Abstract:

Antibiotic resistance genes (ARGs) have been recognized as new pollutants in the environment. Horizontal gene transfer (HGT) is the main way of ARGs transmission and diffusion. The horizontal transfer of ARGs in pure culture system has been reported, and the molecular mechanism of HGT has also been fully explored. However, whether ARGs in the environment can be captured by human pathogenic bacteria under natural conditions is still unknown. In order to investigate whether Salmonella can obtain ARGs from manure-amended soil under natural conditions and survive in soil for a long time, this study applied manure containing a large amount of tetracycline sensitive Salmonella to three types of soil, and treated the soils with or without flooding. After 90 days, tetracycline resistant Salmonella was isolated using selective medium containing tetracycline. The results showed that Salmonella in these soils did not obtain tetracycline resistance gene and survived in the soil for more than 90 days. Three suspected Salmonella strains resistant to tetracycline were isolated from the red clay soil, but they were identified as H₂S producing Escherichia coli by physiological and biochemical analysis as well as 16S sequencing. They all carried multiple ARGs detected by HT-qPCR. Among the three strains, T2 carried the most ARGs, so the whole genome of T2 was sequenced and analyzed. The results showed that T2 carried an IncX1 type plasmid with a length of 40.48 kb and an IncY type plasmid with a length of 93.31 kb, 15 gene islands, and 3807 injection-release genes which were annotated by COG database, including 197 prophages and transposons. A total of 34 ARGs were obtained through the ARDB annotation, including two tetracycline resistance genes located on the IncY type plasmid. By co-culturing T2 with tetracycline sensitive E. coli O157:H7 and applying different concentrations of tetracycline, the tetracycline resistance gene carried by T2 did not transfer to O157 strain. To sum up, it is rare for Salmonella to obtain ARGs from soil or manure in natural environment, and it is also difficult for the resistant E. coli T2 isolated from soil environment to transfer its tetracycline resistance genes to E. coli O157:H7 in pure culture system. These results can provide reference for scientific assessment of the transfer risk of ARGs in soil environment.

Key words: Antibiotic resistance gene, Salmonella, Whole bacterial genome sequencing, Horizontal gene transfer

摘要：

抗生素抗性基因（Antibiotic resistance genes，ARGs）已被公认为环境中的新型污染物，水平基因转移是ARGs传播与扩散的主要途径，纯培养体系下ARGs的水平基因转移（Horizontal gene transfer，HGT）已经多有报道，HGT的分子机制也已被充分挖掘。然而，环境中的ARGs在自然条件下是否能够转移到人类致病菌中目前仍然未知。为了探究沙门氏菌是否能够在自然条件下从施肥土壤中获得ARGs并在土壤中长期存活，将含有大量四环素敏感型沙门氏菌的粪肥施入3种土壤，并对土壤进行淹水或不淹水处理，90 d后分离四环素抗性沙门氏菌。结果表明，沙门氏菌未通过HGT获得四环素抗性基因并在土壤中长期存活。从红壤中分离到3株具有四环素抗性的疑似沙门氏菌，经过生理生化和16S测序鉴定确定它们均为产H₂S的大肠杆菌，经HT-qPCR检测，它们均携带多种ARGs。对其中携带ARGs种类最多的T2菌株进行全基因组测序与分析，结果表明T2携带了两个长度分别为40.48 kb的IncX1型和93.31kb的IncY型质粒，15个基因岛，与COG数据库比对得到了3807个注释基因，包含前噬菌体和转座子共197个；经ARDB注释获得34个ARGs，其中包括了2种四环素抗性基因均位于IncY型质粒上。将T2菌体与四环素敏感型E. coli O157:H7菌株共培养，在施加不同浓度梯度四环素的条件下，T2携带的四环素抗性基因没有向O157菌株转移。综上所述，在自然环境中沙门氏菌难以从施肥土壤中获得ARGs并在土壤中长期存活，从土壤环境中分离的抗性大肠杆菌T2所携带的四环素抗性基因也难以在纯培养体系下转移到E. coli O157:H7中。研究结果可为科学评估ARGs在土壤环境中的转移风险提供参考。

关键词: 抗生素抗性基因, 沙门氏菌, 细菌全基因组测序, 基因转移