犬猫皮肤病细菌性病原分离鉴定及天然产物抑菌效果分析

doi:10.16431/j.cnki.1671-7236.2022.04.038

摘要/Abstract

摘要： 【目的】分离鉴定武汉市患皮肤病犬猫细菌性病原,并探索其对传统抗菌药物与天然活性产物藤黄酸(GA)和6-溴靛玉红-3’-肟(BIO)的敏感性。【方法】对患皮肤病犬猫采样并分离病原,通过生长特性观察、革兰氏染色镜检、PCR等方法鉴定并利用SPF小鼠验证致病性;通过药敏纸片验证其对传统药物的耐药性,并测定天然产物对其最小抑菌浓度(minimum inhibitory concentration,MIC)值。【结果】分离得到2株金黄色葡萄球菌、3株伪中间型葡萄球菌、2株猫葡萄球菌、1株犬链球菌及1株奇异变形杆菌。SPF小鼠皮肤创伤感染验证分离菌株均有致病性。犬链球菌及奇异变形杆菌对各自受试药物均敏感;葡萄球菌对复方新诺明、青霉素、红霉素、四环素、左氧氟沙星、苯唑西林、庆大霉素、克林霉素及氯霉素存在不同程度耐药。天然活性产物GA和BIO对上述9株菌均具有良好抑菌效果,且除分离菌株F5外GA对分离菌株的MIC值均小于BIO。【结论】本研究共分离得到5种、9株犬猫皮肤细菌。犬链球菌、奇异变形杆菌对传统抗菌药物均敏感,部分葡萄球菌存在耐药。GA和BIO对犬猫皮肤病原菌均有明显抑菌活性,显示其可作为防控犬猫细菌性皮肤病的候选药物。

关键词: 犬猫; 皮肤病; 病原菌; 分离鉴定; 天然产物; 抑菌效果

Abstract: 【Objective】 The purpose of the experiment was to isolate and identify the bacterial pathogens of dogs and cats suffering from skin diseases in Wuhan,and to explore their sensitivity to traditional antibiotics and natural active products gambogic acid (GA) and 6-bromoindirubin-3’-oxime (BIO).【Method】 Skin pathogen bacteria from dogs and cats were collected and isolated,as well as growth characteristics,Gram staining microscope observation and PCR were determined.SPF mice were used to verify pathogenicity.The drug resistance to traditional drugs was verified by drug sensitive paper,and the minimum inhibitory concentration (MIC) of natural products was measured.【Result】 2 strains of Staphylococcus aureus,3 strains of Staphylococcus pseudointermediate,2 strains of Staphylococcus felis,1 strain of Streptococcus canis,and 1 strain of Proteus mirabillis were isolated.The pathogenicity was verified by skin infection in SPF mice.Streptococcus canis and Proteus mirabillis were sensitive to the tested drugs.Staphylococcus aureus had different resistance to cotrimoxazole,penicillin,erythromycin,tetracycline,levofloxacin,oxacillin,gentamicin,clindamycin and chloramphenicol.The natural active products of GA and BIO had good antibacterial effects on the pathogenic bacteria,and except strain F5,the MIC of GA was universally lower than that of BIO.【Conclusion】 In the study,a total of 9 pathogenic bacteria were isolated from dogs and cats.Streptococcus canis and Proteus mirabilis were sensitive to traditional antimicrobial agents,and some Staphylococcus strains were resistant to them.GA and BIO had obvious antibacterial effects against skin pathogenic bacteria.It showed that GA and BIO could be used as potential antibacterial drugs to prevent and control skin bacterial disease in dogs and cats.

Key words: dogs and cats; skin disease; pathogenic bacteria; isolation and identification; natural products; antibacterial effect

中图分类号:

S852.61

赵天睿, 邵锦华, 王朝好, 严蕊, 雷镒妃, 刘晓雅, 胡长敏. 犬猫皮肤病细菌性病原分离鉴定及天然产物抑菌效果分析[J]. 中国畜牧兽医, 2022, 49(4): 1556-1567.

