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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 bla ampC , 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.

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.

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.

In-situ surface-enhanced Raman spectroscopy of the headspace above cultures of 6 bacterial species allowed sensitive and selective detection of characteristic bands from chemisorbed methyl sulphide. This marker compound is created by dissociation of adsorbed dimethyl disulphide (DMDS) on the surface of the Au or Ag nanoparticle (NP) films which are used as the enhancing media. DMDS is a well-known fermentative metabolite of bacteria and was also detected here by GC-MS of the headspace of bacteria cultures. Kinetic binding plots of media spiked with DMDS and of live cultures, both showed that the more rapid adsorption of DMDS onto Au based substrates made them more suitable for the rapid detection of bacteria than Ag based substrates. E. coli DH5α was chosen for the proof-of-principal experiments. For this micro-organism, the sensitivity limit was found to be the detection of bacteria in the headspace of a 1.5 x 10⁷ CFU/ml culture. Under our conditions, this corresponds to detection 15 minutes after inoculation of the growth medium, but it will depend on the CFU/ml of the initial inoculum used. 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 model cultures.

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.

Aims The aim of this study was to determine whether the sequential application of povidone iodine-alcohol (PVI) followed by chlorhexidine gluconate-alcohol (CHG) would reduce surgical wound contamination to a greater extent than PVI applied twice in patients undergoing spinal surgery. Patients and Methods A single-centre, interventional, two arm, parallel group randomised controlled trial was undertaken, involving 407 patients who underwent elective spinal surgery. For 203 patients, the skin was disinfected before surgery using PVI (10% [w/w (1% w/w available iodine)] in 95% industrial denatured alcohol, povidone iodine; Videne Alcoholic Tincture) twice, and for 204 patients using PVI once followed by CHG (2% [w/v] chlorhexidine gluconate in 70% [v/v] isopropyl alcohol; Chloraprep with tint). The primary outcome measure was contamination of the wound determined by aerobic and anaerobic bacterial growth from samples taken after disinfection. Results The detection of viable bacteria in any one of the samples taken after disinfection (culture-positive) was significantly lower in the group treated with both PVI and CHG than in the group treated with PVI alone (59 (29.1%) versus 85 (41.7%), p = 0.009; odds ratio 0.574; 95% confidence interval, 0.380 to 0.866). Conclusions Antisepsis of the skin with the sequential application of PVI and CHG more effectively reduces the contamination of a surgical wound than PVI alone. Cite this article: Bone Joint J 2017;99-B:1354–65.

Summary Ubiquitin is highly conserved across eukaryotes and is essential for normal eukaryotic cell function. The bacterium Bacteroides fragilis is a member of the normal human gut microbiota, and the only bacterium known to encode a homologue of eukaryotic ubiquitin. The B. fragilis gene sequence indicates a past horizontal gene transfer event from a eukaryotic source. It encodes a protein (BfUbb) with 63% identity to human ubiquitin which is exported from the bacterial cell. The aim of this study was (i) to determine if there was antigenic cross-reactivity between B. fragilis ubiquitin and human ubiquitin and (ii) to determine if humans produced antibodies to BfUbb. Molecular model comparisons of BfUbb and human ubiquitin predicted a high level (99·8% confidence) of structural similarity. Linear epitope mapping identified epitopes in BfUbb and human ubiquitin that cross-react. BfUbb also has epitope(s) that do not cross-react with human ubiquitin. The reaction of human serum (n = 474) to BfUbb and human ubiquitin from the following four groups of subjects was compared by enzyme-linked immunosorbent assay (ELISA): (1) newly autoantibody-positive patients, (2) allergen-specific immunoglobulin (Ig)E-negative patients, (3) ulcerative colitis patients and (4) healthy volunteers. We show that the immune system of some individuals has been exposed to BfUbb which has resulted in the generation of IgG antibodies. Serum from patients referred for first-time testing to an immunology laboratory for autoimmune disease are more likely to have a high level of antibodies to BfUbb than healthy volunteers. Molecular mimicry of human ubiquitin by BfUbb could be a trigger for autoimmune disease.

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., 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).

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.

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.

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.

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 β-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).

