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Objective:To study the molecular phylogeny and virulence gene profile of Francisella salimarina. Methods:Phylogenetic analysis of Francisella salimarina was performed based on the global genome data of related Francisella species on GenBank database. The consistency in phylogenetic analysis based on single marker genes (such as 16S rRNA gene, rpoB gene and mdh gene) and the core genome as compared. Virulence genes and antibiotic resistance genes were annotated using the virulence factor database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD), respectively. The virulence of Francisella salimarina was analyzed with a Galleria mellonella (greater wax moth) infection model using Francisella philomiragia ATCC 25015 T as reference strain. Results:The phylogenetic analysis revealed that Francisella salimarina was closely related to Francisella philomiragia. The phylogenetic tree based on mdh gene was highly similar to that based on the core genome. Francisella salimarina could be differentiated from other related species by 16S rRNA gene or mdh gene, with the latter being more accurate. Eight Francisella salimarina strains carried multiple virulence genes, mainly involved in secretion, adhesion, immune regulation, motility and stress survival. Moreover, beta-lactam resistance gene blaFPH was identified in all eight strains. Francisella salimarina showed high lethality in the Galleria mellonella infection model, which was similar to Francisella philomiragia ATCC 25015 T. Conclusions:Francisella salimarina was a rare pathogen with similar pathogenicity to Francisella philomiragia. The mdh gene could be used as a molecular target for rapid identification of Francisella salimarina.
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Objective:To identify and characterize two Balneatrix alpica strains isolated from a patient′s blood sample (strain X117) and the natural hot spring water in the patient′s residential district (strain GN-1), and to provide experimental evidence for the pathogenic diagnosis of clinical infection caused by this rare pathogen. Methods:Biochemical phenotypic identification, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), 16S rRNA gene sequencing, phylogenetic analysis, single-nucleotide polymorphism (SNP) analysis, and genome-wide analysis were performed to accurately determine the taxonomic status of the isolates X117 and GN-1 by using Balneatrix alpica DSM 16621 T as a reference. Microdilution broth method was used to test their antimicrobial susceptibility. The virulence genes carried by them were annotated and analyzed using the virulence factor database (VFDB). Results:Strains X117 and GN-1 formed light yellow or tan colonies with mottled surfaces on Columbia blood agar and chocolate agar plates after 4 d of culture. They were Gram-negative rods and positive for oxidase and indole tests, which were consistent with the characteristics of Balneatrix alpica DSM 16621 T. The phylogenetic analysis based on the 16S rRNA gene showed that the isolates X117 and GN-1 were both Balneatrix alpaca. The average nucleotide identity (ANI) values between the two isolates and Balneatrix alpica DSM 16621 T were 98.44% and 98.41%, respectively, and the digital DNA-DNA hybridization (dDDH) values were both 87.1%. The SNP distance between the two strains was 13, indicating that X117 and GN-1 might belong to the same clone. The antibiotic susceptibility testing showed that all of the three Balneatrix alpica strains were sensitive to the commonly used antibiotics against Gram-negative rods. The virulence genes carried by the three Balneatrix alpica strains were mainly involved in adhesion, invasion, flagella and biofilm formation. Conclusions:This study identified a case of bloodstream infection caused by Balneatrix alpica which was closely related to natural hot spring water. Natural hot spring water migh be an important source of clinical infections caused by this species.
