New Therapeutic
Developments against Shiga
Toxin-Producing Escherichia coli
ANGELA R. MELTON-CELSA1 and ALISON D. O’BRIEN1
1
Department of Microbiology and Immunology, Uniformed Services
ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC)
is an etiologic agent of bloody diarrhea. A serious sequela of
disease, the hemolytic uremic syndrome (HUS) may arise in up
to 25% of patients. The development of HUS after STEC infection
is linked to the presence of Stx. STEC strains may produce one or
more Stxs, and the Stxs come in two major immunological
groups, Stx1 and Stx2. A multitude of possible therapeutics
designed to inhibit the actions of the Stxs have been developed
over the past 30 years. Such therapeutics are important because
antibiotic treatment of STEC infections is contraindicated due to
an increased potential for development of HUS. The reason for
the increased risk of HUS after antibiotic treatment is likely
because certain antibiotics induce expression of the Stxs, which
are generally associated with lysogenic bacteriophages. There
are a few potential therapeutics that either try to kill STEC
without inducing Stx expression or target gene expression within
STEC. However, the vast majority of the treatments under
development are designed to limit Stx receptor generation or to
prevent toxin binding, trafficking, processing, or activity within
the cell. The potential therapies described in this review include
some that have only been tested in vitro and several that
demonstrate efficacy in animals. The therapeutics that are
currently the furthest along in development (completed phase I
and II trials) are monoclonal antibodies directed against Stx1
and Stx2.
BACKGROUND
Shiga toxin (Stx)-producing Escherichia coli (STEC)
colonizes the intestine and causes hemorrhagic colitis.
STEC encodes a variety of colonization factors, but
a significant subset of STEC, the enterohemorrhagic
E. coli (EHEC) strains, have the locus of enterocyte
effacement (LEE), the products of which allow the
ASMscience.org/MicrobiolSpectrum
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
University of the Health Sciences, Bethesda, MD 20814
bacteria to intimately adhere to and form attaching
and effacing lesions on intestinal tissue. The O157:H7
strains, which are responsible for the majority of large
outbreaks due to STEC infection, are members of the
EHEC group. All STEC strains make one or more Stxs;
these pathogens may produce two immunologically
distinct but highly similar Stxs, Stx1 and Stx2. These
toxins are briefly described in the section on therapeutics
targeted to the Stxs.
Some individuals infected with STEC manifest a se-
rious sequela called hemolytic uremic syndrome (HUS),
a thrombotic microangiopathy defined by the presence
of hemolytic anemia, thrombocytopenia, and renal fail-
ure. The initial insult leading to the development of the
thrombotic microangiopathy is damage by the Stx(s) to
vascular endothelial cells that express the toxin receptor,
globotriaosylceramide (Gb3). The Stx-mediated injury
to endothelial cells initiates a cascade of events that
lead to the activation of platelets and the formation of
thrombi in the small vessels of the kidney and sometimes
Received: 10 July 2013, Accepted: 21 August 2013,
Published: 12 September 2014
Editors: Vanessa Sperandio, University of Texas Southwestern
Medical Center, Dallas, TX, and Carolyn J. Hovde, University of Idaho,
Moscow, ID
Citation: Melton-Celsa AR, O’Brien AD. 2014. New therapeutic
developments against Shiga toxin-producing Escherichia coli.
Microbiol Spectrum 2(5):EHEC-0013-2013. doi:10.1128
/microbiolspec.EHEC-0013-2013.
Correspondence: Angela R. Melton-Celsa, angela.melton-celsa
.ctr@usuhs.edu
© 2014 American Society for Microbiology. All rights reserved.
1
Melton-Celsa and O’Brien
the central nervous system. Prevention of HUS is im-
portant as HUS can lead to death or long-term conse-
quences such as hypertension and renal disease (1–3).
Because STEC strains are intestinal bacterial pathogens,
the inclination by physicians is to treat with antibiotics
to eliminate the organisms from the gut. However,
several studies show that treatment of STEC-infected
patients with certain antibiotics may lead to an increase
in HUS (antibiotic use is discussed further in the next
section). Therefore, the focus for therapeutics against
STEC and HUS has been to find (i) compounds that act
at the level of the bacterium but do not cause an increase
in Stx production; (ii) receptor mimics or other mole-
cules that alter trafficking of the toxin or the cellular
response to the toxin; (iii) antibodies directed against the
Stxs; or (iv) therapies to prevent or treat the HUS disease
process. The therapies discussed in this article are listed
in Table 1.
THE DIFFICULTIES WITH ANTIBIOTIC
USE FOR STEC INFECTIONS
In the United States, antibiotics are not recommended
for treatment of STEC infections because of the in-
creased risk for the development of HUS (4, 5). How-
ever, even though antibiotics are contraindicated for
those with STEC infection, a recent study at FoodNet
sites found that antibiotics are commonly used in those
with proven O157 infection (6). The issue of the use of
antibiotics to treat STEC infections is confounded by a
number of factors: (i) some, but not all, antibiotics at
sublethal doses increase the expression of Stx by in-
ducing the lysogenic phage that encodes the toxin genes
(see diagram in Fig. 1) (7); (ii) treatment of STEC with a
lethal dose of antibiotics may cause release of a large
bolus of toxin at one time as the bacteria die—a result
that would be bad for a patient; (iii) in some STEC
strains, the Stxs are either chromosomally encoded or
are associated with defective bacteriophages, and thus
antibiotic treatment may not alter Stx expression; (iv)
antibiotics may be used only in the most ill patients, a
fact that may skew the apparent risk for HUS develop-
ment; and (v) published studies on the risk of HUS as-
sociated with antibiotic treatment used different
treatments administered at different stages in the disease
process. In addition, although several studies demon-
strate an increased risk for HUS after treatment with
antibiotics (8, 9), others do not find an increased risk
(10). Conversely, several studies indicate that the use of
β-lactam antibiotics, particularly in the first 2 to 3 days
of illness, and perhaps associated with age <13 years, is
2 ASMscience.org/MicrobiolSpectrum
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
correlated with increased HUS risk (11). In contrast,
many people were treated with fosfomycin during a
large O157:H7 outbreak in Japan without an apparent
increase in HUS, and perhaps even a protective effect
if the antibiotic was given within the first 2 days of illness
(12). However, the low overall HUS rate in the Sakai
outbreak (13) suggests that the causative strain may
(fortunately) have had reduced virulence compared with
O157 strains from other outbreaks, and, as such, would
make that outbreak a poor platform on which to make
generalized decisions about treatment with antibiotics.
During the O104:H4 outbreak in Germany in 2011
caused by an unusual enteroaggregative E. coli (EAEC)
strain that makes Stx2, antibiotics were used in many
patients, some of whom were given up to three anti-
biotics at various times after disease onset (14). Because
of the vastly different protocols used to treat patients
during the outbreak in Germany, it is difficult to make
generalized conclusions about antibiotic use based on
that outbreak; however, overall there was not defini-
tive evidence of a benefit due to antibiotic treatment.
Although one surprising study asserted ciprofloxacin
treatment reduced the HUS incidence in patients, the
number of treated patients was small (n = 5), and in
two of those patients HUS did develop (15). Further-
more, in several patients from the O104:H4 outbreak
HUS was reported to develop after ciprofloxacin or
metronidazole treatment (16, 17). We consider the use
of ciprofloxacin to be especially dangerous because of
the evidence that ciprofloxacin increases Stx expres-
sion in the EAEC O104:H4 strain (18) and STEC O157
strains. Finally, one unique aspect of the outbreak in
Germany was the use of azithromycin to reduce shed-
ding of O104:H4 in patients who appeared to be long-
term carriers (19).
Animal studies also give contradictory information
about the use of antibiotics for the treatment of STEC
infections. In two studies fosfomycin reduced coloniza-
tion and mortality from STEC infection in mice (20, 21),
but another study did not show a protective effect by
that antibiotic (22). In a mouse model in which the mice
are starved for protein calories, the use of trimethoprim-
sulfamethoxazole was detrimental when given between
days 3 and 5 post infection, whereas the same antibiotic
as well as ampicillin and fosfomycin were protective
when given anywhere from days 1 to 5 after infection
(23). Nevertheless, we should note that the use of am-
picillin to treat an infection with an organism (Shigella
dysenteriae or the Stx2+ EAEC O104:H4, respectively)
resistant to that antibiotic appears to be detrimental to
humans and mice (24, 25).
 New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli
Overall, the current data do not support the use
of antibiotics for the treatment of STEC infection in
humans, and the majority of the evidence indicates
that antibiotic treatment is potentially detrimental. The
empirical use of antibiotics could be dangerous, since
some of these treatments could increase the risk of
HUS. Furthermore, since there is no clear demonstra-
ble benefit to the use of antibiotics in STEC-infected
patients, and because antibiotic use itself may pose
possible risk to the patient (allergy or other risks), we
believe antibiotics should not be used to treat STEC
infections.
