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.

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.

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.

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 .

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.

Objective To identify one runny mucoid-like Gram-negative bacteria with pink pigment isolated from clinical pus sample. Methods The pus sample was aseptically extracted from a deep lesions of one patient, then stored in Amies medium at room temperature for transportation. One sheep blood plate and one chocolate plate were used to detect the possible pathogens from the specimens. After inoculation, the plates were placed in a humidified incubator with 5% CO2 at 35 ℃. To identify the obtained isolates, we used the commercial Vitek2 and API systems, combining some traditional morphological examination and classical biochemical and physiological characteristics. For pure cultures, the cellular fatty acids were extracted, methylated, and determined by gas chromatography method. The 16S rRNA gene was amplified and sequenced by a commercial broad-spectrum PCR primers. The phylogenetic tree based on 16S rRNA gene was constructed by Mega 4.1 software using the neighbour-joining methods with 1 000 bootstrap replications. Results One runny mucoid-like Gram-negative bacterium, named K8756, was isolated both on sheep blood and chocolate plates after 72 h incubation. The API 20NE profile was 1245045 after a 3-day culture, which would be identified as Ochrobactrum anthropi with a good confidence of 98% probability. It was identified as Ralstonia pickettii and Bordetella bronchiseptica by VITEK 2 GN kits. However, further comparative 16S rRNA gene sequences showed that strain K8756 was closely related to the valid published Roseomonas mucosa MDA 5527 with 100% identity. Colonial morphologic features, phenotypic characteristics and major cellular fatty acid composition were also with high similarity to Roseomonas mucosa. Conclusions Strain K8756( = GIMCC 1.0030 ) is identified as Roseomonas mucosa by the polyphasic phenotypic and genotypic characteristics. The comparative analysis based on 16S rRNA gene sequences is a useful method for identifying the problematic and newly named bacteria.

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.

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.

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.

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.