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Objective:To analyze the molecular epidemiological characteristics of Campylobacter fetus subsp. testudinum ( Cft). Methods:Fifteen strains of Cft collected in our laboratory from 2010 to 2022 were subjected to whole-genome sequencing. Their epidemiological characteristics were analyzed based on the global genome data of Cft on GenBank database. MLST-GrapeTree software was used to obtain the genetic structure of Cft strains. A phylogenetic tree was constructed using core-genome single nucleotide polymorphism (cgSNP) analysis, and the sequence clusters were identified using rhierBAPS. Virulence genes and drug resistance genes of Cft strains were annotated using CARD, ResFinder and VFDB database. Their susceptibility to antibiotics was tested using E-test method and the results were analyzed using the CLSI-M45 sensitivity standard for Campylobacter jejuni/ Campylobacter coli. Results:Based on average nucleotide identity (ANI) analysis, the genome data of 41 Cft strains including 24 isolated from human, 13 from animals and four of unknown sources were collected from GenBank database. Among the 24 human-derived strains, 20 were linked to Asian descent and only one was linked to Caucasian descent (spouse of Asian descent), showing statistically significant differences in human ethnicity. All of the 13 animal-derived strains were originated from reptilian sources, including six from turtles, four from snakes and three from lizards. MLST revealed that ST46 was the predominant ST in China, while ST15 was the major sequence type in the United States. Grapetree analysis also demonstrated that the genetic diversity in China was greater than that in the United States. The phylogenetic tree constructed based on cgSNP and BAPS identified six distinct sequence clusters. The Chinese isolates were scattered in diverse sequence clusters and closely related to animal-derived strains, while the American isolates mainly belonged to ST15. The genes encoding virulence factors such as flagella, glycosylation systems and adhesins were carried by all of the 41 Cft strains (100.00%). The invasion-related virulence genes, such as the genes encoding the IV type secretion system ( virB4, virB9, virD4) and the resistance-related tetO efflux pump gene were specifically identified in the emerging ST74 clones. In vitro drug susceptibility testing of 15 Chinese isolates revealed 46.67% of the Cft strains were resistant to ciprofloxacin and 100.00% were sensitive to erythromycin. Conclusions:The global sequence clusters of Cft isolates showed a great genetic diversity. Most of the people with Cft infection had basic immune diseases and might have eaten or had contact with reptiles. Notably, the Chinese domestic infection of ST46 and the emerging ST74 should arouse our more attention.
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Objective:To identify and characterize one Spiroplasma strain (designated as DGKH1) isolated from the blood of a patient with sepsis. Methods:The traditional bacterial culture, staining, morphological observation, physiological and biochemical identification, 16S rRNA gene sequencing, phylogenetic analysis, genome sequencing, and the genome-related index analysis were performed to accurately determine the taxonomic status of the strain DGKH1. Antibiotic susceptibility testing was performed using a specific kit for culturing and testing Ureaplasma urealyticum/ Metamycoplasma hominis. Results:The strain DGKH1 could weakly grow on Columbia blood agar, chocolate agar, and Haemophilus chocolate 2 agar. However, it did not grow in liquid culture medium containing tetracycline (4 μg/ml), doxycycline (1 μg/ml), minocycline (1 μg/ml), josamycin (2 μg/ml), roxithromycin (1 μg/ml), clarithromycin (1 μg/ml), or telithromycin (1 μg/ml). DGKH1 resembling Metamycoplasma hominis formed "fried egg-like colonies" on Mycoplasma solid culture medium. DGKH1 could not be stained by Gram staining. When observed under transmission electron microscopy (TEM) using phosphate buffer as the matrix, the bacteria were spiral-shaped. Results of 16S rRNA gene sequence alignment showed that DGKH1 was highly similar (99.85%) to Spiroplasma eriocheiris CCTCC M 207170 T. However, the urea decomposition test was positive, which was different from all of the known Spiroplasma species. The phylogenetic analysis based on whole genome showed that DGKH1 was clustered in a small branch along with Spiroplasma eriocheiris CCTCC M 207170 T. However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 94.14% and 56.00%, respectively, both below the threshold for prokaryotic species identification. Conclusions:DGKH1 represented a potential new species of genus Spiroplasma, closely related to Spiroplasma eriocheiris. Some microbiological characteristics of DGKH1 were similar to Mycoplasmas. However, the natural host and epidemiological data of DGKH1 need to be further studied.