COMPOUNDS DIRECTED TOWARD STEC
Pyocin
A novel strategy to kill O157 strains based on a modified
pyocin (a bactericidal protein, similar to bacteriophage
tail fibers, made by Pseudomonas aeruginosa that usu-
ally targets other P. aeruginosa cells) that consists of
the R2 tail fiber fused to a phage spike protein specific
for O157 was found to kill O157 strains without in-
creasing expression of Stx (26). Subsequent tests dem-
onstrated that a slightly modified version of the pyocin,
AvR2-V10.3, given 3 h after infection and once daily
for the next 2 days to infant rabbits infected with
O157:H7 strain EDL933 reduced colonization and pre-
vented diarrhea at the highest dose of pyocin admin-
istered (27). When the modified pyocin was given to
EDL933-infected infant rabbits that exhibited diarrhea,
the amount of loose stool decreased relative to the
control animals given buffer alone and the numbers of
EDL933 organisms decreased in the intestines and
stools. One problem with the pyocin approach is that it
is specific for serogroup, so other pyocins would have to
be developed for non-O157 STEC strains.
A Small Molecule and a Divalent Cation
Another alternative strategy to antibiotics directed to-
ward the bacterium uses a small molecule, LED209,
which inhibits the activity of the QseC sensor that is
involved in regulating some of the proteins involved in
EHEC adherence and, indirectly, Stx2 expression. Al-
though LED209 inhibited the formation of attaching
and effacing lesions by EHEC on HeLA cells, the com-
pound was not effective in reducing colonization or
disease in infant rabbits (28). The reason for LED209
failing to protect in the rabbits may be that the con-
centration of the compound was not high enough at the
site of EHEC colonization in the gut.
ASMscience.org/MicrobiolSpectrum
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
Similar to LED209, treatment of STEC infection with
the divalent cation zinc reduces adherence to a HeLa
cell monolayer, though through a different mechanism,
perhaps due to decreased expression of E. coli-secreted
proteins EspA and EspB (29). In addition to a reduction
in adherence to cells, zinc treatment also appears to
decrease expression of the stx genes. The potential
therapeutic effect of zinc was tested in a rabbit ileal loop
model, in which rabbit enteropathogenic E. coli strains
transduced with a bacteriophage that encodes stx1 or
stx2 were inoculated into the loop in the presence or
absence of zinc. The presence of 1 mM of zinc reduced
the amount of toxin found in the loops, decreased ad-
herence, and lessened histological damage. The next
avenue to explore for zinc is to determine if the cation
could prevent disease or mortality in an oral infection
model.
THERAPEUTICS THAT INTERFERE
WITH TOXIN BINDING, UPTAKE,
TRAFFICKING, OR FUNCTION
Stx1 and Stx2 are the key STEC virulence factors that
lead to the development of HUS. Therefore, therapeutics
that impede toxin binding, uptake, trafficking, or func-
tion are strong contenders for treatment of STEC
infections. Stx1 and Stx2 share about 56% homology at
the amino acid level but are immunologically distinct.
Structurally, the toxins consist of five identical B
subunits and a single A subunit. The B pentamer binds to
the cellular receptor, Gb3. The A subunit contains the
enzymatic function of the toxin and removes an adenine
residue from the 28S rRNA, an action that destroys
ribosome function. After binding to Gb3, the toxin re-
ceptor complex is taken up by clathrin-dependent and
-independent mechanisms. The toxin then traffics in a
retrograde direction from the endosome to the Golgi
apparatus to the endoplasmic reticulum (ER). The A
subunit of the toxin can be nicked within the Golgi body
such that the enzymatic function (within A1) is separated
by a disulfide bond from the A2 peptide that links A1 to
the B pentamer. The nicked toxin traffics next to the ER
where the disulfide bond between A1 and A2 is reduced.
The A1 subunit then enters the cytoplasm and targets the
ribosome. The damage to the ribosome halts protein
synthesis and can lead to a ribotoxic stress response and
apoptosis (see review in reference 30). A model of Stx
trafficking is shown in Fig. 2.
Stx1 and Stx2 are the major toxin types produced by
STEC that cause human disease. There are subtypes of
both Stx1 and Stx2, however, and toxin nomenclature
3
4

Melton-Celsa and O’Brien

TABLE 1 Therapies directed against STEC, the Stx receptor, Stx function, or the cellular response to Stx

Target (step in Fig. 2 at

which therapeutic acts) Therapeutic Function Model used to test function or efficacy Reference(s)
STEC or STEC pathogenesis Pyocin (AvR2-V10.3) Kills O157 In vitro growth; infant rabbits 26, 27
LED209 Reduces attaching and effacing lesions on HeLa cells; infant rabbits 28
HeLa cells but did not reduce colonization
or disease in rabbits
Zinc Reduces stx transcription; HeLa cells; rabbit ileal loops (using rabbit 29
reduces adherence enteropathogenic E. coli transduced with
phage carrying stx1 or stx2)
Receptor (Gb3) C-9 Receptor generation; glucosylceramide HRTEC; Stx2-intoxicated rats 36, 37
synthesis inhibitor synthase
ASMscience.org/MicrobiolSpectrum

Receptor analogs (B) SYNSORB Pk Receptor analog Phase I and II trials 39, 41
STARFISH Receptor analog Vero cells; Stx1-intoxicated mice 42, 43
(does not protect Stx2-intoxicated mice)
Daisy Receptor analog Vero cells; Stx1- and Stx2-intoxicated mice; 43
B2F1-infected, streptomycin-treated mice
SUPER TWIG (1)6 Receptor analog Vero cells; Stx1- and Stx2-intoxicated mice; 44
O157-infected, protein-calorie-deficient mice
Gb3 polymers Receptor analog Mice 45
TVP (also known as Ac-PPP-tet) Receptor analog, Vero cells; O157-infected, 46, 49, 50
binds Stx2 but not Stx1 protein-calorie-deficient mice,
rabbit ileal loops; baboon
MMA-tet Receptor analog Vero cells; O157-infected, 51
protein-calorie-deficient mice
Probiotic that displays Receptor analog Vero cells; streptomycin-treated mice 47, 52–54
Galα1-4Galβ1-4Glc- infected with an Stx2 or Stx2dact producer
HuSAP Binds Stx2 but not Stx1 Stx1- or Stx2-intoxicated mice 57–59
(only protects Stx2-intoxicated animals)

Downloaded from www.asmscience.org by

IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
ASMscience.org/MicrobiolSpectrum
Antitoxin antibodies Polyclonal anti-Stx2
(B, U, T, or E) cαStx1 (human/mouse chimeric
version of 13C4)
cαStx2 (anti-A subunit,
human/mouse chimeric
of 11E10)
Anti-Stx1 (5-5B)
Urtoxazumab (also called
TMA-15; humanized
version of VTm1.1)
Anti-Stx1 monoclonals
Anti-Stx2 (5C12)
Toxin transport (U or T) Exo2
Retro-2cycl
Chloroquine
Small molecules
Eeyarestatin 1
Nitrobenzyl-thioinosine
Manganese
Toxin processing (N) Furin inhibitor
Toxin function (E) Small molecule
Cellular or host response Imatinib
to toxin (ribotoxic stress Ouabain
response or apoptosis)
Complement factor C5 Eculizumab
5
Neutralize Stx2 Gnotobiotic pigs 62
Block Stx1 binding (B subunit) Vero cells; Stx1-intoxicated mice; 64, 68, 70
phase I and II trials
Alters intracellular trafficking and Vero cells; streptomycin-treated mice; 63, 68–70
inhibits enzymatic function phase I and II trials
Neutralize Stx1 (B subunit) Ramos cells 71
Neutralize Stx2 (B subunit) Human renal 73–75
adenocarcinoma cells; Stx2-intoxicated mice;
B2F1-infected streptomycin-treated mice,
phase I trial
Neutralize Stx1 (most are anti-B subunit) HeLa cells; Stx1-intoxicated mice 76
Neutralize Stx2 (A subunit) HeLa cells; Stx2-intoxicated mice; 77, 78
B2F1-infected streptomycin-treated mice;
O157-infected gnotobiotic piglets
Blocks transport at the level of the early Vero cells 82, 83
endosome/trans-Golgi interface
Toxin transport HeLa cells 85, 86
New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli
Toxin transport HEp-2 cells 87
Toxin transport HeLa cells 88
Intracellular trafficking HeLa cells 90
Stx1 trafficking Human renal cortical epithelial cells 91
(retention in early endosomes)
Protects HeLa cells and BALB/c mice from HeLa cells; Stx1-intoxicated BALB/c mice 92, 93
Stx1-mediated lethality; blocks StxB1 (does not protect Stx2-intoxicated mice)
trafficking; does not protect against Stx2
Cleavage of A subunit to A1 and A2 HEp-2 cells 94
Enzymatic activity Cell-free reporter assay 89
Ribotoxic stress response inhibitor HCT-8 cells; infant rabbit 98
Apoptosis inhibition Rat proximal tubule cells (RPTC) 99
Anticomplement factor C5 STEC-infected patients 101, 105, 106
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
Melton-Celsa and O’Brien

FIGURE 1 Induction of the stx-encoding phage by antibiotics such as ciprofloxacin.

Subinhibitory concentrations of some antibiotics induce the lysogenic stx phage to
enter the lytic cycle, and as consequence, stx-bacteriophage are made and released.