Folliculitis is a common inflammatory skin syndrome. Several microbial organisms have been put forward as causative agents, but few studies visualized microbes directly in inflamed hair follicles. This retrospective study investigated bacterial and fungal colonization of inflamed hair follicles in patients with clinically diagnosed non-infectious folliculitis. Skin biopsies from 39 folliculitis patients and 27 controls were screened by fluorescence in situ hybridization (FISH) using broad-range bacterial and fungal probes and by immunofluorescence microscopy using a monoclonal antibody towards Gram-positive bacteria. Specific monoclonal and polyclonal antibodies towards Staphylococcus spp. and Propionibacterium acnes were applied for further species identification. Inflamed follicles were associated with bacterial colonization in 10 samples (26%) and fungal colonization in three samples (8%). Staphylococcus spp. were observed in inflamed follicles in seven samples (18%). Two samples were positive for P. acnes, which were identified as either type II or type IB/type III. Both Staphylococcus spp. and P. acnes were seen in macrocolonies/biofilm structures. In conclusion, one-third of patients with clinically diagnosed, non-infectious folliculitis exhibited microbial colonization with predominance of Staphylococcus spp.

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.

The gastrointestinal (GI) tract represents the front line of microbial-host interaction by virtue of its immense surface area and constant microbial supply from ingested food. The gastrointestinal immune system shapes the communities of microbes throughout the GI tract, and in turn, the microbiota provide metabolites and other cues to support the development and normal function of the immune system. Emerging research shows that this influence on the immune system encompasses both innate and adaptive immunity and extends beyond the gut to anatomical sites throughout the body. This chapter presents an overview of the microbiology and immunology of the GI tract, examines microbial population dynamics revealed by studies such as the Human Microbiome Project and discusses the potential impact of emerging antimicrobial resistance to the microbiota and human health.

Summary Background Acne vulgaris is a disorder of the sebaceous follicles. Propionibacterium acnes can be involved in inflammatory acne. Objectives This case-control study aimed at investigating ...

Rosacea is a common skin disease in adults affecting mainly the facial skin. Although inflammation appears to play a pathogenic role in rosacea, initiating factors are largely unknown. Microbial involvement in the development of rosacea has been suggested previously. We aimed to visualize Propionibacterium acnes in the skin compartments of rosacea patients. Facial skin biopsies from 82 rosacea patients and 25 controls were stained with a P. acnes-specific monoclonal antibody (QUBPa3). Seven of 82 patients (8.5%) tested positive for P. acnes which was present either as a biofilm (57% of positive) or a microcolony (43%) in colonized patients. Our results suggest that P. acnes does not play a major role in the pathogenesis of rosacea.

The inactivation of Bacteroides fragilis genes encoding putative RecQ helicases recQ1, recQ2 and recQ3 (ORFs BF638R_3282, BF638R_3781, BF638R_3932) was used to determine whether these proteins are involved in cell survival following metronidazole exposure. The effects of the mutations on growth, cellular morphology and DNA integrity were also evaluated. Mutations in the RecQ DNA helicases caused increased sensitivity to metronidazole, with recQ1, recQ2 and recQ3 mutants being 1.32-fold, 41.88-fold and 23.18-fold more sensitive than the wild type, respectively. There was no difference in cell growth between the recQ1 and recQ3 mutants and the wild type. However, the recQ2 mutant exhibited reduced cell growth, aberrant cell division and increased pleiomorphism, with an increase in filamentous forms and chains of cells being observed using light, fluorescence and electron microscopy. There was no spontaneous accumulation of DNA single- or double-strand breaks in the recQ mutants, as compared with the wild type, during normal cell growth in the absence of metronidazole. Bacteroides fragilis RecQ DNA helicases, therefore, enhance cell survival following metronidazole damage. The abnormal cellular phenotype and growth characteristics of recQ2 mutant cells suggest that this gene, or the downstream gene of the operon in which it occurs, may be involved in cell division.

Comparison of the complete genome sequence ofBacteroides fragilis638R, originally isolated in the USA, was made with two previously sequenced strains isolated in the UK (NCTC 9343) and Japan (YCH46). The presence of 10 loci containing genes associated with polysaccharide (PS) biosynthesis, each including a putative Wzx flippase and Wzy polymerase, was confirmed in all three strains, despite a lack of cross-reactivity between NCTC 9343 and 638R surface PS-specific antibodies by immunolabelling and microscopy. Genomic comparisons revealed an exceptional level of PS biosynthesis locus diversity. Of the 10 divergent PS-associated loci apparent in each strain, none is similar between NCTC 9343 and 638R. YCH46 shares one locus with NCTC 9343, confirmed by mAb labelling, and a second different locus with 638R, making a total of 28 divergent PS biosynthesis loci amongst the three strains. The lack of expression of the phase-variable large capsule (LC) in strain 638R, observed in NCTC 9343, is likely to be due to a point mutation that generates a stop codon within a putative initiating glycosyltransferase, necessary for the expression of the LC in NCTC 9343. Other major sequence differences were observed to arise from different numbers and variety of inserted extra-chromosomal elements, in particular prophages. Extensive horizontal gene transfer has occurred within these strains, despite the presence of a significant number of divergent DNA restriction and modification systems that act to prevent acquisition of foreign DNA. The level of amongst-strain diversity in PS biosynthesis loci is unprecedented.