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Objective:To identify a pathogenic strain JM-1 isolated from the pus of a patient stabbed by a sea shrimp and to analyze its antibiotic susceptibility and virulence genes, aiming to provide reference for screening clinically related infections caused by Cysteiniphilum litorale as a rare pathogen and improving prognosis. Methods:Biochemical phenotype identification, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), 16S rRNA gene sequencing, analysis of average nucleotide identity (ANI) and average amino acid identity (AAI) based on the whole genome and phylogenetic analysis of the 16S rRNA gene and the whole genome were performed to accurately determine the taxonomic status of the strain JM-1. E-test was used to detect antibiotic susceptibility, and the results were interpreted according to the interpretation standards of Francisella tularensis in CLSI M45-A3. The virulence factor database (VFDB) was used for genome-wide annotation and analysis of virulence genes. Results:After culturing the strain JM-1 on the Columbia blood plate for 3 d, some grey-white, medium-sized, smooth, round and convex hemolytic colonies were observed. Gram staining result showed lightly colored Gram-negative Coccobacillus. API NH identification results suggested that the isolate JM-1 was Moraxella catarrhalis (biochemical code: 3010), while there was no identification result in Vitek2 system NH card (biochemical code: 0211002121). The EXS3000 mass spectrometer self-built database identified the isolate JM-1 as Cysteiniphilum litorale. The phylogenetic analysis based on the 16S rRNA gene and the whole genome showed that the isolate JM-1 and Cysteiniphilum litorale DSM 101832 T clustered into the same branch, and the ANI and AAI values between the two strains were 95.07% and 95.65%, respectively. The biochemical phenotype identification indicated the isolate JM-1 producing β-lactamase and penicillinase. Antibiotic susceptibility test results showed the strain was resistant to penicillin and sensitive to gentamicin, streptomycin, doxycycline, tetracycline, ciprofloxacin, levofloxacin, and chloramphenicol. Genome annotation suggested the virulence genes of the isolate JM-1 were similar to those of Francisella, including Francisella pathogenicity island (FPI), type Ⅳ fimbriae, capsule and lipopolysaccharide. Conclusions:Cysteiniphilum litorale was a rare pathogen with virulence genes similar to those of Francisella, and its antibiotic susceptibility was also similar to that of Francisella. This study confirmed a case of clinical infection caused by Cysteiniphilum litorale. The self-built MALDI-TOF MS system could be used for its rapid identification.
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Atopic dermatitis (AD) is a chronic, relapsable and pruritic skin disease, commonly found in children and adolescents. The prevalence of AD is increasing worldwide. It is reported that AD is related to many factors such as genetic inheritance, environment, immunity and skin barrier dysfunction, suggesting a very complex pathogenesis. In recent years, high-throughput technologies in the field of genomics and metabolomics have opened up new perspectives on the pathogenesis of AD, and shown potential application prospects in microbiome transplantation therapies for AD. This review summarized the current advances in the relationship between skin microecology and skin health, the pathogenesis and microbiomic characteristics of AD, features of pathogenic microorganisms, and microbiome transplantation therapies for AD. Based on our own practical experience, we put forward a culturomics research protocol to study the human skin microbiome and a method for quantitative microbiological examination, aiming to provide reference for the prevention, clinical treatment and therapeutic monitoring of AD.
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Objective@#To identify and characterize the 4 strains of Prototheca isolated from the clinical samples of skin or ascites samples in China. @*Methods@#The taxonomic position of 4 yeast-like organisms was revealed by polyphasic taxonomic approach, i.e., cultural and morphologic characteristics, commercial biochemical systems of Vitek 2 (YST kit) and Vitek matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) systems in combination with phylogenetic analysis based on the gene sequences of 16S and 28S rRNA. @*Results@#The 4 strains of Prototheca were characterized as cream-white, smooth, moist yeast-like colonies on Sabouraud gentamicin chloramph agar after incubation for 3 days. However, round, oval-shaped or elliptical sporangiums with mulberry-like or strawberry-like endospores were observed by optical microscope, which showed distinct differences from the general yeast species. The 4 isolates were identified as Prototheca wickerhamii with Vitek YST kits by Vitek 2 systems and Vitek MALDI-TOF MS systems. The genome for the 4 isolates was characterized with the existence of the prokaryotic 16S rRNA gene and eukaryotic 28S rRNA gene. The 16S rRNA gene sequence of the 4 strains showed more than 99.7% similarity to that of P. wickerhamii. Sequence analysis of 28S rRNA gene showed that the organisms included multiple copies of different sequences, which showed sequence similarities of 91.9% to 100% even in the same strain. The phylogenetic dendrogram based on 16S rRNA and 28S rRNA gene sequences showed that the 4 strains of Prototheca formed a cluster along with P. wickerhamii. @*Conclusion@#The 4 yeast-like organisms could be identified as P. wickerhamii, and 16S rRNA gene should be the suitable molecular target for the identification.
