Background Bacteria are the most diverse and widely sourced microorganisms in the indoor air of subway stations, where pathogenic bacteria can spread through the air, leading to increased health risks. Objective To understand the status and distribution characteristics of indoor air bacterial pollution in subway stations and compartments in a city of Central South China, and to provide a scientific basis for formulating intervention measures to address indoor air bacteria pollution in subways. Methods Three subway stations and the compartments of trains parking there in a city in Central South China were selected according to passenger flow for synchronous air sampling and monitoring. Temperature, humidity, wind speed, carbon dioxide (CO₂), fine particulate matter (PM_2.5), and inhalable particulate matter (PM₁₀) were measured by direct reading method. In accordance with the requirements of Examination methods for public places-Part 3: Airborne microorganisms (GB/T 18204.3-2013), air samples were collected at a flow rate of 28.3 L·min⁻¹, and total bacterial count was estimated. Bacterial microbial species were identified with a mass spectrometer and pathogenic bacteria were distinguished from non-pathogenic bacteria according to the Catalogue of pathogenic microorganisms transmitted to human beings issued by National Health Commission. Kruskal-Wallis H test was used to compare the subway hygiene indicators in different regions and time periods, and Bonferroni test was used for pairwise comparison. Spearman correlation test was used to evaluate the correlation between CO₂ concentration and total bacterial count. Results The pass rates were 100.0% for airborne total bacteria count, PM_2.5, and PM₁₀ in the subway stations and train compartments, 94.4% for temperature and wind speed, 98.6% for CO₂, but 0% for humidity. The overall median (P₂₅, P₇₅) total bacteria count was 177 (138,262) CFU·m⁻³. Specifically, the total bacteria count was higher in station halls than in platforms, and higher during morning peak hours than during evening peak hours (P<0.05). A total of 874 strains and 82 species were identified by automatic microbial mass spectrometry. The results of identification were all over 9 points, and the predominant bacteria in the air were Micrococcus luteus (52.2%) and Staphylococcus hominis (9.8%). Three pathogens, Acinetobacter baumannii (0.3%), Corynebacterium striatum (0.1%), and Staphylococcus epidermidis bacilli (2.2%) were detected in 23 samples (2.6%), and the associated locations were mainly distributed in train compartments during evening rush hours. Conclusion The total bacteria count in indoor air varies by monitoring sites of subway stations and time periods, and there is a risk of opportunistic bacterial infection. Attention should be paid to cleaning and disinfection during peak passenger flow hours in all areas.

Objective:A nested-PCR assay is developed to detect and identify the genomic DNA of Brucella vaccine A19 strain. Methods:The whole genomic sequences of Brucella vaccine A19 strain and other Brucella spp. strains were compared and analyzed. The primers were designed by nucleotide difference sites. The nested-PCR assay was established to detect and identify Brucella vaccine A19 strain. The genomic DNA of Brucella vaccine A19 strain was extracted and diluted. The diluted template DNA was tested for sensitivity of using nested-PCR assay. And the specificity of nested-PCR assay was tested for the genomic DNA of other Brucella spp. strains and non- Brucella spp. strains. Results:The minimum detection limit of the nested-PCR assay was 3.43 fg. The nested-PCR assay established for amplification of Brucella vaccine A19 strain showed 246 bp electrophoresis bands, while other Brucella spp. strains showed 314 bp electrophoresis bands, and non- Brucella spp. strains did not produce electrophoresis bands. Conclusions:The nested-PCR assay established has the characteristics of high sensitivity and specificity. It can be detected when there is one copy of Brucella vaccine A19 strain genomic DNA in the reaction system. This method is particularly suitable for the detection and identification of trace genomic DNA of Brucella vaccine A19 strain in sample.

