斑节对虾(非洲群体)肠道细菌抗生素耐药性及耐药基因分布特征

Distribution characteristics of antibiotic-resistant bacteria and antimicrobial-resistant genes in the intestine of Penaeus monodon(African)

摘要:

[目的]解析斑节对虾(Penaeus monodon)(非洲群体) (俗称"金刚虾",以下同)携带耐药菌及耐药基因现状。[方法]本研究从山东滨州北海新区采集了金刚虾,对其肠道细菌常用抗生素的耐药菌性质及数量、占比及种类进行检测,通过荧光定量PCR技术分析肠道内容物样品中的4类抗生素的4种耐药性基因分布特征。[结果]肠道中可培养细菌总数约1.45×10⁵- 2.13×10⁶CFU/g,有四环素、萘啶酸、氟苯尼考、庆大霉素4种抗生素耐药菌的检出,其中喹诺酮类萘啶酸耐药菌占比最高,达到35.00%,氨基糖苷类庆大霉素占比最少。10种抗生素药敏性质分析表明,肠道可培养细菌对庆大霉素、氟苯尼考等6种抗生素高度敏感,对四环素、卡那霉素中度敏感,对萘啶酸、青霉素、阿莫西林耐药。从分离的耐药菌鉴定结果可以得出,可培养的抗生素耐药菌主要集中在弧菌属,基于属水平的不同抗生素耐药菌统计显示,不同抗生素耐药菌种类存在明显差异,且同一菌属有耐多种抗生素的情况。荧光定量PCR检测分析,4种耐药基因的丰度不同,tetA基因相对拷贝数和四环素耐药菌比例、floR基因和氟苯尼考耐药菌比例之间存在显著相关性(P<0.01);qnrA基因相对拷贝数和萘啶酮酸耐药菌比例、aadA基因相对拷贝数和庆大霉素耐药菌比例之间均不存在显著相关性(P>0.01)。[结论]本研究说明,金刚虾肠道微生物中存在一定的耐药菌和耐药基因,具有携带风险。

关键词:斑节对虾(非洲群体);肠道;抗生素耐药菌;耐药基因;荧光定量PCR

Abstract:

[Objective] To analyze the antibiotic-resistant bacteria and antimicrobial-resistant genes in Penaeus monodon (African).[Methods]We collected P.monodon from Beihai New Area,Binzhou,Shandong,and then tested the number,proportion,and species of antibiotic-resistant bacteria in the intestine.Meanwhile,we analyzed the distribution of genes related to the resistance to four antibiotics by quantitative reverse transcription PCR (qRT-PCR).[Results] The total number of culturable bacteria in the intestine was about 1.45x10⁵-2.13x10⁶ CFU/g,and bacteria resistant to tetracycline,nalidixic acid,florfenicol,and gentamicin were all detected,among nalidixic acid-resistant bacteria (35.00%) and gentamicin-resistant bacteria took up the largest and smallest proportion,respectively.The sensitivity to 10 antibiotics was analyzed and the result showed that the intestinal bacteria were highly susceptible to six antibiotics such as gentamicin and florfenicol,moderately susceptible to tetracycline and kanamycin and resistant to nalidixic acid,penicillin,and amoxicillin.The culturable antibiotic-resistant bacteria mainly belonged to Vibrio,and the species of bacteria resistant to different antibiotics varied.Moreover,species of the same genus may be resistant to multiple antibiotics.The abundance of genes related to the resistance of the four antibiotics was different.Significant correlation between the relative copy number of tetA gene and the proportion of tetracycline-resistant bacteria and between the relative copy number of floR gene and the proportion of florfenicol-resistant bacteria was detected (P<0.01),but no significant correlation was observed between the relative copy number of qnrA gene and the proportion of nalidixic acid-resistant bacteria,and between the relative copy number of aadA gene and the proportion of gentamicin-resistant bacteria (P>0.01).[Conclusion] The intestine of P.monodon (African) harbors antibiotic-resistant bacteria and antimicrobial-resistance genes.

Key words:Penaeus monodon(African);intestine;antibiotic-resistant bacteria;antimicrobial-resistant genes;real-time quantitative PCR