Bacteroides fragilis is a bacterium that resides in the normal human gastro-intestinal tract; however, it is also the most commonly isolated Gram-negative obligate anaerobe from human clinical infections, such as intra-abdominal abscesses, and the most common cause of anaerobic bacteraemia. Abscess formation is important in bacterial containment, limiting dissemination of infection and bacteraemia. In this study, we investigated B. fragilis binding and degradation of human fibrinogen, the major structural component involved in fibrin abscess formation. We have shown that B. fragilis NCTC9343 binds human fibrinogen. A putative Bacteroides fragilis fibrinogen-binding protein, designated BF-FBP, identified in the genome sequence of NCTC9343, was cloned and expressed in Escherichia coli. The purified recombinant BF-FBP bound primarily to the human fibrinogen Bβ-chain. In addition, we have identified fibrinogenolytic activity in B. fragilis exponential phase culture supernatants, associated with fibrinogenolytic metalloproteases in NCTC9343 and 638R, and cysteine protease activity in YCH46. All nine clinical isolates of B. fragilis examined degraded human fibrinogen; with eight isolates, initial Aα-chain degradation was observed, with varying Bβ-chain and γ-chain degradation. With one blood culture isolate, Bβ-chain and γ-chain degradation occurred first, followed by subsequent Aα-chain degradation. Our data raise the possibility that the fibrinogen-binding protein of B. fragilis, along with a variety of fibrinogenolytic proteases, may be an important virulence factor that facilitates dissemination of infection via reduction or inhibition of abscess formation.

Objectives: The aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance. Methods: The genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays. Results: A metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose. Conclusions: These data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron

Bacteroides thetaiotaomicron is an important human gut commensal, which also causes opportunistic infections outside this environment. It utilises a range of host and diet-related carbohydrates, including rhamnose. In this study, the rha gene cluster, required for rhamnose utilisation, was characterised by transcription analysis, gene targeted mutagenesis and enzyme assays. Growth in the presence of L-rhamnose induced transcription of all the genes of this cluster. The first five genes of the cluster, rhaKIPAO, were transcribed as an operon from a transcriptional start site upstream of rhaK, whereas the sixth gene, rhaR, was transcribed independently. Bioinformatic analysis and mutation of the rhaR gene identified it as encoding the positive transcriptional activator of rhaKIPAO. A rhaR mutant could not utilise rhamnose as the sole carbon source but grew normally on glucose. The rhaO gene encoded a lactaldehyde reductase, and a rhaO mutant produced reduced levels of L-1,2-propanediol during growth in rhamnose, indicating its contribution to rhamnose catabolism in Bacteroides thetaiotaomicron.

Pulmonary infection in cystic fibrosis (CF) is polymicrobial and it is possible that anaerobic bacteria, not detected by routine aerobic culture methods, reside within infected anaerobic airway mucus.To determine whether anaerobic bacteria are present in the sputum of patients with CF.Sputum samples were collected from clinically stable adults with CF and bronchoalveolar lavage fluid (BALF) samples from children with CF. Induced sputum samples were collected from healthy volunteers who did not have CF. All samples were processed using anaerobic bacteriologic techniques and bacteria within the samples were quantified and identified.Anaerobic species primarily within the genera Prevotella, Veillonella, Propionibacterium, and Actinomyces were isolated in high numbers from 42 of 66 (64%) sputum samples from adult patients with CF. Colonization with Pseudomonas aeruginosa significantly increased the likelihood that anaerobic bacteria would be present in the sputum. Similar anaerobic species were identified in BALF from pediatric patients with CF. Although anaerobes were detected in induced sputum samples from 16 of 20 volunteers, they were present in much lower numbers and were generally different species compared with those detected in CF sputum. Species-dependent differences in the susceptibility of the anaerobes to antibiotics with known activity against anaerobes were apparent with all isolates susceptible to meropenem.A range of anaerobic species are present in large numbers in the lungs of patients with CF. If these anaerobic bacteria are contributing significantly to infection and inflammation in the CF lung, informed alterations to antibiotic treatment to target anaerobes, in addition to the primary infecting pathogens, may improve management.

