Pornsukarom Suchawan, Thakur Siddhartha
Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA.
Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, USA
Appl Environ Microbiol. 2017 Sep 29;83(20). doi: 10.1128/AEM.01503-17. Print 2017 Oct 15.
The aim of this study was to characterize the plasmids carrying antimicrobial resistance (AMR) determinants in multiple serotypes recovered from the commercial swine farm environment after manure application on land. Manure and soil samples were collected on day 0 before and after manure application on six farms in North Carolina, and sequential soil samples were recollected on days 7, 14, and 21 from the same plots. All environmental samples were processed for , and their plasmid contents were further characterized. A total of 14 isolates including serotypes Johannesburg ( = 2), Ohio ( = 2), Rissen ( = 1), Typhimurium var5- ( = 5), Worthington ( = 3), and 4,12:i:- ( = 1), representing different farms, were selected for plasmid analysis. Antimicrobial susceptibility testing was done by broth microdilution against a panel of 14 antimicrobials on the 14 confirmed transconjugants after conjugation assays. The plasmids were isolated by modified alkaline lysis, and PCRs were performed on purified plasmid DNA to identify the AMR determinants and the plasmid replicon types. The plasmids were sequenced for further analysis and to compare profiles and create phylogenetic trees. A class 1 integron with an ANT(2″)-Ia- cassette was detected in the 50-kb IncN plasmids identified in Worthington isolates. We identified 100-kb and 90-kb IncI1 plasmids in Johannesburg and Rissen isolates carrying the and (A) genes, respectively. An identical 95-kb IncF plasmid was widely disseminated among the different serotypes and across different farms. Our study provides evidence on the importance of horizontal dissemination of resistance determinants through plasmids of multiple serotypes distributed across commercial swine farms after manure application. The horizontal gene transfer of antimicrobial resistance (AMR) determinants located on plasmids is considered to be the main reason for the rapid proliferation and spread of drug resistance. The deposition of manure generated in swine production systems into the environment is identified as a potential source of AMR dissemination. In this study, AMR gene-carrying plasmids were detected in multiple serotypes across different commercial swine farms in North Carolina. The plasmid profiles were characterized based on serotype donors and incompatibility (Inc) groups. We found that different Inc plasmids showed evidence of AMR gene transfer in multiple serotypes. We detected an identical 95-kb plasmid that was widely distributed across swine farms in North Carolina. These conjugable resistance plasmids were able to persist on land after swine manure application. Our study provides strong evidence of AMR determinant dissemination present in plasmids of multiple serotypes in the environment after manure application.
本研究的目的是对在农田施用猪粪后从商业化养猪场环境中分离出的多种血清型中携带抗菌抗性（AMR）决定簇的质粒进行特征分析。在北卡罗来纳州的六个农场，于施用猪粪前的第0天以及施用猪粪后的第0天采集粪便和土壤样本，并在第7天、第14天和第21天从相同地块再次采集土壤样本。对所有环境样本进行处理，并进一步分析其质粒含量。总共挑选了14株分离株，包括约翰内斯堡血清型（n = 2）、俄亥俄血清型（n = 2）、里森血清型（n = 1）、鼠伤寒沙门氏菌变种5 - （n = 5）、沃辛顿血清型（n = 3）和4,12:i: - （n = 1），代表不同的农场，用于质粒分析。在接合试验后，通过肉汤微量稀释法对14株已确认的转接合子进行针对一组14种抗菌药物的药敏试验。通过改良碱裂解法分离质粒，并对纯化的质粒DNA进行PCR，以鉴定AMR决定簇和质粒复制子类型。对质粒进行测序以进行进一步分析，并比较图谱和构建系统发育树。在沃辛顿分离株中鉴定出的50 kb IncN质粒中检测到一个带有ANT(2″)-Ia - 盒的1类整合子。在约翰内斯堡和里森分离株中分别鉴定出携带blaCTX-M-1和blaTEM(A)基因的100 kb和90 kb IncI1质粒。一个相同的95 kb IncF质粒在不同血清型之间以及不同农场中广泛传播。我们的研究提供了证据，证明在农田施用猪粪后，抗性决定簇通过分布在商业化养猪场的多种血清型的质粒进行水平传播的重要性。位于质粒上的抗菌抗性（AMR）决定簇的水平基因转移被认为是耐药性快速增殖和传播的主要原因。猪生产系统中产生的粪便排入环境被确定为AMR传播的一个潜在来源。在本研究中，在北卡罗来纳州不同的商业化养猪场的多种血清型中检测到携带AMR基因的质粒。根据血清型供体和不相容性（Inc）组对质粒图谱进行了特征分析。我们发现不同的Inc质粒在多种血清型中显示出AMR基因转移的证据。我们检测到一个相同的95 kb质粒在北卡罗来纳州的养猪场中广泛分布。这些可接合的抗性质粒在施用猪粪后能够在土壤中持续存在。我们的研究提供了有力证据，证明在农田施用猪粪后环境中多种血清型的质粒中存在AMR决定簇的传播。
