introduced additional intergenic shufflons encoding divergent Type 1 restriction/modification systems. Conjugative transposon-like genes within a transferred segment and within a putative integrative conjugative element (ICE5) ~45 kb downstream from the transferred segment both encode proteins that may be involved in the observed transfer. These data indicate that chromosomal transfer is a driver of antigenic diversity and nutrient adaptation in Bacteroides that (1) contributes to the dissemination of the extensive B. fragilis pan-genome, (2) allows rapid adaptation to a changing environment and (3) can confer pathogenic characteristics to host symbionts.

Tropical ulcer plant treatments used by Papua New Guinea's Apsokok nomads.

ETHNOPHARMACOLOGICAL RELEVANCE: The tropical ulcer is a debilitating bacterial infection that is common in Papua New Guinea. Deploying healthcare infrastructure to remote and inaccessible rainforest locations is not practical, therefore local plants may be the best treatment option. Here we present an ethnobotanical survey of the tropical ulcer plant medicines used by the semi-nomadic Apsokok who roam the remote central mountains of Papua New Guinea's West New Britain Province. In vitro biological activity in assays relevant to tropical ulcer wound healing is also presented. MATERIALS AND METHODS: Focus groups and semi-structured interviews were used to acquire information on the uses of plants, vouchers of which were identified by comparison with authentic herbarium specimens. Antibacterial disc diffusion assays with Staphylococcus aureus and Fusobacterium ulcerans, MMP-9 enzyme inhibition and dermal fibroblast stimulation assays were carried out on plant saps and aqueous extracts of plant material. LC-MS was used to identify known plant metabolites. RESULTS: The ethnobotanical survey identified sixteen species that were used to treat tropical ulcers, all of which were applied topically. A subset of twelve species were investigated further in vitro. Four species produced zones of inhibition with S. aureus, all 12 species provided low level inhibition of MMP-9 and 8 species stimulated dermal fibroblast proliferation, although cytotoxicity occurred at higher concentrations. The extract of Homalium foetidum Benth. inhibited S. aureus and MMP-9 while at lower sub-cytotoxic concentrations stimulated fibroblast proliferation. Trans-3-O-p-coumaroylquinic acid cis-3-O-p-coumaroylquinic acid were detected in the aqueous extract of H. foetidum. CONCLUSIONS: Topical application of plant saps to wounds results in very high localised concentrations of plant metabolites which is likely to result in inhibition of MMP proteases. H. foetidum is a candidate plant for tropical ulcer treatment in remote areas.

Emendation of Propionibacterium acnes subsp. acnes (Deiko et al. 2015) and proposal of Propionibacterium acnes type II as Propionibacterium acnes subsp. defendens subsp. nov.

Recently, it has been proposed that strains of Propionibacterium acnes from the type III genetic division should be classified as P. acnessubsp. elongatum subsp. nov., with strains from the type I and II divisions collectively classified as P. acnessubsp. acnes subsp. nov. Under such a taxonomic re-appraisal, we believe that types I and II should also have their own separate rank of subspecies. In support of this, we describe a polyphasic taxonomic study based on the analysis of publicly available multilocus and whole-genome sequence datasets, alongside a systematic review of previously published phylogenetic, genomic, phenotypic and clinical data. Strains of types I and II form highly distinct clades on the basis of multilocus sequence analysis (MLSA) and whole-genome phylogenetic reconstructions. In silico or digital DNA-DNA similarity values also fall within the 70-80 % boundary recommended for bacterial subspecies. Furthermore, we see important differences in genome content, including the presence of an active CRISPR/Cas system in type II strains, but not type I, and evidence for increasing linkage equilibrium within the separate divisions. Key biochemical differences include positive test results for ß-haemolytic, neuraminidase and sorbitol fermentation activities with type I strains, but not type II. We now propose that type I strains should be classified as P. acnessubsp. acnes subsp. nov., and type II as P. acnessubsp. defendens subsp. nov. The type strain of P. acnessubsp. acnes subsp. nov. is NCTC 737T (=ATCC 6919T=JCM 6425T=DSM 1897T=CCUG 1794T), while the type strain of P. acnessubsp. defendens subsp. nov. is ATCC 11828 (=JCM 6473=CCUG 6369).

Strains of the Propionibacterium acnes type III lineage are associated with the skin condition progressive macular hypomelanosis.

Progressive macular hypomelanosis (PMH) is a common skin disorder that causes hypopigmentation in a variety of skin types. Although the underlying aetiology of this condition is unclear, there is circumstantial evidence that links the skin bacterium Propionibacterium acnes to the condition. We now describe the first detailed population genetic analysis of P. acnes isolates recovered from paired lesional and non-lesional skin of PMH patients. Our results demonstrate a strong statistical association between strains from the type III phylogenetic lineage and PMH lesions (P = 0.0019), but not those representing other phylogroups, including those associated with acne (type IA1). We also demonstrate, based on in silico 16S rDNA analysis, that PMH isolates previously recovered from patients in Europe are also consistent with the type III lineage. Using comparative genome analysis, we identified multiple genomic regions that are specific for, or absent from, type III strains compared to other phylogroups. In the former case, these include open reading frames with putative functions in metabolism, transport and transcriptional regulation, as well as predicted proteins of unknown function. Further study of these genomic elements, along with transcriptional and functional analyses, may help to explain why type III strains are associated with PMH.

Emergence and evolution of an international cluster of MDR Bacteroides fragilis isolates.

