猪β防御素-2对猪源肠外致病性大肠杆菌的体内外抑菌活性研究

doi:10.16431/j.cnki.1671-7236.2021.02.035

摘要/Abstract

摘要： 本研究旨在评价猪β防御素2（porcine beta defensin 2，PBD-2）在体内外对猪源肠外致病性大肠杆菌（ExPEC）的抗菌效果，为评估PBD-2在抗生素替代品中的应用前景提供参考。首先，在体外检测不同浓度PBD-2对猪源ExPEC PCN033的杀菌活性。随后，选取5周龄，体重在18~22 g之间的雌性昆明小鼠，检测PBD-2处理组和PBS对照组小鼠（n ≥ 5）感染不同剂量的ExPEC PCN033后的存活率，脑、脾脏、肺脏组织和血液中的载菌量、炎性细胞因子白细胞介素-6（IL-6）、IL-12、IL-1β和肿瘤坏死因子-α（TNF-α）的水平及脑、脾脏、肺脏组织的病理变化程度。结果表明，PBD-2在25 μg/mL时即可在体外极显著抑制猪源ExPEC PCN033的生长（P<0.01），且抑制作用随PBD-2的浓度升高而增强。在体内，与PBS对照组相比，PBD-2处理有效降低了小鼠感染不同剂量ExPEC PCN033后的死亡率。提高PBD-2的治疗剂量对降低小鼠死亡率的效果更加明显。腹腔注射和肌内注射的方式在PBD-2降低小鼠死亡率的效果方面优于口服途径。PBD-2治疗在降低小鼠死亡率的效果方面略低于氯霉素治疗。同时，PBD-2治疗极显著降低了小鼠感染ExPEC PCN033 21 h后的脑、脾脏、肺脏组织和血液中的细菌载量（P<0.01），降低了血液中的IL-6、IL-12和IL-1β的含量（P<0.01），减轻了脑、脾脏和肺脏组织的病变程度。上述结果说明PBD-2在体外具有良好的抗猪源ExPEC的活性，同时在小鼠体内对猪源ExPEC感染具有治疗作用，表明PBD-2具有开发成为治疗性药物或者抗生素替代品的潜力。

关键词: 猪β防御素2(PBD-2); 细菌感染; 治疗; 肠外致病性大肠杆菌(ExPEC)

Abstract: The purpose of this study was to evaluate the antibacterial activity of porcine beta defensin 2 (PBD-2) against porcine extraintestinal pathogenic Escherichia coli (ExPEC) in vivo and in vitro,and provide more insights for evaluating the value of PBD-2 as an antibiotic substitutes.Firstly,the bactericidal activity of different concentrations of PBD-2 against the porcine ExPEC PCN033 was tested in vitro.Subsequently,female Kunming mice aged 5 weeks and weighing between 18-22 g infected with different doses of ExPEC PCN033 were treated with PBD-2 or PBS (n ≥ 5).The survival rate,the bacteria loads of brain,spleen,lung tissue and blood,the levels of inflammatory cytokines IL-6,IL-12,IL-1β and TNF-α and the pathological changes of brain,spleen and lung tissues were observed.The results showed that 25 μg/mL of PBD-2 could extremely significantly inhibit the growth of ExPEC PCN033 in a concentration-dependent manner (P<0.01).The in vivo study revealed that PBD-2 treatment could effectively reduce the mortality rate of mice infected with ExPEC PCN033.The effects of PBD-2 were enhanced with increase of treatment dose of PBD-2.The effects of PBD-2 via intraperitoneal injection and intramuscular injection route were better than that of the oral route.PBD-2 treatment was less effective than chloramphenicol treatment in reducing the mortality of mice.At the same time,PBD-2 treatment extremely significantly reduced the bacteria loads in the brain,spleen,lung tissue and blood of mice at 21 h post infection with ExPEC PCN033 (P<0.01).At the same time,PBD-2 treatment extremely significantly reduced the levels of IL-6,IL-12 and IL-1β of mice at 21 h post infection with PCN033 (P<0.01).PBD-2 treatment also significantly reduced the degree of pathological damages of the brain,lung and spleen tissues of mice at 21 h post infection with ExPEC PCN033.These results indicated that PBD-2 had good activity against porcine ExPEC in vitro,and had therapeutic effect on swine derived ExPEC infection in mice,indicating that PBD-2 had the potential to develop into therapeutic drugs or antibiotic substitutes.