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Objective@#Establishing the mass spectrum library of a new Campylobacter- " C.fetus subsp.testudinum" for rapid species identification in clinical microbiology laboratory.@*Methods@#Illumina second generation sequencing platform 2000/miSeq was used to carry out high flux genome sequencing for the strains which were collected to establish mass spectrum library.The analysis oforthologous average nucleotide identity (OrthoANI) between collected strains and reference strains was performed at JAVA 8 operation environment. Then, the mass spectrums ofcollected strains andreference strains were acquired using MALDI-TOF MS. And the mass spectrum library of C. fetus subsp.testudinum. were established and verified.@*Results@#The OrthoANI analysis showed that the OrthoANI value of the collected strains and the reference strain C. fetus subsp.testudinum03-427 was 99.30%-99.96%, while the OrthoANI values of collected strains and C. fetus subsp.venerealisNCTC10354 orC.fetus subsp.fetus82-40 were 91.05%-92.26%. With reference to OrthoANI ≥ 95% as the basis for the determination of the same strain, the strains which collected to establish mass spectrum library was finally identified as " C. fetus subsp.testudinum" . The identification accuracy rate of the mass spectrum library was 100% (consistent with gene sequencing), and the confidence interval was 82.3%-99.9%, identification of the same strain is 100% reproducible.@*Conclusions@#The new" gold standard" based on high throughput sequencing and total genome analysis has provided the ideal reference value for the establishment of mass spectrum library.And the accurate and objective reference spectrum of the" C.fetus subsp.testudinum" provides a new platform for the rapid diagnosis of fetal Campylobacter infection. (Chin J Lab Med, 2018, 41: 583-588)
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Objective Reference standard of the RPOB(rifampin resistance)gene recommended by CLSI-MM18A(Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing) was used to evaluate the ability of MALDI-TOFMS techniques for the identification and classification of non-tuberculous Mycobacterium.Methods Fifty five clinicalstrains were collected from 2012 to 2016 with different sources.The RPOB gene was sequenced, and results were applied to phylogenetics analysis. MALDI-TOF MS technology was implemented to identify the strains, and cluster analysis was conducted based on protein fingerprint.The consistency of two methods for NTM identification and typing was evaluated.Results The RPOB gene method showed a good ability of identification(similarity>99.0%) and subtyping(to subspeciesof the complex level).The French BioMérieux MALDI-TOF MS identified 89.1% of 55 strains to genus level and 78.2% to species level.The phylogeneticsanalysis of protein fingerprint by SARAMS Premium software also showed good typing ability.Conclusions MALDI-TOF MS technology can identify and classify non-tuberculous Mycobacterium effectively,which is rapid and easy.It is complementary to RPOB gene method in laboratory application.
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The perpetual challenge was found for the diagnosis of newly emerging infectious diseases and reemerging infectious diseases .At present , reference laboratories for clinical microbiology were urgently demanded to deal with the challenge in China . Therefore , some suggestions for constructing reference laboratories were put forward in this paper , which may provide guidance for the future design of building comprehensive clinical microbiology laboratory .
