Objective:To analyze the biological characteristics, phylogeny and the taxonomic status of strain 7712 (=CGMCC 1.17031=NBRC 113783=KCTC 15766) isolated from a clinical blood sample.Methods:Strain 7712 was identified by the cultural properties, cellular and colonial morphology, physiological and biochemical reactions, matrix-assisted laser desorption ionization time-of-flight mass spectrometry system, and genome correlation index analysis. The genomic phylogenetic tree was construct to analyze the taxonomic position. The virulence factors and resistance genes of strain 7712 and related strains were then compared by the online virulence factor database and online comprehensive antibiotic research database respectively.Results:Strain 7712 was urease negative, gram-negative nonfermenters, which was identified as Ochrobactrum anthropi by VITEK GN card. The 16S rRNA gene analysis showed that the strain was closely related to the members of genera Ochrobactrum and Brucella. The phylogenetic tree showed that strain 7712 was clustered together with Ochrobactrum teleogrylli LCB8 T and Ochrobactrum haematophilum CCUG 38531 T, along with genus Brucella and other Ochrobactrum species. The genome relatedness indexes analysis showed that the average nucleotide identity between strain 7712 and Ochrobactrum teleogrylli LCB8 T was 98.16%, which was higher than the threshold for prokaryotic species. Genetic prediction showed that strain 7712 carried several virulence-related genes and resistance-related genes, of which the existence of OCH gene might be responsible to the resistance to cephalosporin. Conclusions:A case of human infection caused by Ochrobactrum teleogrylli is identified, which would help promote the understanding of biodiversity of genus Ochrobactrum.

Objective:To evaluate the diagnostic performances of plasma placental extracellular vesicles (pcEV) and their clearance protein (Lactadherin) in predicting adverse pregnancy outcomes in patients with severe preeclampsia (sPE).Methods:This is a retrospective case-control study. 60 patients aged 32 (29, 36) years diagnosed with sPE at 27-37 weeks of pregnancy, who underwent prenatal examinations and delivered between January 31 th, 2018 and January 31 th, 2019, were recruited. According to the occurrence of endpoint events (fetal distress and/or fetal growth restriction), sPE patients were further divided into an event group of 34 cases and a non event group of 26 cases. 33 healthy pregnant women of the same gestational age were selected as the control group, aged 31 (29, 36) years old. 25 non pregnant healthy women were selected as the healthy control group, aged 26 (25, 38) years old. Flow cytometry was used to detect placental alkaline phosphatase antibody positivity as pcEV, while membrane surface expression of phosphatidylserine, i.e. membrane associated protein V (AV) positivity as AV +pcEV. ELISA kits were used to detect the level of Lactadherin. Logistic regression was used to perform multiple correlation analysis. The performances of pcEV and Lactadherin in predicting adverse pregnancy outcomes were evaluated by the receiver operating characteristic (ROC) curve. Survival analyses were performed by the Kaplan Meier curve. The hazard ratios (HR) was calculated by the Cox proportional risk regression model. Results:The plasma AV +pcEV levels in sPE patients were 8 260 (4 991, 16 751)/μl, which were higher than 1 088 (784, 1 871)/μl of healthy pregnant women and 206 (116,256)/μl of healthy controls ( H=94.490, P<0.05). The plasma AV +pcEV levels in sPE patients with endpoint events were 11 225 (7 496, 20 599)/μl, which were higher than 5 199 (2 914, 8 347)/μl of patients without endpoint events ( U=178, P<0.05). The plasma levels of Lactadherin in sPE patients were 2 635 (1876, 3 137) pg/ml, which were higher than 1 597 (1 287, 1 818) pg/ml in healthy pregnant woman and 1 123 (749, 1 405) pg/ml in healthy controls ( H=54.307, P<0.05). ROC showed that the critical value of AV +pcEV predicting fetal distress and/or fetal growth restriction events within 77 days in sPE patients was 6 524/μl and area under the curve(AUC) was 0.799 (95% CI 0.680-0.917). The critical value of Lactadherin was 2 336.5 pg/ml and AUC was 0.702 (95% CI 0.564-0.841). Logistic regression analysis showed that there was a significant correlation between AV +pcEV levels in sPE patients and 24-hour urine protein quantification ( OR=9.288, 95% CI 1.993-43.293), as well as the need for combined antihypertensive therapy ( OR=18.690, 95% CI 1.919-182.077) ( P<0.05). Survival analysis showed that the cumulative probability of fetal distress and/or fetal growth restriction events within 77 days were significantly increased in sPE patients with AV +pcEV levels above the critical value (Log-rank χ 2=21.430, P<0.05). The Cox proportional regression model showed that the levels of AV +pcEV can independently identify fetal distress and/or fetal growth restriction events ( HR=7.983, P<0.05). Conclusions:The changes of pcEV in plasma of pregnant women in late pregnancy were related to the development of PE. High concentrations of pcEV suggested an increased risk of fetal distress and fetal growth restriction, and pcEV could serve as an effective marker for early warning of adverse pregnancy outcomes.

