Characteristics of the Propionibacterium strains isolated from acne patients

Propionibacterium acnes is a component of physiological flora of human skin. It colonizes the outlets of sebaceous glands and participates in the pathogenesis of inflammatory acne. Acne vulgaris is a common skin disease. It is found in more or less exacerbated form in approximately 85% of adolescent population. The main purpose of the research was to confirm the hypothesis of Propionibacterium bacteria participation in the aetiopathogenesis of acne vulgaris. The researches have proved the presence of Propionibacterium acnes on the surface of the skin both of people with acne-related changes and these with whom such changes were not found. Statistically significant differences were found in the number of P. acnes bacteria per 1 square centimeter of healthy and disease-affected skin as well as in the diversity of biochemical types. The highest number of P. acnes bacteria have been found in fresh changes with visible symptoms of inflammation. In order to confirm the hypothesis of the participation of Propionibacterium bacteria in the aetiopathogenesis of acne, a detailed phenotypical analysis of isolated P. acnes strains have been conducted. Type, biotype, resistance pattern, proteolytic and lipolytic properties have been determined.     

Maintenance of the normal flora of human skin grafts transplanted to mice

Full-thickness human cadaver skin was maintained on the dorso-lateral thoracic region of hairless mice whose immune rejection mechanism was suppressed using anti-mouse-thymocyte globulin. The bacterial profile of the pregrafted skin did not differ significantly from the normal human microflora. In contrast, the murine skin exhibited quantitative and qualitative differences from the human flora, in particular by the complete absence of Propionibacterium acnes, the dominant bacterium on sebum-rich areas of human skin. The normal microbial profile of the human grafts was maintained throughout the experimental period despite the novel environmental milieu. There was little contamination of the grafts from the normal murine flora. It was concluded that the grafted human skin would provide a realistic model for studying the ecology of human cutaneous micro-organisms.     

Propionibacterium acnes in the etiology of endocarditis]

Propionibacterium acnes is the gram positive anaerobic bacteria belongs to the normal skin and oral microbial flora. The participation of this microorganism in the infective endocarditis is still controversial. The aim of the study was to perform the diagnostic and therapeutic difficulties in 5 patients with infective endocarditis caused by Propionibacterium acnes. In 3 out of 5 patients the infective endocarditis developed after prosthesis valve replacement, in 2 others on the native valves. The inserted prostheses were mechanical ones, propionibacterium acnes was identified as causative organisms in all of the causes (two positive blood and/or valve culture). The bacterial strains were sensitive to the antibiotics as: penicillins, cephalosporins, clindamycin, and vancomycin, however cephalosporins used at the beginning of the treatment in 3 patients and clindamycin in 1 patient had limited clinical efficacy. Later treatment with timentin, augmentin and tienamycin was successful in 3 patients; one patient was cured with vancomycin. One patient died because of septic, embolic complication in early stage of illness. We conclude the effectiveness of penicillins in combination with clavulanic acid and tienamycin in therapy of infective endocarditis due to Propionibacterium acnes. The treatment should be lasted during 4-6 weeks.     

Eradication of Propionibacterium acnes by its endogenic porphyrins after illumination with high intensity blue light

Propionibacterium acnes is a Gram-positive, microaerophilic bacterium that causes skin wounds. It is known to naturally produce high amounts of intracellular porphyrins. The results of the present study confirm that the investigated strain of P. acnes is capable of producing endogenic porphyrins with no need for any trigger molecules. Extracts from growing cultures have demonstrated emission peaks around 612 nm when excited at 405 nm, which are characteristic for porphyrins. Endogenic porphyrins were determined and quantified after their extraction from the bacterial cells by fluorescence intensity and by elution retention time on high-performance liquid chromatography (HPLC). The porphyrins produced by P. acnes are mostly coproporphyrin, as shown by the HPLC elution patterns. Addition of delta-aminolevulinic acid (ALA) enhanced intracellular porphyrin synthesis and higher amounts of coproporphyrin have been found. Eradication of P. acnes by its endogenic porphyrins was examined after illumination with intense blue light at 407-420 nm. The viability of 24 h cultures grown anaerobically in liquid medium was reduced by less than two orders of magnitude when illuminated once with a light dose of 75 J cm(-2). Better photodynamic effects were obtained when cultures were illuminated twice or three times consecutively with a light dose of 75 J cm(-2) and an interval of 24 h between illuminations. The viability of the culture under these conditions decreased by four orders of magnitude after two illuminations and by five orders of magnitude after three illuminations. When ALA-triggered cultures were illuminated with intense blue light at a light dose of 75 J cm(-2) the viability of the treated cultures decreased by seven orders of magnitude. This decrease in viability can occur even after a single exposure of illumination for the indicated light intensity. X-ray microanalysis and transmission electron microscopy revealed structural damages to membranes in the illuminated P. acnes. Illumination of the endogenous coproporphyrin with blue light (407-420 nm) apparently plays a major role in P. acnes photoinactivation. A treatment protocol with a series of several illuminations or illumination after application of ALA may be suitable for curing acne. Treatment by both pathways may overcome the resistance of P. acnes to antibiotic treatment.     