Furthermore, 10- to 100-fold more toxin is made and released from the cell. doi:10.1128
/microbiolspec.EHEC-0013-2013.f1

was recently updated so that the prototype Stxs are now
designated Stx1a and Stx2a. The less specific Stx1 and
Stx2 designations are used when the toxin subtype is
unknown (31). We use the more general designations for
the toxins elsewhere in this review for simplicity. Besides
the prototypic Stx2a, two Stx2 subtypes are associated
with HUS, Stx2c and Stx2d (32, 33). Stx2c and Stx2d
have two amino acid differences in the B subunit as
compared to Stx2a, and those changes are responsible
for reduced cytotoxicity on Vero cells, though Stx2d is
just as toxic to mice as Stx2a (34). Stx2d has two amino
acid differences from Stx2a and Stx2c in the A subunit,
and those two changes contribute to the capacity of the
toxin to exhibit increased toxicity when treated with
elastase from intestinal mucus (35), a phenotype indi-
cated with the designation Stx2dact.
Receptor Synthesis Inhibitor: C-9
One step at which to stop Stx is at the level of the toxin
receptor. Various laboratories have tried to protect cells
or animals from Stxs with compounds that prevent
Gb3 synthesis or that bind to the toxin to prevent toxin-
receptor interaction. For example, a molecule called
C-9 was used in human renal tubular epithelial cells
(HRTEC) to prevent the conversion of ceramide to glu-
cosylceramide, an early step in Gb3 synthesis. HRTEC
pretreated with C-9 for 24 h exhibited reduced levels
of Gb3 and decreased sensitivity to Stx2 (36). In rats
treated with C-9 for 2 days before intoxication and for 4
days post intoxication, about 50% protection from in-
jection with bacterial supernatants that contained Stx2
was observed (37). Treated rats also showed lower rises
in serum creatinine and urea and reduced renal tubular
injury.
Receptor Analogs
The first drug tested in humans intended for the treat-
ment of HUS was SYNSORB Pk, a silicon dioxide com-
pound that contains the trisaccharide component of
Gb3 (38, 39). The theory behind the use of SYNSORB
Pk is that the compound would bind up free Stx(s)
within the intestines of infected patients and prevent
that toxin from binding to the functional receptor so
that the toxin could not act either locally or systemically.
SYNSORB Pk was shown to neutralize both Stx1 and
Stx2 on human renal adenocarcinoma cells (40). Al-
though SYNSORB Pk was tolerated well in the phase I
trial (41), the double-blind placebo-controlled trial sug-
gested no difference between treated and placebo groups
(39). The reason for the lack of efficacy by SYNSORB Pk
in the latter study may be that the patients were enrolled
after HUS diagnosis, whereas the best time to neutralize
toxin to prevent HUS is most likely before development
of this serious sequela.
A number of other Stx receptor analogs in addition to
SYNSORB Pk have been developed, such as STARFISH
(42), Daisy (43), SUPER TWIG (1)6 (44), Gb3 polymers
(45), and Ac-PPPtet (46), as well as a probiotic that
displays an Stx binder on its surface (47). STARFISH is a
five-“armed” molecule with two receptor mimics at the
end of each arm; the compound appears to bind two B
pentamers at the same time and neutralizes both Stx1

6 ASMscience.org/MicrobiolSpectrum
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
 New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli

FIGURE 2 Cellular trafficking of Stx and points in the pathway where therapeutics func-
tion. Therapeutics can interfere with Stx action at several points as it traffics into and
through the cell. The steps in toxin trafficking are briefly diagramed above and outlined
here. The toxin first binds (B) to the receptor Gb3. The toxin/Gb3 complex is taken up (U)
by both clathrin-dependent and -independent mechanisms, and then traffics (T) from the
early endosome to the late endosome to the trans-Golgi apparatus. Within the Golgi the
toxin A subunit is nicked (N), but the toxin remains intact due to a disulfide bond between
the A1 and A2 subunits. The nicked toxin continues to traffic (T) along the retrograde
pathway to the endoplasmic reticulum. The disulfide bond in the A subunit is reduced (R)
within the endoplasmic reticulum and the A1 subunit enters the cytoplasm where it exerts
its enzymatic (E) attack and depurinates the ribosome. The action of the toxin within the
cell can lead to a ribotoxic stress response (RSR) and apoptosis (APOP). doi:10.1128
/microbiolspec.EHEC-0013-2013.f2

and Stx2 in vitro. However, STARFISH has a higher
avidity for Stx1 than Stx2 and was only able to protect
mice injected with Stx1 but not Stx2 (43). Because of the
lack of protective efficacy by STARFISH in the Stx2-
injection model, a similar but modified divalent trisac-
charide inhibitor called Daisy was synthesized (43).
Daisy protects Vero cells and mice from both Stx1 and
Stx2, and in a streptomycin-treated mouse model,
prevented death due to infection with O91:H21 STEC
strain B2F1 in 50% of the animals (43). The SUPER
TWIG (1)6 identified by Nishikawa’s group carries six
trisaccharides and blocks the binding of both Stx1 and
Stx2 to Vero cells, protects mice from Stx2 when ad-
ministered together with the toxin, and prevents death of
O157:H7-infected mice in a protein-calorie-deficient
animal model when given intravenously after infection
(44). However, one point to note is that SUPER TWIG
(1)12, developed in the latter study, neutralized as well
as SUPER TWIG (1)6 in vitro but did not protect in vivo,
a finding that demonstrates the importance of testing
potential therapeutics in an animal model system. To
find a receptor analog that could be given orally, the
Nishikawa group developed Gb3 polymers that bind to
the Stxs with higher affinity than SUPER TWIG (1)6,
and those polymers, when given by gavage twice daily in
the protein-calorie-deficient mouse model on days 3 to 5
after infection, protect mice from death (45). The strong
neutralization effect observed with SUPER TWIG and
the Gb3 polymers is due to the fact that the B pentamer
of the Stxs contains multiple Gb3-binding sites, so re-
ceptor mimics that display multiple copies of the Gb3
trisaccharide bind the toxin tightly. A similar approach
as described above was used by another group to display
the receptor sugar moiety on chitosan, and they similarly
found that the analog neutralizes in vitro and in vivo
(48).
According to Nishikawa, however, a drawback to the
multiple trisaccharide display approach is the complex-
ity of synthesis (46, 49); therefore, a tetravalent peptide
library was screened to find molecules that bind the Stx2

ASMscience.org/MicrobiolSpectrum 7
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
Melton-Celsa and O’Brien
pentamer (49). In that latter screen, tetravalent peptides
were identified that form a complex with Stx2 and neu-
tralize the toxin on Vero cells but do not prevent the
toxin/peptide moiety from binding to the cell. Rather,
the peptide-bound Stx2 was unable to reach the ER.
Furthermore, O157-infected protein-calorie-deficient mice
gavaged after infection with the peptides were protected
from death; the protection appeared to be dose depen-
dent and treatment had to be started by day 3 post
infection. Further testing of the optimized tetrapeptide,
Ac-PPP-tet (later renamed TVP), demonstrated that
the compound inhibits Stx2-mediated fluid accumu-
lation in rabbit ileal loops (46). However, the tetra-
valent peptide only protects when administered orally
and not intravenously in the protein-calorie-deficient
mouse model, so TVP efficacy was tested in the baboon
model of intoxication (50). Baboons given Stx2 and TVP
simultaneously were protected from death, renal injury,
and thrombocytopenia, but not anemia. TVP also res-
cued 75% of animals that received the drug 24 h post
intoxication and a supplemental dose on days 2, 3, and
4. Untreated but intoxicated baboons died by day 6. The
authors of that study suggest that the advantage of TVP
compared to other Stx binders is that the compound is
cell-permeable. However, it is unclear how TVP would
rescue intoxicated cells since the proposed mechanism
for action is for the Stx2-TVP complex to traffic differ-
ently within the cell than Stx2 alone, and indeed, in
vitro, cells are only protected if the TVP is added to
cells at the same time as Stx2. In a recent study, the
Nishikawa group identified another tetravalent peptide,
MMA-tet, that inhibits both Stx1 and Stx2 on Vero cells
when it is added at the same time as the toxins, and when
administered orally, protects protein-calorie-deficient
mice infected with STEC strain N-9 (51). The mecha-
nism whereby MMA-tet protects cells and animals is
unclear: MMA-tet forms a complex with the Stx1 B
subunit but does not appear to alter binding or traf-
ficking of the MMA-tet–StxB1 through the cell, at least
as far as the ER. However, MMA-tet pretreatment of
Vero cells did prevent protein synthesis inhibition by
Stx1, a finding that may indicate that MMA-tet some-
how prevents the toxin A1 subunit from reaching the
cytoplasm from the ER.
Finally, a novel modification of the receptor analog
approach to bind up Stx specifically within the intes-
tine was designed by Paton’s group: the core trisaccha-
ride from Gb3, Galα1-4Galβ1-4Glc-, was displayed on
the lipopolysaccharide (LPS) structure of a commensal
E. coli to create a probiotic (52). The constructed strain,
CWG308:pJCP-Gb3, neutralizes Stx1, Stx2, Stx2c, and
8 ASMscience.org/MicrobiolSpectrum
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
Stx2d on Vero cells. In addition, streptomycin-treated
mice are protected from infection with either an Stx2-
or Stx2d-producer when fed the Galα1-4Galβ1-4Glc-
probiotic twice daily. What was not clear in that
study was whether the mice would continue to do well
once the probiotic was discontinued. However, the pro-
biotic with a receptor-analog approach might be pos-
sible as long as the infecting strain did not persist for
long-term colonization. Additionally, it may be neces-
sary to monitor shedding of the infecting strain over
time. CWG308:pJCP-Gb3 was found to be effective
even when killed prior to administration; however,
the formaldehyde-treated probiotic had to be adminis-
tered three times daily for complete protection (53).
