Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona

Abstract

Strains of multidrug-resistant Salmonella enterica serovar Typhimurium DT104 (DT104) and S. enterica serovar Agona (Agona) have been found to harbor Salmonella genomic island 1 (SGI1), a 43-kb genomic region that contains many of the drug resistance genes. Such strains are resistant to ampicillin (pse-1), chloramphenicol/florfenicol (floR), streptomycin/spectinomycin (aadA2), sulfonamides (sul1), and tetracycline [tet(G)] (commonly called the ACSSuT phenotype). All five resistance genes are found in a 13-kb multidrug resistance (MDR) region consisting of an unusual class I integron structure related to In4. We examined DT104 and Agona strains that exhibited other resistance phenotypes to determine if the resistance genes were associated with variant SGI1 MDR regions. All strains were found to harbor variant SGI1-like elements by using a combination of Southern hybridization, PCR mapping, and sequencing. Variant SGI1-like elements were found with MDR regions consisting of (i) an integron consisting of the SGI1 MDR region with the addition of a region containing a putative transposase gene (orf513) and dfrA10 located between duplicated qacEDelta1/sulI genes (SGI1-A; ACSSuTTm); (ii) an integron with either an aadA2 (SSu) or a pse-1 (ASu) cassette (SGI1-C and SGI1-B, respectively); (iii) an integron consisting of the SGI1-C MDR region plus an orf513/dfrA10 region as in SGI1-A (SGI1-D; ASSuTm; ampicillin resistance due to a TEM beta-lactamase); and (iv) an integron related to that in SGI1 but which contains a 10-kb inversion between two copies of IS6100, one which is inserted in floR (SGI1-E; ASSuT). We hypothesize that the MDR of SGI1 is subject to recombinational events that lead to the various resistance phenotypes in the Salmonella strains in which it is found.

FIG. 1.

Genetic organization of the MDR regions of the various strains in this study based on Southern hybridization data, PCR mapping, and sequence analysis. A schematic of SGI1 is shown at the top, with the approximate locations of the primers used to detect the left and right junction regions indicated. The cryptic retronphage region is absent in Agona strains. Direction of transcription of genes is indicated by arrows. Locations of PCR products used as probes are indicated (thin black bars), as are the locations of some primers used in mapping. IRi and and IRt are 25-bp imperfect inverted repeats defining the ends of class 1 integrons. Regions sequenced are indicated under the various MDR regions by a thick black bar. The nucleotide sequence of the insertion point of the IS6100 element in the floR gene of S/960081 is shown; the 8-bp region duplicated upon insertion is boxed (coordinates are for the floR gene only). IR-L and IR-R refer to the inverted repeats that define one end of IS6100. The sequence of the orf513/dfrA10 region from Agona 1169SA97 has been assigned accession no. AY049746.

FIG. 2.

Alignment of Orf513, OrfA, and Orf2 proteins. Identical residues in two or three of the proteins are boxed. Coordinates are at the right end of each line.

FIG. 3.

Schematic diagram of the generation of variant SGI1 MDR regions from SGI1. Mechanisms are discussed in the text.