Key words: porcine beta defensin 2 (PBD-2); bacterial infection; treatment; extraintestinal pathogenic Escherichia coli (ExPEC)

中图分类号:

S855.1

王安田, 黄靖, 孙瑜凡, 宋炳晓, 谭臣, 黄琦, 周锐, 黎璐. 猪β防御素-2对猪源肠外致病性大肠杆菌的体内外抑菌活性研究[J]. 中国畜牧兽医, 2021, 48(2): 726-735.

WANG Antian, HUANG Jing, SUN Yufan, SONG Bingxiao, TAN Chen, HUANG Qi, ZHOU Rui, LI Lu. Antibacterial Activity of Porcine Beta Defensin-2 Against Porcine Extraintestinal Pathogenic Escherichia coli in vivo and in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 726-735.

参考文献

[1] ALLEN H K,LEVINE U Y,LOOFT T,et al.Treatment,promotion,commotion:Antibiotic alternatives in food-producing animals[J].Trends in Microbiology,2013,21(3):114-119.
[2] KHURSHID Z,ZAFAR M S,NASEEM M,et al.Human oral defensins antimicrobial peptides:A future promising antimicrobial drug[J].Current Pharmaceutical Design,2018,24(10):1130-1137.
[3] VELDHUIZEN E J,VAN D A,TERSTEEG M H,et al.Expression of beta-defensins pBD-1 and pBD-2 along the small intestinal tract of the pig:Lack of upregulation in vivo upon Salmonella Typhimurium infection[J].Molecular Immunology,2007,44(4):276-283.
[4] VELDHUIZEN E J,RIJNDERS M,CLAASSEN E A,et al.Porcine beta-defensin 2 displays broad antimicrobial activity against pathogenic intestinal bacteria[J].Molecular Immunology,2008,45(42):386-394.
[5] HUANG J,QI Y H,WANG A T,et al.Porcine β-defensin 2 inhibits proliferation of pseudorabies virus in vitro and in transgenic mice[J].Virology Journal,2020,17(1):18.
[6] PENG Z X,WANG A R,XIE L Q,et al.Use of recombinant porcine β-defensin 2 as a medicated feed additive for weaned piglets[J].Scientific Report,2016,6:26790.
[7] LLNDSTEDT B A,FINTON M D,PORCELLATO D,et al.High frequency of hybrid Escherichia coli strains with combined intestinal pathogenic Escherichia coli (IPEC) and extraintestinal pathogenic Escherichia coli (ExPEC) virulence factors isolated from human faecal samples[J].BMC Infectious Disease,2018,18(1):544.
[8] MANGES A R,GEUMH M,GUO A,et al.Global extraintestinal pathogenic Escherichia coli (ExPEC) lineages[J].Clinical Microbiology Reviews,2019,32(3):e00135-18.
[9] TANF X,TAN C,ZHANG X,et al.Antimicrobial resistances of extraintestinal pathogenic Escherichia coli isolates from swine in China[J].Microbial Pathogenesis,2011,50(5):207-212.
[10] TAN C,XU Z,ZHENG H,et al.Genome sequence of a porcine extraintestinal pathogenic Escherichia coli strain[J].Journal of Bacteriology,2011,193(18):5038.
[11] ZONG B,ZHANG Y,WANG X,et al.Characteri-zation of multiple type-Ⅵ secretion system (T6SS) VgrG proteins in the pathogenicity and antibacterial activity of porcine extra-intestinal pathogenic Escherichia coli[J].Virulence,2019,10(1):118-132.
[12] HUANG C,YANG X,HUANG J,et al.Porcine beta-defensin 2 provides protection against bacterial infection by a direct bactericidal activity and alleviates inflammation via interference with the TLR4/NF-κB pathway[J].Frontiers in Immunology,2019,10:1673.
[13] CASTANON J I.History of the use of antibiotic as growth promoters in European poultry feeds[J].Poultry Science,2007,86(11):2466-2471.
[14] JENSEN H M.Health management with reduced antibiotic use-experiences of a Danish pig vet[J].Animal Biotechnology.2006,17(2):189-194.
[15] ZASLOFF M.Antimicrobial peptides of multicellular organisms:My perspective[J].Advances in Experi-mental Medicine and Biology,2019,1117:3-6.
[16] CHEN R B,ZHANG K,ZHANG H,et al.Analysis of the antimicrobial mechanism of porcine beta defensin 2 against E.coli by electron microscopy and differentially expressed genes[J].Scientific Reports,2018,8(1):14711.