To develop a probiotic that might be able to be used in
humans, a K-12 strain that expressed the same Galα1-
4Galβ1-4Glc- epitope on its LPS was created (54). In
that strain, additional mutations were incorporated so
that no antibiotic resistance markers were needed for
plasmid maintenance. The K-12 probiotic that displays
Galα1-4Galβ1-4Glc- neutralizes both in vitro and in
vivo (54).
Human Serum Amyloid Component P (HuSAP)
Reports that there is an Stx2-neutralizing component in
normal human serum that was not immunoglobulin
were published in 1993 (55, 56). Later, HuSAP was
identified as the protein from plasma that can neutral-
ize Stx2, but not Stx1 (57). HuSAP administered intra-
venously protects BALB/c mice given Stx2 1 h later (58).
Another group showed similar results, though they ad-
ministered the HuSAP twice daily intraperitoneally and
the Stx2 by the subcutaneous route (59). This group
then challenged transgenic C57Bl/6 mice that express
HuSAP with two 50% lethal doses of Stx2. The HuSAP-
transgenic mice survived nearly twice as long as the
control mice in that study. The reason that the HuSAP-
injected BALB/c mice survived longer than the HuSAP-
transgenic mice was hypothesized to be because the
circulating levels of HuSAP were higher in the BALB/c
mice injected with HuSAP than in the transgenic ani-
mals. If, however, the HuSAP-transgenic mice were in-
jected with a combination of LPS and Stx2, the animals
were not protected, even though the LPS did not prevent
HuSAP-Stx2 interaction or the neutralization by HuSAP
of Stx2 for Vero cells in vitro (60). Exactly how HuSAP
binds Stx2 is not clear; Marcato et al. reported that the
interaction requires both toxin A and B subunits and
cannot be competed with Daisy (61). The latter finding
suggests that HuSAP does not bind within the Gb3-
binding sites on the B pentamer.
 New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli
ANTIBODIES TO THE Stxs
Because the Stxs are the primary factors responsible for
the development of HUS, neutralization of the toxins
is a critical therapeutic approach for STEC infections.
Polyclonal antiserum against Stx2 protects gnotobiotic
piglets from infection by O157:H7 strain 86-24 (62).
Furthermore, monoclonal antibodies to the toxins are
protective in animal models, and two groups have taken
such therapeutics into phase II safety trials, as detailed
below.
Monoclonal antibodies specific for the Stx1 B subunit
(13C4) and the Stx2 A subunit (11E10) were developed
in the O’Brien laboratory in the mid to late 1980s (63,
64). Those antibodies neutralize the toxins on Vero cells
(65, 66), and the Stx2 monoclonal antibody is protective
in mice (67). The epitopes for both antibodies have been
mapped (65, 66); of note, although 11E10 neutralizes
Stx2 in a cell-free protein synthesis assay, the antibody
also alters the intracellular localization of the toxin such
that the toxin-antibody complex does not reach the cy-
toplasm (65). Both 11E10 and 13C4 were transformed
into human/mouse chimeras by genetic techniques. The
“humanized” versions of anti-Stx1 and anti-Stx2 neu-
tralize the toxins on Vero cells and in either a mouse
intoxication model (Stx1) or the streptomycin-treated
mouse model of infection with strain B2F1 (68). The
humanized versions of the antibodies (cαStx1 for 13C4
and cαStx2 for 11E10) were evaluated in phase I (69, 70)
and II trials. The preliminary results of the phase II trial
indicate that the antibodies were safe and well tolerated
in sick children infected with STEC.
Takeda’s group developed monoclonal antibodies
that neutralize Stx1 or Stx2 by preventing receptor
binding (71, 72). The anti-Stx2 monoclonal was later
humanized and named TMA-15. TMA-15 demonstrates
Vero cell neutralization and protective efficacy in ani-
mal models (73, 74). Antibody TMA-15 was renamed
urtoxazumab and evaluated in safety trials in healthy
adults and STEC-infected children (75). Urtoxazumab
was well tolerated in STEC-infected children, but no
efficacy data are yet published.
Finally, Tzipori’s group generated fully humanized
neutralizing monoclonal anti-Stx1 and -Stx2 antibodies
in transgenic mice (76, 77). The anti-Stx2 monoclonal
antibody, 5C12, was developed further, and shown to
protect piglets and streptomycin-treated mice from
STEC strains that produce Stx2 or Stx2dact, respectively
(77, 78). Similarly to 11E10, antibody 5C12 neutralizes
Stx2 by altering its intracellular trafficking pattern and
also has the capacity to prevent protein synthesis inhi-
bition by Stx2 in a cell-free assay (79, 80). The Tzipori
ASMscience.org/MicrobiolSpectrum
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
group also evaluated isotype variants and Fab and F(ab′)2
fragments of 5C12 in in vitro and in vivo models of ef-
ficacy and found that all of the isotype variants demon-
strate in vitro and in vivo neutralization but the Fab and
F(ab′)2 were only efficacious in the in vitro assay (81).
One point of note from the latter study is that the IgG4
variant of 5C12 was the least protective of the isotype
variants in vitro, but was as protective in vivo as the best
in vitro neutralizer (the IgG3 variant). These latter results
indicate once again that in vitro and in vivo neutraliza-
tion data do not always correlate.
Nonantibody Inhibitors of Toxin Trafficking
or the Cellular Response to Stx
Brefeldin A (BFA), a fungal toxin that disrupts the Golgi
apparatus but also causes tabulation of early endo-
somes, is a known inhibitor of Stx transport; in fact,
the protective effect of BFA against the Stxs helped
define the pathway by which Stx is transported through
the cell. Because BFA is a global inhibitor of protein
transport within the cell, it is not included in Table 1.
Another Golgi disruptor, Exo2, a small molecule that
does not inhibit cholera toxin trafficking, prevents Stx
from reaching the Golgi apparatus (82). Unfortunately,
cell disruptors such as BFA and Exo2 are toxic to cells.
Another drawback to molecules such as BFA and Exo2
is that treated cells recover over time, such that incu-
bation of the cells with the inhibitor and the toxin for
longer periods reduces the effectiveness of the thera-
peutic. Exo2 was recently derivatized to generate an
inhibitor with lower toxicity that still interferes with Stx
trafficking (83). Another small molecule, Retro-2, blocks
Stx from reaching the trans-Golgi network (84, 85) and
is effective against ricin, a toxin that traffics in the same
manner as the Stxs. Recently, a derivative of Retro-2,
Retro-2cycl, was identified. Retro-2cycl is 100-fold more
active on HeLa cells than Retro-2 (86). However, the
drawback to Retro-2cycl and derivatives is that the cells
needed to be pretreated with the drug to observe pro-
tection from the toxin (84, 86). Another compound
known to protect cells from Stx, chloroquine, was also
recently shown to permit the toxin to reach the ER (87).
Since chloroquine does not inhibit the enzymatic activity
of Stx nor degrade the toxin in vitro, the drug likely acts
by inhibiting the toxin from exiting the ER.
Further small-molecule screens have identified other
compounds that inhibit Stx and ricin transport within
Vero cells (88) or the enzymatic activity of both toxins
(which have the same mode of action) (89). Another
small molecule, eeyarestatin I, which interferes with
9
Melton-Celsa and O’Brien
intracellular trafficking among cellular compartments,
was shown to cause a lag in protein synthesis inhibi-
tion in HeLa cells by Stx (90). Finally, nitrobenzylthio-
inosine was shown in 2002 to inhibit the trafficking
of Stx1, trapping the toxin within the early endosome
(91); however, no additional information on the use
of this compound to protect cells or animals has been
published.
A recent report suggests that manganese inhibits in-
tracellular trafficking of the B subunit of Stx (92), a
finding that may or may not extend to the holotoxin.
However, manganese treatment increased the viability
of HeLa cells incubated with Stx1 in that study and
protected BALB/c mice injected with 500 ng of Stx1
and injected with daily doses of manganese (at least
10 mg/kg daily injections were required). Whether such
levels of manganese would be reasonably achievable in
a patient is not clear. In addition, manganese fails to
protect HeLa cells from Stx2 intoxication (93), a finding
that makes the potential use of manganese less likely as
Stx2 is more commonly associated with HUS develop-
ment than Stx1.
A COMPOUND THAT INTERFERES
WITH TOXIN PROCESSING
Because the A subunit must be cleaved by a furin- or
trypsin-like protease so that the A1 or enzymatically
active moiety of the toxin may be separated from the
holotoxin, furin inhibitors were tested in a cell-based
assay for the capacity to protect HEp-2 cells from Stx
(94). Although one of the inhibitors showed moderate
inhibition activity against Stx, the inhibition was over-
come at higher concentrations of Stx. Furthermore, since
the toxin can be cleaved by enzymes in intestinal mucus
(35), as well as by intracellular proteases (95), and be-
cause Stx mutated at the trypsin-sensitive site can still be
cleaved (96, 97), we suspect that it will be difficult to
protect animals from Stx with protease inhibitors.