Immunofluorescence microscopy-based identification of presumptivePropionibacterium acnesisolates, using theP. acnes-specific mAb QUBPa3, revealed five organisms with an atypical cellular morphology. Unlike the coryneform morphology seen withP. acnestypes I and II, these isolates exhibited long slender filaments (which formed large tangled aggregates) not previously described inP. acnes. No reaction with mAbs that labelP. acnestypes IA (QUBPa1) and II (QUBPa2) was observed. Nucleotide sequencing of the 16S rRNA gene (1484 bp) revealed the isolates to have between 99.8 and 99.9 % identity to the 16S rRNA gene of theP. acnestype IA, IB and II strains NCTC 737, KPA171202 and NCTC 10390, respectively. Analysis of therecAhousekeeping gene (1047 bp) did reveal, however, a greater number of conserved nucleotide polymorphisms between the sequences from these isolates and those from NCTC 737 (98.9 % identity), KPA171202 (98.9 % identity) and NCTC 10390 (99.1 % identity). Phylogenetic investigations demonstrated that the isolates belong to a novelrecAcluster or lineage distinct fromP. acnestypes I and II. We now propose this new grouping asP. acnestype III. The prevalence and clinical importance of this novelrecAlineage amongst isolates ofP. acnesremains to be determined.

A putative transcriptional regulator of the AraC/XylS family was identified in a genomic genebank of Bacteroides fragilis Bf-1, which partially relieved the sensitivity of Escherichia coli DNA repair mutants to the DNA-damaging agents, metronidazole and mitomycin C. A homologue of this gene with the same phenotype was identified as BF638R3281 in B. fragilis 638R. Transcription of BF638R3281 was constitutive with respect to exposure to sublethal doses of metronidazole. BF638R3281 was interrupted by single cross-over gene-specific insertion mutation, and the gene disruption was confirmed by PCR and DNA-sequencing analysis. The mutant grew more slowly than the wild type, and the mutation rendered B. fragilis more sensitive to metronidazole and mitomycin C. This indicates that the BF638R3281 gene product plays a role in the survival of B. fragilis following DNA damage by these agents.

Bacteroides is the predominant genus within the lower human intestinal tract, as evidenced by its prevalence in the product of this open-ended culture system, faeces. Within the intestinal tract Bacteroides spp. host molecular interaction can influence host function, for example in relation to immune system development. Given the opportunity to escape from the intestinal tract into the normally uncolonized peritoneal cavity as a result of, for example, either trauma or surgery, selected Bacteroides species, in particular B. fragilis, can cause life-threatening infection including bacteraemia. Although aerotolerance is evident in some Bacteroides, in the medical diagnostic setting they are effectively obligate anaerobes. Bacteroides exhibit an unprecedented level of surface component diversity, both within and between strains, exemplified by B. fragilis surface polysaccharides. Extensive multiple DNA inversion events, conjugative and mobilizable transposons and multiple extracytoplasmic function (ECF) sigma factors contribute to this diversity and the adaptability of Bacteroides. Metronidazole has been an effective antibiotic for both treatment and prophylaxis, but the potential global spread of multidrug resistance is a major cause for concern.

Despite accumulating data implicating Propionibacterium acnes in a variety of diseases, its precise role in infection remains to be determined. P. acnes antigen-specific CD4+ T cells are present in early inflamed acne lesions and may be involved in the inflammatory response; however, little is known about the specific antigens involved. In this study, B cell and T cell antigens from P. acnes expression libraries were cloned and evaluated and the four predominant proteins identified were investigated. Two of these antigens share some homology with an M-like protein of Streptococcus equi and have dermatan-sulphate-binding activity (PA-25957 and 5541). The remaining two antigens, PA-21693 and 4687, are similar to the product of the Corynebacterium diphtheriae htaA gene from the hmu ABC transport locus, although only one of these (PA-21693) is encoded within an hmu-like operon and conserved amongst a range of clinical isolates. All four proteins contain an LPXTG motif, although only PA-21693 contains a characteristic sortase-sorting signal. Variation in the expression of PA-4687, 25957 and 5541 is evident amongst clinical isolates and is generated both by frameshifts associated with the putative signal peptide and by variable numbers of repeat regions toward the carboxy-terminus, potentially generating heterogeneity of molecular mass and antigenic variation. In addition, in the case of PA-25957, a frameshift in a C-rich region at the extreme carboxy-terminus eliminates the LPXTG motif in some isolates. For the dermatan-sulphate-binding PA-25957, IgG1 antibody in serum from acne-positive donors was shown to be specific for the amino-terminal region of the protein, which also contains a CD4+ T cell epitope. In contrast, serum from acne-negative donors shows an IgG2 and IgG3 antibody subclass response to the carboxy-terminal region. These data have implications for the potential role of P. acnes in inflammatory acne and other diseases.