Objective:To establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. Methods:Based on the differences in the entire genome sequence between Brucella S2 vaccine strain and other reference strains of Brucella, primers and probes were designed to establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. The DNA of 22 reference strains of Brucella and 8 non- Brucella control strains were obtained from the National Institute for Infectious Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. At the same time, environmental samples were obtained from the brucellosis vaccine manufacturers, and bacterial DNA from environmental samples was extracted using a blood/tissue genomic DNA extraction kit. The obtained DNA was pre-amplified by conventional PCR, and then subjected to quantitative real-time PCR secondary amplification (nested fluorescence quantitative PCR) using the amplified PCR product as a template. The specific fluorescence curve and corresponding number of cycles (Ct value) were observed, and the sensitivity was tested. Results:The quantitative real-time PCR detection system established did not detect specific fluorescence curves (without Ct values) for 21 reference strains of Brucella and 8 non- Brucella control strains, except for S2 vaccine strains. The established detection system had a minimum detection limit of 4.34 fg (genomic DNA) for detecting the DNA of Brucella S2 vaccine strain; DNA of Brucella S2 vaccine strain was detected in 3 of the 14 environmental samples collected. Conclusion:The quantitative real-time PCR detection system established can detect Brucella S2 vaccine strain in samples, with good sensitivity and specificity.

Objective:To establish a real-time quantitative PCR assay to detect the copy number of IS711 transposase gene (orfA) in Brucella genome, and the assay is applied to identify the species and biovars of Brucella. Methods:To establish an orfA gene copy number detection system based on Taqman real-time quantitative PCR technique. Primers and probes of bcsp31 and orfA genes were designed, the contents of bcsp31 gene and orfA gene in the same strain with the same DNA concentration were simultaneously detected by real-time quantitative PCR assay, and cycle number (CT value) of the two genes were obtained. According to the differences of CT values of bcsp31 gene and orfA gene, the copy number of orfA gene in Brucella genome was calculated. At the same time, the DNA of Brucella 16M strain was double decreasing dilution to verify the stability of the detection system. Results:A real-time quantitative PCR assay was used to detect bcsp31 gene and orfA gene simultaneously, when the DNA concentration difference of 16M strain was 2 times, the mean difference of CT values measured was 1.00, 95% confidence interval was 0.95-1.05, standard deviation was 0.17, and coefficient of variation was 0.17. The orfA gene copy number of 30 Brucella strains was detected by this detection system. It was found that there were 6, 9, and 7 copy numbers in the biovars 1-3 of Brucella melitensis, respectively. The strain of Brucella suis biovar 2 had 10 copy numbers, which were different from those of the other 4 strains of biovars 1, 3-5. There were 37 copy numbers in Brucella ovis strain. The copy numbers were stable at 5-6 copies in 8 biovars (1-7, 9) of Brucella abortus strains. Conclusions:A real-time quantitative PCR assay for detection of orfA gene copy number in Brucella DNA has been established. This method could identify some Brucella species and biovars strains.

Objective:To understand the molecular characteristics and correlation among isolated strains of Brucella melitensis (BM) so as to improve the strategies on prevention and control of the disease in Jiangxi province. Methods:A total of 25 strains of BM isolated from human in 17 counties of Jiangxi province were analyzed by multiple locus variable-number tandem repeat analysis (MLVA) method.Results:A total of 25 strains of BM were classified into 24 independent genotypes with similarities between 67.00% and 100.00% and Simpson index between 0.000 and 0.773. There were 3 genotypes in MLVA8, including 60.00% (15/25) as 42 genotype, 32.00% (8/25) as 43 genotype, and 8.00% (2/25) as 63 genotype, respectively. There were 7 genotypes in MLVA11 identified, with 116 genotype and 125 genotype the main genotypes, accounting for 56.00% (14/25) of all the identified strains.Conclusions:Genes from all the 25 strains of BM that isolated from human being were with high genetic diversities, and various, genotypes. However, no obvious epidemiological correlation was noticed among these strains, indicating the complexity of the source of infection on Brucella in Jiangxi province.

