Distribution of Non-Locus of Enterocyte Effacement Pathogenic Island-Related Genes in Escherichia coli carrying eae from Patients with Diarrhea and Healthy Individuals in Japan ▿

The relationship to diarrhea of genes located on the pathogenicity islands (PAI) other than the locus of enterocyte effacement (LEE) was investigated. Enteropathogenic Escherichia coli (EPEC), the retention of espC on the EspC PAI, the OI-122 genes (efa1/lifA, nleB), the phylogenetic marker gene yjaA, and the bundle-forming pilus gene bfpA on the EPEC adherence factor (EAF) plasmid were studied. E. coli strains carrying the intimin gene (eae) without the Shiga toxin gene, isolated from patients with diarrhea (n = 83) and healthy individuals (n = 38) in Japan, were evaluated using PCR. The genotypes of eae and espC were identified by heteroduplex mobility assay (HMA). The proportions of strains isolated from individuals with and without diarrhea that carried these genes were as follows: bfpA, 13.3 and 7.9%, respectively; espC, 25.3 and 36.8%; efa1/lifA, 32.5 and 13.2%; nleB, 63.9 and 60.5%; yjaA, 42.2 and 55.3%. Statistical significance (P < 0.05) was achieved only for efa1/lifA. The proportion of strains lacking espC and carrying efa1/lifA was higher for patient-derived strains (30.1%) than for strains from healthy individuals (13.2%), but the difference was not significant. Strains carrying both espC and efa1/lifA were rare (2 strains from patients). Statistical analyses revealed significant relationships between espC and yjaA and between efa1/lifA and nleB, as well as significant inverse relationships between espC and efa1/lifA and between efa1/lifA and yjaA. espC was found in eae HMA types a1, a2, and c2, whereas efa1/lifA was found in types b1, b2, and c1. In addition, 6 polymorphisms of espC were found. The espC, yjaA, efa1/lifA, and nleB genes were mutually dependent, and their distributions were related to eae type, findings that should be considered in future epidemiological studies

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

Intimin Types Determined by Heteroduplex Mobility Assay of Intimin Gene (eae)-Positive Escherichia coli Strains

We developed a quick genetic approach to screen variants of the intimin gene (eae) by using a heteroduplex mobility assay (HMA) that targets the 5′ conserved region of eae. The eae variants were categorized into 4 major HMA types and 10 minor subtypes

stx_2f PCR-positive droppings from pigeons from 12 locations in Kyushu, Japan (experiment 1).

<p>stx_2f PCR-positive droppings from pigeons from 12 locations in Kyushu, Japan (experiment 1).</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

Characteristics of Shiga toxin 2f-producing <i>Escherichia coli</i> isolates that were isolated in this study.

<p>*<i>astA</i>, heat-stable toxin 1; <i>lpfA</i>O26, major subunit of long polar fimbriae of STEC O26; <i>sfpA</i>, major fimbrial subunit of Sfp fimbriae; <i>irp</i>₂, iron-repressible protein 2; <i>fyuA</i>, ferric yersiniabactin uptake receptor; <i>cdt</i>, cytolethal distending toxin; EHEC-<i>hlyA</i>, enterohemorrhagic <i>E. coli</i> hemolysin: <i>bfpA</i>, major pilin structural unit bundling; <i>iha</i>, iron-regulated gene A homolog adhesin; <i>espP</i>, extracellular serine protease; <i>efa1</i>, enterohemorrhagic <i>E. coli</i> factor for adherence (Efa-1); <i>pagC</i>, outer membrane invasion protein.</p>†<p>MMC, mitomycin C.</p>‡<p>Type A is as same as the sequence of O128:H2 isolates (AJ270998 and AJ 010730) in Europe, compared across 1,230 bp; Type B is as same as the sequence of an O63 isolate (AB232172) from Nagasaki, Japan, compared across 1,230 bp.</p>§<p>+, positive; −, negative.</p>‖<p>OUT, untypeable in O serogroup.</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

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