一种羊种布鲁氏菌复方新诺明耐药株荧光定量PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2025.03.044

Abstract

Abstract:

In recent years, strains of Brucella spp. resistant to trimethoprim-sulfamethoxazole have been isolated worldwide. Rapid detection of these resistant strains in samples can guide clinical medication. Currently, there are no rapid detection methods for Brucella trimethoprim-sulfamethoxazole-resistance strains. The aim of this study is to establish a rapid detection method for trimethoprim-sulfamethoxazole-resistance strains of B. melitensis. Based on previous studies, the isolates of B. melitensis with higher minimum inhibitory concentrations were selected. Whole genome sequencing was used to analyze SNPs and InDels, and variation sites were selected for developing a quantitative real-time PCR detection method using MGB modified probes. The results showed that B. melitensis bv.2 strain 63/9 could be the reference genome to analysis resistance strains, and 64 unique SNPs and InDels have been passed. Among them, twelve mutation sites were identified that could be used to establish a rapid detection method for resistant strains. A quantitative real-time PCR detection method developed using the A/G polymorphism at site 2 014 308 of chromosome 1 showed good specificity and could detect as low as 2×10¹ CFU, making it suitable for detecting trimethoprim-sulfamethoxazole-resistance strains in clinical samples. In conclusion, this study established a quantitative real-time PCR detection method for trimethoprim-sulfamethoxazole-resistance B. melitensis strains, providing a scientific basis for the prevention and control of drug resistant strains in China, and can also serve as a reference for clinical medication.

Key words: Brucella melitensis, trimethoprim-sulfamethoxazole, whole genome SNPs, MGB modified probe

CLC Number:

S852.614

YANG Xiaowen, NING Wenqing, ZHOU Shizhong, YUAN Yaqin, HOU Xuexin, DING Jiabo. Establishment of a Quantitative Real-time PCR Detection Method for Trimethoprim-Sulfamethoxazole-resistance Strains of Brucella melitensis[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(3): 1465-1472.