Therapies That Interfere with
the Cellular Response to Stx
Inhibitors of mitogen-activated protein kinase pathways
were used to assess potential protective efficacy against
the Stx2-mediated ribotoxic stress response in HCT-
8 cells or for oral gavage of Stx2 into infant rabbits (98).
The authors observed modest protective capacity by
imatinib in HCT-8 cells and against some aspects of Stx2
intoxication in the animals, specifically heterophil infil-
tration into the intestine. That study indicates that the
infant rabbit model of Stx2 gavage may prove useful for
10
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
the evaluation of therapeutics. However, such a model
would be expensive, and determining effective thera-
peutic doses and the timing for those doses might prove
challenging.
A recent paper suggests that an inhibitor of apoptosis,
ouabain, protects rat renal proximal tubules from Stx-
mediated cell death (99). Kidneys from ouabain-treated
mice that were given about four 50% lethal doses of
Stx2 showed reduced podocyte depletion as compared
to phosphate-buffered saline–treated mice. However, no
data were reported on whether the ouabain could pro-
tect the mice from Stx2-mediated lethality.
TREATMENT ONCE HUS IS DIAGNOSED
What do you do for patients who already have HUS? We
leave discussion of specific clinical interventions such as
dialysis and apheresis for medical experts. In terms of a
therapeutic that may interfere in the etiology of HUS,
Lapeyraque and colleagues reported in 2011 the use of
eculizumab (an antibody directed against complement
protein C5 used to treat atypical HUS, a disease that
may arise due to complement dysregulation) in three
children with HUS due to STEC infection (100). Al-
though the sick children exhibited improvements in
platelet counts and reductions in lactate dehydrogenase,
plasma exchange and hemodialysis were used concur-
rently. In addition, no children in the small study re-
ceived just antibody infusion alone. Eculizumab was
also used to treat many patients with HUS during the
O104:H4 outbreak in Germany in 2011. Although there
was no evidence of efficacy for eculizumab in treating
HUS from the outbreak in Germany, the cohorts are
difficult to compare because the patients received other
concurrent treatments, such as plasma exchange, anti-
biotic therapy, immunoglobulin G immunoadsorption,
and dialysis (14, 101–105). In addition, many of the
HUS patients given eculizumab were quite ill and had
neurological complications. Therefore, a randomized
controlled trial is necessary to answer the question
about the efficacy of eculizumab for the treatment of Stx-
associated HUS.
CONCLUSION
Since the first outbreak of STEC infection in the United
States in 1982, much research has focused on ways to
neutralize the action of the Stxs. At this time, only
SYNSORB Pk, urtoxazumab, and the Shiga monoclonal
antibodies cαStx1 and cαStx2 have been tested in phase I
and II trials intended to treat or prevent HUS. Efforts to
ASMscience.org/MicrobiolSpectrum
 New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli
prevent STEC infection, such as elimination from the
food supply and proper food handling, are important as
well as we try to stop the development of the potentially
deadly HUS. Lastly, consideration should be given to the
development of a vaccine against the Stxs, minimally for
those in research or clinical labs who are exposed to
STEC.
ACKNOWLEDGMENT
We declare no conflicts of interest with regard to the manuscript.
REFERENCES
1. Rosales A, Hofer J, Zimmerhackl LB, Jungraithmayr TC, Riedl M,
Giner T, Strasak A, Orth-Holler D, Wurzner R, Karch H. 2012. Need for
long-term follow-up in enterohemorrhagic Escherichia coli-associated
hemolytic uremic syndrome due to late-emerging sequelae. Clin Infect Dis
54:1413–1421.
2. Palermo MS, Exeni RA, Fernandez GC. 2009. Hemolytic uremic syn-
drome: pathogenesis and update of interventions. Expert Rev Anti Infect
Ther 7:697–707.
3. Spinale JM, Ruebner RL, Copelovitch L, Kaplan BS. 2013. Long-term
outcomes of Shiga toxin hemolytic uremic syndrome. Pediatr Nephrol
28:2097–2105.
4. Thielman NM, Guerrant RL. 2004. Clinical practice. Acute infectious
diarrhea. N Engl J Med 350:38–47.
5. Molbak K, Mead PS, Griffin PM. 2002. Antimicrobial therapy in
patients with Escherichia coli O157:H7 infection. JAMA 288:1014–1016.
6. Nelson JM, Griffin PM, Jones TF, Smith KE, Scallan E. 2011. Anti-
microbial and antimotility agent use in persons with Shiga toxin-
producing Escherichia coli O157 infection in FoodNet sites. Clin Infect
Dis 52:1130–1132.
7. Karch H, Goroncy-Bermes P, Opferkuch W, Kroll H-P, O’Brien A.
1985. Subinhibitory concentrations of antibiotics modulate amount of
Shiga-like toxin produced by Escherichia coli, p 239–245. In Adam D,
Hahn H, Opferkuch W (ed), The Influence of Antibiotics on the Host-
Parasite Relationship II. SpringerVerlag, Berlin, Germany.
8. Wong CS, Jelacic S, Habeeb RL, Watkins SL, Tarr PI. 2000. The risk of
the hemolytic-uremic syndrome after antibiotic treatment of Escherichia
coli O157:H7 infections. N Engl J Med 342:1930–1936.
9. Slutsker L, Ries AA, Malone K, Wells JG, Greene KD, Griffin PM.
1998. A nationwide case-control study of Escherichia coli O157:H7 in-
fection in the United States. J Infect Dis 177:962–966.
10. Panos GZ, Betsi GI, Falagas ME. 2006. Systematic review: are
antibiotics detrimental or beneficial for the treatment of patients with
Escherichia coli O157:H7 infection? Aliment Pharmacol Ther 24:731–742.
11. Smith KE, PWilke PR, Reiter PL, Hedican EB, Bender JB, Hedberg
CW. 2012. Antibiotic treatment of Escherichia coli O157 infection and
the risk of hemolytic uremic syndrome, Minnesota. Pediatr Infect Dis J
31:37–41.
12. Ikeda K, Ida O, Kimot K, Takatorige T, Nakanishi N, Tatar K. 1999.
Effect of early fosfomycin treatment on prevention of hemolytic uremic
syndrome accompanying Escherichia coli O157:H7 infection. Clin
Nephrol 52:357–362.
13. Fukushima H, Hashizume T, Morita Y, Tanaka J, Azuma K,
Mizumoto Y, Kaneno M, Matsuura M, Konma K, Kitani T. 1999.
Clinical experiences in Sakai City Hospital during the massive outbreak of
enterohemorrhagic Escherichia coli O157 infections in Sakai City, 1996.
Pediatr Int 41:213–217.
14. Menne J, Nitschke M, Stingele R, Abu-Tair M, Beneke J, Bramstedt J,
Bremer JP, Brunkhorst R, Busch V, Dengler R, Deuschl G, Fellermann K,
Fickenscher H, Gerigk C, Goettsche A, Greeve J, Hafer C, Hagenmuller F,
ASMscience.org/MicrobiolSpectrum
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
Haller H, Herget-Rosenthal S, Hertenstein B, Hofmann C, Lang M,
Kielstein JT, Klostermeier UC, Knobloch J, Kuehbacher M, Kunzendorf
U, Lehnert H, Manns MP, Menne TF, Meyer TN, Michael C, Munte T,
Neumann-Grutzeck C, Nuernberger J, Pavenstaedt H, Ramazan L,
Renders L, Repenthin J, Ries W, Rohr A, LRump LC, Samuelsson O, Sayk
F, Schmidt BM, Schnatter S, Schocklmann H, Schreiber S, von Seydewitz
CU, Steinhoff J, Stracke S, Suerbaum S, van de Loo A, Vischedyk M,
Weissenborn K, Wellhoner P, Wiesner M, Zeissig S, Buning J, Schiffer M,
Kuehbacher T. 2012. Validation of treatment strategies for entero-
haemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic
syndrome: case-control study. BMJ 345:e4565.
15. Geerdes-Fenge HF, Lobermann M, Nurnberg M, Fritzsche C,
Koball S, Henschel J, Hohn R, Schober HC, Mitzner S, Podbielski A,
Reisinger EC. 2013. Ciprofloxacin reduces the risk of hemolytic uremic
syndrome in patients with Escherichia coli O104:H4-associated diarrhea.
Infection 41:669–673.
16. Binks S, Regan K, Richenberg J, Chevassut T. 2012. Microbes without
frontiers: severe haemolytic-uraemic syndrome due to E coli O104:H4.
BMJ Case Rep 2012.
17. Colic E, Dieperink H, Titlestad K, Tepel M. 2011. Management of an
acute outbreak of diarrhoea-associated haemolytic uraemic syndrome
with early plasma exchange in adults from southern Denmark: an ob-
servational study. Lancet 378:1089–1093.
18. Bielaszewska M, Idelevich EA, Zhang W, Bauwens A, Schaumburg F,
Mellmann A, Peters G, Karch H. 2012. Effects of antibiotics on Shiga
toxin 2 production and bacteriophage induction by epidemic Escherichia
coli O104:H4 strain. Antimicrob Agents Chemother 56:3277–3282.
