Miranda Alfonso, Ávila Bárbara, Díaz Patricia, Rivas Lina, Bravo Karen, Astudillo Javier, Bueno Constanza, Ulloa María T, Hermosilla Germán, Del Canto Felipe, Salazar Juan C, Toro Cecilia S
Programa de Microbiología y Micología, Facultad de Medicina, Instituto de Ciencias Biomédicas, Universidad de Chile Santiago, Chile.
Front Cell Infect Microbiol. 2016 Jul 20;6:77. doi: 10.3389/fcimb.2016.00077. eCollection 2016.
The most common mechanism of trimethoprim (TMP)-resistance is the acquisition of dihydrofolate reductase enzyme resistant to this drug. Previous molecular characterization of TMP-genes resistance in Chilean isolates of Shigella sonnei searching for dfrA1 and dfrA8, showed solely the presence of dfrA8 (formerly dhfrIIIc). However, these genetic markers were absent in S. sonnei strains further isolated during an outbreak in 2009. To identify the TMP-resistance gene in these strains, a genomic DNA library from a TMP-resistant (TMP(R)) S. sonnei representative strain for the outbreak was used to clone, select and identify a TMP-resistance marker. The TMP(R) clone was sequenced by primer walking, identifying the presence of the dfrA14 gene in the sul2-strA'-dfrA14-'strA-strB gene arrangement, harbored in a native 6779-bp plasmid. The same plasmid was isolated by transforming with a ~4.2 MDa plasmid extracted from several TMP(R) S. sonnei strains into Escherichia coli. This plasmid, named pABC-3, was present only in dfrA14-positive strains and was homologous to a previously described pCERC-1, but different due to the absence of an 11-bp repetitive unit. The distribution of dfrA1, dfrA8, and dfrA14 TMP-resistance genes was determined in 126 TMP(R) S. sonnei isolates. Most of the strains (96%) carried only one of the three TMP-resistance genes assessed. Thus, all strains obtained during the 2009-outbreak harbored only dfrA14, whereas, dfrA8 was the most abundant gene marker before outbreak and, after the outbreak dfrA1 seems have appeared in circulating strains. According to PFGE, dfrA14-positive strains were clustered in a genetically related group including some dfrA1- and dfrA8-positive strains; meanwhile other genetic group included most of the dfrA8-positive strains. This distribution also correlated with the isolation period, showing a dynamics of trimethoprim genetic markers prevalent in Chilean S. sonnei strains. To our knowledge, dfrA14 gene associated to a small non-conjugative plasmid was detected for the first time in Shigella. Apparently, the strain causing the outbreak must have been introduced, changing drastically the genetic distribution of trimethoprim resistance in Chilean S. sonnei strains.
甲氧苄啶（TMP）耐药的最常见机制是获得对该药物耐药的二氢叶酸还原酶。先前在智利宋内志贺氏菌分离株中对TMP基因耐药性进行分子特征分析以寻找dfrA1和dfrA8时，仅发现了dfrA8（以前称为dhfrIIIc）。然而，在2009年一次疫情中进一步分离的宋内志贺氏菌菌株中没有这些遗传标记。为了鉴定这些菌株中的TMP耐药基因，使用来自疫情中一株TMP耐药（TMP（R））宋内志贺氏菌代表性菌株的基因组DNA文库来克隆、筛选和鉴定一个TMP耐药标记。通过引物步移对TMP（R）克隆进行测序，发现在sul2-strA'-dfrA14-'strA-strB基因排列中存在dfrA14基因，该基因位于一个天然的6779 bp质粒中。通过将从几株TMP（R）宋内志贺氏菌菌株中提取的约4.2 MDa质粒转化到大肠杆菌中，分离出了相同的质粒。这个质粒命名为pABC - 3，仅存在于dfrA14阳性菌株中，并且与先前描述的pCERC - 1同源，但由于缺少一个11 bp的重复单元而有所不同。在126株TMP（R）宋内志贺氏菌分离株中测定了dfrA1、dfrA8和dfrA14 TMP耐药基因的分布。大多数菌株（96%）仅携带所评估的三个TMP耐药基因中的一个。因此，在2009年疫情期间获得的所有菌株仅携带dfrA14，而dfrA8是疫情前最丰富的基因标记，疫情后dfrA1似乎已出现在流行菌株中。根据脉冲场凝胶电泳（PFGE）分析，dfrA14阳性菌株聚集在一个遗传相关的组中，包括一些dfrA1和dfrA8阳性菌株；同时其他遗传组包括大多数dfrA8阳性菌株。这种分布也与分离时期相关，显示了智利宋内志贺氏菌菌株中甲氧苄啶遗传标记的动态变化。据我们所知，首次在志贺氏菌中检测到与一个小的非接合性质粒相关的dfrA14基因。显然，导致疫情的菌株一定是被引入的，这极大地改变了智利宋内志贺氏菌菌株中甲氧苄啶耐药性的遗传分布。