正常菌群对常见致病菌拮抗作用的研究

生物拮抗作用是微生物种群关系研究的一个侧面,实验中我们使用从皮肤上分离的常住菌——痤疮丙酸杆菌(A14-1)和表皮葡萄球菌(F65)对常见致病菌如金黄色葡萄球菌(C189)、绿脓杆菌(C8514)和埃希氏大肠杆菌(C1356)的体外拮抗试验表明,它们具有明显的拮抗作用,拮抗作用一般从48小时开始,72小时明显。而皮肤常住菌间无拮抗作用,相反它们还有协同作用,这对于皮肤的自净以及维持皮肤的微生态平衡具有重要的作用。此外,实验中还进行了乳杆菌和双歧杆菌等正常菌群分离株与上述常见致病菌的体外拮抗试验,结果表明也     

Severe infections caused by Propionibacterium acnes: an underestimated pathogen in late postoperative infections.

Propionibacterium acnes belongs to the cutaneous flora of humans and is rarely considered a pathogen in human diseases. It is a frequent contaminant in blood cultures; however, in some patients it has been identified as the causative agent of life-threatening infections. Within the last years we have observed an abrupt increase in severe P. acnes infections which prompted us to study in detail the clinical and microbiological features, risk factors, and outcomes of these cases. In a retrospective review of microbiological records of 905 Propionibacterium isolates from a five-year period (1990-95), 70 were identified from 20 patients with clinical and microbiological evidence of a P. acnes infection. The clinical syndromes included endocarditis (7 patients), post-craniotomy infections (6 patients), arthritis and spondylodiscitis (4 patients), endophthalmitis (2 patients) and pansinusitis (1 patient). The predominant predisposing conditions were previous surgery preceding the infection from 2 weeks to 4 years and implantation of foreign bodies such as prosthetic heart valves, intraocular lenses and ventriculo-peritoneal shunts. Therapy consisted of intravenous antibiotics in all cases and surgical procedures to remove infected tissue in eighteen patients. The outcome was favorable in sixteen patients (80 percent) who had a complete recovery. These data confirm the pathogenic potential of P. acnes in late post-surgical infections, in particular after implantation of a foreign body, and suggest a combined therapeutic approach with intravenous antibiotics and surgical removal of the infected tissue.     

Isolation of Propionibacterium acnes from central nervous system infections

Propionibacterium acnes is a common skin colonizer and its involvement in central nervous system (CNS) infections may be related with previous neurosurgical procedures. P. acnes was isolated in pure or mixed cultures from ten patients with CNS infections during a 5-year period. The clinical presentation, treatment and outcome were retrospectively reviewed. Nine out of 11 patients had CNS infections after a neurosurgical procedure. The clinical presentation was: brain abscess (five patients), subdural or epidural empyema (four patients) and shunt meningitis (one patient). Three patients had also secondary meningitis. All patients received antibiotic therapy and all abscesses and empyemas were drained. The patient with shunt meningitis cured without catheter removal. Only one patient with a brain abscess by P. acnes died, but several months thereafter and as a consequence of a Gram-negative superinfection. P. acnes is a pathogen for the CNS and infections must be surgically managed under adequate antibiotic treatment.     

Selective antibacterial action of 2-mercaptoethanol on propionibacteria in skin cultures.

2-Mercaptoethanol applied to the surface of agar medium had a selective antibacterial effect on Propionibacterium acnes and Propionibacterium granulosum without interfering with the growth of Peptococcus saccharolyticus or staphylococci in anaerobic cultures of skin or in pure cultures. In aerobic broth culture, 2-mercaptoethanol inhibited aerobes and stimulated anaerobes, consistent with its action as a reducing agent.     