19. Nitschke M, Sayk F, Hartel C, Roseland RT, Hauswaldt S, Steinhoff J,
Fellermann K, Derad I, Wellhoner P, Buning J, Tiemer B, Katalinic A,
Rupp J, Lehnert H, Solbach W, Knobloch JK. 2012. Association between
azithromycin therapy and duration of bacterial shedding among patients
with Shiga toxin-producing enteroaggregative Escherichia coli O104:H4.
JAMA 307:1046–1052.
20. Isogai E, Isogai H, Hayashi S, Kubota T, Kimura K, Fujii N, Ohtani
T, Sato K. 2000. Effect of antibiotics, levofloxacin and fosfomycin, on a
mouse model with Escherichia coli O157 infection. Microbiol Immunol
44:89–95.
21. Zhang X, McDaniel AD, Wolf LE, Keusch GT, Waldor MK,
Acheson DW. 2000. Quinolone antibiotics induce Shiga toxin-encoding
bacteriophages, toxin production, and death in mice. J Infect Dis 181:
664–670.
22. Yoshimura K, Fujii J, Taniguchi H, nd S. Yoshida S. 1999. Chemo-
therapy for enterohemorrhagic Escherichia coli O157:H infection in a
mouse model. FEMS Immunol Med Microbiol 26:101–108.
23. Kurioka T, Yunou Y, Harada H, Kita E. 1999. Efficacy of antibiotic
therapy for infection with Shiga-like toxin-producing Escherichia coli
O157:H7 in mice with protein-calorie malnutrition. Eur J Clin Microbiol
Infect Dis 18:561–571.
24. Zangari T, Melton-Celsa AR, Panda A, Boisen N, Smith MA, Taratov
I, De Tolla LJ, Nataro JP, O’Brien AD. 2013. Virulence of the Shiga toxin
type 2-expressing Escherichia coli O104:H4 German outbreak isolate in
two animal models. Infect Immun 81:1562–1574.
25. Al-Qarawi S, Fontaine RE, Al-Qahtani MS. 1995. An outbreak of
hemolytic uremic syndrome associated with antibiotic treatment of hos-
pital inpatients for dysentery. Emerg Infect Dis 1:138–140.
26. Scholl D, Cooley M, Williams SR, Gebhart D, Martin D, Bates A,
Mandrell R. 2009. An engineered R-type pyocin is a highly specific and
sensitive bactericidal agent for the food-borne pathogen Escherichia coli
O157:H7. Antimicrob Agents Chemother 53:3074–3080.
27. Ritchie JM, Greenwich JL, Davis BM, Bronson RT, Gebhart D,
Williams SR, Martin D, Scholl D, Waldor MK. 2011. An Escherichia coli
O157-specific engineered pyocin prevents and ameliorates infection by
E. coli O157:H7 in an animal model of diarrheal disease. Antimicrob
Agents Chemother 55:5469–5474.
11
Melton-Celsa and O’Brien
28. Rasko DA, Moreira CG, Li de R, Reading NC, Ritchie JM, Waldor
MK, Williams N, Taussig R, Wei S, Roth M, Hughes DT, Huntley JF,
Fina MW, Falck JR, Sperandio V. 2008. Targeting QseC signaling and
virulence for antibiotic development. Science 321:1078–1080.
29. Crane JK, Byrd IW, Boedeker EC. 2011. Virulence inhibition by zinc
in Shiga-toxigenic Escherichia coli. Infect Immun 79:1696–1705.
30. Tesh VL. 2012. The induction of apoptosis by Shiga toxins and ricin.
Curr Top Microbiol Immunol 357:137–178.
31. Scheut F, Teel LD, Beutin L, Pierard D, Buvens G, Karch H, Mellmann
A, Caprioli A, Tozzoli R, Morabito S, Strockbine NA, Melton-Celsa AR,
Sanchez M, Persson S, O’Brien AD. 2012. Multicenter evaluation of a
sequence-based protocol for subtyping Shiga toxins and standardizing Stx
nomenclature. J Clin Microbiol 50:2951–2963.
32. Bielaszewska M, Friedrich AW, Aldick T, Schurk-Bulgrin R, Karch H.
2006. Shiga toxin activatable by intestinal mucus in Escherichia coli iso-
lated from humans: predictor for a severe clinical outcome. Clin Infect Dis
43:1160–1167.
33. Friedrich AW, Bielaszewska M, Zhang WL, Pulz M, Kuczius T,
Ammon A, Karch H. 2002. Escherichia coli harboring Shiga toxin 2 gene
variants: frequency and association with clinical symptoms. J Infect Dis
185:74–84.
34. Lindgren SW, Samuel JE, Schmitt CK, O’Brien AD. 1994. The specific
activities of Shiga-like toxin type II (SLT-II) and SLT-II-related toxins of
enterohemorrhagic Escherichia coli differ when measured by Vero cell
cytotoxicity but not by mouse lethality. Infect Immun 62:623–631.
35. Melton-Celsa AR, Darnell SC, O’Brien AD. 1996. Activation of Shiga-
like toxins by mouse and human intestinal mucus correlates with virulence
of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected,
streptomycin-treated mice. Infect Immun 64:1569–1576.
36. Silberstein C, Copeland DP, Chiang W-L, Repetto HA, Ibarra C.
2008. A glucosylceramide synthase inhibitor prevents the cytotoxic effects
of Shiga toxin-2 on human renal tubular epithelial cells. J Epithel Biol
Pharmacol 1:71–75.
37. Silberstein C, Lucero MS, Zotta E, Copeland DP, Lingyun L, Repetto
HA, Ibarra C. 2011. A glucosylceramide synthase inhibitor protects rats
against the cytotoxic effects of Shiga toxin 2. Pediatr Res 69:39–394.
38. Armstrong GD, Fodor E, Vanmaele R. 1991. Investigation of Shiga-
like toxin binding to chemically synthesized oligosaccharide sequences.
J Infect Dis 164:1160–1167.
39. Trachtman H, Cnaan A, Christen E, Gibbs K, Zhao S, Acheson DW,
Weiss R, Kaskel FJ, Spitzer A, Hirschman GH. 2003. Effect of an oral
Shiga toxin-binding agent on diarrhea-associated hemolytic uremic syn-
drome in children: a randomized controlled trial. JAMA 290:1337–1344.
40. Takeda T, Yoshino K, Adachi E, Sato Y, Yamagata K. 1999. In vitro
assessment of a chemically synthesized Shiga toxin receptor analog at-
tached to chromosorb P (Synsorb Pk) as a specific absorbing agent of Shiga
toxin 1 and 2. Microbiol Immunol 43:331–337.
41. Armstrong GD, Rowe PC, Goodyer P, Orrbine E, Klassen TP,
Wells G, MacKenzie A, Lior H, Blanchard C, Auclair F, et al. 1995. A
phase I study of chemically synthesized verotoxin (Shiga-like toxin) Pk-
trisaccharide receptors attached to chromosorb for preventing hemolytic-
uremic syndrome. J Infect Dis 171:1042–1045.
42. Kitov PI, Sadowska JM, Mulvey G, Armstrong GD, Ling H, Pannu
NS, Read RJ, Bundle DR. 2000. Shiga-like toxins are neutralized by tai-
lored multivalent carbohydrate ligands. Nature 403:66–672.
43. Mulve GL, Marcato P, Kitov PI, Sadowska J, Bundle DR, Armstrong
GD. 2003. Assessment in mice of the therapeutic potential of tailored,
multivalent Shiga toxin carbohydrate ligands. J Infect Dis 187:640–649.
44. Nishikawa K, Matsuoka K, Kita E, Okabe N, Mizuguchi M, Hino K,
Miyazawa S, Yamasaki C, Aoki J, Takashima S, Yamakawa Y, Nishijima
M, Terunuma D, Kuzuhara H, Natori Y. 2002. A therapeutic agent
with oriented carbohydrates for treatment of infections by Shiga toxin-
producing Escherichia coli O157:H7. Proc Natl Acad Sci USA 99:7669–
7674.
12
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
45. Watanabe M, Matsuoka K, Kita E, Igai K, Higashi N, Miyagawa A,
Watanabe T, Yanoshita R, Samejima Y, Terunuma D, Natori Y,
Nishikawa K. 2004. Oral therapeutic agents with highly clustered
globotriose for treatment of Shiga toxigenic Escherichia coli infections.
J Infect Dis 189:360–368.
46. Watanabe-Takahashi M, Sato T, Dohi T, Noguchi N, Kano F,
Murata M, Hamabata T, Natori Y, Nishikawa K. 2010. An orally ap-
plicable Shiga toxin neutralizer functions in the intestine to inhibit the
intracellular transport of the toxin. Infect Immun 78:177–183.
47. Paton AW, Morona R, Paton JC. 2001. Neutralization of Shiga toxins
Stx1, Stx2c, and Stx2e by recombinant bacteria expressing mimics of
globotriose and globotetraose. Infect Immun 69:1967–1970.
48. Li X, Wu P, Cheng S, Lv X. 2012. Synthesis and assessment of
globotriose-chitosan conjugate, a novel inhibitor of Shiga toxins produced
by Escherichia coli. J Med Chem 55:2702–2710.
49. Nishikawa K, Watanabe M, Kita E, Igai K, Omata K, Yaffe MB,
Natori Y. 2006. A multivalent peptide library approach identifies a novel
Shiga toxin inhibitor that induces aberrant cellular transport of the toxin.
