Multi-locus sequence typing of Salmonella enterica subsp. enterica serovar Enteritidis strains in Japan between 1973 and 2004

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) was responsible for a worldwide pandemic during the 1980s and 1990s; however, changes in the dominant lineage before and after this event remain unknown. This study determined S. Enteritidis lineages before and after this pandemic event in Japan using multilocus sequence typing (MLST). Thirty S. Enteritidis strains were collected in Japan between 1973 and 2004, consisting of 27 human strains from individual episodes, a bovine strain, a liquid egg strain and an eggshell strain. Strains showed nine phage types and 17 pulsed-field profiles with pulsed-field gel electrophoresis. All strains had homologous type 11 sequences without any nucleotide differences in seven housekeeping genes. These MLST results suggest that S. Enteritidis with the diversities revealed by phage typing and pulsed-field profiling has a highly clonal population. Although type 11 S. Enteritidis may exhibit both pleiotropic surface structure and pulsed-field type variation, it is likely to be a stable lineage derived from an ancestor before the 1980s and/or 1990s pandemic in Japan

<Preliminary>An O-Methyltransferase (OMT) cDNA Clone in Japanese Red Pine (Pinus densiflora) Seedlings

この論文は国立情報学研究所の学術雑誌公開支援事業により電子化されました

Isolation and characteristics of Shiga toxin 2f-producing Escherichia coli among pigeons in Kyushu, Japan.

An increasing number of Shiga toxin 2f-producing Escherichia coli (STEC2f) infections in humans are being reported in Europe, and pigeons have been suggested as a reservoir for the pathogen. In Japan, there is very little information regarding carriage of STEC2f by pigeons, prompting the need for further investigation. We collected 549 samples of pigeon droppings from 14 locations in Kyushu, Japan, to isolate STEC2f and to investigate characteristics of the isolates. Shiga toxin stx 2f gene fragments were detected by PCR in 16 (2.9%) of the 549 dropping samples across four of the 14 locations. We obtained 23 STEC2f-isolates from seven of the original samples and from three pigeon dropping samples collected in an additional sampling experiment (from a total of seven locations across both sampling periods). Genotypic and phenotypic characteristics were then examined for selected isolates from each of 10 samples with pulsed-field gel electrophoresis profiles. Eight of the stx 2f gene fragments sequenced in this study were homologous to others that were identified in Europe. Some isolates also contained virulence-related genes, including lpfA O26, irp 2, and fyuA, and all of the 10 selected isolates maintained the eae, astA, and cdt genes. Moreover, five of the 10 selected isolates contained sfpA, a gene that is restricted to Shiga toxin-producing E. coli O165:H2 and sorbitol-fermenting Shiga toxin-producing E. coli O157:NM. We document serotypes O152:HNM, O128:HNM, and O145:H34 as STEC2f, which agrees with previous studies on pigeons and humans. Interestingly, O119:H21 was newly described as STEC2f. O145:H34, with sequence type 722, was described in a German study in humans and was also isolated in the current study. These results revealed that Japanese zoonotic STEC2f strains harboring several virulence-related factors may be of the same clonal complexes as some European strains. These findings provide useful information for public health-related disease management strategies in Japan

Phylogenetic tree showing <i>stx</i>_2f nucleotide sequence clusters in this study and <i>stx</i>_2f sequences in other <i>Escherichia coli</i>.

<p>The <i>stx</i>_2f sequence in this study belonged to the branch of <i>stx</i>_2f genogroups, based on approximately 1.23 kb of sequence from the start codon of subunit A to the stop codon of subunit B. Accession numbers for reference sequences are in parentheses. Nucleotide sequence clusters identified in the current study are indicated in bold. The scale bar indicates the number of nucleotide substitutions per site. Cluster analysis was performed using an unweighted pair-group method with arithmetic average using MEGA 4 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086076#pone.0086076-Tamura1" target="_blank">[18]</a>. “Type A” and “Type B” are described in footnote “‡” of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086076#pone-0086076-t002" target="_blank">Table 2</a>.</p

Results of multi-locus sequence typing of Shiga toxin 2f-producing <i>Escherichia coli</i> isolates.

<p>(a) Phylogenetic tree showing nucleotide sequence clusters of selected isolates with multi-locus sequence typing. Allele sequences for each strain were concatenated in the order <i>adk</i>–<i>fumC</i>–<i>gyrB</i>–<i>icd</i>–<i>mdh</i>–<i>purA</i>–<i>recA</i> for a final composite length of 3,423 bp. Reference sequence types (ST) 20, ST 382, ST 583, ST 722, ST 582, and ST 585 are available from the study by Prager <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086076#pone.0086076-Prager1" target="_blank">[1]</a>. Other reference sequences were tested in the present study. The scale bar indicates the number of nucleotide substitutions per site. <i>Escherichia coli</i> type strain ATCC11775^T and <i>Escherichia albertii</i> type strain LMG20976^T are included as reference. (b) A minimum spanning tree was also constructed using Prim's algorithm from the PubMLST site (<a href="http://pubmlst.org/" target="_blank">http://pubmlst.org/</a>, accessed May 2, 2013).</p

Comparison of pulsed-field gel electrophoresis analysis of fingerprints from Shiga toxin 2f-producing <i>Escherichia coli</i> isolates digested with <i>Xba</i>I.

<p>Isolates 65-4 to H4-2 were tested with Tris-borate-EDTA buffer. Isolates 17-8 to O1-3 were tested using buffer with 50 µM thiourea because they showed degradation in the presence of Tris.</p