Transitional Forms of Corynebacterium acnes in Disease

A clear-cut triad of sequential Corynebacterium acnes transitional forms from disease has been discovered. This entity includes three major forms which are capable of stabilization in culture, the spherical, the intermediate, and the definitive C. acnes. During conversion or reversion among the three forms, a variety of forms with mixed characteristics was observed. The spherical form was gram-negative and osmotically fragile, but it possessed a vestigial cell wall and mesosomes which excluded it from the L forms. In lieu of the L-form designation, the term "transitional" was adopted for all forms leading up to the definitive C. acnes. Culture of the spherical form was successful only on Mycoplasma-type media. The intermediate form was gram-negative, had mixed spherical and filamentous morphology, and bore a striking resemblance to Streptobacillus moniliformis. Like the spherical form, it was nutritionally exacting. The definitive form of C. acnes was preceded by gram-positive transitional forms of C. acnes morphology. It lacked, however, the carbohydrases and proteinases of C. acnes and susceptibility to C. acnes bacteriophages. Reversion was often blocked at this stage. A series of blood cultures from a patient with endocarditis was studied. Postmortem stain sections of the heart-valve lesion included intracellular masses of gram-negative spherical organisms. Indirect fluorescent antibody staining of these masses was strongly positive with antiserum to the spherical form and weakly positive with antiserum to the intermediate form.     

Evaluation of microbial flora of the eye during wear of soft contact lenses.

The microflora of the eye has been monitored in 21 patients during a 6-month period to study changes resulting from wear of soft contact lenses. A minimum of 20 cul-de-sac cultures were taken from each patient. Fifty-one percent of cultures taken prior to lens wear were positive for microbial growth, whereas, after lens wear, positive cultures ranged from 14.3% to 30.9" over the 6-month period. Staphylococcus epidermidis was the most frequently isolated organism, followed by Micrococcus spp., diphtheroids, and Bacillus spp. Nonfermentative, gram-negative rods and fungi were isolated spordically. Bacterial growth was sparse from all specimens, but individual differences were found. The microflora of the eye appears to resemble that of the skin, suggesting that the eye has no real flora of its own, but has a transient flora supplied from the skin, possibly the eyelid.     

Evaluation of microbial flora of the eye during wear of soft contact lenses.

The microflora of the eye has been monitored in 21 patients during a 6-month period to study changes resulting from wear of soft contact lenses. A minimum of 20 cul-de-sac cultures were taken from each patient. Fifty-one percent of cultures taken prior to lens wear were positive for microbial growth, whereas, after lens wear, positive cultures ranged from 14.3% to 30.9" over the 6-month period. Staphylococcus epidermidis was the most frequently isolated organism, followed by Micrococcus spp., diphtheroids, and Bacillus spp. Nonfermentative, gram-negative rods and fungi were isolated spordically. Bacterial growth was sparse from all specimens, but individual differences were found. The microflora of the eye appears to resemble that of the skin, suggesting that the eye has no real flora of its own, but has a transient flora supplied from the skin, possibly the eyelid.     

Antibody response to crude cell lysate of propionibacterium acnes and induction of pro-inflammatory cytokines in patients with acne and normal healthy subjects

Propionibacterium acnes (P. acnes) plays an important role in the disease pathogenesis of acne vulgaris, a disorder of pilosebaceous follicles, seen primarily in the adolescent age group. In the present study, the presence of antibodies against P. acnes (MTCC1951) were detected in acne patient (n=50) and disease free controls (n=25) using dot-ELISA and Western blot assay. The ability of P. acnes to induce pro-inflammatory cytokines by human peripheral blood mononuclear cells (PBMCs), obtained from acne patients and healthy subjects, were also analysed. The patients (n=26) who were culture positive for skin swab culture, were found to have a more advanced disease and higher antibody titres (1:4000 to > 1:16000) compared to the P. acnes negative patients (n=24) and normal controls (n=25). An analysis of patients' sera by western blot assay recognized a number of antigenic components of P. acnes, ranging from 29 to 205 kDa. The major reactive component was an approximately 96 kDa polypeptide, which was recognised in 92% (24 of 26) of the patients sera. Further, the P. acnes culture supernatant, crude cell lysate and heat killed P. acnes whole cells, obtained from 72-h incubation culture, were observed to be able to induce significant amounts of IL-8 and tumor necrosis factor alpha (TNF-alpha) by the PBMCs in both the healthy subjects and patients, as analysed by cytokine-ELISA. The levels of cytokines were significantly higher in the patients than the healthy subjects. A major 96 kDa polypeptide reactant was eluted from the gel and was found to cause dose dependent stimulation of the productions of IL-8 and TNF-alpha. Thus, the above results suggest that both humoral and pro-inflammatory responses play major roles in the pathogenesis of acne.     