Bacterial infection primarily with Staphylococcus spp. and Propionibacterium acnes remains a significant complication following total hip replacement. In this in vitro study, we investigated the efficacy of gentamicin loading of bone cement and pre- and postoperative administration of cefuroxime in the prevention of biofilm formation by clinical isolates. High and low initial inocula, representative of the number of bacteria that may be present at the operative site as a result of overt infection and skin contamination, respectively, were used. When a high initial inoculum was used, gentamicin loading of the cement did not prevent biofilm formation by the 10 Staphylococcus spp. and the 10 P. acnes isolates tested. Similarly, the use of cefuroxime in the fluid phase with gentamicin-loaded cement did not prevent biofilm formation by four Staphylococcus spp. and four P. acnes isolates tested. However, when a low bacterial inoculum was used, a combination of both gentamicin-loaded cement and cefuroxime prevented biofilm formation by these eight isolates. Our results indicate that this antibiotic combination may protect against infection after intra-operative challenge with bacteria present in low numbers as a result of contamination from the skin but would not protect against bacteria present in high numbers as a result of overt infection of an existing implant.

Failure to treat and eradicate prosthetic hip infection with systemic antibiotic regimens is usually due to the fact that the infection is associated with biofilm formation and that bacterial cells growing within a biofilm exhibit increased resistance to antimicrobial agents. In this in vitro study, we investigated the susceptibility of prosthetic hip Propionibacterium acnes and Staphylococcus spp. isolates growing within biofilms on polymethylmethacrylate (PMMA) bone cement to a range of antibiotics. All P. acnes isolates in the biofilm mode of growth demonstrated considerably greater resistance to cefamandole, ciprofloxacin and vancomycin. In contrast, only four of the eight P. acnes isolates demonstrated an increase in resistance to gentamicin. All ten Staphylococcus spp. isolates in the biofilm mode of growth exhibited large increases in resistance to gentamicin and cefamandole with eight of the ten isolates also exhibiting an increase in resistance to vancomycin. However, only three of the ten Staphylococcus spp. isolates exhibited an increase in resistance to ciprofloxacin. Biofilms were also formed on three different titanium alloys and on PMMA bone cement using P. acnes, Staphylococcus epidermidis and Staphylococcus aureus strains to determine if the underlying biomaterial surface had an effect on biofilm formation and the antimicrobial susceptibility of the bacteria growing within biofilms. Although differences in the rate at which the three strains adhered to the different biomaterials were apparent, no differences in biofilm antibiotic resistance between the biomaterials were observed. In the light of these results, it is important that the efficacy of other antibiotics against P. acnes and Staphylococcus spp. prosthetic hip isolates growing within biofilms on orthopaedic biomaterials be determined to ensure optimal treatment of orthopaedic implant infection.

Physiological studies on M. parvicella have been conducted to determine the rate of growth of this organism in pure culture. The organism displayed a doubling time of 128 days despite its profuse abundance in a local Wastewater Treatment Plant (WWTW). An extensive survey has been ongoing since February 2000 into the extent of M. parvicella in the WWTW. A suite of monoclonal and polyclonal antibodies has been developed to detect and quantify M. parvicella.

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.

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.

