Adaptation of acneic and non acneic strains of Cutibacterium acnes to sebum-like environment.

Cutibacterium acnes, former Proprionibacterium acnes, is a heterogeneous species including acneic bacteria such as the RT4 strain, and commensal bacteria such as the RT6 strain. These strains have been characterized by metagenomic analysis but their physiology was not investigated until now. Bacteria were grown in different media, brain heart infusion medium (BHI), reinforced clostridial medium (RCM), and in sebum like medium (SLM) specifically designed to reproduce the lipid rich environment of the sebaceous gland. Whereas the RT4 acneic strain showed maximal growth in SLM and lower growth in RCM and BHI, the RT6 non acneic strain was growing preferentially in RCM and marginally in SLM. These differences were correlated with the lipophilic surface of the RT4 strain and to the more polar surface of the RT6 strain. Both strains also showed marked differences in biofilm formation activity which was maximal for the RT4 strain in BHI and for the RT6 strain in SLM. However, cytotoxicity of both strains on HaCaT keratinocytes remained identical and limited. The RT4 acneic strain showed higher inflammatory potential than the RT6 non acneic strain, but the growth medium was without significant influence. Both bacteria were also capable to stimulate ß-defensine 2 secretion by keratinocytes but no influence of the bacterial growth conditions was observed. Comparative proteomics analysis was performed by nano LC-MS/MS and revealed that whereas the RT4 strain only expressed triacylglycerol lipase, the principal C. acnes virulence factor, when it was grown in SLM, the RT6 strain expressed another virulence factor, the CAMP factor, exclusively when it was grown in BHI and RCM. This study demonstrates the key influence of growth conditions on virulence expression by C. acnesand suggest that acneic and non acneic strains are related to different environmental niches.

Structural basis of branched-chain fatty acid synthesis by Propionibacterium acnes ß-ketoacyl acyl Carrier protein synthase.

Propionibacterium acnes is an anaerobic gram-positive bacterium found in the niche of the sebaceous glands in the human skin, and is a causal pathogen of inflammatory skin diseases as well as periprosthetic joint infection. To gain effective control of P. acnes, a deeper understanding of the cellular metabolism mechanism involved in its ability to reside in this unique environment is needed. P. acnes exhibits typical cell membrane features of gram-positive bacteria, such as control of membrane fluidity by branched-chain fatty acids (BCFAs). Branching at the iso- or anteiso-position is achieved by incorporation of isobutyryl- or 2-methyl-butyryl-CoA via ß-ketoacyl acyl carrier protein synthase (KAS III) from fatty acid synthesis. Here, we determined the crystal structure of P. acnes KAS III (PaKAS III) at the resolution of 1.9 Šfor the first time. Conformation-sensitive urea polyacrylamide gel electrophoresis and tryptophan fluorescence quenching experiments confirmed that PaKAS III prefers isobutyryl-CoA as the acetyl-CoA, and the unique shape of the active site cavity complies with incorporation of branched-short chain CoAs. The determined structure clearly illustrates how BCFA synthesis is achieved in P. acnes. Moreover, the unique shape of the cavity required for the branched-chain primer can be invaluable in designing novel inhibitors of PaKAS III and developing new specifically targeted antibiotics.

Highly efficient and biocompatible nanoparticle-based photosensitizer for treatment of acne vulgaris.

AIM: Nanoparticle-based photosensitizers containing silver core and mesoporous silica shell with hematoporphyrin IX embedded were developed to treat vulgaris photodynamically. MATERIALS & METHODS: The hybrid photosensitizers were dispersed in 30% polyethylene glycol (PEG-200) solution and used for the photodynamic treatment of Staphylococcus epidermidis and Propionibacterium acnes under the illumination of a portable LED (∼410 nm). RESULTS: After a 5 min illumination by the LED, the hybrid photosensitizers of 50 µg/ml displayed killing efficacy of approximately 5-log for S. epidermidis and approximately 4-log for P. acnes. Results indicated that hybrid photosensitizers in PEG-200 matrix perform better than in deionized (DI) water (∼1-log increase in killing efficacy). CONCLUSION: Under short illumination of a portable LED, hybrid photosensitizers demonstrated immense potential for treatment of acne vulgaris without involving antibiotics.

Is Human Sebum the Source of Skin Follicular Ultraviolet-Induced Red Fluorescence? A Cellular to Histological Study.