OBJECTIVES: The aim of this study was to examine the antibiotic resistance profiles, antibiotic resistance mechanisms and possible 'clonal' nature of some MDR Bacteroides fragilis strains that simultaneously harboured cfiA, nimB, IS1186 and IS4351. METHODS: Antibiotic susceptibilities were determined by Etests and antibiotic resistance genes and different genetic elements were detected by applying PCR methods. The environments of the cfiA and nimB genes were also determined by sequencing. The transferability of the cfiA, nimB and tet(Q) genes was tested by conjugation. The genetic relatedness of the test strains was tested by ERIC-PCR or PFGE. The complete genome sequences of two strains (B. fragilis BF8 and O:21) were determined by next-generation sequencing. RESULTS: Most of the seven B. fragilis strains tested displayed multidrug resistance phenotypes; five strains were resistant to at least five types of antibiotics. Besides the common genetic constitution, ERIC-PCR implied high genetic relatedness. Similarities in some of the antibiotic resistance mechanisms [carbapenems (cfiA) and metronidazole (nimB)] also confirmed their common origin, but some other resistance mechanisms {MLSB [erm(F)] and tetracycline [tet(Q)]} and PFGE typing revealed differences. In B. fragilis BF8 and O:21, erm(F) and tet(X) genes were found with IS4351 borders, thus constituting Tn4351. All the strains were tet(Q) positive and transferred this gene in conjugation experiments, but not the cfiA and nimB genes. CONCLUSIONS: An international cluster of MDR B. fragilis strains has been identified and characterized. This 'clone' may have emerged early in the evolution of division II B. fragilis strains, which was suggested by the low-complexity ERIC profiles and differences in the PFGE patterns.

A single chromosome assembly of Bacteroides fragilis strain BE1 from Illumina and MinION nanopore sequencing data.

BACKGROUND: Second and third generation sequencing technologies have revolutionised bacterial genomics. Short-read Illumina reads result in cheap but fragmented assemblies, whereas longer reads are more expensive but result in more complete genomes. The Oxford Nanopore MinION device is a revolutionary mobile sequencer that can produce thousands of long, single molecule reads. RESULTS: We sequenced Bacteroides fragilis strain BE1 using both the Illumina MiSeq and Oxford Nanopore MinION platforms. We were able to assemble a single chromosome of 5.18 Mb, with no gaps, using publicly available software and commodity computing hardware. We identified gene rearrangements and the state of invertible promoters in the strain. CONCLUSIONS: The single chromosome assembly of Bacteroides fragilis strain BE1 was achieved using only modest amounts of data, publicly available software and commodity computing hardware. This combination of technologies offers the possibility of ultra-cheap, high quality, finished bacterial genomes.

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 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.

Deficiency of the ferrous iron transporter FeoAB is linked with metronidazole resistance in Bacteroides fragilis.

BACKGROUND: Metronidazole is the most commonly used antimicrobial for Bacteroides fragilis infections and is recommended for prophylaxis of colorectal surgery. Metronidazole resistance is increasing and the mechanisms of resistance are not clear. METHODS: A transposon mutant library was generated in B. fragilis 638R (BF638R) to identify the genetic loci associated with resistance to metronidazole. RESULTS: Thirty-two independently isolated metronidazole-resistant mutants had a transposon insertion in BF638R_1421 that encodes the ferrous transport fusion protein (feoAB). Deletion of feoAB resulted in a 10-fold increased MIC of metronidazole for the strain. The metronidazole MIC for the feoAB mutant was similar to that for the parent strain when grown on media supplemented with excess iron, suggesting that the increase seen in the MIC of metronidazole was due to reduced cellular iron transport in the feoAB mutant. The furA gene repressed feoAB transcription in an iron-dependent manner and disruption of furA resulted in constitutive transcription of feoAB, regardless of whether or not iron was present. However, disruption of feoAB also diminished the capacity of BF638R to grow in a mouse intraperitoneal abscess model, suggesting that inorganic ferrous iron assimilation is essential for B. fragilis survival in vivo. CONCLUSIONS: Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis.

Identification of a collagen type I adhesin of Bacteroides fragilis.

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼ 31 and ∼ 34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼ 31 kDa and the ∼ 34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼ 31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.

Propionibacterium acnes and Staphylococcus lugdunensis cause pyogenic osteomyelitis in an intramedullary nail model in rabbits.

Propionibacterium acnes and coagulase-negative staphylococci (CoNS) are opportunistic pathogens implicated in prosthetic joint and fracture fixation device-related infections. The purpose of this study was to determine whether P. acnes and the CoNS species Staphylococcus lugdunensis, isolated from an "aseptically failed" prosthetic hip joint and a united intramedullary nail-fixed tibial fracture, respectively, could cause osteomyelitis in an established implant-related osteomyelitis model in rabbits in the absence of wear debris from the implant material. The histological features of P. acnes infection in the in vivo rabbit model were consistent with localized pyogenic osteomyelitis, and a biofilm was present on all explanted intramedullary (IM) nails. The animals displayed no outward signs of infection, such as swelling, lameness, weight loss, or elevated white blood cell count. In contrast, infection with S. lugdunensis resulted in histological features consistent with both pyogenic osteomyelitis and septic arthritis, and all S. lugdunensis-infected animals displayed weight loss and an elevated white blood cell count despite biofilm detection in only two out of six rabbits. The differences in the histological and bacteriological profiles of the two species in this rabbit model of infection are reflective of their different clinical presentations: low-grade infection in the case of P. acnes and acute infection for S. lugdunensis. These results are especially important in light of the growing recognition of chronic P. acnes biofilm infections in prosthetic joint failure and nonunion of fracture fixations, which may be currently reported as "aseptic" failure.