While the association of the Gram-positive anaerobic bacterium Propionibacterium acnes with the inflammatory skin condition acne vulgaris has been known for over a century, its potential role in other human infections and clinical conditions has undoubtedly been underestimated. This is likely to reflect inadequate anaerobic processing methods and culture of clinical specimens (>7d incubation), combined with the traditionally held view that P. acnes in a biological sample simply reflects contamination from the resident skin microflora or is clinically irrelevant due to a low level of virulence [1]. On such basis, it is likely that an unknown number of P. acnes infections go undiagnosed and unrecognised. In addition, subtle clinical signs and symptoms that are often associated with P. acnes infections may further lead to the conclusion that an infection is absent, particularly in relation to prosthetic joints and other indwelling medical devices [2]. Despite the historical failure to recognise the pathogenic potential of P. acnes, there is now a growing awareness, as evidenced in the scientific literature, that the bacterium is an important cause of opportunistic human infection and should not be readily dismissed as a contaminant/passive presence in clinical samples [2–6]. Within the last decade, advances in our understanding of the population genetic structure of P. acnes combined with whole genome sequence (WGS) data now available for a large number of strains, mostly derived from the Human Microbiome Project, have provided a platform for hypothesis-driven research into the exact role of P. acnes in human health and disease and the possibility of developing vaccines and other novel therapeutic treatments [7–11]. In particular, the availability of WGS information has enabled both genomic and transcriptomic differences between lineages from phylogroups associated mainly with health or disease to be examined, thus identifying potential genetic determinants and mechanisms of virulence [11, 12]. Despite its ability to behave as an opportunistic pathogen, it must not be forgotten that P. acnes is part of the normal human microbiota and, consequently, also plays a role in human health via occupation of niches that could be colonized by other, more pathogenic, microorganisms. Furthermore, the powerful immunomodulatory properties of P. acnes have long been investigated for their capacity to stimulate protective host responses against various human cancers, and more generally against Th-2-mediated diseases. In light of the growing interest and recognition of the role of P. acnes in human health and disease, this special issue aims to provide a description of recent developments and advances in different areas of P. acnes research via original research and review articles, and thus provide a framework to discuss and identify key issues that need to be addressed in future studies. Developments in our understanding of P. acnes bacteriophages are reviewed in this special issue by H. Bruggemann and R. Lood. They describe both historical and recent developments in the field and highlight how genomic analyses have provided insights into P. acnes phage biology, as well as the identification of phage sequence signatures in clustered regularly interspaced short palindromic repeats (CRISPRs) in P. acnes. They also discuss bacteriophages as a therapeutic strategy to combat P. acnes-associated diseases, although there are currently formidable regulatory barriers to this approach. In a research paper, G. Kasimatis et al. describe a comparative genomics study on the acne-derived type IA1 strain, HL096PA1, which they found harbours the first described linear plasmid in P. acnes, pIMPLE-HL096PA1. Homologues of pIMPLE-HL096PA1 appear to be widely distributed in ribotype 4 and 5 strains of P. acnes which are tetracycline resistant and associated with acne [11]. These plasmids carry a tight adhesion locus (Tad) that may enhance binding to host cell surfaces. Identification of this novel plasmid also provides a potentially new opportunity for genetic manipulation of P. acnes and possibly targeted therapy against specific disease-associated strains. HL096PA1 also contains fivefold more pseudogenes than the type IB strain KPA171202 to which it was compared, as well as island-like regions unique to the strain; type IB strains have rarely been isolated from acne lesions. Collectively, this paper provides additional data on P. acnes virulence properties and host adaptation mechanisms that may be relevant to our understanding of acne pathogenesis. Still on the theme of acne, E. A. Eady et al. in a second review paper hypothesise that the availability of water and possibly one or more water soluble micronutrients limits microbial growth within the follicular microenvironment of the skin and that reduction of water activity with biocompatible solutes of very high solubility may represent a novel and safe therapeutic approach in acne management. In an era of growing antibiotic resistance and the continuing widespread use of oral and topical antibiotics for the first line treatment of acne, the development of alternative therapeutic approaches for this extremely common skin condition is highly desirable. In this special issue, we also have a research paper by N. Fischer et al. which describes a study into the intracellular fate of P. acnes in macrophages. This is an important study as the ability to persist in macrophages and other cell types as an intracellular pathogen may be important in the context of P. acnes-related diseases, particularly acne, sarcoidosis, and prostate cancer, to which the bacterium has been associated. This study confirmed the ability of P. acnes to survive phagocytosis in vitro, although no intracellular replication or escape from THP-1 host cells were observed. There was also no evidence of the P. acnes-containing phagosome fusing with lysosomes. The association of P. acnes with sarcoidosis, a systemic granulomatous disease, is extensively reviewed in a paper by Y. Eishi. While sarcoidosis is a disease of unknown etiology, infectious granulomas are commonly caused by intracellular pathogens and P. acnes is currently the only microorganism isolated from sarcoid lesions by culture. The review proposes mechanisms of granuloma formation in response to P. acnes in subjects with sarcoidosis and introduces a new concept of endogenous infection caused by hypersensitivity to indigenous bacteria. Interestingly, acne is also a delayed type hypersensitivity reaction, and if the condition is left untreated granuloma formation can occur [13]; this is rarely seen today due to clinical intervention. In a research paper by I. Dekio et al., the intracellular proteomes of different phylogroups were investigated and variations in protein expression profiles between strains grown under different oxygen tensions were examined. A good correlation was observed between genomic and proteomic profiles for the major genetic divisions of P. acnes. Differences were also noted in the intracellular proteome of strains grown under anaerobic/microaerophilic and aerobic culture conditions, which could be relevant in the context of P. acnes transferring from the skin surface to hypoxic/anoxic environments associated with deep seated systemic infections. The role of P. acnes as a cause of infections related to indwelling medical devices is now increasingly recognised, especially in relation to prosthetic shoulder implants [2, 14]. A study from the Mayo Clinic, described by M. J. Karau et al., highlights how application of a vortexing-sonication technique for the detection of bacterial biofilm identifies Propionibacterium species, likely P. acnes, as common colonizers of breast tissue expanders. The presence of these colonised expanders in clinically uninfected subjects may present an increased risk factor for future infection or capsular contracture. The role of P. acnes in a wide range of implant-associated infections, along with its clinical management, is further discussed in a review by M. Portillo et al. They also describe how sonication of explanted prosthetic material improves the diagnosis of such infections and suggest that molecular methods may further increase the sensitivity of P. acnes detection in the resulting sonicate. In a paper by J. Rollason et al., P. acnes isolates from surgically excised lumbar disc herniations are characterised for phylogroup and antimicrobial susceptibility. Previous studies have suggested a role for P. acnes in the pathophysiology of disc degeneration and herniation, and antibiotic treatment does appear to have a positive effect upon patients with chronic back conditions [15–17]. In this study, type II and III strains were the predominant phylotypes isolated. Since strains from these genetic divisions are infrequently recovered from the skin, this study suggests that the recovery of P. acnes from excised disc material should not be readily dismissed as skin contamination in all cases. Furthermore, isolates from excised discs were susceptible to a range of antimicrobials commonly used to treat P. acnes-related infections. In summary, this special issue covers a range of diverse topics related to P. acnes and disease, thus highlighting the widespread opportunistic behaviour of the bacterium in relation to infection. We hope the papers published will serve to further highlight the pathogenic potential of P. acnes, as well as stimulating further research into the role of the bacterium in human health and disease. In particular, future studies aimed at providing firm evidence of a role in chronic diseases, such as prostate cancer and disc disease, may provide exciting new opportunities for novel patient treatment and management strategies.

