Hemolytic-uremic syndrome associated with enterohemorrhagic Escherichia coli O26:H infection and consumption of unpasteurized cow's milk

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Shiga Toxin-Mediated Hemolytic Uremic Syndrome: Time to Change the Diagnostic Paradigm?

Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic Escherichia coli (EHEC) which possess genes encoding Shiga toxin (stx), the major virulence factor, and adhesin intimin (eae). However, the frequency of stx-negative/eae-positive E. coli in stools of HUS patients and the clinical significance of such strains are unknown.Between 1996 and 2006, we sought stx-negative/eae-positive E. coli in stools of HUS patients using colony blot hybridization with the eae probe and compared the isolates to EHEC causing HUS. stx-negative/eae-positive E. coli were isolated as the only pathogens from stools of 43 (5.5%) of 787 HUS patients; additional 440 (55.9%) patients excreted EHEC. The majority (90.7%) of the stx-negative/eae-positive isolates belonged to serotypes O26:H11/NM (nonmotile), O103:H2/NM, O145:H28/NM, and O157:H7/NM, which were also the most frequent serotypes identified among EHEC. The stx-negative isolates shared non-stx virulence and fitness genes with EHEC of the corresponding serotypes and clustered with them into the same clonal complexes in multilocus sequence typing, demonstrating their close relatedness to EHEC.At the time of microbiological analysis, approximately 5% of HUS patients shed no longer the causative EHEC, but do excrete stx-negative derivatives of EHEC that lost stx during infection. In such patients, the EHEC etiology of HUS is missed using current methods detecting solely stx or Shiga toxin; this can hamper epidemiological investigations and lead to inappropriate clinical management. While maintaining the paradigm that HUS is triggered by Shiga toxin, our data demonstrate the necessity of considering genetic changes of the pathogen during infection to adapt appropriately diagnostic strategies

Phylogenetic relatedness of <i>stx</i>-negative and <i>stx</i>-positive <i>E. coli</i> strains within serotypes O26:H11/NM, O103:H2/NM, O121:H19, O145:H28/NM, and O157:H7/NM.

<p>Unrooted neighbor-joining tree was generated from allelic profiles of seven housekeeping genes (<i>adk</i>, <i>fumC</i>, <i>gyrB</i>, <i>icd</i>, <i>mdh</i>, <i>purA</i>, <i>recA</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001024#pone.0001024-Wirth1" target="_blank">[30]</a> using the Phylip software package (<a href="http://evolution.genetics.washington.edu/phylip.html" target="_blank">http://evolution.genetics.washington.edu/phylip.html</a>). ST, sequence type; CC, clonal complex (at least six identical alleles); NM, non-motile; <i>stx</i>, Shiga toxin-encoding gene; <i>stx</i>−, <i>stx</i>-negative; <i>stx</i>+, <i>stx</i>-positive; SF, sorbitol-fermenting; NSF, non-sorbitol-fermenting. Strains of serotype O121:H19 differ by at least 4 alleles from all known sequence types and have therefore no assigned clonal complex. Scale bar, 5% estimated evolutionary distance.</p

Comparison of phenotypes of <i>stx</i>-negative and <i>stx</i>-positive <i>E. coli</i> strains of serotypes O26:H11/NM, O103:H2/NM, O121:H19, O145:H28/NM, and O157:H7/NM.

a<p>The phenotypes were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001024#s2" target="_blank">Materials and Methods</a>.</p>b<p>NM, nonmotile; <i>stx</i>−, <i>stx</i>-negative; <i>stx</i>+, <i>stx</i>-positive. n, number of strains tested; +, all strains tested (n) expressed the phenotype; −, none of the strains tested (n) expressed the phenotype; if a subset of the strains expressed the phenotype, the percentage is given in parenthesis.</p>c<p>The CDT-V titers were 1∶4–1∶16 in both <i>stx</i>-negative and <i>stx</i>-positive strains as determined by Chinese hamster ovary cell assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001024#pone.0001024-Janka1" target="_blank">[26]</a>.</p

Numbers of HUS patients from whom <i>stx</i>-negative/<i>eae</i>-positive <i>E. coli</i> or EHEC strains were isolated and serotypes of the isolates.

a<p>NM, nonmotile; NSF, non-sorbitol-fermenting; SF, sorbitol-fermenting.</p>b<p>Serotypes (number of isolates, if more than one, in parenthesis): O121:H19, ONT:H6 (2), ONT:H7; ONT, O antigen not typeable with antisera against <i>E. coli</i> O antigens 1 to 181.</p>c<p>Serotypes (number of isolates, if more than one, in parenthesis): O4:NM, O55:H7, O55:HNT, O70:H8, O73:H18, O76:H19, O91:H21 (2), O98:NM, O104:H4, O112:NM, O113:H21 (2), O119:H2, O121:H19 (2), O128:H2, O136:HNT, O145:H25 (2), O163:H19, O174:H21, Orough:H2, Orough:H11, Orough:NM, ONT:H21, ONT:NM, ONT:HNT; Orough, autoagglutinable strains; HNT, H antigen not typeable with antisera against <i>E. coli</i> H antigens 1 to 56.</p>d<p>In none of the 483 culture-positive patients <i>stx</i>-negative and s<i>tx</i>-positive (EHEC) strains were found in the same stool sample.</p>e<p>Four patients shed two different EHEC serotypes including <u>O157:H7</u> and O145:NM; <u>O157:H7</u> and O103:H2; <u>SF O157:NM</u> and O145:NM; <u>O26:H11</u> and O145:NM (the underlined serotypes which prevailed in the stools and were isolated as the first are included in the table).</p