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Objectives To identify and characterize 10 strains of Francisella philomiragia-like organisms isolated from blood samples and environmental water.Methods The 10 clinical and environmental isolates were identified by traditional morphological examination and biochemical characterization,matrix-assisted laser desorption/ionization time of flight(MALDI-TOF) mass spectrometry(MS) systems and sequencing based on 16S rRNA gene.The minimum inhibitory concentrations were tested by E-test methods.Results All the 10 isolates were gram-negative coccobacilli appearing tiny and faint counterstain of safranin,negative for urease,nitrate reduction and X and/or V factor requirement,but positive for oxidase and catalase.The isolates grew rapidly in sheep blood agar,chocolate agar and BCYE plate forming white opaque,colorless transparent or gray smooth colonies with about 2-mm diameters,but did not grow in M-H agar and MacConkey agar.The sequencing for 16S rRNA gene indicated that the 10 isolates shared more than 99.6％ similarity to Francisella philomiragia,and fell into the same clusters of Francisella philomiragia on phylogenetic tree.The MALDI-TOF MS analysis also showed the typical peaks with 6 153 m/z,5 180 m/z,7 757 m/z and 9 392 m/z which were similar to Francisella philomiragia ATCC 25015.However,they may be misidentified to be Sphingomonas paucimobilis by using Vitek 2 GN cards,Neisseria cinerea by using Vitek 2 NH cards,Myroides odoratimimus by using API 20NE strips and Haemophilus by using API NH cards.The results of antimicrobial susceptibility showed that they were all sensitive to chloramphenicol,doxycycline,tetracycline,gentamicin,ofloxacin and ciprofloxacin.Conclusion The 10 isolates could be identified as Francisella philomiragia,so we should pay more attention to the infrequent pathogen for its inactive biochemical reaction and the misidentification by commercial detection systems.
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Objective To identify and analyze the homology of Ochrobactrum isolated from clinical blood samples of children.Methods The 26 strains of Ochrobactrum anthropi were identified by Vitek 2 Compact and test strips of API 20 NE bacterial identification system.The biochemical phenotypes were identified by manual tests.The 16S rRNA and recA gene were amplified by PCR and sequenced.The drug sensitivity tests of Ochrobactrum anthropi were performed by Vitek 2 Compact and matched GN13 card.The homology was analyzed by pulsed field gel electrophoresis.Results Based on the identification of the instruments and the manual tests for biochemical phenotype,all the 26 experimental strains were Ochrobactrum anthropi.The results of sequencing for 16S rRNA and recA gene amplification products showed 25 strains were Pseudochrobactrum saccharolyticum and the other 1 was O.grignonensein.Drug sensitivity analysis showed that the all the 26 strains were resistant to aztreonam,but the sensitive rates to quinolones,aminoglycosides,trimethoprim sulfamethoxazole,four generation of cephalosporins and the antibiotics compound of piperacillin/tazobactam were all more than 80％.Pulsed field gel electrophoresis analysis showed that the 25 strains were highly homologous with differences of only 1 to 3 bands in fingerprint profiles.Conclusion Based on the biochemical phenotype and the sequencing of 16S rRNA and recA gene,the Ochrobactrum-like bacteria could be identified to the level of species.The highly homologous strains of Pseudochrobactrum saccharolyticum may be sourced from a clustered infection.
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Objectives To identify the Francisella strain isolated from blood of a patient with drowning-associated pneumonia.Methods The whole genome of the strain,designated Wenzhou1,was sequenced using the high throughput sequencing technology by 2000/miSeq system of Illumina platform,and the obtained genome draft was assembled by MicrobeTrakr Plus software.The phylogenetic neighbors of Wenzhou1 were obtained by NCBI BLAST analysis from GenBank database for the gene sequences of 16S rRNA,malate dehydrogenase(mdh),DNA-directed RNA polymerase subunit beta (rpoB) and succinate dehydrogenase subunit alpha (sdhA).The average nucleotide identity(ANI) between Wenzhou1 and its phylogenetic neighbors was analyzed by the software OrthoANI using NCBI BLAST search under the Java Runtime Environment Version 8.Results The genome size of Wenzhou1 was 1.96 × 106 bp,containing 74 contigs.The genomic G + C mol％ of Wenzhou1 was 32.1％,which was similar to the other species of genus Francisella and Allofranicella.Based on the analysis of NCBI BLAST of GenBank for the similarities of 16S rRNA gene,mdh gene,rpoB gene and sdbA gene sequences,Wenzhou1 was most closely related to F.hispaniensis FSC454 and Francisella cf.novicida 3523.The ANI of Wenzhou1 was 97.8％ to F.hispaniensis FSC454,97.5％ to 97.6％ to Francisella cf.novicida 3523,but only 91.3％ to 91.5％ to the four subspecies of F.tularensis.Conclusion ANI analysis based on whole genome sequence should be an accurate,effective method for bacterial identification.Wenzhou1 could be identified as F.hispaniensis by ANI with high-throughput whole genome sequencing technology.