Our aim was to determine if the detection rate of infection of total hip replacements could be improved by examining the removed prostheses. Immediate transfer of prostheses to an anaerobic atmosphere, followed by mild ultrasonication to dislodge adherent bacteria, resulted in the culture of quantifiable numbers of bacteria, from 26 of the 120 implants examined. The same bacterial species were cultured by routine microbiological techniques from only five corresponding tissue samples. Tissue removed from 18 of the culture-positive implants was suitable for quantitative tissue pathology and inflammatory cells were present in all samples. Furthermore, inflammatory cells were present in 87% of tissue samples taken from patients whose implants were culture-negative. This suggests that these implants may have been infected by bacteria which were not isolated by the techniques of culture used. The increased detection of bacteria from prostheses by culture has improved postoperative antibiotic therapy and should reduce the need for further revision.

The anaerobic skin commensalPropionibacterium acnesis an underestimated cause of human infections and clinical conditions. Previous studies have suggested a role for the bacterium in lumbar disc herniation and infection. To further investigate this, five biopsy samples were surgically excised from each of 64 patients with lumbar disc herniation.P. acnesand other bacteria were detected by anaerobic culture, followed by biochemical and PCR-based identification. In total, 24/64 (38%) patients had evidence ofP. acnesin their excised herniated disc tissue. UsingrecAand mAb typing methods, 52% of the isolates were type II (50% of culture-positive patients), while type IA strains accounted for 28% of isolates (42% patients). Type III (11% isolates; 21% patients) and type IB strains (9% isolates; 17% patients) were detected less frequently. The MIC values for all isolates were lowest for amoxicillin, ciprofloxacin, erythromycin, rifampicin, tetracycline, and vancomycin (≤1mg/L). The MIC for fusidic acid was 1-2 mg/L. The MIC for trimethoprim and gentamicin was 2 to ≥4 mg/L. The demonstration that type II and III strains, which are not frequently recovered from skin, predominated within our isolate collection (63%) suggests that the role ofP. acnesin lumbar disc herniation should not be readily dismissed.

The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis.

Surmmary A monospecific polyclonal antiserum, prepared against Bacteroides fragilis common polysaccharide antigen purified by polyacrylamide gel immunoblot detected B. fragilis, B. thetaiotaomicron, B. ovatus and Prevotella melaninogenica in pus samples from various anatomical sites by immunofluorescence microscopy of the pus. With standard clinical laboratory culture methods, 36% of 147 samples were positive for one or more of the above bacteria. Of these, B. fragilis accounted for 33%. By immunofluorescent labelling of pus with the common antigen antiserum the detection of these bacteria in the samples increased to 50%. All nine of the blood cultures in which B. fragilis was detected by culture contained bacteria positive for the common antigen. Immunofluorescent labelling of pus samples with a selection of monoclonal antibodies specific for surface polysaccharides which are known to be antigenically variable in culture in vitro and in an animal model of infection showed that these polysaccharides are also variable in natural infection. The results indicate that the common polysaccharide antigen, in contrast to the variable surface polysaccharides, is a suitable target for the immunodetection of B. fragilis in clinical samples from a range of anatomical sites.