BACKGROUND: The ultraviolet-induced red fluorescence (UVRF) from human skin follicles was suggested to be a result of Propionibacterium acnes and was used for the monitoring of acne. More recent studies suggested that the UVRF may be more related to sebum rather than to microorganisms. OBJECTIVE: To clarify whether human sebum or follicular microorganisms are the source of UVRF. METHODS: We examined the fluorescence of human-derived SZ95 sebocytes, human sebaceous glands, sebum extracted from the sebaceous glands, and bacteria isolated from human hair follicles under ultraviolet light. RESULTS: SZ95 sebocytes, human sebaceous glands, and sebum do not emit UVRF. Two types of UVRF peaking at about 635 nm and at about 620 nm were detected in P. acnes and Staphylococcus epidermidis, respectively. This is the first report that S. epidermidis emits UVRF when it is anaerobically cultured and then exposed to air. CONCLUSION: Human follicular UVRF is emitted by resident bacteria, not by sebum. Therefore, UVRF may be used to monitor certain species of skin microorganisms.

Characterization of a Propionibacterium acnes Surface Protein as a Fibrinogen-Binding Protein.

Propionibacterium acnes (P. acnes) is a major skin-associated bacterium that was long considered commensal, until several studies revealed it to be an opportunistic pathogen. We investigated the ability of P. acnes surface proteins to recognize ECM proteins and showed that a 58 kDa P. acnes surface protein was specifically recognized by human fibrinogen (hFg). The 58 kDa protein was further characterized by two-dimensional (2-D) electrophoresis and MALDI-ToF as a P. acnes host cell-surface attachment protein, PA25957, recognizing dermatan sulfate (DsA1). This protein sequence contains 432 amino acids with the presence of three structurally different domains: an N-terminal signal peptide, a C-terminal LPXTG motif, and a PT repeat region. DsA1 is mostly produced during stationary phase. It appears to be highly glycosylated, containing GalNAc residues. Purified DsA1 strongly recognizes the Aα and Bß subunits of hFg, and specific enzymatic deglycosylation of hFg demonstrated the involvement of the protein backbone in the recognition process. The Bß subunit of hFg was cloned in four peptide fractions (Fg1-Fg4). The N-terminal Fg1 peptide of hFg was recognized by DsA1, and priming DsA1 with Fg1 inhibited DsA1/hFg recognition. We describe here for the first time, the characterization of a P. acnes surface glycoprotein recognizing human fibrinogen.

Immunoproteomic Identification of In Vivo-Produced Propionibacterium acnes Proteins in a Rabbit Biofilm Infection Model.

Propionibacterium acnes is well-known as a human skin commensal but can also act as an invasive pathogen causing implant-associated infections. In order to resolve these types of P. acnes infections, the implants must be removed, due to the presence of an established biofilm that is recalcitrant to antibiotic therapy. In order to identify those P. acnes proteins produced in vivo during a biofilm infection, we established a rabbit model of implant-associated infection with this pathogen. P. acnes biofilms were anaerobically grown on dextran beads that were then inoculated into the left tibias of rabbits. At 4 weeks postinoculation, P. acnes infection was confirmed by radiograph, histology, culture, and PCR. In vivo-produced and immunogenic P. acnes proteins were detected on Western blot using serum samples from rabbits infected with P. acnes after these bacterial proteins were separated by two-dimensional gel electrophoresis. Those proteins that bound host antibodies were then isolated and identified by tandem mass spectrometry. Radiographs and histology demonstrated a disruption in the normal bone architecture and adherent biofilm communities in those animals with confirmed infections. A total of 24 immunogenic proteins were identified; 13 of these proteins were upregulated in both planktonic and biofilm modes, including an ABC transporter protein. We successfully adapted a rabbit model of implant-associated infection for P. acnes to identify P. acnes proteins produced during a chronic biofilm-mediated infection. Further studies are needed to evaluate the potential of these proteins for either a diagnostic test or a vaccine to prevent biofilm infections caused by P. acnes.

Treatment of common warts with the immune stimulant Propionium bacterium parvum.