Objective:To analyze the epidemiological and molecular characteristics of human brucellosis in Qinghai province from 2005 to 2019 and provide basic data for brucellosis prevention and control.Method:The data about human brucellosis in Qinghai from 2005 to 2019 were collected from the information system of China CDC to describe the spatial, population and time distributions of human brucellosis cases in Qinghai. The isolated strains were identified and typed with traditional methods, BCSP31-PCR, AMOS-PCR and multi-locus variablenumber tandem repeat (MLVA-16).Results:A total of 577 human brucellosis cases were reported in Qinghai from 2005 to 2019, the average prevalence rate was 0.07 per 100 000 person, there were statistic differences among different years. The disease occurred all the year around, but mainly during March-October. The 577 cases were distributed in 31 counties (cities/districts) from 6 autonomous prefectures (cities). The prevalence rats of five counties were high, i.e. Menyuan Hui autonomous county (22.88 %, 132/577), Tianjun county (10.57 %, 61/577)、Xining city (10.57 %, 61/577), Henan Mongol Autonomous County (10.51 %, 58/577) and Haiyan county (9.53 %, 55/577). Age of the cases ranged from 8 years to 82 years, and the male to female ratio of the cases was 1.8∶1 (374/203). The prevalence rate in herdsman (47.83 %, 276/577) was highest among different occupational populations. Ten isolates were all Brucella melitensis strains, belonging to biovar 3, and clustering analysis indicated that the 10 strains had 5 genotypes, in which 2 were distinct, the remaining 3 were same. MLVA-16 analysis indicated that the 10 strains had close relationship with 26 B. melitensis strains isolated in Qinghai previously. Conclusions:The prevalence of brucellosis increased in Qinghai in recent years, we should strengthen the population based brucellosis surveillance and reporting. MLVA-16 indicated the gene diversity of the Brucella strains, suggesting that MLVA-16 can be used for genetic diversity analysis and molecular epidemiology survey to improve brucellosis surveillance.

Objective To screen the most suitable medium for Brucella drug susceptibility test, and observe the resistance of human derived Brucella to different antibiotics. Methods Totally 180 strains of Brucella isolated from 25 provinces (municipalities, autonomous regions) in recent years were taken as observation objects. Mueller-Hinton ( MH ) agar , MH blood agar and Brinell agar were used to carried out the drug susceptibility test in vitro, and to compare the results of drug susceptibility test of different medium; the most suitable Brucella drug susceptibility test medium was used to detect the resistance of human derived Brucella to Doxycycline, Rifampicin, Streptomycin, Levofloxacin, Moxifloxacin, Ceftriaxone sodium, Co-trimoxazole and Amoxicillin/Clavulanic acid by K-B drug sensitive paper, and to observe the formation of antibacterial ring around the drug sensitive paper. Results The growth of Brucella on the MH agar and MH blood agar were slower than that on the Brinell agar, and the antibacterial rings were not obvious. All the 180 strains of Brucella were sensitive to seven antibiotics such as Doxycycline, Rifampicin, Streptomycin, Levofloxacin, Moxifloxacin, Ceftriaxone sodium, and Amoxicillin/Clavulanic acid; and 70 strains of Brucella were resistant to Co-trimoxazole, accounting for 39％ (70/180); Brucella strains resistant to Co-trimoxazole were found in 21 provinces. Conclusions Brinell agar is the most suitable medium for Brucella susceptibility test. The human derived Brucella is resistant to Co-trimoxazole; the resistant strains are distributed in 21 provinces ( municipalities , autonomous regions ) . It is recommended that relevant departm ents of the province ( municipalities , autonomous regions ) carry out epidemiological investigations on the resistance of Brucella, and strengthen the monitoring of drug resistance in clinical drugs of brucellosis patients.

