Concordance between phenotypical features and PCR-REA for the identification of Malassezia spp

The genus Malassezia has been recently revised and nowadays includes 11 species that cannot always be differentiated from each other by physiological and morphological tests. This study was aimed to evaluate the correlation between a molecular method and conventional phenotypic features in the identification of Malassezia spp. To achieve this aim, 92 Argentinean clinical strains isolated between 2001 and 2005 were analyzed along with three reference strains (Malassezia furfur CBS 7019, Malassezia sympodialis CBS 7222 and Malassezia slooffiae CBS 7956). By using PCR and restriction enzyme analysis with three different DNA endonucleases (PCR-REA), the molecular method consistently identified all three reference strains and all 92 clinical isolates as follows: 63 M. sympodialis, 18 M. furfur, 10 Malassezia globosa and one Malassezia obtusa. Phenotypic studies undentified 85 clinical isolates and two of the reference strains (total agreement > 91%). In particular for M. sympodialis, M. furfur and M. globosa, the species more frequently involved in human pathology, the agreement ranged between 84 and 96%. This result suggests that phenotypic studies are suitable for the presumptive identification of important Malassezia species in the clinical medical mycology laboratories where molecular methodologies are not available.     

Differentiation of Paenibacillus larvae subsp. larvae,the cause of American foulbrood of honeybees,by using PCR and restriction fragment analysis of genes encoding 16S rRNA

A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources.     

Differentiation of non-pylori Helicobacter species based on PCR–restriction fragment length polymorphism of the 23S rRNA gene

Phenotypic identification of non-pylori Helicobacter species has always been problematic and time-consuming in comparison with many other bacteria. We developed a rapid two-step identification assay based on PCR–restriction fragment length polymorphism (PCR–RFLP) analysis of the 23S rRNA gene for differentiating between non-pylori Helicobacter species. A new genus-specific primer pair based on all available complete and partial 23S rRNA sequences of Helicobacter species was designed. In silico restriction analysis of variable regions of the 23S rRNA gene suggested SmaI and HindIII endonucleases would provide a good level of differentiation. Analysis of the obtained 23S rRNA RFLP patterns divided all Helicobacter study strains into three species groups (groups A–C) and 12 unique restriction patterns. Wolinella succinogenes also gave a unique pattern. Our proposed PCR–RFLP method was found to be as a valuable tool for routine identification of non-pylori Helicobacter species from human or animal samples.     

PCR-酶切技术快速鉴定军团菌

[目的]建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值.[方法]根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法.对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序.[结果]该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的Hinf Ⅰ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致.[结论]PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测.     

Amplified ribosomal DNA restriction analysis for the characterization of Azotobacteraceae: a contribution to the study of these free-living nitrogen-fixing bacteria

A 16S rRNA gene-based fingerprinting method was developed for the identification of Azotobacteraceae and tested onto 48 soil isolates and 28 reference strains belonging to the free-living nitrogen-fixing bacterial group and to the most common species found in soil samples. According to this method, the 16S rRNA gene was amplified using universal primers for Eubacteria and PCR products were subsequently digested with RsaI, HhaI, HpaII, FnuDII, and AluI. The analysis of the restriction profiles obtained showed that the method is able to define a unique species-specific phylotype (SSP) for each of the eight Azotobacteraceae species tested. Cluster analysis was successfully employed for the identification of members of the family Azotobacteraceae, being assignation into species of the isolates confirmed by means of partial 16S rRNA gene sequencing.     

Rapid identification of Enterococcus italicus by PCR with primers targeted to 16S rRNA gene

AIMS: To develop a species-specific PCR assay with primers targeted to 16S rRNA gene for the identification of Enterococcus italicus, a new species of Enterococcus, involved in the production of Italian cheeses. METHODS AND RESULTS: The type strain of E. italicus (DSM 15952(T) - 16S rRNA gene accession no. AJ582753) and other strains of the species were subjected to a rapid identification by PCR using primer pairs located within the 16S rRNA gene. A species-specific PCR product of approximately 323 bp was obtained after amplification of all E. italicus strains tested. The specificity of the primers was validated with representatives of the most closely related genera and species and a number of other bacterial species. In addition, the technique enabled the recognition of E. italicus from cheeses. CONCLUSIONS: The protocol was highly efficient and sensitive, enabling the identification of E. italicus from cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific PCR offers a reliable and rapid alternative to conventional phenotypic methods for the identification of E. italicus within the heterogeneous genus Enterococcus.     

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.     

Differentiation of Bartonella species using restriction endonuclease analysis of PCR-amplified 16S rRNA genes

Abstract Differentiation of the four Bartonella species which were formerly classified as Rochalimaea using restriction endonuclease analysis of PCR-amplified citrate synthase gene fragments has previously been described. However, attempts to extend this method to include all members of Bartonella were confounded when amplification of the gene fragment from strains of B. bacilliformis each yielded two products of differing sizes. An alternative differentiation scheme for Bartonella species was developed based on restriction endonuclease analysis of their 16S rRNA genes. As the complete 16S rRNA gene sequences of all extant Bartonella species are available, the usefulness of specific endonucleases could be theoretically predetermined rather than discovered empirically. The potential usefulness of the restriction enzymes Ddel and Mnll was established using this approach, and this potential was confirmed in practice as all eight species could be distinguished from each other.     

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.     

