Genetic basis of resistance in Propionibacterium acnes strains isolated from diverse types of infection in different European countries

The purpose of the study was to characterize the resistance mechanism of 36 clindamycin (CL) and erythromycin (EM) resistant Propionibacterium acnes strains and 27 tetracycline (TET) resistant P. acnes isolates, collected from nine European countries, both from acne patients and from patients with different infections. PCR and sequencing of the genes encoding domain V of 23S rRNA for CL and EM resistant strains and 16S rRNA for TET resistant strains were performed. Pulsed-field gel electrophoresis was used as a typing method to establish the relationship between resistance genotypes and pulsed-field types. Several unique resistant genotypes were found to be distributed throughout Europe. P. acnes CL and EM resistant strains carrying one of the mutations within the 23S rRNA were predominantly isolated from Swedish acne patients (64%) compared to other infections (43%), OR=2.33 [CI=1.16-4.69]. Of 36 P. acnes isolates tested, none was found to carry the erm(X) resistance gene. Forty-four percent of TET resistant strains were found to carry a G-C transition in the 16S rRNA of the small ribosomal subunit and all these strains were isolated from Swedish acne patients. MIC of TET among all strains carrying this G-C mutation (n=12) was 32 mg/L and the MIC range for the strains where no mutation was detected ranged from 2 to 8 mg/L. The MIC values of TET were unaffected by the presence of reserpine, a well-known inhibitor of efflux pumps. Those TET resistant strains harbouring the mutation at 16S rRNA were clustered in one pulsotype. For TET resistant strains where no mutation was found, greater variability was noticed. No correlation was noticed between different resistance genotypes of CL and EM resistant strains and pulsed-field types.     

Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction

Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.     

Effects of dilution rate on biomass and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture

Propionibacterium acnes, P. avidum and P. granulosum were grown in continuous culture at a range of dilution rates on a semi-synthetic medium. Dilution rates were chosen to allow the bacteria to grow at the same relative growth rates as compared to their respective mumax values. The steady-state levels and production rates of biomass and extracellular enzymes were determined. The lipase and hyaluronate lyase of P. granulosum and the proteolytic activity of P. acnes and P. avidum were growth linked enzymes (i.e. they were produced at constant amounts per unit of biomass). In contrast, the lipase, hyaluronate lyase and acid phosphatase of P. acnes and the lipase of P. avidum were shown to be non-growth linked enzymes.     

Effects of pH on biomass, maximum specific growth rate and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture

Three cutaneous propionibacteria, Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum, were grown in chemostats using semi-synthetic medium at various pH values. Growth occurred between pH 4.5 and 7.5 for P. acnes and pH 5.0 and 8.0 for P. avidum and P. granulosum. The highest mumax was at pH 6.0 for the three species. Maximum biomass production was obtained at pH 6.0 for P. acnes and P. avidum and at pH 7.5 for P. granulosum. Extracellular enzyme production occurred over the entire pH growth range when denaturation of the enzymes was taken into account. However, detectable activity of the enzymes was found in a narrower range of pH due to the denaturation of the enzymes at low or high pH values. The highest production of enzymes occurred at pH values between 5.0 and 6.0, apart from the production of hyaluronate lyase of P. granulosum (pH 6.0 to 7.0) and the proteinase of P. acnes and P. avidum (pH 5.0 to 7.5). Propionibacterium acnes produced a lipase, hyaluronate lyase, phosphatase and proteinase activity. Propionibacterium avidum produced a lipase and proteinase activity. Propionibacterium granulosum produced a lipase and hyaluronate lyase.     

Effects of tetracyclines on the production of extracellular proteins by members of the propionibacteriaceae

The effect of tetracyclines on the synthesis of proteins in Propionibacterium avidum and P. acnes was examined. Synthesis of an extracellular lipase by P. avidum was slightly more sensitive to inhibition by tetracycline than total (cellular and extracellular) protein synthesis. The effect of tetracycline and other analogues on the synthesis of secreted proteins was also examined in P. avidum and P. acnes by protein radiolabelling experiments. In all cases the synthesis of secreted proteins was only about 2-fold more sensitive to inhibition by tetracyclines than total protein synthesis. These results contrast with previously published findings in Escherichia coli which show that synthesis of secreted proteins is highly susceptible to inhibition by tetracycline. The implications of these findings in relation to inhibition of membrane bound ribosomes by tetracyclines are discussed.     

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.     