Objective: To study the molecular-epidemiological characteristics of Brucella species isolated from different countries, using the multiple locus tandem-repeat (MLVA) analysis. Methods: Eleven variable-number tandem-repeat (VNTR) loci were selected. VNTR strains of Brucella isolated from 48 different countries in 1953-2013, were analyzed by using the BioNumerics software. Unweighted Paired Arithmetic Average method was used to cluster and draw phylogenetic tree as well as the minimum spannin. Results: The evolutionary relationship of Brucella phylogenetic tree was consistent with the classical biological typing method. However, the Brucella suis biovar 5 strains were different from the other Brucella suis biovars 1, 2, 3 and 4. Brucella ceti strains were divided into two parts and different from each other. Worldwide epidemics of brucellosis were emerged from 2005 to 2008 under the MLVA11 Orsay analysis. China has been a brucellosis-prone regions, with Brucella melitensis as the main epidemic Brucella species, followed by Brucella abortus. Brucella suis was mainly identified in the southern provinces, but Brucella canis was mainly found in dogs. No human cases were found. Conclusion Molecular-epidemiological characteristics of the Brucella strains were related to factors as time, region and hosts of isolation, which are important to setting up prevention and control programs on brucellosis.

Objective To analyze the etiological characteristics of human Brucella strains isolated, and to improve the precision in control and prevention of brucellosis. Methods In 2016, blood samples were collected from patients in Jingyuan County Gansu Province, and tested via the Rose-Bengal Plate Agglutination Test (RBPT) and the tube agglutination test methods,and serological positive blood samples were inoculated to bidirectional blood culture bottle for culturing, and further identified by traditional biological classification method and the Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis species-specific PCR (AMOS-PCR). Multiple-locus variable number tandem repeat sequence analysis (MLVA) -16 was used to detect molecular typing and do cluster analysis. Results The isolated strain was identified by the traditional biological classification method, bacteria could grow in thionine and reddened dye, A and M factors agglutination tests were positive, Bk2phage treatment of bacterial strain cracking, but Tb, Wb phages were not cracked. AMOS-PCR amplification result showed a 731 bp band, which was a strain of Brucella melitensis. The results of MLVA-16 showed that there was a difference in the number of repeats on some Variaable Number of Tandem Repeat(VNTR)sites of the isolated strain. Clustering analysis showed that, the isolated strain was clusted into the same clade with the clustering of Brucella melitensis type 3 from GS-201605 in Gansu. And the clustering was similar compared with that of Zhejiang, Guangdong, Fujian and Yunnan. Conclusion Human brucellosis is a inputting transmission in Gansu Province, there is a genetic variation of genotype 3 sheep Brucella between Gansu Province and other domestic provinces.

Objective: To compare the safety of continued warfarin therapy and bridging anticoagulation therapy during hospital stay in patients undergoing percutaneous coronary intervention (PCI). Methods: We retrospectively analyzed patients on warfarin therapy referred for PCI in Beijing Anzhen Hospital from January 2008 to December 2016. The patients were divided into continued warfarin therapy (n=195) or bridging anticoagulation therapy (n=311) groups. After Propensity Score Matching, data from matched patients (n=123 in each group) were analyzed. Bleeding complications and major adverse cardiac events including death, myocardial infarction, target vessel revascularization, and stent thrombosis were assessed. Results: There were no significant difference in the rate of death (2.4%(3/123) vs. 1.6%(2/123),P=0.54), acute myocardial infarction (4.1%(5/123) vs. 4.9%(6/123), P=0.78),re-revascularization (0.8%(1/123) vs. 1.6%(2/123),P=0.16), stent thrombosis (1.6%(2/123) vs. 1.6%(2/123),P=1.00) and stroke between the two groups. Prevalence of minor bleeding complications was significantly higher in the bridging therapy group (15.4%(19/123) vs. 9.8%(12/123),P=0.01). Rate of access-site complications (hematoma:4.1%(5/123) vs. 2.4%(3/123),P=0.20; pseudoaneurysm:2.4%(3/123) vs. 2.4%(3/123),P=1.00; arteriovenous fistula:0.8%(1/123) vs. 1.6%(2/123),P=0.09; and retroperitoneal hematoma:0(0/123) vs. 0.8%(1/123),P=0.23) were similar between the two groups. Conclusion For patients receiving chronic warfarin therapy, the uninterrupted oral anticoagulant treatment is as safe as bridging therapy in PCI patients.