鄱阳湖候鸟保护区白鹤粪便细菌分离鉴定与耐药性分析

Isolation, identification, and antibiotic resistance analysis of bacteria from feces of Grus leucogeranus in Poyang Lake Migratory Bird Reserve

摘要:

背景 白鹤等重要候鸟以鄱阳湖作为主要越冬地，其健康状态是评估当地生态系统安全的重要指标。近年来，随着越冬地周边人类活动的加剧，抗生素通过医疗、农业和水产养殖等途径进入环境，可能使白鹤暴露于抗生素残留及耐药菌的选择压力之下，对其生存与健康构成潜在威胁。目的 通过分析白鹤粪便中条件致病菌的耐药特征，积累该珍稀物种相关的微生物本底数据，并探讨其对当地生物健康与生态安全的潜在影响，为生物安全监测与生态健康保护提供科学依据。方法 采集保护区内60份白鹤新鲜粪便，经LB肉汤增殖后，分别接种于麦康凯、肠球菌和葡萄球菌选择性培养基中分离目标菌株，通过菌落形态、PCR鉴定确认，并开展最小抑菌浓度(minimum inhibitory concentration, MIC)药敏试验。对多重耐药菌株采用PacBio三代测序进行全基因组分析。结果 共分离出大肠杆菌(Escherichia coli) 28株(46.67%)、肠球菌(Enterococcus) 36株(60.00%)和金黄色葡萄球菌(Staphylococcus aureus) 13株(21.67%)。药敏试验显示，大肠杆菌对多黏菌素E (57.14%)、磺胺异噁唑(42.86%)和四环素(46.43%)耐药率较高；肠球菌对头孢噻呋(75.00%)、头孢西丁(86.11%)、苯唑西林(88.89%)和磺胺异噁唑(80.56%)高度耐药；金黄色葡萄球菌对四环素(69.23%)和氟苯尼考(53.85%)耐药突出。共发现9株大肠杆菌、27株肠球菌和1株金黄色葡萄球菌为多重耐药菌。基因组测序显示，多重耐药菌株携带多种耐药基因，如五重耐药的屎肠球菌(Enterococcus faecium)携带17种耐药基因。结论 白鹤粪便中分离菌呈现较高的耐药性，并且存在多重耐药菌株及多种耐药基因。这些结果丰富了珍稀候鸟耐药菌的本底数据库，也为进一步探究耐药基因在环境中的传播与归趋提供了重要线索。

Abstract:

Background The Poyang Lake serves as a major wintering ground for important migratory birds such as the Siberian crane (Grus leucogeranus). The health status of these birds is a key indicator for assessing the safety of the local ecosystem. In recent years, increased human activities around the wintering areas have led to the introduction of antibiotics into the environment through medical, agricultural, and aquaculture practices. This may expose the Siberian cranes to antibiotic residues and selective pressure from drug-resistant bacteria, posing a potential threat to their survival and health.Objective This study analyzed the antibiotic resistance characteristics of conditionally pathogenic bacteria in Siberian crane feces and discusses the potential effects of these bacteria on local biological health and ecological safety, aiming to enrich the microbial data of local rare and precious species and provide a basis for biological safety monitoring and ecological health protection.Methods 60 fresh fecal samples were collected from Siberian cranes within the reserve. After enrichment in LB broth, they were inoculated onto MacConkey, Enterococcus-selective, and Staphylococcus-selective media to isolate target strains. Bacteria were identified by colony morphological characterization and PCR. Antimicrobial susceptibility testing was performed via the minimum inhibitory concentration method. PacBio third-generation sequencing was conducted to reveal the whole genomes of multidrug-resistant (MDR) strains.Results A total of 28 (46.67%) Escherichia coli strains, 36 (60.00%) Enterococcus strains, and 13 (21.67%) Staphylococcus aureus strains were isolated. Antimicrobial susceptibility testing revealed that E. coli showed high resistance rates to polymyxin E (57.14%), sulfisoxazole (42.86%), and tetracycline (46.43%). Enterococcus exhibited high resistance rates to ceftiofur (75.00%), cefoxitin (86.11%), oxacillin (88.89%), and sulfisoxazole (80.56%). S. aureus showed high resistance rates to tetracycline (69.23%) and florfenicol (53.85%). Nine E. coli strains, 27 Enterococcus strains, and one S. aureus strain were identified as MDR strains. Genomic sequencing revealed that these multidrug-resistant strains carried various resistance genes. For instance, a pentadrug-resistant Enterococcus faecium strain carried 17 resistance genes.Conclusion The results indicate that bacteria isolated from Siberian crane feces exhibit high resistance rates, with the presence of multidrug-resistant strains carrying diverse resistance genes. These findings enrich the baseline database of drug-resistant bacteria in rare migratory birds and provide important clues for further research on the transmission and fate of resistance genes in the environment.