DNA base alignment and taxonomic study of genus Malassezia based upon partial sequences of mitochondrial large subunit ribosomal RNA gene

The sequences of the large subunit of mitochondrial ribosomal RNA (LsmtrRNA) gene of Malassezia species were analysed. The sequences of the seven species of Malassezia are well separated in each species. Therefore the LsmtrRNA gene is thought to be one of the gene targets for species identification in the genus Malassezia. The dendrogram obtained from this gene supports the previous study of Malassezia species based upon the chromosomal genes. This is the first report of taxonomic analysis of Malassezia species based upon the mitochondrial gene.     

Evaluation of ribosomal RNA gene restriction patterns for the classification of Bacillus species and related genera

AIMS: To identify Bacillus species and related genera by fingerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classification of Bacillus species and related genera. METHODS AND RESULTS: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from five newly created Bacillus-related genera, and five species from five phenotypically related genera were tested. A total of 77 distinct 16S rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene fingerprinting and the 23S and 5S rRNA genes fingerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. CONCLUSIONS: The Bacillus species along with the species from related genera were identified successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, but does not duplicate, phylogenies based on 16S rRNA gene sequences.     

Evaluation of a molecular identification scheme based on 23S rRNA gene polymorphisms for differentiating canine and feline gastric Helicobacter spp

A scheme for the rapid identification of Helicobacter spp. using restriction fragment length polymorphism digestion profiles of PCR amplified 23S rRNA genes is described. The efficacy of this scheme for speciation of the closely related gastric species H. felis, H. bizzozeronii and H. salomonis was evaluated. It was difficult to distinguish between some RFLP profiles obtained and often, more than one profile was seen with each species examined. Some evidence was found that the 23S rRNA gene copies of these species may not be identical. Moreover, the identification scheme was ineffective in discriminating these species from each other, although they could be differentiated, as a group, from other Helicobacter spp. The results indicate that this scheme should be carefully evaluated with a number of isolates if it is to be applied to additional, highly related Helicobacter spp.     

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri , Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii . Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.     

Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.     

Species identification of clinically important Aeromonas spp. by restriction fragment length polymorphism of 16S rDNA

AIMS: The aim of the study was to characterize 16S rDNA of Aeromonas spp. to rapidly identify clinically important species of these bacteria. METHODS AND RESULTS: Sequence analysis of published 16S rDNA for unique restriction sites revealed prospect of species identification. Extraction of genomic DNA followed by amplification and step-by-step restriction endonuclease digestion of 16S rDNA was able to identify Aeromonas spp. of medical significance. Validation of the method was performed by subjecting 53 Aeromonas strains of multiple origin to similar treatment. Results of the study were in agreement with corresponding species of the isolates. CONCLUSIONS: The method developed offers an easily interpretable tool for the identification of Aeromonas spp. of clinical relevance. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed methodology should facilitate routine laboratory diagnosis of Aeromonas spp. from clinical cases to species level.     

我国代表地区须癣毛癣菌复合体的分子鉴定与分型研究

目的对我国代表地区的须癣毛癣菌菌株进行分子再鉴定和分型研究。方法选取我国南北方8个省市地区经表型鉴定的须癣毛癣菌菌株47株,通过再培养形态观察、生理试验;PCR扩增核糖体DNA(rDNA)的内转录间隔区(ITS)和核糖体大亚基(LSU)D1-D2区,测序后利用数据库进行序列比对,对须癣毛癣菌复合体进行再鉴定;PCR扩增rDNA非转录间隔区(NTS)的三个串联重复亚单位S0、S1和S2区,进行种内分型,并比较不同部位来源菌株型别的差异性。结果我国南北方8个省市地区47株须癣毛癣菌中3株鉴定为断发毛癣菌,6株鉴定为无性型苯海姆节皮菌,其余均鉴定为万博节皮菌中的亲人型趾间毛癣菌;三对不同引物扩增38株趾间型毛癣菌和2株苯海姆节皮菌NTS区,共产生28种特征性带型。带型和菌株来源及发生部位无相关性。结论我国分离自人类须癣毛癣菌复合体的主要组成菌种为趾间毛癣菌;ITS区结合LSU D1-D2区测序有助于鉴定须癣毛癣菌复合体至种水平;NTS区的三个串联重复亚单位所产生的特征性指纹图提供了一种快速、稳定的分子生物学种内分型方法,可应用于趾间毛癣菌感染的流行病学研究。     

Chitin synthase 2 gene sequence of Malassezia species.

Nucleotide sequences of the chitin synthase 2 (CHS2) gene of seven species, Malassezia furfur, M. globosa, M. obtusa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS2 gene were amplified from these Malassezia species by polymerase chain reaction (PCR) and sequenced. The CHS2 nucleotide sequences of these Malassezia species showed more than 95% similarity between the species. A phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of seven Malassezia species revealed that the species were genetically distinct from each other.     

New lipid-dependent Malassezia species from parrots

BackgroundAll the currently recognized Malassezia species have been isolated from mammals. However, only a few of them have been isolated from birds. In fact, birds have been less frequently studied as carriers of Malassezia yeasts than mammals.AimIn this study we describe two new taxa, Malassezia brasiliensis sp. nov. and Malassezia psittaci sp. nov.MethodsThe isolates studied in this publication were isolated from pet parrots from Brazil. They were characterized using the current morphological and physiological identification scheme. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene, the ITS-5.8S rRNA gene sequences and the β-tubulin gene were also performed.ResultsThe strains proposed as new species did not completely fit the phenotypic profiles of any the described species. The validation of these new species was supported by analysis of the genes studied. The multilocus sequence analysis of the three loci provides robust support to delineate these species.ConclusionsThese studies confirm the separation of these two new species from the other species of the genus Malassezia, as well as the presence of lipid-dependent Malassezia yeasts on parrots.