AUTHOR=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. 

TITLE=Emergence of Plasmid-Borne dfrA14 Trimethoprim Resistance Gene in Shigella sonnei

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=6

YEAR=2016

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2016.00077

DOI=10.3389/fcimb.2016.00077

ISSN=2235-2988

ABSTRACT=<p>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 <italic>Shigella sonnei</italic> searching for <italic>dfrA1</italic> and <italic>dfrA8</italic>, showed solely the presence of <italic>dfrA8</italic> (formerly <italic>dhfrIIIc</italic>). However, these genetic markers were absent in <italic>S. sonnei</italic> 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<sup>R</sup>) <italic>S. sonnei</italic> representative strain for the outbreak was used to clone, select and identify a TMP-resistance marker. The TMP<sup>R</sup> clone was sequenced by primer walking, identifying the presence of the <italic>dfrA14</italic> gene in the <italic>sul2-strA'-dfrA14-‘strA-strB</italic> 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<sup>R</sup><italic>S. sonnei</italic> strains into <italic>Escherichia coli</italic>. This plasmid, named pABC-3, was present only in <italic>dfrA14</italic>-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 <italic>dfrA1, dfrA8</italic>, and <italic>dfrA14</italic> TMP-resistance genes was determined in 126 TMP<sup>R</sup><italic>S. sonnei</italic> 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 <italic>dfrA14</italic>, whereas, <italic>dfrA8</italic> was the most abundant gene marker before outbreak and, after the outbreak <italic>dfrA1</italic> seems have appeared in circulating strains. According to PFGE, <italic>dfrA14-positive</italic> strains were clustered in a genetically related group including some <italic>dfrA1</italic>- and <italic>dfrA8</italic>-positive strains; meanwhile other genetic group included most of the <italic>dfrA8</italic>-positive strains. This distribution also correlated with the isolation period, showing a dynamics of trimethoprim genetic markers prevalent in Chilean <italic>S. sonnei</italic> strains. To our knowledge, <italic>dfrA14</italic> gene associated to a small non-conjugative plasmid was detected for the first time in <italic>Shigella</italic>. Apparently, the strain causing the outbreak must have been introduced, changing drastically the genetic distribution of trimethoprim resistance in Chilean <italic>S. sonnei</italic> strains.</p>