FASEB J 20:2597–2599.
50. Stearns-Kurosawa DJ, Collins V, Freeman S, Debord D, Nishikawa K,
Oh SY, Leibowitz CS, Kurosawa S. 2011. Rescue from lethal Shiga toxin
2-induced renal failure with a cell-permeable peptide. Pediatr Nephrol
26:2031–2039.
51. Tsutsuki K, Watanabe-Takahashi M, Takenaka Y, Kita E, Nishikawa
K. 2013. Identification of a peptide-based neutralizer that potently inhibits
both Shiga toxins 1 and 2 by targeting specific receptor-binding regions.
Infect Immun 81:2133–2138.
52. Paton AW, Morona R, Paton JC. 2000. A new biological agent for
treatment of Shiga toxigenic Escherichia coli infections and dysentery in
humans. Nat Med 6:265–270.
53. Paton JC, Rogers TJ, Morona R, Paton AW. 2001. Oral adminis-
tration of formaldehyde-killed recombinant bacteria expressing a mimic of
the Shiga toxin receptor protects mice from fatal challenge with Shiga-
toxigenic Escherichia coli. Infect Immun 69:1389–-1393.
54. Pinyon RA, Paton JC, Paton AW, JBotten JA, Morona R. 2004.
Refinement of a therapeutic Shiga toxin-binding probiotic for human
trials. J Infect Dis 189:1547–1555.
55. Bitzan M, Klemt M, Steffens R, Muller-Wiefel DE. 1993. Differences
in verotoxin neutralizing activity of therapeutic immunoglobulins and sera
from healthy controls. Infection 21:140–145.
56. Bitzan M, Ludwig K, Klemt M, Konig H, Buren J, Muller-Wiefel DE.
1993. The role of Escherichia coli O157 infections in the classical
(enteropathic) haemolytic uraemic syndrome: results of a Central Euro-
pean, multicentre study. Epidemiol Infect 110:183–196.
57. Kimura T, Tani S, Matsumoto Yi Y, Takeda T. 2001. Serum amyloid
P component is the Shiga toxin 2-neutralizing factor in human blood.
J Biol Chem 276:41576–41579.
58. Kimura T, Tani S, Motoki M, Matsumoto Y. 2003. Role of Shiga toxin
2 (Stx2)-binding protein, human serum amyloid P component (HuSAP), in
Shiga toxin-producing Escherichia coli infections: assumption from in vitro
and in vivo study using HuSAP and anti-Stx2 humanized monoclonal an-
tibody TMA-15. Biochem Biophys Res Commun 305:1057–1060.
59. Armstrong GD, Mulvey GL, Marcato P, Griener TP, Kahan MC,
Tennent GA, Sabin CA, Chart H, Pepys MB. 2006. Human serum amy-
loid P component protects against Escherichia coli O157:H7 Shiga toxin 2
in vivo: therapeutic implications for hemolytic-uremic syndrome. J Infect
Dis 193:1120–1124.
60. Griener TP, Strecker JG, Humphries RM, Mulvey GL, Fuentealba C,
Hancock RE, Armstrong GD. 2011. Lipopolysaccharide renders trans-
genic mice expressing human serum amyloid P component sensitive to
Shiga toxin 2. PLoS One 6:e21457.
61. Marcato P, Vander Helm K, Mulvey GL, Armstrong GD. 2003. Serum
amyloid P component binding to Shiga toxin 2 requires both a subunit and
B pentamer. Infect Immun 71:607–6078.
ASMscience.org/MicrobiolSpectrum
 New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli
62. Donohue-Rolfe A, Kondova I, Mukherjee J, Chios K, Hutto D,
Tzipori S. 1999. Antibody-based protection of gnotobiotic piglets infected
with Escherichia coli O157:H7 against systemic complications associated
with Shiga toxin 2. Infect Immun 67:3645–3648.
63. Perera LP, Marques LR, O’Brien AD. 1988. Isolation and character-
ization of monoclonal antibodies to Shiga-like toxin II of entero-
hemorrhagic Escherichia coli and use of the monoclonal antibodies in a
colony enzyme-linked immunosorbent assay. J Clin Microbiol 26:2127–
2131.
64. Strockbine NA, Marques LR, Holmes RK, O’Brien AD. 1985.
Characterization of monoclonal antibodies against Shiga-like toxin from
Escherichia coli. Infect Immun 50:69–700.
65. Smith MJ, Melton-Celsa AR, Sinclair JF, Carvalho HM, Robinson
CM, O’Brien AD. 2009. Monoclonal antibody 11E10, which neutralizes
shiga toxin type 2 (Stx2), recognizes three regions on the Stx2 A subunit,
blocks the enzymatic action of the toxin in vitro, and alters the overall
cellular distribution of the toxin. Infect Immun 77:2730–2740.
66. Smith MJ, Carvalho HM, Melton-Celsa AR, O’Brien AD. 2006. The
13C4 monoclonal antibody that neutralizes Shiga toxin type 1 (Stx1)
recognizes three regions on the Stx1 B subunit and prevents Stx1 from
binding to its eukaryotic receptor globotriaosylceramide. Infect Immun
74:6992–6998.
67. Sauter KA, Melton-Celsa AR, Larkin K, Troxell ML, O’Brien AD,
Magun BE. 2008. Mouse model of hemolytic-uremic syndrome caused by
endotoxin-free Shiga toxin 2 (Stx2) and protection from lethal outcome by
anti-Stx2 antibody. Infect Immun 76:4469–4478.
68. Edwards AC, Melton-Celsa AR, Arbuthnott K, Stinson JR, Schmitt
CK, Wong HC, O’Brien AD. 1998. Vero cell neutralization and mouse
protective efficacy of humanized monoclonal antibodies against Esche-
richia coli toxins Stx1 and Stx2, p 388–392. In Kaper JB, O’Brien AD (ed),
Escherichia coli O157:H7 and Other Shiga Toxin-Producing E. coli
Strains. ASM Press, Washington, DC.
69. Dowling TC, Chavaillaz PA, Young DG, Melton-Celsa A, O’Brien A,
Thuning-Roberson C, Edelman R, Tacket CO. 2005. Phase 1 safety and
pharmacokinetic study of chimeric murine-human monoclonal antibody c
alpha Stx2 administered intravenously to healthy adult volunteers. Anti-
microb Agents Chemother 49:1808–1812.
70. Bitzan M, Poole R, Mehran M, Sicard E, Brockus C, Thuning-
Roberson C, Riviere M. 2009. Safety and pharmacokinetics of chimeric
anti-Shiga toxin 1 and anti-Shiga toxin 2 monoclonal antibodies in healthy
volunteers. Antimicrob Agents Chemother 53:3081–3087.
71. Nakao H, Kataoka C, Kiyokawa N, Fujimoto J, Yamasaki S, Takeda
T. 2002. Monoclonal antibody to Shiga toxin 1, which blocks receptor
binding and neutralizes cytotoxicity. Microbiol Immunol 46:777–780.
72. Nakao H, Kiyokawa N, Fujimoto J, Yamasaki S, Takeda T. 1999.
Monoclonal antibody to Shiga toxin 2 which blocks receptor binding and
neutralizes cytotoxicity. Infect Immun 67:5717–5722.
73. Kimura T, Co MS, Vasquez M, Wei S, Xu H, Tani S, Sakai Y,
Kawamura T, Matsumoto Y, Nakao H, Takeda T. 2002. Development of
humanized monoclonal antibody TMA-15 which neutralizes Shiga toxin
2. Hybrid Hybridomics 21:161–168.
74. Yamagami S, Motoki M, Kimura T, Izumi H, Takeda T, Katsuura Y,
Matsumoto Y. 2001. Efficacy of postinfection treatment with anti-Shiga
toxin (Stx) 2 humanized monoclonal antibody TMA-15 in mice lethally
challenged with Stx-producing Escherichia coli. J Infect Dis 184:738–742.
75. Lopez EL, Contrini MM, Glatstein E, Gonzalez Ayala S, Santoro R,
Allende D, Ezcurra G, Teplitz E, Koyama T, Matsumoto Y, Sato H, Sakai
K, Hoshide S, Komoriya K, Morita T, Harning R, Brookman S. 2010.
Safety and pharmacokinetics of urtoxazumab, a humanized monoclonal
antibody, against Shiga-like toxin 2 in healthy adults and in pediatric
patients infected with Shiga-like toxin-producing Escherichia coli. Anti-
microb Agents Chemother 54:239–243.
76. Mukherjee J, Chios K, Fishwild D, Hudson D, O’Donnell S, Rich SM,
Donohue-Rolfe A, Tzipori S. 2002. Production and characterization of
ASMscience.org/MicrobiolSpectrum
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
protective human antibodies against Shiga toxin 1. Infect Immun 70:
5896–5899.
77. Mukherjee J, Chios K, Fishwild D, Hudson D, O’Donnell S, Rich SM,
Donohue-Rolfe A, Tzipori S. 2002. Human Stx2-specific monoclonal
antibodies prevent systemic complications of Escherichia coli O157:H7
infection. Infect Immun 70:612–619.
78. Sheoran AS, Chapman S, Singh P, Donohue-Rolfe A, Tzipori S. 2003.
Stx2-specific human monoclonal antibodies protect mice against lethal
infection with Escherichia coli expressing Stx2 variants. Infect Immun
71:3125–3130.