Propionibacterium acnes , a non-spore-forming, anaerobic Gram-positive bacterium, is most notably recognized for its association with acne vulgaris (I. Kurokawa et al., Exp. Dermatol. 18:821–832, 2009). We now present the draft genome sequence of an antibiotic-resistant P. acnes strain, PRP-38, isolated from an acne patient in the United Kingdom and belonging to the novel type IC cluster.

The resident microbiota of the human gastrointestinal (GI) tract is comprised of ~2000 bacterial species, the majority of which are anaerobes. Colonization of the GI tract is important for normal development of the immune system and provides a reservoir of catabolic enzymes that degrade ingested plant polysaccharides. Bacteroides fragilis is an important member of the microbiota because it contributes to T helper cell development, but is also the most frequently isolated Gram-negative anaerobe from clinical infections. During the annotation of the B. fragilis genome sequence, we identified a gene predicted to encode a homolog of the eukaryotic protein modifier, ubiquitin. Previously, ubiquitin had only been found in eukaryotes, indicating the bacterial acquisition as a potential inter-kingdom horizontal gene transfer event. Here we discuss the possible roles of B. fragilis ubiquitin and the implications for health and disease.

Propionibacterium acnes is an anaerobic Gram-positive bacterium that has been linked to a wide range of opportunistic human infections and conditions, most notably acne vulgaris (I. Kurokawa et al., Exp. Dermatol. 18:821–832, 2009). We now present the whole-genome sequences of three P. acnes strains from the type IA 2 cluster which were recovered from ophthalmic infections (A. McDowell et al., Microbiology 157:1990–2003, 2011).

In the complete genome sequences of Bacteroides fragilis NCTC9343 and 638R, we have discovered a gene, ubb, the product of which has 63 % identity to human ubiquitin and cross-reacts with antibodies raised against bovine ubiquitin. The sequence of ubb is closest in identity (76 %) to the ubiquitin gene from a migratory grasshopper entomopoxvirus, suggesting acquisition by inter-kingdom horizontal gene transfer. We have screened clinical isolates of B. fragilis from diverse geographical regions and found that ubb is present in some, but not all, strains. The gene is transcribed and the mRNA is translated in B. fragilis, but deletion of ubb did not have a detrimental effect on growth. BfUbb has a predicted signal sequence; both full-length and processed forms were detected in whole-cell extracts, while the processed form was found in concentrated culture supernatants. Purified recombinant BfUbb inhibited in vitro ubiquitination and was able to covalently bind the human E1 activating enzyme, suggesting it could act as a suicide substrate in vivo. B. fragilis is one of the predominant members of the normal human gastrointestinal microbiota with estimates of up to >1011 cells per g faeces by culture. These data indicate that the gastro-intestinal tract of some individuals could contain a significant amount of aberrant ubiquitin with the potential to inappropriately activate the host immune system and/or interfere with eukaryotic ubiquitin activity. This discovery could have profound implications in relation to our understanding of human diseases such as inflammatory bowel and autoimmune diseases.

Propionibacterium acnes is an anaerobic Gram-positive bacterium that forms part of the normal human cutaneous microbiota and is thought to play a central role in acne vulgaris, a chronic inflammatory disease of the pilosebaceous unit (I. Kurokawa et al., Exp. Dermatol. 18: 821-832, 2009). Here we present the whole genome sequence of P. acnes type IB strain 6609, which was recovered from a skin sample from a woman with no recorded acne history and is thus considered a nonpathogenic strain (I. Nagy, Microbes Infect. 8: 2195-2205, 2006).

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64 % of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

The inactivation of Bacteroides fragilis genes encoding putative RecQ helicases recQ1, recQ2 and recQ3 (ORFs BF638R_3282, BF638R_3781, BF638R_3932) was used to determine whether these proteins are involved in cell survival following metronidazole exposure. The effects of the mutations on growth, cellular morphology and DNA integrity were also evaluated. Mutations in the RecQ DNA helicases caused increased sensitivity to metronidazole, with recQ1, recQ2 and recQ3 mutants being 1.32-fold, 41.88-fold and 23.18-fold more sensitive than the wild type, respectively. There was no difference in cell growth between the recQ1 and recQ3 mutants and the wild type. However, the recQ2 mutant exhibited reduced cell growth, aberrant cell division and increased pleiomorphism, with an increase in filamentous forms and chains of cells being observed using light, fluorescence and electron microscopy. There was no spontaneous accumulation of DNA single- or double-strand breaks in the recQ mutants, as compared with the wild type, during normal cell growth in the absence of metronidazole. Bacteroides fragilis RecQ DNA helicases, therefore, enhance cell survival following metronidazole damage. The abnormal cellular phenotype and growth characteristics of recQ2 mutant cells suggest that this gene, or the downstream gene of the operon in which it occurs, may be involved in cell division.