BACKGROUND: Warts are epithelial proliferations in the skin and mucous membrane caused by various types of HPV. They can decrease spontaneously or increase in size and number according to the patient's immune status. The Propionium bacterium parvum is a strong immune stimulant and immune modulator and has important effects in the immune system and it is able to produce antibodies in the skin. OBJECTIVE: To show the efficacy of the Propionium bacterium parvum in saline solution in the treatment of skin warts. METHODS: A randomized double-blind study. Twenty patients with multiple warts were divided into two groups: one received 0,1 ml intradermal injection of placebo solution in just one of the warts and the other received 0,1 ml of saline solution of Propionium bacterium parvum, one dose a month, for 3 to 5 months. RESULTS: Among the 20 patients who participated in the study, ten received the placebo and ten received the saline solution with Propionium bacterium parvum. In 9 patients treated with the Propionium bacterium parvum solution the warts disappeared without scars and in 1 patient it decreased in size. In 9 patients who received the placebo no change to the warts was observed and in 1 it decreased in size. CONCLUSIONS: The immune modulator and immune stimulant Propionium bacterium parvum produced antibodies in the skin which destroyed the warts without scars, with statistically significant results (P<0,001), and cured 90 % of the patients. We suggest the use of the immune stimulant in the treatment of warts.

Treatment of common warts with the immune stimulant Propionium bacterium parvum / Tratamento das verrugas vulgares com o imunoestimulante Propionium bacterium parvum

BACKGROUND: Warts are epithelial proliferations in the skin and mucous membrane caused by various types of HPV. They can decrease spontaneously or increase in size and number according to the patient's immune status. The Propionium bacterium parvum is a strong immune stimulant and immune modulator and has important effects in the immune system and it is able to produce antibodies in the skin. OBJECTIVE: To show the efficacy of the Propionium bacterium parvum in saline solution in the treatment of skin warts. METHODS: A randomized double-blind study. Twenty patients with multiple warts were divided into two groups: one received 0,1ml intradermal injection of placebo solution in just one of the warts and the other received 0,1 ml of saline solution of Propionium bacterium parvum, one dose a month, for 3 to 5 months. RESULTS: Among the 20 patients who participated in the study, ten received the placebo and ten received the saline solution with Propionium bacterium parvum. In 9 patients treated with the Propionium bacterium parvum solution the warts disappeared without scars and in 1 patient it decreased in size. In 9 patients who received the placebo no change to the warts was observed and in 1 it decreased in size. CONCLUSIONS: The immune modulator and immune stimulant Propionium bacterium parvum produced antibodies in the skin which destroyed the warts without scars, with statistically significant results (P<0,001), and cured 90 % of the patients. We suggest the use of the immune stimulant in the treatment of warts.

FUNDAMENTOS: Verrugas são proliferações epiteliais na pele e mucosas causadas por diversos tipos de HPV. Elas podem involuir espontaneameme ou aumentar em número e tamanho de acordo com estado imunitário do paciente. O Propionium bacterium parvum é urn potente imunoestimulador e imunomodulador e tem efeitos importantes no sistema imune e é capaz de produzir anticorpos na pele. OBJETIVO: Mostrar a eficácia do Propionium bacterium parvum diluído em solução salina no tratamento de verrugas cutâneas. MÊTODOS: Estudo duplo-cego randomizado. Vinte pacientes com verrugas múltiplas foram divididos em dois grupos, um recebeu aplicação intradérmica do placebo em uma (1) única verruga e o outro da solução salina com Propionium bacterium parvum, uma dose por mês por 3 a 5 meses. RESULTADOS: Dos 20 pacientes do estudo, dez receberam placebo e 10 de solução salina com Propionium bacterium parvum. Dos pacientes tratados com Propionium bacterium parvum nove (9) foram curados e um teve diminuição das lesões. Do grupo do placebo nove (9) não apresentaram alterações e 1 (um) apresentou diminuição das lesões. CONCLUSÔES: O imunomodulador e imunoestimulador Propionium bacterium parvum produz anticorpos na pele que destroem as verrugas sem cicatrizes e mostrou uma significância de P<0,001, com cura de 90% dos pacientes submetidos à terapia. Sugerimos a utilização de imunoestimulante para o tratamento de verruga vulgar.

NCPP treatment alleviates ConA-induced hepatitis via reducing CD4+T activation and NO production.