Objective To monitor the susceptibility of clinical isolates to antimicrobial agents in tertiary hospitals in major regions of China in 2022.Methods Clinical isolates from 58 hospitals in China were tested for antimicrobial susceptibility using a unified protocol based on disc diffusion method or automated testing systems.Results were interpreted using the 2022 Clinical &Laboratory Standards Institute(CLSI)breakpoints.Results A total of 318 013 clinical isolates were collected from January 1,2022 to December 31,2022,of which 29.5％were gram-positive and 70.5％were gram-negative.The prevalence of methicillin-resistant strains in Staphylococcus aureus,Staphylococcus epidermidis and other coagulase-negative Staphylococcus species(excluding Staphylococcus pseudintermedius and Staphylococcus schleiferi)was 28.3％,76.7％and 77.9％,respectively.Overall,94.0％of MRSA strains were susceptible to trimethoprim-sulfamethoxazole and 90.8％of MRSE strains were susceptible to rifampicin.No vancomycin-resistant strains were found.Enterococcus faecalis showed significantly lower resistance rates to most antimicrobial agents tested than Enterococcus faecium.A few vancomycin-resistant strains were identified in both E.faecalis and E.faecium.The prevalence of penicillin-susceptible Streptococcus pneumoniae was 94.2％in the isolates from children and 95.7％in the isolates from adults.The resistance rate to carbapenems was lower than 13.1％in most Enterobacterales species except for Klebsiella,21.7％-23.1％of which were resistant to carbapenems.Most Enterobacterales isolates were highly susceptible to tigecycline,colistin and polymyxin B,with resistance rates ranging from 0.1％to 13.3％.The prevalence of meropenem-resistant strains decreased from 23.5％in 2019 to 18.0％in 2022 in Pseudomonas aeruginosa,and decreased from 79.0％in 2019 to 72.5％in 2022 in Acinetobacter baumannii.Conclusions The resistance of clinical isolates to the commonly used antimicrobial agents is still increasing in tertiary hospitals.However,the prevalence of important carbapenem-resistant organisms such as carbapenem-resistant K.pneumoniae,P.aeruginosa,and A.baumannii showed a downward trend in recent years.This finding suggests that the strategy of combining antimicrobial resistance surveillance with multidisciplinary concerted action works well in curbing the spread of resistant bacteria.

Objective: To optimize the sample pretreatment and establish an ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-MS) assay based on the parallel reaction monitoring (PRM) mode for determination of antibiotic residues in chicken meat. Methods: Blank matrix-spiked chicken meat samples were extracted with 95% acetonitrile aqueous solution containing Na2EDTA and formic acid. The extraction solutions were cleaned up using different combinations of C18, PSA and GCB fillers, and the combinations with a higher antibiotic recovery rate was screened. The residues of 32 antibiotics were determined using UPLC-Q-Orbitrap-MS based on the PRM mode. Results: If the extraction solution was cleaned up using the C18 filler, the largest number of antibiotics with a spiked recovery rate of >80% was seen, with matrix effects of 82.2% to 112.6%. The detection limits of 32 antibiotics were 0.8 to 5.8 μg/kg, with linear correlation coefficients of >0.99, spiked recovery rates of 71.3% to 111.5% and relative standard deviations of 3.2% to 14.2%. Conclusion The UPLC-Q-Orbitrap-MS assay is suitable for determination and quantitative analysis of multiple antibiotics in chicken meat. Key words： high-resolution mass spectrometry orbitrap antibiotic residue parallel reaction monitoring

