Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (16): 3394-3403.doi: 10.3864/j.issn.0578-1752.2020.16.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Construction of lpxM Gene Deletion Strain of Haemophilus parasuis and It's Some Biological Characteristics

YANG Jun1,2(),CHU PinPin2,SONG Shuai2,CAI RuJian2,YANG DongXia2,BIAN ZhiBiao2,GOU HongChao2,LI Yan2,JIANG ZhiYong2,LI ChunLing2(),YAN He1()   

  1. 1School of Food Sciences and Engineering, South China University of Technology, Guangzhou 510640
    2Institute of Animal Health, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of Livestock Disease Prevention/Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province , Guangzhou 510640
  • Received:2019-09-03 Accepted:2019-12-05 Online:2020-08-16 Published:2020-08-27
  • Contact: ChunLing LI,He YAN E-mail:1048494572@qq.com;lclclare@163.com;573790583@qq.com

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Abstract:

【Objective】The effects of lpxM, a gene related to lipopolysaccharide synthesis of Haemophilus parasuis, on its some biological characteristics such as growth, biofilm formation ability, antibacterial ability against 50% porcine serum, virulence to macrophage and antibiotic susceptibility were studied, which laid a certain theory for revealing the pathogenesis of HPS and the construction of lpxM gene deletion vaccine, and also provided a basis for the selection of drugs for the prevention and treatment of HPS on pig farms.【Method】The highly pathogenic serotype 5 of HPS local isolate H45 was used as the research object, and the suicide plasmid PK18mobsacB was used as a vector. The constructed recombinant plasmid was transformed into H45 by natural transformation method, and homologous recombination occurred under the pressure of antibiotics. Finally, the lpxM gene deleted strain H45-ΔlpxM was obtained by antibiotic screening, and verified by PCR and sequencing. The differences in biological characteristics between the two were compared. The OD600-t curve of the two was used to compare the growth conditions. The ability of the two to form biofilm formation after 24 hours of culture was compared by crystal violet staining. The survival rate of the two in 50% porcine serum was compared, and the antiserum complements of the two were compared. The two both simultaneously stimulated mouse monocyte macrophage RAW264.7 cells for 6, 12 and 24h, and detected the release of LDH in cell culture supernatant for comparing the virulence of macrophages. The difference in antibiotic susceptibility was studied by KB diffusion method. Antibiotics included 13 antibiotics in clinical practice, such as ampicillin and polymyxin B antibiotics. The sensitivity to antibiotics was determined by measuring the diameter of the inhibition zone according to the resistance standard. 【Result】 The lpxM gene deletion strain H45-ΔlpxM was successfully constructed. The growth of the deleted strain was found to be slower than the wild-type strain in the early growth stage, but they were consistent after 8 hours. The results showed that the lpxM gene deletion could inhibit the growth of H45 to some extent. The two both could form biofilm, but the deleted strain was weaker than the wild strain. The wild strain had a survival rate of 16.1% in 50% porcine serum, while the deletion strain was only 0.71%, and the deletion strain was significantly lower than the wild strain. The two both could cause macrophage death. The lethal rate of wild-type plants was 6.63%, 10.86% and 22.17%, after 6, 12 and 24h, respectively, while the lethal rate of the deleted strain was 2.62%, 6.35% and 18.01%, respectively. With the prolongation of action time, the virulence effect was more obvious, and had a certain time dependence. At each time point, the virulence of the deleted strains was lower than that of the wild strain. The antibiotic sensitivity results showed sensitive to ten antibiotics such as thiophene and showed resistance to enrofloxacin. But the resistance to amoxicillin clavulanic acid, sulfamethoxazole and ampicillin changed greatly. Amoxicillin clavulanic acid and sulfamethoxazole became sensitive from resistance and ampicillin also changed from intermediate to sensitive. The results showed that lpxM gene deletion had certain influence on some antibiotic sensitivity of H45.【Conclusion】 The deletion of lpxM gene could inhibit growth of HPS to a certain extent, reduce its biofilm formation ability, anti-serum bactericidal ability and virulence to macrophages, and increase sensitivity to some commonly used antibiotics, revealing that lpxM gene might be the virulence gene of HPS and be closely related to the pathogenic ability of HPS, but the specific mechanism needed further study.