Corynebacterium parvum (CP), a kind of immunomodulator, has been well documented in many diseases. Non-cell C. parvum product (NCPP) is a newly-found nano-preparation. To investigate the effect of NCPP on Con A-induced murine severe hepatitis, we pretreated mice with NCPP intraperitoneally. After 12 h, ConA (25 µg/g body wt) was injected intravenously to provoke severe hepatitis and the degree of liver injury was evaluated by serum transaminase analysis and heptatic tissue pathology. Results have shown that levels of serum transaminase and degree of liver injury in ConA/NCPP groups had significantly declined than those in ConA/PBS groups. Notably, results of flow cytometry have demonstrated that activation of CD4+T cells in ConA/NCPP groups has been down-regulated, compared with ConA/PBS groups. Further, levels of serum and KC-related nitric oxide (NO) was displayed significantly lower in ConA/NCPP groups than those in ConA/PBS groups. The results indicate that NCPP may alleviate ConA-induced hepatitis by reducing CD4+T activation and NO production.

The polysaccharide fraction of Propionibacterium acnes modulates the development of experimental focal segmental glomerulosclerosis.

The pathogenesis of focal segmental glomerulosclerosis (FSGS) appears to be associated with type-2 cytokines and podocyte dysfunction. In this study, we tested the hypothesis that immunization with the polysaccharide fraction of Propionibacterium acnes (PS), a pro-Th1 agonist, may subvert the type-2 profile and protect podocytes from adriamycin-induced glomerulosclerosis. Adriamycin injection resulted in albuminuria and increased serum creatinine in association with loss of glomerular podocin and podoplanin expression, which is consistent with podocyte dysfunction. Renal tissue analysis revealed the expression of transcripts for GATA3 and fibrogenic-related proteins, such as TGF-ß, tissue inhibitor of metalloproteinase-1 (TIMP-1) and metalloproteinase 9 (MMP9). In association with the expression of fibrogenic transcripts, we observed peri-glomerular expression of α-smooth muscle actin (α-SMA), indicating epithelial-to-mesenchymal transition, and increased expression of proliferating cell nuclear antigen (PCNA) in tubular cells, suggesting intense proliferative activity. Previous immunization with PS inhibited albuminuria and serum creatinine in association with the preservation of podocyte proteins and inhibition of fibrogenic transcripts and the expression of α-SMA and PCNA proteins. Tissue analysis also revealed that PS treatment induced expression of mRNA for GD3 synthase, which is a glycosiltransferase related to the synthesis of GD3, a ganglioside associated with podocyte physiology. In addition, PS treatment inhibited the influx of inflammatory CD8(pos) and CD11b(pos) cells to kidney tissue. Finally, PS treatment on day 4 post-ADM, a period when proteinuria was already established, was able to improve renal function. Thus, we demonstrate that the PS fraction of P. acnes can inhibit FSGS pathogenesis, suggesting that immunomodulation can represent an alternative approach for disease management.

Heme oxygenase-1 alleviates mouse hepatic failure through suppression of adaptive immune responses.

Heme oxygenase-1 (HO-1) has protective effects on liver damage induced by noxious stimuli. The mechanism of action of HO-1 is not well understood. In the present study, we investigate the effect of HO-1 in a model of fulminant hepatic failure induced by Propionibacterium acnes and lipopolysaccharide (LPS). The expression of HO-1 mRNA and protein in the liver was increased after repeated administration of the HO-1 inducer cobalt protoporphyrin IX. We found that HO-1 protected mice from acute liver damage induced by P. acnes/LPS and prolonged survival. On the contrary, administration of the HO-1 inhibitor zinc protoporphyrin IX increased liver damage induced by P. acnes/LPS. Subsequently, to investigate the underlying mechanisms of HO-1 in the acute liver injury model, we primed mice with P. acnes only. We found that the expression of HO-1 mRNA and protein in dendritic cells (DCs) was increased after the administration of cobalt protoporphyrin IX. HO-1 decreased the mature markers major histocompatibility complex II and CD80 on liver DCs. The expression of CCR7, CCL2, and CCL22 mRNA, which are expressed by mature DCs, was also reduced. These liver DCs could not efficiently stimulate CD4+ T cell activation and proliferation. Consequently, HO-1 inhibited the activation, proliferation, and T helper 1 polarization of liver-infiltrating CD4+ T cells and reduced the production of serum alanine aminotransferase and proinflammatory cytokines such as interferon-γ and tumor necrosis factor-α. Taken together, our data suggest that HO-1 alleviates P. acnes/LPS-induced fulminant hepatic failure, probably by inhibiting DC-induced adaptive responses.

