牦牛瘤胃微生物抗生素抗性基因对3种外源性刺激因子的响应

通过给牦牛投喂硫酸头孢喹肟(CEF)、盐酸二氟沙星(DIF)和黄曲霉毒素B1(AFB1),并进行瘤胃微生物宏基因组测序,旨在揭示这3种外源性刺激因子对抗生素抗性基因(ARGs)种类、抗性类型、抗性机制等的影响,对于深入研究微生物抗性组特征和抗性机制具有重要价值。选取15头牦牛,随机分5组。Cef组和Dif组分别根据说明书推荐剂量按体重计算、灌服CEF 1 mg·kg^-1和DIF 1 mL·kg^-1;E1组和E2组分别按采食量投喂AFB120、60μg·kg^-1;C组为对照组。处理7 d后,采集瘤胃液,提取DNA,Illumina HiSeq测序,对reads counts进行标准化得到TPM值,并进行方差分析。结果显示,对照组共获得132种ARGs,分属30种抗性类型,其中,四环素类tetQ和tetW基因丰度较高;Cef组tetW基因丰度增加(P<0.05),Dif组tetQ丰度增加(P<0.05);Cef组四环素类和头孢菌素类抗性基因丰度增加(P<0.05),Dif组四环素类和氨基香豆素类抗性基因丰度增加(P<0.05),E1组氨基香豆素和青霉烯类抗性基因丰度增加(P<0.05),E2组青霉烯类、头孢菌素类等9类抗性基因丰度均增多(P<0.05);Dif组Erm基因23S核糖体RNA甲基转移酶丰度增加(P<0.05),E2组中ATP结合盒超家族等3种抗性机制相关基因的丰度增加(P<0.05);3种因子均显著增加四环素类ARGs宿主的种类。结论:瘤胃是蕴含丰富ARGs的储藏库,其中,四环素类抗生素抗性基因tetQ和tetW是主要的ARGs。不仅CEF和DIF使部分ARGs的种类、抗性类型以及耐药机制相关酶等的丰度升高,增加瘤胃微生物的耐药性,而且AFB1也具有类似作用,且高剂量AFB1对抗性类型的影响范围较抗生素大。这3种因子还导致携带四环素类ARGs宿主微生物的种类数量增加,从而强化横向转移机制,加快ARGs传播,增强微生物对四环素类的耐药性。

Response of Antibiotic Resistance Genes (ARGs) in Rumen of Yak to Three Exogenous Stimulating Factors

This study aimed to analyze the effects of three exogenous stimulating factors on the species, resistance types, and resistance mechanisms, etc. of antibiotic resistance genes (ARGs) by feeding yak with cefquinome (CEF), difloxacin (DIF), and aflatoxin B₁ (AFB₁), and sequence the rumen microorganisms by metagenomics. It is important to study the characteristics and resistance mechanisms of microbial antibiotic resistome or resistance reservoirs. Fifteen yaks were selected and randomly divided into 5 groups. According to the recommended dosage of the instructions, the Cef group and the Dif group were orally administered with CEF 1 mg·kg^-1 and DIF 1 mL·kg^-1, respectively. E1 groups and E2 groups were fed AFB₁ 20 and 60 μg·kg^-1, respectively. Group C was the control group. The rumen fluid samples of yaks were collected 7 days after treatment and DNA was extracted. The DNA was sequenced by Illumina HiSeq. After standardizing the reads counts, the TPM was obtained. An analysis of variance was performed on the TPM. The results showed that 132 ARGs were obtained in the control group, belonging to 30 resistance types, among which the abundance of tetracycline tetQ and tetW genes was higher. The abundance of tetW gene in Cef group increased (P<0.05). The abundance of tetQ in Dif group increased (P<0.05). The abundance of tetracycline antibiotic and cephalosporin antibiotic resistance genes in Cef group increased (P<0.05). The abundance of tetracycline antibiotic and aminocoumarin antibiotic resistance genes in Dif group increased (P<0.05). The abundance of aminocoumarin antibiotic and penem antibiotic resistance genes in the E1 group increased (P<0.05). The abundances of 9 resistance genes such as penems antibiotic and cephalosporins antibiotic in E2 group increased (P<0.05). The abundance of Erm 23S ribosomal RNA methyltransferase in Dif group increased (P<0.05). The abundance of three resistance mechanisms such as ATP-binding cassette (ABC) antibiotic efflux pump in E2 group increased (P<0.05). All three treatments significantly increased the host species of tetracycline antibiotic resistance genes. In conclusion:The rumen is a rich library of ARGs, in which tetQ and tetW are dominant ARGs. Not only CEF, DIF increase the abundance of some ARGs species, resistance types and enzymes related to resistance mechanisms, thereby increasing rumen microbial resistance, but also AFB₁ has a similar effect. Moreover, the high-dose AFB₁ has a greater range of effects on antibiotic resistance than antibiotics. These three factors also increase types of host microorganisms carrying tetracycline antibiotic resistance gene, thereby enhancing the horizontal gene transfer, accelerating the spread of ARGs, and enhancing the resistance of microorganisms to tetracycline antibiotics.