笼养绿尾虹雉大肠埃希菌耐药特征与毒力基因分析

Antibiotic resistance characteristics and virulence gene analysis of Escherichia coli in caged Chinese monals (Lophophorus lhuysii

  • 摘要: 【目的】分析笼养绿尾虹雉大肠埃希菌的耐药特征与毒力基因携带状况,了解笼养绿尾虹雉大肠埃希菌耐药性及潜在风险,为绿尾虹雉驯养繁育过程中抗生素的合理使用及动物源细菌耐药性的遏制提供理论依据。【方法】于2022年1月、3月、4月、7月和10月采集四川蜂桶寨国家级自然保护区管护中心笼养绿尾虹雉新鲜粪便样本116份,进行细菌分离鉴定,通过药敏试验测定分离获得的大肠埃希菌对18种抗生素的耐药表型,筛选鉴定产超广谱β-内酰胺酶(ESBL)的菌株,通过PCR扩增对13种耐药基因和9种毒力基因进行检测,并对代表性耐药表型与相关耐药基因进行关联分析。【结果】116份样本共分离获得84株大肠埃希菌,分离率为72.41%,其中有12株不耐受任何抗生素,耐受3种及以上抗生素的耐药菌株有53株,占63.10%,菌株的耐药数量最高为12种,耐药谱以氨苄青霉素、甲氧苄氨嘧啶和庆大霉素为主,平均耐药率分别为78.57%、75.00%和45.24%。耐药基因检测结果显示,多黏菌素类耐药基因pmrA的平均检出率最高,为80.95%。其他平均检出率达50.00%的耐药基因还有β-内酰胺类耐药基因blaTEM(70.24%)、氯霉素类耐药基因floR(57.14%)和磺胺类耐药基因sulⅠ(50.00%);庆大霉素耐药性与氨基糖苷类耐药基因aadA1具有极显著关联性(P<0.01),而氨苄青霉素耐药性与blaTEM基因、甲氧苄氨嘧啶耐药性与sulI基因、环丙沙星耐药性与喹诺酮类耐药基因qnrS均无显著关联性(P>0.05,下同)。筛选鉴定出产ESBL菌36株,产ESBL菌对氨苄青霉素与甲氧苄氨嘧啶的耐药率均为100.00%,但产ESBL菌与blaTEM基因无显著关联性。对7月和10月粪便样本中分离的39株大肠埃希菌进行毒力基因检测,其中蛋白分泌物调节基因(escV)的检出率最高(17.95%),其次为肠定植因子基因(pic)(15.38%),而侵袭性质粒调节基因(invE)未检出。【结论】笼养绿尾虹雉粪便中的大肠埃希菌具有多重耐药性并携带部分毒力基因,具有潜在致病性,耐药谱以耐氨苄青霉素、甲氧苄氨嘧啶和庆大霉素为主。在笼养绿尾虹雉的细菌性疾病防控治疗中,应控制对β-内酰胺类、磺胺类、氨基糖苷类及喹诺酮类抗生素的使用。

     

    Abstract: 【Objective】To analyze the antibiotic resistance characteristics and virulence gene carriage status of Escherichia coli in caged Chinese monals (Lophophorus lhuysii), explore the antibiotic resistance and potential risks of E. coli in caged Chinese monals, which could provide theoretical basis for the rational use of antibiotics and containment of drug resistance in animal-derived bacteria in the breeding process of Chinese monals. 【Method】A total of 116 fresh fecal samples were collected from the artificial breeding ground at the Sichuan Fengtongzhai National Nature Reserve Administration in January, March, April, July and October 2022. Conducted bacterial isolation and identification, determined the antibiotic resistance patterns of E. coli strains obtained through susceptibility testing against 18 types of antibiotics, screened for strains producing extended-spectrum beta-lactamases(ESBLs), and used PCR amplification to detect 13 types of resistance genes and 9 types of virulence genes. Additionally, performed a correlation analysis between representative resistance phenotypes and associated resistance genes. 【Result】A total of 84 strains of E. coli were isolated from 116 samples, with a detection rate of 72.41%. Among them, 12 strains were sensitive to all tested antibiotics, while 53 strains were resistant to 3 or more tested antibiotics, accounting for 63.10%. The highest number of antibiotic resistance phenotype in the isolates was 12, and a majority of the observed antibiotic resistance phenotype were ampicillin, trimethoprim and gentamicin, with verage resistance rates of 78.57%, 75.00% and 45.24%. The antibiotic resistance gene detection results showed that the average detection rate of the polymyxins resistance gene pmrA was the highest(80.95%). Other antibiotic resistance genes with a detection rate exceeding 50.00% included β-lactams resistance gene blaTEM(70.24%), chloramphenicol resistance gene floR(57.14%) and sulfonamides resistance gene sulI(50.00%). The resistance to gentamicin was significantly associated with the aminoglycoside resistance gene aadA1P<0.01), while there was no significant correlation between the resistance to ampicillin and the blaTEM gene, nor between the resistance to trimethoprin and the sulI gene, or between the resistance to ciprofloxacin and the qnrS gene(P>0.05, the same below). A total of 36 ESBL-producing strains were screened and identified. The resistance rates of ESBL-producing strains to ampicillin and trimethoprim were both 100.00%, but there was no significant correlation between ESBL-producing strains and blaTEM gene. Among the 39 E. coli strains isolated from fecal samples in July and October, the protein secretion regulator gene(escV) had the highest detection rate (17.95%), followed by the intestinal colonization factor gene(pic) (15.38%), while the invasion plasmid regulator gene (invE) was not detected. 【Conclusion】The E. coli isolated from the feces of caged Chinese monals exhibits multiple antibiotic resistances and carries certain virulence genes, posing a potential pathogenic risk. The resistance spectrum is primarily characterized by resistance to ampicillin, trimethoprin, and gentamicin. The use of β-lactam, sulfanilamide, aminoglycoside and quinolone antibiotics should be controlled in the prevention and treatment of bacterial diseases in caged Chinese monals.

     

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