Extensive genetic variability linked to IS26 insertions in the fljB promoter region of atypical monophasic variants of Salmonella enterica serovar Typhimurium

Abstract

Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:- and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS26 composite transposon insertion along with a local genomic rearrangement. Several other IS26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.

FIG 1

Map of the fljBA operon region of the monophasic variant strain VAR-2009/08643/1 (83,173 bp). IS26 copies are drawn with hatched arrows. Black arrows delineate antibiotic resistance genes. Gray boxes indicate regions homologous to Salmonella Typhimurium LT2 (GenBank accession number AE006468). The dotted box is homologous to a region found on the E. coli plasmid pO111_1 (GenBank accession number AP010961). Antibiotic resistance regions 1 (RR1) and 2 (RR2) are similar to a Salmonella 4,[5],12:i:− chromosomal sequence (GenBank accession number HQ331538) except for (i) a deletion covering partial merE, urf2, and tniAΔ1, (ii) an inverted IS26 element, (iii) a complete IS10 copy, and (iv) an additional region (dotted box) in RR2. Δ′ and Δ″ refer to deletions noticed in the 3′ or 5′ part of some genes, respectively. Boxed nucleotides refer to IS26-associated repeats. The insertion of a 26-kb transposon consisting of the RR1 and RR2 elements occurred at an intermediate stage and was followed by a genomic rearrangement between the first and fourth IS26 copies, leading to an inverted DNA sequence in between. This is supported by the finding of inverted repeats bordering the first and fourth IS elements (boxed nucleotides) and the orientation of the genes within the inverted segment (gray boxes). The arrowheads at the bottom point to primers used to demonstrate the presence of IS26 elements (possibly as part of the 26-kb transposon). Double-headed arrows show the corresponding amplicons with their sizes (TLJ, transposon left junction; IS26LJ, IS26 left junction; TRJ, transposon right junction; IS26RJ, IS26 right junction).

FIG 2

Map of the hin-iroB intergenic sequence of various monophasic variants. The intergenic sequence lying between the hin and iroB genes and observed in the Salmonella Typhimurium LT2 reference strain as well as in some representative isolates of the monophasic variants described in this work is shown as a black box. IS26 elements are indicated by hatched arrows. Nucleotide positions in the intergenic sequence bordering IS26 elements are given in white boxes above vertical arrows and follow the nucleotide sequence numbering of Salmonella Typhimurium LT2 (AE006468). Compared to S. Typhimurium LT2 (panel a), the hin-iroB intergenic sequence was deleted by 536 (panel b), 482 (panel c), or 451 (panel d) bp. LJ and RJ indicate the left and right junctions, respectively, amplified by the junction PCRs described in Materials and Methods. The dotted line in panel d delineates the large (56.6-kb) intervening region detailed in Fig. 1.