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

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.

Genomic epidemiology of Shigella in the United Kingdom shows transmission of pathogen sublineages and determinants of antimicrobial resistance

“…This isolate also contained the small resistance plasmid pABC-3, typical of Latin America IIIa S . sonnei 7 , 31 , and also found in various MSM-associated Shigella sublineages (specifically the intercontinentally-distributed MSM-associated S . flexneri 3a 13 and the larger sublineage of MSM-associated S .…”

“…sonnei 6 , 32 pABC-3 dfrA14 sul2 strB ^ Trimethoprim Sulphonamides — S . sonnei 7 , 31 S . flexneri 3a 13 S .…”

Closed Genome and Comparative Phylogenetic Analysis of the Clinical Multidrug Resistant Shigella sonnei Strain 866

“…2 A ), which renders this strain ß-lactamase ( blaTEM-1A ), sulfonamide ( sul2 ), tetracycline ( tetB ), aminoglycoside ( aadA1 ), and trimethoprim ( dfrA ) resistant ( Zankari et al. 2012 ; Miranda et al. 2016 ).…”

Whole genome sequencing of Shigella sonnei through PulseNet Latin America and Caribbean: advancing global surveillance of foodborne illnesses

“…The dominant AMR profiles in each of IIIa and IIIb were determined by the presence of chromosomal and plasmid-encoded AMR genes conferring resistance to multiple antimicrobial classes ( Table 3 ). Specifically, sublineage IIIb carried the chromosomal Int2/Tn7 resistance determinant and the SpA plasmid, and sublineage IIIa carried the chromosomal Shigella resistance locus (SRL) and a variant plasmid of SpA, pABC-3, on which the aminoglycoside resistance gene strA has been interrupted by the acquisition of a trimethoprim resistance-conferring gene, dfrA14 [28] . In contrast to the presence of a dominant AMR profile in sublineages IIIa and IIIb, sublineages IIa, IIb and lineage V contained at least three AMR profiles that were present in ≥15% of the isolates ( Supplementary Table S1 ).…”

“…Similarly, sublineage IIIa contains the chromosomally integrated SRL and pABC-3, two of the major resistance determinants reported in a recent sublineage of Shigella flexneri 3a which transmitted globally among men who have sex with men from a possible LA origin [29] . Notably, pABC-3 has been reported in epidemic S. sonnei in Chile [28] , but the SRL, known as an important AMR determinant in S. flexneri and Shigella dysenteriae [30] , [31] , has not been commonly reported in S. sonnei and thus represents a worrying addition to the armaments of this pathogen.…”