[17] DENG Z,WANG J,LYU W T,et al.Development of a cell-based high-throughput screening assay to identify porcine host defense peptide-inducing compounds[J].Journal of Immunology Research,2018,2018:5492941.
[18] 张炜,杭柏林,司素锦,等.抗菌肽BSN-37的抑菌活性及其稳定性分析[J].中国畜牧兽医,2019,46(1):287-295. ZHANG W,HANG B L,SI S J,et al.Antibacterial activity and stability analysis of antibacterial peptide BSN-37[J].China Animal Husbandry & Veterinary Medicine,2019,46(1):287-295.(in Chinese)
[19] 单玉雪,杨娜,滕达,等.抗菌肽NZ2114对来源于奶牛乳房炎的无乳链球菌及其生物膜的消杀作用[J].中国畜牧兽医,2020,47(7):2284-2294. SHAN Y X,YANG N,TENG D,et al.Antibacterial effect of antibacterial peptide NZ2114 on Streptococcus agalactiae and its biofilm derived from dairy cow mastitis[J].China Animal Husbandry & Veterinary Medicine,2020,47(7):2284-2294.(in Chinese)
[20] BROGDEN K A.Antimicrobial peptides:Pore formers or metabolic inhibitors in bacteria?[J].Nature Reviews. Microbiology,2005,3(3):238-250.
[21] TOKE O.Antimicrobial peptides:New candidates in the fight against bacterial infections[J].Biopolymers,2005,80(6):717-735.
[22] KANG H K,KIM C,SEO C H,et al.The therapeutic applications of antimicrobial peptides (AMPs):A patent review[J].Journal of Microbiology,2017,55(1):1-12.
[23] RIVAS-SANTIAGO B,CASTANEDA-DELGADO J E,RIVAS-SANTIAGO C E,et al.Ability of innate defence regulator peptides IDR-1002,IDR-HH2 and IDR-1018 to protect against Mycobacterium tuberculosis infections in animal models[J].PLoS One,2013,8(3):e59119.
[24] 赵飞,滕达,杨娜,等.抗菌肽NZ2114对多重耐药2型猪链球菌感染小鼠的治疗效果评价[J].中国畜牧兽医,2018,45(4):1089-1097. ZHAO F,TENG D,YANG N,et al.Evaluation of the therapeutic effect of antimicrobial peptide NZ2114 on mice with multi-drug resistant Streptococcus suis infection[J].China Animal Husbandry & Veterinary Medicine,2018,45(4):1089-1097.(in Chinese)
[25] 史瑞军,高静.抗菌肽Gal-4对细菌感染小鼠的预防及治疗研究[J].当代畜牧,2019,4:53-54. SHI R J,GAO J.Antibacterial peptide Gal-4 on the prevention and treatment of bacterial infection in mice[J].Contemporary Animal Husbandry,2019,4:53-54.(in Chinese)
[26] WANG S,ZENG X,YANG Q,et al.Antimicrobial peptides as potential alternatives to antibiotics in food animal industry[J].International Journal of Molecular Sciences,2016,17(5):603.
[27] 李瑞芳,李超楠,陆志方,等.抗菌肽CGA-N12对腹腔注射热带念珠菌小鼠的治疗作用[J].动物医学进展,2017,38(4):62-67. LI R F,LI C N,LU Z F,et al.Therapeutic effect of antibacterial peptide CGA-N12 on mice injected with Candida tropicalis[J].Progress in Veterinary Medicine,2017,38(4):62-67.(in Chinese)
[28] HAN F F,ZHANG H W,XIA X,et al.Porcine β-defensin 2 attenuates inflammation and mucosal lesions in dextran sodium sulfate-induced colitis[J].Journal of Immunology,2015,194(4):1882-1893.
[29] 丁一.猪源肠外致病性大肠杆菌流行病学调查及Ydiv功能研究[D].武汉:华中农业大学,2018. DING Y.Epidemiological survey of porcine extraintestinal pathogenic Escherichia coli and Ydiv function study[D].Wuhan:Huazhong Agricultural University,2018.(in Chinese)
[30] LIU C,CHEN Z,TAN C,et al.Immunogenic characterization of outer membrane porins OmpC and OmpF of porcine extraintestinal pathogenic Escherichia coli[J]. FEMS Microbiology Letters,2012,337(2):104-111.
[31] 李悦.猪源肠外致病性大杆菌耐药相关研究[D].武汉:华中农业大学,2016. LI Y.Study on drug resistance of extraintestinal pathogenic Escherichia coli[D].Wuhan:Huazhong Agricultural University,2016.(in Chinese)
[32] BENINCASA M,PELILLO C,ZORZET S,et al.The proline-rich peptide Bac7(1-35) reduces mortality from Salmonella Typhimurium in a mouse model of infection[J].BMC Microbiology,2010,10:178.