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Objective To identify one acid‐fasting bacteria isolated from wound secretion of breast abscess .Methods The acid‐fasting strain was identified by the morphological characteristics ,API 20A strips ,classical biochemical reaction ,and 16S rRNA gene sequencing .Results Cells of the strain was anaerobic ,non‐spore‐forming ,pleomorphic ,straight or curved rods ,which Gram and acid‐fast stain both were positive .After incubation for 5 days on sheep blood agar ,colonies were than 2 mm in diameter ,circular , smooth ,entire ,bump ,rice cream‐like withβ‐hemolysis .The 16S rRNA gene sequences were 100% identity to Propionibacterium av‐idum .The API 20A profile was 44365062 with positive Voges‐Proskauer test ,which was also consistent to Propionibacterium avi‐dum .Conclusion The pathogens of breast abscess is Proionibacterium avidum ,which is the first acid‐fasting Propionibacterium re‐ported in China .
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Objective To identify the biotype and analyze the epidemiological characteristics of twentyfour Brurella strains from the primary hospitals in Guangdong Province.Methods The twenty-four Brucella strains,collected from Oct.2009 to Oct.2015,were identified by routine biochemical methods,VITEK 2 COMPACT automatic microbial identification analyzer,16S rRNA gene sequencing and biology phenotype based on serological and bacteriophages lysis test.The etiology was analyzed based on clinical data,biotypes of the isolates and other clinical information.Results All of the twenty-four strains were Gram-negative coccobacilli,including two strains of Brucella suis biotype Ⅱ,four strains of Brucella melitensis biotype Ⅰ and eighteen strains of Brucella melitensis biotype Ⅲ.By GN card of VITEK 2 COMPACT automatic microbial identification analyzer,one strain was mistaken as Bordetella bronchiseptica and two strains were mistaken as Ochrobaetrum anthropi.The 16S rRNA gene sequencing showed they were high homology to Ochrobactrum intermedium and Ochrobaetrum anthropi,which completely excluded the possibility of Bordetella bronchiseptica.Tbe clinical data showed that all of the twenty-four patients were adults with an average age of 49.0 years old,men and women were twelve people respectively,with no significant gender differences and no occupational exposure,which presenting a wide and diverse range of nonspecific clinical signs and symptoms,but brucellosis was not aware of by the physician.Conclusion Brucella melitensis biotype Ⅲ is the main pathogen of brucellosis,with the characteristics of sporadic outbreak and occult infection in the primary hospitals in Guangdong Province.
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Objective To test a snapback primer for the identification of Legionella and Legionella pneumophila ( L.pneumophila) strains in a one-step real-time PCR assay.Methods A novel primer was designed with a pair of genus-specific primers of Legionella strains.The species-specific probe sequences of L.pneumophila strains were linked at the 5′end of the reverse primer.The sensitivity and specificity of the novel PCR assay were tested with 43 types of Legionella and non-Legionella strains.The established PCR as-say was used to identify 186 wild Legionella strains isolated from 11 provinces of China and 15 environmental water samples.Results The amplicon melting peak of Legionella strains was detected at 85-86℃.The snapback melting peak of L.pneumophila was detected at 71℃.No melting peak of non-Legionella strains was detected.The sensitivity of the standard strains and simulated water samples were 1 ng/μl DNA tem-plates and (1×103-1×104 )/ml, respectively.186 Legionella strains and 44 L.pneumophila strains isolated from environmental water samples were successfully identified with the snapback primer.Twealve Legionella strains and 4 L.pneumophila strains were identified from 15 environmental water samples with the snapback primer as compared with 8 Legionella strains identified with the culture method.Conclusion The snapback primer mediated one-step PCR assay could be used for the identification of Legionella and L.pneumophila strains with the advantages of high specificity and sensitivity.
