稻蛙共养系统中大肠杆菌耐药性及其传播风险

Evaluation of antibiotics resistance and transmission risk of Escherichia coli in rice-frog coculture system

摘要:

为了解浙江地区稻蛙共养模式下大肠杆菌耐药情况及其传播风险，本研究从浙江省4个不同地区的稻蛙共养基地采集分离泥土、田间水及蛙粪便中的大肠杆菌，进行了16S rRNA测序鉴定，纸片扩散法(Kirby-Bauer,K-B)测定耐药表型，PCR法鉴定耐药基因型与整合子，分析了大肠杆菌的耐药情况，并通过接合转移评估耐药传播特性。结果显示，分离得到的82株大肠杆菌对四环素、磺胺异噁唑、阿莫西林和红霉素表现出较高的耐药率，大多数为多重耐药菌，且富阳分离株的耐药率高于其他3个地区。PCR鉴定结果表明，耐药基因sul1检出率最高，为63.41%，其次是bla_TEM、tetA和tetB。在检测的16种抗生素耐药基因(antibiotic resistance genes,ARGs)中，富阳分离株有9种ARGs的检出率高于其他地区。整合酶基因intI1的检出率最高，整合酶阳性菌中共14株菌(34.15%)携带基因盒，存在4种不同的基因盒组成方式，以dfrA1-aadA1和dfrA17-aadA5最常见。接合转移结果显示，14株携带基因盒的供体菌中有4株接合转移成功，接合转移频率在4.32×10⁻⁵−7.13×10⁻⁴之间。研究表明，4个地区中富阳分离株的耐药情况较为严重为整合子介导大肠杆菌对多种抗生素耐药，并且耐药基因盒可能通过整合子在不同细菌之间传播。本研究对浙江省稻蛙共养环境中大肠杆菌的耐药情况和传播特性进行分析，为保障食品安全提供理论基础。

Abstract:

In order to assess the antibiotic resistance of Escherichia coli and its transmission risk in a rice-frog coculture system in Zhejiang Province, this study collected E. coli from isolated soil, field water, and frog feces from the rice-frog coculture systems in four different areas of Zhejiang Province. The collected isolates were identified by 16S rRNA sequencing, while their antibiotic-resistant phenotypes were determined by Kirby-Bauer (K-B) method. PCR was used to identify the antibiotic-resistant genotypes and integrons, while conjugative transfer experiments were used to assess resistance transmission characteristics. The results showed a high prevalence of antibiotic resistance in the 82 strains of E. coli tested, primarily against tetracycline, sulfisoxazole, amoxicillin, and erythromycin. Most of these strains exhibited multidrug resistance, with the Fuyang area demonstrating the highest resistance rate compared to the other three areas. Further PCR analysis identified the sul1 gene as the most frequently detected resistance gene (63.41%), followed by bla_TEM, tetA, and tetB. Among the 16 antibiotic resistance genes (ARGs) detected, the Fuyang isolates consistencly exhibited higher detection rate of 9 ARGs in comparison to the other regions. Additionally, the integrase gene intI1 displayed the highest detection rate, with 14 strains (34.15%) of integrase-positive bacteria carrying gene cassettes. Four different gene cassette compositions were observed, with dfrA1-aadA1 and dfrA17-aadA5 being the most common combinations. Conjugative transfer experiments demonstrated successful transfer of gene cassettes in 4 out of 14 donor bacteria, with conjugation transfer frequencies ranging from 4.32×10⁻⁵ to 7.13×10⁻⁴. These findings revealed the severity of resistance in the Fuyang area among the four regions. Integrons play a significant role in mediating the resistance to multiple antibiotics in E. coli, facilitating the potential spread of resistance gene cassettes between different bacteria. Overall, this study provides valuable insights into the resistance status and transmission characteristics of E. coli in the rice-frog coculture system in Zhejiang Province, providing a theoretical basis for ensuring the food safety of rice crops.