Objective: To determine human tear proteins using nanoliter liquid chromatography coupled to quadrupole orbitrap mass spectrometry (NanoLC-Q-Orbitrap-MS), and perform a bioinformatics analysis of main proteins. Methods: Human tear samples were collected with capillary, transferred to 3 kDa ultrafiltration tubes containing 400 μL of superpure water and centrifuged at 12 000×g for 15 min. Repeated extraction of tear proteins were performed four times, and following digestion with trypsin, the proteins were separated using the Waters NanoAcquity peptide BEH C18 column (1.7 μm, 100 μm×100 mm) and determined using NanoLC-Q-Orbitrap-MS with the mobile phase of 0.1% formic acid aqueous solution and acetonitrile (0.1% formic acid) in the full MS/dd-MS2 mode. The types of proteins were characterized in the Uniprot database using the software Proteome Discoverer version 2.1 and verified using bovine serum albumin. The tear proteins were subjected to gene annotation analysis using the String database. Results: A total of (387±160) human tear proteins were yielded, with a relative standard deviation (RSD) of 4.13%, and there were 25 types of proteins with a relative high abundance, including lipocalin 1, lysozyme and lactoferrin. The peptide sequence coverage of bovine serum albumin was (86.08±2.61)%, with a RSD of 3.03%. The 25 major tear proteins were involved in substance transduction among cells, homeostasis process, negative regulation of the endopeptidase activity, detection of chemical stimulants and humoral immune responses, and the 16 proteins had close interactions. Lacritin, lipocalin 1, lactoferrin, lysozyme and zinc-α 2-glycoprotein, which had a relative high abundance, had close biological connections. Conclusion NanoLC-Q-Orbitrap-MS is stable, reliable and feasible for detection of multiple proteins in tears.

Objective: To optimize the enzymatic digestion conditions of trypsin, so as to improve the testing capacity of mass spectrometry for shrimp allergens. Methods: The enzymatic digestion test was carried out by response surface methodology for optimizing pH, temperature and time. After enzymatic hydrolysis, the peptides were separated by chromatography and determined by high-resolution mass spectrometry with Q-orbitrap. The allergen protein was identified and quantified by UniProt database and MaxQuant software. Results: Two allergen proteins, tropomyosin and arginine kinase, were isolated from shrimp, and their intensities ranged from 100.2×106 to 436.5×106. Response surface analysis showed that when the digestion time was 4.29 hours, the temperature was 44.15 ℃ and pH value was 6.55, the maximal intensity of the allergen proteins was 457.48×106. The experiment was validated with the digestion time of 4.2 h, pH value of 6.5, and temperature of 44 ℃, then resulted in the average intensity of 448.1×106. The deviation from the predicted value was 2.1%. Conclusions The conditions of enzymatic digestion can be optimized by response surface methodology. The enzyme may have the best performance with the pH value of 6.5, temperature of 44 ℃ and digestion time of 4.2 hours.

Objective : To develop an analytical method of ibotenic acid (IBA) and muscimol (MUS) in wild mushroom by dansyl chloride (DNSCl) derivatization-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to provide technical support for etiological identification of mushroom poisoning events. Methods : The sample was extracted with hydrochloric acid solution, derived by bimolecular DNSCl, diluted and inorganic salts precipitated with acetonitrile. The extract was separated by a waters XBridgeTM BEH C18 column and measured by LC-MS/MS. Results : The limits of detection for IBA and MUS in wild mushroom were 0.15 mg/kg and 0.1 mg/kg, respectively. Good linear relationship was obtained for IBA and MUS at the range of 0.5-250 mg/kg with the correlation coefficient of 0.997 and 0.999, respectively. The average recoveries at three spiking levels were 84.5%-102.0% with relative standard deviations (RSDs, n=6) of 4.7%-8.6% for IBA. The average recoveries were 88.6%-95.4% with RSDs (n=6) of 4.9%-7.5% for MUS. Conclusion The optimized sample extraction and bimolecular DNSCl derivatization conditions can achieve rapid and accurate analysis of IBA and MUS in wild mushroom poisoning sample.

