Characterization of Salmonella enterica Serovar Typhimurium Isolates Harboring a Chromosomally Encoded CMY-2 β-Lactamase Gene Located on a Multidrug Resistance Genomic Island

Abstract

Since 2004, extended-spectrum cephalosporin (ESC)-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates have been detected from cattle in the northern major island of Japan, Hokkaido. Resistance to ESCs was found to be mediated by CMY-2 type β-lactamase among 22 epidemiologically unrelated isolates showing indistinguishable pulsed-field gel electrophoresis patterns. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that the CMY-2 β-lactamase gene (bla(CMY-2)) was integrated in a 2.5-Mb chromosomal fragment. Genetic analysis of S. Typhimurium isolate L-3553 indicated that bla(CMY-2) was located on a unique 125-kb genomic island, GI-VII-6, which consists of 140 open reading frames. Pairwise alignment of GI-VII-6 and Escherichia coli plasmid pAR060302 (size, 167 kb) revealed that a large proportion of GI-VII-6 (99%) shows a high sequence similarity (>99%) with pAR060302. GI-VII-6 contains 11 antimicrobial resistance genes including sul1, qacEΔ1, aadA2, and dfrA12 in the aadA2 region; sugE1 and bla(CMY-2) in the bla(CMY-2) region; and sul2, strA, strB, tet(A), and floR in the floR region. Two directly repeated IS26 copies were present at both ends of GI-VII-6. Junction regions of GI-VII-6 were flanked by an 8-bp direct repeat, indicating that GI-VII-6 was acquired by transposition involving IS26 transposase. PCR scanning revealed that the overall structure of GI-VII-6 was almost identical in the 22 isolates. Phylogenetic analysis suggested that S. Typhimurium isolates harboring GI-VII-6 belong to a different genomic lineage than other whole-genome-sequenced S. Typhimurium strains. These data indicate that a particular clone of S. Typhimurium harboring GI-VII-6 has spread among the cattle population in Hokkaido, Japan.

Salmonella Pathogenicity and Host Adaptation in Chicken-Associated Serovars

“…A case in point was highlighted by a study by Shahada et al, which identified a unique 125-kb genomic island among isolates of a dominant clone of S. Typhimurium from cattle in Japan. This island, named GI-VII-6, displayed striking similarities to portions of IncA/C plasmids containing three copies of bla CMY-2 (144).…”

Genetic Analysis of Non-Hydrogen Sulfide-Producing Salmonella enterica Serovar Typhimurium and S. enterica Serovar Infantis Isolates in Japan

“…Aminoglycoside and trimethoprim resistance genes were also identified (Table 1). A plasmid carrying the bla CMY-2 gene was detected by S1 nuclease-PFGE and Southern blot analysis (12), indicating that it appears to have been an independent acquisition by two different serovars (Fig. 2).…”

Distribution and Relationships of Antimicrobial Resistance Determinants among Extended-Spectrum-Cephalosporin-Resistant or Carbapenem-Resistant Escherichia coli Isolates from Rivers and Sewage Treatment Plants in India

“…To obtain the draft genome sequence, pulsed-field gel electrophoresis (PFGE) and next-generation sequencing (NGS) were performed. Briefly, plasmid DNA was purified from S1 nuclease-digested genomic DNA that had been separated by PFGE, as previously described (15), and the bands were visualized with SYBR Safe gel stain (Life Technologies Japan, Tokyo, Japan) under a blue-light transilluminator, followed by purification using a ZR-96 Zymoclean gel DNA recovery kit (Zymo Research, Irvine, CA, USA). A DNA sequencing library (insert size, 750 to 1,000 bp) was prepared using a Nextera XT DNA sample preparation kit (Illumina, Inc., San Diego, CA) for sequencing on an Illumina MiSeq sequencer (Illumina) according to the manufacturer's instructions.…”