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.

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.

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.

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.

OBJECTIVE:To establish quality standard for Zhuang medicine Homalocladium platycladum. METHODS:TLC method was used for qualitative identification. HPLC method was used to determine the content of quercetrin in samples:Agilent Zorbax SB-C18 column,mobile phase consisted of acetonitrile-0.1% acetic acid(20:80,V/V),flow rate of 1.0 mL/min,detection wavelength of 360 nm,column temperature of 30 ℃,injection volume of 10 μL. The contents of moisture,total ash and extract in samples were determined. referring to Chinese Pharmacopiea(2015 edition). RESULTS:TLC spots of H. platycladum were clear and well-separated without interference from negative control. The linear range of quercetin were 0.023-0.46 mg/mL(r=0.9991). RSDs of precision,stability and repeatability tests were all lower than 2.0%. The average recoveries were 98.01%-103.05%(RSD=1.71%,n=6). The contents of moisture,total ash,extract and quercetrin in samples were 6.39%-9.78%,2.82%-8.07%, 0.02%-0.27%,14.14%-28.45%,0.09%-0.50% respectively. CONCLUSIONS:Established trial can be used for quality control of Zhuang medicine H. platycladum.

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 optimize the content determination conditions of total alkaloids from Zhuang medicine Munronia delavayi,and to determine the content of total alkaloids in M. delavayi from different producing areas. METHODS:With the con-tent of total alkaloids as index,using solvent amount,ultrasonic time,ultrasonic extraction times and pH value of buffer as fac-tors,the content determination conditions of total alkaloids from Zhuang medicine M. delavayi were optimized by orthogonal test. Optimized content determination conditions were adopted to determine the content of total alkaloids in M. delavayi from 18 produc-ing areas in different harvest time. RESULTS:The optimum content determination conditions were as follows as the amount of sol-vent(CHCl3)20 ml,ultrasonic processing for 3 times,lasting for 15 min each time,pH value of buffer 4.5. The contents of total alkaloids in M. delavayi from 18 producing areas were between 0.6-11.98 mg/g,showing great difference. M. delavayi from Long-lin county and Tianlin county harvested in Oct. had the lowest content of total alkaloids. CONCLUSIONS:Optimized content deter-mination condition can be used for the content determination of total alkaloids in Zhuang medicine M. delavayi and quality control of it. The content determination of total alkaloids in M. delavayi is related to producing area and harvest time.

To established an effective GC-MS /MS method for the contents determination of the residual DEHP in injection equipment, and investigate the effect of the pretreatment on the measurement. To simulate the clinical conditions of use, under the condition of 37 degrees C balance extraction, extract liquor by chloroform extraction, then the extract followed by analysis of GC-MS /MS. The method was simple, rapid, sensitive and accurate. The limits of quantitation (LOQ, S/N = 5) of cyclohexanone was 0.075 μg/mL, The spiked average recoveries ranged from 92% to 98%. The relative standard deviations (RSDs) of the method ranged from 1.01% to 1.61%, The method was simple, fast, sensitive and accurate, and may serve as a mass control method for residual DEHP in injection equipment.
Cyclohexanones ; chemistry ; Diethylhexyl Phthalate ; chemistry ; Equipment Contamination ; Gas Chromatography-Mass Spectrometry ; Injections ; instrumentation ; Limit of Detection ; Plasticizers ; chemistry

To established an effective GC-MS /MS method for the contents determination of the residual DEHP in injection equipment, and investigate the effect of the pretreatment on the measurement. To simulate the clinical conditions of use, under the condition of 37oC balance extraction, extract liquor by chloroform extraction, then the extract folowed by analysis of GC-MS /MS. The method was simple, rapid, sensitive and accurate. The limits of quantitation( LOQ, S/N = 5) of cyclohexanone was 0.075mg/mL, The spiked average recoveries ranged from 92% to 98%.The relative standard deviations( RSDs) of the method ranged from 1.01% to 1.61%, The method was simple, fast,sensitive and accurate,and may serve as a mass control method for residual DEHP in injection equipment.