Masahiro Kusumoto

Identification of an Insertion Sequence, IS1203 Variant, in a Shiga Toxin 2 Gene of Escherichia coli O157:H7

An insertion sequence composed of 1310 bp was found in Shiga toxin 2 genes of some isolates of Escherichia coli O157:H7. This insertion sequence showed extremely high homology with IS1203 of E. coli O111:H(-). This IS1203 variant was inserted in the region encoding the amino-terminus of the B subunit with a duplication of 3 bp at the target site, resulting in inactivation of the Shiga toxin 2 gene.

Reactivation of Insertionally Inactivated Shiga Toxin 2 Genes of Escherichia coli O157:H7 Caused by Nonreplicative Transposition of the Insertion Sequence

ABSTRACT IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes of Escherichia coliO157:H7. We analyzed the transpositional mechanism of IS1203v in order to investigate whether the Shiga toxin 2 genes inactivated by IS1203v could revert to the wild type. When the transposase activity of IS1203v was enhanced by artificial frameshifting, IS1203v was obviously excised from the Shiga toxin 2 gene in a circular form. The IS1203v circle consisted of the entire IS1203v, but an extra 3-bp sequence (ATC) intervened between the 5′ and 3′ ends of IS1203v. The extra 3-bp sequence was identical to a direct repeat which was probably generated upon insertion. Moreover, we detected the Shiga toxin 2 gene with a precise excision of IS1203v. In the wild-type situation, the transposition products of IS1203v could be observed by PCR amplification. These results show that IS1203v can transpose in a nonreplicative manner and that the Shiga toxin gene inactivated by this insertion sequence can revert to the wild type.

Identification of Shiga Toxin-Producing Escherichia coli Possessing Insertionally Inactivated Shiga Toxin Gene

We have investigated the Shiga toxin genes of Shiga toxin‐producing Escherichia coli (STEC) strains, using polymerase chain reaction (PCR) amplifying the full lengths of these genes. As a result, we found the Shiga toxin 2 gene which was insertionally inactivated by an insertion sequence (IS). This IS element was identical to IS1203v which has been also found in inactivated Shiga toxin 2 genes, and was inserted at the same site as in the previous paper. On the other hand, both Shiga toxin 2 genes were different (98.3% identity). These suggested that IS1203v independently inserted into each Shiga toxin 2 genes, and STEC strains possessing the insertionally inactivated Shiga toxin genes are most likely to have a wide distribution. Amplification of the full length of the Shiga toxin gene is one of the effective methods to detect the gene no matter where the IS element is included, i.e., the insertion can be reflected in the size of amplicon.

Spontaneous reactivation of Shiga toxins in Escherichia coli O157:H7 cells caused by transposon excision.

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes (stx2) of Escherichia coli O157:H7. Using PCR amplification, we detected the wild-type stx2 genes in colonies of E. coli O157:H7 which possessed stx2 genes inactivated by insertion of IS1203v. This suggests that IS1203v is excised from the inactivated stx2 genes in E. coli O157:H7. We isolated the cells possessing the wild-type stx2 genes, and confirmed Stx2 productivities by reversed passive latex agglutination. We also analyzed the frequency of the appearance of the Stx2-producing cells using a quantitative PCR method. As a result, the frequency was 3.00 x 10(-6) with culturing for 24 h at 37 degrees C, and this increased to 8.83 x 10(-5) when E. coli O157:H7 possessing the inactivated stx2 genes was transformed by an expression plasmid harboring the IS1203v transposase. These results showed that some Stx2-nonproducing E. coli O157:H7 strains could be spontaneously changed into Stx2-producing cells.

Host-dependent activation of IS1203v excision in shiga toxin-producing Escherichia coli

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.

Phylogenetic Characterization of Salmonella enterica Serovar Typhimurium and Its Monophasic Variant Isolated from Food Animals in Japan Revealed Replacement of Major Epidemic Clones in the Last 4 Decades

ABSTRACT Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) and its monophasic variant (Salmonella 4,[5],12:i:−) are the major causes of gastroenteritis in both humans and animals. Pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis have been used widely as subtyping methods for these pathogens in molecular epidemiological analyses, but the results do not precisely reflect phylogenetic information. In this study, we performed a phylogenetic analysis of these serovars using whole-genome sequencing data and identified nine distinct genotypic clades. Then, we established an allele-specific PCR-based genotyping method detecting a clade-specific single nucleotide polymorphism to rapidly identify the clade of each isolate. Among a total of 815 isolates obtained from cattle in Japan between 1977 and 2017, clades 1, 7, and 9 contained 77% of isolates. Obvious replacement of the dominant clone was observed five times in this period, and clade 9, which mostly contains Salmonella 4,[5],12:i:−, is currently dominant. Among 140 isolates obtained from swine in Japan between 1976 and 2017, clades 3 and 9 contained 64% of isolates. Clade 9 is the latest clone as is the case in cattle isolates. Clade 9 is similar to an epidemic clone from Europe, which is characterized by sequence type 34 (ST34), chromosomal Salmonella genomic island 3, and a composite transposon containing antimicrobial resistance genes. The increased prevalence of clade 9 among food animals in Japan might be a part of the pandemic of the European Salmonella 4,[5],12:i:− clone.