ZHAO Tianrui, SHAO Jinhua, WANG Chaohao, YAN Rui, LEI Yifei, LIU Xiaoya, HU Changmin. Isolation and Identification of Skin Pathogenic Bacteria from Dogs and Cats and Analysis of Antibacterial Effect of Natural Products[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1556-1567.

参考文献

[1] 杨涵琪,唐锋莉,马新武,等.西安市970例犬皮肤病的病例分析[J].动物医学进展,2020,41(4):127-131. YANG H Q,TANG F L,MA X W,et al.Case analysis of 970 dog dermatosis cases in Xi’an[J].Progress in Veterinary Medicine,2020,41(4):127-131.(in Chinese)
[2] DEVRIESE L A,VANCANNEYT M,BAELE M,et al.Staphylococcus pseudintermedius sp.nov.,a coagulase-positive species from animals[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(Pt 4):1569-1573.
[3] MORIELLO K A.Zoonotic skin diseases of dogs and cats[J].Animal Health Research Reviews,2003,4(2):157-168.
[4] HAMMOND D,CHAUDHRY A,ANDERSON D,et al.Postsurgical pyoderma gangrenosum after breast surgery:A plea for early suspicion,diagnosis,and treatment[J].Aesthetic Plastic Surgery,2020,44(6):2032-2040.
[5] 艾景彪.犬猫常见皮肤病及防治[J].畜牧兽医科技信息,2019,6:149-150. AI J B.Common skin diseases in dogs and cats and their prevention and treatment[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2019,6:149-150.(in Chinese)
[6] 张瑜.宠物皮肤致病菌抗菌肽的筛选及纳米乳的制备[D].长春:吉林大学,2018. ZHANG Y.Screening of antimicrobial peptides from pathogenic bacteria of pet skin and preparation of nanoemulsion[D].Changchun:Jilin University,2018.(in Chinese)
[7] 曹珍,薛璇玑,张新新,等.临床常见耐药细菌及其天然抗生素增效剂的研究进展[J].中草药,2020,51(22):5868-5876. CAO Z,XUE X J,ZHANG X X,et al.Research progress of common clinical resistant bacteria and natural antibiotic synergists[J].Chinese Traditional and Herbal Drugs,2020,51(22):5868-5876.(in Chinese)
[8] 黄梅,谭余庆,罗俊,等.植物类中药抗细菌耐药性的研究进展[J].中国实验方剂学杂志,2018,24(23):218-224. HUANG M,TAN Y Q,LUO J,et al.Research progress on antibacterial resistance of plant Chinese medicine[J].Chinese Journal of Experimental Traditional Medical Formulae,2018,24(23):218-224.(in Chinese)
[9] PERIASAMY H,ISWARYA S,PAVITHRA N,et al.In vitro antibacterial activity of plumbagin isolated from Plumbago zeylanica L.against methicillin-resistant Staphylococcus aureus[J].Letters in Applied Microbiology,2019,69(1):41-49.
[10] HAMOUD R,REICHLING J,WINK M.Synergistic antibacterial activity of the combination of the alkaloid sanguinarine with EDTA and the antibiotic streptomycin against multidrug resistant bacteria[J].Journal of Pharmacy and Pharmacology,2015,67(2):264-273.
[11] AELENEI P,RIMBU C M,GUGUIANU E,et al.Coriander essential oil and linalool-interactions with antibiotics against Gram-positive and Gram-negative bacteria[J].Letters in Applied Microbiology,2019,68(2):156-164.
[12] 彭清,孙柳,苗雪圆,等.天然产物化合物作为肿瘤免疫调节剂的研究进展[J].中国中西医结合外科杂志,2021,27(1):136-140. PENG Q,SUN L,MIAO X Y,et al.Research progress of natural product compounds as tumor immunomodulators[J].Chinese Journal of Surgery of Integrated Traditional and Western Medicine,2021,27(1):136-140.(in Chinese)
