Abstract
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 (TMPR) S. sonnei representative strain for the outbreak was used to clone, select and identify a TMP-resistance marker. The TMPR 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 TMPR 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 TMPR 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.
Highlights
Shigella sonnei has become a major problem of public health due to the increasing multidrug-resistance to antibiotic (MDR) worldwide (Ashkenazi et al, 2003; De Lappe et al, 2003; Seol et al, 2006; Vrints et al, 2009)
To characterize the molecular determinants of TMP-resistance in these Chilean S. sonnei strains, we randomly chose 126 TMPR strains obtained from different periods, divided as 3 groups: 45 strains before the outbreak, the 41 strains during the outbreak (2008–2009), and 40 strains isolated after the outbreak (2010– 2013)
Characterizing TMPR S. sonnei strains belonged to the 2009-oubreak, we cloned the TMP-resistance genetic dfrA14 in Shigella sonnei determinant identified as dfrA14, formerly named dhfrlb (Recchia and Hall, 1995) and harbored in a small plasmid (6779 bp)
Summary
Shigella sonnei has become a major problem of public health due to the increasing multidrug-resistance to antibiotic (MDR) worldwide (Ashkenazi et al, 2003; De Lappe et al, 2003; Seol et al, 2006; Vrints et al, 2009). This pathogen, one of the most frequent etiologic agents of foodborne diseases in industrialized countries, is responsible for shigellosis, an acute enteric disease for which antimicrobial therapy is usually recommended to manage infection and reduce fecal excretion of the bacterium to prevent further dissemination. The outbreak (739 cases) mainly affected children under 10 years from Región Metropolitana, and the source of infection was not clearly established (Instituto de Salud Pública, 2009)
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