酯酶试验与聚合酶链反应鉴别中间普氏菌的比较研究

目的 研究脂酶试验和常规生化方法与聚合酶链反应比较在鉴别中间普氏菌Pi中的敏感度和特异度。方法 对207株牙周临床分离的产黑色素G厌氧杆菌分别进行脂酶试验、常规生化法鉴定和16srRNA特异引物PCR鉴定。结果 207株实验菌中PCR鉴定出Pi97株,脂酶试验检测出Pi126株,其中有85株PCR阳性,其敏感度为87．6％,特异度为63．7％。常规生化方法和脂酶试验共同鉴定出Pi80株,其中有55株PCR为阳性,其敏感度为67％,特异度为86．4％。结论 脂酶与常规生化方法对Pi的鉴别能力低于PCR（ P＜0．05）,尚不能作为Pi菌种的可靠鉴定方法。     

Electrophoretic protein patterns and enzyme mobilities in anaerobic coryneforms.

The soluble protein patterns and electrophoretic mobilities of malate and succinate dehydrogenases and catalase have been examined in 25 strains of Propionibacterium acnes, P. granulosum, and P. avidum. A distinctive protein pattern for each species was found, and it was possible also to distinguish the serotypes within P. acnes and P. avidum. Strains of P. acnes, P. granulosum, and P. avidum could be differentiated by the mobilities of their malate dehydrogenases. Catalase activity was detected in the soluble fractions of all strains. Catalases from P. acnes and P. avidum strains had the same mobility, whereas that from P. granulosum was slightly slower. Under the conditions used, succinate dehydrogenase activity could be detected, but the patterns were not distinctive.     

人工神经原网络处理PCR数据在细菌鉴定中的应用

目的16SrRNA和16S-23SrRNA间区片段是常用细菌分类鉴定靶点,本研究探讨人工神经原网络（ANN）对上述位点PCR扩增产物数据分析在细菌快速鉴定方面的价值。方法2对15SrRNA基因荧光引物和1对16S-23SrRNA区间基因引物用于扩增血液标本中分离出的317株细菌。相关毛细管电泳（CE）限制性片段长度多态性（RFLP）和单链构象多态性（SSCP）数据进行人工神经原网络分析。结果16S-23SrRNA基因的RFLP数据对未知菌鉴定的准确率高于16SrRNA基因的SSCP数据,分别为98．0％和79．6％。结论实验证明了人工神经原网络作为一种模式识别方法对于简化细菌鉴定十分有价值。     

Vaccination targeting a surface sialidase of P. acnes: implication for new treatment of acne vulgaris

Background

Acne vulgaris afflicts more than fifty million people in the United State and the severity of this disorder is associated with the immune response to Propionibacterium acnes (P. acnes). Systemic therapies for acne target P. acnes using antibiotics, or target the follicle with retinoids such as isotretinoin. The latter systemic treatment is highly effective but also carries a risk of side effects including immune imbalance, hyperlipidemia, and teratogenicity. Despite substantial research into potential new therapies for this common disease, vaccines against acne vulgaris are not yet available.

Methods and Findings

Here we create an acne vaccine targeting a cell wall-anchored sialidase of P. acnes. The importance of sialidase to disease pathogenesis is shown by treatment of a human sebocyte cell line with recombinant sialidase that increased susceptibility to P. acnes cytotoxicity and adhesion. Mice immunized with sialidase elicit a detectable antibody; the anti-sialidase serum effectively neutralized the cytotoxicity of P. acnes in vitro and P. acnes-induced interleukin-8 (IL-8) production in human sebocytes. Furthermore, the sialidase-immunized mice provided protective immunity against P. acnes in vivo as this treatment blocked an increase in ear thickness and release of pro-inflammatory macrophage inflammatory protein (MIP-2) cytokine.

Conclusions

Results indicated that acne vaccines open novel therapeutic avenues for acne vulgaris and other P. acnes-associated diseases.     

Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes

Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division Verrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil.     