Figures/Tables 5

Table 1

Fig. 1

Fig. 2

Table 2

Fig. 3

References 23

1	NELSON-JONES A . Brucellosis[J]. Postgrad Med J, 1952, 28 (324): 529- 534. doi: 10.1136/pgmj.28.324.529
2	BUKHARI E E . Pediatric brucellosis. An update review for the new millennium[J]. Saudi Med J, 2018, 39 (4): 336- 341. doi: 10.15537/smj.2018.4.21896
3	PAPPAS G , PAPADIMITRIOU P , AKRITIDIS N , et al. The new global map of human brucellosis[J]. Lancet Infect Dis, 2006, 6 (2): 91- 99. doi: 10.1016/S1473-3099(06)70382-6
4	LIU Z G , GAO L P , WANG M , et al. Long ignored but making a comeback: a worldwide epidemiological evolution of human brucellosis[J]. Emerg Microbes Infect, 2024, 13 (1): 2290839. doi: 10.1080/22221751.2023.2290839
5	WANG H Y , LIU H Y , ZHANG Q R , et al. Natural history of and dynamic changes in clinical manifestation, serology, and treatment of brucellosis, China[J]. Emerg Infect Dis, 2022, 28 (7): 1460- 1465.
6	《中华传染病杂志》编辑委员会. 布鲁菌病诊疗专家共识[J]. 中华传染病杂志, 2017, 35 (12): 705- 710. doi: 10.3760/cma.j.issn.1000-6680.2017.12.001
	Editorial Board of Chinese Journal of Infectious Diseases . Expert consensus on the diagnosis and treatment of brucellosis[J]. Chinese Journal of Infectious Diseases, 2017, 35 (12): 705- 710. doi: 10.3760/cma.j.issn.1000-6680.2017.12.001
7	DESHMUKH A , HAGEN F , SHARABASI O A , et al. In vitro antimicrobial susceptibility testing of human Brucella melitensis isolates from Qatar between 2014- 2015[J]. BMC Microbiol, 2015, 15, 121.
8	BARBOSA PAULETTI R , REINATO STYNEN A P , PINTO DA SILVA MOL J , et al. Reduced susceptibility to rifampicin and resistance to multiple antimicrobial agents among Brucella abortus isolates from cattle in Brazil[J]. PLoS One, 2015, 10 (7): e0132532. doi: 10.1371/journal.pone.0132532
9	TORKAMAN ASADI F , HASHEMI S H , ALIKHANI M Y , et al. Clinical and diagnostic aspects of brucellosis and antimicrobial susceptibility of Brucella isolates in Hamedan, Iran[J]. Jpn J Infect Dis, 2017, 70 (3): 235- 238. doi: 10.7883/yoken.JJID.2016.133
10	DADAR M , ALAMIAN S , BRANGSCH H , et al. Determination of virulence-associated genes and antimicrobial resistance profiles in Brucella isolates recovered from humans and animals in Iran using NGS technology[J]. Pathogens, 2023, 12 (1): 82. doi: 10.3390/pathogens12010082
11	杨晓雯, 姜霞, 安翠红, 等. 羊种布鲁氏菌分离株体外抗生素敏感性研究[J]. 疾病监测, 2021, 36 (12): 1286- 1290.
	YANG X W , JIANG X , AN C H , et al. Sensitivity of Brucella melitensis isolates to antibiotics in vitro[J]. Disease Surveillance, 2021, 36 (12): 1286- 1290.
12	杨晓雯, 韩秀瑞, 朴东日, 等. 一株羊种变异布鲁氏菌分离株遗传特征分析[J]. 中国人兽共患病学报, 2020, 36 (11): 894- 899.
	YANG X W , HAN X R , PIAO D R , et al. Genetic characteristics of the Brucella melitensis strain with a rough phenotype[J]. Chinese Journal of Zoonoses, 2020, 36 (11): 894- 899.
13	PRJIBELSKI A , ANTIPOV D , MELESHKO D , et al. Using SPAdes de novo assembler[J]. Curr Protoc Bioinformatics, 2020, 70 (1): e102.
14	DARLING A C E , MAU B , BLATTNER F R , et al. Mauve: multiple alignment of conserved genomic sequence with rearrangements[J]. Genome Res, 2004, 14 (7): 1394- 1403.
15	BOUNAADJA L , ALBERT D , CHÉNAIS B , et al. Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes[J]. Vet Microbiol, 2009, 137 (1-2): 156- 164.
16	SKÖLD O . Sulfonamide resistance: mechanisms and trends[J]. Drug Resist Updat, 2000, 3 (3): 155- 160.
17	田国忠, 崔步云, 赵鸿雁, 等. 一起羊种布鲁氏菌病暴发的流行病学和分子特征研究[J]. 疾病监测, 2017, 32 (3): 211- 215.
	TIAN G Z , CUI B Y , ZHAO H Y , et al. Studies on epidemiology and molecular characteristics of Brucella melitensis during an outbreak of brucellosis[J]. Disease Surveillance, 2017, 32 (3): 211- 215.
18	梁英风, 吴金英, 刘岩, 等. 62株临床分离布鲁氏菌对四种常用抗生素药物敏感性实验[J]. 现代检验医学杂志, 2020, 35 (4): 116-117, 129.
	LIANG Y F , WU J Y , LIU Y , et al. Sensitivity test of 62 strains of Brucella isolated from clinical to four common antibiotics[J]. Journal of Modern Laboratory Medicine, 2020, 35 (4): 116-117, 129.
19	MA H R , XU H J , WANG X , et al. Molecular characterization and antimicrobial susceptibility of human Brucella in Northeast China[J]. Front Microbiol, 2023, 14, 1137932.
20	YUAN H T , WANG C L , LIU L N , et al. Epidemiologically characteristics of human brucellosis and antimicrobial susceptibility pattern of Brucella melitensis in Hinggan League of the Inner Mongolia Autonomous Region, China[J]. Infect Dis Poverty, 2020, 9 (1): 79.
21	刘志国, 王妙, 李晓燕, 等. 内蒙古乌兰察布地区布鲁氏菌体外药物敏感性及耐药机制分析[J]. 中国人兽共患病学报, 2019, 35 (5): 393- 398.
	LIU Z G , WANG M , LI X Y , et al. In vitro drugs susceptibility and resistance mechanism investigation of Brucella isolates from Ulanqab region of Inner Mongolia[J]. Chinese Journal of Zoonoses, 2019, 35 (5): 393- 398.
22	LIU Z G , DI D D , WANG M , et al. In vitro antimicrobial susceptibility testing of human Brucella melitensis isolates from Ulanqab of Inner Mongolia, China[J]. BMC Infect Dis, 2018, 18 (1): 43.
23	赵忠智, 崔步云, 姜海, 等. 人源布鲁菌的药敏试验研究[J]. 中华地方病学杂志, 2019, 38 (7): 536- 540.
	ZHAO Z Z , CUI B Y , JIANG H , et al. Drug sensitivity test of human derived Brucella[J]. Chinese Journal of Endemiology, 2019, 38 (7): 536- 540.

菌株 Strains	组装后长度/bp Assembly	N50长度/bp N50 length	contigs数量 Number	最大contigs长度/bp Max contigs	测序深度(×) Coverage (×)	可用reads比例/% Used reads ratio
RSXT015	3 288 879	298 831	26	609 534	234	97.60
RSXT019	3 288 710	296 830	26	609 502	230	97.01
RSXT089	3 288 684	296 861	26	609 517	229	95.69