Objective: To establish the ultra-high performance liquid chromatography coupled with quadrupole orbitrap mass spectrometry ( UPLC-Q-Orbitrap-MS ) for the analysis of allergen protein in Macrobrachium. Methods: Based on the strategy of bottom-up protein analysis, the proteins in Macrobrachium samples were extracted by Tris-HCl, digested by trypsin at 40 ℃ for 6 hours, separated by chromatography, and analyzed by Q-Orbitrap-MS spectrometry ( Full MS/dd-MS2, TopN=10 ) . Allergen proteins were identified with UniProt protein database and Proteome Discoverer software. Results: Four kinds of allergen proteins were obtained, which were tropomyosin, arginine kinase, sarcoplasmic calcium binding protein and hemocyanin. The coverage rates of peptides in proteins were 53%, 36%, 12% and 12%, respectively. Post translation modifications were methylation of aspartic acid (D), deacylylation of aspartic acid ( N ) , glutamyl ammonia ( Q ) and oxidation of methionine ( M ) . Conclusions The UPLC-Q-Orbitrap-MS can identified abundant peptide and fragment information with high sensitivity and resolution, which provides a technical support for the analysis of shrimp allergens.

Objective To investigate the risk factors for cerebral microbleeds (CMBs) in patients with non-disabling ischemic cerebrovascular events (NICE).Methods From January 2017 to September 2018,patients with NICE admitted to the Department of Neurology,the Second Affiliated Hospital of Kunming Medical University were enrolled.The relevant clinical data were collected and the cranial MRI examinations were completed.CMBs were detected by susceptibility-weighted imaging.The demographic and clinical data of the CMBs group and non-CMBs group were compared.Multivariate logistic regression analysis was used to determine the independent risk factors for CMBs.Multivariate linear regression analysis was used to determine the independent influencing factors of the number of CMBs.Results A total of 159 patients were enrolled,including 73 (45.9％) in the CMBs group and 86 (54.1％) in the non-CMBs group.There were significant differences in hypertension,diabetes mellitus,past stroke or transient ischemic attack (TIA) history,carotid atherosclerosis,NICE classification (TIA,mild stroke) and the proportion of patients taking drugs before onset,as well as diastolic blood pressure and white matter Fazekas score between the CMBs group and the non-CMBs group (all P＜ 0.05).Multivariate logistic regression analysis showed higher diastolic blood pressure (odds ratio 1.047,95％ confidence interval 1.016-1.079;P =0.002) and higher Fazekas score (odds ratio 1.825,95 ％ confidence interval 1.465-2.273;P ＜ 0.001) were the independent risk factors for CMBs.Multiple linear regression analysis showed that there was an independent positive correlation between the white matter Fazekas score and the number of CMBs (r =0.273,P ＜ 0.001).Conclusion In patients with NICE,CMBs were associated with higher diastolic blood pressure level and higher white matter Fazekas score,and the white matter Fazekas score was positively correlated with the severity of CMBs.

Objective: To quickly determine bongkrekic acid（BKA）in plasma qualitatively and quantitatively by liquid chromatography-tandem mass spectrometry（LC-MS/MS）,and to provide technical support for etiological identification of food poisoning events. Methods: The plasma sample was protein precipitated with acetonitrile,diluted with water and purified with anion exchange solid phase extraction cartridge of PAX. The sample extract was separated by an XBridgeTM BEH C18 chromatographic column. Gradient elution was conducted with the mobile phase of 0.01 %（v/v）ammonia and methanol. Then BKA was detected by LC-MS/MS. Results: The equation of linear regression was y=16 509x+3 134.3. Good linear relationship was obtained for BKA at a range from 1 to 400 ng/mL in plasma,with the correlation coefficient of 0.999 3. The limit of detection（LOD）was 0.5 ng/mL and the limit of quantitation（LOQ）was 1 ng/mL. The average recoveries were 76.0%-96.7% with relative standard deviations（RSDs,n=6）of 5.2%-12.8% at three spiking levels of 1（LOQ）,10（10 times of LOQ）and 200 ng/mL（medium of linear range）. The concentrations of BKA in plasma obtained from two patients suffering from food poisoning were 394 and 92.3 ng/mL. Conclusion The optimized sample pretreatment and chromatographic separation conditions can achieve rapid,accurate,qualitative and quantitative analysis of BKA in plasma.