青少年痤疮面部皮肤微生物群落结构变化

【背景】青少年痤疮是一种最常见的慢性炎症性损容性皮肤病,与痤疮丙酸杆菌的异常增殖有关。【目的】探究痤疮皮损区与附近无明显皮损区微生物组成与健康对照的差异,为从微生态角度防治痤疮提供理论基础。【方法】利用细菌16S rRNA基因V1-V2区和真菌TIS1高通量测序技术分析北京地区16岁青少年面部痤疮皮肤细菌和真菌群落结构,将痤疮皮损区与附近无明显皮损区微生物组成与健康组进行比较,寻找差异菌群。【结果】痤疮患者面部皮损区与附近无明显皮损区细菌多样性(Shannon指数)较健康对照组显著性降低(P0.001),主要与丙酸杆菌(痤疮丙酸杆菌)和葡萄球菌(表皮葡萄球菌PM221)显著性上升相关,而痤疮皮损区与附近未明显皮损区细菌组成无显著性差异。痤疮患者皮损区与附近无明显皮损区较健康对照组真菌丰富度(Chao1指数)显著性上升(P0.05),与限制性马拉色菌的显著上升相关。【结论】面部皮肤微生物变化与青少年痤疮的发生相关。本研究为从微生物角度防治痤疮提供理论依据。     

Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems

PCR amplification of the rRNA gene is the most popular method for assessing microbial diversity. However, this molecular marker is often present in multiple copies in cells presenting, in addition, an intragenomic heterogeneity. In this context, housekeeping genes may be used as taxonomic markers for ecological studies. However, the efficiency of these protein-coding genes compared to 16S rRNA genes has not been tested on environmental data. For this purpose, five protein marker genes for which primer sets are available, were selected (rplB, pyrG, fusA, leuS and rpoB) and compared with 16S rRNA gene results from PCR amplification or metagenomic data from aquatic ecosystems. Analysis of the major groups found in these ecosystems, such as Actinobacteria, Bacteroides, Proteobacteria and Cyanobacteria, showed good agreement between the protein markers and the results given by 16S rRNA genes from metagenomic reads. However, with the markers it was possible to detect minor groups among the microbial assemblages, providing more details compared to 16S rRNA results from PCR amplification. In addition, the use of a set of protein markers made it possible to deduce a mean copy number of rRNA operons. This average estimate is essentially lower than the one estimated in sequenced genomes.     

Species-specific PCR for identification of common contaminant mollicutes in cell culture.

Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5' end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5' end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.     

Improved detection of bifidobacteria with optimised 16S rRNA-gene based pyrosequencing

The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, 'bifidobacteria-optimised' universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.     

Identification and analysis of PCB dechlorinating anaerobic enrichments by amplification: accuracy of community structure based on restriction analysis and partial sequencing of 16S rRNA genes

The composition of polychlorinated biphenyl (PCB) dechlorinating mixed communities was analysed by restriction fragment length polymorphism of PCR amplified rDNAs (ARDRA) and partial sequencing of 16S rRNA genes amplified from PCB degrading enrichments. Restriction analysis confirms that the 16S rRNA genes amplified from PCB dechlorinating communities vary depending on the PCB congener dechlorinated. Comparison of 16S rRNA sequences to published ribosomal databases indicates that the two most abundant Operational Taxonomic Units (OTUs) appear to be species of the genus Clostridium . The amount that the amplification procedure contributed to this result was determined by varying the amplification procedure and by creating an artificial template mixture. Varying the amount of template by sixfold in the amplification did not affect the distribution of OTUs but the number of OTUs observed decreased with decreasing template concentration. Comparison of products amplified from mixtures of 16S rDNA clones indicates that the more abundant Clostridium OTU did not amplify more efficiently than those of less abundant OTUs. Hybridization to a probe designed to detect the most abundant OTUs indicates that two other OTUs are closely related to this Clostridium species.     

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.     

Development of a rapid identification method for Aeromonas species by multiplex-PCR

Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.     

PCR differentiation of seventeen genospecies of Acinetobacter

Abstract In the present study, strains of 17 reference Acinetobacter genospecies were investigated by using the polymerase chain reaction (PCR). We used primers to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the tested acinetobacters into 15 groups. The genospecies 5 ( Acinetobacter junii ), 7 ( Acinetobacter johnsonii ) and 10 produced the same characteristic PCR patterns, suggesting the identity of these three genospecies. A preliminary evaluation of the proposed scheme for PCR diagnostics was carried out. Using the proposed scheme, tested clinical strains were identified correctly to the genospecies level, and the identifications confirmed by conventional biochemical tests. On the basis of our results, PCR amplification of the 16S–23S spacer region shows significant promise as a tool for the simple identification of genospecies belonging to Acinetobacter sp. The nucleotide sequences of our primers are sufficiently highly conserved among these organisms as to permit PCR reactions to be carried out with a single set of reaction conditions and amplification parameters irrespective of species or genus.