[13] YI T F,YI Z F,CHO S G,et al.Gambogic acid inhibits angiogenesis and prostate tumor growth by suppressing vascular endothelial growth factor receptor 2 signaling[J].Cancer Research,2008,68(6):1843-1850.
[14] JANG S W,OKADA M,SAYEED I,et al.Gambogic amide,a selective agonist for TrkA receptor that possesses robust neurotrophic activity,prevents neuronal cell death[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(41):16329-16334.
[15] JIE C,LUO Z,CHEN H,et al.Indirubin,a bisindole alkaloid from isatis indigotica,reduces H1N1 susceptibility in stressed mice by regulating MAVS signaling[J].Oncotarget,2017,8(62):105615-105629.
[16] GUO D H,CHENG L Z,SHEN Y,et al.6-bromoindirubin-3'-oxime (6BIO) prevents myocardium from aging by inducing autophagy[J].Aging,2020,12(24):26047-26062.
[17] 赖金伦.中药单体靛玉红抗LPS诱导的小鼠乳腺炎作用及机制研究[D].武汉:华中农业大学,2017. LAI J L.Anti-tiflammtory effect and mechanism of indiruby in LPS-induced mouse mastitis[D].Wuhan:Huazhong Agricultural University,2017.(in Chinese)
[18] 唐鑫.藤黄酸对LPS诱导的小鼠乳腺炎的作用及机制研究[D].武汉:华中农业大学,2020. TANG X.Effect and mechanism of gambogic acid on LPS-induced mastitis in mice[D].Wuhan:Huazhong Agricultural University,2020.(in Chinese)
[19] MCCLURE J A,CONLY J M,LAU V,et al.Novel multiplex PCR assay for detection of the staphylococcal virulence marker Panton-Valentine leukocidin genes and simultaneous discrimination of methicillin-susceptible from -resistant staphylococci[J].Journal of Clinical Microbiology,2006,44(3):1141-1144.
[20] 毕水莲.食源性变形杆菌属PCR检测方法的研究[D].广州:暨南大学,2008. BI S L.Detectation methods in food-brone Proteus by polymerase chain reation[D].Guangzhou:Jinan University,2008.(in Chinese)
[21] 邹君,王楷宬,田莉莉,等.链球菌猪主要致病种和型多重PCR检测方法的建立[J].中国兽医科学,2011,41(7):701-706. ZOU J,WANG K C,TIAN L L,et al.Establishment of multiplex PCR detection method for main pathogenic species and types of Streptococcus suis[J].Chinese Veterinary Science,2011,41(7):701-706.(in Chinese)
[22] 赵霞玲,肖琦,钱雯娴,等.藏猪产色葡萄球菌分离鉴定及生物学特性研究[J].中国畜牧兽医,2020,47(9):3014-3021. ZHAO X L,XIAO Q,QIAN W X,et al.Isolation,identification and biological characteristics of Staphylococcus chromogenes from Tibetan pigs[J].China Animal Husbandary & Veterinary Medicine,2020,47(9):3014-3021.(in Chinese)
[23] 葛强,马东鑫,周雨晴,等.猪源奇异变形杆菌的分离鉴定及特征分析[J].中国畜牧兽医,2021,48(5):1804-1815. GE Q,MA D X,ZHOU Y Q,et al.Isolation,identification and characterization of Proteus mirabilis from pigs[J].China Animal Husbandary & Veterinary Medicine,2021,48(5):1804-1815.(in Chinese)
[24] 文德亮,郭志波,何随彬,等.猪群链球菌携带情况和关节病分离株毒力基因分析[J].上海畜牧兽医通讯,2016,5:6-10. WEN D L,GUO Z B,HE S B,et al.Carrying status of Streptococcus suis and virulence gene analysis of joint disease isolates[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2016,5:6-10.(in Chinese)
[25] LWENSTEIN C.Pyoderma in dogs[J].Tierrztl Prax,2011,39(6):405-417.