序号 Number	序列(5′→3′) Sequence	功能 Function
seq1	5′-TCTTTGTGGGCGGCTATCC-3′	扩增布鲁氏菌检测序列 Amplification sequences of Brucella spp.
seq2	5′-CCGTTCGAGATGGCCAGTT-3′
seq3	5′Cy5-ACGGGCGCAATCT-3′BHQ-1	布鲁氏菌检测探针 Probe for Brucella spp. detection
seq4	5′- CAGGATTTTCCGATCGACCTT-3′	扩增耐药株检测序列 Amplification sequences of resistance strain
seq5	5′- AAGCTGTTCTAAGCGGCACATAG-3′
seq6	5′-VIC- CGGTGGCGACAGA-3′ MGB	敏感株检测探针 Probe for sensitive strain detection
seq7	5′-FAM- TGGCGGCAGATAT-3′ MGB	耐药株检测探针 Probe for resistance strain detection

[1]	WANG Hengtai, LU Lang, JIANG Hui, CHENG Junsheng, LIU Minghe, CHU Yuefeng, XU Jian, LI Peng, DING Jiabo. Biological Function of MgtC Protein in Brucella abortus to Low-Mg²⁺ Resistant Environment [J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 365-377.
[2]	XU Zhenyu, DENG Xiaoyu, WANG Yueli, SUN Can, WU Aodi, CAO Jian, YI Jihai, WANG Yong, WANG Zhen, CHEN Chuangfu. Biological Characteristics of Brucella abortus A19ΔBtpA Deletion Strain and Its Immunogenicity Study [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2135-2145.
[3]	ZHAO Canqi, FENG Yu, LÜ Lang, LI Yanjun, WEI Yulei, DING Jiabo, CHEN Xiang, JIANG Hui. Study on Purification of Bovine Brucellosis by Competitive ELISA and Indirect ELISA [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2146-2153.
[4]	XI Jing, WANG Yueli, DENG Xiaoyu, YANG Qin, LI Peidong, ZHANG Jiangwei, SUN Tianhao, ZHU Liangquan, YI Jihai, CHEN Chuangfu. Effect of STAT6 Mediated Macrophage Polarization on Intracellular Survival of Brucella [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 263-271.
[5]	JIANG Hui, FENG Yu, LI Xiaoying, FAN Xuezheng, PENG Xiaowei, DING Jiabo. Comparative Research of Four High Throughput Antibody Detection Methods for Brucella [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3208-3214.
[6]	MEI Li, WANG Yingchao, CHENG Rujia, YU Guoji, FAN Xuezheng, GAO Xiaolong, GAO Min, QIN Yuming, LI Xiaoying, LI Qiaoling, ZHU Liangquan, FENG Xiaoyu. A Droplet Digital PCR Method for Detection of Brucella [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1753-1759.
[7]	WANG Shuli, ZHANG Huiru, BI Yanqi, WANG Dejuan, CHEN Lin, ZHANG Xiaoting, LI Zhiqiang. Analysis of Immune Responses Induced by Brucella Transcriptional Regulatory Factor HFQ [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1977-1984.
[8]	ZHOU Yucheng, GUO Mengnan, CHENG Shipeng, ZHANG Haiwei, ZHOU Manli, QIAO Lianjiang, YANG Yanling. Differential Analysis of Ubiquitination Modification of Host Immune-related Proteins after Brucella 16M Infection [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(11): 2290-2301.
[9]	NIU Kai, CHENG Rujia, XU Guanlong, FENG Yu, SUN Shijing, FAN Xuezheng, PENG Xiaowei, ZHU Liangquan, QIN Yuming, DING Jiabo, CHANG Weishan, JIANG Hui. Identification and Complete Genomic Analysis of a Brucella Strain Isolated from Deer [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1666-1675.
[10]	YANG Yan-ling, CHENG Yue-ning, ZHANG Ping, ZHOU Yu-cheng, ZHANG Hai-wei, CHENG Shi-peng. Screening and Preliminary Identification of in vivo-induced Genes of Brucella melitensis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1979-1986.
[11]	PENG Wu-li， JI Xin-cheng，YU Xue-hui， SHI Qian,WANG Ke-ke， LI Na. A Multiplex PCR Method for Detecting Brucella, Chlamydia psittaci and Coxiella burnetii of Dairy Cows [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(1): 123-128.
[12]	LIU Qian-hong, HAN Wen-yu. Deep Sequenceing-based Early Transcriptome Analysis of the RAW264.7 Cell with the Brucella Infection [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(11): 1810-1817.
[13]	XIAN Dong-Bao, GAO Ming-Chun, CAO Di-Fei, WANG Xiao-Dong, WANG Jun-Wei. Identification of Linear B-cell Epitope of Structural Protein BP26 of Brucella abortus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(9): 1444-1448.
[14]	ZHANG Yu, ZHANG Hui, ZHANG Yan, MENG Ru, WANG Zhen, CHEN Rui-Hua, ZHANG Jun-Bo, LU Zhi-Zi, CHEN Chuang-Fu. The Effect of Cellular Inflammatory Response by Glycosyltransferase of Brucella [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(9): 1437-1443.

Establishment of a Quantitative Real-time PCR Detection Method for Trimethoprim-Sulfamethoxazole-resistance Strains of Brucella melitensis

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 5

References 23

Related Articles 14

Recommended Articles

Metrics

Comments