Augmentation of gene expression and production of promatrix metalloproteinase 2 by Propionibacterium acnes-derived factors in hamster sebocytes and dermal fibroblasts: a possible mechanism for acne scarring.

Aberrant extracellular matrix (ECM) remodeling in sebaceous glands and pilosebaceous units in the skin is associated with scar formation under acne conditions. To investigate the involvement of Propionibacterium acnes (P. acnes), a Gram-positive anaerobic microbial species, in ECM remodeling in sebaceous glands and pilosebaceous units, we examined the effects of P. acnes culture media, formalin-fixed P. acnes, and peptidoglycan (PGN) from Gram-positive bacteria walls on the production of promatrix metalloproteinase 2 (proMMP-2)/progelatinase A in hamster sebocytes and dermal fibroblasts. When hamster sebocytes (1.8×10(5) cells) and dermal fibroblasts (1×10(5) cells) were treated with P. acnes culture media and formalin-fixed P. acnes (corresponding to 1×10(6) and 1×10(7) bacterial cells), the production of proMMP-2 was augmented. In addition, PGN (5-50 µg/ml) dose-dependently augmented the production of proMMP-2 in both cells. Furthermore, the PGN (50 µg/ml)-augmented proMMP-2 production was resulted from an increase of its transcript. In contrast, there were no changes in cell proliferative activity in either the P. acnes or PGN-treated sebocytes and dermal fibroblasts, indicating that the augmented proMMP-2 production was not due to an increase in cell numbers. Therefore, these results provide novel evidence that PGN transcriptionally up-regulates the production of proMMP-2 in hamster sebocytes and dermal fibroblasts. Given an increase in the quantity of Gram-positive bacteria, including P. acnes in acne lesions, the aberrant ECM degradation may progress in sebaceous glands and pilosebaceous units, which is associated with acne scar formation.

Identification, typing and characterisation of Propionibacterium strains from healthy mucosa of the human stomach.

Forty two Propionibacterium isolates were recovered from biopsy samples of the gastric mucosa of eight out of 12 healthy people. Of these, 41 were identified as belonging to Propionibacterium acnes; the remaining isolate was identified as belonging to Propionibacterium granulosum. Repetitive extragenic palindromic (REP)-PCR typing suggested that up to four strains might be present in the mucosa of the same individual. Sequence analysis of either recA, tly or camp5 genes of P. acnes isolates revealed two distinct phylogenetic lineages. As per the recA, most isolates belonged to type I, while the remainder of the isolates belonged to type II. Phenotypic analyses of representative isolates showed the different strains to have diverse biochemical properties. For example, large differences were seen in carbohydrate fermentation patterns, the results of qualitative and quantitative enzymatic profiling, and survival at acidic pH. In contrast, the patterns of resistance/susceptibility to a series of 16 antibiotics were rather similar, with no atypical resistances observed. The examined strains showed limited-if any-enzymatic activities that could be ultimately related to pathogenicity (lipolytic, proteolytic or haemolytic activity). This suggests that, in the gastric ecosystem, some Propionibacterium spp. genotypes and/or phenotypes can be considered true commensals.

MIS416, a non-toxic microparticle adjuvant derived from Propionibacterium acnes comprising immunostimulatory muramyl dipeptide and bacterial DNA promotes cross-priming and Th1 immunity.

Propionibacterium acnes was modified using biochemical extraction methods generating a suspension of microparticles (MIS416) comprising a minimal cell wall skeleton rich in immunostimulatory crosslinked muramyl dipeptide repeats and native bacterial DNA fragments, each which have known adjuvant activity. In vitro studies demonstrated that MIS416 was readily internalized by human myeloid and plasmacytoid DC inducing cytokine secretion and cell activation/maturation. Vaccination studies in mice using OVA as a model antigen demonstrated that MIS416 acts as a Th1 adjuvant, promoting cross-priming of cytotoxic CD8(+) T cell responses and enhanced anti-tumour immunity. Covalent attachment of OVA to MIS416 enabling simultaneous delivery of antigen and adjuvant to the antigen presentation system resulted in a dose-sparing vaccine formulation. Preclinical GLP toxicology studies demonstrated that MIS416 has a favorable safety profile in mouse and rabbit supporting its use in human vaccine formulations.