79. Krautz-Peterson G, Chapman-Bonofiglio S, Boisvert K, Feng H,
Herman IM, Tzipori S, Sheoran AS. 2008. Intracellular neutralization of
Shiga toxin 2 by an a subunit-specific human monoclonal antibody. Infect
Immun 76:1931–1939.
80. Jeong KI, Chapman-Bonofiglio S, Singh P, Lee J, Tzipori S, Sheoran
AS. 2010. In vitro and in vivo protective efficacies of antibodies that
neutralize the RNA N-glycosidase activity of Shiga toxin 2. BMC
Immunol 11:16.
81. Akiyoshi DE, Sheoran AS, Rich CM, Richard L, Chapman-Bonofiglio
S, Tzipori S. 2010. Evaluation of Fab and F(ab′)2 fragments and isotype
variants of a recombinant human monoclonal antibody against Shiga
toxin 2. Infect Immun 78:1376-1382.
82. Spooner RA, Watson P, Smith DC, Boal F, Amessou M, Johannes L,
Clarkson GJ, Lord JM, Stephens DJ, Roberts LM. 2008. The secretion
inhibitor Exo2 perturbs trafficking of Shiga toxin between endosomes and
the trans-Golgi network. Biochem J 414:471–484.
83. Guetzoyan LJ, Spooner RA, Boal F, Stephens DJ, Lord JM, Roberts
LM, Clarkson GJ. 2010. Fine tuning Exo2, a small molecule inhibitor of
secretion and retrograde trafficking pathways in mammalian cells. Mol
Biosyst 6:2030–2038.
84. Stechmann B, Bai SK, Gobbo E, Lopez R, Merer G, Pinchard S,
Panigai L, Tenza D, Raposo G, Beaumelle B, Sauvaire D, Gillet D,
Johannes L, Barbier J. 2010. Inhibition of retrograde transport protects
mice from lethal ricin challenge. Cell 141:231–242.
85. Park JG, Kahn JN, Tumer NE, Pang YP. 2012. Chemical structure of
Retro-2, a compound that protects cells against ribosome-inactivating
proteins. Sci Rep 2:631.
86. Noel R, Gupta N, Pons V, Goudet A, Garcia-Castillo MD, Michau A,
Martinez J, Buisson DA, Johannes L, Gillet D, Barbier J, Cintrat JC. 2013.
N-methyl dihydroquinazolinones derivatives of Retro-2 with enhanced
efficacy against Shiga toxin. J Med Chem 56:3404–3413.
87. Dyve Lingelem AB, Bergan J, Sandvig K. 2012. Inhibitors of
intravesicular acidification protect against Shiga toxin in a pH-indepen-
dent manner. Traffic 13:443–454.
88. Saenz JB, Doggett TA, Haslam DB. 2007. Identification and charac-
terization of small molecules that inhibit intracellular toxin transport.
Infect Immun 75:4552–4561.
89. Wahome PG, Bai Y, Neal LM, Robertus JD, Mantis NJ. 2010.
Identification of small-molecule inhibitors of ricin and Shiga toxin using a
cell-based high-throughput screen. Toxicon 56:313–323.
90. Aletrari MO, McKibbin C, Williams H, Pawar V, Pietroni P, Lord
JM, Flitsch SL, Whitehead R, Swanton E, High S, Spooner RA. 2011.
Eeyarestatin 1 interferes with both retrograde and anterograde intracel-
lular trafficking pathways. PLoS One 6:e22713.
91. Sekino T, Kiyokawa N, Taguchi T, Ohmi K, Nakajima H, Suzuki T,
Furukawa S, Nakao H, Takeda T, Fujimoto J. 2002. Inhibition of Shiga
toxin cytotoxicity in human renal cortical epithelial cells by nitrobenzyl-
thioinosine. J Infect Dis 185:785–796.
92. Mukhopadhyay S, Linstedt AD. 2012. Manganese blocks intracellular
trafficking of Shiga toxin and protects against Shiga toxicosis. Science
335:332–335.
93. Mukhopadhyay S, Redler B, Linstedt AD. 2013. Shiga toxin-binding
site for host cell receptor GPP130 reveals unexpected divergence in toxin-
trafficking mechanisms. Mol Biol Cell 24:2311–2318.
13
Melton-Celsa and O’Brien
94. Becker GL, Lu Y, Hardes K, Strehlow B, Levesque C, Lindberg I,
Sandvig K, Bakowsky U, Day R, Garten W, Steinmetzer T. 2012. Highly
potent inhibitors of proprotein convertase furin as potential drugs for
treatment of infectious diseases. J Biol Chem 287:21992–22003.
95. Garred O, van Deurs B, Sandvig K. 1995. Furin-induced cleavage and
activation of Shiga toxin. J Biol Chem 270:10817–10821.
96. Garred O, Dubinina E, Holm PK, Olsnes S, van Deurs B, Kozlov JV,
Sandvig K. 1995. Role of processing and intracellular transport for opti-
mal toxicity of Shiga toxin and toxin mutants. Exp Cell Res 218:39–49.
97. Burgess BJ, Roberts LM. 1993. Proteolytic cleavage at arginine
residues within the hydrophilic disulphide loop of the Escherichia coli
Shiga-like toxin I A subunit is not essential for cytotoxicity. Mol Microbiol
10:171–179.
98. Stone SM, Thorpe CM, Ahluwalia A, Rogers AB, Obata F, Vozenilek
A, Kolling GLKan AV, Magun BE, Jandhyala DM. 2012. Shiga toxin 2-
induced intestinal pathology in infant rabbits is A-subunit dependent and
responsive to the tyrosine kinase and potential ZAK inhibitor imatinib.
Front Cell Infect Microbiol 2:135.
99. Burlaka I, Liu XL, Rebetz J, Arvidsson I, Yang L, Brismar H, Karpman
D, Aperia A. 2013. Ouabain protects against Shiga toxin-triggered apo-
ptosis by reversing the imbalance between Bax and Bcl-xL. J Am Soc
Nephrol 24:1413–1423.
100. Lapeyraque AL, Malina M, Fremeaux-Bacchi V, Boppel T,
Kirschfink M, Oualha M, Proulx F, Clermont MJ, Le Deist F, Niaudet P,
Schaefer F. 2011. Eculizumab in severe Shiga-toxin-associated HUS. N
Engl J Med 364:2561–2563.
101. Greinache A, Friesecke S, Abel P, Dressel A, Stracke S, Fiene M, Ernst
F, Selleng K, Weissenborn K, Schmidt BM, Schiffer M, Felix SB, Lerch
MM, Kielstein JT, Mayerle J. 2011. Treatment of severe neurological
deficits with IgG depletion through immunoadsorption in patients with
14
Downloaded from www.asmscience.org by
IP: 202.86.180.130
On: Thu, 19 Sep 2019 02:14:16
Escherichia coli O104:H4-associated haemolytic uraemic syndrome: a
prospective trial. Lancet 378:1166–1173.
102. Ullrich S, Bremer P, Neumann-Grutzeck C, Otto H, Ruther C, von
Seydewitz CU, Meyer GP, Ahmadi-Simab K, Rother J, Hogan B, Schwenk
W, Fischbach R, Caselitz J, Puttfarcken J, Huggett S, Tiedeken P, Pober J,
Kirkiles-Smith NC, Hagenmuller F. 2013. Symptoms and clinical course
of EHEC O104 infection in hospitalized patients: a prospective single
center study. PLoS One 8:e55278.
103. Loos S, Ahlenstiel T, Kranz B, Staude H, Pape L, Hartel C, Vester U,
Buchtala L, Benz K, Hoppe B, Beringer O, Krause M, Muller D, Pohl M,
Lemke J, Hillebrand G, Kreuzer M, Konig J, Wigger M, Konrad M,
Haffner D, Oh J, Kemper MJ. 2012. An outbreak of Shiga toxin-pro-
ducing Escherichia coli O104:H4 hemolytic uremic syndrome in
Germany: presentation and short-term outcome in children. Clin Infect
Dis 55:753–759.
104. Hauswaldt S, Nitschke M, Sayk F, Solbach W, Knobloch JK. 2013.
Lessons learned from uutbreaks of Shiga toxin producing Escherichia coli.
Curr Infect Dis Rep 15:9.
105. Kielstein JT, Beutel G, Fleig S, Steinhoff J, Meyer TN, Hafer C,
Kuhlmann U, Bramstedt J, Panzer U, Vischedyk M, Busch V, Ries W,
Mitzner S, Mees S, Stracke S, Nurnberger J, Gerke P, Wiesner M, Sucke B,
Abu-Tair M, Kribben A, Klause N, Schindler R, Merkel F, Schnatter S,
Dorresteijn EM, Samuelsson O, Brunkhorst R. 2012. Best supportive care
and therapeutic plasma exchange with or without eculizumab in Shiga-
toxin-producing E. coli O104:H4 induced haemolytic-uraemic syndrome:
an analysis of the German STEC-HUS registry. Nephrol Dial Transplant
27:3807–3815.
106. Trachtman H, Austin C, Lewinski M, Stahl RA. 2012. Renal and
neurological involvement in typical Shiga toxin-associated HUS. Nat Rev
Nephrol 8:658–669.
ASMscience.org/MicrobiolSpectrum