[26] 金亮,戴鹏秀,张欣珂,等.派乐菲魔法洗和洗必灵对犬猫皮肤致病菌的抑菌效果研究[J].动物医学进展,2019,40(2):124-130. JIN L,DAI P X,ZHANG X K,et al.Study on Anti-microbial Effects of LOVET HexZole Shampoo and LOVET Ultra Hex on skin pathogenic fungi and bacteria in dogs and cats[J].Progress in Veterinary Medicine,2019,40(2):124-130.(in Chinese)
[27] BAUMER W,JACOBS M,TAMAMOTO M C.Efficacy study of a topical treatment with a plant extract with antibiofilm activities using an in vivo model of canine superficial pyoderma[J].Veterinary Dermatology,2020,31(2):86-89.
[28] PESAVENTO P A,BANNASCH M J,BACHMANN R,et al.Fatal Streptococcus canis infections in intensively housed shelter cats[J].Veterinary Pathology,2007,44(2):218-221.
[29] IANCU G M,OCNEANU A,ROTARU M.Hydroxyurea-induced superinfected ulcerations:Two case reports and review of the literature[J].Experimental and Therapeutic Medicine,2020,20(6):191.
[30] MISTRY R D,SCOTT H F,ALPERN E R,et al.Prevalence of Proteus mirabilis in skin abscesses of the axilla[J].Journal of Pediatrics,2010,156(5):850-851.
[31] 孙凯.五倍子溶液对犬真菌性皮肤病的疗效研究[D].成都:四川农业大学,2018. SUN K.Effect of Galla chinensis solution on fungal skin disease of dogs[D].Chengdu:Sichuan Agricultural University,2018.(in Chinese)
[32] BEA N,BESSA L J,MENDES ,et al.Coagulase-positive Staphylococcus:Prevalence and antimicrobial resistance[J].Journal of the American Animal Hospital Association,2015,51(6):365-371.
[33] 胡付品,郭燕,朱德妹,等.2017年CHINET中国细菌耐药性监测[J].中国感染与化疗杂志,2018,18(3):241-251. HU F P,GUO Y,ZHU D M,et al.Detection of bacterial resistance in China by CHINET 2017[J].Chinese Journal of Infection and Chemotherapy,2018,18(3):241-251.(in Chinese)
[34] 张文皓.犬猫口腔疾病病例分析及口腔细菌分离鉴定与耐药性分析[D].武汉:华中农业大学,2019. ZHANG W H.The analysis of canine and feline oral disease cases and isolation, identification and drug resistance analysis of oral bacteria[D].Wuhan:Huazhong Agricultural University,2019.(in Chinese)
[35] HUA X,JIA Y,YANG Q,et al.Transcriptional analysis of the effects of gambogic acid and neogambogic acid on methicillin-resistant Staphylococcus aureus[J].Frontiers in Pharmacology,2019,10:986.
[36] 王丹,刘延平,田春昊,等.GA衍生物的合成与抗菌活性研究及其对拓扑异构酶的影响[J].辽宁大学学报(自然科学版),2019,46(2):188-192. WANG D,LIU Y P,TIAN C H,et al.Synthesis and antibacterial activity of gambogic acid derivatives and their effects on topoisomerase[J].Journal of Liaoning University (Natural Science Edition),2019,46(2):188-192.(in Chinese)
[37] CZELEN P,SZEFLER B.Molecular dynamics study of the inhibitory effects of ChEMBL474807 on the enzymes GSK-3β and CDK-2[J].Journal of Molecular Modeling,2015,21(4):74.
[38] 林健,林志立,陈良善,等.靛玉红对耐甲氧西林金黄色葡萄球菌的抗菌作用[J].九江学院学报(自然科学版),2017,32(1):92-94. LIN J,LIN Z L,CHEN L S,et al.Antibacterial effect of indirubin on methicillin-resistant Staphylococcus aureus[J].Journal of Jiujiang University (Natural Science Edition),2017,32(1):92-94.(in Chinese)