Key words: Haemophilus parasuis, lpxM gene, biofilm, anti-serum bactericidal, cytotoxicity, antibiotic resistance

Table 1

Primers sequence"

引物Primers 序列(5′→3′) Sequence 长度 Length
kan-F CATTGCACAAGATAAAAATATAT 909 bp
kan-R CAATTAACCAATTCTGATTAG
lpxM-up-EcoRI-F CCGGAATTCACCGCTTGT CCTACAACCTGTTTCGCC 489 bp
lpxM-up-kan-R TATTTTTATCTTGTGCAATG CAACATTGCCAGGGCTAG
lpxM-kan-down -F CTAATCAGAATTGGTTAATTG AGGTCGCCAGCTTATCAC 593 bp
lpxM-down-PstI-R TAACTGCAGACAAGCGGT CCGTATTCCGATTTATGTG
HPS-F F1：TATCGGGAGATGAAAGAC
F2：GTAATGTCTAAGGACTAG		 1090 bp
HPS-R CCTCGCGGCTTCGTC
sacB-F GCAGGAGGCGCAACTCAA 986 bp
sacB-R ATTTTAAAGACGTTCGCGC

Fig. 1

Construction of recombinant fragments marker;1: kanR resistance expression cassette (909 bp); 2: lpxM-up upstream homology arm (489 bp); 3: lpxM-down downstream homology arm (593 bp); 4: M-UKD recombinant fragment (1991 bp)"

Fig. 2

Identification of recombinant plasmid PKM-UKD by double enzyme digestion marker;1,2,3,4：Double digestion vector (5719 bp) and fragment(1991 bp)"

Fig. 3

Identification of the lpxM gene deletion strain M: DL2000 DNA marker; 1-3: Identification of the deletion fragments, the deletion strain, wild strain and positive control, primer lpxM-up-EcoRI-F/ lpxM-down-PstI-R ; 4-6: Identification of the kanR resistance expression cassette, the deletion strain ,wild strain and positive control, primer kanR-F/R; 7-9: Identification of plasmid elimination, the deletion strain, wild strain and positive control, primer sacB-F/R"

Fig. 4

Identification of the HPS M: DL2000 DNA marker; 1-3: the deletion strain; +: wild strain, primer HPS-F/R"

Fig. 5

Growth curves of wild and deletion strains"

Fig. 6

Biofilm formation 1-3: wild strains、deletion strains and blank control"

Fig. 7

Biofilm OD630 value"

Fig. 8

Survival of HPS treated with porcine serum"

Fig. 9

Cell cytotoxicity test"

Table 2

Results of drug sensitivity (antimicrobial zone diameter mm)"

抗生素名称
Antimicrobial agents		 H45 H45-△lpxM 抗生素敏感性判断标准
Standard for antibiotic sensitivity
氨苄西林ampicillin 20.6（I） 26（S） R：<=18;I：19-21;S：>22
阿莫西林克拉维酸amoxicillin clavulantic acid 16.8（R） 22.2（S） R：<=19;I：--;S：>20
头孢噻吩cephalothin 25.5（S） 37（S） R：<=14;I：15-17;S：>=18
头孢氨苄cephalexin 19（S） 25（S） R：<=14;I：15-17;S：>=18
头孢唑啉cefazolin 23（S） 30（S） R：<=14;I：15-17;S：>=18
头孢拉定cefradine 20（S） 27.5（S） R：<=14;I：15-17;S：>=18
头孢噻肟cefotaxime 35（S） 26（S） R：<=14;I：15-17;S：>=18
庆大霉素gentamicin 22（S） 26.8（S） R：<=12;I：13-14;S：>=15
多西环素doxycycline 20（S） 22.4（S） R：<=12;I：13-15;S：>=16
磺胺二甲异噁唑sulfisoxazole 10（R） 19（S） R：<=12;I：13-16;S：>=17
氟苯尼考florfenicol 29（S） 35（S） R：<=12;I：13-17;S：>=18
阿莫西林amoxicillin 20（S） 23（S） R：--;I：--;S：19-25
恩诺沙星enrofloxacin 16（R） 21（R） R：--;I：--;S：28-36
多粘菌素B polymyxin B 18.9（S） 23.4（S） R：<=8;I：8-11;S：>=12
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