Bacterial Typing Techniques ; methods ; Brucella ; classification

To investigate the the possibility to utilize the cellular fatty acid (CFA) information as a method in Brucella typing, 90 Brucella strains were subjected to the study on CFAs, and all the experimental strains were inoculated on Brucella Agar plates for 48 hours. After that, cells were harvested, saponificated, methylated and extracted to provide fatty acids methylesters for gas chromatography analysis. Based on the CFAs data matrix, dendrogram of 90 experimental strains was generated by SPSS11.5 software package. As shown in the dendrogram, 90 Brucella strains could be divided into 5 clusters. The first cluster included some species of Brucella abortus,Brucella melitensis,Brucella suis, Brucella ovis; and some of the variant strains of Brucella abortus and Brucella melitensis and the typical strain of Brucella neotomae. The second cluster included typical strains of Brucella suis (1,2,3 and 5 types); vaccine strains of Brucella suis S2; vaccine strains of Brucella melitensis M28、Rev.1 and typical strain of Brucella ovis. The third cluster included some of Brucella melitensis; some of the variant strains of Brucella melitensis; some of Brucella abortus(3,6 types); Brucella canis and Brucella ovis. The fourth cluster was the typical strain of Brucella canis.and the fifth cluster included some of Brucella melitensis(1 type); some of Brucella abortus (1 type); some of the variant strains of Brucella melitensis and Brucella suis(1,3 type). It is apparent that CFAs information can be used in brucella typing. and Brucella suis and Brucella canis can be distinguished by the difference in the CFA contents of 3 fatty acids 19:0CYCLOω8c, 18:1ω7c and 16:0. The results of CFAs typing in Brucella species show that Brucella canis includes 2 biovars at least and the high homologization of Brucella abortus (3 type) and Brucella abortus(6 type) can be found.

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 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 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.