Identification of Propionibacterium acnes by polymerase chain reaction for amplification of 16S ribosomal RNA and lipase genes

Propionibacterium acnes belongs to the cutaneous flora and is present in sebaceous follicles. The fatty acids that are released from sebum triglycerides by the action of this bacterial lipase play an important role in the pathogenesis of acne vulgaris. P. acnes is also involved in postoperative disorders and opportunistic infections in immunosuppressed hosts. Recently, it has been proposed that P. acnes causes sarcoidosis. Therefore, rapid isolation and identification of P. acnes is important. This study evaluated the polymerase chain reaction (PCR) for the detection of the 16S rRNA and lipase genes of P. acnes. The PCR used to detect the 16S rRNA gene could amplify the gene of P. acnes, but not the genes of the other tested strains of P. avidum, P. granulosum, P. lymphophilum, P. jensenii, P. acidipropionici and P. thoenii. The PCR to detect the lipase gene of P. acnes, however, could amplify not only the gene of P. acnes but also that of P. avidum. The PCR product of this lipase gene was not found in the strains of the other species tested. Therefore, the organism that has both the 16S rRNA gene and lipase gene was identified as P. acnes, while the strain with the lipase gene but not the 16S rRNA gene of P. acnes was characterized as P. avidum. These findings were confirmed by the conventional biochemical tests including lipase activity. Furthermore, out of the seven clinical isolates from acne vulgaris, four were identified as P. acnes and three as P. avidum by the PCR method and biochemical tests. The combination of two PCR, one for the detection of the 16S rRNA and the other of lipase genes was shown to be an easier, faster and more accurate method to identify P. acnes and P. avidum than conventional methods.     

A rapid screening method for streptococcal plasmids yielding plasmid DNA suitable for restriction enzyme analysis

Abstract Anaerobically and aerobically grown cultures of Propionibacterium acnes have been used to demonstrate the presence of respiration, respiration-driven proton translocation and stimulation of membrane potential formation by O₂ in such cultures. The results obtained indicate that P. acnes has the capacity to carry out oxidative phosphorylation.     

部分正常菌群分离菌株的超氧化歧化酶活性的测定

为了进一步研究部分正常菌群的生理和病理机制,我们测定了这些菌株的SOD活性和脂质过氧化物(LPO)变化,发现皮肤常住菌的分离株的混合培养时,SOD活性增加,说明它们具有协同作用。此外,对其它菌株SOD活性测定结果可知,类杆菌和双歧杆菌的SOD活性最低,其它菌株也有一定的SOD活性,如乳杆菌的SOD活性为47.47～98.98μg/ml,大肠杆菌的SOD活性为127.12μg/ml肠球菌的SOD活性为172.5μg/ml。     

Prophylactic antibiotics in neonates with umbilical artery catheter placement: a prospective study of 137 patients

To analyze the risk of cannula sepsis from indwelling umbilical arterial catheters and the indication for prophylactic antibiotics, 137 catheterized neonates with respiratory distress were prospectively placed into either antibiotic-treated (penicillin 50,000U/kg/day and kanamycin 15 mg./kg./day) or non-treated groups. Although bacteria were frequently isolated from blood and catheter tip cultures obtained upon removal of the catheter, especially among non-antibiotic treated infants, these isolates were predominantly non-pathogens and probably skin flora. Corresponding peripheral blood cultures were usually sterile. No cases of cannula-associated sepsis occurred among treated and non-treated newborns. The risk of bacteriologically proven sepsis resulting from an indwelling umbilical artery catheter appears insufficient to justify prophylactic antibiotics.     