Proteomic identification of secreted proteins of Propionibacterium acnes.

BACKGROUND: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. RESULTS: Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, ß-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. CONCLUSIONS: Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates. Thus, our data presents a rich resource for guiding much-needed investigations on P. acnes virulence factors and host interacting properties.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the soluble domain of PPA0092, a putative nitrite reductase from Propionibacterium acnes.

The soluble domain (residues 483-913) of PPA0092, a putative copper-containing nitrite reductase from Propionibacterium acnes KPA171202, has been overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.4 A. The crystal belonged to space group P2(1)3, with unit-cell parameters a = b = c = 108.63 A. Preliminary diffraction data show that one molecule is present in the asymmetric unit; this corresponds to a V(M) of 2.1 A(3) Da(-1).

Adjuvant effect of the Propionibacterium acnes and its purified soluble polysaccharide on the immunization with plasmidial DNA containing a Trypanosoma cruzi gene.

In the present work we investigated the role of killed Propionibacterium acnes or a soluble polysaccharide extracted from bacterium cell wall in modulated experimental immunization with plasmidial DNA. We used a plasmid, p154/13, containing a gene-encoding catalytic domain of Trypanosoma cruzi (T. cruzi) trans-sialidase. As previously described, immunization of BALB/c mice with p154/13 elicited humoral, cell-mediated and protective immune responses against T. cruzi infection. In this study we describe that both P. acnes and its soluble polysaccharide fraction have the ability to modulate the immune response elicited by p154/13. Treatment with these adjuvants enhanced specific trans-sialidase Th1 immune response, as revealed by a lower IgG1/IgG2a ratio and stronger in vitro IFN-gamma synthesis by CD4+ T cells. The most important fact was that treatment with P. acnes or its soluble polysaccharide fraction in the presence of p154/13 significantly reduced the peak of parasitemia observed 7 to 8 days after T. cruzi challenge. These data suggest that P. acnes or its soluble polysaccharide fraction may improve the protective potential of a DNA vaccine against experimental T. cruzi infection.

Purification and characterisation of lipoglycan macroamphiphiles from Propionibacterium acnes.

Lipidated macroamphiphiles such as the lipoteichoic acids and mycobacterial lipoarabinomannans are cell envelope components of Gram-positive bacteria that have been extensively associated with the pathogenesis of disease. In order to study such associations, purification of these macroamphiphiles is essential for resolving their structures and diverse biological effects. We describe herein a method for purification of lipoglycan components from Propionibacterium acnes. This method uses the existing phenol-water extraction, followed by hydrophobic interaction chromatography and an additional purification step that utilises preparative electrophoresis for the separation of two lipoglycan components. Analysis of these lipoglycans revealed evidence for a lipid anchor based on fatty acids whilst the polysaccharide moiety contained significant amounts of mannose, glucose and galactose, together with an amino sugar suspected of being a diaminohexuronic acid. These latter components have been previously identified as components of the P. acnes cell wall polysaccharide. Consequently, it is proposed that there may be a relationship between the structures of these distinctive cell envelope polymers.

Identification of anaerobic nonsporeforming gram-positive bacilli by biochemical tests and gas-liquid chromatography.

There are many methods to identify anaerobic nonsporeforming bacilli: histological, bacteriological (biochemical test, microsystem API 20 A), serological, cell wall composition analysis, molecular methods and gas-liquid chromatography (GLC). A comparison between biochemical tests and gas-liquid chromatography was made in this study for the identification of this group of microorganisms. GLC conditions were established with the aid of reference strains. These conditions were then applied to ten strains which were previously identified by biochemical tests. Strains were grown in PYG broth and fermentation end products were analyzed, volatile and non volatile fatty acids. Their qualitative determination was made by comparing the retention time of known standards and the chromatographic pattern of reference strains. In addition, a semiquantitative analysis was made. The results of identification by biochemical tests were: five strains belonged to Actinomyces genus; three were Propionibacterium acnes; one Propionibacterium granulosum and one P. propionicum. By the GLC only seven strains were identified: four were Actinomyces and three P. acnes. Only six strains showed identification correlation by both biochemical tests and GLC. GLC is a presumptive identification method that can be used along with other complementary tests for a definitive identification at genus level.