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Francisella guangzhouensis was used as an example to illustrate how to utilize the bioinformatics resources to identify the novel species, how to name the novel species with Latinized letters, and how to validate a novel species in the clinical microbiology laboratories.Some related rules of International Code of Nomenclature of Bacteria was also introduced.
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Objectives Use ITS gene sequence analysis to identify 15 strains of dematiaceous fungi , to learn the types of pathogenic strains and clinical treatment. Methods By observing the colony morphology and microscope morphological of the dematiaceous fungi isolated from superficial mycoses , and identified by ITS gene sequence analysis. Results 15 strains were identified by morphological observation as dematiaceous fungi.The amplified bands were identified by Tanon-3500 gel imaging system between 500 ~ 700 bp. Blast sequencing results show that 2 strains Alternaria alternate , 2 strains Cladosporium sphaerospermum. 2 strains Exophiala dermatitis, 1 strains Cladosporium cladosporioides, Curvularia lunata, Talaromyces rugulosus, Phaeobotryon cupressi, Cladosporium tenuissimum, Fonseceea pedrosoi, Exophiala werneckii, Exophiala oligosperma and Fonsecaea monophora. Conclusion ITS gene sequence analysis can identify dematiaceous fungi effectively , avoided undetected and misdiagnose cause by the lack of clinical experience.
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Objective To indentify Streptobacillus moniliformis isolated from the knee joint pus by 16S rRNA gene sequencing and biochemical reactions and explore the clinical value of the method. Methods The bacterial 16S rRNA gene sequence-based identification, bacterial morphology, VITEK 2 automate systems, API 20NE strips, API 20E strips and API 50CH were performed to identify the rare bacteria. Results The bacteria grew slow on blood agar and chocolate agar and were inhibited on Maconkey agar. The bacterial colony on blood agar tookes the form of 1~2 mmomelette, which was translucent and moist with circular protrusion and smooth edges. They were Gram-staining negative and in catenation, its thalli 1~3μm, round, oval or fusiform. Vitek 2 GN-13, API 20NE and API 20E were unable to reach the identification of the bacteria. 16S rRNA gene sequencing showed the bacteria were similar to streptobacillus moniliformis by 100%. Conclusion The rare bacteria isolated from left knee joint are streptobacillus moniliformis. 16S rRNA gene sequences combined with the biochemical reactions is accurate in the identification of these bacteria.
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ObjectivesTo identified the strain 1012 from the National Center of Clinical Laboratory of China for microbe inter-laboratory quality assessment in 2010, and study the taxonomic status of strain 1012 and related species in the genus Actinomyces. Methods The bacterial traditional morphological characteristics, commercial API systems, and 16S rRNA gene sequence analysis were applied to identify the problematic culture of strain 1012. The phylogenetic tree based on the remote information of the prokaryotes systems was constructed to study the taxonomic status and evolutionary relationship of the genus Actinomyces and related species in the family Actinomycetaceae. Results Strain 1012 was determined as a kind of facuhative anaerobic,non-spot-forming,Gram-positive coryneformbacteria,which was identifiedto Actinomyces turicensis for the phenotypic biochemical characteristics of more than 60 items, The comparative study of 16S rRNA gene showed the strain 1012 with 99. 8％ similarities to Actinomyces turicensis, but only 90. 6％ to the type species of Actinomyces bovis in the genus Actinomyces. However, the comparative study of 16S rRNA gene showed the strain 1012 with only 90. 6％ homology to the type species of Actinomyces bovis in the genus Actinomyces. Further phylogenetic analysis showed that nine independent clusters were grouped in the family Actinomycetaceae, of which four clusters were separately represented the genera Varibaculum,Mobiluncus, Actinobaculum and Arcanobacterium, while other five clusters all were designated to the genus Actinomyces. The study showed strain 1012 was located in genus Ⅲ of Actinomyces, yet with a relatively long genetic distance to Actinomyces bovis. ConclusionThe genus Actinomyces may be reclassified as one genus Actinomyces sensu stricto and several new genera for the genotypic characteristics.