Multilocus sequence typing and repetitive-sequence-based PCR (DiversiLab) for molecular epidemiological characterization of Propionibacterium acnes isolates of heterogeneous origin

Propionibacterium acnes is a gram-positive bacillus predominantly found on the skin. Although it is considered an opportunistic pathogen it is also been associated with severe infections. Some specific P. acnes subtypes are hypothesized to be more prone to cause infection than others. Thus, the aim of the present study was to investigate the ability to discriminate between P. acnes isolates of a refined multilocus sequence typing (MLST) method and a genotyping method, DiversiLab, based on repetitive-sequence-PCR technology. The MLST and DiversiLab analysis were performed on 29 P. acnes isolates of diverse origins; orthopedic implant infections, deep infections following cardiothoracic surgery, skin, and isolates from perioperative tissue samples from prostate cancer. Subtyping was based on recA, tly, and Tc12S sequences. The MLST analysis identified 23 sequence types and displayed a superior ability to discriminate P. acnes isolates compared to DiversiLab and the subtyping. The highest discriminatory index was found when using seven genes. DiversiLab was better able to differentiate the isolates compared to the MLST clonal complexes of sequence types. Our results suggest that DiversiLab can be useful as a rapid typing tool for initial discrimination of P. acnes isolates. When better discrimination is required, such as for investigations of the heterogeneity of P. acnes isolates and its involvement in different pathogenic processes, the present MLST protocol is valuable.     

Activation of toll-like receptor 2 in acne triggers inflammatory cytokine responses

One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes; yet, the molecular mechanism by which P. acnes induces inflammation is not known. Recent studies have demonstrated that microbial agents trigger cytokine responses via Toll-like receptors (TLRs). We investigated whether TLR2 mediates P. acnes-induced cytokine production in acne. Transfection of TLR2 into a nonresponsive cell line was sufficient for NF-kappa B activation in response to P. acnes. In addition, peritoneal macrophages from wild-type, TLR6 knockout, and TLR1 knockout mice, but not TLR2 knockout mice, produced IL-6 in response to P. acnes. P. acnes also induced activation of IL-12 p40 promoter activity via TLR2. Furthermore, P. acnes induced IL-12 and IL-8 protein production by primary human monocytes and this cytokine production was inhibited by anti-TLR2 blocking Ab. Finally, in acne lesions, TLR2 was expressed on the cell surface of macrophages surrounding pilosebaceous follicles. These data suggest that P. acnes triggers inflammatory cytokine responses in acne by activation of TLR2. As such, TLR2 may provide a novel target for treatment of this common skin disease.     

Requirement for TLR9 in the immunomodulatory activity of Propionibacterium acnes

Propionibacterium acnes (formerly Corynebacterium parvum) is part of the human flora and, as such, is associated with several human pathologies. It possesses strong immunomodulatory activities, which makes this bacterium interesting for prophylactic and therapeutic vaccination. The bacterial component(s) and the host receptor(s) involved in the induction of these activities are poorly understood. We show in this study that TLR9 is crucial in generating the characteristic effects of killed P. acnes priming in the spleen, such as extramedullary hemopoiesis and organ enlargement, and granuloma formation in the liver. Furthermore, the ability to overproduce TNF-alpha and IFN-gamma in response to LPS, lipid A, synthetic lipopeptide Pam(3)CysK(4), or whole killed bacteria was present in P. acnes-primed wild-type, but not TLR9(-/-), mice. Finally, P. acnes priming failed to induce enhanced resistance to murine typhoid fever in TLR9(-/-) mice. Thus, TLR9 plays an essential role in the induction of immunomodulatory effects by P. acnes. Because IFN-gamma is a key mediator of these effects, and enhanced IFN-gamma mRNA expression was absent in spleen and liver of P. acnes-primed TLR9(-/-) mice, we conclude that TLR9 is required for the induction of IFN-gamma by P. acnes.     

Aerobic growth and toxigenicity ofClostridium botulinum types A and B

Clostridium botulinum types A and B cultured in association with avian skin flora, had similar growth patterns under both aerobic and anaerobic conditions. The selective “C. botulinum isolation” (CBI) medium was found to be especially useful for the recovery and quantitation of small numbers of type A or type B organisms from the mixed cultures. Enzyme immunoassay in conjunction with conventional mouse biossay provided a practical means for the quantitation of toxigenicity ofC. botulinum in avian skin cultures. The amount of toxin produced by type A was always higher than that produced by type B strains. The aerobically incubated type A or type B cultures appeared to be less toxigenic than cultures incubated anaerobically.