AUTHOR=Hauser Jocelyn R. , Atitkar Rama R. , Petro Courtney D. , Lindsey Rebecca L. , Strockbine Nancy , O'Brien Alison D. , Melton-Celsa Angela R. 

TITLE=The Virulence of Escherichia coli O157:H7 Isolates in Mice Depends on Shiga Toxin Type 2a (Stx2a)-Induction and High Levels of Stx2a in Stool

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=10

YEAR=2020

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2020.00062

DOI=10.3389/fcimb.2020.00062

ISSN=2235-2988

ABSTRACT=<p>In this study we compared nine Shiga toxin (Stx)-producing <italic>Escherichia coli</italic> O157:H7 patient isolates for Stx levels, <italic>stx</italic>-phage insertion site(s), and pathogenicity in a streptomycin (Str)-treated mouse model. The strains encoded <italic>stx</italic><sub>2a</sub>, <italic>stx</italic><sub>1a</sub> and <italic>stx</italic><sub>2a</sub>, or <italic>stx</italic><sub>2a</sub> and <italic>stx</italic><sub>2c</sub>. All of the strains elaborated 10<sup>5</sup>-10<sup>6</sup> cytotoxic doses 50% (CD<sub>50</sub>) into the supernatant after growth <italic>in vitro</italic> as measured on Vero cells, and showed variable levels of increased toxin production after growth with sub-inhibitory levels of ciprofloxacin (Cip). The <italic>stx</italic><sub>2a</sub>+<italic>stx</italic><sub>2c</sub>+ isolates were 90–100% lethal for Str-treated BALB/c mice, though one isolate, JH2013, had a delayed time-to-death. The <italic>stx</italic><sub>2a</sub>+ isolate was avirulent. Both an <italic>stx</italic><sub>2a</sub> and a <italic>recA</italic> deletion mutant of one of the <italic>stx</italic><sub>2a</sub>+<italic>stx</italic><sub>2c</sub>+ strains, JH2010, exhibited at least a three-log decrease in cytotoxicity <italic>in vitro</italic> and both were avirulent in the mice. Stool from Str-treated mice infected with the highly virulent isolates were 10- to 100-fold more cytotoxic than feces from mice infected with the clinical isolate, JH2012, that made only Stx2a. Taken together these findings demonstrate that the <italic>stx</italic><sub>2a</sub>-phage from JH2010 induces to higher levels <italic>in vivo</italic> than does the phage from JH2012. The <italic>stx</italic><sub>1a</sub>+<italic>stx</italic><sub>2a</sub>+ clinical isolates were avirulent and neutralization of Stx1 in stool from mice infected with those strains indicated that the toxin produced <italic>in vivo</italic> was primarily Stx1a. Treatment of mice infected with Stx1a+Stx2a+ isolates with Cip resulted in an increase in Stx2a production <italic>in vivo</italic> and lethality in the mice. Our data suggest that high levels of Stx2a in stool are predictive of virulence in mice.</p>